Synthesis, Characterization, and Antimicrobial Activity of Water … · Synthesis,...

Synthesis, Characterization, and Antimicrobial Activity of

Water-soluble, Tri-carboxylato Amphiphiles

Eko Winny Sugandhi

Dissertation submitted to the Faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

in

Chemistry

Richard D. Gandour, Chair Paul R. Carlier

Brian E. Hanson David G. I. Kingston

James M. Tanko

January 31, 2007 Blacksburg, Virginia

Keywords: multi-tailed, multi-headed, tri-carboxylato, dendritic, amphiphile, solubility, microorganism, biological and intrinsic activity

Copyright 2007, Eko W. Sugandhi

Synthesis, Characterization, and Antimicrobial Activity of Water-soluble, Tri-carboxylato Amphiphiles

Eko Winny Sugandhi

ABSTRACT

Many previous studies of biological activity in a homologous series of

amphiphiles have shown a cut-off effect, where the biological activity increases with an

increase in chain length, after which the activity plateaus or weakens. One factor

suspected to cause this problem is solubility issues. We have designed several series of

very hydrophobic, water-soluble amphiphiles to overcome this problem. Three

homologous series containing mobile hydrophobic moieties and two series of epimers

containing rigid cholestane moieties have been synthesized; the hydrophobic moiety is

connected to the first-generation, Newkome-type dendron via a ureido linker. We have

demonstrated that as tris(triethanolammonium) salts, these amphiphiles show excellent

solubility in water. The solubilities in aqueous solution of the three series containing

mobile hydrophobic moieties are 19,500 to 25,700 µM depending on the formula weight

of the homolog, while those containing rigid cholestane moieties are 18,900 and 17,400

μM.

Having eliminated the solubility issue, the antimicrobial activity against a broad

spectrum of microorganisms has been screened. We have demonstrated that the

antimicrobial activity depends on the amphiphile-series, species, chain-length, or epimer

specificities, as well as hydrophobicity. The one-tailed, tri-carboxylato amphiphiles are

iii

generally better than the other series, with two exceptions. First, the two-tailed tri-

carboxylato amphiphiles, 3CUr1(11)2 and 3CUr1(12)2, are more active against

Cryptococcus neoformans; in fact, both amphiphiles (MICs are 6.9 and 7.2 μM,

respectively) are considered to display good antifungal activity. Second, amphiphile

3CUr-β-cholestane, whose MIC is 27 μM, is more active against Staphylococcus

aureus. Overall, these new tri-carboxylato amphiphiles only exhibit moderate activity

with two promising leads.

Furthermore, we have demonstrated the intrinsic activity (MIC0) of the one-tailed,

tri-carboxylato amphiphile series (3CUrn) against Mycobacterium smegmatis. All the

MIC0‘s observed are at least 8-fold lower than the corresponding CMCs. Amphiphile

3CUr16 is the most active; the MIC0 is 100-fold smaller than the CMC. With this

consideration, we have suggested that the mechanism of action of the antimycobacterial

activity in amphiphile 3CUr16 is not related to detergency.

iv

ACKNOWLEDGMENTS

I would like to express my deepest appreciation to my research advisor, Dr.

Richard D. Gandour for his guidance, ideas, encouragement, and continual patience

throughout the duration of my graduate years. His method of gentle guidance made this

very difficult time in my life a little less stressful. I also acknowledge my committee

members⎯Dr. Paul Carlier, Dr. Brian Hanson, Dr. David Kingston, and Dr. James

Tanko⎯for their advice and assistance.

I thank Dr. Joseph Falkinham, III, and Ms. Myra Williams for their generous

assistance in doing biological assays. Beginning with no experience in handling

microorganisms, I learned a great deal in preparing the media, growing the culture, safe

handling, storing, running the assays, and finally interpreting the results.

I thank Mr. Tom Glass and Mr. William Bebout for their help and support in the

analytical services, and Mrs. Carla Slebodnick for her help in the X-ray crystallography

services. Many thanks go to Mrs. Kay Castagnoli and Mrs. Janice McGinty for their

special friendship. Special thanks also to Dr. Yang Yanyan, and current Gandour group

members, especially André William, Richard Macri, Brett Kite, Shauntrece Hardrict, for

making my stay a most enjoyable one.

Finally I would like to thank my husband Ernest for his unconditional love and

sacrifices in order to make this work a reality. Also, my parents and the rest of my

family, who are always loving and supporting me in their characteristic style, deserve

more gratitude than I can ever express.

v

DEDICATION

To my parents,

my husband Ernest, and my little Stanley.

vi

Table of Contents

List of Figures……………………………………………………………………………ix List of Schemes…………………………………………………………………………..xi List of Tables…………………………………………………………………………….xii Chapter I Background of Amphiphiles and Their Antimicrobial Activity .................... 1

I.1 Introduction to Amphiphiles ................................................................................ 1 I.2 Designing Amphiphiles as Antimicrobial Agents ............................................... 2

I.2.1 Detergency ..................................................................................................... 3 I.2.2 Hydrophilic Moiety of an Amphiphile .......................................................... 4 I.2.3 Hydrophobic Moiety of an Amphiphile......................................................... 5 I.2.4 Amphiphiles of Interest.................................................................................. 6

I.3 Antimicrobial Properties of Amphiphiles............................................................ 7 I.3.1 Overview on Previous Studies in Gandour’s Laboratory .............................. 7 I.3.2 The Cut-off Effect........................................................................................ 10

I.4 Literature Review in Designing Amphiphiles of Interest .................................. 11 I.4.1 Multi-headed Amphiphiles .......................................................................... 12 I.4.2 Newkome Dendrons..................................................................................... 13 I.4.3 Ureido Linker............................................................................................... 15 I.4.4 One-tailed Tri-carboxylato Amphiphile ...................................................... 18 I.4.5 Two-tailed Tri-carboxylato Amphiphile...................................................... 19

I.5 Cholestane-based Polyanionic Compounds Derived from Natural Product...... 22 I.6 Tri-carboxylato Amphiphile Containing Rigid Cholestane-based Hydrophobic

Moiety ................................................................................................................ 26 I.7 Microorganisms in Antimicrobial Screening..................................................... 26

I.7.1 Bacteria ........................................................................................................ 27 I.7.2 Fungi ............................................................................................................ 28

I.8 Summary ............................................................................................................ 29 References for Chapter 1 .................................................................................................. 29 Chapter II Synthesis of One-Tailed, Tri-headed Amphiphiles (3CUrn) ...................... 39

II.1 Preparation of WeisocyanateTM (3).................................................................... 40 II.2 Preparation of Alkan-1-amines .......................................................................... 41

II.2.1 Literature Review on Syntheses of Long-chain Alkan-1-amines (4) .......... 41 II.2.2 Literature Review on Syntheses of 1-Azidoalkanes (8) .............................. 43 II.2.3 Literature Review on Syntheses of 1-Methanesulfonyloxyalkanes (9) ....... 46 II.2.4 Literature Review on Syntheses of 1-Bromoalkanes (11) ........................... 46 II.2.5 Preferred Starting Material⎯Alkan-1-ols (12) or 1-Bromoalkanes (11) .... 47 II.2.6 Synthesis of Long-chain Alkan-1-amines (4) .............................................. 47

II.3 Synthesis of the 3EUrn Series (2a−e) ............................................................... 50 II.4 Synthesis of the 3CUrn Series (1a−e)............................................................... 51 II.5 X-ray Crystal Structure of Tri-tert-butyl Ester 3EUr16.................................... 51 II.6 CMCs (Critical Micelle Concentration) of Amphiphiles 3CUrn...................... 53 II.7 Experimental Procedures ................................................................................... 55

References for Chapter II.................................................................................................. 65 Chapter III Synthesis of Two-Tailed, Tri-headed Amphiphiles (3CUr(n)2 and

3CUr1(n)2) .................................................................................................. 73

vii

III.1 The 3CUr(n)2 Homologous Series .................................................................... 74 III.1.1 Preparation of Secondary Amines ............................................................... 75

III.1.1.1 Literature Review on Syntheses of N-Alkylalkan-1-amines (4a, 4c, and 4e) ....................................................................................................... 75

III.1.1.2 Literature Review on Syntheses of N-Alkylalkanamides (5) ............. 77 III.1.2 Synthesis of N-Alkylalkan-1-amines (4a, 4c, and 4e) ................................. 78 III.1.3 Synthesis of Two-tailed, Tri-headed 3CUr(n) 2 Homologous Series.......... 79

III.2 The 3CUr1(n)2 Homologous Series .................................................................. 81 III.2.1 Preparation of Primary Amines (10a−f) ...................................................... 82

III.2.1.1 Literature Review on Syntheses of 1-Alkylalkan-1-amines (10a−f) .. 82 III.2.1.2 Literature Review on Syntheses of Azidoalkanes .............................. 85 III.2.1.3 Literature Review on Syntheses of Methanesulfonyloxyalkanes ....... 86 III.2.1.4 Literature Review on Syntheses of Secondary Alkanols.................... 87

III.2.2 Synthesis of Amines (10a−f) ....................................................................... 89 III.2.3 Synthesis of Two-tailed, Tri-headed 3CUr1(n)2 Homologous Series......... 91

III.3 Comparison of Trends in Melting Temperatures (Tri-tert-butyl Esters vs Tri-carboxylic Acids) ............................................................................................... 93

III.4 Comparison of NMR Spectra of 3EUr(n)2 vs 3EUr1(n)2................................. 94 III.5 Experimental Procedures ................................................................................... 97

References for Chapter III .............................................................................................. 115 Chapter IV Synthesis of Tri-headed Amphiphiles Containing Cholestane-based

Structure on the Hydrophobic Moiety ....................................................... 122 IV.1 The 3CUr-z-cholestane Series........................................................................ 123

IV.1.1 Preparation of 5α-Cholestan-3-amine (4a−b) ........................................... 125 IV.1.1.1 Literature Review on Syntheses of Amines (4a−b).......................... 125 IV.1.1.2 Literature Review on Syntheses of 3-Azidocholestane (5a−b) ........ 127 IV.1.1.3 Literature Review on Syntheses of 3-Methanesulfonyloxy-5α-

cholestane (6a–b).............................................................................. 129 IV.1.1.4 Literature Review on Synthesis of 3α-Bromo-5α-cholestane (7) .... 129

IV.1.2 Synthesis of 5α-Cholestan-3-amines (4a−b) ............................................. 131 IV.1.3 Synthesis of 3CUr-z-cholestane (1a−b) ................................................... 133 IV.1.4 X-ray Structure of 3EUr-α-cholestane (2a) ............................................. 134

IV.2 The 3CUrnEs-z-cholestane Homologous Series............................................ 137 IV.2.1 Preparation of 3α- and 3β- Epimer of 5α-Cholestan-3-yl Aminoethanoate

(11a−b) ...................................................................................................... 138 IV.2.1.1 Literature Review on the Synthesis of 5α-Cholestan-3α-yl

Aminoethanoates (11a−b) ................................................................ 139 IV.2.1.2 Literature Review on the Synthesis of 5α-Cholestan-3α-yl

Azidoethanoate (12a−b) ................................................................... 140 IV.2.1.3 Literature Review on the Synthesis of 5α-Cholestan-3α-yl

Chloroethanoate (13a−b) .................................................................. 140 IV.2.2 Synthesis of 5α-Cholestan-3α-yl Aminoethanoate (11a−b) ..................... 141 IV.2.3 Synthesis of 3CUr1Es-z-cholestane (9a−b)............................................... 143

IV.3 Experimental Procedures ................................................................................. 144 References for Chapter IV .............................................................................................. 162

viii

Chapter V Antimicrobial Activity of Various Tri-headed Amphiphiles..................... 168 V.1 Introduction to Biological Assays.................................................................... 168

V.1.1 Measurement of Antimicrobial Activity.................................................... 168 V.1.2 Goals .......................................................................................................... 169

V.2 Recent Studies of Solubility of One-tailed Tri-carboxylato Amphiphiles....... 170 V.3 Results of Biological Assays ........................................................................... 171

V.3.1 Range of Concentration of Active Amphiphiles........................................ 174 V.3.2 The Most Active Amphiphiles within Each Homologous Series .............. 175 V.3.3 Various Specificities Patterns of Antimicrobial Activity .......................... 177

V.3.3.1 One- and Two-tailed Amphiphiles................................................... 177 V.3.3.2 Cholestane-based Amphiphiles........................................................ 179

V.3.4 Comparing Hydrophobicity and Antimicrobial Activity........................... 180 V.3.4.1 One- and Two-tailed Amphiphiles.................................................... 180 V.3.4.2 Cholestane-based Amphiphiles.......................................................... 182

V.4 Comparison with Prior Work........................................................................... 182 V.5 Conclusions...................................................................................................... 184 V.6 Experimental Procedures ................................................................................. 185

V.6.1 Preparation of Tri-carboxylato Amphiphiles Solutions in Aqueous Triethanolamine ......................................................................................... 185

V.6.2 Microbial Strains, Culture Conditions, and Preparations of Inocula for Susceptibility Testing................................................................................. 185

V.6.3 Measurement of MICs ............................................................................... 187 References for Chapter V................................................................................................ 188 Chapter VI Intrinsic Activity of the 3CUrn Homologus Series Against Mycobacterium

smegmatis................................................................................................... 192 VI.1 The “inoculum effect”...................................................................................... 194 VI.2 Intrinsic Activity of Homologous Amphiphiles .............................................. 194 VI.3 Results and Discussions................................................................................... 197

VI.3.1 MICs of the 3CUrn Series......................................................................... 197 VI.3.2 Comparison with Previous Work............................................................... 199 VI.3.3 Comparison of MICs and CMCs ............................................................... 199

VI.4 Conclusions...................................................................................................... 201 VI.5 Experimental Procedures ................................................................................. 201

VI.5.1 Microbial Strain, Culture Conditions, and Preparations of Inoculum for Susceptibility Testing................................................................................. 201

VI.5.2 Measurement of MICs ............................................................................... 202 References for Chapter VI .............................................................................................. 203 Chapter VII Accomplishments, Conclusions, and Future Work.................................... 205

VII.1 Accomplishments............................................................................................. 205 VII.2 Conclusions...................................................................................................... 206 VII.3 Future Work ..................................................................................................... 207

References for Chapter VII............................................................................................. 208 APPENDICES ................................................................................................................ 209 VITA............................................................................................................................... 354

ix

List of Figures Figure I.1 Common representations of an individual amphiphile ............................... 1 Figure I.2 Representation of amphiphile aggregates ................................................... 2 Figure I.3 Mono-, di-, and tri-ionic amphiphiles ......................................................... 5 Figure I.4 Schematic representations of proposed multi-headed amphiphiles ............ 7 Figure I.5 Plots of log MEC (M) vs chain length in spermicidal (left) and log MIC

(M) vs chain length in antifungal (right) assays of Z-n.............................. 8 Figure I.6 Plots of log MEC vs chain length in spermicidal assays of 1(p) and 2(p) 10 Figure I.7 Various multi-headed amphiphiles with anti-HIV properties................... 12 Figure I.8 Various Newkome-type dendrons (first and second generations) associated

with ferrocene, dansyl, pyrene, and viologen ........................................... 14 Figure I.9 Structures of one-tailed tri-carboxylato amphiphiles having amido and

carbamato linkers ...................................................................................... 15 Figure I.10 Various compounds containing ureido functional groups ........................ 17 Figure I.11 General representation and structure of one-tailed tri-carboxylato

amphiphiles ............................................................................................... 18 Figure I.12 Various fatty acids tested for skin penetration rates and skin irritation.... 21 Figure I.13 General representations of two-tailed, tri-headed amphiphile series........ 22 Figure I.14 Polyol and polyamine dendrimers associated with cholestane moieties .. 24 Figure I.15 General structures of cholestane-based, tri-headed amphiphile series ..... 26 Figure II.1 X-ray crystal structure of tri-tert-butyl ester 3EUr16, showing the packing

diagram and the intermolecular hydrogen bonding to water and to a neighboring molecule. Hydrogen-bond Donor(D)–Acceptor(A) distances and approximate ∠DHA: O(8)···N(2), 2.863(3) Å and 159.1°; O(15)···O(1), 2.718(4) Å and 170(4)°; O(15)···O(3), 2.843(4) Å and 169(4)°; N(4)···O(15), 2.832(3) Å and 153.4°. The other nitrogens on the ureido linker are just outside the range for significant hydrogen bonding. N(1)···O(8), 3.201(3) Å and 145.6°; N(3)···O(15), 3.074(3) Å and 141.6°................................................................................................................... 53

Figure III.1 General representation of two-tailed, tri-headed amphiphiles.................. 73 Figure IV.1 Structural representations of 5α- and 5β-cholestane .............................. 122 Figure IV.2 General structures of tri-headed amphiphiles series containing cholestane

moiety ..................................................................................................... 123 Figure IV.3 X-ray crystal structure of 3EUr-α-cholestane (2a), showing the packing

and the intermolecular hydrogen bonding to ethanol. Hydrogen-bond Donor(D)–Acceptor(A) distances and approximate ∠DHA: O(16)···O(1), 2.621(7) Å and 158.1°; O(15)···O(8), 2.625(6) Å and 161.0°; O(4)···O(16), 2.865(7) Å and 155(7)°; N(2)···O(15), 2.849(8) Å and 159.6°. The other nitrogens on the ureido linker are just outside the range for significant hydrogen bonding. N(3)···O(16), 3.035(7) Å and 141.6°; N(1)···O(15), 3.084(8) Å and 146.0°............................................................................. 135

Figure V.1 Relationship between log MIC of the 3CUrn series vs chain length for E. coli, M. smegmatis (left) and C. albicans, C. neoformans, and S. cerevisiae (right). Error bars (not shown for clarity) are ± 0.3. Lines connecting points are eye guides............................................................................... 175

x

Figure V.2 Relationship between log MIC of the 3CUr(n)2 and 3CUr1(n)2 series vs chain length for C. neoformans (left) and C. albicans (right). Error bars (not shown for clarity) are ± 0.3. Lines connecting points are eye guides................................................................................................................. 176

Figure V.3 Relationship between log MIC of one- and two-tailed amphiphiles vs logD for M. smegmatis (left) and C. albicans (right). Error bars (not shown for clarity) are ± 0.3...................................................................................... 181

Figure V.4 Relationship between log MIC and logD for C. neoformans (left) and S. cerevisiae (right). Error bars (not shown for clarity) are ± 0.3............... 182

Figure VI.1 Effect of initial cell density on inhibition of growth of M. smegmatis for the 3CUrn series. Combined data of two separate assays⎯3.2 to 3.2 × 107 CFU/mL and 500 to 5.0 × 108 CFU/mL. Error bars (not shown for clarity) are ± 0.3................................................................................................... 197

Figure VI.2 Effect of chain length on inhibition of growth of M. smegmatis of the 3CUrn series as function of the initial cell density. Error bars (not shown for clarity) are ± 0.3................................................................................. 198

Figure VI.3 Comparison of log CMC and log MIC of the 3CUrn series at the highest and lowest initial cell densities of M. smegmatis.................................... 200

xi

List of Schemes Scheme I.1 Synthesis of isocyanate monomer of Newkome-type dendron................. 18 Scheme II.1 Retrosynthesis of one-tailed, tri-headed amphiphiles (3CUrn) ............... 39 Scheme II.2 Synthetic scheme of the synthesis of isocyanate 3 ................................... 40 Scheme II.3 Retrosynthesis of alkan-1-amines ............................................................. 41 Scheme II.4 Synthetic scheme of amines 4d and 4e ..................................................... 49 Scheme II.5 The synthetic scheme of one-tailed, tri-headed amphiphiles (3CUrn) .... 50 Scheme III.1 Retrosynthesis of the 3CUr(n)2 homologous series ................................. 74 Scheme III.2 Preparation of N-heptylheptan-1-amines from heptan-1-amine3 and N-

heptylheptanamide14 ................................................................................. 76 Scheme III.3 Synthetic scheme of N-alkylalkan-1-amines 4a, 4c, and 4e ..................... 79 Scheme III.4 Synthetic scheme of the 3CUr(n)2 homologous series............................. 80 Scheme III.5 Retrosynthesis of 3CUr1(n)2 series.......................................................... 82 Scheme III.6 Retrosynthesis of amines 10a−f................................................................ 84 Scheme III.7 Synthetic scheme of the alkanamines 10a−f............................................. 90 Scheme III.8 Synthetic scheme of the preparation of 3CUr1(n)2 .................................. 92 Scheme IV.1 Retrosynthesis of 3CUr-z-cholestane .................................................... 124 Scheme IV.2 Retrosynthesis of epimer 5α-cholestan-3-amines................................... 125 Scheme IV.3 Synthetic scheme of the preparation of epimeric amines (4a−b) ........... 133 Scheme IV.4 Retrosynthesis of the 3CUrnEs-z-cholestane homologous series......... 138 Scheme IV.5 Retrosynthesis of 3α- and 3β- epimer of 5α-cholestan-3-yl

aminoethanoate ....................................................................................... 139 Scheme IV.6 Synthetic scheme of the preparation of amines 11a−b ........................... 143 Scheme V.1 Structures of dendritic tri-carboxylato amphiphiles ............................... 172 Scheme VI.1 Simplified kinetic model of inhibition of cell growth ............................ 195

xii

List of Tables Table I.1 Physical differences between Gram-negative and Gram-positive bacteria

................................................................................................................... 27 Table II.1 Preparations of amines 4 from different precursors.................................. 43 Table II.2 Preparations of azides 8 from different precursors ................................... 45 Table II.3 Preparation of bromoalkanes 11 from alcohols 12 ................................... 47 Table II.4 Isolated yields and melting ranges of the tri-tert-butyl esters 3EUrn ...... 50 Table II.5 Isolated yields and melting ranges of the triacids 3CUrn ........................ 51 Table II.6 CMCs of the 3CUrn homologous series .................................................. 54 Table II.7 Crystal data and structure refinement of 3EUr16..................................... 61 Table III.1 Melting ranges of the 3EUr(n)2 and 3CUr(n)2 homologous series ......... 81 Table III.2 Preparations of 1-alkylalkan-1-amines from different precursors ............ 83 Table III.3 Preparation of secondary azidoalkanes from secondary

methanesulfonyloxyalkanes...................................................................... 86 Table III.4 Preparation of secondary methanesulfonyloxyalkanes from secondary

alcohols ..................................................................................................... 87 Table III.5 Preparations of secondary alcohols 13a−f from different precursors....... 88 Table III.6 Isolated yields and melting ranges of intermediates and alkanamines 10a−f

................................................................................................................... 90 Table III.7 Melting ranges of the 3EUr1(n)2 and 3CUr1(n)2 .................................... 93 Table III.8 Comparison of trends in melting temperatures (tri-tert-butyl esters vs tri-

carboxylic acids) ....................................................................................... 93 Table IV.1 Preparation of amines 4a−b from different precursors........................... 126 Table IV.2 Preparation of azides 5a and 5b from various precursors....................... 128 Table IV.3 Preparations of 6a−b from 8a−b............................................................. 129 Table IV.4 Preparations of bromoalkane 7 from 8b and 8b’s derivatives................ 130 Table IV.5 Preparations of 5α-cholestan-3α-yl chloroethanoate from 5α-cholestan-

3β-ol (13a−b).......................................................................................... 141 Table IV.6 Crystal data and structure refinement for 3EUr-α-cholestane (2a)....... 152 Table V.1 MICs of tri-carboxylato amphiphiles against bacteria, yeasts, and a

filamentous fungus.................................................................................. 173

1

Chapter I Background of Amphiphiles and Their Antimicrobial Activity

I.1 Introduction to Amphiphiles

An amphiphile contains two distinct parts, which are in an opposite nature.1 The

word is translated from the Greek, amphi means ‘on both sides’ and phileein means ‘to

love’. Consequently, amphiphiles preferentially absorb at an interface of two immiscible

phases.

Depending on the solvent system used, the two opposite parts of an amphiphile

are referred to as solvent-loving (solvophilic) and solvent-hating (solvophobic). In

aqueous solutions, these two parts are known as hydrophilic (water-loving) and

hydrophobic (water-hating) moieties, respectively; the character of the former is polar,

which generally is depicted as a circle, and that of the latter is nonpolar, which generally

is depicted as a straight or wavy line(s) (Figure I.1).1

Figure I.1 Common representations of an individual amphiphile

Because of this unique property of absoption in the interface of two immiscible

phases, amphiphiles will distribute themselves so that the nonpolar moiety associates

with the nonpolar phase, and vice versa. When surrounded in one phase, for example in

an aqueous environment, amphiphiles tend to form the ‘other phase’ by assemblying their

nonpolar moieties. In other words, the hydrophobic tails cluster together to create a

nonpolar environment, while the polar moieties are still interacting with the polar

environment. At a given concentration and above, the amphiphiles associate with

a b

a. polar hydrophilic head; b. nonpolar hydrophobic tail

a b

2

themselves to form aggregates (Figure I.2); these aggregates are named based on the

shape of the aggregates form. One-tailed amphiphiles tend to form micelles, while two-

tailed amphiphiles tend to form bilayers.1 Higher concentrations of amphiphiles

eventually cause aggregation (crystalization or precipitation) where amphiphiles are no

longer soluble. The concentration at which micellar aggregates start to form is defined as

the critical micelle concentration (CMC).

micelle bilayer Figure I.2 Representation of amphiphile aggregates

Variation in temperatures affects the aggregation of an amphiphile. When a

solution of amphiphiles is cooled, the amphiphiles reach the point where they are not

soluble; this temperature is known as the Kraft temperature. Above the Kraft

temperature, the solubility of the amphiphile monomers increases. When the amphiphile

solution is heated up, it eventually reaches to a point where aggregation begins.1,2

I.2 Designing Amphiphiles as Antimicrobial Agents

Amphiphiles have been used as antimicrobial agents.3 Some advantages of using

amphiphiles as microbicides have been presented;4 they are inexpensive, fast-acting, and

have a broad spectrum of biological activity. However, there are also some drawbacks,

especially with those that can function as detergents.

3

I.2.1 Detergency

Often, the word amphiphile and surfactant are used synonymously. The word

surfactant itself comes from the phrase SURFace ACTive AgeNT, whose main traditional

application is as a soap and a detergent in various cleaning processes. The word

detergent is generally reserved for cleaning products containing surfactants and other

materials; these other materials function by improving the detergency or by adding

appealing properties, such as color (dye) or aroma (perfume). As cleaning agents,

surfactants remove the unwanted material, such as oils, greases, along with dust and clay,

from the surface of a solid. Based on the various physicochemical and mechanical

methods, this process is known as detergency.1 Detergency involves the micellar solution

of a surfactant, where the unwanted nonpolar materials interact with the hydrocarbon

core of the micelles. This interaction basically causes the unwanted material to be

solubilized within the core of the micelles. As micellar formation is needed, good

detergents must contain surfactants that have a low CMC. In other words, detergency

occurs at concentrations close to the CMC.1,5

In a recent study,6 detergency is claimed to cause irritation, which is a

disadvantage in any topical antimicrobial agent. As detergency occurs at concentrations

close to the CMC,5 controlling the CMC is necessary. Recently, it has been pointed out

that the active form of a good non-irritating amphiphile as an antimicrobial agent should

be in the monomeric form, not the micellar form.4 Consequently, it becomes our goal to

design amphiphiles that are not detergents, but antimicrobial agents. This means that the

amphiphiles of our interest should have high CMCs.

4

I.2.2 Hydrophilic Moiety of an Amphiphile

A hydrophilic moiety of a amphiphile is usually called the headgroup, and it is

either polar or charged. Depending on the type of their headgroup, amphiphiles are

usually classified as anionic, cationic, zwitterionic, and nonionic. The largest type of

amphiphiles in general use today falls in the anionic class, which constitutes 70−75% of

total amphiphile consumption.2 Anionic amphiphiles are mostly found in soaps,

detergents, toiletries, while cationic amphiphiles are often found in bactericides and hair

conditioners. Zwitterionic amphiphiles, which are also referred as amphoteric

amphiphiles, are generally milder on the skin than anionic or cationic; their main

applications are in shampoo and cosmetics. Nonionic amphiphiles are often found as

foam enhancers, detergents, and emulsifiers.1

The solubility and CMC of an amphiphile are affected by the type and number of

headgroups. In aqueous solutions, ionic amphiphiles are more soluble than the

corresponding nonionic amphiphiles. In studies of the effect of multi-headed ionic

amphiphiles on the CMCs, cationic7-9 and anionic10,11 amphiphiles, where chain length

are maintained equal, have been utilized. In these studies, the CMCs of mono-, di-, and

tri-ionic amphiphiles are measured (Figure I.3); compared to their mono-ionic

counterparts (1, 4), di- and tri-ionic amphiphiles (2, 3, 5, 6) display higher CMCs. In

other words, an increase in number of headgroups causes an increase in the CMCs. It is

suggested that higher CMCs are achieved because there are more dissociable group.7-9

5

ON

O

Br

O

ON

N

O

O

Br

Br

O

O N

O O

N

Br

Br

Br

1313

13

O N

O

O

O Kn

O

O Kn

O O K

O

O

K

O O K

O OK

nn = 7, 9, 11, 13, 15

1

2

3

4 5 6

Figure I.3 Mono-, di-, and tri-ionic amphiphiles

I.2.3 Hydrophobic Moiety of an Amphiphile

A hydrophobic moiety of an amphiphile is typically called the tail. This tail is

most commonly represented by a linear hydrocarbon. The chain length of the tail affects

the solubility and the CMC of an amphiphile. In the one-tailed amphiphile, an increase in

chain length causes a linear decrease in the log CMC, following the so-called Klevens

equation.2,12 In this equation, the relationship of log CMC vs the number of carbons in

the chain (nc), is stated as log10 CMC = A−Bnc. In this mathematical equation, the

intercept A and slope B are constants specific to the homologous series under constant

temperature and pressure.2 The intercept A depends on the ionic strength and type of

headgroups, while the slope B depends on the number of hydrophilic headgroups and the

number of hydrophobic alkyl chains.

The linear relationship of log CMC vs chain length has been observed

6

previously.13-15 A slope of –0.3 is observed with potassium salts of natural saturated fatty

acids.11 The CMCs of triethanolammonium salts of natural saturated fatty acids show a

dependence on chain length;16 the slopes have values of –0.06 for octanoate to

tetradecanoate and –0.6 for tetradecanoate to octadecanoate. Generally, one-headed

amphiphiles have slopes ranging from –0.26 to –0.33 for anionic and cationic

amphiphiles, and –0.45 to –0.52 for nonionic and zwitterionic amphiphiles.17

In different studies,10,11 log CMC of homologous series with different number of

headgroups are investigated. In these studies, the log CMC also decreases linearly as the

chain length increases in every homologous series. Among the different homologous

series compared, the slope of the log CMC vs chain length also changes with the different

number of headgroup attached.10 However, only slight changes in slope are observed in

the di- and tri-headed amphiphiles; diheaded amphiphiles RCH(COOK)2 and tri-headed

amphiphiles RCH(COOK)CH(COOK)2 have slopes of –0.22 to –0.23, respectively.10,11

I.2.4 Amphiphiles of Interest

We have discussed that the ideal active form of amphiphiles as antimicrobial

agents is the monomeric form; thus, the amphiphiles should have high CMCs.

Furthermore, CMCs have been shown to be affected by the hydrophilic and hydrophobic

moieties. Based on the previous discussion, we have designed multi-headed amphiphiles.

In addition, another modification made is to add a linker between the hydrophilic and

hydrophobic moieties.

In order to enrich the multi-headed amphiphiles series, we plan on attaching

different hydrophobic moieties (Figure I.4). Two natural sources of hydrophobic

moieties, such as one-tail hydrocarbons (a) and a steroidal backbone (d, e), will be

7

employed; a spacer will be introduced between the linker and the steroidal moiety. As

for the unnatural source of hydrophobic moiety, two symmetrical hydrocarbon chains

will be used in the design (b, c); furthermore, modification will be made on the chain

topology.

linker

head

head

head

tail

linker

head

head

head

tail

tail

linker

head

head

head

tail

tail

steroidalmoiety

spacerlinker

head

head

head

steroidalmoiety linker

head

head

head

a

b c

d e

Figure I.4 Schematic representations of proposed multi-headed amphiphiles

I.3 Antimicrobial Properties of Amphiphiles I.3.1 Overview on Previous Studies in Gandour’s Laboratory

A previous study18 in Gandour’s laboratory have shown that hydrocarbon chain

length directly affects biological activity. Homologous long-chain zwitterionic

amphipiles that are derived from acylcarnitine have been synthesized; these Z-n

amphiphiles display spermicidal and antifungal activity.18

8

N

OCO2

ROH

Z-nR = n-CnH2n+1, n = 10−18

NO

O O

acylcarnitine

OR

In the spermicidal assays (Figure I.5, left), the plot of the activity is represented as

log MEC (Minimum Effective Concentration) vs chain length. MEC is defined as the

lowest concentration of the amphiphile required to stop all sperm motility in 20 seconds.

In general, as the chain length increases, the activity of these amphiphiles increases,

represented by lower MECs. However, a breaking point is observed in the amphiphiles

with longer chains; in the spermicidal assays, the activity of Z-14 and Z-15 is similar. In

a recent report,4 MECs of Z-16−Z-18 cannot be determined because of solubility issues.

Figure I.5 Plots of log MEC (M) vs chain length in spermicidal (left) and log MIC (M) vs chain length in antifungal (right) assays of Z-n

In the antifungal assays (Figure I.5, right), the plot of the activity is represented as

log MIC vs chain length, where MIC is defined as the lowest concentration of amphiphile

-4

-3.5

-3

-2.5

-2

9 10 11 12 13 14 15

chain length

log

[MEC

] (M

)

-5.5

-5

-4.5

-4

-3.5

11 12 13 14 15

chain length

log

[MIC

] (M

)

9

required to inhibit the growth of Candida albicans. MICs of Z-10 and Z-11 are greater

than 3 × 10-4 M (data not shown); the exact MICs are not measured.4 Clearly, among Z-

12−Z-15, Z-15 shows the best antifungal activity. Within the plotted data, it is apparent

that as the chain becomes longer, the activity is better. No breaks or plateaus in activity

are observed within the homologs tested. It will be interesting to see how homologs with

longer chain lengths affect the antifungal activity. Unfortunately, under the assays

condition, Z-16−Z-18 are not soluble enough to give reliable MICs.4

In more recent studies,19,20 homologous series of di- and trihydroxylated cationic

amphiphiles, namely 2-hydroxy-N-(2-hydroxyethyl)-N,N,-dimethyl-1-alkaminium

bromide (1(p)) and 2-hydroxy-N,N-bis(2-hydroxyethyl)-N-methyl-1-alkaminium bromide

(2(p)), have been synthesized and investigated for spermicidal activity.

N

HOHO R

N

HOHO R

OHBr Br

1(p) 2(p)

R = n-CnH2n+1, p = n+2

Spermicidal assays of these series show similar behavior (Figure I.6). Initially,

the activity of these amphiphiles increases as the chain length increases. After a certain

point, the activity plateaus or weakens. Comparison of the two longest homologs 1(p)

show that the activity of 1(18) is slightly stronger than that of 1(17); however, both have

very similar activity. On the other homologous series, the longest homolog, 2(18), is

weaker than 2(17). MICs of 1(p) and 2(p), except 2(18), are observed to be lower than

their CMCs.

10

These behaviors observed in both spermicidal assays represent a condition where

a cut-off effect21 is observed. As the chain length becomes longer, solubility of the

amphiphiles may encounter a problem, as it is pointed out later.4 When such a problem

occurs, it can be suggested that the cut-off effect is actually due to the physicochemical

factor, not the biological factor.

-4

-3.5

-3

-2.5

-2

11 12 13 14 15 16 17

chain length

log

[MEC

] (M

)

1(p)2(p)

Figure I.6 Plots of log MEC vs chain length in spermicidal assays of 1(p) and 2(p)

I.3.2 The Cut-off Effect

In a homologous series of compounds, antimicrobial activity often increases up to

an optimal point as chain length increases; after that, the activity decreases;21,22 such a

phenomenon is referred to as the cut-off effect.21 Several factors are suggested to be

responsible for such a phenomenon. These factors include solubility (monomer

concentration), size discrimination, kinetic effect, and free volume.21 In order to

eliminate solubility issues, the amphiphiles must be completely soluble and in the

monomeric form. For this reason, we attempted to design series of homologous water-

11

soluble amphiphiles.

Theoretically, micelles are easier to form as chain length increase. The

concentration of monomer decreases when micelles form. Supposedly, the monomer is

the active form of the amphiphile, a decrease in monomer concentration certainly

weakens the activity of an amphiphile. Size discrimination involves the partitioning of

the compound into the membrane. Increasing the chain length should increase

partitioning into the membrane; however, there are evidences that increasing the chain

length will eventually restrict partitioning into the membrane. Kinetic effect is related to

the rate of an amphiphile partitioning itself into the membrane, where increasing the

chain length reduces the rate. The free volume changes when an amphiphile is inserted

into the membrane. Insertion of an amphiphile into the membrane increases the free

volume. The maximum biological effect is achieved when the total free volume is

maximized without causing the membrane to solubilize.

I.4 Literature Review in Designing Amphiphiles of Interest

In a recent review,4 it is suggested that amphiphiles can inactivate pathogens by

acting as micelles, or monomers via several mechanisms. However, because irritation

results from detergency,6 and detergency itself occurs at the concentrations close to the

CMC,5 amphiphiles as topical microbicides should be in their active form as monomers,

but not micelles. As is mentioned above, one goal in this project is to design amphiphiles

that have excellent activity that is not related to detergency, consequently, amphiphiles of

interest should have high CMCs.

12

I.4.1 Multi-headed Amphiphiles

Multi-headed amphiphiles, such as alkyl malonates11 and alkane-1,1,2-

tricarboxylic acid,10 have been studied. With regards to biological matters,

polycarboxylate amphiphiles (8, 9) anti-HIV activity are synthesized based on

aurintricarboxylic acid (Figure I.7).23,24 Cushman et al.25 have shown how the

carboxylate groups play an important role in inhibiting the attachment of the virus to a

cell. Polymerizable monomers with multiple headgroups (10−14) have also display anti-

N

O OR

O O

ClCl

OHO OHO

8, R = Me, Et

O

OH

OHO OHO

OH

OH O

OH

HO

O

HO

OH

O OH

O9

NH8

OO OH

O

OHn

NH8

OO

O

O

OH

O

OH

O

OH

O NH8

ON

OH

OHN

OH

OO OH

OO OH

O

H

NH8

O OSO3H

OH

O

9

10 11, n = 1, 2 12

13 14

Figure I.7 Various multi-headed amphiphiles with anti-HIV properties

13

HIV activity (Figure I.7).26 In addition to anionic amphiphiles, incorporation of multiple

cationic headgroups in amphiphiles has been shown to lead to impressive antibacterial

activity.27

We have mentioned how multiple headgroups affect solubility; multiple

headgroup increases the solubility. In addition, previous studies (see Section I.3.1) in our

laboratory have shown how insolubility has become a problem in exploring the biological

activity of compounds with very hydrophobic moiety. With all the advantages offered by

having multiple headgroups in the compounds, we have designed and synthesized multi-

headed amphiphiles that can possibly overcome the insolubility issues of longer chains.

This will ultimately give us a chance to explore biological activity of new classes of

amphiphiles with very long chains and very hydrophobic moieties.

I.4.2 Newkome Dendrons

The presence of Newkome-type, 1→3 C-branched dendrons28,29 in amphiphiles

with very hydrophobic groups have been shown to improve amphiphile solubility in

aqueous solutions. As shown by Kaifer and collaborators, Newkome-type dendrons

(first, second, and third generation) that terminate with carboxylate groups impart water

solubility on hydrophobic groups—those containing a ferrocene (15, 16),30 dansyl (17),31

pyrene (18),32 and viologen (19) (Figure I.8).33 In the case of the dansyl group, the first

generation dendron provides the most polar microenvironment.31 Novel of multi-headed,

multi-tailed amphiphiles containing two second-generation Newkome-type dendritic

heads and lipophilic tails attached to calixarene34 and fullerene35 have also been

established. In a study36 of froth flotation, amphiphiles containing first- and second-

14

generation Newkome-type dendrons have been synthesized and shown to improve the

packing density of the adsorption layer on the mineral particle.

Fe

NH

OHO

O

OH

OH

O

HOFe

NH

HN

O

O

NH

O

HO

OH

O

OH

HN

OH

O

3

3

3O

NH

OH

O

OH

OH

O

HO

SO

O

N

NH

OHO

O

OH

OH

O

HO

NH

OHO

O

OH

OH

O

HO

NN

15 16

17 18

19

Figure I.8 Various Newkome-type dendrons (first and second generations) associated with ferrocene, dansyl, pyrene, and viologen

With all the examples above, Newkome-type, 1→3 C-branched dendrons are

potential candidates as hydrophilic moieties in designing water-soluble amphiphiles that

have a very long chain in the hydrophobic moiety. Current group members André

Williams37,38 and Richard Macri37 have made a homologous series of water-soluble

amphiphiles containing the Newkome-type first-generation dendron and linear alkyl

15

chains with twenty or more carbons (Figure I.9)—ultra-long chains,39 which have been

rarely studied in amphiphile chemistry.40-45

NH

O

n-1 NH

O

n-1O

n = 14, 16, 18, 20, 22n = 13−17, 19, 21

O OH

OH

O

OHO

O OH

O

OH

OHO

Figure I.9 Structures of one-tailed tri-carboxylato amphiphiles having amido and carbamato linkers I.4.3 Ureido Linker

The term linker is mostly found in combinatorial chemistry. In combinatorial

chemistry,46 a linker is a bifunctional chemical moiety that functions to attach a

compound to a solid support. This linker is always attached during a multistep synthesis;

in the final step, the linker is cleaved to release the product. The linker utilized in this

project is different from those utilized in combinatorial chemistry. We apply the term

linker as a functional chemical moiety to connect the hydrophobic and hydrophilic

moieties.

Having investigated the chemistry associated with the Newkome-type first-

generation dendron, several homologous series of water-soluble amphiphiles, each of

which containing a particular linker, can be designed. Three different linkers, namely,

amido, carbamato, and ureido, have been employed in our laboratory. One linker

proposed in this project is a ureido (−HNCONH−) linker. The origin of the name is

derived from a urea; compounds containing such a linker are disubstituted ureas.

16

NH

O

NH

O

O NH

O

NH

amido carbamato ureido

A recent review47 points out that ureides, compounds that incorporate urea as a

substructural component, are one among the oldest classes of bioactive compounds. Urea

and its derivatives have displayed antibacterial activity against Gram-negative bacteria,

and to the less extent to Gram-positive bacteria.48 A number of compounds (20−22)

containing a ureido linker, even though they are not amphiphiles, have been shown to

have antifungal and antibacterial activity; most of them contains heterocyclic moieties. A

study49 of a gemini amphiphile (23) containing a ureido linker and two carboxylate

groups has been conducted in the field of cosmetics. In another study,50 mono- and bis-

urea substituted ferrocene receptors, containing a ureido linker and the first-generation

Newkome-type dendron, have been synthesized for an investigation of electrochemical

recognition and sensing of anionic guest (24, 25) (Figure I.10).

17

NH

N N NH

OO O

OOOHHO

OO

1212

NH

NH

OO

O

O

O

O

O

Fe

NH

NH

OO

O

O

O

O

O

Fe

HN

HN

OO

O

O

O

O

O

HN

HN

O

ClClCl

H2N NH

NH

HNO

20 21

23 24

25

NH

NH

O

Cl22

NN

Figure I.10 Various compounds containing ureido functional groups

In a recent study,51 alkylureas, which are monosubstituted ureas, have displayed

unique properties that render them as potential candidates for microbicide formulations.

Furthermore, the author suggests that alkylureas with longer alkyl chains may have

greater selective microbicidal activity.

Having presented all this evidence on how a ureido functional group may give

some advantages in topical microbicides, we were inspired to utilize the ureido linker in

our project.

18

I.4.4 One-tailed Tri-carboxylato Amphiphile

Based on the background in designing multi-headed amphiphiles derived from the

first generation of Newkome-type dendron, a straight hydrocarbon as the hydrophobic

moiety can be attached via a ureido linker (Figure I.11). Formation of the ureido

functional groups have recently been reviewed;52 one commonly applied method is the

reaction of amines and isocyanates. The synthesis of the isocyanates derived from tri-

headed first-generation Newkome-type dendron has been well developed via a three-step

synthesis.29,53 Such a synthesis begins with commercially available nitromethane and

tert-butyl acrylate (Scheme I.1).

linker

head

head

head

tail NH

NH

COOH

COOH

COOH

R

O

R = n-CnH2n+1

Figure I.11 General representation and structure of one-tailed tri-carboxylato amphiphiles

Scheme I.1 Synthesis of isocyanate monomer of Newkome-type dendron

O2NOtBu

O 3

CH3O2N H2NOtBu

O 3

OCNOtBu

O 3

i ii iii

i. tert-butyl acrylate, Triton B, dimethoxyethane; b. Raney Ni, H2, EtOH; c. BOC2(O), DMAP, CH2Cl2

As shown by previous studies,54-59 the isocyanate monomer reacts readily with a

variety of primary amines. Based on this chemistry, a hydrophobic moiety⎯a long chain

hydrocarbon⎯can be attached to the hydrophilic moiety by combining an isocyanate and

long chain alkan-1-amines, which in turn will afford a ureido linker. This strategy will

allow us to develop a rapid synthesis to generate homologous series of a new class of

19

amphiphiles with a common headgroup. Having these multiple headgroups in

amphiphiles with very long hydrocarbon chain should increase the solubility in aqueous

solutions, which in turn allow us to explore the biological activity of the new class of the

amphiphiles.

I.4.5 Two-tailed Tri-carboxylato Amphiphile

As the solubility of an amphiphile in aqueous solutions is often a problem,

extensive attempts have been made to design amphiphiles that are more stable in aqueous

solution. In addition, when amphiphiles are intended as topical microbicides, they must

be non-irritating. The level of irritancy of amphiphiles is affected by the chemical

structure, steric factors, and relative polarity.60 Structural modification, such as

branching, can be used as tool to improve solubilitation and minimize the level of

irritancy.

The concept of branching does not necessarily mean insertions of an alkyl group

to the backbone.61 Rather, branching can be considered as indications of the amphiphile

bulkiness or reduced packing density.62 These include introduction of unsaturation,

addition of alkyl groups, modification on the attachment to the hydrophilic and

hydrophobic moieties, or even substitutions of hydrogen atoms in the alkyl group with

other functional groups.61

As suggested by Wormuth and Zushma,63 branching can be classified as methyl

branching, double-tail branching⎯equal and unequal tails, and multiple branching.

Studies64-68 of double-tail branched amphiphiles indicate when the tails are designed

more equal, Kraft temperature decreases,64 the CMC increases,67 solubilization in

cyclohexane increases,66,68 and the amphiphiles is more effective at reducing the air-water

20

surface tension.65

Langhals et al.69-71 have investigated and proven that solubility of perylene

bisimide dyes is improved by attaching a long chain through the middle carbon. A

compound containing this kind of tail is also referred to as a “swallowtail”.72 In this

study, a compound containing a “swallowtail” improve the solubility and has the ability

to form a Langmuir−Blodgett monolayer.72 Derivatives of dioctadecylamine are

functioning as precursors to liposomes,73 lipid anchors for ion channel mimics,74 artificial

lipid clusters,75 and organic gelators.76 These suggest that compounds with two equal

tails, as well as those with a single tail, are also able to pack themselves or associate with

a bilayer membrane that might cause biological activities.

Aungst77 has demonstrated how the structure of fatty acid isomers⎯branched vs

unbranched saturated fatty acids (26−39) and cis- vs trans- unsaturated fatty acids (40,

41)⎯act as skin penetration enhancher (Figure I.12). No significant difference exists

between cis- vs trans- unsaturated fatty acids in the skin penetration rates; however,

compared to saturated fatty acids, unsaturated fatty acids with the same chain length

increased the skin penetration rates. Neodecanoic acid (35) and elaidic acid (41) showed

less skin irritation compared to lauric acid (37) and stearic acid (39), respectively.

Several other studies78-81 also reported that structure modification by having alkyl

branched compounds results in less or no irritative effect when applied to the animal and

human skin.

21

O

OH16OH

O

14

8 OH

O

5

O

OH7

O

OH5

O

OH

O

OH

O

OHO

OH

O

OH

O

OH8

O

OH4 OH

O

OH

O

7 77 OH

O

7

26 27 28 29

30 31 32 33

5

34 35 36 37

38 39 40

O

OH10

41

Figure I.12 Various fatty acids tested for skin penetration rates and skin irritation

We are interested in constructing two-tailed tri-headed amphiphiples with a ureido

group and a Newkome-type dendron as the linker and the hydrophilic headgroup,

respectively. Long, straight alkyl chains are employed as the hydrophobic moieties.

More specifically, both chain lengths of the alkyl on each tail are the same. In this

project, we present double-tail branching in two different series with different geometry

on how the hydrophobic tails are attached to the linker. One is where a nitrogen on the

linker is connected to two chains that are equal in length, and the other is where a

nitrogen on the linker is connected to a long chain through the middle carbon.

22

linker

head

head

head

tail

tail

linker

head

head

head

tail

tail

Figure I.13 General representations of two-tailed, tri-headed amphiphile series I.5 Cholestane-based Polyanionic Compounds Derived from Natural Product

Natural products are often used as a source in molecular diversity to drug

discovery and development. Approximately 60% of the currently available antitumor and

anti-infective agents are of natural product origin.82 Before investigations were focused

on biological targets, natural product-based drugs were originally discovered by the

traditional in-vitro assays, including antibacterial, antifungal, antiviral, antiparasitic, and

cytotoxic assays.83

Several natural products contain the steroidal skeleton. Steroids are a class of

lipids that have the same chemical skeleton of four fused rings⎯three cyclohexanes and

a cyclopentane. These four rings are designated as A, B, C, and D. The large number of

C−H bonds in a steroid makes this molecule nonpolar. Compounds containing a steroidal

skeleton have many advantages for studies in biology because the skeleton contains

unique properties, such as chirality and structural rigidity.84 A couple examples of

steroids found in the human body include cholesterol and sex hormones, such as

testosterone and estrogen. Because cholesterol is a building block of cell membranes,

cholesterol derivatives are often synthesized for studies as cell membrane models.84

23

A B

C D

HO

H

HH

H

21

43

6

5

8

7

10

9

HO

H

HH

H

21

43

6

5

8

7

10

9

steroidal skeleton cholesterol 5α-cholestane

H

The C3 hydroxy group of cholesterol has been the key portion for structural

modification, where different functional groups such as esters or ethers are introduced to

the steroidal moiety. These modified compounds are widely found in the fields of

toiletries, cosmetics, pharmaceuticals, and in the chemical industry.79 Recently, another

structural modification has been studied. When hydrazone derivatives85 and a hydroxyl

group86 are introduced to C6 or C7, new compounds with antifungal properties are

generated.

Synthetic steroids are often found in medical drugs. Compounds with steroidal

skeletons, such as a cholestane moiety, are synthesized, modified, and investigated for

any biological activity. Introduction of a heterocyclic ring to the 2,3-position of various

steroids generates new compounds with anabolic,87 anti-inflamatory,88,89

antiprogestational,90 contraceptive,91 anticancer,92 and antimicrobial93,94 properties.

Modification on the steroidal skeleton itself has been explored. Compounds with

antimicrobial properties are generated when a carbon (C4) is substituted by nitrogen.95,96

Many sites in the steroidal skeleton can be explored; the steroidal skeleton is a potential

building block in structural modification.

Cholestane moiety has been involved in the syntheses of different types of

dendrimers (Figure I.14). In a study97 of transmembrane ion transport, a dendrimer of

24

amphiphilic polyamine (42) anchored to a hydrophobic cholestane moiety is involved.

Dendrimers of polyol (43, 44) anchored to cholestane moiety are synthesized and

employed in the study of the interaction of liposome with a blood complement system.98

H

HH

H

21

43

6

5

8

7

10

9

HNN

N

H3N

N

H3N

H3N

H3N

H

H

H H H

8TFA

42

H

HH

H

21

43

6

5

8

7

10

9

HO

HOO

O

HO

43

H

HH

H

21

43

6

5

8

7

10

9

HO

OO

O

HO

HO

HO

HO

44

O

Figure I.14 Polyol and polyamine dendrimers associated with cholestane moieties

Cosalane, a synthetic anti-HIV (Human Immunodeficiency Virus) agent derived

from aurintricarboxylic acid, contains a steroidal cholestane moiety. Because of its

25

HOOC

HO

ClOH

HOOC

Cl

HOOC

HO

ClOH

HOOC

Cl

cosalane dichlorinated disalicylmethane fragment

cholestane moiety

H

H

ability to inhibit multiple sites in the retroviral replication, it is considered as a potent

inhibitor in the treatment of AIDS.99 In cosalane, a cholestane moiety is connected

through a three-carbon linker chain to a dichlorinated disalicylmethane fragment of the

polymeric anti-HIV agent aurintricarboxylic acid.

A number of cosalane analogs have been synthesized to improve its potency as an

anti-HIV agent. A study100 has investigated the role of the steroidal cholestane; this

moiety serves as a lipophilic accessory appendage to escort the dichlorodisalicylmethane

pharmacophore to a lipid environment. In other words, the steroidal cholestane functions

as nonpolar hydrophobic moiety as any hydrocarbon chain in most amphiphiles.

Maintaining the steroidal cholestane moiety, most modifications are made on the three-

carbon linker, carboxylic acid, and hydroxyl groups. A hypothetical model is proposed

by Cushman et al.25 that the two negatively charged carboxylate group of cosalane

interact with the positively charged Arg-58 and Arg-59 residues of gp120, a large

glycoprotein molecule. Consequently, this blocks the virus attachment to the primary

binding site, CD4, of the cell. This hypothetical model shows the crucial factor of having

the carboxylic acid groups.

26

I.6 Tri-carboxylato Amphiphile Containing Rigid Cholestane-based Hydrophobic Moiety

In our tri-headed amphiphile series, the triheadgroups are tricarboxylic acids. So

far, the different series are presented based on the modification of the alkyl chain on the

hydrophobic moiety. Hydrophobic moieties are not limited to alkyl chains. Any moiety

containing nonpolar hydrocarbon, such as steroidal skeleton, may serve as a hydrophobic

moiety as well. It becomes our interest to incorporate steroidal skeleton as a hydrophobic

moiety into our tri-headed amphiphiles.

When the ureido linker is located between the hydrophilic moiety and C3 of

cholestane moiety, only two compounds that are epimeric are generated. To expand our

study on this new cholestane-based amphiphiles, we are challenged to make

modifications so that a number of compounds are generated within a series. In order to

fulfill this need, we put a spacer between the nitrogen on the ureido linker and C3 of

cholestane moiety (Figure I.15).

linker

head

head

head

head

head

head

C3cholestane moiety

linker spacerC3cholestane moiety

Figure I.15 General structures of cholestane-based, tri-headed amphiphile series I.7 Microorganisms in Antimicrobial Screening

Our very goal in this project is to design amphiphiles that are not detergents, but

antimicrobial agents. As we have synthesized the amphiphiles, we need to screen them

for any antimicrobial activity. What microorganisms are targeted?

27

Microorganisms or microbes, which are considered neither as plant nor animal,

are defined organisms that in general are too small to be seen with an aided eye. They

can be both useful in human life and also responsible for many infectious diseases.

Microorganisms that can cause diseases are known as pathogens. Based on their physical

appearance of their cells microorganisms can be classified into six major

categories⎯protozoa, microscopic algae, fungi, bacteria, cyanobacteria, and viruses.

Protozoa, microscopic algae, and fungi are classified as eucaryotes, while bacteria and

cyanobacteria are classified as prokaryotes. Viruses are considered neither. Eucaryotic

cells contains a nucleus surrounded by a membrane, on the other hand, procaryotic cells

contains no nucleus, thus lacks of nuclear membrane.101 In this project we narrow our

scope to employ just bacteria and fungi.

I.7.1 Bacteria

In general, bacteria are classifed based on their responses to Gram-staining, which

basically are due to differences in the physical structure of their cell walls.101 This results

in Gram-negative and Gram-positive bacteria (Table I.1). An obvious difference between

the two is the presence of an outer membrane in the cell wall. Gram-negative bacteria

posses an outer membrane, on the other hand, Gram-positive bacteria lack an outer

membrane.

Table I.1 Physical differences between Gram-negative and Gram-positive bacteria Gram-negative Gram-positive thinner cell walls ( ~ 100−150 Å) thicker cell walls ( ~ 200 Å) surrounded by an outer membrane lack of an outer membrane low amino sugar content ( ≤ 10%) high amino sugar content (10−30%) high lipid content (10−20%) low lipid content ( ≤ 2%)

Another class of bacteria is mycobacteria, which is not classified as neither Gram-

28

negative nor Gram-positive bacteria.101 As found in Gram-negative bacteria, an outer

membrane is found in mycobacteria, however, the outer membrane in mycobacteria is

thicker than that in Gram-negative bacteria. The outer membrane of mycobacteria

contains mycolic acids, which are α-alkyl, β-hydroxy fatty acids; mycolic acids contain

60 to 90 carbons, and α-branches contain 20 to 24 carbons.102 In fact, it is suggested that

the resistance of mycobacteria to any lethal environment is caused by the low

permeability of the mycolic acids in the mycobacterial cell wall.103-105

OH

OOH

R'R"

general structure of mycolic acids

Two Gram-negative bacteria employed include Escherichia coli and Klebsiella

pneumoniae, and Gram-positive bacteria employed include Lactobacillus plantarum,

Micrococcus luteus, Staphyllococcus aureus, and methicillin-resistant Staphyllococcus

aureus. Mycobacterium smegmatis will be used as a model of mycobacteria.

I.7.2 Fungi

Fungi are characterized by the presence of cell walls, the lack of motility, and the

absence of photosynthesis.101 Some fungi develop as single cells, or produce networks of

filaments. The former is called yeast, and the later is called molds. Even though some

yeasts have been known for their useful action in life, some yeasts are pathogens. We

include three different yeasts, namely Saccharomyces cerevisiae, Candida albicans, and

Cryptococcus neoformans. The first is non-pathogenic yeast; S. cerevisiae is known as

brewer’s yeast and it is used in bread-making process. The last two yeasts are classified

29

as pathogenic yeasts. In addition, we also include Aspergillus niger, which is a

filamentous fungus.

I.8 Summary

Finally, we intend to achieve two goals in this project. First, we will synthesize

several homologous series of tri-headed amphiphiles with different type of hydrophobic

moieties, namely mobile and rigid hydrocarbon chain. In the series containing mobile

hydrocarbon moieties, two different series, in this case one- vs two-tail, will be presented

while the numbers of carbons on the chain are maintained equal. In addition, the two-tail

will be modified based on the topology of the tails. Second, we will screen the

antimicrobial activity of the tri-headed amphiphiles against a broad array of

microorganisms to explore the structure-relationship activity. We hope to discover leads

for development as topical microbicides.

References for Chapter 1 1 Clint, J. H. Surfactant aggregation; Chapman and Hall, Inc.: New York, NY,

1992, p 1-12, 120-122, 147-149 2 Myers, D. Surfactant science and technology; 3rd ed.; John Wiley & Sons, Inc.:

Hoboken, NJ, 2006, p 48-50, 108-110, 131-140. 3 Lambert, P. A. Membrane-active antimicrobial agents. Prog. Med. Chem. 1978,

15, 87-124. 4 Gandour, R. D. Toward a design of affordable, topical microbicides:

Acylcarnitine analogues. Curr. Pharm. Design 2005, 11, 3757-3767. 5 le Maire, M.; Champeil, P.; Moller, J. V. Interaction of membrane proteins and

lipids with solubilizing detergents. Biochim. Biophys. Acta 2000, 1508, 86-111.

30

6 Fichorova, R. N.; Tucker, L. D.; Anderson, D. J. The molecular basis of nonoxynol-9-induced vaginal inflamation and its possible relevance to human immunodeficiency virus type 1 transmission. J. Infect. Dis. 2001, 184, 418-28.

7 Bhattacharya, S.; Haldar, J. Molecular design of surfactants to tailor its

aggregation properties. Colloids Surf. A 2002, 205, 119-126. 8 Haldar, J.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. Molecular modulation of

surfactant aggregation in water: effect of the incorporation of multiple headgroups on micellar properties. Angew. Chem., Int. Ed. 2001, 40, 1228-1232.

9 Haldar, J.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. Role of incorporation of

multiple headgroups in cationic surfactants in determining micellar properties. Small-Angle-Neutron-Scattering and fluorescence studies. J. Phys. Chem. B 2001, 105, 12803-12808.

10 Shinoda, K. The critical micelle concentrations in aqueous solutions of potassium

alkane tricarboxylates. J. Phys. Chem. 1956, 60, 1439-1441. 11 Shinoda, K. The critical micelle concentrations in aqueous solutions of potassium

alkyl malonates. J. Phys. Chem. 1955, 59, 432-435. 12 Klevens, H. B. Structure and aggregation in dilute solution of surface-active

agents. J. Am. Oil Chem. Soc. 1953, 30, 74-80. 13 Scott, A. B.; Tartar, H. V. Electrolytic properties of solutions of paraffin-chain

quarternary ammonium salts. J. Am. Chem. Soc. 1943, 65, 692-698. 14 Hartley, G. S. Ion aggregation in solutions of salts with long paraffin chains.

Kolloid Z. 1939, 88, 22-40. 15 Stauff, J. Equilibrium between molecularly dispersed and coloidal substances in

aqueous soap solutions. II. Temperature dependency of the equilibrium and the solubility curve of soaps. Z.Physik. Chem. A 1939, 183, 45-59.

16 Bashura, A. G.; Klimenko, O. I.; Nurimbetov, K. N.; Gladukh, E. V. Colloidal-

micellar properties of some new surfactants. Farmatsevt. Zh. (Kiev) 1989, 3, 57-58.

17 Holmberg, K.; Jonsson, B.; Kronberg, B.; Lindman, B. Surfactants and polymers

in aqueous solution; 2nd ed.; John Wiley & Sons, Ltd.: New York, 2002. 18 Savle, P. S.; Doncel, G. F.; Bryant, S. D.; Hubieki, M. P.; Robinette, R. G.;

Gandour, R. D. Acylcarnitine analogues as topical, microbicidal spermicides. Bioorg. Med. Chem. Lett. 1999, 9, 2545-2548.

31

19 Wong, Y.-L.; Curfman, C. L.; Doncel, G. F.; Hubieki, M. P.; Dudding, T. C.; Savle, P. S.; Gandour, R. D. Spermicidal, anti-HIV, and micellar properties of di- and trihydroxylated cationic surfactants. Tetrahedron 2002, 58, 45-54.

20 Wong, Y.-L.; Hubieki, M. P.; Curfman, C. L.; Doncel, G. F.; Dudding, T. C.;

Savle, P. S.; Gandour, R. D. A structure-activity study of spermicidal and anti-HIV properties of hydroxylated cationic surfactants. Bioorg. Med. Chem. Lett. 2002, 10, 3599-3608.

21 Balgavý, P.; Devinsky, F. Cut-off effects in biological activities of surfactants.

Adv. Colloid Interface Sci. 1996, 66, 23-63. 22 Hansch, C.; Clayton, J. M. Lipophilic character and biological activity of drugs.

II. The parabolic case. J. Pharm. Sci. 1973, 62, 1-21. 23 Xu, G. Z.; Kannan, A.; Hartman, T. L.; Wargo, H.; Watson, K.; Turpin, J. A.;

Buckheit, R. W.; Johnson, A. A.; Pommier, Y.; Cushman, M. Synthesis of substituted diarylmethylenepiperidines (DAMPs), a novel class of anti-HIV agents. Bioorg. Med. Chem. Lett. 2002, 10, 2807-2816.

24 Wang, P.; Kozlowski, J.; Cushman, M. Isolation and structure elucidation of low

molecular weight components of aurintricarboxylic acid (ATA). J. Org. Chem. 1992, 57, 3861-3816.

25 Cushman, M.; Shabana, I.; Ruell, J. A.; Schaeffer, C. A.; Rice, W. G. Synthesis of

a cosalane analog with an extended polyanionic pharmacophore conferring enhanced potency as anti-HIV agent. Bioorg. Med. Chem. Lett. 1998, 8, 833-836.

26 Leydet, A.; Barthelemy, P.; Boyer, B.; Lamaty, G.; Roque, J.-P. Polyanion

inhibitors of huan immunodeficiency virus. Part IV. Polymerized anionic surfactants: influence of the density and distribution of anionic groups on the antiviral activity. Bioorg. Med. Chem. Lett. 1996, 6, 397-402.

27 Haldar, J.; Kondaiah, P.; Bhattacharya, S. Synthesis and antibacterial properties of

novel hydrolyzable cationic amphiphiles. Incorporation of multiple head groups leads to impressive antibacterial activity. J. Med. Chem. 2005, 48, 3823-3831.

28 Newkome, G. R.; Weis, C. D.; Moorefield, G. R.; Baker, B.; Childs, J.; Epperson,

J. Isocyanate-based dendritic building blocks: combinatorial tier construction and macromolecular-property modification. Angew. Chem., Int. Ed. Engl. 1998, 37, 307-310.

29 Newkome, G. R.; Behera, R. K.; Moorefield, C. N.; Baker, G. R. Chemistry of

micelles. 18. Cascade polymers: syntheses and characterization of one-directional arborols based on adamantane. J. Org. Chem. 1991, 56, 7162-7167.

32

30 Cardona, C. M.; McCarley, T. D.; Kaifer, A. E. Synthesis, electrochemistry, and interactions with ß-cyclodextrin of dendrimers containing a single ferrocene subunit located "off-center". J. Org. Chem. 2000, 65, 1857-1864.

31 Cardona, C. M.; Alvarez, J.; Kaifer, A. E.; McCarley, T. D.; Pandey, S.; Baker, G.

A.; Bonzagni, N. J.; Bright, F. V. Dendrimers functionalized with a single fluorescent dansyl group attached "off-center": Synthesis and photophysical studies. J. Am. Chem. Soc. 2000, 122, 6139-6144.

32 Cardona, C. M.; Wilkes, T.; Ong, W.; Kaifer, A. E.; McCarley, T. D.; Pandey, S.;

Baker, G. A.; Kane, M. N.; Baker, S. N.; Bright, F. V. Dendrimers functionalized with a single pyrene label: Synthesis, photophysics, and fluorescence quenching. J. Phys. Chem. B 2002, 106, 8649-8656.

33 Ong, W.; Kaifer, A. E. Unusual electrochemical properties of unsymmetric

viologen dendrimers. J. Am. Chem. Soc. 2002, 124, 9358-9359. 34 Kellermann, M.; Bauer, W.; Hirsch, A.; Schade, B.; Ludwig, K.; Bottcher, C. The

first account of a structurally persistent micelle. Angew. Chem. Int. Ed. 2004, 43, 2959-2962.

35 Burghardt, S.; Hirsch, A.; Schade, B.; Ludwig, K.; Bottcher, C. Switchable

supramolecular organization of structurally defined micelles based on an amphiphilic fullerene. Angew. Chem., Int. Ed. Engl. 2005, 44, 2976-2979.

36 Müller, P. U.; Akpo, C. C.; Stöckelhuber, K. W.; Weber, E. Novel amphiphiles

with preorganized functionalities formation of Langmuir-films and efficiency in mineral flotation. Adv. Coll. Interface Sci. 2005, 114-115, 291-302.

37 Williams, A. A.; Sugandhi, E. W.; Macri, R. V.; Falkinham, J. O. I.; Gandour, R.

D. Antimicrobial activity of long-chain, water-soluble, dendritic tri-carboxylato amphiphiles. J. Antimicrob. Chemother. 2007, in press.

38 Williams, A. A.; Day, B. S.; Kite, B. L.; McPherson, M. K.; Slebodnick, C.;

Morris, J. R.; Gandour, R. D. Homologous, long-chain alkyl dendrons form homologous thin films on silver oxide surfaces. Chem. Commun. 2005, 5053-5055.

39 Brode, P. F. Adsorption of ultra-long-chain zwitterionic surfactants on a polar

solid. Langmuir 1988, 4, 176-180. 40 Zana, R.; Schmidt, J.; Talmon, Y. Tetrabutylammonium alkyl carboxylate

surfactants in aqueous solution: Self-association behavior, solution nanostructure, and comparison with tetrabutylammonium alkyl sulfate surfactants. Langmuir 2005, 21, 11628-11636.

33

41 Hasegawa, T.; Nishijo, J.; Watanabe, M.; Umemura, J.; Ma, Y. Q.; Sui, G. D.; Huo, Q.; Leblanc, R. M. Characteristics of long-chain fatty acid monolayers studied by Infrared external-reflection spectroscopy. Langmuir 2002, 18, 4758-4764.

42 Bongiovanni, R.; Ottewill, R. H.; Rennie, A. R.; Laughlin, R. G. Interaction of

small ions with the micellar surface of an ultralong chain zwitterionic surfactant. Langmuir 1996, 12, 4681-4690.

43 Ottewill, R. H.; Rennie, A. R.; Laughlin, R. G.; Bunke, G. M. Micellar structure

in solutions of an ultralong chain zwitterionic surfactant. Langmuir 1994, 10, 3493-3499.

44 Menger, F. M.; Yamasaki, Y. Hyperextended amphiphiles. Bilayer formation

from single-tailed compounds. J. Am. Chem. Soc. 1993, 115, 3840-3841. 45 Hodge, D. J.; Laughlin, R. G.; Ottewill, R. H.; Rennie, A. R. Micellization of

ultralong chain surfactants. Langmuir 1991, 7, 878-884. 46 Kiely, J. S.; Hayes, T. K.; Griffith, M. C.; Pei, Y. A practical guide to

combinatorial chemistry; American Chemical Society: Washington DC, 1997. 47 Batra, S.; Tusi, Z.; Madapa, S. Medicinal chemistry of ureido derivatives as anti-

infectives. Anti-Infect. Agents Med. Chem. 2006, 5, 135-160. 48 Weinstein, L.; McDonald, A. Effect of urea, urethane, and other carbamates on

bacterial growth. Science 1945, 101, 44-45. 49 Tsubone, K. e. a. Cosmetics containing anionic or nonionic amphiphatic

surfactants having amide bonds.Jpn. Kokai Tokkyo Koho 1996, JP08301747 50 Beer, P. D.; Michelle, D. P. Anion regognition and sensing by mono- and bis-urea

substituted ferrocene receptors. Polyhedron 2003, 22, 649-653. 51 Rubinstein, A. Preclinical studies of alkylureas as anti-HIV-1 contraceptive. Curr.

Pharm. Design 2005, 11, 3769-3778. 52 Sartori, G.; Maggi, R. Acyclic and cyclic ureas. Sci. of Synthesis 2005, 18, 665-

758. 53 Newkome, G. R.; Weis, C. D.; Childs, J. Syntheses of 1→3 branched isocyanate

monomers to dendritic construction. Des. Monomers Polym. 1998, 1, 3-14. 54 Newkome, G. R. Design, synthesis, and characterization of conifer-shaped

dendritic architectures. Chem.––Eur. J. 2006, 12, 3726-3724.

34

55 Sun, H.; Kaifer, A. E. Unsymmetric dendrimers containing a single ureidopyrimidine unit: generation-dependent dimerization via hydrogen bonding. Org. Lett. 2005, 7, 3845-3848.

56 Wang, P.; Moorefield, C. N.; Newkome, G. R. Synthesis, x-ray structure, and

self-assembly of functionalized bis-(2,2':6',2''-terpyridinyl)arenes. Org. Lett. 2004, 6, 1197-1200.

57 Pratt, M. D.; Beer, P. D. Anion recognition and sensing by mono- and bis-urea

substituted ferrocene receptors. Polyhedron 2003, 22, 649-653. 58 Newkome, G. R.; Yoo, K. S.; Kabir, A.; Malik, A. Synthesis of benzyl-terminated

dendrons for use in high-resolution capillary gas chromatography. Tetrahedron Lett. 2001, 42, 7537-7541.

59 Newkome, G. R.; Godinez, L. A.; Moorefield, C. N. Molecular recognition using

β-cyclodextrin-modified dendrimers: novel building blocks for convergent self-assembly. Chem. Commun. 1998, 17, 1821-1822.

60 Charaf, U. K.; Hart, G. L. Phospholipid liposomes/surfactant interactions as

predictors of skin irritation. J. Soc. Cosmet. Chem. 1991, 42, 71-85. 61 Ezrahi, S.; Tuval, E.; Aserin, A.; Garti, N. The efffect of structural variation of

alcohols on water solubilization in nonionic microemulsions. 1. From linear to branched amphiphiles-General considerations. J. Colloid Interface Sci. 2005, 291, 263-272.

62 Bourrel, M.; Schechter, R. S. Microemulsions and related systems-formulation,

solvency and physical properties; Dekker: New York/Basel, 1988; Vol. 30. 63 Wormuth, K. R.; Zushma, S. Phase behavior of branched surfactants in oil and

water. Langmuir 1992, 7, 2048-2053. 64 Valint, P. L.; Bock, J.; Kim, M.-W.; Robbins, M. L.; Steyn, P.; Zushma, S. Krafft

points and microemulsion phase behavior of some alkylarenesulfonates. Colloids Surf. 1987, 26, 191-203.

65 Lascaux, M. P.; Dusart, O.; Granet, R.; Piekarski, S. Surface properties of

branched sodium hexadecylbenzenesulfonates at air-water interface. J. Chim. Phys. 1983, 80, 615-620.

66 Shinoda, K.; Sagitani, H. Conceptual diagram on solution properties in dialkyl

type surfactants. Principle of changing hydrophile/lipophile balance of ionic surfactant. J. Phys. Chem. 1983, 87, 2018-2020.

35

67 Graciaa, A.; Barakat, Y.; El-Emary, M.; Fortney, L.; Schechter, R. S.; Yiv, S.; Wade, W. H. HLB, CMC, and phase behavior as related to hydrophobe branching. J. Colloid Interface Sci. 1982, 89, 209-216.

68 Sagitani, H.; Suzuki, T.; Nagai, M.; Shinoda, K. Solubilization of cyclohexane in

aqueous solutions of sodium a-alkylalkanoates. J. Colloid Interface Sci. 1982, 87, 11-17.

69 Langhals, H. Control of the interactions in multichromophores: novel doncepts.

Perylene bis-imides as components for larger functional units. Helv. Chim. Acta 2005, 88, 1309-1343.

70 Langhals, H.; Jona, W. Intense dyes through chromophore-chromophore

Interactions: bi- and trichromophoric perylene-3,4:9,10-bis(dicarboximide)s. Angew. Chem., Int. Ed. Engl. 1998, 37, 952-955.

71 Langhals, H.; Demmig, S.; Potrawa, T. The relation between packing effects and

solid-state fluorescence of dyes. J. Prakt. Chem. 1991, 333. 72 Wescott, L. D.; Mattern, D. L. Donor-α-acceptor molecules incorporating a

nonadecyl-swallowtailed perylenediimide acceptor. J. Org. Chem. 2003, 68, 10058-10066.

73 Neumann, H. C.; Ringsdorf, H. Peptide liposomes from amphiphilic amino acids.

J. Am. Chem. Soc. 1986, 108, 487-490. 74 Pajewski, R.; Ferdani, R.; Pajewska, J.; Djedovič, N.; Schlesinger, P. H.; Gokel,

G. W. Evidence for dimer formation by an amphiphilic heptapeptide that mediates chloride and carboxyfluorescein release from liposomes. Org. Biomol. Chem. 2005, 3, 619-625.

75 Ariga, K.; Urakawa, T.; Michiue, A.; Kikuchi, J. i. Spider-web amphiphiles as

artificial lipid clusters: design, synthesis, and accommodation of lipid components at the air-water interface. Langmuir 2004, 20, 6762-6769.

76 Abdallah, D. J.; Lu, L.; Weiss, R. G. Thermoreversible organogels from alkane

gelators with one heteroatom. Chem. Mater. 1999, 11, 2907-2911. 77 Aungst, B. J. Structure/effect studies of fatty acid isomers as skin penetration

enhancers and skin irritants. Pharm. Res. 1989, 6, 244-247. 78 Kim, M.-S.; Park, K.-M.; Chang, I.-S.; Kang, H.-H.; Sim, Y.-C. β-(1,6)-Branched

β-(1,3)-glucan in skin care. Cosmet. Toiletries 2000, 115, 89-86.

36

79 Urata, K.; Takaishi, N. Cholesteryl ester compounds containing alkyl branched acyl groups. Their preparations, properties, and applications. Fett/Lipid 1997, 99, 327-332.

80 Suzuki, T.; Nakamura, M.; Sumida, H.; Shigeta, A. Liquid crystal makeup

remover: conditions of formation and its cleansing mechanisms. J. Soc. Cosmet. Chem. 1992, 43, 21-36.

81 MacMillan, F. S. K.; Reller, H. H.; Synder, F. H. The antiperspirant action of

topically applied anticholinergics. J. Invest. Dermatol. 1964, 43, 363-378. 82 Cragg, G. M. Natural products in drug discovery and development. J. Nat. Prod.

1997, 60, 52-60. 83 Shu, Y.-Z. Recent natural products based drug development: a pharmaceutical

industry prespective. J. Nat. Prod. 1998, 61, 1053-1071. 84 Urata, K.; Takaishi, N. Cholesterol as synthetic building blocks for artificial lipids

with characteristic physical, chemical and biological properties. Eur. J. Lipid Sci. Technol 2001, 103, 29-39.

85 Loncle, C.; Brunel, J. M.; Vidal, N.; Dherbomez, M.; Letourneux, Y. Synthesis

and antifungal activity of cholesterol-hydrazone derivatives. Eur. J. Med. Chem. 2004, 39, 1067-1071.

86 Brunel, J. M.; Loncle, C.; Vidal, N.; Dherbomez, M.; Letourneux, Y. Synthesis

and antifungal activity of oxygenated cholesterol derivatives. Steroids 2005, 70, 907-912.

87 Clinton, R. O.; Manson, A. J.; Stonner, F. W.; Neumann, H. C.; Christianse, R.

G.; Clarke, R. L.; Ackerman, J. H.; Page, D. F.; Dickinson, W. B.; Carabateas, C. Steroidal [3,2-c]pyrazoles. II. Androstanes, 19-norandrostanes, and their unsaturated analogs. J. Am. Chem. Soc. 1961, 83.

88 Gupta, R.; Singh, I.; Jindall, D. P. Synthesis and study of

steroidal[16,17]triazoles. Indian J. Chem., Sect. B 1996, 35, 1017-1020. 89 Kwon, T.; Heiman, A. S.; Oriaku, E. T.; Yoon, K.; Lee, H. J. New steroidal

antiinflammatory antedrugs: Steroidal [16α ,17α -d]-3'-carbethoxyisoxazolines. J. Med. Chem. 1995, 38, 1048-1051.

90 Tamaya, T.; Furuta, N.; Motoyama, T.; Boku, S.; Ohono, Y.; Okada, H.

Mechanism of antiprogestational action of synthetic steroids. Act. Endocrinol. (Copenhagen) 1978, 88, 190-198.

37

91 Schane, H. P.; Anzalone, A. J.; Potts, G. O. Fertility in the rhesus monkey following long-term inhibition of ovarian function with danazol. Fertil. Steril. 1978, 29, 692-701.

92 Peters, T. G.; Lewis, J. D. Treatment of breast cancer with danazol. Surg. Forum.

1976, 1976, 97-98. 93 Pettit, R. K.; Cage, G. D.; Pettit, G. R.; Liebman, J. A. Antimicrobial and cancer

cell growth inhibitory activities of 3β-acetoxy-17β-(L-prolyl)amino-5α-androstane in vitro. J. Antimicrobial Agents 2000, 15, 299-304.

94 Solomons, W. E.; Doorenbos, N. J. Synthesis and antimicrobial properties of 17β

-amino-4-aza-5α -androstane and derivatives. J. Pharm. Sci. 1974, 63, 19-23. 95 Norman, F. P.; Doorenbos, N. J. The 4-(β -hydroxyethyl)-4-aza-5α -cholestane -

an antimicrobial agent. J. of the Mississippi Acad. Sci. 1976, 21, 23-26. 96 Aboul-Enein, H. Y.; Doorenbos, N. J. Synthesis and antimicrobial activity of 4-

aza-5α -sitostane and the 4-methyl derivative. Pharm. Acta Helv. 1974, 49, 320-324.

97 Sakai, N.; Matile, S. Transmembrane ion transport mediated by amphiphilic

polyamine dendrimers. Tetrahedron Lett. 1997, 38, 2613-2616. 98 Nemoto, H.; Kikuishi, J.; Yanagida, S.; Kawano, T.; Yamada, M.; Harashima, H.;

Kiwada, H.; Shibuya, M. Design and synthesis of cholestane derivatives bearing a cascade-type polyol and the effect of their property on a complement system in rat serum. Bioorg. Med. Chem. Lett. 1999, 9, 205-208.

99 Cushman, M.; Golebiewski, W. M.; McMohan, J. B.; Buckeit, J., R. W.; Clanton,

D. J.; Weislow, O.; Haugwitz, R. D.; Bader, J. P.; Graham, L.; Rice, W. G. Design, synthesis and biological evalutation ofa cosalane, a novel anti-HIV agent which inhibits multiple features of virus reproduction. J. Med. Chem. 1994, 37, 3040-3050.

100 Keyes, R. F.; Golebiewski, W. M.; Cushman, M. Correlation of anti-HIV potency

with lipophilicity in a series of cosalane analogs having normal alkenyl and phosphodiester chains as cholestane replacements. J. Med. Chem. 1996, 39, 508-514.

101 McKane, L.; Kandel, J. In Microbiology. Essentials and applications; McGraw-

Hill Book Co.: Toronto, 1985, p 1-151. 102 O'Leary, W. M. Practical handbook of microbiology; CRC Press: Boston, 2000, p

481.

38

103 Quemard, A.; Laneelle, M.-A.; Marrakchi, H.; Prome, D.; Dubnau, E.; Daffe, M. Structure of a hydroxymycolic acid potentially involved in the synthesis of oxygenated mycolic acids of the Mycobacterium tuberculosis complex. Eur. J. Biochem. 1997, 250, 758-763.

104 Liu, J.; Barry III, C. E.; Besra, G. S.; Nikaido, H. Mycolic acid structure

determines the fluidity of the mycobacterial cell wall. J. Biol. Chem. 1996, 271, 29545-29551.

105 Yuan, Y.; Lee, R. E.; Besra, G. S.; Belisle, J. T.; Barry III, C. E. Identification of

a gene involved in the biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6630-6634.

39

Chapter II Synthesis of One-Tailed, Tri-headed Amphiphiles (3CUrn)

To construct a homologous series of one-tailed, tri-headed amphiphiles, we have

chosen to use amines as the hydrophobic moiety and a tricarboxylic dendron as the

hydrophilic moiety. The hydrophobic moiety is attached to the hydrophilic moiety by

forming a ureido linker, which is attached to the Newkome-type dendron by combining

an isocyanate and a variety of amines.1-6 This homologous series is abbreviated as

3CUrn (Scheme II.1), where “3C” represents three carboxylic groups on the hydrophilic

moiety, “Ur” represents a ureido linker (b) between the hydrophobic (c) and hydrophilic

(a) moieties, and “n” represents the number of carbons of the n-alkyl on the hydrophobic

moiety. Based on this chemistry, the synthesis of 3CUrn requires two steps⎯addition

and removal of tert-butyl groups⎯and begins with WeisocyanateTM (3) and alkan-1-

amines (4a−e) (Scheme II.1). Similar to the naming of the 3CUrn series, the

Scheme II.1 Retrosynthesis of one-tailed, tri-headed amphiphiles (3CUrn)

HN

HN

O

OCNO

O3

R NH2

R

O

O

O

O

O

O

H

H

HN

HN

O

R

O

O

O

O

O

O

+

4a-e

3

3CUrn 3EUrn

R = n-CnH2n+1

1 2

c b a

a. a hydrophilic moiety; b. a ureido linker, c. a hydrophobic moiety

a, n=14b, n=16c, n=18d, n=20e, n=22

H

40

homologous series in which the tert-butyl groups will be removed, is abbreviated as

3EUrn, where “3E” represents three tert-butyl esters on the dendron.

II.1 Preparation of WeisocyanateTM (3)

WeisocyanateTM (3) was prepared according to the published

procedure7⎯combining Behera’s amine (5, 0.25M), DMAP, and (Boc)2O in

dichloromethane. Newkome et al.7 reported that the reaction was run for 30 minutes,

95% crude yield, and no purification yield. These conditions produced side products in

our laboratory.

Scheme II.2 Synthetic scheme of the synthesis of isocyanate 3

DMAP, (Boc)2O

CH2Cl2rt

3 +

7

NH

O

O 3

NH

OO

O 3

NH

O

O 3

O

O

+

6

H2NO

O 35

Based on the mechanistic study8 of the reaction of various amines and (Boc)2O in

the presence of DMAP, the desired isocyanate 3 was obtained along with the side

products⎯the t-Boc-protected amine 6 and urea 7. We speculated from the 1H NMR

spectrum that the only side product was urea 7. When the reaction solution was run at a

more dilute concentration (i.e., 0.12 M of amine 5 for two hours), isocyanate 3 and urea 7

based on its 1H NMR spectrum, were obtained in 76% and 24% yields, respectively.

When the reaction was run for a shorter reaction time, the side products 6 and 7 (as

41

shown by the 1H NMR spectrum) were not observed. The optimal reaction time was 15

minutes. The crude product was recrystallized from hexane to give isocyanate 3 (84%

yield).

II.2 Preparation of Alkan-1-amines

We needed five even-numbered long-chain alkan-1-amines (4a−e)⎯tetradecan-,

hexadecan-, octadecan-, icosan-, and docosanamine⎯to construct a homologous series.

However, amines 4d−e are not commercially available. Thus, the synthesis of both was

necessary. We sought to design the synthesis beginning with inexpensive commercially

available materials.

II.2.1 Literature Review on Syntheses of Long-chain Alkan-1-amines (4)

There are few literature procedures describing the synthesis of long-chain

alkan-1-amines (n > 18). Retrosynthesis (Scheme II.3) shows different commercially

available materials can be employed as precursors.

Scheme II.3 Retrosynthesis of alkan-1-amines

4 8 9

10 11 12

R NH2 R N3 R OMs

R OHR Br

R = n-CnH2n+1

4d, n = 204e, n = 22

n = 14, 16, 18, 20, 22

letters "d" and "e" refer to the corresponding amines

NH2

O

n-2

42

Amines 4 can be simply obtained from reduction of the corresponding amides 10

or azides 8 (Table II.1). In the literature,9-17 there are a variety of reagents reported to

reduce azides 8 to the corresponding amines 4. The reducing agents may have some

limitations such as selectivity, ready availability, and convenience of operation. In our

case, reagent selectivity is not considered because there is no other functional group in

the precursors being reduced.

Various reducing agents of 1-azidoalkanes have been documented. These include

lithium aluminum hydride,9,10,17 and different forms of borohydride,11,13,18-23 tributyltin

hydride,12 tributyltin hydride in the presence of nickel dichloride

diphenylphosphinoethane,24 tin dichloride and aluminum trichloride in methanol,25 tin(II)

complex,26 catalytic hydrogenation,16,27 N,N-dimethylhydrazine/ferric chloride in

acetonitrile,15 magnesium in methanol,28 metallic zinc in ethanol,29 methanol,30,31

tetrahydrofuran in the presence of cobaltdichloride hexahydrate,32 powdery zinc in

tetrahydrofuran in the presence of nickeldichloride hexahydrate,33 tetracarbonylhydrido-

ferrate,34 borane or dichloroborane-dimethylsulfide,35 aluminum triiodide,36 ferric

chloride and sodium iodide in acetonitrile,14 metallic samarium and samarium iodide in

various alcohols,37-41 indium in aqueous hydrochloric acid,42 and tellurium in water.43

43

Table II.1 Preparations of amines 4 from different precursors Precursor Reagent, Solvent Yield (%) Reference

18NH2

O

LiAlH4, Et2O

not reported 9

N319

LiAlH4

not reported 10

N315

NaBH4, H2O, C16H33PBu3Br, PhCH3

88

11

N315

Te, H2O

91 43

N314

LiAlH4, Et2O

not reported 17

N311

AIBN, Bu3SnH, PhSiH3, n-PrOH AIBN, Bu3SnH, PhMS, n-PrOH

94 95

12

N311

(Sn(SPh)3)(Et3NH), PhH

98 26

N311

BHCl2.SMe2, THF or Et2O

78 35

N310

FeCl3/NaI, CH3CN

85 14

N310

Zn(BH4)2, silica gel, DME

92 13

N310

AlI3, PhH

75 36

N39

SnCl2, AlCl3, MeOH

88 25

N39

In, aq. HCl, THF

92

42

N39

Bu3SnH, NidppeCl2, THF dpee = diphenylphosphinoethane

80 24

N39

Mg, MeOH

95 28

II.2.2 Literature Review on Syntheses of 1-Azidoalkanes (8)

Azides 8 are usually synthesized from the corresponding alcohols 12 or

44

bromoalkanes 11. Starting with alcohols 12, the first step involves the conversion of the

hydroxyl group into a better leaving group. This leaving group is typically a bromide or

a sulfonate ester. In some cases, bromoalkanes 11 are commercially available. The next

step involves a direct nucleophilic substitution (SN2) with an azide acting as the

nucleophile.

Several examples of the synthesis of azides 8 from the corresponding

bromoalkanes 11 and sulfonate esters are presented in Table II.2.

45

Table II.2 Preparations of azides 8 from different precursors Precursor Reagent, solvent Yield (%) Reference

Br19

azide not specified

not reported 10

Brn-1

n = 8, 16

(C8H17)3NCH3Cl, NaN3, H2O, 80 °C

89 (n=16) 85 (n=8)

11

Br10

NaN3, dry DMF, 80 °C

75 13

Br7

NaN3, DMSO, rt

99 40

OSO2CH3n-1

n = 8, 10, 15

NaN3, DMF, 90 °C

not reported (n=15)

n = 15,17 10,24 8.24

OSO2CH3n-1

n = 6, 8

[hexmim][N3], various solvents, 60 °C solvents: PhCl, DMSO, MeOH, [hexmim][ClO4], and [hexmim][PF6] [hexmim] = [1-hexyl-3-methylimidazolium]

not reported 44

OSO2CH37

(C8H17)3NCH3Cl, NaN3, H2O, 80 °C

89 11

OSO2CH37

[cis-Dicyclohexane-18-crown-6][K]+[N3]- or C16H33NBu3N3 in PhCl, H2O, 60 °C

not reported 45

OTs11

Me3SiN3, Bu4NF, THF, rt

96 46

OTs7

NaN3 and [bmim][PF6] NaN3 and [bmim][N(Tf)2] NaN3 and [hpyr][N(Tf)2] [bmim] = [1-butyl-3-methylimidazolium] [hpyr] = [1-hexylpyridinium]

>90 >90 >90

47

OTs7

NaN3, CH3CN, heating

not reported 48

OTs7

NaN3, H2O, 90 °C NaN3, DMF, 90 °C, microwave Irradiation

92 94

49

OTs7

C16H33PBu3N3, MeOH, 60 °C

not reported 50

OTs7

n = 5−7

NaN3, DMF, 80 °C

not reported 51

46

II.2.3 Literature Review on Syntheses of 1-Methanesulfonyloxyalkanes (9)

As mentioned above, a sulfonate ester is one of several typical leaving groups in

many SN2 reactions. When SN2 reactions are performed with alcohols as precursors,

transformation of the alcohols into the corresponding sulfonate esters is often required.

As sulfonate esters⎯mesylate and tosylate⎯are good leaving groups, such a

transformation is almost routine in organic synthesis. In addition, not only is the reaction

run under mild conditions, but it also gives excellent yield. The transformation of many

alcohols into the corresponding 1-methanesulfonyloxyalkanes,24,52-59 several of which are

long-chain alcohols 12 (dodecan-,52 tetradecan-,53 octadecan-,58 and icosan-56), has been

reported to give good to excellent yields.

II.2.4 Literature Review on Syntheses of 1-Bromoalkanes (11)

In general, bromoalkanes 11 are commercially available. However, should

synthesis be required, the most common precursor is the corresponding alcohols 12.

Alcohols 12 are mostly commercially available at low cost. One-step syntheses of

bromoalkanes 11 have been well documented. Many of them employ triphenylphosphine

in combination with carbon tetrabromide,60-62 bromine,63,64 N-bromosuccinimide,65

2,4,4,6-tetrabromo-2,5-cyclohexadien-1-one,66 and 1,2-dibromotetrachloroethane.67

Several ultra long-chain bromoalkanes 11,68 such as -tetracosane, -octacosane, and -

pentatriacontane, are obtained in 56–63% from the corresponding alcohols 12 in the

presence of 48% aqueous hydrobromic acid and concentrated sulfuric acid; these

procedures are typically run by heating at high temperature (≥ 80 °C). On the other hand,

bromoalkanes 11 can also be obtained under mild conditions in several steps (Table II.3).

47

Table II.3 Preparation of bromoalkanes 11 from alcohols 12 Precursor Reagent Yield (%) Reference

OH21

1.ClCH2CH2SO2Cl (or CH2=CHSO2Cl), Me3N; 2. Me2NH; 3. CH3SO3F; 4. KBr

67

69,70

OH15

1.ClCH2CH2SO2Cl, Me3N; 2. Me2NH; 3. MeBr

83 69,70

OH19

1. Et3N, MsCl, CH2Cl2; 2. LiBr, THF

77 56

OH15

1. MsCl; 2. NaBr

not reported 71

II.2.5 Preferred Starting Material⎯Alkan-1-ols (12) or 1-Bromoalkanes (11)

Both bromoalkanes 11 and alcohols 12 can be employed as the precursors for the

synthesis of azides 8 via SN2 reactions. Beginning with bromoalkanes 11, the synthesis

of azides 8 is only a one-step reaction. On the other hand, should alcohols 12 be utilized,

the synthesis of azides 8 requires a two-step reaction⎯transformation to the

corresponding sulfonate esters, such as 1-methanesulfonyloxyalkanes, followed by SN2

reactions. As sulfonate esters are better leaving groups than bromide,72 theoretically, the

SN2 reaction of 1-methanesulfonyloxyalkanes that leads to the formation of the

corresponding azides 8 should be easier than that of bromoalkanes 11.

II.2.6 Synthesis of Long-chain Alkan-1-amines (4)

At the beginning, we repeated the reduction procedure9 with lithium aluminum

hydride and technical grade of docosanamide (amide 10e) as the starting material, which

was commercially available at a low cost. Attempts to purify the technical grade amide

10e were not successful because of low solubility in many organic solvents. As the

reaction yield was low and there were several unidentified side products based on the 1H

NMR spectrum, no attempt was made to isolate the desired product.

48

We then switched our strategy by utilizing bromoalkane 11d−e as the precursors.

Initially, we followed the procedure 11 for phase-transfer catalysis reaction. Some

modifications were made; cetyltrimethylammonium bromide (CTAB) was used as the

catalyst instead of trioctylmethylammonium chloride, and the reaction was sonicated

prior to reflux. When the reaction was conducted without sonication in a variety of high

boiling-point solvents, such as tert-butyl alcohol and toluene, we observed no reaction.

We found out that sonication for three hours prior to reflux was very critical for the

transformation. Compared to the published procedure11 for 1-azidohexadecane (8c) and

1-azidooctane, in which the reaction time was only eight hours, the reactions for the

syntheses of 1-azidoicosane (8d) and 1-azidodocosane (8e) were refluxed for three days

to get complete conversions. The overall yields of azides 8d and 8e from the

corresponding bromoalkanes 11 were 87% and 75%, respectively.

In order to overcome the long reaction time above, we decided to use alcohols as

our precursors. Alcohols 12d10 and 12e73 were transformed into the corresponding

methanesulfonyloxyalkanes 9 in excellent yields. Methanesulfonyloxyalkane 9e was

characterized by comparing its 1H NMR spectrum to published values,73 and used

without further purification. Again, the resulting methanesulfonyloxyalkane 9e could be

transformed into bromoalkane 11e as we wished. However, we attempted to directly

convert them into azide 8e according to the published procedure.13 Because

methanesulfonate was a better leaving group than bromide, we expected that the reaction

would be completed in less time than that in the procedure13 followed. On the other

hand, because we employed a longer chain, the reaction could require a longer time. We

monitored the reactions by thin layer chromatography, and after several trials on our scale

49

(7.82 mmol of methanesulfonylalkanes 9), we concluded the reaction was completed

within 3–4 hours. The crude azides 8 were purified by flash column chromatography in

hexane. Melting temperatures of the desired products were not taken because we

obtained “glue-like” solids. The desired products were characterized by comparing their

IR and NMR to published data.69

Because of convenience (ease of work-up and fewer side products), the desired

azides 8 were reduced via hydrogenation with 10% palladium on carbon as a catalyst.16

The resulting amines 4 were purified by recrystallization from ethyl acetate. Amines 4

were characterized by comparing the mp, IR, and NMR spectra to published data.10,74,75

The general synthesis and the yield of 1-alkanamines (4d and 4e) are presented in

Scheme II.4. The overall yields of 4d and 4e obtained from the corresponding

bromoalkanes 11 in two steps were 65% and 53%, respectively, while those from the

corresponding alcohols 12 in three steps were about 60% and 57%, respectively. Amine

4d was obtained in a higher yield from bromoalkane 11d, while amine 4e was obtained in

a higher yield from alcohol 12e.

Scheme II.4 Synthetic scheme of amines 4d and 4e

i. MsCl, Et3N, CH2Cl2, rt, 3h (9d−e, 92–95%)ii. NaN3, DMF, reflux, 4h (8d, 86%; 8e, 87%)iii. Pd/C, H2, hexane, rt, 3h (4d, 75%; 4e, 70%)iv. LiBr, dry THF, rt, overnight (11d, 84%; 11e, 65%)v. CTAB, H2O, , for 3 h; NaN3, 80–90 °C, 3 d (8d, 87%; 8e, 75%)

N3OSO2CH3

) ))

NH2OH

Br

i ii iii

ivv

R R R R

R

4

R = n-CnH2n+1d, n = 20e, n = 22

8912

11

50

II.3 Synthesis of the 3EUrn Series (2a−e)

The tri-tert-butyl esters 3EUrn were generated by combining amines 4 and

isocyanate 3 in dichloromethane at ambient temperature (Scheme II.5). Purification was

carried out by either recrystallization (from ethanol−water) or flash column

chromatography. Both purifications gave pure compounds in good to excellent yields

(Table II.4). The trend (Table II.4) observed in the melting ranges data was expected; the

longer the chain, the higher the melting ranges.

Scheme II.5 The synthetic scheme of one-tailed, tri-headed amphiphiles (3CUrn)

HN

HN

O

OHO

n-13

HCOOHrt, 9 h

HN

HN

O

OtBuO

n-1+

CH2Cl2

rt, overnight

33EUrn 3CUrn

4 3

Table II.4 Isolated yields and melting ranges of the tri-tert-butyl esters 3EUrn

n Isolated yield (%) Melting range (°C) 14 86 55.0–55.6 16 72 59.1–59.8 18 92 65.0–65.8 20 69 66.5–67.9 22 84 67.9–68.8

Interestingly, the recrystallized tri-tert-butyl ester 3EUr16 contained one water

molecule for two molecules of 3EUr16 as shown by the X-ray crystal structure. The

presence of water in the recrystallized compounds was supported by both elemental and

thermogravimetric analyses. Elemental analysis showed there was a water molecule for

every two ester molecules, and thermogravimetric analysis showed 1.3% weight loss

from 40–80 °C corresponds to loss of ½H2O. We presumed that ½H2O would be present

in the other 3EUrn since X-ray crystal structure showed that it was trapped between two

of 3EUr16 molecules. Indeed, the elemental analyses from tri-tert-butyl esters 3EUr18,

51

3EUr20, and 3EUr22 supported this hypothesis. However, elemental analysis of tri-tert-

butyl ester 3EUr14 did not support the hypothesis. Unlike the other 3EUrn, 3EUr14

was purified by flash column chromatography because attempts to recrystallize 3EUr14

were not successful. In this case, elemental analysis showed there was no water molecule

in the purified 3EUr14.

II.4 Synthesis of the 3CUrn Series (1a−e)

Removal of the tert-butyl group is a routine procedure in organic synthesis.

Newkome et al.76-82 employed formic acid to remove such a group; the reaction condition

was mild and the yield was excellent. Formolysis of the tri-tert-butyl esters 3EUrn

produced the triacids, 3CUrn, in good yields of recrystallized products (Table II.5). In

contrast to the melting range trend observed in the tri-tert-butyl esters 3EUrn series,

there was no particular trend in the melting range of the triacids 3CUrn series (Table

II.5). Triacid 3CUr16 was observed to have the highest melting range, after which the

melting range slightly decreases for longer chain lengths. All tri-tert-butyl esters and

triacids—3EUrn and 3CUrn— were fully characterized.

Table II.5 Isolated yields and melting ranges of the triacids 3CUrn n Isolated yield (%) Melting range (°C) 14 84 159.0–159.5 16 83 162.4–162.8 18 85 160.7–161.5 20 82 160.3–161.2 22 74 157.3–158.3

II.5 X-ray Crystal Structure of Tri-tert-butyl Ester 3EUr16

The crystal structure of tri-tert-butyl ester 3EUr16 (Figure II.1) reveals how these

molecules that have bulky head groups assemble in the solid state. The molecule can be

considered in three parts: a head group (the three esters), a linker (ureido), and a tail

52

(alkyl chain). Figure II.1 illustrates the closely packed alkyl chains and hydrogen bonds,

which are essential for self-assembly. The molecules are arranged in the lattice in the

typical manner that is seen for compounds with polar head groups and long alkyl chains.

There are two crystallographically independent molecules arranged in parallel;

inversion symmetry generates the antiparallel chains. The hydrogen bonding among the

headgroups and the extensive overlap of the interdigitated alkyl chains suggest that both

are essential for self assembly. Antiparallel chains, which are separated by carbon-to-

carbon distances ranging from 3.9 to 4.4 Å, pack more closely than parallel chains, which

are separated by carbon-to-carbon distances ranging from 4.9 to 5.4 Å. To achieve this

close packing of antiparallel chains, the C(3)–C(4)–C(5)–C(6) and the C(42)–C(43)–

C(44)–C(45) torsion angles adopt gauche conformations, ±67.4(3)° and ±60.9(3)°,

respectively. The hydrogen bonds involve primarily the linker atoms on neighboring

molecules; a water molecule connects alternate pairs of neighbors. In one hydrogen

bond, the carbonyl oxygen [O(8)] on the ureido linker accepts a hydrogen bond from the

amido nitrogen [N(2)] connected to the long alkyl chain. In the other hydrogen bond, the

carbonyl oxygen [O(1)] of the ureido linker accepts a hydrogen bond from a water

[O(15)], which in turn accepts a hydrogen bond from the amido nitrogen [N(4)]

connected to the long alkyl chain. The water also donates a hydrogen bond to an ester-

carbonyl oxygen [O(3)]. The hydrogen bond between the carbonyl oxygen [O(1)] of the

ureido linker and the water [O(15)] is the shortest. The other amido nitrogens in the

ureido linkers, N(1) and N(3), form weak hydrogen bonds (donor–acceptor distances >

3.0 Å) with O(8) and O(15), respectively.

53

Figure II.1 X-ray crystal structure of tri-tert-butyl ester 3EUr16, showing the packing diagram and the intermolecular hydrogen bonding to water and to a neighboring molecule. Hydrogen-bond Donor(D)–Acceptor(A) distances and approximate ∠DHA: O(8)···N(2), 2.863(3) Å and 159.1°; O(15)···O(1), 2.718(4) Å and 170(4)°; O(15)···O(3), 2.843(4) Å and 169(4)°; N(4)···O(15), 2.832(3) Å and 153.4°. The other nitrogens on the ureido linker are just outside the range for significant hydrogen bonding. N(1)···O(8), 3.201(3) Å and 145.6°; N(3)···O(15), 3.074(3) Å and 141.6° II.6 CMCs (Critical Micelle Concentration) of Amphiphiles 3CUrn

One of our current goals in designing the new series of amphiphiles, 3CUrn, is to

have a more water-soluble amphiphile. In the case of amphiphiles, the concept of

solubility is often involved with critical micelle concentration (CMC); CMC is defined as

the concentration in which micellar aggregates start to form. The number of headgroups

and carbons in the chain lengths affect the CMCs.83,84 The number of headgroups affects

CMCs in a proportional manner.83,85-87 On the other hand, the number of carbons in the

hydrophobic moiety is inversely proportional to the CMCs.84 Based on that concept, we

present a series of long-chain multi-headed (tri-headed) amphiphiles, 3CUrn.

54

The choice of the counterion followed from a study88 of N-lauroyl-L-glutamate in

water; the triethanolammonium salt dissolves to a much greater concentration than did

the potassium salt. As chain length can affect the pKa of fatty acids because of

aggregation,89 we explored different conditions to achieve the maximum solubility in

aqueous solutions of triethanolamine (pKa 7.76).90 The 3CUrn homologus series

dissolved readily in a solution of triethanolamine/water [~ 5% (wt/vol)]; the final solution

contained ≥ 9:1 molar equivalents of triethanolamine : 3CUrn.

The CMCs measurement of the homologous 3CUrn series was performed by

Richard V. Macri (Table II.6). Using a pendant-drop analyzer to measure surface

tensions, the CMCs of 3CUr16–3CUr22 were measured in triethanolamine/water [~ 6%

(wt/vol)]. The pH of the solutions ranged from 9.3 to 10.0 depending on the

concentration of the amphiphiles.

Table II.6 CMCs of the 3CUrn homologous series Compound CMCs (mM)

3CUr14 N/A 3CUr16 3.4 ± 0.17 3CUr18 2.2 ± 0.11 3CUr20 1.4 ± 0.070 3CUr22 0.92 ± 0.046

The slope of the plot of log CMC vs chain length is ~ 0.1; this value contrasts

with a value of 0.3 for a similar plot of the CMCs of potassium salts of fatty acids.83

Slopes of similar plots for di- and tri-carboxylato amphiphiles83,87 have values of 0.22.

Unlike the others, amphiphile 3CUr14 did not show a leveling off of the surface tension

with increasing concentrations up to 10 mM suggesting that it failed to form micelles.

The CMC for the longest homolog, 3CUr22, is 1 mM, which is quite high for an ultra-

long chain.

55

II.7 Experimental Procedures Materials and Methods.

Chemicals were obtained from Aldrich, Acros and TCI; they were used without further

purification. Solvents were reagent grade or HPLC grade; they were used as received

unless otherwise specified. THF was distilled from sodium/benzophenone ketyl.

WeisocynateTM was prepared as described7 with a shorter reaction time as mentioned

above. Analytical thin layer chromatography was performed by polyester-coated silica

gel 60 Å and detected by treating with 10% ethanolic phosphomolybdic acid reagent (20

wt. % solution in ethanol) followed by heating. Flash column chromatography was

carried out on silica gel (60 Å); column diameter × height (13/4 × 6 in), eluted samples

varied between ~ 2.00−4.00 g, flow rate (~ 1.5−2 in/min) was controlled by air pressure.

Solutions were concentrated by rotary evaporation. Melting ranges, determined in open

capillary tubes, were uncorrected. NMR spectra were recorded on an INOVA at 400 and

100 MHz for 1H and 13C, respectively, and reported in ppm relative to the known solvent

residual peak. Resonances were reported in the order of chemical shift (δ), followed by

the splitting pattern, and the number of protons. Abbreviations used in the splitting

pattern were as the following: s = singlet, d = doublet, t = triplet, q = quartet, quin =

quintet, m = multiplet, and b = broad. IR spectra were recorded on neat samples with an

FTIR equipped with a diamond ATR system, and reported in cm-1. HRMS data were

obtained on a dual-sector mass spectrometer in FAB mode with 2-nitrobenzylalcohol as

the proton donor. Elemental analyses were performed by Atlantic Microlabs, Inc. in

Norcross, GA.

56

Preparation of alkyl mesylate (9d−e)56

OMsn-1

OHn-1 MsClEt3N+ +

DCMrt, 3h

To a solution of alcohol 12 (16.2 mmol) and Et3N (5.23 mL, 37.3 mmol) in CH2Cl2 was

added MsCl (2.00 mL, 25.7 mmol) through a syringe. The resulting solution was stirred

for 3 h. The resulting yellow reaction was washed successively with water (14 mL), HCl

(2 M, 14 mL), water (14 mL), satd NaHCO3 (14 mL), and water (14 mL). The organic

solution was dried with MgSO4 and concentrated to give a white-yellow solid (92–95%).

The 1H NMR spectra of the respective crude products—icosyl mesylate and docosyl

mesylate73—suggested that the material was suitable for the next step without further

purification.

Preparation of 1-bromoicosane56

Br19

OMs19

+ LiBrdry THF

rt, overnight

To a solution of 9d (6.10 g, 16.2 mmol) in dry THF (50 mL) was slowly added LiBr

(3.57 g, 40.7 mmol). The cloudy yellow suspension was stirred at rt for 14 h, after which

the solution was concentrated to dryness to give an off-white solid. This solid was

treated with hexane (60 mL); most of the solid dissolved. After filtration, the filtrate was

concentrated to dryness to leave a light yellow solid, which was recrystallized from hot

EtOH to give a white solid (60%); mp 36.0–36.4 °C (lit.91 mp 36.2–37.0 °C); 1H NMR

(CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 32H), 1.42 (m, 2H), 1.86 (quin, 2H), 3.41 (t, 2H)

(lit.92 200 MHz); 13C NMR (CDCl3) δ 14.3, 22.9, 28.4, 29.0, 29.6, 29.7, 29.82, 29.86,

29.89, 32.1, 33.0, 34.21; IR 2915, 2847, 1472, 1462, 730, 719 (lit.92 in CHCl3).

57

General procedure for the preparation of 1-azidoalkane (8d−e)

Method A.11

N3n-1

Brn-1 + NaN3

CTAB, H2O))) , 4h

80–90 °C, 3 d

A mixture of bromoalkane 11 (6.64 mmol), CTAB (243 mg, 0.661 mmol), NaN3 (1.12 g,

17.0 mmol), and water (3 mL), was sonicated for 4 h at 60 °C. The resulting emulsion

was refluxed (80–90 °C) for 3 d. The milky reaction mixture was treated with hexane

and some solid formed. After gravity filtration, the filtrate was concentrated to give a

sticky white solid (75–87%).

Method B.

N3n-1

OMsn-1

+ NaN3

85 °C, 4hDMF

NaN3 (1.28 g, 19.5 mmol) was added to a solution of methanesulfonyloxyalkane 9 (7.82

mmol) in DMF (52 mL). The mixture was stirred at rt for 30 min, and then refluxed at 85

°C for 4 h. Afterwards, the reaction was cooled before portions of hexane (104 mL) and

water (10.5 mL) were added. The organic layer was separated and washed successively

with satd NaHCO3 (10.5 mL) and satd NaCl (10.5 mL). The organic layer was dried with

Na2SO4 and concentrated to give a crude product, which was purified by flash column

chromatography in hexane to give a sticky white solid (82–86%).

1-Azidoicosane (8d)

The procedures described above afforded a sticky white solid (1.87 g, 87%, Method A;

2.37 g, 86%, Method B); 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.40 (bm, 34H), 1.60

(quin, 2H), 3.25 (t, 2H); 13C NMR (CDCl3) δ 14.3, 22.9, 26.9, 29.0, 29.4, 29.56, 29.68,

58

29.74, 29.83, 29.89, 32.1, 51.7; IR 2915, 2848, 2093, 1467, 1260.

1-Azidodocosane (8e)

The procedures described above afforded a sticky white solid (lit.69 mp 41–42 °C); 1H

NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.40 (bm, 38H), 1.60 (p, 2H), 3.26 (t, 2H) (lit.69 60

MHz); 13C NMR (CDCl3) δ 14.3, 22.9, 26.9, 29.0, 29.4, 29.6, 29.69, 29.75, 29.83, 29.86,

29.90, 32.1, 51.7; IR 2915, 2848, 2093, 1466, 1253 (lit.69 in CHCl3).

General procedure for the preparation of alkan-1-amine (4d−e):16

NH2n-1

N3n-1

Pd/C, H2

hexane, rt, 3 h

To a solution of azide 8 (5.38 mmol) in hexane (25 mL) was added 10% Pd/C (3% w/w

of azide 8). The resulting mixture was hydrogenated at 62 psi at rt for 3 h. Following

gravity filtration, the filtrate was concentrated to give off-white solid, which was

recrystallized from ethyl acetate (70–75%).

Icosan-1-amine (4d)

The procedures described above afforded a white solid (1.20 g, 75%); mp 58.3–59.0 °C

(lit.74 mp 58.5 °C); 1H NMR (CDCl3) δ 0.89 (t, 3H), 1.20–1.35 (bm, 36H; 2H exchange

with D2O), 1.44 (m, 2H), 2.69 (t, 3H) (lit.10); 13C NMR (CDCl3) δ 14.3, 22.9, 27.1, 29.55,

29.72, 29.85, 29.89, 32.1, 34.1, 42.5; IR 3328, 2915, 2849, 1486, 1472.

Docosan-1-amine (4e)

The procedures described above afforded a white solid (1.23g, 70%); mp 62.9–63.8 °C

(lit.75 mp 62.7 °C); 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 40H; 2H exchange

with D2O), 1.44 (m, 2H), 2.68 (t, 3H); 13C NMR (CDCl3) δ 14.3, 22.9, 27.1, 29.55, 29.71,

29.83, 29.85, 29.89, 32.1, 34.1, 42.5; IR 3300, 2915, 2848, 1472, 1462.

59

General procedure for the preparation of long chain tri tert-butyl esters 3EUrn (2a−e)

HN

HN

O

OtBuO

n-1OCN

OtBu

O 3

NH2n-1 +

DCMrt, overnight

3

An amine 4 (5.43 mmol) was added slowly to a solution of isocyanate 3 (5.43 mmol) in

CH2Cl2 (45 mL). The resulting colorless solution was stirred at rt. After stirring

overnight, the solvent was removed to leave a crude yellow oil (n = 14) or a crude white

solid (n = 16, 18, 20, 22). The crude yellow oil was purified by flash chromatography

(hexane:ethyl acetate = 5:1), while the crude white solid was purified by recrystalization

with EtOH–H2O. Both purification methods gave a pure white solid (69–92%).

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3-tetradecylureido)heptanedioate,

3EUr14

The general procedure described above afforded a white solid (3.07 g, 86%); mp 55.0–

55.6 °C; 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 22H), 1.35–1.50 (bm, 29H),

1.94 (t, 6H), 2.24 (t, 6H), 3.14 (q, 2H), 4.08 (m, 1H), 4.57 (s, 1H); 13C NMR (CDCl3) δ

14.1, 22.7, 26.9, 28.1, 29.3, 29.58, 29.60, 29.64, 29.65, 29.9, 30.2, 30.6, 31.9, 40.6, 56.4,

80.5, 156.8, 173.1; IR 3327, 2919, 2850, 1721, 1673, 1646, 1559, 1148; HRMS: for

C37H71N2O7 (M + H)+calcd 655.5246, found 655.5261. Anal. Calcd for C37H70N2O7: C,

67.85; H, 10.77; N, 4.28. Found: C, 68.01; H, 10.92; N, 4.27.

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3-hexadecylureido)heptanedioate,

3EUr16




60

14.1, 22.7, 26.9, 28.1, 29.3, 29.58, 29.60, 29.64, 29.7, 29.9, 30.2, 30.6, 31.9, 40.6, 56.5,

80.5, 156.8, 173.1; IR 3310, 2917, 2851, 1719, 1700, 1645, 1563, 1147; HRMS: for

C39H75N2O7 (M + H)+ calcd 683.5574, found 683.5540. Anal. Calcd for

C39H74N2O7·½H2O: C, 67.69; H, 10.92; N, 4.05. Found: C, 67.92; H, 11.03; N, 4.04.

Thermogravimetric analysis: ~ 1.3 % weight loss from 40–80 °C corresponds to loss of

½H2O.

X-ray analysis of amphiphile 3EUr16

Colorless needles (~3 × 0.2 × 0.1 mm3) were crystallized from EtOH–H2O at rt. The

chosen crystal was cut (0.37 x 0.12 x 0.07 mm3) and mounted on a nylon CryoLoop™

(Hampton Research) with Krytox® Oil (DuPont) and centered on the goniometer of a

Oxford Diffraction XCalibur2™ diffractometer equipped with a Sapphire 2™ CCD

detector. The data collection routine, unit cell refinement, and data processing were

carried out with the program CrysAlis.93 The Laue symmetry was consistent with the

triclinic space group P1̄. The structure was solved by direct methods and refined with the

SHELXTL NT program package.94 The asymmetric unit of the structure comprises two

crystallographically independent molecule of amphiphile 3EUr16 and one water

molecule. One tert-butyl group of molecule two showed evidence of rotational disorder

and the methyl groups were modeled as occupying two conformations with relative

occupancies of 0.728 and 0.272. The final refinement model involved anisotropic

displacement parameters for non-hydrogen atoms. A riding model was used for the

aromatic and alkyl hydrogens. Hydrogen atom positions for the water molecule were

located from the electron density map and subsequently restrained to be 0.84 Å from the

61

oxygen. The program package SHELXTL NT94 was used for molecular graphics

generation.

Table II.7 Crystal data and structure refinement of 3EUr16 Category Crystal data and structure refinement Empirical formula C39H74N2O7 • 0.50H2O Formula weight 692.01 Temperature 100(2) K Wavelength 0.71073 Å Crystal system triclinic Space group P1̄ Unit cell dimension a = 11.362(3) Å; b = 12.2865(19); c = 32.335(3) Å;

α = 88.141(12)°; β = 59.584(9)°; γ = 67.804(19)° Volume 4177.1(13) Å3 Z 4 Density (calculated) 1.100 Mg/m

3

Absorption coefficient 0.075 mm-1 F(000) 1532 Crystal size 0.37 × 0.12 × 0.07 mm3 Theta range for data collection 2.78 to 24.13° Index ranges −13 ≤ h ≤ 10, −14 ≤ k ≤ 14, −36 ≤ l ≤ 37 Reflections collected 21062 Independent reflections 13197 [R(int) = 0.0378] Completeness to theta = 25.07° 99.2 % Absorption correction None Refinement method Full-matrix least-squares on F

2

Data / restraints / parameters 13197/2/932 Goodness-of-fit on F2 0.949 Final R indices [I>2σ(I)] R1 = 0.0517, wR2 = 0.1207 R indices (all data) R1 = 0.0956, wR2 = 0.1392 Largest diff. peak and hole 0.719 and −0.523 e.Å

-3

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3-octadecylureido)heptanedioate,

3EUr18




14.1, 22.7, 26.9, 28.0, 29.3, 29.58, 29.62, 29.66, 29.9, 30.2, 30.6, 31.9, 40.5, 56.4, 80.5,

156.9, 173.1; IR 3310, 2917, 2850, 1721, 1700, 1646, 1565, 1149; HRMS: for

62



Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3-icosylureido)heptanedioate,

3EUr20




14.2, 22.7, 26.9, 28.1, 29.4, 29.63, 29.65, 29.69, 29.73, 29.9, 30.2, 30.6, 32.0, 40.6, 56.5,

80.5, 156.8, 173.2; IR 3310, 2917, 2851, 1721, 1701, 1646, 1567, 1150; HRMS: for



Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3-docosylureido)heptanedioate,

3EUr22

The general procedure described above afforded a white solid (3.49g, 84%); mp 67.9–



14.1, 22.7, 26.9, 28.1, 29.3, 29.59, 29.61, 29.7, 29.9, 30.2, 30.6, 31.9, 40.6, 56.5, 80.5,

156.8, 173.1; IR 3310, 2916, 2850, 1723, 1701, 1646, 1565, 1152; HRMS: for



General procedures for preparation of long-chain triacids, 3CUrn (1a−e)

HN

HN

O

OHO

n-1HCO2Hrt, 9 h

HN

HN

O

OtBuO

n-1

3 3

63

A tri tert-butyl ester 2 (5.02 mmol) was added to HCOOH (20 mL). The resulting

mixture was stirred to give a transparent solution. The mixture might need to be warmed

slightly to get all the 3CUrn to dissolve. The transparent solution was stirred at rt for 9

h; the complete reaction was identified by the formation of milky solution. The solution

was concentrated to yield a white solid, which was recrystallized with HOAc−hexane to

give a white solid (74–85%).

4-(2-Carboxyethyl)-4-(3-tetradecylureido)heptanedioic acid, 3CUr14

The general procedure described above afforded a white solid (2.06 g, 84%); 159.0–159.5

°C; 1H NMR (CD3OD) δ 0.90 (t, 3H), 1.25–1.35 (bm, 22H), 1.44 (bm, 2H), 1.95 (t, 6H),

2.27 (t, 6H), 3.05 (t, 2H); 13C NMR (DMSO-d6) δ 14.0, 22.1, 26.4, 28.2, 28.7, 28.8,

29.04, 29.09, 30.0, 31.3, 38.8, 55.0, 157.0, 174.5; IR 3395, 3352, 2916, 2849, 1709,

1693, 1610, 1559; HRMS: for C25H47N2O7 (M + H)+ calcd 487.3383, found 487.3384.

Anal. Calcd for C25H46N2O7: C, 61.70; H, 9.53; N, 5.76. Found: C, 61.94; H, 9.55; N,

5.70.

4-(2-Carboxyethyl)-4-(3-hexadecylureido)heptanedioic acid, 3CUr16


162.8 °C; 1H NMR (CD3OD) δ 0.90 (t, 3H), 1.25–1.35 (bm, 26H), 1.44 (bm, 2H), 1.95 (t,

6H), 2.27 (t, 6H), 3.05 (t, 2H); 13C NMR (DMSO-d6) δ 14.4, 22.6, 26.9, 28.6, 29.2, 29.3,

29.5, 30.47, 30.54, 31.8, 39.3, 55.5, 157.5, 175.0; IR 3395, 3355, 2916, 2849, 1701,

1692, 1610, 1560; HRMS for C27H51N2O7 (M + H)+ calcd 515.3696, found 515.3669.


5.33.

64

4-(2-Carboxyethyl)-4-(3-octadecylureido)heptanedioic acid, 3CUr18


161.5 °C; 1H NMR (CD3OD) δ 0.90 (t, 3H), 1.25–1.35 (bm, 30H), 1.44 (bm, 2H), 1.95 (t,

6H), 2.27 (t, 6H), 3.05 (t, 2H); 13C NMR (DMSO-d6) δ 14.6, 22.8, 27.1, 28.8, 29.4, 29.5,

29.71, 29.74, 30.7, 32.0, 39.5, 55.7, 157.7, 175.2; IR 3396, 3348, 2914, 2849, 1709,



5.12.

4-(2-Carboxyethyl)-4-(3-icosylureido)heptanedioic acid, 3CUr20


161.2 °C; 1H NMR (CD3OD) 1H NMR (CD3OD) δ 0.90 (t, 3H), 1.25–1.35 (bm 34H),

1.44 (bm, 2H), 1.95 (t, 6H), 2.28 (t, 6H), 3.05 (t, 2H); 13C NMR (DMSO-d6) δ 14.4, 22.6,

26.9, 28.6, 29.2, 29.3, 29.5, 30.48, 30.54, 31.8, 39.3, 55.5, 157.5, 175.0; IR 3394, 3348,

2914, 2848, 1708, 1691, 1608, 1560; HRMS: for C31H59N2O7 (M + H)+ calcd 571.4322,

found 571.4290. Anal. Calcd for C31H58N2O7: C, 65.23; H, 10.24; N, 4.91. Found: C,

65.08; H, 10.28; N, 4.80.

4-(2-Carboxyethyl)-4-(3-docosylureido)heptanedioic acid, 3CUr22


158.3 °C; 1H NMR (CD3OD) 1H NMR (CD3OD) δ 0.90 (t, 3H), 1.25–1.35 (bm, 38H),

1.44 (bm, 2H), 1.95 (t, 6H), 2.27 (t, 6H), 3.05 (t, 2H); 13C NMR (DMSO-d6) δ 13.9, 22.1,

26.4, 27.7, 28.1, 28.7, 28.8, 29.0, 30.0, 31.3, 38.8, 55.0, 157.0, 174.5; IR 3395, 3347,



65

66.44; H, 10.60; N, 4.57.

References for Chapter II 1 Newkome, G. R. Design, synthesis, and characterization of conifer-shaped

dendritic architectures. Chem.––Eur. J. 2006, 12, 3726-3724. 2 Sun, H.; Kaifer, A. E. Unsymmetric dendrimers containing a single

ureidopyrimidine unit: generation-dependent dimerization via hydrogen bonding. Org. Lett. 2005, 7, 3845-3848.

3 Wang, P.; Moorefield, C. N.; Newkome, G. R. Synthesis, x-ray structure, and

self-assembly of functionalized bis-(2,2':6',2''-terpyridinyl)arenes. Org. Lett. 2004, 6, 1197-1200.

4 Pratt, M. D.; Beer, P. D. Anion recognition and sensing by mono- and bis-urea

substituted ferrocene receptors. Polyhedron 2003, 22, 649-653. 5 Newkome, G. R.; Yoo, K. S.; Kabir, A.; Malik, A. Synthesis of benzyl-terminated

dendrons for use in high-resolution capillary gas chromatography. Tetrahedron Lett. 2001, 42, 7537-7541.

6 Newkome, G. R.; Godinez, L. A.; Moorefield, C. N. Chem. Commun. 1998, 17,

1821-1822. 7 Newkome, G. R.; Weis, C. D.; Childs, B. J. Syntheses of 1→3 branched

isocyanate monomers for dendritic construcion. Des. Monomers. Polym. 1998, 1, 3-14.

8 Hassner, A.; Basel, Y. Di-tert-butyl dicarbonate and 4-(dimethylamino)pyridine

revisited. Their reactions with amines and alcohols. J. Org. Chem. 2000, 65, 6368-6380.

9 Nagase, A.; Kuwahara, Y.; Tominaga, Y.; Sugawara, R. Nematicidal activity of

N-substituted and N,N-disubstituted alkylamines against the pine wood nematode, Bursaphelenchus xylophilus. Agric. Biol. Chem. 1983, 47, 53-58.

10 Cuvillier, N.; Rondelez, F. Kinetics of ion exchange under a charged surface.

Langmuir 1999, 15, 5547-5554. 11 Rolla, F. Sodium borohydride reactions under phase-transfer conditions:

reduction of azides to amines. J. Org. Chem. 1982, 47, 4327-4329.

66

12 Hays, D. S.; Fu, G. C. Development of Bu3SnH-catalyzed processes: efficient

reduction of azides to amines. J. Org. Chem. 1998, 63, 2796-2797. 13 Ranu, B. C.; Sarkar, A.; Chakraborty, R. Reduction of azides with zinc

borohydride. J. Org. Chem. 1994, 59, 4114-4116. 14 Kamal, A.; Ramana, K. V.; Ankati, H. B.; Ramana, A. V. Mild and efficient

reduction of azides to amines: synthesis of fused [2.1-b]quinazolinones. Tetrahedron Lett. 2002, 43, 6861-6863.

15 Kamal, A.; Reddy, B. S. N. A mild and facile reduction of azides to amines by

N,N-dimethylhydrazine and catalytic ferric chloride. Chem. Lett. 1998, 593. 16 Gartizer, T.; Selve, C.; Delpuech, J.-J. Reduction d'azide en amines par le

formiate d'ammonium par "transfert d'hydrogene catalyse". Tetrahedron Lett. 1983, 24, 1609-1610.

17 Vandevoorde, S.; Tsuboi, K.; Ueda, N.; Jonsson, K.-O.; Fowler, C. J.; Lambert,

D. M. Esters, retroesters, and a retroamide of palmitic acid: pool for the first selective inhibitors of N-palmitoylethanolamine- selective acid amidase. J. Med. Chem. 2003, 46, 4373 - 4376.

18 Ram, S. R.; Chary, K. P.; Salahuddin, S.; Iyengar, D. S. An efficient

chemoselective production of amines from azides using AlCl3/NaBH4. Indian J. Chem. Sect. B 2003, 42, 935-937.

19 Ram, S. R.; Chary, K. P.; Iyengar, D. S. An efficient and chemoselective

deoxygenation of heterocyclic N-oxides using LiCl/NaBH4. Synth. Commun. 2000, 30, 3511-3515.

20 Chary, K. P.; Ram, S. R.; Salahuddin, S.; Iyengar, D. S. A novel, chemoselective

and efficient production of amines from azides using ZrCl4/NaBH4. Synth. Commun. 2000, 30, 3559-3564.

21 Sim, T. B.; Yoon, N. M. Reducing characteristics of borohydride exchange resin-

CuSO4 in methanol. Bull. Chem. Soc. Jpn. 1997, 70, 1101-1108. 22 Rao, H. S. P.; Siva, P. Facile reduction of azides with sodium

borohydride/copper(II) sulfate system. Synth. Commun. 1994, 24, 549-556. 23 Yoon, N. M.; Choi, J.; Shon, Y. S. Reduction of azides to amines with

borohydride exchange resin-nickel acetate. Synth. Commun. 1993, 23, 3047-3054. 24 Malanga, C.; Mannucci, S.; Lardicci, L. Nickel mediated reduction of azides by

Bu3SnH. J. Chem. Res. Synop. 2000, 6, 0701-0715.

67

25 Maiti, S. N.; Singh, M. P.; Micetich, R. G. Facile conversion of azides to amines.

Tetrahedron Lett. 1986, 27, 1423-1424. 26 Bartra, M.; Urpi, F.; Vilarrasa, J. New synthetic tricks. [Et3NH][Sn(SPh)3] and

Bu2SnH2, two useful reagents for the reduction of azides to amines. Tetrahedron Lett. 1987, 28, 5941-5944.

27 Kantam, M. L.; Chowdari, N. S.; Rahman, A.; Choudary, B. M. MCM-silylamine

Pd(II) complex, a heterogeneous catalyst for selective azide reductions. Synlett 1999, 9, 1413-1414.

28 Maiti, S. N.; Spevak, P.; Reddy, A. V. N. Alkaline earth metal-mediated reduction

of azides to amines. Synth. Commun. 1988, 18, 1201-1206. 29 Pathak, D.; Laskar, D. D.; Prajapati, D.; Sandhu, J. S. A novel and chemoselective

protocol for the reduction of azides using FeCl3-Zn system. Chem. Lett. 2000, 7, 816-817.

30 Prasad, H. S.; Srinivasa, G. R.; Abiraj, K.; Gowda, D. C. Hydrazinium

monoformate mediated facile preparation of amines from azides catalyzed by zinc. Indian J. Chem. Sect. B 2004, 43, 1787-1789.

31 Srinivasa, G. R.; Nalina, L.; Abiraj, K.; Gowda, D. C. Zinc/ammonium formate.

A chemoselective and cost-effective protocol for the reduction of azides to amines. J. Chem. Res., Synop. 2003, 10, 630-631.

32 Baruah, M.; Hussain, A.; Prajapati, D.; Sandhu, J. S. A new and efficient method

for the chemoselective reduction of azidoarenes and aroylazides: use of zinc-cobalt chloride. Chem. Lett. 1997, 8, 789-790.

33 Boruah, A.; Baruah, M.; Prajapati, D.; Sandhu, J. S. The efficient chemoselective

reduction of azides to primary amines. Synlett 1997, 11, 1253-1254. 34 Shim, S. C.; Choi, K. N. Amination of organic azides using

tetracarbonylhydridoferrate, HFe(CO)4- as a highly selective reducing agent.

Tetrahedron Lett. 1985, 26, 3277-3278. 35 Salunkhe, A. M.; Ramachandran, P. V.; Brown, H. C. Selective reductions. Part

60: Chemoselective reduction of organyl azides with dichloroborane-dimethyl sulfide. Tetrahedron 2002, 58, 10059-10064.

36 Barua, A.; Bez, G.; Barua, N. C. A facile procedure for reduction of azides to

amines with aluminum triiodide. Indian J. Chem. Sect. B 1999, 38, 128-129.

68

37 Wu, H.; Chen, R.; Zhang, Y. A facile reduction of azides to the corresponding amines with Sm/NiCl2

.6H2O system. Synth. Commun 2002, 32, 189-194. 38 Wu, H.; Chen, R.; Zhang, Y. A novel method for the chemoselective reduction of

azides to amines with a Sm/CoCl2.6H2O system. J. Chem. Res. Synop. 2000, 5,

248-249. 39 Huang, Y.; Zhang, Y.; Wang, Y. Reduction of arylsulfonyl derivatives with the

Sm/Cp2TiCl2 system. A novel method for preparation of disulfides. Tetrahedron Lett. 1997, 38, 1065-1066.

40 Alvarez, S. G.; Alvarez, M. T. A practical procedure for they synthesis of alkyl

azide at ambient temperature in dimethylsulfoxide in high purity and yield. synthesis 1997, 413-414.

41 Huang, Y.; Zhang, Y. Reduction of azides to amines with SmI2 or Cp2TiCl2-Sm

system. Synth. Commun. 1996, 26, 2911-2916. 42 Lee, J. G.; Choi, K. I.; Koh, H. Y.; Kim, Y.; Kang, Y.; Cho, Y. S. Indium-

mediated reduction of nitro and azide groups in the presence of HCl in aqueous media. Synthesis 2001, 1, 81-84.

43 Wang, L.; Li, P.; Chen, J.; Yan, J. A novel reduction of azides to amines with

tellurium metal in near-critical water. Phosphorus, Sulfur Silicon Relat. Elem. 2003, 178, 1807-1814.

44 Landini, D.; Maia, A. Anion nucleophilicity in ionic liquids: a comparison with

traditional molecular solvents of different polarity. Tetrahedron Lett. 2005, 46, 3961-3964.

45 Montanari, F. Anion activation in nonpolar media: a comparison among the

reactivities of lipophilic complexed macrocyclic and macrobicyclic polyethers and quaternary onium salts. Cron. Chim. 1982, 71, 3-19.

46 Ito, M.; Koyakumaru, K.-i.; Ohta, T.; Takaya, H. A simple and convenient

synthesis of alkyl azide under mild conditions. Synthesis 1995, 4, 376-378. 47 Chiappe, C.; Pieraccini, D.; Saullo, P. Nucleophilic displacement reactions in

ionic liquids: Substrate and solvent effect in the reaction of NaN3 and KCN with alkyl halides and tosylates. J. Org. Chem. 2003, 68, 6710-6715.

48 Barrett, I. C.; Kerr, M. A. A "one-pot" procedure for the oxidative conversion of

alkyl halides and tosylates to N,N-dimethylhydrazones. Synlett 2000, 11, 1673-1675.

69

49 Park, S. H. Acceleration of azidation by microwave irradiation. Bull. Korean Chem. Soc. 2003, 24, 253-255.

50 Landini, D.; Maia, A.; Montanari, F.; Rolla, F. Effect of the leaving group and

solvent in nucleophilic aliphatic substitutions promoted by quaternary onium salts. J. Org. Chem. 1983, 48, 3774-3777.

51 Biagi, G.; Giorgi, I.; Livi, O.; Scartoni, V.; Lucacchini, A. Evaluation of the

quantitative contribution of an aryl group on C(2) of 8-azaadenines to binding with adenosine deaminase: a new syntesis of 8-azaadenosine. Farmaco 1992, 47.

52 Arpicco, S.; Canevari, S.; Ceruti, M.; Galmozzi, E.; Rocco, F.; Cattel, L.

Synthesis, characterization and transfection activity of new saturated and unsaturated cationic lipids. Farmaco 2004, 59, 869-878.

53 Pijper, D.; Bulten, E.; Smisterova, J.; Wagenaar, A.; Hoekstra, D.; Engberts, J. B.

F. N.; Hulst, R. Novel biodegradable pyridinium amphiphiles for gene delivery. J. Org. Chem. 2003, 22, 4406-4412.

54 Hong, Y.-R.; Gorman, C. B. Synthetic approaches to an isostructural series of

redox-active, metal tris(bipyridine) core dendrimers. J. Org. Chem. 2003, 68, 9019-9025.

55 Adam, G. C.; Cravatt, B. F.; Sorensen, E. J. Profiling the specific reactivity of the

proteome with non-directed activity-based probes. Chem. Biol. 2001, 8, 81-95. 56 Hahn, S.; Stoilov, I. L.; Ha, T. B. T.; Raederstorff, D.; Doss, G. A.; Li, H. T.;

Djerassi, C. J. Biosynthetic studies of marine lipids. 17. The course of chain elongation and desaturation in long-chain fatty acids of marine sponges. J. Am. Chem. Soc. 1988, 110, 8117-8124.

57 Robinson, P. L.; Barry, C. N.; Kelly, J. W.; Evans, S. A.

Diethoxytriphenylphosphorane: a mild, regioselective cyclodehydrating reagent for conversion of diols to cyclic ethers: stereochemistry, synthetic utility, and scope. J. Am. Chem. Soc. 1985, 107, 5210-5219.

58 Prinz, H.; Six, L.; Ruess, K.-P.; Lieflaender, M. Stereoselective synthesis of long-

chain 1-O-(β-D-maltosyl)-3-O-alkyl-sn-glycerols (alkyl glyceryl ether lysoglycolipids). Liebigs Ann. Chem. 1985, 2, 217-225.

59 Huffman, J. W.; Desai, R. C. A procedure for alcohol inversion using cesium

acetate. Synth. Commun. 1983, 13, 553-558. 60 Kim, H. S.; Lee, S. M.; Ha, K.; Jung, C.; Lee, Y.-J.; Chun, Y. S.; Kim, D.; Rhee,

B. K.; Yoon, K. B. Aligned inclusion of hemicyanine dyes into silica zeolite films for second harmonic generation. J. Am. Chem. Soc. 2004, 126, 673-682.

70

61 Easton, C. J.; Xia, L.; Pitt, M. J.; Ferrante, A.; Poulos, A.; Rathjen, D. A.

Polyunsaturated nitroalkanes and nitro-substituted fatty acids. Synthesis 2001, 3, 451-457.

62 Keyes, R. F.; Golebiewski, W. M.; Cushman, M. Correlation of anti-HIV potency

with lipophilicity in a series of cosalane analogs having normal alkenyl and phosphodiester chains as cholestane replacements. J. Med. Chem. 1996, 39, 508-514.

63 Pissas, D.; Dais, P.; Mikros, E. Quantitative treatment of the rotational dynamics

of flexible-chain molecules. 13C NMR relaxation study of hydrocarbon chains attached to the fluorene anchor. Magn. Reson. Chem. 1994, 32, 263-275.

64 Kocian, O.; Stransky, K.; Zavada, J. Unsaturated analogs of 1-triacontanol.

Collect. Czech. Chem. Commun. 1982, 47, 1346-1355. 65 Dulayymi, J. a. R. A.; Baird, M. S.; Roberts, E. The synthesis of a single

enantiomer of a major a-mycolic acid of M. tuberculosis. Tetrahedron 2005, 61, 11939-11951.

66 Tanaka, A.; Oritani, T. A mild and efficient method for converting alcohols and

tetrahydropyranyl ethers to bromides with inversion of configuration. Tetrahedron Lett. 1997, 38, 1955-1956.

67 Bringmann, G.; Schneider, S. Improved methods for dehydration and

hydroxy/halogen exchange using novel combinations of triphenylphosphine and halogenated ethanes. Synthesis 1983, 2, 139-141.

68 Menger, F. M.; Yamasaki, Y. Hyperextended amphiphiles. Bilayer formation

from single-tailed compounds. J. Am. Chem. Soc. 1993, 115, 3840-3841. 69 King, J. F.; Loosmore, S. M.; Aslam, M.; Lock, J. D.; McGarrity, M. J. Betylates.

3. Preparative nucleophilic substitution by way of [2]-, [3]-, and [4]betylates. Stoichiometric phase transfer and substrate-reagent ion-pair (SRIP) reactions of betylates. J. Am. Chem. Soc. 1982, 104, 7108-7122.

70 King, J. F.; Loosmore, S. M.; Lock, J. D.; Aslam, M. Mixed phenazine-N-

methylphenazinium7,7,8,8-tetracyano-p-quinodimethanide. A quasi-one-dimensional "metal-like" system with variable band filling. J. Am. Chem. Soc. 1978, 100, 1639-1641.

71 Jeong, T.-S.; Kim, E. E.; Lee, C.-H.; Oh, J.-H.; Moon, S.-S.; Lee, W. S.; Oh, G.-

T.; Lee, S.; Bok, S.-H. Hypocholesterolemic activity of hesperetin derivatives. Bioorg. Med. Chem. Lett. 2003, 13, 2663-2665.

71

72 Smith, M. B.; March, J. Advanced organic chemistry: reactions, mechanisms, and structure; John Wiley & Sons, Inc:, 2001.

73 Deckert, A. A.; Farrell, C.; Ross, J.; Waddell, R.; Stubna, A. Binding of

myoglobin to thin films. Langmuir 1999, 15, 5578-5583. 74 Watanabe, A.; Ishizawa, A.; Kosaka, M.; Goto, R. Synthesis and physical

properties of long chain compounds. X. Studies of the x-ray diffraction patterns of normal higher primary amines. Bull. Chem. Soc. Jpn. 1969, 42, 1360-1363.

75 Zvejnieks, A. Preparation and properties of some pure fatty and rosin amines.

Sven. Kem. Tidskr 1954, 66, 316-322. 76 Newkome, G. R.; He, E.; Godinez, L. A.; Baker, G. R. Electroactive

metallomacromolecules via tetrabis(2,2':6',2''-terpyridine)ruthenium(II) complexes: dendritic nanonetworks toward constitutional isomers and neutral species without external counterions. J. Am. Chem. Soc. 2000, 122, 9993-19996.

77 Newkome, G. R.; Patri, A. K.; Godinez, L. A. Design, syntheses, complexation,

and electrochemistry of polynuclear metallodendrimers possessing internal metal binding loci. Chem. ___Eur. J. 1999, 5, 1445-1451.

78 Newkome, G. R.; Gross, J.; Moorefield, C. N.; Woosley, B. D. Approaches

towards specifically functionalized cascade macromolecules: dendrimers with incorporated metal binding sites and their palladium(II) and copper(II) complexes. Chem. Commun. 1997, 6, 515-516.

79 Newkome, G. R.; Woosley, B. D.; He, E.; Moorefield, C. N.; Guether, R.; Baker,

G. R.; Escamilla, G. H.; Merrill, J.; Luftmann, H. Supramolecular chemistry of flexible, dendritic-based structures employing molecular recognition. Chemical Communications. Chem. Commun. 1996, 24, 2737-2738.

80 Newkome, G. R.; Guther, R.; Moorefield, C. N.; Cardullo, F.; Echegoyen, L.;

Perez-Cordero, E.; Luftmann, H. Chemistry of micelles. Routes to dendritic networks: bis-dendrimers by coupling of cascade macromolecules through metal centers. Angew. Chem., Int. Ed. Engl. 1995, 34, 2023-2036.

81 Newkome, G. R.; Nayak, A.; Behera, R. K.; Moorefield, C. N.; Baker, G. R.

Chemistry of micelles series. 22. Cascade polymers: synthesis and characterization of four-directional spherical dendritic macromolecules based on adamantane. J. Org. Chem. 1992, 57, 358-362.

82 Newkome, G. R.; Behera, R. K.; Moorefield, C. N.; Baker, G. R. Chemistry of

micelles. 18. Cascade polymers: syntheses and characterization of one-directional arborols based on adamantane. J. Org. Chem. 1991, 56, 7162-7167.

72

83 Shinoda, K. The critical micelle concentrations in aqueous solutions of potassium alkyl malonates. J. Phys. Chem. 1955, 59, 432-435.

84 Shinoda, K. The effect of alcohols on the critical micelle concentrations of fatty

acid soaps and the critical micelle concentration of soap mixtures. J. Phys. Chem. 1954, 58, 1136-1141.

85 Bhattacharya, S.; Haldar, J. Molecular design of surfactants to tailor its

aggregation properties. Colloids Surf. A 2002, 205, 119-126. 86 Haldar, J.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. Molecular modulation of

surfactant aggregation in water: effect of the incorporation of multiple headgroups on micellar properties. Angew. Chem., Int. Ed. 2001, 40, 1228-1232.

87 Shinoda, K. The critical micelle concentrations in aqueous solutions of potassium

alkane tricarboxylates. J. Phys. Chem. 1956, 60, 1439-1441. 88 Kaneko, D.; Olsson, U.; Sakamoto, K. Self-assembly in some N-lauroyl-L-

glutamate/water systems. Langmuir 2002, 18, 4699-4703. 89 Kanicky, J. R.; Shah, D. O. Effect of premicellar aggregation on the pKa of fatty

acid soap solutions. Langmuir 2003, 19, 2034-2038. 90 Bates, R. G.; Allen, G. F. Acid dissociation constant and related thermodynamic

quantities for triethanolammonium ion in water from 0° to 50°. J. Research Natl. Bur. Standards 1960, 64A, 343-346.

91 Hesse, J. E. Melting point and index of refraction data for n-alkyl bromides,

thiols, and disulfides. J. Chem. Eng. Data 1967, 12, 266. 92 Gonzalez, A. G.; Barrera, J. B.; Perez, E. M. R. Synthesis of hierridin, a phenol

from the lichen, Ramalina hierrensis. Phytochemistry 1992, 31, 1436-1439. 93 CrysAlis v1. 170. Oxford Diffraction Wroclaw, Poland, 2002. 94 Sheldrick, G. M. SHELXTL NT ver. 6.12; Bruker Analytical X-ray Systems, Inc.:

Madison, MI, 2001.

73

Chapter III Synthesis of Two-Tailed, Tri-headed Amphiphiles (3CUr(n)2 and 3CUr1(n)2)

Two new series of two-tailed, tri-headed amphiphiles have been designed. These

series share common parts with the previous 3CUrn series (Chapter II), where a ureido

and a tricarboxylic Newkome-type dendron are still applied as the linker and the

hydrophilic moeity, respectively. Long, straight alkyl chains are still employed as the

hydrophobic moieties. Instead of one tail, the new amphiphiles contain two tails.

Another modification is made based on how the alkyl chains on the hydrophobic moiety

are connected to the linker. In order to correspond with the previous single-tailed, tri-

headed amphiphiles 3CUrn series, the total number of the carbons on the hydrophobic

moiety is equal as that in the 3CUrn series. Both alkyl chain lengths on each tail are also

equal. By doing this, two homologous series, which have different geometries of

hydrophobic moieties, are designed.

Based on how the tails are connected to the linker, we present these two different

series. How do these two differ? One is where two tails are directly connected to the

same nitrogen on the ureido linker; the other is where two tails are attached to one carbon

that is bonded to a nitrogen on the ureido linker (Figure III.1). In other words, the middle

linker

head

head

head

tail

tail

linker

head

head

head

tail

tail

Figure III.1 General representation of two-tailed, tri-headed amphiphiles

74

carbon of a long alkyl chain is connected to the nitrogen on the linker. This kind of tail is

later referred to a “swallowtail”.1

III.1 The 3CUr(n)2 Homologous Series

A Newkome-type dendron has been generated from the reaction of

WeisocyanateTM (3) and the cyclic amine piperidine.2 Based on the same chemistry,

acyclic secondary amines and 3 should react readily. In the first homologous series

(Scheme III.1), two tails are directly attached to the same nitrogen on the ureido linker.

This homologous series is abbreviated as 3CUr(n)2 (Scheme III.1), where “3C”

represents three carboxylic groups on the hydrophilic moiety, “Ur” represents a ureido

linker between the hydrophobic and hydrophilic moieties, “n” represents the number of

carbons of the alkyl group on the symmetrical tail, and subscript “2” represents the two

tails attached to the linker. In the same manner as the 3EUrn naming, the homologous

series of tri-tert-butyl esters is abbreviated as 3EUr(n)2. Retrosynthesis (Scheme III.1)

Scheme III.1 Retrosynthesis of the 3CUr(n)2 homologous series

NHN

O

OCNO

O3

O

O

O

O

O

O

H

H

H

NHN

O

O

O

O

O

O

O

+

4a−e

3

3CUr(n)2 3EUr(n)2

R = CnH2n+1

1 2 a, n=7b, n=8c, n=9d, n=10e, n=11

R

R

R

RHN

R R

75

shows that beginning with 3 and necessary secondary amines, the addition reaction takes

place to give the 3EUr(n)2 series. The tert-butyl groups of the 3EUr(n)2 series are later

removed to generate the 3CUr(n)2 series.

III.1.1 Preparation of Secondary Amines

Five secondary amines containing two symmetrical alkyl chains (4a−e)⎯N-

heptylheptan-, N-octyloctan-, N-nonylnonan-, N-decyldecan-, and N-undecylundecan-1-

amine⎯ were utilized to construct the 3CUr(n)2 series. Amines 4a−e were

commercially available. The secondary amines containing even number of carbons on

each tail were available at low cost. On the other hand, those containing an odd number

of carbons on each tail⎯4a, 4c, and 4e⎯ were commercially available only in milligram

quantities and expensive. This motivated us to develop our own synthesis of such

compounds.

III.1.1.1 Literature Review on Syntheses of N-Alkylalkan-1-amines (4a, 4c, and 4e)

Studies documented that compounds 4a, 4c, and 4e could be generated from the

corresponding primary amines,3-6 oximes,7-9 1-aminoalcohol,9 aldehyde,10 nitrile,11,12

ester,13 and amides.14-16 In most of these reported procedures, secondary amines were

obtained as side products when generating primary amines. Only the work of Murahashi

et al.3 and Djedovič et al.14 (Scheme III.2) reported the desired secondary amines were

obtained in excellent yields. These two procedures utilized ruthenium3 complex catalyst

and lithium aluminum hydride,14 respectively.

Alkylation of a primary amine is not the best way to generate a secondary amine,

because the resulting secondary amine is more reactive than the primary amine; the

76

reaction proceeds to give the tertiary amine as a major side product. Alkylation of

heptan-1-amine6 and decan-1-amine5 with the corresponding bromoalkanes produced a

low yield of the N-heptylheptan-1-amine and N-decyldecan-1-amine (yield not reported),

respectively.

Scheme III.2 Preparation of N-heptylheptan-1-amines from heptan-1-amine3 and N-heptylheptanamide14

NH2RuH2(PPh3)4, EtOH, H2

HN

HN

O5 6

LiAlH4, THF

yield 98%

yield 99%

+OH

The ruthenium complex, RuH2(PPh3)4, is selective for the oxidation of the

alcohol. When a reaction of RNH2 and R′OH is performed in the presence of

RuH2(PPh3)4, the N-alkyl iminium ion complex is formed, and the secondary amine

RR′NH is formed selectively.3 Murahashi et al.3 have shown that hydrogenation of

heptanal oxime in the presence of RuH2(PPh3)4 leads to the formation of heptan-1-amine

and the corresponding imine. These latter two also react to release ammonia gas and the

corresponding secondary imine. Hydrogenation of N-alkyl imine gives the desired N-

heptylheptan-1-amine in an excellent yield. Besides hydrogenation, reduction of heptanal

oxime is also performed with a mild reducing agent (sodium borohydride in the presence

of copper (II) sulfate) where an imine is reported to be the key intermediate.7 An imine is

also reported as the key intermediate when heptanal10 and heptanitrile12 are employed as

77

precursors, in which the presence of ammonia minimizes the formation of N-

heptylheptanamine to less than 5%.

Hydrogenation of heptanamide15 and N,N-diethylheptanamide 16 in the presence

of copper-chromium catalyst gives N-heptylheptan-1-amine as the side product, which is

isolated in 25−50% and 58% yields, respectively. On the other hand, N-

heptylheptanamide is reduced with lithium aluminum hydride to give N-heptylheptan-1-

in an excellent yield.14

We developed our procedure on the reduction of the N-alkylalkanamide with

lithium aluminum hydride based on the published procedure17 for the synthesis of

unsymmetrical secondary amines and N-undecylundecanamide. Hence, we needed to

synthesize the necessary N-alkylalkanamides. Later, Djedovič et al.14 reported an

excellent yield of N-alkylalkanamine from a reduction of N-alkylalkanamide with the

same reducing agent.

III.1.1.2 Literature Review on Syntheses of N-Alkylalkanamides (5)

Amide bonds are usually made from the reaction of amines and carbonyl

compounds, such as carboxylic acids, acid halides, and anhydrides. In the literature, a

number of preparations of long-chain N-alkylalkanamide beginning with primary amines

have been documented.14,17-32 However, only few contain the synthesis of our interest

(5), where the alkyl attached to the nitrogen atom contains one more carbon than the alkyl

attached to the carbonyl carbon.

78

RHN R'

O

5

R = CnH2n+1, n>6R' = CnH2n+1, n>6

5a,R = C7H15, R' = C6H135c, R = C9H19, R' = C8H175e, R = C11H23, R' = C10H21

letter "a", "c", and "e" refer to the corresponding amines

Most procedures for generating N-alkylalkanamides in the literature are

performed by standard methods. Long-chain carboxylic acid,21,28 acid

chloride,14,17,19,23,26,27,29,31,32 nitrile,30 methyl ester,24,25 ethyl ester,18 4-nitrophenyl ester,20

and 1-(5,7-dinitro-2,3-dihydroindol-1yl)-dodecanone22 react with necessary primary

amines to give amides 5.

III.1.2 Synthesis of N-Alkylalkan-1-amines (4a, 4c, and 4e)

Following the published procedures,26,27 acid chlorides, which are very reactive

and commercially available at low cost, were employed as acylating reagents of the

required primary amines with triethylamine as a base (Scheme III.3). The formation of

amide 5a was confirmed by comparing the 1H NMR and IR spectra to published values.31

Djedovič et al.14 later reported that amide 5a can be obtained in excellent yield from the

reaction of heptan-1-amine, heptanoyl chloride, triethylamine, and N,N-dimethyl-4-

aminopyridine as a catalyst in dichloromethane. The 1H NMR chemical shifts of amides

5c and 5e were compared to those of 5a, and necessary adjustments were made for the

proton integration of the higher homologs.

79

Scheme III.3 Synthetic scheme of N-alkylalkan-1-amines 4a, 4c, and 4e

R NH2R' Cl

O

R' NH

O

R R' NH

R+ i ii

6a, R = C7H156c, R = C9H196e, R = C11H23

6 7 45

7a, R' = C6H137c, R' = C8H177e, R' = C10H21

i. Et3N, dry THF, rt, 15 min (5a, 85%; 5c, 83%; 5e, 71%)ii. LiAlH4, Et2O, reflux, overnight (4a, 84%; 4c, 83% 4e, 80%)

RHN

R

Amides 5a, 5c, and 5e were reduced to the corresponding amines 4 with lithium

aluminum hydride in good yields. The reduction was confirmed by the disappearance of

the amide I and amide II bands absorption (1640 cm-1 and 1555 cm-1, respectively) in the

IR spectra. The 1H NMR spectrum of amine 4a was compared to that of published

values7 before being used in the subsequent step. Similar chemical shifts were observed

in the 1H NMR spectra of the other homologs 4c and 4e. Amines 4a and 4c were

obtained as colorless liquids, in which some portions started solidifying. Later, Djedovič

et al.14 reported amine 4a as a solid with a low melting temperature.

III.1.3 Synthesis of Two-tailed, Tri-headed 3CUr(n) 2 Homologous Series

Similar to the synthesis of the 3CUrn series, the synthesis of 3CUr(n)2 begins

with 3 and amines 4. The reactions were run in dichloromethane at room temperature

overnight (Scheme III.4). In the first step, the tri-tert-butyl ester 3EUr(n)2 series is

obtained. After purifications performed by flash column chromatography (hexane:ethyl

acetate, 5:1), the 3EUr(n)2 homologs were obtained in good yields.

80

Scheme III.4 Synthetic scheme of the 3CUr(n)2 homologous series

NHN

O

O tBuO

OCNO tBu

O 3

RHN

+

3

3EUr(n)2

3

R

NHN

O

OHO

3CUr(n)2

3R

R R

R

R = CnH2n+1

4a−e

a, n=7b, n=8c, n=9d, n=10e, n=11

i ii

i. CH2Cl2, rt, overnight; a, 79%; b, 60%; c, 70%; d, 85%; e, 73%ii. HCO2H, rt, 9 h; a, 90%; b, 86%; c, 88%; d, 86%; e, 77%

Removal of the tert-butyl groups on the 3EUr(n)2 series to generate 3CUr(n)2

series was accomplished by stirring the tri-tert-butyl esters in formic acid at room

temperature for nine hours. The resulting 3CUr(n)2 series was then recrystallized from

ethyl acetate to give good yields of pure materials. All compounds, 3EUr(n)2 and

3CUr(n)2, were fully characterized.

The melting temperatures of the 3EUr(n)2 homologous series increased up to

3EUr(9)2, then the melting temperatures decreased for longer chain (Table III.1). This

trend was different from that found in the 3CUrn series, where homologs with longer

chain melted at higher temperature. Conversion of the tri-tert-butyl esters into the

corresponding tricarboxylic acids caused a big difference in melting temperatures; in

general, melting temperatures of the 3CUr(n)2 series are higher than those of the

3EUr(n)2 series. In addition, the trend observed in the 3CUr(n)2 series suggested that

melting temperatures appeared to be determined by the interactions of the carboxylic

acids and not the chain length.33

81

Table III.1 Melting ranges of the 3EUr(n)2 and 3CUr(n)2 homologous series n Melting range (°C)

3EUr(n)2 3CUr(n)2 7 53.5–54.1 125.3–125.9 8 56.8–57.5 120.4–120.8 9 62.6–63.4 121.3–121.7

10 49.7–50.4 120.9–121.2 11 42.9–43.5 121.4–122.1

III.2 The 3CUr1(n)2 Homologous Series

The second homologous series is abbreviated as 3CUr1(n)2 (Scheme III.5), where

“3C” represents three carboxylic groups on the hydrophilic moiety, “Ur” represents a

ureido linker between the hydrophobic and hydrophilic moieties, “1” represents there is

one carbon separating the linker and the two symmetrical tails, “n” represents the number

of carbons of the alkyl group on the symmetrical tail, and subscript “2” represents the two

tails attached to the carbon next to the linker. In other words, a long tail is connected to a

nitrogen on the ureido linker via the midcarbon (R2CH−). The series with the tri-tert-

butyl esters is abbreviated as 3EUr1(n)2.

Retrosynthesis (Scheme III.5) shows that beginning with WeisocyanateTM (3) and

amines 10a−f, the addition reaction takes place to give 3EUr1(n)2 series. The chemistry

of this series is very similar to that of the 3CUrn series, where primary alkylamines were

allowed to react with isocyanate 3. The tert-butyl groups of the 3EUr1(n)2 series are

later removed to generate 3CUr1(n)2 series.

82

Scheme III.5 Retrosynthesis of 3CUr1(n)2 series

HN

HN

O

OCNO

O3

O

O

O

O

O

H

H

H

HN

HN

O

O

O

O

O

O+

10a−f

3

3CUr1(n)2 3EUr1(n)2

R = CnH2n+18 9a, n = 7b, n = 8c, n = 9d, n = 10e, n =11f, n = 12

R R

R

R

R

R

O ONH2

a long tail connected to a nitrogen on the linker via the midcarbon

III.2.1 Preparation of Primary Amines (10a−f)

We utilized six primary amines where two symmetrical carbon chains are

connected to the carbon attached to the nitrogen atom. These are amines 10a−f

⎯pentadecan-8-, heptadecan-9-, nonadecan-10-, henicosan-11, tricosan-12-, and

pentacosan-13-amine. As they were not commercially available, syntheses were

required.

III.2.1.1 Literature Review on Syntheses of 1-Alkylalkan-1-amines (10a−f)

There are several procedures1,5,34-38 for the synthesis of compounds 10a−f

reported in the literature (Table III.2). Most precursors used in the synthesis of these

amines are ketones1,5,35-38 and azidoalkanes.34 Ketones, such as pentadecan-8-one, react

with an amine to give an imine, which then is converted into the corresponding amine in

moderate yield. Another way is to transform ketones to other intermediates, such as the

corresponding N-alkylformamide and oxime derivatives, following which are converted

83

into alkanols of interest in low to moderate yields. The reaction of ketones and

formamide is performed at 160−170 °C for 15h,37 and 180−185 °C for 6−8h.36 The N-

alkylformamides are hydrolyzed with hydrochloric acid and the free amines are released

after reaction with aqueous sodium hydroxide. The yields are improved when

nitrobenzene is added to increase the solubility of ketones.36 Reduction of ketoximes

with sodium in ethanol5 gave a very low yield (≤ 5%), on the other hand, reduction of

ketoximes with RedAl1 improve the yield to 84%.

Table III.2 Preparations of 1-alkylalkan-1-amines from different precursors Precursors Reagents Remarks References

O

6 6

H2O, EtOH, Raney nickel, NH3, H2

150 °C, 56% yield 5

O

6 6

1. NH2CHO 2. concd. HCl 3. aq. NaOH

Step 1 reaction condition not specified, yield not reported Step 2 was run at 160−170 °C, 15h, 40%

37

O

n-1 n-1

n = 7, 8, 9, 11

1. NH2CHO, PhNO2 2. concd. HCl 3. aq. NaOH

Step 1 was run at 180−185 C, 4h Step 2 was run by heating for 6−8 h. 65% (n = 7), 60% (n = 8), 63% (n = 9), 50% (n = 11)

36

O

6 6

1. HO−NH2, ROH5,38 2. Na, EtOH5

Step 1 was run at rt for 2 d, no yield reported Step 2. 5%5

5,38

O

8 8

1. EtOH, HO−NH2

.HCl,1,352. PhCH3, RedAl, aq. HCl, concd. HCl1

84%1 1,35

N3

3 3

Bu3SnH, NidppeCl2 95% 34

Based on the starting materials employed (Table III.2), both azidoalkane and

oxime derivatives of ketone can be utilized as precursors for the synthesis. Thus, two

84

different retrosynthesis pathways are constructed (Scheme III.6). The first pathway of

retrosynthesis of amines 10a−f shows that the necessary ketones 16a−f can be

transformed to amines 10a−f via the oxime derivatives 15a−f. The second, where one of

the intermediates is the corresponding azidoalkanes 11a−f, shows that the synthesis can

begin with commercially available alcohols 13a−f.

Scheme III.6 Retrosynthesis of amines 10a−f

R R

NH2

R R

N3

R R

OMs

R R

OHRBr

H OEt

O+R = n-CnH2n+1

10a−f

11a−f

12a−f

13a−f 14a−f10a, n = 710b, n = 810c, n = 910d, n = 1010e, n = 1110f, n = 12

R R

O

16a−f

R R

N

15a−f

OH

CO+n-3

Most ketones (16a−e) and alcohols (13a−e) are commercially available, but they

are very expensive. Thus, we are required to synthesize the required precursor⎯either

ketones or alcohols. In general, ketones are generated from alkenes, and the reactions are

run in special conditions, such as extremely high pressures and temperatures. Because of

the harsh conditions required for generating ketones, these reactions are not easily

performed in our laboratory. Thus, we consider the second alternative route, where

alcohols are utilized as the precursors.

Alcohols 13a−f can be easily synthesized via Grignard reaction starting with

85

bromoalkanes 14a−f and ethyl formate. Based on the consideration on cost-per mole

basis, bromoalkanes 14a−f are one-tenth the price of ketones 16a−e, and the reactions for

generating alcohol 13a−f are easily performed with standard laboratory glasswares.

Thus, we have chosen this second alternative route in order to synthesize amines 10a−f.

Following this route, bromoalkaness 14a−f are converted into amines 10a−f in at least

three steps⎯Grignard reaction, azidation, and reduction.

III.2.1.2 Literature Review on Syntheses of Azidoalkanes

Very few procedures for generating long chain azidoalkanes are documented in

the literature. The syntheses of 10-azidoicosane39 and 11-azidodocosane40 are achieved

from the corresponding alcohols via Mitsunobu reaction utilizing diphenylphosphorazide

and triphenylphosphine in the presence of diisopropylazodicarboxylate (DIAD),39

diethylazodicarboxylate (DEAD).40

R R'

OH

R R'

N3(PhO)2PN3, PPh3

DIAD or DEAD

R = C9H19, R' = C10H21, 86−93%R = C10H21, R' = C11H23

O

Unfortunately, the synthesis of compounds 11a−f has never been reported.

Despite of the advantages offered by the Mitsunobu reaction, including the fact that it is a

one-pot reaction and thus a shorter route, and the products are obtained in good to

excellent yield on the example above, all the reagents needed for such a transformation

are only commercially available at high cost. Because of this reason, a different

alternative route is presented. Considering low-cost commercially available

86

methanesulfonyl chloride, and the success of the synthesis of both 10-azidoicosane and

11-azidodocosane utilizing sodium azide in N,N-dimethylformamide, we elected to apply

the same procedure for the transformation of alcohols 13a−f into azides 11a−f. Even

though that this means the synthesis is one-step longer, the reagents needed for the

transformations are available in much lower costs. In addition, the side

products⎯triethylamine hydrochloride and sodium methanesulfonyloxide⎯are easier to

handle, and yet the yields of the reactions are just as high.

Based on examples found in the literature,34,40 two secondary alcohols are

converted into the corresponding methanesulfonyloxyalkanes. These

methanesulfonyloxyalkanes are then allowed to react with sodium azide in two different

solvent, dimethylsulfoxide41 and N,N-dimethylformamide,34 to produce the corresponding

azides (Table III.3). Following this procedure, methanesulfonyloxyalkanes as precursors

are required.

R R'

OMs NaN3

R R'

N3

various solvent

Table III.3 Preparation of secondary azidoalkanes from secondary methanesulfonyloxyalkanes

R R' solvent condition yields (%) C2H5 C3H7 DMSO 34−40 °C, 6 h not reported41 C2H5 C4H9 DMF 60 °C, 6 h 8034

III.2.1.3 Literature Review on Syntheses of Methanesulfonyloxyalkanes

Transformation of secondary alcohols into the corresponding

methanesulfonyloxyalkanes is routinely performed in organic synthesis. Utilizing low-

cost reagents, the transformation is simply performed in a mild condition and short

87

reaction time. Moreover, the products are obtained in quantitative yields. However,

there were not many examples involving symmetrical straight chain alkyls. Based on

examples found in the literature,42-44 three symmetrical secondary alcohols are converted

into the corresponding methanesulfonyloxyalkanes (Table III.4).

R R

OH MsCl

base R R

OMs

Table III.4 Preparation of secondary methanesulfonyloxyalkanes from secondary alcohols

R base condition yield (%) C3H3 pyridine 0−5 °C, 3.5 h 4344 C4H9 Et3N 0 °C→rt, 12 h not reported34

C16H33 Et3N 15−60 min 6642 III.2.1.4 Literature Review on Syntheses of Secondary Alkanols

Secondary alkanols 13a−f are synthesized via different ways (Table III.5). Most

of them are routinely generated from ketones or combination of haloalkanes⎯mostly

bromoalkanes⎯and carbonyl compounds, such as aldehydes and ethyl formate. Less

common precursors are alkenes and primary alcohols.

Ketones are reduced by hydrogenation in the presence of Raney nickel,43,45 Raney

nickel with alcohol as a solvent serving as the hydrogen source,46 sodium,38,47

hydrides,48,49 and aluminum isopropoxide,43 which is known as the Meerwein-Pondorff-

Varley method. Haloalkanes are typically converted into the corresponding Grignard

reagents prior to the reaction with the carbonyl compound of interest.

When primary alkanols are employed as precursors, two mechanisms of the

transformation into the corresponding secondary alkanols in the presence of calcium

hydroxide have been proposed. The first involves dehydrogenation, aldol condensation,

88

Table III.5 Preparations of secondary alcohols 13a−f from different precursors Precursors Reagents Yield (%) Reference

O

n-1n-1n = 7−9

H2, Ni or Raney nickel

No reported yield 43,45

11 11

O

PhCH3, iPrOH, Raney nickel

80 46

O

n-1 n-1n = 7, 8

Na, EtOH

No yield reported 38

7 7

O

Na, TiCl3, THF

5 47

O

77

Li+iPrO-

No reported yield 43

7 7

O

LiAlH4, THF

90 48

6 6

O

iBu2AlH

No yield reported 49

Brn-1

n = 7−11

H

O

n-1

Mg ,

No reported yield 50,51

Xn-1

X = Br, n = 7, 8 ,10, 1252,53 X = Cl, n = 7, 8

H

OMg ,

9053, 8452 No reported yield

X = Br50-54 X = Cl43

5 1. LiAlH(OCH3)3, THF, CO 2. HCl 3. NaOH/H2O2

78 55

5 1. borane−THF; 1,3-dithiane, n-BuLi; HgCl2 2. NaOH, H2O2

74

56

7OH

Ca(OH)2 55−67 57

89

decarbonylation, and neutralization. The second involves oxidation by water, ketonic

decomposition, hydrogenation, and neutralization. With this method, the transformation

of oct-1-ene into heptadecan-9-ol is obtained in a moderate yield.57

Beginning with oct-1-ene, carbonylation of the corresponding trialkylborane

using trimethoxyaluminohydride produced an intermediate; this intermediate is

successively treated with concentrated hydrochloric acid and peroxide to give the desired

heptadecan-9-ol in a good yield.55 Another way is to treat the corresponding

trialkylborane with lithiated 1,3-dithiane in the presence of mercury chloride, followed by

treatment with peroxide.56

III.2.2 Synthesis of Amines (10a−f)

We began the synthesis with Grignard reaction of bromoalkanes 14a−f and ethyl

formate, which Mr. Richard V. Macri and Ms. Erika Bechtold optimized. In order to get

the optimal result, the reaction was run for three days (Scheme III.7). After

recrystallization from ethyl acetate, 13C NMR spectra of pentadecan-9-ol and heneicosan-

11-ol were compared to published values.58 The chemical shifts on the 1H NMR of

compounds 13a−f were compared to those42 of another homolog, namely tritriacontan-

17-ol. In addition, the melting temperature data were compared to published values58-61

before being used for the following step.

Alkanols 13a−f were then converted into the corresponding

methanesulfonyloxyalkanes 12a−f. It is to our advantage that the reaction time was short

and the products were obtained in near quantitative yields. The formation of products

was verified by 1H NMR spectra. The chemical shifts of the hydrogen on the carbon

bonded to a new electron-withdrawing group (−OSO2CH3) were shifted from δ 3.58 ppm

90

to 4.60 ppm; this confirmed that the corresponding methanesulfonylalkanes (12a−f) were

formed. As 1H NMR spectra of 12a−f were not in the literature, we compared the

chemical shifts of 12a−f to those42 of corresponding longer-chain homolog tritriacontan-

17-ol. Necessary adjustment of the proton integration was made depending on the chain

length of the homolog. Based on that analysis, 12a−f were used without purification for

the next step.

Azides 11a−f were generated via an SN2 reaction with sodium azide. The reactive

site on this SN2 reaction was a secondary carbon. Thus, compared to the SN2 reaction for

the formation of 1-azidoalkanes (Chapter II), whose reactive site was a primary carbon,

we expected this reaction would require a longer time. However, after several trials, we

Scheme III.7 Synthetic scheme of the alkanamines 10a−f

R R

NH2

R R

N3

R R

OMs

R R

OHRBr

H OEt

O+

R = n-CnH2n+1

10a−f

11a−f12a−f13a−f14a−f

14a, n = 714b, n = 814c, n = 914d, n = 1014e, n = 1114f, n = 12

i ii iii

ivi. Mg, dry THF, 60 °C, 3 dii. MsCl, Et3N, dry THF, rt, 15 miniii. NaN3, DMF, 80 °C, 4 hiv. Pd/C, H2, EtOH, rt, 3 h

Table III.6 Isolated yields and melting ranges of intermediates and alkanamines 10a−f i ii iii iv n

yield (%)

melting range (°C)

yield (%)

yield (%)

melting range (°C)

yield (%) melting range (°C)

7 49 53.5−54.3 90−95 78 oil 66 oil 8 52 61.7−62.2 92−96 83 oil 75 oil 9 54 67.0−67.4 90−96 92 oil 84 oil

10 55 71.1−72.0 94−98 97 oil 88 oil 11 52 76.8−77.6 91−96 84 oil 81 65.5−64.3 12 56 80.2−81.0 91−94 90 36.8−37.4 86 79.7−80.4

91

concluded the reaction was completed in 4 hours, the same reaction time as that in the

synthesis of 1-azidoalkanes. Thin layer chromatography (in hexane) and the absorption

(~ 2100 cm-1) in the infrared spectra verified the presence of new products containing

azido groups. Azides 11a−f were obtained in good to excellent yields after purification

by flash column chromatography with hexane as the eluent. The 1H NMR chemical

shifts of the hydrogen on the carbons bonded to the azido groups were shifted upfield (δ

~ 3.22 ppm). Azides 11a−f were fully characterized.

Finally, conversion of azides 11a−f into amines 10a−f was performed by

hydrogenation in the presence of 10% palladium/carbon catalyst (Scheme III.7, Table

III.6). The absorption at ~ 2100 cm-1 in the infrared spectrum disappeared; this

confirmed that azido groups had been reduced. Compounds 10a−f were obtained in good

yields and used without purification.

III.2.3 Synthesis of Two-tailed, Tri-headed 3CUr1(n)2 Homologous Series

The 3EUr1(n)2 series was obtained by stirring isocyanate 3 and amines 10a−f in

dichloromethane at room temperature overnight (Scheme III.8). Purification by flash

column chromatography (hexane:ethyl acetate, 5:1) generated white solids in good yields.

Repeated attempts to recrystallize 3EUr1(n)2 series from acetonitrille in order to generate

a crystal for X-ray analysis were not successful.

92

Scheme III.8 Synthetic scheme of the preparation of 3CUr1(n)2

HN

HN

O

OtBuO

+

3EUr1(n)2

3HN

HN

O

OHO

3CUr1(n)2

3

R = n-CnH2n+1

10a−f

a, n = 7b, n = 8c, n = 9d, n = 10e, n = 11 f, n = 12

,

i iiR

R

R

R

i. CH2Cl2, rt, overnight; a, 60%; b, 70%, c, 85%, d, 77%; e, 86%; f, 87%ii. HCO2H, rt, 9 h; a, 85%; b, 86%, c, 83%, d, 80%; e, 85%; f, 87%

3

Removal of the tert-butyl groups on the 3EUr1(n)2 was performed by stirring in

formic acid at room temperature for about 9 hours. The crude material was recrystallized

from acetic acid-hexane to give good yields of pure 3CUr1(n)2 homologs.

In 3EUr1(n)2 homologs, homologs containing longer alkyl chain melted at lower

temperature. Melting temperatures of 3EUr1(n)2 homologs decreased irregularly as

chain lengths increased (Table III.7). These melting temperatures were lower compared

to those of 3CUr1(n)2 homologs, suggesting that the presence of the carboxylic acid

groups caused the raise in the melting temperatures. The observed changes in melting

temperatures do show a particular trend in compounds containing either odd or even alkyl

chains. Compounds containing odd-numbered alkyl chain have lower melting

temperatures as the chain length increases (Table III.7). The same trend was observed for

those containing even-numbered alkyl chain. In both cases, it appeared that the longer

the chain length, the less significant is the difference in the melting temperatures.

93

Table III.7 Melting ranges of the 3EUr1(n)2 and 3CUr1(n)2 homologous series

n Melting range (°C) 3EUr1(n)2 3CUr1(n)2

7 109.0–109.8 168.5–169.2 8 108.5–109.3 164.3–165.1 9 100.4–101.2 165.1–165.7

10 87.0–87.6 163.8–164.3 11 86.1–86.9 164.6–165.1 12 73.8–74.3 163.3–163.9

III.3 Comparison of Trends in Melting Temperatures (Tri-tert-butyl Esters vs Tri-carboxylic Acids)

Each tri-tert-butyl esters and tricarboxylic acids presents unique characteristic

(Table III.8). Melting temperatures of 3EUrn homologs (Chapter II) were affected by

the chain length; the longer the chain, the higher the melting temperatures. This trend

differed from those observed in both 3EUr(n)2 and 3EUr1(n)2 homologs. Melting

temperatures of 3EUr(n)2 homologs appeared to fit a parabolic figure, where they

increased up to 3EUr(9)2 and decreased for a longer homologs. The trend observed in

the 3EUr1(n)2 homologs⎯melting temperatures decrease as the chain length

increase⎯was in contrast to that observed in the 3CUrn homologs. In addition,

compared to both 3EUrn and 3EUr(n)2 homologs, 3EUr1(n)2 homologs melted at higher

temperature.

Table III.8 Comparison of trends in melting temperatures (tri-tert-butyl esters vs tri-carboxylic acids)

Trends in melting temperatures tri-tert-butyl esters tricarboxylic acids

3EUrn: increases as chain lengths increases 3CUrn: increases and then decreases, parabolic figure; 3CUr16 melts at the highest temperature

3EUr(n)2: increases and then decreases, parabolic figure; 3EUr(9)2 melts at the highest temperature

3CUr(n)2: irregular trend, within 120-126 °C

3EUr1(n)2: decreases as chain length increases 3CUr1(n)2: decreases as chain length increases in compounds containing odd- or even-numbered alkyl chains

94

All the tricarboxylic acid homologs melt at significantly higher temperature than

the corresponding tri-tert-butyl esters. The high melting temperatures accompanied by

small and irregular changes with chain length suggested that interaction of the carboxyl

groups were dominant than the chain length.

III.4 Comparison of NMR Spectra of 3EUr(n)2 vs 3EUr1(n)2

In the 1H NMR spectra of the 3EUr(n)2 homologous series, symmetrical protons

on the hydrophobic moieties showed to have identical chemical shifts. Protons Has and

Hbs appeared at ~ δ 3.1 and 1.5 ppm, respectively, and both set showed as multiplet.

Chemical shifts of protons on the rest of the methylene groups, δ ~ 1.2−1.35 ppm, were

clearly differentiated from those on tert-butyl groups, δ ~ 1.4 ppm.

Fourteen different chemical shifts appeared in the 13C NMR spectrum of

3EUr(7)2. This suggested that symmetrical carbons on the alkyl chains had identical

chemical shifts. Thus the rotation of the C−N bond, formed between the carbonyl carbon

and the nitrogen bonded to the two alkyl chain, was fast on an NMR time scale compared

to that of an N,N-dialkylamide.62,63 The significant difference of restricted rotational

barrier of carbon−nitrogen in compounds containing amido and ureido functional groups,

which are within 60−100 kJ/mol and < 40 kJ/mol, respectively, caused different

observation in the NMR spectroscopy.64 Two factors contributed to lower the rotational

N

O HN

n-3 n-3

3

n = 7−11

O

O

H HH H

H H H H

a aaa

b bbb

95

barrier on C−N bonds in a ureido group were cross-conjugation, which is the competition

between both nitrogen for conjugation with the carbonyl group, and steric hindrance of

the substituents on nitrogens, which prevents the planarity for conjugation of the

N−CO−N group.65

As expected, when alkyl chain on each tail was longer by one methylene, an

additional peak was observed in the 13C NMR spectra. This behavior was observed in the

spectra of tri-tert-butyl ester 3EUr(7)2−3EUr(10)2. The chemical shifts of each

individual carbon in different environment were observed. However, the homolog with

the longest chain⎯3EUr(11)2 ⎯did not follow this observation. Eighteen different

chemical shifts were expected in the 13C NMR spectra of tri-tert-butyl ester 3EUr(11)2,

and yet only seventeen different chemical shifts were observed.

Unlike 1H NMR spectra of tri-tert-butyl esters 3EUr(n)2, protons bonded to the

symmetrical carbons on the hydrophobic moieties were not completely in the same

environment. Each set⎯Has, Hbs, Hcs, Hds, etc⎯of symmetrical protons on the two

symmetrical tails were in the same environment. The methylenes showed two distinct

chemical shifts⎯1.43 and 1.20–1.35ppm.

HN HH H

HHH H

H

HN

O

H

a ab b

c cdd

n-3n-3

x

O

O

3

96

From proton integrations of all 3EUr1(n)2 homologs, we noticed that two protons

were buried along with the protons of the tert-butyl groups at δ 1.43 ppm, raising the

proton integration to 29 protons. Protons Has, as well as Hbs, are in the same

environment, as only a set of two protons was observed at δ 1.43 ppm. Thus, the

chemical shifts at δ 1.43 ppm must have arisen from either Has or Hbs. The other two

protons⎯either Has or Hbs⎯appeared at δ ~ 1.20−1.35 ppm along with the rest of the

methylene groups. So, geminal protons Ha and Hb appeared to be diastereotopic. This

evidence was supported by a further experiment on the two-dimensional 1H−1H COSY

NMR. Proton Hx appeared at δ ~ 3.5 ppm. The two-dimensional 1H−1H COSY NMR

spectrum showed that proton Hx appeared to couple to other protons at 1.20–1.35 and

1.43 ppm. It was obvious that Hx coupled to protons on the neighboring carbon, Has and

Hbs. This suggested that the geminal protons Ha and Hb were diastereotopic, thus found

in different chemical shifts.

The 13C NMR spectrum of tri-tert-butyl ester 3EUr1(7)2 showed fifteen different

chemical shifts. This suggested each symmetrical carbons on the alkyl chains was in the

same environment, thus they appeared to have identical chemical shifts. As each tail was

longer by one methylene, an additional chemical shift was observed in the 13C NMR

spectrum. This behavior was observed in the first four 3EUr1(n)2 homologs. As the

chain became longer, carbons on the symmetrical chain began overlapping, which caused

fewer peaks observed in the 13C NMR spectra. Thus, the two highest homologs

⎯3EUr1(11)2 and 3EUr1(12)2⎯did not appear to follow the pattern.

The chemical shifts in 1H NMR spectra of the 3CUr(n)2 series followed the same

pattern as those of the 3EUr(n)2 series. Symmetrical carbons on both tails appeared to

97

have identical chemical shifts. Protons Has and Hbs appeared at δ ~ 3.1 and 1.38 ppm,

respectively.

As shown in the 1H NMR spectra of the 3CUr1(n)2 homologs, geminal protons

Has and Hbs remained diastereotopic; the chemical shift appeared at δ ~ 1.20−1.40, along

with the rest of the methylene protons, and 1.44 ppm. Unlike 3EUr1(7)2−3EUr1(10)2,

where all carbons in different environment were observed in the 13C NMR spectra, fewer

carbons with different chemical shift were observed in the 3CUr1(n)2 series.

III.5 Experimental Procedures Materials and Methods. Chemicals were obtained from Aldrich, Acros, Lancaster, and TCI; they were used

without further purification. Solvents were reagent grade or HPLC grade; they were used

as received unless otherwise specified. THF was distilled from sodium/benzophenone

ketyl. WeisocyanateTM was prepared as described66 with a shorter reaction time as

mentioned above. Analytical thin layer chromatography was performed by polyester-

coated silica gel 60 Å and detected by treating with 10% ethanolic phosphomolybdic acid

reagent (20 wt. % solution in ethanol) followed by heating. Flash column

chromatography was carried out on silica gel (60 Å); samples were loaded as a

concentrated solution in the solvent system needed; column diameter × height (13/4 × 6

inches), eluted sample varied between ~ 2.00−4.00 g, flow rate (~ 1.5−2 inches/min) was

controlled by air pressure. Solutions were concentrated by rotary evaporation. Melting

ranges, determined in open capillary tubes, were uncorrected. NMR spectra were

recorded on an INOVA at 400 and 100 MHz for 1H and 13C, respectively, and reported in

ppm relative to the known solvent residual peak. Resonances were reported in the order

98

of chemical shift (δ), followed by the splitting pattern, and the number of protons.

Abbreviations used in the splitting pattern were as the following: s = singlet, d = doublet,

t = triplet, q = quartet, q = quin, m = multiplet, and b = broad. IR spectra were recorded

on neat samples with an FTIR equipped with a diamond ATR system, and reported in

cm-1. HRMS data were obtained on a dual-sector mass spectrometer in FAB mode with

2-nitrobenzylalcohol as the proton donor. Elemental analyses were performed by

Atlantic Microlabs, Inc. in Norcross, GA.

General procedure for the preparation of N-alkylalkanamides (5a, 5c, 5e)

NH

O

n-2 n-1

O

n-2 ClNH2

n-1+

NEt3, dry THF

rt, 15 min

An amine 6 (13.0 mmol) and Et3N (15.6 mmol) were combined and dissolved in dry THF

(38 mL), and stirred in the ice bath. An acid chloride 7 (14.3 mmol) was added dropwise

to the cold solution. After the addition, the ice bath was removed, and the reaction was

stirred for another 15 min at rt. The resulting reaction was washed successively with 1M

HCl (7.5 mL), satd aq NaHCO3 (12 mL), and water (12 mL). The organic layer was

dried with Na2SO4 and concentrated to dryness to give a white solid, which was purified

by flash column chromatography to give a pure white solid, which gave a single spot on

TLC in 2%MeOH in CH2Cl2 (Rf = 0.23−0.31).

N-Heptylheptanamide (5a) The general procedure described above afforded a white solid (2.50 g, 85%); mp 45.0–

45.7 °C (lit.14 mp 44–46 °C); 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 14H), 1.50

(m, 2H), 1.62 (m, 2H), 2.17 (t, 2H), 3.24 (m, 2H), 5.69 (bs, 1H) (lit.14,31 300 MHz14); 13C

NMR (CDCl3) δ 14.19, 14.20, 22.69, 22.75, 26.0, 27.1, 29.15, 29.17, 29.9, 31.7, 31.9,

99

37.1, 39.7, 173.3 (lit.14 75 MHz); IR 3287, 2955, 2921, 2851, 1640, 1555, 1467 (lit.31

neat). HRMS: for C14H30NO (M + H)+ calcd 228.2327, found 228.2328.

N-Nonylnonamide (5c) The general procedure described above afforded a white solid (3.05 g, 83%); mp 62.7–

63.3.9 °C (lit.67 mp 52−55 °C); 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 22H),

1.48 (m, 2H), 1.62 (m, 2H), 2.15 (t, 2H), 3.25 (m, 2H), 5.36 (bs, 1H); 13C NMR (CDCl3)

δ 14.1, 22.66, 22.68, 25.9, 26.9, 29.18, 29.25, 29.32, 29.35, 29.5, 29.7, 31.84, 31.87, 37.0,

39.5, 173.03; IR 3287, 2955, 2918, 2849, 1638, 1551, 1467; HRMS: for C18H38NO (M +

H)+ calcd 284.2953, found 284.2940.

N-Undecylundecanamide (5e)


74.7 °C (lit.68 mp 73 °C); 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 32H), 1.49 (m,

2H), 1.62 (m, 2H), 2.15 (t, 2H), 3.24 (m, 2H), 5.36 (bs, 1H); 13C NMR (CDCl3) δ 14.3,

22.9, 26.1, 27.2, 29.53, 29.54, 29.59, 29.73, 29.77, 29.79, 29.80, 29.9, 32.1, 37.2, 39.7,

173.3; IR 3312, 2953, 2914, 2849, 1634, 1544, 1471 (lit.19 bands); HRMS: for C22H46NO

(M + H)+ calcd 340.3579, found 340.3584.

General procedure for the preparation of N-alkylalkan-1-amines (4a, 4c, and 4e)

NH

O

n-2 n-1+

reflux, overnightLiAlH4

Et2O HN

n-1 n-1

An N-alkylalkanamide 5 (9.58 mmol) was added slowly to a stirred suspension of LiAlH4

(574 mg, 14.7 mmol) in Et2O (50 mL). Addition took place in an ice bath. After the

addition, the reaction was warmed to rt and then heated to reflux. After being refluxed

overnight, the resulting reaction was cooled to rt. Successive addition of water (2 mL),

100

NaOH (10 M, 2.5 mL), and water (4 mL) yields a sticky white solid. The ether layer was

decanted, and the sticky white solid was washed once with ether (10 mL). The ether

layers were combined, dried with Na2SO4, and concentrated to give a clear liquid (n = 7,

9), and a white solid (n = 11) in of 80–85% yield. The resulting N-alkylalkan-1-amine

(4a, 4c, 4e) was used for the next step as it was without purification.

N-Heptylheptan-1-amine (4a)

The general procedure described above afforded a clear liquid (1.72 g, 84%) (lit.14 mp

30−31 °C); 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 16H), 1.48 (m, 4H), 2.58 (t,

4H) (lit.14 300 MHz); 13C NMR (CDCl3) δ 14.3, 22.8, 27.6, 29.5, 30.4, 32.0, 50.4 (lit.7,14

60 MHz,7 75 MHz14); IR 2955, 2922, 2853, 1457, 1377, 1129 (lit.7 neat); HRMS: for

C14H32N (M + H)+ calcd 214.2535, found 214.2538.

N-Nonylnonan-1-amine (4c)

The general procedure described above afforded a clear liquid (2.14 g, 83%); 1H NMR

(CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 24H), 1.47 (m, 4H), 2.58 (t, 4H); 13C NMR

(CDCl3) δ 14.3, 22.9, 27.7, 29.5, 29.79, 29.82, 30.5, 32.1, 50.4; IR 2954, 2921, 2852,

1466, 1377, 1130; HRMS: for C18H40N (M + H)+ calcd 270.3161, found 270.3148.

N-Undecylundecan-1-amine (4e)


48.3 °C (lit.11 51.5–52.5); 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 32H), 1.47

(m, 4H), 2.58 (t, 4H); 13C NMR (CDCl3) δ 14.3, 22.9, 27.7, 29.6, 29.8, 30.5, 32.1, 50.4;

IR 2955, 2913, 2827, 1469, 1375, 1127; HRMS: for C22H48N (M + H)+ calcd 326.3787,

found 326.3775.

101

General procedure for the preparation of long chain tri-tert-butyl esters, 3EUr(n)2

NHN

O

OtBuO

OCNOtBu

O 3R

HN +R R

RCH2Cl2, rt

overnight3

A dialkylamine 4 (4.95 mmol) was added slowly to a solution of isocyanate 3 (4.71

mmol) in CH2Cl2 (20 mL). The resulting transparent solution was stirred at rt. After

stirring overnight, the solution was concentrated to leave a crude white solid. The crude

solid was purified by flash column chromatography with a mixture of hexane:EtOAc to

give a white solid (60–85%), which gave a single spot on TLC (hexane:EtOAc, 5:1, Rf =

0.18−0.25)

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3,3-diheptylureido)heptanedioate,

3EUr(7)2


54.1 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 16H), 1.43 (bs, 27H), 1.51 (m,

4H), 1.96 (m, 6H), 2.22 (m, 6H), 3.12 (m, 4H), 4.56(s, 1H); 13C NMR (CDCl3) δ 14.0,

22.5, 27.0, 28.0, 28.7, 29.1, 29.9, 30.6, 31.8, 47.2, 56.6, 80.3, 156.1, 173.1; IR 3368,



67.89; H, 10.84; N, 4.30.

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3,3-dioctylureido)heptanedioate,

3EUr(8)2


57.5 °C; 1H NMR (CDCl3) δ 0.89 (t, 6H), 1.20−1.35 (bm, 20H), 1.43 (s, 27H), 1.50 (m,

4H), 1.96 (m, 6H), 2.21 (m, 6H), 3.11 (m, 4H), 4.55 (s, 1H); 13C NMR (CDCl3) δ 14.1,

102

22.6, 27.0, 28.1, 28.7, 29.27, 29.44, 29.9, 30.6, 31.8, 47.3, 56.6, 80.4, 156.1, 173.2; IR

3366, 2923, 1732, 1724, 1615, 1365, 1144; HRMS: for C39H75N2O7 (M + H)+ calcd

638.5574, found 638.5540. Anal. Calcd for C39H74N2O7: C, 68.58; H, 10.92; N, 4.10.

Found: C, 68.55; H, 11.07; N, 4.10.

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3,3-dinonylureido)heptanedioate,

3EUr(9)2


63.4 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 24H), 1.44 (bs, 27H), 1.51 (bm,

4H), 1.97 (m, 6H), 2.22 (m, 6H), 3.12 (m, 4H), 4.57 (s, 1H); 13C NMR (CDCl3) δ 14.1,

22.6, 27.0, 28.1, 28.7, 29.2, 29.49, 29.58, 29.9, 30.6, 31.8, 47.3, 56.6, 80.4, 156.1,

173.15; IR 3377, 2922, 1732, 1725, 1617, 1365, 1144; HRMS: for C41H79N2O7 (M + H)+

calcd 711.5887, found 711.5893. Anal. Calcd for C41H78N2O7: C, 69.25; H, 11.06; N,

3.94. Found: C, 69.34; H, 11.27; N, 3.96.

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3,3-didecylureido)heptanedioate,

3EUr(10)2


50.4 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20−1.35 (bs, 28H), 1.43 (s, 27H), 1.50 (m,

4H), 1.96 (m, 6H), 2.21 (m, 6H), 3.11 (m, 4H), 4.55(s, 1H); 13C NMR (CDCl3) δ 14.1,

22.7, 27.0, 28.1, 28.7, 29.3, 29.49, 29.55, 29.63, 29.9, 30.6, 31.9, 47.3, 56.6, 80.4, 156.1,



3.79. Found: C, 70.02; H, 11.41; N, 3.80.

103

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3,3-diundecylureido)heptanedioate,

3EUr(11)2


43.5 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 32H), 1.43 (bs, 27H), 1.50 (bm,

4H), 1.96(m, 6H), 2.21 (m, 6H), 3.11 (m, 4H), 4.56(s, 1H); 13C NMR (CDCl3) δ 14.1,

22.65, 27.03, 28.1, 28.7, 29.3, 29.49, 29.59, 29.62, 29.9, 30.6, 31.9, 47.3, 56.6, 80.35,

156.1, 173.1; IR 3372, 2923, 1734, 1725, 1616, 1365, 1143; HRMS: for C45H87N2O7 (M

+ H)+ calcd 767.6513, found 767.6498. Anal. Calcd for C45H86N2O7: C, 70.45; H, 11.30;

N, 3.65. Found: C, 70.45; H, 11.46; N, 3.71.

General procedure for the preparation of long chain triacids, 3CUr(n)2

NHN

O

OO 3

R

R

rt, 9hN

HN

O

OtBuO 3

R

RHCOOH H

A tri-tert-butyl ester 3EUr(n)2 (3.33 mmol) were dissolved in 99% HCOOH so that its

concentration is 0.1 M. The mixture might need to be warmed slightly to get all the

3EUr(n)2 to dissolve. Once dissolved to give a transparent solution, the solution was

stirred at rt. After 9 h, the resulting milky white solution was concentrated and the

resulting residue was recrystallized from EtOAc to yield a white solid (77–90%).

4-(2-Carboxyethyl)-4-(3,3-diheptylureido)heptanedioic acid, 3CUr(7)2


125.9 °C; 1H NMR (DMSO-d6) δ 0.83 (t, 6H), 1.10–1.30 (bm, 16H), 1.38 (m, 4H), 1.81

(m, 6H), 2.09 (m, 6H), 3.09 (m, 4H), 5.07 (s, 1H), 12.00 (bs, 3H); 13C NMR (DMSO-d6)

δ 14.4, 22.5, 26.7, 28.5, 28.7, 29.0, 30.2, 31.7, 46.3, 56.4, 156.3, 175.1; IR 2926, 1735,

1701, 1590, 1527, 1285, 1174; HRMS: for C25H47N2O7 (M + H)+ calcd 487.3383 found

104

487.3391. HRMS: for C39H74N2O7 calcd 683.5574, found 683.5540. Anal. Calcd for

C25H46N2O7: C, 61.70; H, 9.53; N, 5.76. Found: C, 61.61; H, 9.50; N, 5.75.

4-(2-Carboxyethyl)-4-(3,3-dioctylureido)heptanedioic acid, 3CUr(8)2


120.8 °C; 1H NMR (DMSO-d6) δ 0.80 (t, 6H), 1.10–1.30 (bm, 20H), 1.38 (m, 4H),

1.81(m, 6H), 2.08 (m, 6H), 3.09 (m, 4H), 5.06 (s, 1H), 12.00 (bs, 3H); 13C NMR (DMSO-

d6) δ 13.9, 22.0, 26.2, 28.0, 28.2, 28.7, 28.8, 29.7, 31.2, 45.8, 55.9, 155.9, 174.7; IR 2921,



62.98; H, 9.79; N, 5.40.

4-(2-Carboxyethyl)-4-(3,3-dinonylureido)heptanedioic acid, 3CUr(9)2




δ 14.6, 22.8, 27.0, 28.7, 29.0, 29.3, 29.58, 29.66, 30.4, 32.0, 46.6, 56.6, 156.6, 175.4; IR

3446, 2919, 1712, 1694, 1608, 1534, 1293, 1175; HRMS: for C29H55N2O7 (M + H)+ calcd


Found: C, 63.93; H, 10.02; N, 5.15.

4-(2-Carboxyethyl)-4-(3,3-didecylureido)heptanedioic acid, 3CUr(10)2


121.2 °C. 1H NMR (DMSO-d6) δ 0.83 (t, 6H), 1.10–1.30 (bm, 28H), 1.37 (m, 4H), 1.81


δ 14.4, 22.5, 26.7, 28.5, 28.7, 29.1, 29.32, 29.40, 29.46, 30.2, 31.7, 46.3, 56.4, 156.3,

105



4.91. Found: C, 65.33; H, 10.34; N, 4.93.

4-(2-Carboxyethyl)-4-(3,3-diundecylureido)heptanedioic acid, 3CUr(11)2




δ 14.4, 22.6, 26.7, 28.5, 28.7, 29.17, 29.33, 29.46, 30.2, 31.8, 46.4, 56.4,156.3, 175.2; IR

2917, 1713, 1694, 1608, 1535, 1295, 1185; HRMS: for C33H63N2O7 (M + H)+ calcd


Found: C, 66.17; H, 10.40; N, 4.69.

General procedure for the preparation of dialkylcarbinols (13a−f)

Br 1. Mg, catalytic I2, dry THF

2. HCO2Et, dry THF, 65−70 °Cn-1n-1 n-1

OH

To a three-neck round bottom flask containing Mg turnings (2.34 g, 100 mmol) was

added several chips of I2. After the flask was flushed with N2, the mixture was stirred for

a couple minutes until the I2 sublimed. A portion of THF (12 mL) was added, following

a solution of bromoalkanes (48.1 mmol) in THF (36 mL) in a dropwise (1 drop / 3

seconds) manner through a dropping funnel. After the addition, ethyl formate (1.43 g,

19.0 mmol) in dry THF (24 mL) was added in the same rate through a dropping funnel.

The reaction was stirred heated (65−70 °C) for 3 d. The reaction mixture was diluted

with THF (36 mL), followed by successive addition of MeOH (4 mL) and satd NH4Cl

(30 mL). The organic layer was separated and washed once with satd NaCl (65 mL).

106

The organic layer was dried and concentrated to give a light yellow solid, which was

recrystallized from EtOAc to give a pure white solid in moderate yields (60–75%); 1H

NMR (CDCl3) δ 0.88 (t, 6H), 1.20−1.50 (bm, various number of Hs), 3.58 (bm, 1H); mp

53.5−54.3 °C (n = 7), lit.61 52−52.6 °C; 61.7−62.2 °C (n = 8), lit.61 60.8−61.2 °C; 67.0−

67.4 °C (n = 9), lit.59 65.9−66.1; 71.1−72.0 °C (n = 10), lit.58,61 71.3−72.5 °C; 76.8−77.6

°C (n = 11), lit.61 75.5−75.7; 80.2−81.0 °C (n = 12), lit.60 79.5−80.5.

General procedure for the preparation of methanesulfonyloxyalkanes (12a−f)

+OH

n-1 n-1MsCl

dry THF

rt, 15 min

OMs

n-1 n-1Et3N +

To a solution of dialkylcarbinol 13 (8.96 mmol) and Et3N (9.86 mmol) in THF (59 mL)

was added mesyl chloride (25.7 mmol) dropwise rate through a syringe at rt. In the

middle of the addition, the clear solution started getting cloudy. After the addition of

mesyl chloride, the reaction was stirred for another 15 min. The resulting suspension was

filtered; the filtrate was washed with water (8 mL) and satd NaCl (2 mL). The organic

layer was dried with Na2SO4 and concentrated to give a yellowish liquid (90–98%). The

product was used for the next step without further purification; 1H NMR (CDCl3) δ 0.88

(t, 6H), 1.20−1.50 (bm, 24−44Hs), 1.60−1.75 (m, 4H), 3.00 (s, 3H), 4.70 (quin, 1H).

General procedure for the preparation of azidoalkanes (11a−f)

+OMs

n-1 n-1NaN3

DMF85 °C, 4h

N3

n-1 n-1

To a stirred solution of methanesulfonyloxylalkane 12 (7.41 mmol) in DMF (49 mL) at rt

was slowly added NaN3 (36.3 mmol). The suspension was heated to 85 °C for 4 h. After

the resulting yellow solution was cooled to rt, hexane (98 mL) and water (16 mL) were

107

added. The organic layer was separated and washed successively with satd NaHCO3 (8

mL) and satd NaCl (8 mL). The organic layer was dried with Na2SO4 and concentrated

to give a colorless oil, which was purified by flash column chromatography with hexane

to give a pure colorless oil (n = 6–10) and a white solid (n = 11), which gave a single spot

on TLC with hexane (Rf = 0.41), in 78–97% yield.

8-Azidopentadecane (11a)

The general procedure described above afforded a colorless liquid (1.46 g, 78%); 1H

NMR (CDCl3) δ 0.89 (m, 6H), 1.20–1.53 (bm, 24H), 3.22 (quin, 1H); 13C NMR (CDCl3)

δ 14.3, 22.9, 26.4, 29.4, 29.6, 32.0, 34.6, 63.4; IR 2924, 2855, 2093; HRMS: for

C15H32N3 (M + H)+ calcd 254.2596, found 254.2597. Anal. Calcd for C15H31N3: C,

71.09; H, 12.33; N, 16.58. Found: C, 71.16; H, 12.44; N, 16.44.

9-Azidoheptadecane (11b)


NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.53 (bm, 28H), 3.22 (quin, 1H); 13C NMR (CDCl3)

δ14.3, 22.9, 26.4, 29.5, 29.68, 29.70, 32.1, 34.6, 63.4; IR 2923, 2854, 2091; HRMS: for


72.54; H, 12.53; N, 14.93. Found: C, 72.83; H, 12.68; N, 14.86.

10-Azidononadecane (11c)


NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.54 (bm, 32H), 3.22 (quin, 1H); 13C NMR (CDCl3) δ

14.3, 22.9, 26.4, 29.5, 29.65, 29.72, 32.1, 34.6, 63.3; IR 2922, 2853, 2093; HRMS: for


73.73; H, 12.70; N, 13.57. Found: C, 73.91; H, 12.77; N, 13.42.

108

11-Azidohenicosane (11d)



14.3, 22.9, 26.3, 29.5, 29.65, 29.71, 29.76, 29.78, 32.1, 34.6, 63.4; IR 2922, 2853, 2093;

HRMS: for C21H44N3 (M + H)+ calcd 310.3474, found 310.3467. Anal. Calcd for

C21H43N3: C, 74.71; H, 12.84; N, 12.45. Found: C, 74.86; H, 12.98; N, 12.44.

12-Azidotricosane (11e)



14.3, 22.9, 26.4, 29.6, 29.7, 29.75, 29.78, 29.85, 29.86, 32.1, 34.6, 63.4; IR 2921, 2852,

2094; HRMS: for C23H48N3 (M + H)+ calcd 338.3787, found 338.3794. Anal. Calcd for

C23H47N3: C, 75.55; H, 12.96; N, 11.49. Found: C, 75.78; H, 13.10; N, 11.49.

13-Azidopentacosane (11f)

The general procedure described above afforded a white solid (2.63 g, 90%); mp 36.8−

37.4 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.26–1.52 (bm, 44H), 3.22 (quin, 1H); 13C

NMR (CDCl3) δ 14.3, 22.9, 26.4, 29.6, 29.67, 29.75, 29.8, 29.86, 29.88, 32.1, 34.6, 63.4;

IR 2913, 2848, 2085; HRMS: for C25H52N3 (M + H)+ calcd 366.4100, found 366.4090.

Anal. Calcd for C25H51N3: C, 72.67; H, 13.06; N, 10.67. Found: C, 76.35; H, 13.14; N,

10.63.

General procedure for the preparation of alkanamines (10a−f)

+N3

n-1 n-1H2

Pd/C, hexane

rt, 3 h

NH2

n-1 n-1

To a solution of azidoalkane 11 (7.03 mmol) in hexane (35 mL) was added 10% Pd/C

109

(3% weight of azidoalkane). The resulting suspension was shaken and hydrogenated

under 62 psi at rt for 3 h. After sitting overnight, the suspension was filtered. The filtrate

was concentrated to give a colorless liquid (n = 6–10) and a white solid (n = 11, 12) in

75–88% yield. The product was used in the next step without purification.

Pentadecan-8-amine (10a)


NMR (CDCl3) δ 0.88 (t, 6H), 1.20−1.45 (bm, 26H; 2Hs exchange with D2O), 2.67 (bm,

1H); 13C NMR (CDCl3) δ 14.3, 22.8, 26.4, 29.5, 30.0, 32.0, 38.4, 51.40; IR 2955, 2921,

2852, 1464, 797.

Heptadecan-9-amine (10b)


NMR (CDCl3) δ 0.88 (t, 6H), 1.20−1.45 (bm, 30H), 2.67 (bm, 1H); 13C NMR (CDCl3) δ

14.3, 22.9, 26.4, 29.5, 29.8, 30.0, 32.1, 38.2, 51.4; IR 2955, 2921, 2852, 1464, 800.

Nonadecan-10-amine (10c)


NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.45 (bm, 34H; 2Hs exchange with D2O), 2.68 (bm,

1H) (lit.1 500 MHz); 13C NMR (CDCl3) δ 14.3, 22.9, 26.4, 29.5, 29.8, 29.9, 30.0, 32.1,

38.4, 51.4; IR 2955, 2921, 2852, 1464, 797 (lit.1 KBr pellet).

Henicosan-11-amine (10d)


NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.45 (bm, 38H; 2Hs exchange with D2O), 2.67 (bm,

1H); 13C NMR (CDCl3) δ 14.3, 22.9, 26.4, 29.5, 29.83, 29.85, 29.87, 30.0, 32.1, 38.3,

51.4; IR 2955, 2920, 2852, 1464, 792.

110

Tricosan-12-amine (10e)


66.4 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.13 (s, 2H), 1.20–1.45 (bm, 42H; 2Hs

exchange with D2O), 2.67 (bm, 1H); 13C NMR (CDCl3) δ 14.3, 22.9, 26.4, 29.6, 29.86,

29.89, 30.1, 32.1, 33.3, 51.4; IR 2953, 2913, 2848, 1468, 720.

Pentacosan-13-amine (10f)


80.4 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.45 (m br, 46H; 2Hs exchange with

D2O), 2.67 (bm, 1H); 13C NMR (CDCl3) δ 14.3, 22.9, 26.4, 29.6, 29.87, 29.89, 29.91,

30.1, 32.1, 38.4, 51.4; IR 2952, 2914, 2848, 1467, 720.

General procedure for the preparation of long chain tri-tert-butyl esters, 3EUr1(n)2

3OCN

OtBu

O

n-1 n-1

CH2Cl2rt, overnight

+NH2

n-1 n-1

HN NH

OtBuO

O3

An amine 10 (4.95 mmol) was added slowly to a stirred solution of isocyanate 3 (4.71

mmol) in CH2Cl2 (20 mL). After stirring overnight at rt, the solution was concentrated to

afford a crude white solid. This crude was purified by flash column chromatography,

which gave a single spot on TLC (hexane:EtOAc, 5:1, Rf = 0.17−0.30) to give a white

solid. The isolated solid was then recrystallized from CH3CN (60–87%).

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(1-heptyloctyl)ureido]-

heptanedioate, 3EUr1(7)2


109.8 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20−1.35 (bm, 22H), 1.43 (bs, 29H), 1.94

111

(m, 6H), 2.23 (m, 6H), 3.52 (bm, 1H), 3.76 (d, 1H), 4.41 (s, 1H); 13C NMR (CDCl3) δ

14.0, 22.6, 25.8, 28.0, 29.2, 29.6, 29.8, 30.6, 31.8, 35.7, 50.2, 56.4, 80.4, 156.4, 173.0; IR



67.96; H, 10.91; N, 4.26.

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(1-octylnonyl)ureido]-




6H), 2.23 (m, 6H), 3.51 (bm, 1H), 3.75 (d, 1H), 4.41 (s, 1H); 13C NMR (CDCl3) δ 14.0,

22.6, 25.8, 28.0, 29.2, 29.5, 29.7, 29.8, 30.6, 31.8, 35.7, 50.2, 56.4, 80.4, 156.4, 173.0; IR



69.06; H, 11.17; N, 4.06.

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(1-nonyldecyl)ureido]-





22.6, 25.9, 28.0, 29.3, 29.55, 29.56, 29.7, 29.9, 30.7, 31.8, 35.7, 50.2, 56.4, 80.4, 156.4,

173.0; IR 3328, 2922, 2852, 1726, 1651, 1148; HRMS: for C42H81N2O7 (M + H)+ calcd


Found: C, 69.60; H, 11.22; N, 3.85.

112

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(1-decylundecyl)ureido]-

heptandioate, 3EUr1(10)2




22.6, 25.9, 28.02, 28.04, 29.3, 29.57, 29.61, 29.68, 29.9, 30.6, 31.9, 35.7, 50.2, 56.4, 80.4,

156.4, 173.0; IR 3376, 2919, 2850, 1730, 1675, 1146; HRMS: for C44H85N2O7 (M + H)+


3.72. Found: C, 69.89; H, 11.33; N, 3.70.

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(1-undecyldodecyl)ureido]-




6H), 2.24 (m, 6H), 3.52 (bm, 1H), 3.83 (m, 1H), 4.45 (s, 1H); 13C NMR (CDCl3) δ 14.0,

22.6, 25.9, 28.0, 29.3, 29.58, 29.60, 29.7, 29.9, 30.7, 31.9, 35.7, 50.2, 56.4, 80.4, 156.4,

173.0; IR 3374, 2920, 2850, 1722, 1676, 1148; HRMS: for C46H89N2O7 (M + H)+ calcd


Found: C, 70.49; H, 11.37; N, 3.67.

Di-tert-butyl 4-(2-tert-butoxycarbonyl-ethyl)-4-[3-(1-dodecyltridecyl)-ureido]-





113

22.7, 25.9, 28.0, 29.3, 29.61, 29.63, 29.66, 29.70, 29.85, 30.6, 31.9, 35.7, 50.2, 56.4, 80.4,

156.4, 173.1; IR 3319, 2917, 2851, 1730, 1654, 1147; HRMS: for C48H93N2O7 (M + H)+


3.46. Found: C, 71.43; H, 11.58; N, 3.50.

General procedure for the preparation of long chain triacids, 3CUr1(n)2

OtBuNHN

O

O

n-1

HCO2H

rt, 9 h

n-1

HOH

NHNO

O

n-1 n-1

H3 3

A tri-tert-butyl ester 3EUr1(n)2 (3.00 mmol) were dissolved in 99% HCOOH so that the

concentration was 0.1 M. Some mixtures needed warming to completely dissolve the

3EUr1(n)2. Once dissolved to give a transparent solution, the mixture was stirred at rt.

After stirring 9 h, the resulting milky white solution was concentrated in vacuo. The

white solid was recrystallized from HOAc−hexane to yield a white solid (77–90%).

4-(2-Carboxyethyl)-4-[3-(1-heptyloctyl)ureido]heptanedioic acid, 3CUr1(7)2


169.2 °C; 1H NMR (CD3OD) δ 0.89 (t, 6H), 1.20−1.40 (bm, 24H), 1.44 (m, 2H), 1.95 (m,

6H), 2.28 (m, 6H), 3.55 (bm, 1H); 13C NMR (DMSO-d6) δ 14.4, 22.5, 25.8, 28.6, 29.2,

29.4, 30.5, 31.7, 35.8, 48.3, 55.4, 157.4, 175.0; IR 3405, 2924, 2855, 1732, 1700, 1585;

HRMS: for C26H49N2O7 (M + H)+ calcd 501.3540, found 501.3562. Anal. Calcd for

C26H48N2O7: C, 62.37; H, 9.66; N, 5.60. Found: C, 62.28; H, 9.57; N 5.52.

4-(2-Carboxyethyl)-4-[3-(1-octylnonyl)ureido]heptanedioic acid, 3CUr1(8)2


165.1 °C; 1H NMR (CD3OD) δ 0.89 (t, 6H), 1.20–1.40 (bm, 28H), 1.44 (m, 2H), 1.95 (m,


114

29.69, 29.73, 30.8, 31.9, 36.0, 48.5, 55.6, 157.6, 175.2; IR 3404, 2921, 2852, 1730, 1699,

1556; HRMS: for C28H53N2O7 (M + H)+ calcd 529.3853, found 529.3837. Anal. Calcd

for C28H52N2O7: C, 63.61; H, 9.91; N, 5.30. Found: C, 63.65; H, 9.94; N 5.20.

4-(2-Carboxyethyl)-4-[3-(1-nonyldecyl)ureido]heptanedioic acid, 3CUr1(9)2




29.36, 29.43, 30.5, 31.7, 35.7, 48.3, 55.4, 157.3, 174.9; IR 3405, 2921, 2852, 1730, 1699,



4-(2-Carboxy-ethyl)-4-[3-(1-decylundecyl)ureido]heptanedioic acid, 3CUr1(10)2

The general procedure described above afforded a white solid (1.42, 81%); mp 162.8–

163.5 °C; 1H NMR (CD3OD) δ 0.86 (t, 6H), 1.20–1.35 (bm, 34H), 1.41 (m, 2H), 1.87 (t,

6H), 2.25 (t, 6H), 3.51 (bm, 1H); 13C NMR (DMSO-d6) δ 14.4, 22.5, 25.8, 28.5, 29.1,

29.41, 29.43, 30.5, 31.7, 35.6, 48.3, 55.4, 157.3, 174.9; IR 3404, 2921, 2852, 1731, 1699,



4-(2-Carboxyethyl)-4-[3-(1-undecyldodecyl)ureido]heptanedioic acid, 3CUr1(11)2




29.41, 29.44, 30.5, 31.7, 35.6, 48.2, 55.3, 157.3, 174.9; IR 3404, 2921, 2852, 1732, 1699,


115


4-(2-Carboxyethyl)-4-[3-(1-dodecyltridecyl)ureido]heptanedioic acid, 3CUr1(12)2


163.9 °C. 1H NMR (CD3OD) δ 0.86 (t, 6H), 1.20–1.35 (bm, 42H), 1.41 (m, 2H), 1.95 (m,


29.67, 29.71, 29.75, 30.8, 32.0, 35.9, 48.5, 55.6, 157.6, 175.2; IR 3403, 2920, 2851,


Anal. Calcd for C36H68N2O7: C, 67.46; H, 10.69; N, 4.37. Found: C, 67.31; H, 10.62, N

4.34.

References for Chapter III 1 Wescott, L. D.; Mattern, D. L. Donor-α-acceptor molecules incorporating a

nonadecyl-swallowtailed perylenediimide acceptor. J. Org. Chem. 2003, 68, 10058-10066.

2 Newkome, G. R.; Weis, C. D.; Moorefield, C. N.; Fronczek, F. R. Chemistry of

micelles. 66. A useful dendritic building block: di-tert-butyl 4-[(2-tert-butoxycarbonyl)ethyl]-4-isocyanato-1,7-heptanedicarboxylate. Tetrahedron Lett. 1997, 38, 7053-7056.

3 Murahashi, S.; Kondo, K.; Hakata, T. Ruthenium-catalyzed synthesis of

secondary or tertiary amines from amines and alcohols. Tetrahedron Lett. 1982, 23, 229-232.

4 Haedicke, E.; Schlenk, W., Jr. Length and conformation of guest molecules in

urea inclusion channels. Justus Liebigs Ann. Chem. 1972, 764, 103-111. 5 Borrows, E. T.; Hargreaves, B. M. C.; Page, J. E.; Resuggan, J. C. L.; Robinson,

F. A. Preparation and properties of some long-chain aliphatic amines. J. Chem. Soc. 1947, 197-202.

116

6 Davis, T. L.; Elderfield, R. C. The determination of the ionization constants of guanidine and some of its alkylated derivatives. J. Am. Chem. Soc. 1934, 55, 1499-1503.

7 Rao, H. S. P.; Bharathi, B. Reduction of oximes with sodium borohydride-copper

(II) sulfate in methanol. Indian J. Chem. B 2002, 41, 1072-1074. 8 Paul, R. Transposition of aldoximes under the influence of Raney nickel catalyst.

Bull. Soc. Chim. Fr. 1937, 4, 1115-1121. 9 Winans, C. F.; Adkins, H. The preparation of amines by catalytic hydrogenation

of derivatives of aldehydes and ketones. J. Am. Chem. Soc. 1933, 55, 2051-2058. 10 Skita, A.; Keil, F. Formation of bases from carbonyl compound. Chem. Ber. 1928,

61B, 1452-1459. 11 Wright, J. B.; Elderfield, R. C. 9-Alkylamino carbinols derived from 9-acyl-

1,2,3,4-tetrahydrophenanthrene derivatives. J. Org. Chem. 1946, 11, 111-122. 12 Schwoegler, E. J.; Adkins, H. Preparation of certain amines. J. Am. Chem. Soc.

1939, 61, 3499-3502. 13 Kliger, G. A. Hydroammonolysis of diheptyl ether and heptanoate on an iron

catalyst. Bull. Acad. Sci. USSR Div. Chem. Sci. (Engl. Transl.) 1986, 35, 2383-2384.

14 Djedovič, N.; Ferdani, R.; Harder, E.; Pajewska, J.; Pajewski, R.; Weber, M. E.;

Schlesinger, P. H.; Gokel, G. W. The C-and N-terminal residues of synthetic heptapeptide ion channels influence transport efficacy through phospholipid bilayers. New J. Chem. 2005, 29, 291-305.

15 Adkins, H.; Wojcik, B. Hydrogenation from amides to amines. J. Am. Chem. Soc.

1934, 56, 247. 16 Wojcik, B.; Adkins, H. Catalytic hydrogenation of amides to amines. J. Am.

Chem. Soc. 1934, 2419-2422. 17 Nagase, A.; Kuwahara, Y.; Tominaga, Y.; Sugawara, R. Nematicidal activity of

N-substituted and N,N-disubstituted alkylamines against the pine wood nematode, Bursaphelenchus xylophilus. Agric. Biol. Chem. 1983, 47, 53-58.

18 Perreux, L.; Loupy, A.; Delmotte, M. Microwave effects in solvent-free esters

aminolysis. Tetrahedron 2003, 59, 2185-2189.

117

19 Yoshioka, Y.; Tashiro, K. Structural changes in phase transitions of nylon model compounds. 1. Transition behavior of model compounds of R−NHCO−R' type. J. Phys. Chem. B 2003, 107, 11835-11842.

20 Mirgorodskaya, A. B.; Kudryavtseva, L. A.; Zuev, Y. F.; Vylegzhanina, N. N.

Effect of micellar surfactant solutions on the reactivity of long-chain amines. Zh. Fiz. Khim. 2002, 76, 2033-2036.

21 Perreux, L.; Loupy, A.; Volatron, F. Solvent-free preparation of amides from

acids and primary amines under microwave irradiation. Tetrahedron 2002, 58, 2155-2162.

22 Helgen, C.; Bochet, C. G. Preparation of secondary and tertiary amides under

neutral conditions by photochemical acylation of amines. Synlett 2001, 12, 1968-1970.

23 Tomioka, K.; Sumiyoshi, T.; Narui, S.; Nagaoka, Y.; Iida, A.; Miwa, Y.; Taga, T.;

Nakano, M.; Handa, T. Molecular assembly and gelating behavior of didodecanoylamides of alpha,omega-alkylidenediamines. J. Am. Chem. Soc. 2001, 123, 11817-11818.

24 Kuroki, Y.; Ishihara, K.; Hanaki, N.; Ohara, S.; Yamamoto, H. Metal-templated

macrolactamization of triamino and tetraamino esters. Facile synthesis of macrocyclic spermidine and spermine alkaloids, (S)-(+)-dihydroperiphylline, (±)-buchnerine, (±)-verbacine, (±)-verbaskine, and (±)-verbascenine. Bull. Chem. Soc. Jpn. 1998, 71, 1221-1230.

25 Ishihara, K.; Kuroki, Y.; Hanaki, N.; Ohara, S.; Yamamoto, H. Antimony-

templated macrolactamization of tetraamino esters. Facile synthesis of macrocyclic spermine alkaloids (±)-buchnerine, (±)-verbacine, (±)-verbaskine, and (±)-verbascenine. J. Am. Chem. Soc. 1996, 118, 1569-1570.

26 Lu, L. D.; Weiss, R. G. New lyotropic phases (thermally-reversible organogels) of

simple tertiary amines and related tertiary and quaternary ammonium halide salts. Chem. Commun. 1996, 2029-2030.

27 Ghirlanda, G.; Scrimin, P.; Tecilla, P.; Tonellato, U. A hydrolytic reporter of

Cu(II) availability in artificial liposomes. J. Org. Chem. 1993, 58, 3025-3029. 28 Jursic, B. S.; Zdravkovski, Z. A simple preparation of amides from acids and

amines by heating of their mixture. Synth. Commun. 1993, 23, 2761-2770. 29 Kiuchi, F.; Nishizawa, S.; Kawanishi, H.; Kinoshita, S.; Ohsima, H.; Uchitani, A.;

Sekino, N.; Ishida, M.; Kondo, K.; Tsuda, Y. Studies on crude drugs effective on visceral larva migrans. XVI. Nematocidal activity of long alkyl chain amides,

118

amines, and their derivatives on dog roundworm larvae. Chem. Pharm. Bull. 1992, 40, 3234-3244.

30 Gushchin, N. V.; Sokolov, V. P.; Smirnov, V. A.; Gusev, G. M.; Zelenaya, S. A.;

Onishchenko, P. P. Hydrogenation of nitriles to secondary amines. Zh. Prikl. Khim. (Leningrad) 1984, 57, 1109-1115.

31 Troyanskii, E. I.; Ioffe, V. A.; Mizintsev, V. V.; Nikishin, G. I. Oxidative

cyanation of aminyl radicals in the sodium peroxydisulfate-sodium cyanide system. Izv. Akad. Nauk SSSR, Ser. Khim. 1984, 8, 1814-1822.

32 Robson, P.; Speakman, P. R. H. Generation of nitrenes from alkanamidates and

alkanesulfonamidates. J. Chem. Soc. B 1968, 4, 463-467. 33 Williams, A. A.; Day, B. S.; Kite, B. L.; McPherson, M. K.; Slebodnick, C.;

Morris, J. R.; Gandour, R. D. Homologous, long-chain alkyl dendrons form homologous thin films on silver oxide surfaces. Chem. Commun. 2005, 5053-5055.

34 Malanga, C.; Mannucci, S.; Lardicci, L. Nickel mediated reduction of azides by

Bu3SnH. J. Chem. Res. Synop. 2000, 6, 701-715. 35 Demmig, S.; Langhals, H. Very soluble and photostable perylene fluorescent

dyes. Chem. Ber. 1988, 121, 225-230. 36 Semenov, V. A.; Skorovarov, D. I. Application of the Leuckart reaction for the

synthesis of hygher primary aliphatic amines. Zh. Prg. Khim. 1969, 5, 41-43. 37 Crossley, F. S.; Moore, M. L. Studies in the Leuckart reaction. J. Org. Chem.

1944, 9, 529-536. 38 Kipping, F. S. Action of phosphoric anhydride on fatty acids. Part III. J. Chem.

Soc. 1893, 63. 39 Myrtil, E.; Zarif, L.; Greiner, J.; Riess, J. G. Double-tailed perfluoroalkyl

telomeric surfactants derived from tris(hydroxymethyl)acrylamidomethane for medical application. Macromol. Chem. Phys. 1994, 195, 1289-1304.

40 Guedj, C.; Pucci, B.; Zarif, L.; Coulomb, C.; Riess, J. G.; Pavia, A. A. Vesicles

and other supramolecular systems from biocompatible synthetic glycolipids with hydrocarbon and/or fluorocarbon chains. Chem. Phys. Lipids 1994, 72, 153-173.

41 Zwierzak, A. Triethyl phosphite in organic synthesis. A facile, one-pot conversion

of alcohols into amines. Phosphorus, Sulfur Silicon Relat. Elem. 1993, 75, 51-54.

119

42 Meekel, A. A. P.; Wagenaar, A.; Smisterova, J.; Kroeze, J. E.; Haadsma, P.; Bosgraaf, B.; Stuart, M. C. A.; Brisson, A.; Ruiters, M. H. J.; Hoekstra, D.; Engberts, J. B. F. N. Synthesis of pyridinium amphiphiles used for transfection and some characteristics of amphiphile/DNA complex formation. Eur. J. Org. Chem. 2000, 4, 665-674.

43 Carrington, R. A. G.; Evans, H. C. Alkyl sulfates. II. Spectra of the

cyclohexylammonium salts in the 800 cm-1 region. J. Chem. Soc. 1957, 1701-1709.

44 Williams, H. R.; Mosher, H. S. Organic peroxides. II. Secondary alkyl

hydroperoxides. J. Am. Chem. Soc. 1954, 76, 2987-2990. 45 Dreger, E. E.; Keim, G. I.; Miles, G. D.; Shedlovsky, L.; Ross, J. Sodium alcohol

sulfates. Properties involving surface activity. Ind. Eng. Chem 1944, 36. 46 Kleiderer, E. C.; Kornfeld, E. C. Raney nickel as an organic oxidation-reduction

catalyst. J. Org. Chem. 1948, 13, 455-458. 47 Fuerstner, A.; Seidel, G. High-surface sodium as a reducing agent for TiCl3.

Synthesis 1995, 1, 63-68. 48 Fyles, T. M.; Knoy, R.; Mullen, K.; Sieffert, M. Membrane activity of isophthalic

acid derivatives: ion channel formation by a low molecular weight compound. Langmuir 2001, 17, 6669-6674.

49 Orita, A.; Tanahashi, C.; Kakuda, A.; Otera, J. Highly powerful and practical

acylation of alcohols with acid anhydride catalyzed by Bi(OTf)3. J. Org. Chem. 2001, 66, 8926-8934.

50 Polas, A.; Wilton-Ely, J. D. E. T.; Slawin, A. M. Z.; Foster, D. F.; Steynberg, P.

J.; Green, M. J.; Cole-Hamilton, D. J. Limonene-derived phosphines in the cobalt-catalysed hydroformylation of alkenes. Dalton Trans. 2003, 24, 4669-4677.

51 Granet, R.; Piekarski, S. Surface tension and micellization of symmetrical double-

tailed sodium alkylsulfonates. J. Chim. Phys. Phys. Chim. Biol. 1986, 83, 131-136.

52 Boal, A. K.; Das, K.; Gray, M.; Rotello, V. M. Monolayer exchange chemistry of

γ-Fe2O3 nanoparticles. Chem. Mater. 2002, 14, 2628-2636. 53 Zakeeruddin, S. M.; Nazeeruddin, M. K.; Humphry-Baker, R.; Pechy, P.;

Quagliotto, P.; Barolo, C.; Viscardi, G.; Graetzel, M. Design, synthesis, and application of amphiphilic ruthenium polypyridyl photosensitizers in solar cells based on nanocrystalline TiO2 films. Langmuir 2002, 18, 952-954.

120

54 Dusart, O. Synthesis and surface properties of symmetric secondary sodium alkylsulfnates at water-air and water-alkane interfaces. J. Chim. Phys. Phys. Chim. Biol. 1984, 81, 491-496.

55 Hubbard, J. L.; Brown, H. C. Hydride-induced carbonylation of trialkylbranes

followed by treatment with acid and oxidation. A new synthesis of dialkylcarbinols under mild conditions. Synthesis 1978, 9, 676-677.

56 Hughes, R. J.; Ncube, S.; Pelter, A.; Smith, K.; Negishi, E.; Yoshida, T. Synthesis

of secondary or tertiary alcohols by reactions of trialkylboranes with acyl carbanion equivalents. J. Chem. Soc. Perkin Trans. 1 1977, 10, 1172-1176.

57 Burgoyne, E. E.; Condon, F. E. Condensation of 1-alkanols to dialkylcarbinols. J.

Am. Chem. Soc. 1952, 74, 5592-5595. 58 Overmars, F. J. J.; Engberts, J. B. F. N.; Weringa, W. D. Synthesis of and vesicle

formation from phosphorylcholine amphiphiles with one symmetrically branched alkyl chain. Recl. Trav. Chim. Pays-Bas 1994, 113, 293-296.

59 Meakins, R. J.; Sack, R. A. The dielectric properties of symmetrical long-chain

secondary alcohols in the solid state. Australian J. Sci. Research 1951, 4A, 213-228.

60 Baykut, F. The bihomologous series of the symmetrical dialkyl ketones and

dialkylcarbinols, up to C41. Rev. fac. sci. univ. Istanbul 1954, 19, 121-124. 61 Breusch, F. L.; Sokullu, S. Isomeric and homologous series. IV. Synthesis of dl-

hydroxyparaffins with 14-23 carbon atoms. Chem. Ber. 1953, 86, 678-684. 62 Oki, M. Applications of dynamic NMR spectroscopy to organic chemistry; VCR

Publisher, Inc.: Deerfield, 1985. 63 Stewart, W. E.; Siddall, I., T. H. Nuclear magnetic resonance studies of amides.

Chem. Rev. 1970, 70, 517-551. 64 Hanson, P.; Williams, D. A. R. Restricted carbon-nitrogen bond rotation in some

ureas, thioureas, and thiuronium salts. J. Chem. Soc. Perkin Trans. II 1973, 2162-2165.

65 Siddall, I., T. H.; Stewart, W. E. Slow rotation in a thiourea detected by proton

magnetic resonance. J. Org. Chem. 1967, 32, 3261. 66 Newkome, G. R.; Weis, C. D.; Childs, B. J. Syntheses of 1→3 branched


121

67 Gall, R.; Bosies, E. Preparation of diphosphonic acid derivatives for treating calcium metabolic disorders.1988, DE 3623397 A1, 12pp.

68 Froger, C.; Parisot, A. The melting points of the N-monosubstituted amides.

Compt. rend 1954, 238, 1589-1591.

122

Chapter IV Synthesis of Tri-headed Amphiphiles Containing Cholestane-based Structure on the Hydrophobic Moiety

Two new series of tri-headed amphiphiles containing cholestane moiety have

been designed. Both series still contains a ureido linker and a tricarboxylic Newkome-

type dendron. Instead of a long, straight alkyl chains, these new series contains a

nonpolar cholestane moiety as the hydrophobic tail. With the consideration of

commercial availability, only the 5α-cholestane moiety is utilized as a starting material.

In one series, C3 of the cholestane moiety is directly connected to the linker. In the other

series, a spacer −(CH2)nCOO−, where n = 1, is constructed between C3 of the cholestane

moiety and the linker (Figure IV.2).

H

5α-cholestane 5β-cholestane

H

H

H

H1

2

34

5

H

H

H

H1

2

34

5A A

B

C D C D

B

H H

H H

Figure IV.1 Structural representations of 5α- and 5β-cholestane

123

linker

head

head

head

head

head

head

C3cholestane moiety

linker spacerC3cholestane moiety

Figure IV.2 General structures of tri-headed amphiphiles series containing cholestane moiety

The linker and spacer can be attached through two different configurations⎯

axial and equatorial at C3 of cholestane. These configurations are referred as α- and β-

stereochemistry, respectively. Based on how either the linker or spacer is connected to

C3 of cholestane, each series contains a pair of epimers.

IV.1 The 3CUr-z-cholestane Series

The first series is abbreviated as 3CUr-z-cholestane, where “3C” represents three

carboxylic groups on the hydrophilic moiety, “Ur” represents a ureido linker between the

hydrophobic and hydrophilic moieties, and “z-cholestane” represents the

configuration⎯α or β⎯ on how a nitrogen on the linker is attached to the C3 of 5α-

cholestane. When a nitrogen on the ureido linker is connected to C3 of 5α-cholestane via

the α-configuration, the amphiphile is abbreviated as 3CUr-α-cholestane. In a similar

manner, when connection is made via the β-configuration, the abbreviation is 3CUr-β-

cholestane. In a similar manner to the one-tail and two-tail series, the series containing

the tert-butyl esters is abbreviated as 3EUr-z-cholestane.

The chemistry on the 3CUr-z-cholestane was very similar to that of 3CUr1(n)2

(Chapter III). In a similar manner as how the previous series had been synthesized,

retrosynthesis of 3CUr-z-cholestane shows the synthesis begins with WeisocyanateTM

124

(3) and the cholestan-3-amines (4) (Scheme IV.1).

Scheme IV.1 Retrosynthesis of 3CUr-z-cholestane

NH

NH

OH

O

OH

OH

O

O

O

NH

NH

OO

O

OO

O

O

NCOO

O 3+

H

H

H

H1

23

45

H2N

1a, 3CUr-α-cholestane1b, 3CUr-β-cholestane

3

4

4a, 5α-cholestan-3α-amine4b, 5α-cholestan-3β-amine

a b c

a. hydrophilic moiety, b. a ureido linker,c. a hydrophobic 5α-cholestane moiety

2a, 3EUr-α-cholestane2b, 3EUr-β-cholestane

H

H

H

H1

2

34

5

A

C D

B

H

H

When 5α-cholestane moiety is drawn in the chair conformation for the A ring, α-

and β-configurations of C3 correspond to axial and equatorial positions, respectively. As

the only difference is the C3 configuration on the cholestane moiety, 3CUr-z-cholestane

and 3EUr-z-cholestane are a pair of epimers.

HX

Y

X is in axial or 3α-positionY is in equatorial or 3β-position

H

125

IV.1.1 Preparation of 5α-Cholestan-3-amine (4a−b)

In order to synthesize the epimeric pair 1a and 1b, we required amines 4a and 4b,

respectively, to react with 3. As both amines are not commercially available, syntheses

of such were necessary.

IV.1.1.1 Literature Review on Syntheses of Amines (4a−b)

Retrosynthesis (Scheme IV.2) of amines 4a−b shows the synthesis can begin with

cholestanol 8a−b as precursors. However, as only 5α-cholestan-3β-ol (8b) is

commercially available, we sought to design the synthesis beginning with 8b.

Scheme IV.2 Retrosynthesis of epimer 5α-cholestan-3-amines

3

H2N

3

N3

3

MsO3

HO

3

H2N3

N3

3

Y3

HO

4a5a 6b 8b

4b 5b 8a6a, Y = OMs7, Y = Br

H

H

H H

H3

Most reported syntheses of cholestanamine 4a−b begin with the corresponding 3-

azidocholestane (5a−b),1-7 5α-cholestan-3-one oxime, 8-12 5α-cholestan-3-one, 13,

sulfonate ester derivatives of 5α-cholestan-3-ols (6a−b) (Table IV.1).14,15 Lithium

aluminum hydride1,2,4-7 and catalytic hydrogenation in the presence of ammonia3 were

employed to convert azides 5a−b into amines 4a−b with retention of stereochemistry at

126

C3 in good yields. When a particular epimer 5 (5a or 5b), 8 (8a or 8b), or sulfonate ester

derivatives of 8a−b is employed as precursors, the corresponding cholestanamine, either

4a or 4b, is obtained exclusively via an SN2 reaction. However, when 5α-cholestan-3-

Table IV.1 Preparation of amines 4a−b from different precursors Precursors Reagents Product(s); remarks References

N3

LiAlH4, Et2O

H2N

Reaction run under reflux1,2,6 and rt5,7 Yield: 65%1, 77% 5

1,2,4-7

N3

LiAlH4, Et2O

H2N68%

1

N3

H2, NH3, Pd/C

H2N89%

No solvent reported

3

O

H2, NH3, M black M = Ru, Pd

H2N H2N+

For M = Ru, ratio = 26:54 (3α:3β); addition of NH4Cl yields ratio = 16:82 (3α:3β) For M = Pd, ratio= 19:81(3α:3β) addition of NH4Cl yields ratio = 21:79 (3α:3β)

13

NHO

LiAlH4, Et2O, reflux

H2N H2N+

10,11

NHO

n-C5H11OH, Na

H2N

8,9,12

NHO

1. H2, PtO, HOAc 2. HCl

H2N H2N+

9

FG FG = −OTs,14,15 −OMs15

Anhydrous NH3

H2N

FG = −OTs, 70%;15 56%14 FG = −OMs, 63%

14,15

127

one oxime and 5α- cholestan-3-one are utilized, a mixture of epimers, 4a and 4b, is

obtained in various ratios. Reduction of 5α-cholestan-3-one oxime performed with

lithium aluminum hydride results in a mixture of the same epimers, in almost equal

amounts.10,11 Interestingly, the reduction performed with sodium in pentan-1-ol yields

amine 4b exclusively.8,9,12 When reduction of cholestan-3-one oxime is carried out by

hydrogenation in acetic acid followed by hydrolysis of the N-acetyl derivative with

concentrated hydrochloric acid, amine 4a is obtained as the major product.9 Sodium

borohydride and hydrogenation in dioxane are ineffective.9

IV.1.1.2 Literature Review on Syntheses of 3-Azidocholestane (5a−b)

Both azides 5a−b are usually synthesized starting from the corresponding alcohol

by using derivatives such as a sulfonate ester, 6a−b, and a haloalkane (Table IV.2). The

syntheses involve SN2 reactions with different azide reagents, and the corresponding

azides are mostly obtained in good to excellent yields. Azides 5b and 5a are prepared

directly from alcohols 8b and 8a, respectively, via a Mitsunobu-type substitution. The

traditional approach is where alcohols are converted into the corresponding sulfonate or

halide derivative prior to a nucleophilic displacement of the corresponding sulfonate ester

or halide by azide. In any case, conversion of alcohol 8 into azide 5 results in inversion

of the stereochemistry at C3.

Compared to the traditional approach, a Mitsunobu-type substitution is more

convenient because such a reaction can be performed in one-pot; it is a shorter route. In

addition, most of these Mitsunobu-type reactions give good to excellent yields. Only one

procedure1 following a Mitsunobu-type reaction reported that azide 5a was obtained in

slightly low yield.

128

Table IV.2 Preparation of azides 5a and 5b from various precursors Precursors Reagents Product(s); remarks References

HO

PPh3, DEAD, THF, various azides Azides: nicotinoyl azide, benzoyl azide N3

94%

16

HO

2,4,4,6-tetrabromo-2,5-cyclohexadienone, PPh3, Zn(N3)2

.2Py, PhCH3, CH3CN N3 94%

17

HO

PPh3, DEAD, (PhO)2PON3, THF

N3 51%1

1,18

MsO

Dry DMPU, NaN3

N3 75%

19

HO

PPh3, DEAD, THF, various azides Azides: nicotinoyl azide, benzoyl azide N3

nicotinoyl azide (96%) benzoyl azide (91%)

16

HO

PPh3, DEAD, HN3, PhH

N3 77%

20

Z

Z = I, 1 OMs21

NaN3, DMSO

N3 Z = I (81%);1 OMs (75%)21

1,21

Z

Z = OMs,22 OTs,2,6,23 p-oxopyridinium tosylate5

NaN32,6,22,23 or LiN3

5 ,22 Solvents: HMPT,2 DMA, 6 DMF22,23 N3

95% without purification; 2 75%23

2,5,6,22,23

129

IV.1.1.3 Literature Review on Syntheses of 3-Methanesulfonyloxy-5α-cholestane (6a–b)

Conversion of alcohols 8a−b into the corresponding sulfonate esters, such as a

methanesulfonyloxy- and toluenesulfonyloxyalkane, is widely utilized in organic

synthesis. Even though methanesulfonyloxy is a better leaving group than

toluenesulfonyloxy, toluenesulfonyloxy derivatives are more widely found in the

literature. The conversion of 8 into 6 is usually achieved by the standard method utilizing

toluenesulfonyl or methanesulfonyl chloride15 ,21,22,24,25 and pyridine as a base; the

corresponding product is obtained with retention of stereochemistry (Table IV.3). On the

other hand, when the conversion into methanesulfonyloxy derivative is prepared with a

Mitsunobu-type reaction, the product is obtained with inversion of stereochemistry.

Table IV.3 Preparations of 6a−b from 8a−b Precursors Reagents Remarks References

HO

MsCl, pyridine

MsO 100%,24 60%25

21,22,24,25

HO

PPh3, DEAD, MsOH, THF

MsO no yield reported

19

HO

MsCl, pyridine

MsO 60%,25 50%15

15,25

IV.1.1.4 Literature Review on Synthesis of 3α-Bromo-5α-cholestane (7)

As depicted on the retrosynthesis of amine 4b (Scheme IV.3), the synthesis of

bromoalkane 7 is required. Several procedures, most of which involve phosphine,26-29

have been reported to give 7 in good to excellent yields. Employing the phosphine-free

130

method, 30,31 the traditional catalyst cuprous chloride (CuCl) works as well as cupric

triflate (Cu(OTf)2) in the conversion of the primary alcohols. However, when secondary

alcohols are employed, Cu(OTf)2 is superior to the traditional catalyst CuCl. When CuCl

is employed for such a conversion, the reaction takes longer time and the isolated product

is identified as a complex mixture. Acetyl bromide, N-bromosuccinimide (NBS), lithium

bromide, and 2,4,4,6-tetrabromocyclohexanone are commonly used brominating agents

in such a conversion (Table IV.4). Lithium bromide as a bromide source needs to be

added in large excess (10 equiv) in order to obtain the product in excellent yield.28

Table IV.4 Preparations of bromoalkane 7 from 8b and 8b’s derivatives Precursors Reagents Remarks References

HO

Cu(OTf)2, DIC, brominating agents (AcBr, 30 NBS31)

80%30,31 solvent : THF,30dioxane31 minor elimination products observed

30,31

HO

PPh3Br2, DMF 80%

29

HO

DEAD, PPh3, LiBr, THF 96%

28

RO

R = H, THP

PPh3,Br

Br

Br

Br

O , CH3CN

86% (R = H), 91% (R = THP)

26

OEt3Si PPh3,

Br

Br

Br

Br

O , CH3CN

CaCO3, THF

86% 27

131

IV.1.2 Synthesis of 5α-Cholestan-3-amines (4a−b)

We began our synthesis with commercially available alcohol 8b. In order to

synthesize the epimeric pair 4a−b, the first step was to convert alcohol 8b into the

corresponding sulfonate ester derivatives 6a−b or bromoalkane 7 (Scheme IV.3).

Considering availability, better reactivity, and successful conversion into the intermediate

sulfonate ester in the previous homologous series (Chapter II and III), we decided to

transform alcohol 8b into the corresponding methanesulfonyloxy derivative (6b). Both

cholestanol 8a and bromoalkane 7 could be employed as an intermediate in the synthesis

of amine 4b. As cholestanol 8b was easily converted into bromoalkane 7 via 6b, we

chose to synthesize bromoalkane 7 instead of cholestanol 8a as an intermediate to

generate amine 4b.

As expected, the conversion of alcohol 8b into the corresponding derivative 6b

retained the stereochemistry on C3. The reaction was completed in a relatively short time

(15 minutes), and the product was obtained in quantitative yield. Completion of the

reaction was indicated by the appearance of a singlet at δ 3.00 ppm (3H on the CH3SO2−)

in the 1H NMR spectrum and a multiplet at 4.62 ppm (H-α). These chemical shifts

compared favorably to published values.25 After comparing the proton integration of the

axial hydrogen on C3, the methyl group on sulfur (CH3SO2−), and the rest of the

cholestanyl hydrogens in the 1H NMR spectrum of the crude product, we decided that the

crude product was suitable for use without purification for the next step.

The crude derivative 6b was allowed to react with lithium bromide (2.5 mol eq.)

via an SN2 reaction to give 3α-bromo-5α-cholestane 7. The reaction was refluxed in

tetrahydrofuran, and completion of the reaction was shown by the 1H NMR spectrum.

132

After purification, the product 7 was obtained in 74% yield based on 6b. In the 1H NMR

spectrum, the chemical shifts of the methyl group (CH3SO2−, s, δ 3.00 ppm) and the α-H

(m, δ 4.62 ppm) on C3 of the starting 6b were no longer observed. An SN2 reaction took

place at C3 to give a β-H, which was observed at δ 4.73 ppm (lit. 60 MHz32 δ 4.63 ppm,

60 MHz33 δ 4.71 ppm) as a triplet. It was intriguing in the beginning to find that the

reported32 chemical shift of the β-H on C3 of bromocholestane 7 was actually much

closer to that of the starting 6b. However, we noticed the change in the splitting pattern

at the reactive C3, caused by the change of the proton position⎯axial H (α-H) to

equatorial H (β-H). Finally, comparison of the 1H NMR spectrum to another published

value33 verified that we did obtain 3α-bromo-5α-cholestane (7).

Azides 5a and 5b were obtained via an SN2 reaction of 6b and 7, respectively,

with sodium azide as the nucleophile. Purification of the resulting azides was carried out

by flash column chromatography in hexane in good yields. The absorption at 2100 cm-1

in the IR spectra showed the presence of azido groups. The configuration⎯α or β⎯of

the proton bonded to C3 on the products was confirmed by change in both splitting

pattern and chemical shift in the 1H NMR spectra.

The last step was reduction of azides 5. Reduction was performed via two

different ways⎯catalytic hydrogenation in hexane and application of reducing agent

lithium aluminum hydride. Both methods gave amines 4a−b. The absorptions at 2100

cm-1 in the IR spectra were no longer present. Both chemical shifts of the α- or β-H on

C3 were shifted downfield ~ 0.7 ppm, however, their splitting patterns were not changed,

indicating the stereochemistry at C3 was retained in both compounds.

The former method, catalytic hydrogenation, was more convenient⎯ease of

133

work-up⎯than the latter. Even though their NMR spectra were the same, the product

generated from the latter method was obtained in higher yields, higher melting

temperatures, and whiter in color. Multiple gravity filtrations on the products generated

from the former method, namely hydrogenation, were attempted. However, the color of

the products obtained did not improve much. Even worse, multiple gravity filtrations

caused material loss during transfers. Other attempts to improve the quality of the color

included filtering through a Celite® pad in vacuo and adding charcoal in the beginning of

the recrystallization process met with no success. Regardless of the convenience,

considering all the advantages offered by lithium aluminum hydride, we decided to carry

out the reduction of azides 5 with such a reducing agent in future experiments.

Scheme IV.3 Synthetic scheme of the preparation of epimeric amines (4a−b)

MsOHO N3 H2N

Br N3 H2N

i ii iii

iv

ii iii

i. MsCl, Et3N, dry DCM; quantitative yieldii. NaN3, DMF, 90 °C, 4h; 3a, 85%; 3b 79%iii. Pd/C, hexane; 3a, 68%; 3b, 65% or LiAlH4, THF, reflux; 3a, 80%; 3b, 86%iv. LiBr, THF, 70 °C, 14h; 74%

8b

7

6b 5a

5b

4a

4b

IV.1.3 Synthesis of 3CUr-z-cholestane (1a−b)

The tri-tert-butyl esters 2a−b were generated by combining 3 and amines 4 in

dichloromethane at ambient temperature (Scheme IV.1). Purification was carried out by

134

flash column chromatography (hexane:THF:MeOH=1:1:0.04) to give a white solid. Tri-

tert-butyl esters 2a and 2b were obtained in 78% and 74% yields, respectively, and the

melting temperatures were 179.9−180.5 °C and 187.4−187.8 °C, respectively. Both 2a

and 2b were fully characterized.

Recrystallization from ethanol was performed in order to obtain a crystal for X-

ray analysis; an X-ray crystal structure of 2a was obtained. Ethanol was observed in the

crystal lattice (see below). On the other hand, repeated attempts to recrystallize 2b in

order to generate a crystal for X-ray analysis were not successful.

Formolysis of the compounds 2a and 2b at room temperature produced the

triacids 1a and 1b, respectively. Triacids 1a and 1b were obtained in 73% and 78%

yields, respectively. As for the melting temperature observation, decomposition of both

compounds was observed at 199.0 °C and 194.3 °C, respectively. Both 1a and 1b were

fully characterized.

IV.1.4 X-ray Structure of 3EUr-α-cholestane (2a)

The crystal structure of 3EUr-α-cholestane (2a) shows how the molecules with a

bulky dendritic polar moiety and extremely rigid hydrophobic moiety assemble in the

solid state. Two different crystallographic independent molecules are found in the X-ray

crystal structure arranged in parallel; inversion symmetry generates the antiparallel

packing. In general, molecules that are antiparallel are packed more closely than

molecules that are parallel. The closest interaction in the antiparallel orientation ranges

from 3.8 to 4.1 Å involving the side chain of one cholestane moiety and both ring B and

C, including the methyl substituents at the junction of ring A-B and ring C-D on the other

cholestane moiety. The closes interaction in the parallel orientation ranges from 4.0 to

135

4.1 Å involving the side chain of one cholestane moiety and ring B, C, D, including the

methyl substituents at the junction of ring A-B of the other cholestane moiety. Ring A of

this cholestane moiety displays short intermolecular contacts to the headgroup of the

other cholestane moiety, ranging from 3.4 to 4.1 Å.

Symmetrically equivalent molecules are separated by 12.12 Å from each other. In

the two independent molecules, all six-membered rings are found in full chair

conformations, while all five-membered rings are found in twisted envelope

conformations. The torsion angles in two headgroups of each independent molecule

Figure IV.3 X-ray crystal structure of 3EUr-α-cholestane (2a), showing the packing and the intermolecular hydrogen bonding to ethanol. Hydrogen-bond Donor(D)–Acceptor(A) distances and approximate ∠DHA: O(16)···O(1), 2.621(7) Å and 158.1°; O(15)···O(8), 2.625(6) Å and 161.0°; O(4)···O(16), 2.865(7) Å and 155(7)°; N(2)···O(15), 2.849(8) Å and 159.6°. The other nitrogens on the ureido linker are just outside the range for significant hydrogen bonding. N(3)···O(16), 3.035(7) Å and 141.6°; N(1)···O(15), 3.084(8) Å and 146.0°

136

adopt anti conformations, while the torsion angle of the other headgroup adopts a gauche

conformation. Torsion angles of the two headgroups in the first molecule are –170.1(6)°

[C(1)−C(37)−C(38)−C(39) and +177.2(6)° [C(1)−C(44)−C(45)−C(46)], and those in the

second molecule are –170.5(6)° [C(51)−C(80)−C(81)−C(82) and +166.8(7)°

[C(51)−C(94)−C(95)−C(96)]. Torsion angles of the third headgroup adopt gauche

conformations, however, in different direction; those in the first and second molecules are

−96.9(5)° [C(1)−C(30)−C(31)−C(32)] and +91.6(7)° [C(51)−C(87)−C(88)−C89)],

respectively.

The presence of ethanol molecules is very important in the packing. Each 3EUr-

α-cholestane (2a) molecule is close to an ethanol molecule; they are arranged in alternate

manner and connected by hydrogen bonds; the short contacts within any different

molecules range from 1.823 Å [O(1)−H(16)] to 3.171 Å [O(1)−C(103)]. Each ethanol

molecule serves as both donor and acceptor for the hydrogen bonds formed; it serves as a

hydrogen bond donor to one crystallographic molecule of 3EUr-α-cholestane (2a) and a

hydrogen bond acceptor to the other. As hydrogen bond donors, they donate hydrogen

bonds, from namely O(16) and O(15), to the carbonyl-oxygen on the linker, namely O(1)

and O(8), respectively (Figure IV.1). As hydrogen bond acceptors, ethanol molecules

[O(16) and O(15)] accept hydrogen bonds from the amido nitrogens [N(4) and N(2),

respectively] connected to the cholestane moiety. In addition, the other amido nitrogens

[N(3) and N(1)] in the linker form weak hydrogen bonds with the same ethanol molecules

(donor−acceptor distance > 3.0 Å). The hydrogen bond between the solvent ethanol and

the carbonyl oxygen of the linker is the shortest. These last two interactions are similar

to those previously observed in the X-ray crystal structure of 3EUr16 (Chapter II) where

137

water, instead of ethanol, is used as the solvent in recrystallization. The formation of

weak hydrogen bonds between the solvent and the amido nitrogen attached to the

headgroup suggests that the bulkiness of the headgroups can possibly obstruct the solvent

to get close enough to the amido nitrogen attached to the headgroup, resulting weak

hydrogen bonds. Compared to the X-ray crystal structure of 3EUr16, there is

unfortunately no hydrogen bond involving the participation of the headgroups.

Intramolecular hydrogen bonds are observed in the X-ray crystal structure of 3EUr16

where the carbonyl oxygen of the linker and an ester carbonyl oxygen accept hydrogen

bonds from water; this is possible as each water molecule contains two hydrogens.

IV.2 The 3CUrnEs-z-cholestane Homologous Series

The second homologous series is abbreviated as 3CUrnEs-z-cholestane (Scheme

IV.5), where “3C” represents three carboxylic groups on the hydrophilic moiety, “Ur”

represents a ureido linker between the hydrophobic and hydrophilic moieties, “n”

represent the number of methylene between the ureido linker and the carbonyl carbon of

the ester functional group, which is represented as “Es”, and finally “z-cholestane”

represents the configuration⎯α- or β-configuration⎯ on how the oxygen on the ester

group is attached to the C3 of 5α-cholestane. As the 3CUrnEs-z-cholestane

homologous series is generated from the corresponding tri-tert-butyl esters, the

homologous series containing the tert-butyl groups is abbreviated as 3EUrnEs-z-

cholestane.

Retrosynthesis of 3CUrnEs-z-cholestane shows that the synthesis can begin with

Weisocyanate (3) and 5α-cholestan-3-yl ω-aminoalkanoates (Scheme IV.4). In this

preliminary study, only the shortest homologs (9, n = 1)⎯3CUr1Es-z-cholestane⎯are

138

synthesized. Thus, a synthesis of both 3α- and 3β- epimers of 5α-cholestan-3-yl

aminoethanoate (11) is necessary.

Scheme IV.4 Retrosynthesis of the 3CUrnEs-z-cholestane homologous series

NCOO

O 3

H

H

H

H12

34

5

O 3

OHN

HN

O

H

H

H

H

3

9, n = 19a, 3CUr1Es-α-cholestane9b , 3CUr1Es-β-cholestane

3

OHO

HO

HO

O

O

O

a b d c

n

a. a hydrophilic moiety, b. a ureido linker,c. a −(CH2)nCO2− spacer, d. a hydrophobic linker

H2NO

+

OHN

HN

O

10, n = 110a, 3EUr1Es-α-cholestane10b, 3EUr1Es-β-cholestane

3

OO

O

O

O

O

O

n

11, n = 111a, 5α-cholestan-3α-yl aminoethanoate11b, 5α-cholestan-3β-yl aminoethanoate

n

3CUrnEs-z-cholestane 3EUrnEs-z-cholestane

5α-cholestan-3-yl ω-aminoalkanoate

H

H

A

B

C D

IV.2.1 Preparation of 3α- and 3β- Epimer of 5α-Cholestan-3-yl Aminoethanoate (11a−b)

In order to synthesize 11a−b (Scheme IV.5), we employed retrosynthesis

following our strategy for the synthesis of amines in the previous 3CUrn (Chapter II) and

3CUr1(n)2 (Chapter III) series. We chose a route so that amines 11 were generated from

the corresponding azides 12. As we sought to construct the synthesis beginning with

139

commercially available cholestanol 8b, syntheses of intermediates 13a−b and 8a would

be required.

Scheme IV.5 Retrosynthesis of 3α- and 3β- epimer of 5α-cholestan-3-yl aminoethanoate

OCl

O

ON3

O

OH2N

O

OCl

O

ON3

O

OH2N

O

HO

HO

11b

11a 12a

12b

13a

13b 8b

8a

IV.2.1.1 Literature Review on the Synthesis of 5α-Cholestan-3α-yl Aminoethanoates (11a−b)

Only one procedure34 on the synthesis of 5α-cholestan-3α-yl aminoethanoate has

been reported. Matsumoto et al.34 generated cholestanol (8b) esters of a various amino

acids, including the simplest amino acid glycine. Beginning with glycine, fusion of such

an amino acid and cholestanol (8b) was achieved by passing hydrogen chloride into the

two reactants. Free amine 11b was then released from the resulting hydrochloride ester.

H2NO

HO

+ 8b 11b HClHCl, CHCl3:MeOH (10:1)

.160 °C, 1.5 h

11banhydrous Na2CO3

With this procedure, a couple intermediates (Scheme IV.6) would not be needed

because 11b could be synthesized in one-step reaction from 8b. However, one drawback

on this procedure was that the products from various amino acids were obtained in

140

20−44% yield; 11b was obtained in 44%. Regardless it was a one-step reaction, and

necessary reagents were commercially available, we did not attempt to follow the

reported procedure because of the resulting low yield. We then followed our original

strategy (Scheme IV.6) to synthesize azides 12a−b.

IV.2.1.2 Literature Review on the Synthesis of 5α-Cholestan-3α-yl Azidoethanoate (12a−b)

The procedure on the synthesis of 5α-cholestan-3α-yl azidoethanoate has never

been documented. However, we have demonstrated that the transformation of

haloalkanes into the corresponding azidoalkanes (in the previous 3CUrn homologous

series) performed by heating the haloalkanes and sodium azide in N, N-

dimethylformamide, gives good yields of azidoalkanes.

It is well documented that several alkyl azidoethanoates,35-40 where the alkyls

contain carbacyclic moieties including cholestane moiety,35 are transformed into the

corresponding amines in good to excellent yields. In these reactions, sodium azide is

again utilized; most reactions are carried out in various solvents⎯ N, N-

dimethylformamide,35,36,40 acetone,37 acetone−water39 and dimethylsulfoxide38⎯ by

heating35-37,39 or stirring at ambient temperature.40 In any case, the ester functional

groups are not affected. With this consideration, we decide to adopt our previous

procedure for the transformation of 5α-cholestan-3α-yl chloroethanoate (13) into 5α-

cholestan-3α-yl azidoethanoate (12).

IV.2.1.3 Literature Review on the Synthesis of 5α-Cholestan-3α-yl Chloroethanoate (13a−b)

Compounds 13a−b are commercially available, however, in milligram quantities,

141

thus syntheses are required. Several procedures41-45 on the syntheses of 13a−b have been

reported (Table VI.5). All syntheses begin with commercially available 8b. Both

chloroacetic acid and chloroacetyl chloride were utilized as the acylating agents.

Beginning with chloroacetic acid, the inverted product is obtained in the presence

of Mitsunobu reagents.43 Chloroacetyl chloride is mostly combined with a base to give

products with retention of stereochemistry. 41,42,44,45

Table IV.5 Preparations of 5α-cholestan-3α-yl chloroethanoate from 5α-cholestan-3β-ol (13a−b) Precursors Reagents Remarks References

HO

ClCH2CO2H, DEAD, PPh3, THF

rt, 24 h, inverted product, 85%, inversion ratio >99%

43

HO

ClCH2CO2H, TMAD, Bu3P, PhH

43%, mixture of α:β=28:72

43

HO

ClCH2COCl, Et3N, PhH reflux at 80 °C, overnight41 or 24 h,44 retention product, 65%

41,44

HO

ClCH2COCl, DMAP, Et3N, PhH

rt, overnight, retention product, 78%

42

HO

ClCH2COCl, CHCl3 retention product, no yield reported

45

IV.2.2 Synthesis of 5α-Cholestan-3α-yl Aminoethanoate (11a−b)

We began our synthesis with cholestanol 8b as it was commercially available

(Scheme IV.6). Conversion of 8b into 8a was carried out following the literature

procedure;46 8a was obtained in 80% yield. Cholestanol 8a and 8b were converted into

13a and 13b, respectively. The formation of the products was confirmed by the

appearance of a singlet at δ ~4.0 ppm (ClCH2CO2−) in 1H NMR spectra. No change in

142

the splitting pattern on the proton bonded to C3 confirmed that the products were

obtained with retention of configuration at C3.

5α-Cholestan-3-yl chloroethanoates (13a−b) were converted into the

corresponding 5α-cholestan-3-yl azidoethanoate (12a−b) via an SN2 reaction. The

reaction was carried out by heating sodium azide in a solution of 13 in N,N-

dimethylformamide. Substitutions of the halogen by azide were confirmed by 1H NMR

spectra; the chemical shifts of the methylene protons (−CH2CO2−) shifted from δ ~4.0 to

δ ~ 3.8 ppm. The absorption at ~ 2100 cm-1 in the IR spectra also confirmed the presence

of an azido group. Compounds 12a−b were obtained in 47−49% yield.

In the synthesis of the amine intermediates of 3CUr-z-cholestane (see Section

IV.1.3), reduction of the azides was carried out with both lithium aluminum hydride and

hydrogenation. From the product characteristics, such as higher melting temperatures

and higher isolated yields, we concluded reduction with lithium aluminum hydride is the

better way to perform such a transformation

Choices of reducing agents become limited when other functional groups present.

The epimers of 3CUr1Es-z-cholestane contain both azido and ester functional groups.

The powerful reducing agent lithium aluminum hydride reduces azido as well as ester

functional groups.47-49 Thus, we performed the conversion of the azides into the

corresponding amines by hydrogenation. Hydrogenation reduces azido groups but does

not affect ester groups.50-54

With this consideration, hydrogenation reduced azides 12a−b to form amines

11a−b, which were identified by the appearance of weak absorptions at ~ 3400 cm-1 in

the IR spectra. The transformation of azides 12a−b into amines 11a−b was also

143

supported by the disappearance of the absorptions of the azido group (~ 2100 cm-1) in the

IR spectra. In the 1H NMR spectra, protons on the methylene (−CH2CO2−) shifted from

~ 3.8 ppm to ~ 3.4 ppm. The products 11a and 11b were obtained in 88 and 76% yields,

respectively.

Scheme IV.6 Synthetic scheme of the preparation of amines 11a−b

OCl

O

ON3

O

OH2N

O

OCl

O

ON3

O

OH2N

O

HO

HO

11b

11a12a

12b

13a

13b8b

8a

i

i

ii

ii

iii

iii

iv

i. ClCH2COCl, Et3N, PhH, rt, 24 h; 13a, 68%; 13b, 57%ii. NaN3, DMF, ref lux; 12a, 47%; 12b, 49%iii. Pd/C, CH2Cl2, H2, 69 °C, 4 h; 11a, 88%; 11b, 76%iv. DIAC, Et3N, TFA, PPh3, dry THF, rt, overnight, then MeOH 50 °C, overnight; 8a, 80% IV.2.3 Synthesis of 3CUr1Es-z-cholestane (9a−b)

The tri-tert-butyl esters 10a−b were generated by combining 3 and amines 11 in

dichloromethane at ambient temperature (Scheme IV.4). Purification was carried out by

flash column chromatography (hexane:ethyl acetate = 2.5:1) to give a white solid, which

gave a single spot on TLC (Rf = 0.33−0.35) in the solvent system above. The product was

further purified by recrystallization from ethanol. The tri-tert-butyl esters 10a and 10b

were obtained in 64% and 61% yields, respectively, and the melting temperatures were

82.9−83.3 °C and 195.8−196.2 °C, respectively. Assignments in the 1H NMR spectra

144

were supported by the two-dimensional 1H−1H COSY NMR.

In 10a and 10b, the geminal protons on the methylene of the ethanoates coupled

to the neighboring N−H (δ 4.65 and δ 4.62 ppm, respectively). In 10a, these methylene

protons were not interchangeable, thus observed to be diastereotopic protons. These

diasterotopic protons (δ 3.94 and 3.89 ppm) were not equivalent; each showed a doublet

of doublets. The bigger group at C3 was on axial position, which possibly restricted the

free rotation about CH2−CO bond. Consequently, the methylene protons were in

different environment, thus giving different chemical shifts. On the other hand, this

behavior was not observed in 10b, where the bigger group at C3 was in equatorial

position; the methylene protons (δ 3.84 ppm) showed a doublet, which suggested that the

free rotation about CH2−CO bond was fast on an NMR time scale. The axial hydrogen at

C3 of 10a and the equatorial hydrogen at C3 of 10b (δ 5.08 and 4.70 ppm, respectively)

coupled to some of the cholestanyl protons (δ 0.60−2.00 ppm).

Formolysis of the compounds 10a−b at room temperature produced the triacids

9a−b, respectively. Triacids 9a−b were obtained in 51% and 57% yields, respectively.

As for the melting temperature observation, compound 9a melted at 192.5−192.9 °C,

while 9b decomposed at 194.2 °C; the large difference in melting temperature observed

between 10a and 10b disappeared when the tert-butyl groups were removed. Triesters

10a−b and triacids 9a−b were fully characterized.

IV.3 Experimental Procedures Material and Methods.

Chemicals were obtained from Aldrich, Acros and TCI; they were used without further

145

purification. Solvents were reagent grade or HPLC grade; they were used as received

unless otherwise specified. THF was distilled from sodium/benzophenone ketyl.

WeisocyanateTM was prepared as described55 with a shorter reaction time as mentioned

above. Analytical thin layer chromatography was performed by polyester-coated silica

gel 60 Å and detected by treating with 10% ethanolic phosphomolybdic acid reagent (20

wt. % solution in ethanol) followed by heating. Flash column chromatography was

carried out on silica gel (60 Å); samples were loaded as concentrated solutions in the

solvent system needed; column diameter × height (13/4 × 6 in), eluted samples varied

between ~ 2.00−4.00 g, flow rate (~ 1.5−2 in/min) was controlled by air pressure.

Solutions were concentrated by rotary evaporation. Melting ranges, determined in open

capillary tubes, were uncorrected. NMR spectra were recorded on an INOVA at 400 and

100 MHz for 1H and 13C, respectively, and reported in ppm relative to the known solvent

residual peak. Resonances were reported in the order of the chemical shifts (δ), followed

by the splitting pattern, and the number of protons. Abbreviations used in the splitting

pattern were as the following: s = singlet, d = doublet, t = triplet, q = quartet, m =

multiplet, and b = broad. IR spectra were recorded on neat samples with an FTIR

equipped with a diamond ATR system, and reported in cm-1. Optical rotations were

measured at the sodium line by using a Perkin-Elmer 241 polarimeter in a 1-dm tube.

HRMS data were obtained on a dual-sector mass spectrometer in FAB mode with 2-

nitrobenzylalcohol as the proton donor. Elemental analyses were performed by Atlantic

Microlabs, Inc. in Norcross, GA.

146

Preparation of 3α-methanesulfonyloxy-5α-cholestane (6b)

MsOHO+ MsCl

Et3N, dry DCM

15 min 0 °C

A 100-mL round-bottomed flask containing a stirred solution of 8b (5.12 g, 12.9 mmol)

and Et3N (2.55 mL, 18.2 mmol) in dry CH2Cl2 was cooled in an ice bath. When the

temperature reached 0 °C, MsCl (1.2 mL, 2.96 g, 15.4 mmol) was added slowly through a

syringe. The resulted reaction was stirred for another 15 min while maintaining the low

temperature. The resulting reaction was quenched by washing successively with water (5

mL), HCl (2 M, 5 mL), water (5 mL), satd NaHCO3 (5 mL), and water (5 mL). The

organic solution was dried and concentrated to give an off-white solid (5.71 g, 12.2

mmol, 94%), which gave a single spot on TLC in CH2Cl2 (Rf = 0.70). The 1H NMR

spectrum of the respective crude product suggested that the material was suitable for the

next step without further purification. 1H NMR (CDCl3) δ 0.60–2.00 (m, 46H), 3.00 (s,

3H), 4.62 (m, 1H) (lit.25 200 MHz); 13C NMR (CDCl3) δ 12.1, 12.2, 18.7, 21.2, 22.6,

22.8, 23.8, 24.2, 28.0, 28.2, 28.5, 28.7, 31.9, 35.2, 35.3, 35.4, 35.8, 36.2, 36.8, 38.9, 39.5,

39.9, 42.6, 44.9, 54.1, 56.3, 56.4, 82.3.

Preparation of 3α-bromo-5α-cholestane (7)

BrMsOLiBr, dry THF

70 °C, 14 h

To a stirred solution of crude mesylated product 6b (4.37 g, 9.36 mmol) in dry THF (40

mL) was added LiBr (2.06 g, 23.5 mmol) slowly. The suspension was refluxed at 70 °C

for 14 h. The resulting reaction was concentrated, and the residue was extracted into

hexane (50 mL); the undissolved solid was filtered off and the filtrate was concentrated to

147

give a white solid, which was recrystallized from EtOH to give product 7 (3.15 g, 74%);

mp 103.5–103.9 °C (lit.29 mp 100–102 °C); 1H NMR (CDCl3) δ 0.60–2.00 (m, 46H), 4.74

(m, 1H) (lit.33 60 MHz); 13C NMR (CDCl3) δ 12.1, 13.3, 18.7, 20.8, 22.6, 22.8, 23.9,

24.2, 27.9, 28.0, 28.2, 31.0, 31.8, 32.9, 35.4, 35.8, 36.17, 36.23, 37.3, 39.5, 39.95, 40.13,

42.6, 53.9, 56.16, 56.24, 56.4; IR 2942, 2923, 2844, 694; [α]25D +29.4° (c 1.62, CHCl3)

(lit.29 [α]D +26° (c 1.2, CHCl3); HRMS: for C27H47Br calcd 450.2861, found 450.2851.

Preparation of 3-azido-5α-cholestanes (5a−b)

a. Preparation of 3α-azido-5α-cholestane (5a)

N3MsONaN3, dry DMF

90 ºC, 4 h

To a 250-mL round-bottomed flask containing solution of crude 6a (4.97 g, 10.65 mmol)

in DMF (75 mL) was slowly added NaN3 (3.49 mg, 53.2 mmol). The resulted suspension

was heated at 90 ºC for 4 h. Then, the reaction was cooled to rt before hexane (150 mL)

and water (25 mL) were added. The organic layer was separated and washed

successively with satd NaHCO3 (25 mL) and satd NaCl (25 mL). The organic layer was

dried with Na2SO4 and concentrated to give a crude product, which is purified by flash

chromatography in hexane to give a white solid (3.73 g, 85%); mp 64.4–64.8 °C (lit.6

62.5−63.5 °C); 1H NMR (CDCl3) δ 0.60–2.00 (m, 46H), 3.88 (bs, 1H) (lit.1 300 MHz);

13C NMR (CDCl3) δ 11.6, 12.1, 18.7, 20.8, 22.6, 22.9, 23.9, 24.2, 25.7, 28.0, 28.3, 28.4,

31.9, 32.6, 32.9, 35.5, 35.8, 35.9, 36.2, 39.5, 40.0, 40.1, 42.6, 54.2, 56.3, 56.5, 58.3; IR

2931, 2865, 2852, 2084 (lit.1 film); [α]26D +18.6° (c 1.52, CHCl3) (lit.6 [α]26

D +18.6° (c

1.01, CHCl3)).

148

b. Preparation of 3β-azido-5α-cholestane (6b)

Br N390 ºC, 4 h

NaN3, DMF

To a 250-mL round-bottomed flask containing solution of 3α-bromocholestane 7 (2.36 g,

5.22 mmol) in DMF (36 mL) was slowly added NaN3 (1.72 mg, 26.1 mmol). The

resulted suspension was heated at 90 °C for 5 hours. The reaction was cooled to rt before

hexane (72 mL) and water (15 mL) were added. The organic layer was separated and

washed successively with satd NaHCO3 (15 mL) and satd NaCl (15 mL). The organic

layer was dried with Na2SO4 and concentrated to give a crude product, which is purified

by flash chromatography in hexane to give a white solid (1.70 g, 79%); mp 66.3–66.8 °C

(lit.16 mp 71.5–72 °C, lit.21 65–66 °C); 1H NMR (CDCl3) δ 0.60–2.00 (t, 47H), 3.25 (m,

1H) (lit.1 300 MHz); 13C NMR (CDCl3) δ 12.1, 12.4, 18.7, 21.2, 22.6, 22.8, 23.8, 24.2,

28.0, 28.3, 38.8, 32.1, 32.7, 35.55, 35.56, 35.8, 36.2, 37.7, 39.51, 39.53, 40.1, 42.6, 45.6,

51.2, 54.5, 56.3, 56.6; IR 2965, 2929, 2848, 2090, 1463, 1256 (lit.1 film); [α]25D +25.6° (c

1.84, CHCl3) (lit.21 [α]20D +26° (c 1.00, CHCl3), lit.20 [α]20

D +25.5° (c 1.00, CHCl3)).

General procedure for the preparation of epimeric 5α-cholestan-3-amines (4)

a. Preparation of 5α-cholestan-3α-amine (4a)

Method A.

H2NN3

Pd/C, hexane

rt, 6 h

To a 250-mL hydrogenation bottle was added an azide 5, 10% Pd/C (4% weight of

azide), and hexane so that the concentration of the azide was ~ 0.13 M. The resulting

suspension was shaken and hydrogenated at 60 psi at rt for 6 h. After sitting overnight,

149

the resulting suspension was filtered; the filtrate was concentrated to give an off-white

solid.

Method B.

H2NN3 reflux, 4h

LiAlH4, THF

A 250mL round-bottomed-flask containing a suspension of LiAlH4 in dry THF was put

in an ice bath. When the temperature reached 0 °C, an azide 5 was added slowly to the

flask, so that its concentration was ~ 0.80 M. The resulting suspension was then refluxed

for 4 hours. After being refluxed, the mixture was diluted with THF so that the volume

doubled, and cooled in an ice bath once again. Water (one-eighth of THF volume),

NaOH (10 M, one-fifth of THF volume), and water (one-eighth of THF volume) were

added successively to the resulting mixture. The solid that formed was filtered; the

filtrate was dried with Na2SO4, filtered, and concentrated to give a white solid.

5α-Cholestan-3α-amine (4a)

Method A. The general procedure for the synthesis of 4 above was followed: Azide 5a

(1.84 g, 4.46 mmol), 10% Pd/C (82.8 mg), and hexane (35 mL) were combined and

treated as above. Amine 4a was obtained as an off-white solid (1.18 g, 3.06 mmol; 68%);

mp 84.6−85.4 °C (lit.11 88−89 °C).

Method B. The general procedure for the synthesis of 4 above was followed: LiAlH4

(320 mg, 8.01 mmol), dry THF (10 mL), and azide 5a (1.00 g, 2.42 mmol) were

combined and treated as above. Amine 4a was obtained as a white solid (755 mg, 1.95

mmol; 80%); 88.4–89.2 °C (lit.11 88−89 °C); 1H NMR (CDCl3) δ 0.60–2.00 (m, 47H),

3.18 (bs, 1 H) (lit.1 300 MHz); 13C NMR (CDCl3) δ 11.4, 12.1, 18.7, 20.8, 22.6, 22.8,

150

23.6, 24.2, 28.0, 28.3, 28.8, 29.3, 32.1, 32.2, 35.6, 35.8, 36.2, 36.37, 36.41, 39.2, 39.5,

40.1, 42.6, 45.8, 54.6, 56.3, 56.6; IR 2930, 2849, 1465, 1380 (lit.1 film); [α]25D +26.4° (c

1.81, CHCl3) (lit.4 [α]28D +28° (c 1.1, CHCl3)).

5α-Cholestan-3β-amine (4b)

Method A. The general procedure for the synthesis of 4 above was followed: azide 5b

(1.38 g, 3.35 mmol), 10% Pd/C (54.3 mg), and hexane (26 mL) were combined and

treated as above. Amine 4b was obtained as an off-white solid (849 mg, 2.19 mmol;

65%); 85.8–86.4 °C (lit.1 104–106 °C).

Method B. The general procedure for the synthesis of 4 above was followed: LiAlH4

(851 mg, 21.3 mmol), dry THF (26 mL), and azide 5b (2.94 g, 7.10 mmol) were

combined and treated as above. Amine 4b was obtained as a white solid (2.36 g, 6.09

mmol; 85.8 %); mp 91.6–92.1 °C (lit.1 104–106 °C); 1H NMR (CDCl3) δ 0.60–2.00 (m,

47H), 2.64 (m, 1 H) (lit.1 300 MHz); 13C NMR (CDCl3) δ 12.1, 12.4, 18.7, 21.2, 22.6,

22.8, 23.8, 24.2, 28.0, 28.3, 28.8, 32.1, 32.6, 35.53, 35.55, 35.8, 36.2, 37.7, 39.4, 39.5,

40.1, 42.6, 45.6, 51.2, 54.5, 56.3, 56.5; IR 2931, 2849, 1465, 1381(lit.1 film); [α]25D

+24.6° (c 1.80, CHCl3) (lit.10 [α]D +29° (c 0.9, CHCl3)).

General procedure for the preparation of 3EUr-z-cholestane (2)

NH

NH

OO

O

H

H

H

H12

34

5 3

+

3

DCM

rt, 24hOCN

OtBu

O 3

H

H2N

To a 50-mL round-bottomed flask containing a solution of 3 in CH2Cl2 was added an

amine 4 slowly and stirred at rt for 24 h. The reaction was concentrated to give colorless

151

oil that was solidified under vacuum. Purification was carried out via flash column

chromatography (hexane:THF:MeOH=1:1:0.04) to give a white solid, which TLC gave a

single spot (Rf = 0.56−0.63), in 74−78% yield.

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(5α-cholestan-3α-

yl)ureido]heptanedioate, 3EUr-α-cholestane (2a)

The general procedure for the synthesis of tri-tert-butyl ester 2 above was followed: 3

(1.15 g, 2.61 mmol), CH2Cl2 (10 mL), and amine 4a (1.01 g, 2.61 mmol) were combined

and treated as above. The tri-tert-butyl ester 2a was obtained in a white solid (2.04 g,

1.69 mmol, 78%); mp 179.9–180.6 °C; 1H NMR (CDCl3) δ 0.60–2.00 (m, 46H), 1.44 (s,

27H), 1.95 (m, 6H), 2.23 (m, 6H), 3.67 (bs, 1H), 4.38–4.42 (m, 2H); 13C NMR (CDCl3) δ

11.4, 12.1, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.6, 28.0, 28.1, 28.3, 28.6, 30.0, 30.7, 32.0,

33.1, 33.5, 35.5, 35.8, 36.0, 36.2, 39.5, 40.1, 40.7, 42.6, 45.4, 54.5, 56.3, 56.5, 56.6, 80.5,

156.2, 173.1; IR 2929, 2864, 1726, 1628, 1551, 1456, 1366, 1247, 1146; [α]27D +15.6° (c

1.41, CHCl3); HRMS: for C50H89N2O7 (M + H)+ calcd 829.6670, found 829.6675. Anal.

Calcd for C50H88N2O7: C, 72.42; H, 10.70; N, 3.38. Found: C, 72.18; H, 10.79; N, 3.38.

X-ray analysis of 3EUr-α-cholestane (2a)

Colorless plates (0.055 x 0.47 x 0.55 mm3) were crystallized from ethanol. The chosen

crystal was mounted on a nylon CryoLoop™ (Hampton Research) with Krytox® Oil

(DuPont) and centered on the goniometer of an Oxford Diffraction Xcalibur™

diffractometer equipped with a Sapphire 2™ CCD. The data collection routine, unit cell

refinement, and data processing were carried out with the program CrysAlis.56 The Laue

symmetry and systematic absences were consistent with the monoclinic space groups P21

and P21/m. As the sample was known to be enantiomerically pure from the synthetic

152

procedure, the chiral space group P21 was chosen. Because there are no heavy atoms, the

absolute configuration could not be determined from the Friedel pairs; the Friedel pairs

were therefore merged for the final refinement. The structure was solved by direct

methods and refined using the SHELXTL NT program.57 The asymmetric unit of the

structure comprises two crystallographically independent cholesterol derivatives and two

ethanol molecules. The final refinement model involved anisotropic displacement

parameters for non-hydrogen atoms and a riding model for all hydrogen atoms. The

program SHELXTL NT was used for molecular graphics generation.57

Table IV.6 Crystal data and structure refinement for 3EUr-α-cholestane (2a) Category Crystal data and structure refinement

Empirical formula C50H88N2O7•CH3CH2OH Formula weight 875.29 Temperature 100(2) K Wavelength 0.71073 Å Crystal system monoclinic Space group P21 Unit cell dimension a = 12.1220(16) Å; b = 12.8097(16); c = 35.589(3) Å;

α = 90°; β = 99.166(9)°; γ = 90° Volume 5455.7(11) Å3 Z 4 Density (calculated) 1.066 Mg/m

3

Absorption coefficient 0.070 mm-1 F(000) 1936 Crystal size 0.550 x 0.465 x 0.055 mm

3

Theta range for data collection 2.81 to 25.07° Index ranges −14 ≤ h ≤ 14, −14 ≤ k ≤ 15, −42 ≤ l ≤ 41 Reflections collected 25907 Independent reflections 10121 [R(int) = 0.0573] Completeness to theta = 25.07° 99.6 % Absorption correction None Refinement method Full-matrix least-squares on F

2

Data / restraints / parameters 10121 / 1 / 1119 Goodness-of-fit on F2 0.967 Final R indices [I>2sigma(I)] R1 = 0.0734, wR2 = 0.1820 R indices (all data) R1 = 0.1180, wR2 = 0.2131 Absolute structure parameter −2.9(17) Largest diff. peak and hole 0.686 and -0.273 e.Å

-3

153

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(5α-cholestan-3β-

yl)ureido]heptanedioate, 3EUr-β-cholestane (2b)

The general procedure for the synthesis of tri-tert-butyl ester 2 above was followed: 3

(1.39 mg, 3.15 mmol), CH2Cl2 (14 mL), and 4b (1.22 mg, 3.16 mmol) were combined

and treated as above. Tri-tert-butyl ester 2b was obtained as a white solid (2.35 g, 1.94

mmol, 74%); mp 187.4–187.8 °C; 1H NMR (CDCl3) δ 0.60–2.0 (m, 46H), 1.43 (s, 27H),

1.93 (m, 6H), 2.23 (m, 6H), 3.87 (sb, 1H), 3.88 (d, 1H), 4.46 (s, 1H); 13C NMR (CDCl3) δ

12.1, 12.3, 18.7, 21.2, 22.6, 22.8, 23.9, 24.2, 28.0, 28.1, 28.3, 28.6, 29.6, 30.0, 30.7, 32.0,

35.4, 35.5, 35.8, 36.2, 36.3, 37.6, 39.5, 40.0, 42.6, 45.1, 49.8, 54.4, 56.3, 56.5, 80.5,

156.2, 173.2; IR 2929, 2864, 1724, 1625, 1558, 1457, 1365, 1308, 1148; [α]27D +9.6° (c

1.49, CHCl3); HRMS: for C50H89N2O7 (M + H)+ calcd 829.6670, found 829.6638. Anal.

Calcd for C50H88N2O7: C, 72.42; H, 10.70; N, 3.38. Found: C, 72.67; H, 10.77; N, 3.35.

General procedure for the preparation of 3CUr-z-cholestane (1)

NH

NH

OO

O 3

HCOOH

rt, 24hNH

NH

OH

O

O 3

To a 50-mL round-bottomed flask containing the tri-tert-butyl ester 2 was added

HCOOH; the flask was warmed in a hot-water bath until a transparent solution appeared.

The resulting solution was stirred overnight at rt. The complete reaction was identified

by the formation of milky solution. The solvent was evaporated to leave a white solid,

which was recrystallized with AcOH/hexane successively to give a white solid.

4-(2-Carboxyethyl)-4-[3-(5α-cholestan-3α-yl)ureido]heptanedioic acid, 3CUr-α-

cholestane (1a)

The general procedure for the synthesis of the triacid 1 above was followed: 2a (1.53 g,

154

1.90 mmol) and HCOOH (30 mL) were combined and treated as above. Triacid 1a was

obtained as a white solid (922 mg, 1.39 mmol; 73%); dec. at 199.0 °C; 1H NMR

(DMSO-d6) δ 0.60–2.00 (m, 51H), 2.09 (m, 6H), 3.67 (bs, 1H), 5.50 (s, 1H), 5.97 (d, 1H),

12.00 (s, 3H); 13C NMR (DMSO-d6) δ 11.6, 12.3, 18.9, 20.8, 22.8, 23.1, 22.8, 23.1, 23.7,

24.2, 26.7, 27.8, 28.2, 28.6, 30.6, 32.2, 33.0, 33.7, 35.4, 35.7, 36.0, 36.1, 39.4, 42.6, 44.0,

54.5, 55.5, 56.2, 56.7, 157.0, 175.0; IR 3373, 1288, 2925, 2865, 1705, 1635, 1559;

[α]27D +19.9° (c 0.87, EtOH); HRMS: for C38H65N2O7 (M + H)+ calcd 661.4792, found

661.4780. Anal. Calcd for C38H64N2O7: C, 69.06; H, 9.76; N, 4.24. Found: C, 69.10; H,

9.80; N, 4.22.

4-(2-Carboxyethyl)-4-[3-(5α-cholestan-3β-yl)ureido]heptanedioic acid, 3CUr-β-

cholestane (1b)

The general procedure for the synthesis of triacid 1 above was followed: 2b (907 mg,

1.09 mmol) and HCOOH (26 mL) were combined and treated as above. Triacid 1b was

obtained as a white solid (560 mg, 0.85 mmol; 78%); dec. at 194.3 °C; 1H NMR (DMSO-

d6) δ 0.60–2.00 (m, 51H), 2.08 (m, 6H), 3.23 (bs, 1H), 5.38 (s, 1H), 5.50 (d, 1H), 12.02

(s, 3H); 13C NMR (DMSO-d6) δ 12.3, 12.4, 19.0, 21.2, 22.8, 23.1, 23.7, 24.2, 27.8, 28.3,

28.6, 28.7, 29.5, 30.5, 32.1, 35.5, 35.7, 36.1, 36.2, 37.7, 42.6, 45.6, 48.9, 54.2, 55.5, 56.2,

56.4, 156.9, 175.0; IR 3405, 2930, 2847, 1704, 1613, 1559, 1295; [α]27D +14.8° (c 0.83,

EtOH); HRMS: for C38H65N2O7 (M + H)+ calcd 661.4792, found 661.4794. Anal. Calcd

for C38H64N2O7: C, 69.06; H, 9.76; N, 4.24. Found: C, 68.85; H, 9.71; N, 4.20.

155

Preparation of 5α-cholestan-3α-ol46 (8a)

HOHO+ DIAD + TFA + PPh3 +

1. dry THF, rt, overnight

2. MeOH, 50 C, overnight

O

O Na

To a 100-mL round bottom flask containing a colorless solution of 10b (4.90 g, 12.4

mmol) in dry THF (24 mL) was added DIAD (2.45 mL, 15.6 mmol). The resulting

solution turned from colorless to orange. Addition was continued with trifluoroacetic

acid (1.20 mL, 15.6 mmol), PPh3 (4.09 g, 15.5 mmol), and sodium benzoate (2.25 g, 15.5

mmol), successively. The resulting colorless solution was stirred at rt overnight. The

solution was concentrated to give an off-white solid. After diluted with MeOH (24 mL),

the resulting solution was heated at 50 °C overnight. The solvent was concentrated; the

residue was partitioned between CH2Cl2 (150 mL) and water (15 mL). The organic layer

was washed once again with water (15 mL) and dried with MgSO4. Concentration of the

solution gave a white solid, which was purified by flash column chromatography in

CH2Cl2 to yield a white solid (3.84 g, 80%); mp 185.2−185.7 °C (lit.46 mp 182−185 °C);

1H NMR (CDCl3) δ 0.60−2.00 (m, 46H), 4.04 (bs, 1H) (lit.46 300 MHz); 13C NMR

(CDCl3) δ 11.2, 12.1, 18.7, 20.8, 22.6, 22.8, 23.8, 24.2, 28.0, 28.3, 28.6, 29.0, 32.0, 32.2,

35.5, 35.8, 35.9, 36.1, 36.2, 39.2, 39.5, 40.1, 42.6, 54.3, 56.2, 56.6, 66.7; IR 3375, 2928,

2848, 1002 (lit.58 bands); [α]27D +25.5° (c 1.78, CHCl3) (lit.59 [α]26.5

D +27° (c 1.92,

CHCl3)).

General preparation of 5α-cholestan-3-yl chloroethanoate (13)

HO

ClO

ODMAP, Et3N, PhHCl

O

Cl+

rt, overnight

156

Chloroacetyl chloride was added dropwise to a 100-mL round bottom flask containing a

solution of a cholestanol (8), Et3N, and DMAP in benzene. The resulting solution was

stirred at rt overnight. The reaction was filtered; the filtrate was concentrated to give the

crude product, which was purified by flash column chromatography to give a white solid,

which gave a single spot on TLC in CH2Cl2:hexane (1:1, Rf = 0.33−0.41), in 57−68%

yield.

5α-Cholestan-3α-yl chloroethanoate (13a)

The general procedure for the synthesis 13 was followed: 8a (3.80 g, 9.78mmol), Et3N

(1.50 mL, 10.7 mmol), DMAP (60.0 mg, 0.49 mmol), benzene (58 mL) and chloroacetyl

chloride (1.80 g, 15.6mmol) were combined and treated as above. The compound above

was obtained as a white solid (2.59 mg, 8.56 mmol, 57%); mp 134.7–135.2 °C; 1H NMR

(CDCl3) δ 0.65–2.00 (m, 46H), 4.07 (s, 2 H), 5.12 (m, 1H); 13C NMR (CDCl3) δ 11.3,

12.1, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.0, 28.0, 28.25, 28.32, 31.9, 32.7, 32.8, 35.5,

35.76, 35.82, 36.2, 39.5, 40.0, 41.4, 42.6, 54.2, 56.3, 56.5, 72.7, 166.8; IR 2932, 2864,

2849, 1754, 1194, 1185; [α]27D +24.5° (c 1.18, CHCl3).

5α-Cholestan-3β-yl chloroethanoate (13b)

The general procedure for the synthesis 13 was followed: 8b (5.00 g, 12.6 mmol), Et3N

(1.90 mL, 13.5 mmol), DMAP (77.3 mg, 0.63 mmol), benzene (94 mL) and chloroacetyl

chloride (1.804 g, 15.6 mmol) were combined and treated as above. The compound

above was obtained as a white solid (3.98 mg, 8.56 mmol, 68%), mp 176.1–176.9 °C (lit.

mp 178−179 °C,45 180−181 °C41); 1H NMR (CDCl3) δ 0.60–2.00 (t, 46H), 4.02 (s, 2H),

4.79 (m, 1H); 13C NMR (CDCl3) δ 12.1,12.2, 18.7, 21.2, 22.6, 22.8, 23.8, 24.0, 27.3,

28.0, 28.2, 28.6, 31.9, 33.8, 35.43, 35.45, 35.8, 36.2, 36.7, 39.5, 40.0, 41.3, 42.6, 44.6,

157

54.2, 56.3, 56.4, 76.1, 166.9; IR 2935, 2917, 2848, 1747, 1205, 1180; [α]24D +11.3° (c

0.36, CHCl3).

General procedure for the preparation of 5α-cholestan-3-yl azidoethanoate (12)

To a 0.20-M solution of 13 in DMF was added slowly NaN3. The mixture was heated at

85−90 °C for 10 h. The resulting mixture was yellow. After it cooled to rt, hexane (as

much as the volume of DMF) and water (one-third volume of DMF) were added. Two

layers formed; the organic layer was separated. The organic layer was washed with satd

NaHCO3 and satd NaCl successively. Each aqueous solution added was the same as the

volume of the water added. The light yellow organic layer was dried and concentrated to

give a light yellow solid. Purification was carried out by flash column chromatography

to give a white solid, which gave a single spot on TLC in hexane (Rf = 0.15−0.21).

5α-Cholestan-3α-yl azidoethanoate (12a)

The general procedure for the synthesis 12 was followed: NaN3 (1.24 g, 18.9 mmol), 13a

(1.76 g, 3.79 mmol), DMF (19 mL) were combined and treated as above. Compound 12a

was obtained as a white solid (846 mg, 1.80 mmol, 47%), mp 114.9–115.6 °C; 1H NMR

(CDCl3) δ 0.65–2.00 (m, 46H), 3.86 (s, 2H), 5.16 (m, 1H); 13C NMR (CDCl3) δ 11.4,

12.1, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.1, 28.0, 28.2, 28.3, 31.9, 32.81, 32.83, 35.5,

35.76, 35.81, 36.2, 39.5, 39.99, 40.04, 42.6, 50.7, 54.2, 56.3, 56.5, 72.6, 167.8; IR 2926,

2865, 2104, 1733, 1218, 1159, 975; [α]27D +29.8° (c 1.14, CHCl3); HRMS: for

C29H49N3O2 (M+) calcd 471.3825, found 471.3812. Anal. Calcd for C29H49N3O2: C,

N3 O

OCl

O

O NaN3, DMF

85−90 °C, 10 h

158

73.84; H, 10.47; N, 8.91. Found: C, 73.89; H, 10.53; N, 8.91.

5α-Cholestan-3β-yl azidoethanoate (12b)

The general procedure for the synthesis 12 was followed: NaN3 (798 mg, 12.2 mmol),

13b (1.85 g, 3.98 mmol), DMF (20 mL) were combined and treated as above. Compound

12b was obtained as a white solid (915 mg, 1.94 mmol, 49%), mp 124.2–124.9 °C; 1H

NMR (CDCl3) δ 0.60–2.00 (m, 46H), 3.83 (s, 2H), 4.81 (m, 1H); 13C NMR (CDCl3) δ

12.3, 12.5, 18.9, 21.4, 22.8, 23.1, 26.1, 24.4, 27.6, 28.2, 28.5, 28.8, 32.2, 34.1, 35.67,

35.68, 36.0, 36.4, 36.9, 39.7, 40.2, 42.8, 44.9, 50.8, 54.4, 56.5, 56.6, 76.0, 168.1; IR

2934, 2849, 2108, 1738, 1218; [α]27D +14.5° (c 1.00, CHCl3); HRMS: for C29H49N3O2

(M+) calcd 471.3825, found 471.3809. Anal. Calcd for C29H49N3O2: C, 73.84; H, 10.47;

N, 8.91. Found: C, 73.90; H, 10.60; N, 8.92.

General procedure for the preparation of 5α-cholestan-3-yl aminoethanoate (11)

A cholestanyl azidoethanoate (12) was added to a hydrogenator vessel containing a

suspension of 10% Pd/C (4% weight of azide 12) in CH2Cl2 so that the concentration of

the ethanoate was ~ 0.11 M. The mixture was shaken and hydrogenated at 60 psi at low

temperature (~ 60 °C) for 4 h. The reaction was cooled to rt and filtered through a pad of

Celite® in vacuo; the filtrate was concentrated to give an off-white solid.

5α-Cholestan-3-yl aminoethanoate (11a)

The general procedure for the synthesis 11 was followed: 12a (736 mg, 1.56 mmol), 10%

Pd/C (29.7 mg), CH2Cl2 (15 mL) were combined and treated as above. Compound 11a

was obtained as a white solid (615 mg, 1.38 mmol, 88%), mp 109.2–110.1 °C; 1H NMR

N3 O

OH2N O

OPd/C, DCM

60 °C, 4h

159

(CDCl3) δ 0.65–2.00 (t, 49H), 3.43 (s, 2H), 5.08 (m, 1H); 13C NMR (CDCl3) δ 11.3, 12.1,

18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.2, 28.0, 28.3, 28.4, 31.9, 32.87, 32.92, 35.5, 35.79,

35.82, 36.2, 39.5, 40.03, 40.05, 42.6, 44.3, 54.2, 56.3, 56.5, 70.9, 173.8; IR 3400, 2931,

2865, 1728, 1206; [α]27D +28.2° (c 0.94, CHCl3); HRMS: for C29H52NO2 (M + H)+ calcd

446.3998, found 446.4001. Anal. Calcd for C29H51NO2: C, 78.15; H, 11.53; N, 3.14.

Found: C, 78.17; H, 11.67; N, 3.12.

5α-Cholestan-3β-yl aminoethanoate (11b)

The general procedure for the synthesis 11 was followed: 12b (2.70 g, 5.72 mmol), 10%

Pd/C (110 mg), CH2Cl2 (50 mL) were combined and treated as above. Compound 11b

was obtained as an off-white solid (1.93 mg, 4.34 mmol, 76%); mp 157.5−158.3 °C (lit.34

178−180 °C); 1H NMR (CDCl3) δ 0.65–2.00 (m, 49H), 3.38 (s, 2H), 4.75 (m, 1H); 13C

NMR (CDCl3) δ 12.0, 12.2, 18.6, 21.2, 22.5, 22.8, 23.8, 24.2, 27.4, 27.97, 28.03, 28.2,

28.6, 31.9, 34.0, 35.4, 35.8, 36.1, 36.7, 39.5, 39.9, 42.5, 44.0, 44.6, 54.2, 56.2, 56.4, 74.4,

173.8; IR 3400, 2933, 2849, 1725, 1220; [α]24D + 12.2° (c 0.43, CHCl3); HRMS: for

C29H52NO2 (M + H)+ calcd 446.3998, found 446.4003. Anal. Calcd for C29H51NO2: C,

78.15; H, 11.53; N, 3.14. Found: C, 78.18; H, 11.57; N, 3.10.

General procedure for the preparation of 3EUr1Es-3α-cholestane (10a−b)

OHN

HN

O

O

3O

ODCMrt, 5 hH2N

O

O

OtBuOCN

O

3

+

An amine 11 was added to a solution containing 3 in CH2Cl2. The solution was stirred at

rt for 5 h. The resulting transparent solution was concentrated to give a white solid.

Flash column chromatography was performed to purify the crude product to give a white

solid, which gave a single spot on TLC (hexane:EtOAc, 2.5:1, Rf = 0.33−0.35) in

160

61−64% yield.

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(5α-cholestan-3α-

yloxycarbonylmethyl)ureido]heptanedioate, 3EUr1Es-α-cholestane (10a)

The general procedure for the synthesis of 10 above was followed: 11a (542 mg, 1.22

mmol), isocyanate 3 (534 mg, 1.21 mmol), CH2Cl2 (6 mL) were combined and treated as

above. Compound 10b was obtained as a white solid (691 mg, 0.78 mmol, 64.0%): mp

82.9–83.3 °C; 1H NMR (CDCl3) δ 0.60–2.00 (m, 79H), 2.24 (m, 6H), 3.94 (dd, 1H), 3.89

(dd, 1H), 4.65 (m, 1H; exchange with D2O), 4.79 (s, 1H; exchange with D2O), 5.08 (m,

1H); 13C NMR (CDCl3) δ 11.3, 12.1, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.1, 28.0, 28.1,

28.2, 28.3, 29.9, 30.6, 31.9, 32.8, 35.5, 35.7, 35.8, 36.2, 39.5, 39.9, 40.0, 42.3, 42.6, 54.1,

56.3, 56.5, 56.7, 71.6, 80.5, 156.1, 170.5, 173.0; IR 2928, 1727, 1365, 1203, 1149, 1100;

[α]27D +6.3° (c 0.80, CHCl3); HRMS: for C52H91N2O9 (M + H)+ calcd 887.6725, found


10.30; N, 3.17.

Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(5α-cholestan-3β-

yloxycarbonylmethyl)ureido]heptanedioate, 3EUr1Es-β-cholestane (10b)

The general procedure for the synthesis of 10 above was followed: 11b (1.09 g, 2.44

mmol), 3 (989 mg, 2.24 mmol), CH2Cl2 (20 mL) were combined and treated as above.

Compound 10b was obtained as a white solid (1.21g, mmol, 61%); mp 195.8–196.2 °C;

1H NMR (CDCl3) δ 0.60–2.00 (m, 79H), 2.20 (m, 6H), 3.84 (d, 2H), 4.62 (t, 1H;

exchanges with D2O), 4.70 (s, 1H; exchanges with D2O), 4.75 (m, 1H); 13C NMR

(CDCl3) δ 11.7, 11.8, 18.3, 20.8, 22.2, 22.4, 23.5, 23.8, 27.0, 27.6, 27.7, 27.8, 28.2, 29.4,

30.1, 31.6, 33.5, 35.0, 35.1, 35.4, 35.8, 36.3, 39.1, 39.6, 41.8, 42.2, 44.2, 53.8, 55.9, 56.0,

161

56.1, 74.4, 80.0, 156.0, 170.4, 172.6; IR 2927, 1726, 1365, 1147, 1199, 1097; [α]27D

+11.6° (c 1.50, CHCl3); HRMS: for C52H91N2O9 (M + H)+ calcd 887.6725, found


10.31; N, 3.16.

General procedure for the preparation of 3CUr1Es-cholestane (9)

OHN

HN

O

O

3O

O

HOHN

HN

O

O

3O

OHCOOHrt, 9 h

To a 50-mL round-bottomed flask containing a tri-tert-butyl ester 10 was added HCOOH;

the flask was warmed in a hot-water bath until a transparent solution appeared. The

resulting solution was removed from the hot-water bath and stirred at rt for 5 h. The

resulting reaction was concentrated to dryness to leave a white solid. Recrystallization in

HOAc−hexane was carried out to purify the crude white solid in 50−57% yield.

4-(2-Carboxyethyl)-4-[3-(5α-cholestan-3α-yloxycarbonylmethyl)ureido]heptanedioic

acid, 3CUr1Es-α-cholestane (9a)

The general procedure for the synthesis of 9 above was followed: HCOOH (6 mL) and

10a (891 mg, 0.472 mmol) were combined and treated as above. The triacid 9a was

obtained as a white solid (174 mg, 0.242 mmol, 51%); mp 192.5–192.9 °C; 1H NMR

(DMSO-d6) δ 0.60–2.10 (m, 53H), 3.66 (d, 2H), 4.87 (s, 1H), 5.86 (s, 1H), 5.91 (t, 1H),

11.97 (bs, 1H); 13C NMR (DMSO-d6) δ 11.7, 12.5, 19.2, 21.0, 23.1, 23.3, 23.9, 24.4,

26.2, 28.0, 28.5, 28.6, 28.8, 30.7, 32.1, 33.0, 33.1, 35.6, 35.9, 36.0, 36.3, 39.6, 42.1, 42.8,

54.2, 55.9, 56.4, 56.6, 70.5, 157.5, 171.2, 175.1; IR 3409, 2933, 1751, 1702, 1559, 1204,

1158; [α]27D +15.7° (c 0.65, EtOH); HRMS: for C40H67N2O9 (M + H)+ calcd 719.4847,


162

66.96; H, 9.37; N, 3.89.

4-(2-Carboxyethyl)-4-[3-(5α-cholestan-3β-yloxycarbonylmethyl)ureido]heptanedioic

acid, 3CUr1Es-β-cholestane (9b)

The general procedure for the synthesis of 9 above was followed: HCOOH (10 mL) and

10b (891.4 mg, 1.00 mmol) were combined and treated as above. The triacid 9b was

obtained as a white solid (409 mg, 0.569 mmol, 57%); dec at 194.2 °C; 1H NMR

(DMSO-d6) δ 0.50–2.10 (m, 59H), 3.62 (d, 2H), 4.54 (h, 1H), 5.84 (s, 1H), 5.91 (t, 1H),

11.96 (bs, 1H); 13C NMR (DMSO-d6) δ 12.5, 12.6, 19.2, 21.4, 23.0, 23.3, 23.9, 24.5,

27.7, 28.0, 28.4, 28.7, 30.6, 32.2, 34.3, 35.6, 35.9, 36.3, 36.8, 42.1, 42.8, 44.6, 54.2, 55.9,

56.4, 56.6, 74.1, 157.4, 171.2, 175.1; IR 3410, 2933, 1728, 1704, 1564, 1221, 1187;

[α]27D +9.8° (c 0.58, EtOH); HRMS: for C40H67N2O9 (M + H)+ calcd 719.4847, found

719.4851. Anal. Calcd for C40H66N2O9: C, 66.82; H, 9.25; N, 3.90. Found: C, 66,72; H,

9.41; N, 3.87.

References for Chapter IV 1 Casimiro-Garcia, A.; De Clercq, E.; Pannecouque, C.; Witvrouw, M.; Stup, T. L.;

Turpin, J. A.; Buckheit, R. W. Synthesis and anti-HIV activity of cosalane analogues incorporating nitrogen in the linker chain. Bioorg. Med. Chem. 2000, 8, 191-200.

2 Krieg, R.; Wyrwa, R.; Mollmann, U.; Gorls, H.; Schonecker, B. Novel (N-

ferrocenylmethyl)amines and (N-ferrocenylmethylen)imines derived from vicinal steroid amino alcohols and amines: synthesis, molecular structure, and biological activity. Steroids 1998, 63, 531-541.

3 Sakai, N.; Matile, S. Transmembrane ion transport mediated by amphiphilic

polyamine dendrimers. Tetrahedron Lett. 1997, 38, 2613-2616.

163

4 Smith, H. E.; Taylor, J., Clinton A.; McDonagh, A. F.; Chen, F.-M. Optically active amines. 30. Application of the salicylidenimino chirality rule to aliphatic and alicyclic amines. J. Org. Chem. 1982, 47, 2525-2531.

5 Hojo, K.; Kobayashi, S.; Soai, K.; Ikeda, S.; Mukaiyama, T. New synthetic

reactions based on 1-methyl-2-fluoropyridinium salts. Stereospecific preparation of primary amines from alcohols. Chem. Lett. 1977, 635-636.

6 Bose, A. K.; Kistner, J. F.; Farber, L. A convenient synthesis of axial amines. J.

Org. Chem. 1962, 2925-2927. 7 Hertler, W. R.; Corey, E. J. A novel preparation of isonitrile. J. Org. Chem. 1958,

23, 1221-1222. 8 Marples, B. A. Steroids. Part III. Some steroidal ketimines and amines. J. Chem.

Soc 1968, 3015-3017. 9 Shoppee, C. W.; Lack, R. E.; Ram, P. Steroids and Walden Inversion. Part LVIII.

The deamination of 3β- and 3α-amino-5α-cholestane. J. Chem. Soc. C 1966, 1018-1023.

10 Shoppee, C. W.; Richards, H. C.; Summers, G. H. R. Steroids and Walden

inversion. Part XXX. The epimeric coprostan-3-ylamines and cholest-4-en-3-ylamines. 1956, 1649-1655.

11 Labler, L.; Cerny, V.; Sorm, F. Steroid. XII. Determination of the configuration

of 3-dimethylamino derivatives of cholestane. Collect. Czech. Chem. 1954, 19, 1249-1257.

12 Dodgson, D., P.; Haworth, R. D. Some basic steroid derivatives. J. Chem. Soc

1952, 67-70. 13 Ikenaga, T.; Matsushita, K.; Shinozawa, J.; Yada, S.; Takagi, Y. The effects of

added ammonium chloride in the reductive amination of some carbonyl compounds over Ru and Pd catalysts. Tetrahedron 2005, 61, 2105-2109.

14 Pinkus, J. L.; Cohen, T. Di-5α-cholestan-3α-ylamine, a diaxial bis steroidal

amine. J. Org. Chem. 1968, 33, 3538-3541. 15 Pinkus, J. L.; Pinkus, G.; Cohen, T. A convenient stereospecific synthesis of axial

amines in some steroidal, decalyl, and cyclohexyl systems. J. Org. Chem. 1962, 27, 4356-4360.

16 Papeo, G.; Posteri, H.; Vianello, P.; Varasi, M. Nicotinoyl azide (NCA)-mediated

Mitsunobu reaction: an expedient one-pot transformation of alcohols into azides. Synthesis 2004, 2886-2892.

164

17 Saito, A.; Saito, K.; Tanaka, A.; Oritani, T. An efficient method for converting

alcohols to azides with 2,4,4,6-tetrabromo-2,5-cyclohexadienone/PPh3/Zn(N3)2

.2Py. Tetrahedron Lett. 1997, 38, 3955-3958. 18 Lal, B.; Pramanik, B. N.; Manhas, M. S.; Bose, A. K. Diphenylphosphoryl azide.

A novel reagent for the stereospecific synthesis of azides from alcohols. Tetrahedron Lett. 1977, 23, 1977-1980.

19 Davis, A. P.; Dresen, S.; Lawless, L. J. Mitsunobu reactions with methanesulfonic

acid; the replacement of equatiorial hydroxyl droups by axide with net retention of configuration. Tetrahedron Lett. 1997, 38, 4305-4308.

20 Loibner, H.; Zbiral, E. Reactions using triphenylphosphane/azodicarboxylate. 2.

Reactions with organophosphorus compounds. XLII. Nucleophilic substitution reactions of hydroxysteroids using triphenylphosphane/diethylazodicarboxylate. Helv. Chim. Acta 1977, 60, 417-425.

21 Djerassi, C.; Moscowitz, A.; Ponsold, K.; Steiner, G. Optical rotary dispersion

studies. CIX. An octant rule for the azide chromophore. J. Am. Chem. Soc. 1967, 89, 346-352.

22 Shimizu, T.; Ohzeki, T.; Hiramoto, K.; Hori, N.; Nakata, T.

Chloromethanesulfonate as an efficient leaving group: rearrangement of the carbon−carbon bond and conversion of alcohols into azides and nitriles. Synthesis 1999, 8, 1373-1385.

23 Henbest, H. B.; Jackson, W. R. The use of aprotic solvents for nucleophilic

substitution reaction at C(3) and C(17) in steroids. 1962, 954-959. 24 Shimizu, T.; Hiranuma, S.; Nakata, T. Efficient method for inversion of

secondary alcohols by reaction of chloromethanesulfonates with cesium acetate. Tetrahedron Lett. 1996, 37, 6145-6148.

25 Erker, G.; Mollenkopf, C. Group 4 metallocene complexes bearing cholestanol-

derived substituents at the Cp-rings−their synthesis and use in propene polymerization catalysis. J. Organometallic Chem. 1994, 483, 173-181.

26 Tanaka, A.; Oritani, T. A mild and efficient method for converting alcohols and

tetrahydropyranyl ethers to bromides with inverstion of configuration. Tetrahedron Lett. 1997, 38, 1955-1956.

27 Tanaka, A.; Oritani, T. Direct transformation of sterically less hindered silyl

ethers to the corresponding bromides with inversion of configuration. Tetrahedron Lett. 1997, 38, 7223-7224.

165

28 Manna, S.; Falck, J. R.; Mioskowski, C. A convenient preparation of alkyl halides and cyanides from alcohols by modification of the Mitsunobu procedure. Synth. Commun. 1985, 15, 663-668.

29 Levy, D.; Stevenson, R. The action of triphenylphosphine dibromide on sterol and

bile acid derivatives. J. Org. Chem. 1965, 30, 3469-3472. 30 Crosignani, S.; Nadal, B.; Li, Z.; Linclau, B. A novel stereoselective one-pot

converstion of alcohols into alkyl halides mediated by N,N'-diisopropylcarbodiimide. Chem. Commun. 2003, 260-261.

31 Li, Z.; Crosignani, S.; Linclau, B. A mild, phosphine-free method for the

conversion of alcohols into halides (Cl, Br, I) via the corresponding O-alkyl isoureas. Tetrahedron Lett. 2003, 44, 8143-8147.

32 Nickon, A. Chemical shifts of axial and equatorial α-protons in the N.M.R of

steroidal α-haloketones. J. Am. Chem. Soc. 1963, 85, 2185-2186. 33 Loibner, H.; Zbiral, E. Reactions with organophosphorus compounds. XLI. New

synthetic aspects of the triphenylphosphine-diethyl azodicarboxylate-hydroxy compound system. Helv. Chim. Acta 1976, 59, 2100-2113.

34 Matsumoto, Y.; Shirai, M.; Minagawa, M. Cholestanol esters of amino acid. Bull.

Chem. Soc. Jpn. 1967, 40, 1650-1655. 35 Lee, M. H.; Yoo, C. L.; Lee, J. S.; Cho, I.-S.; Kim, B. H.; Cha, G. S.; Nam, H.

Tweezer-type neutral carrier-based calcium-selective membrane electrode with drastically reduced anionic interference. Anal. Chem. 2002, 74, 2603-2607.

36 Ilankumaran, P.; Kisanga, P.; Verkade, J. G. Facile synthesis of a chiral auxiliary

bearing the isocyanide group. Heteroat. Chem. 2001, 12, 561-562. 37 Achatz, O.; Grandl, A.; Wanner, K. T. Asymmetric synthesis employing a chiral

5-methoxy-1,4-oxazin-2-one derivative. Preparation of enantiomerically pure a -quaternary a -amino acids. Eur. J. Org. Chem. 1999, 8, 1967-1978.

38 Solladie-Cavallo, A.; Quazzotti, S. An efficient synthesis of (+)-8-phenylmenthyl

isocyanoacetate. Tetrahedron: Asymmetry 1992, 3, 39-42. 39 Genet, J. P.; Kopola, N.; Juge, S.; Ruiz-Montes, J.; Antunes, O. A. C.; Tanier, S.

Double asymmetric synthesis: palladium chiral complexes in the alkylation of chiral Schiff bases derived from glycine. Tetrahedron Lett. 1990, 31, 3133-3136.

40 Venuti, M. C.; Alvarez, R.; Bruno, J. J.; Strosberg, A. M.; Gu, L.; Chiang, H. S.;

Massey, I. J.; Chu, N.; Fried, J. H. Inhibitors of cyclic AMP phosphodiesterase. 4. Synthesis and evaluation of potential prodrugs of lixazinone (N-cyclohexyl-N-

166

methyl-4-[(1,2,3,5-tetrahydro-2-oxoimidazo[2,1-b]quinazolin-7-yl)oxy]butyramide, RS-82856). J. Med. Chem. 1988, 31, 2145-2152.

41 Topala, C.; Meltzer, V.; Pincu, E.; Draghici, C. Synthesis of cholestero

derivatives. III. Diphenyl-thioether alkanoates. Rev. Roum. Chim. 2002, 47, 539-542.

42 Murillo, O.; Watanabe, S.; Nakano, A.; Gokel, G. W. Synthetic models for

transmembrane channels: structural vatiations that alter cation flux. J. Am. Chem. Soc. 1995, 117, 7665-7679.

43 Tsunoda, T.; Yamamiya, Y.; Kawamura, Y.; Ito, S. Mitsunobu acylation of

sterically congested secondary alcohols by N,N,N',N'-tetramethylazodicarboxamide-tributylphosphine reagents. Tetrahedron Lett. 1995, 35, 14.

44 Gokel, G. W.; Hernandez, J. C.; Viscariello, K. A. A.; Campana, C. F.;

Echegoyen, L.; Fronczek, F. R.; Gandour, R. D.; Morgan, C. R.; Trafton, J. E.; Miller, S. R.; Minganti, C.; Eiband, D.; Schultz, R. A.; Tamminen, M. Steroidal lariat ethers: a new class of macrocylces and the crystal structure of N-(cholesteryloxycarbonyl)aza-15-crown-5. J. Org. Chem. 1987, 52, 2963-2968.

45 Ellis, G. W.; Gardner, J. A. β-Cholestanol, some of its derivatives and oxidation

products. Biochem. J. 1918, 12, 72-80. 46 Varasi, M.; Walker, K. A. M.; Maddox, M. L. A revised mechanism for the

Mitsunobu reaction. J. Org. Chem. 1987, 52, 4235-4238. 47 Jordan, A. M.; Osborn, H. M. I.; Stafford, P. M.; Tzortzis, G.; Rastall, R. A.

Synthesis of unsaturated aminopyranosides as possible transition state mimics for glycosidases. J. Carbohydr. Chem. 2003, 22, 705-717.

48 Balo, C.; Blanco, J. M.; Fernandez, F.; Lens, E.; Lopez, C. Synthesis of novel

carbocyclic nucleosides with a cyclopentenyl ring: homocarbovir and analogs. Tetrahedron 1998, 54, 2833-2842.

49 Balo, C.; Fernandez, F.; Lens, E.; Lopez, C. Novel carbocyclic nucleoside

containing a cyclopentyl ring and uracil. Chem. Pharm. Bull. 1998, 46, 687-689. 50 Appeldoorn, C. C. M.; Joosten, J. A. F.; Ait el Maate, F.; Dobrindt, U.; Hacker, J.;

Liskamp, R. M. J.; Khan, A. S.; Pieters, R. J. Novel multivalent mannose compounds and their inhibition of the adhesion of type 1 fimbriated uropathogenic E. coli. Tetrahedron: Asymmetry 2005, 16, 361-372.

167

51 Hayes, W.; Osborn, H. M. I.; Osborne, S. D.; Rastall, R. A.; Romagnoli, B. One-pot synthesis of multivalent arrays of mannose mono- and disaccharides. Tetrahedron 2003, 59, 7983-7996.

52 Roy, R.; Kim, J. M. Cu(II)-Self-assembling bipyridyl-glycoclusters and

dendrimers bearing the Tn-antigen cancer marker: syntheses and lectin binding properties. Tetrahedron 2003, 59, 3881-3893.

53 Corbell, J. B.; Lundquist, J. J.; Toone, E. J. A comparison of biological and

calorimetric analyses of multivalent glycodendrimer ligands for concanavalin A. Tetrahedron: Asymmetry 2000, 11, 95-111.

54 Arimoto, M.; Yamaguchi, H.; Fujita, E.; Nagao, Y.; Ochiai, M. Diazidation of

allylsilanes with a combination of iodosylbenzene and trimethylsilyl azide, and synthesis of allyl azides. Chem. Pharm. Bull. 1989, 37, 3221-3224.

55 Newkome, G. R.; Weis, C. D.; Childs, B. J. Syntheses of 1→3 branched


56 CrysAlis v1. 171. Oxford Diffraction Wroclaw, Poland, 2004. 57 Sheldrick, G. M. SHELXTL NT ver. 6.12; Bruker Analytical X-ray Systems, Inc.:

Madison, WI, 2001. 58 Joris, L.; Schleyer, P. v. R.; Osawa, E. Infrared spectral consequences of

conformational heterogeneity in saturated alcohols. Tetrahedron 1968, 24, 4759-4777.

59 Linstead, R. P. Preparation of cholestanol glucosides with all four possible

configurations of the glucoside link. J. Am. Chem. Soc. 1940, 62, 1766-1770.

168

Chapter V Antimicrobial Activity of Various Tri-headed Amphiphiles

V.1 Introduction to Biological Assays

As several homologous tri-carboxylato amphiphiles had been synthesized, we

aimed to fulfill our next goal, which was to explore the structure-activity relationship of

these amphiphiles. However, our laboratory was not equipped to do any biological

assays. In doing so, we performed the biological assays in collaboration with the

Department of Biological Sciences, Virginia Tech, under Dr. Joseph O. Falkinham’s

supervision.

V.1.1 Measurement of Antimicrobial Activity

In these assays, the antimicrobial activity is measured as MIC (minimum

inhibitory concentration), defined as the lowest minimum concentration of a drug, an

amphiphile in this case, required to inhibit the growth of a microorganism. MICs of

amphiphiles dissolved in aqueous triethanolamine (see Section V.2) are measured by

broth microdilution in 96-well microtiter plates,1 where the concentration of an individual

amphiphile is varied through a series of two-fold dilutions. A suspension of

microorganisms is added to each well of the microtiter plates. These assays require

tedious attention to details; the suspension of the organisms must not be contaminated

and any spattering to any other well should be avoided. After the plates are incubated at

a specific temperature for 4−7 days, the plates are screened visually for any inhibition,

indicated by turbidity, compared to the positive (drug-free) control of growth; this

positive control only contains broth and microorganism, where microorganisms should

grow. Theoretically, growth of microorganisms causes the well to look turbid, on the

other hand, transparent wells means no growth of microorganisms or complete inhibition.

169

It gets more complicated when turbidity with decreasing intensity is observed on all wells

for a specific amphiphile. In this case, we define the result as incomplete inhibition. As

these assays are designed by varying the amphiphile concentration in a series of two-fold

dilutions, we have no ability to determine our reading (MIC interpretation) as the exact

MICs. With this limitation, the experimental error in reading the MICs is within ±1 well

of the recorded wells.

The homologous amphiphile series are screened for their general antimicrobial

activity against a broad spectrum of microorganisms. These included Gram-negative

bacteria (Escherichia coli and Klebsiella pneumoniae), Gram-positive bacteria

(Micrococcus luteus, Lactobacillus plantarum, Staphylococcus aureus, and methicillin-

resistant Staphylococcus aureus (MRSA)), Mycobacterium smegmatis, yeasts

(Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans), and

filamentous fungus (Aspergillus niger).

V.1.2 Goals

For this study, as we have no previous knowledge whether the amphiphiles

display any antimicrobial activity, firstly, we intend to find a range of concentration at

which the amphiphiles are active against a broad spectrum of microorganisms. Secondly,

we identify the most active amphiphile in the homologous series. And thirdly, as we

have designed several homologous series having similar hydrophobicities, but different

tail topology, we will explore if the biological activity is affected by hydrophobicity or

the tail topology.

170

V.2 Recent Studies of Solubility of One-tailed Tri-carboxylato Amphiphiles

As it was initially investigated by a group member, André Williams,2 the 3CAmn

homologous series was dissolved in phosphate buffer (pH = 7.2). Comparison of the

resulting solubilities of the 3CAmn series to those of long chain fatty acids (pH = 7.4)

reveals that the presence of multiple headgroups did improve the solubilities. The

solubilities of 3CAm13, 3CAm15, and 3CAm17 were 6900, 3400 and 1700 μM,

respectively; the solubility of the corresponding long chain fatty acids3⎯myristic (n =

13), palmitic (n = 15), and stearic (n = 17) acids⎯ are 20−30, ~1, and <<1 μM,

respectively. However, as the chains become longer, 3CAm19 and 3CAm21, the

solubilities fell to 140 and 79 μM, respectively. Because of these low solubilities, a

different aqueous solution was considered.2

NH

COOH

COOH

COOH

O

n-1

3CAmn

OH

O

n-1

long-chain fatty acid

The choice of the counterion followed from a study4 of N-lauroyl-L-glutamate in

water; the triethanolammonium salt dissolved to a much greater concentration than did

the potassium salt. Another group member, Richard Macri, explored different conditions

to achieve the maximum solubility of the amphiphile in aqueous triethanolamine.

We initiated this biological study by solubilizing 25 mg of 3CAm13 in 2 mL of

5% (w/v) aqueous triethanolamine. The molar ratio of 3CAm13:triethanolamine was

1:12. Based on this result, we followed the procedure, where an individual amphiphile

(25 mg) was dissolved in 5% (w/v) aqueous triethanolamine solution (2 mL).

171

We successfully dissolved the amphiphiles into aqueous triethanolamine

solutions. These aqueous solutions of the single- and two-tailed amphiphiles (3CUrn,

3CUr(n)2, and 3CUr1(n)2) ranged from 19,500 to 25,700 µM depending on the formula

weight of the homolog, while those of the 3CUr-z-cholestane, and 3CUr1Es-z-

cholestane were 18,900 and 17,400 μM, respectively. We did not pursue the maximum

solubilities of these amphiphiles in the aqueous triethanolamine solution because any

compound with an MIC at this concentration is not worth developing as a lead. The

molar ratio of the one- and two-tailed amphiphiles:triethanolamine ranged from 1:13 to

1:17 depending on the formula weight of the amphiphile, while those of 3CUr-z-

cholestane:triethanolamine and 3CUr1Es-z-cholestane:triethanolamine were 1:17 and

1:19, respectively.

V.3 Results of Biological Assays

Table V.1 presents the MICs for all the amphiphiles. The MICs are recorded as

complete and incomplete inhibitions. As indicated, incomplete inhibitions are observed

in most cases. The MICs are presented in mg/L, following the standard representation of

MICs. However, the MICs will be discussed in term of μM as the formula weights of the

amphiphiles in a series differ by 28 amu between adjacent homologs.

172

Scheme V.1 Structures of dendritic tri-carboxylato amphiphiles

FG NH

COOH

COOH

COOH

O

+ NOH

33 FG N

HCOO

COO

COO

O

HNOH

3

HNOH

3

HNOH

3

3CUrn, FG = −NHR, R = CnH2n+1, n = 14, 16, 18, 20, 223CUr(n)2, FG = −NR2, R = CnH2n+1, n = 7−113CUr1(n)2, FG = −CHNR2, R = CnH2n+1, n = 7−12

NH O

HN

O3CUr-z-cholestane , FG = 3CUr1Es-z-cholestane , FG =

The five series of dendritic tri-carboxylato amphiphiles did not display a uniform

pattern of activity against the broad spectrum of microorganisms tested, but displayed

amphiphile-series-, species-, chain-length-, or epimer-specific patterns. Although the

MIC values obtained were not in the range of 1 mg/L or lower (e.g., ≤ 1 µM), some MICs

were below 10 mg/L (Table V.1).

In general, L. plantarum, S. aureus, M. luteus and A. niger were simply not

inhibited to any degree by any of the amphiphiles (Table V.1). Lack of antimicrobial

activity against L. plantarum is advantageous because lactobacilli, members of the skin

microflora (e.g., vagina), provide some protection to acquisition of infections (e.g.,

sexually transmitted pathogens).5 Some exceptions are observed for several

microorganisms. MRSA is not inhibited by the one- and two-tailed series, however it is

by the amphiphiles with cholestane moieties. None of the yeasts are inhibited to any

degree by the amphiphiles with cholestane moieties. K. pneumoniae also display an

exception; it is weakly inhibited only by the 3CUrn series.

173

Table V.1 MICs of tri-carboxylato amphiphiles against bacteria, yeasts, and a filamentous fungus MIC (mg/L) Amphiphile E

coli K. pneumoniae

L. plantarum

S. aureus

MRSA M. luteus

M. smegmatis

S. cerevisiae

C. albicans

C. neoformans

A. niger

3CUr14b 140 140a 3100a 3100a 3100a >6300 280a 280 780a 780a 3100 3CUr16b 71 140a 3100a 390a 3100a >6300 71a 280 24a 24a 1600 3CUr18b 71 140a 780a 780a 390a >6300 18a 71 780a 49a 780 3CUr20b 36 140a 780a 780a 780a >6300 36a 71 1600a 1600a 390 3CUr22b 71 280a 560a 780 780a >6300 71a 18 1600a 3100a 3100 3CUr(7)2 1600a >560 3100a >6300 1600a 3100a 1600a 390a 780a 570a 390a 3CUr(8) 2 390a >560 780a >6300 1600a 1600a 780a 200a 98a 280a 200a 3CUr(9) 2 780a >560 6300a >6300 6300a 6300a 780a 390a >6300 140a 3100a 3CUr(10) 2 1600a >560 6300a >6300 6300a 6300a 390a 1600a >6300 36a 6300a 3CUr(11) 2 1600a >560 6300a >6300 6300a 6300a 390a 3100a >6300 >560 6300a 3CUr1(7) 2 1600a >6300 3100a >6300 >6300 >6300 1600a 3100a 1600a 560a 3100 3CUr1(8) 2 780a >6300 3100a >6300 >6300 >6300 780a 1600a 390a 140a 3100 3CUr1(9) 2 200a >6300 3100a >6300 >6300 >6300 200a 1600a >6300 36a 1600 3CUr1(10) 2 1600a >6300 3100a >6300 >6300 >6300 200a 1600a >6300 8.9a 6300 3CUr1(11) 2 1600 >6300 6300a >6300 >6300 >6300 390a 780a >6300 4.4a 6300 3CUr1(12) 2 1600 >6300 6300a >6300 >6300 >6300 780a 390a >6300 4.4 a 6300 A 140 560 1600a 6300a 200a 3100 390a >6300a >6300a >6300a 1600 B 140 560 1600a 390a 18a 780 390a >6300a >6300a >6300a 1600 C 560 560 3100a 6300a 200a 6300 390a >6300a >6300a >6300a 6300 D c 560 3100a 160a 780a 6300 390a >6300a >6300a >6300a 6300 aIncomplete inhibition bMIC data from reference 2 cnot measured

A, 3CUr-α-cholestane; B, 3CUr-β-cholestane; C, 3CUr1Es-α-cholestane; D, 3CUr1Es-β-cholestane

174

V.3.1 Range of Concentration of Active Amphiphiles

In general, the 3CUrn series are more active than the rest of the series (3CUr(n)2,

3CUr1(n)2, 3CUr-z-cholestane and 3CUr1Es-z-cholestane). The 3CUrn homologs are

active against a Gram-negative bacterium E. coli and M. smegmatis within 62−290 μM

and 33−580 μM, respectively. The former is observed for complete inhibition, while the

latter is for incomplete inhibition. Against the three yeasts, the concentration of the

3CUrn homologs vary; 30−580, 47−2700, and 47−5200 μM for S. cerevisiae, C.

albicans, and C. neoformans, respectively. Complete inhibitions are observed against S.

cerevisiae, but not observed against C. albicans and C. neoformans. For the rest of the

microorganisms, the 3CUrn homologs are active at concentrations above 250 μM.

Based on the MICs reported for the two-tailed series, the only microorganism that

attracts our attention in these series is C. neoformans. These results showed that the two-

tailed series are very specific against a particular microorganism, i.e., C. neoformans.

The range of the concentration of the 3CUr(n)2 and 3CUr1(n)2 series are 62−1200 and

6.9−1100 μM, respectively. The wide ranges of concentration are observed, especially

in the 3CUr1(n)2.series To us, this can simply mean that particular homologs can be

much more active than other homologs. For the rest of the microorganisms, the

3CUr(n)2 and 3CUr1(n)2 homologs are active at concentrations above 190 μM.

Compared to the one- and two-tailed series, the two last series, 3CUr-z-

cholestane and 3CUr1Es-z-cholestane, are not as active against the microorganisms

tested, with an exception to MRSA. All homologs but 3CUr1Es-β-cholestane are more

active than any homolog in the first three series. The active concentration ranges of the

3CUr-z-cholestane and 3CUr1Es-z-cholestane series are 27−300 and 270−1100 μM,

175

respectively. It is apparent that 3CUr-z-cholestane displays more activity than the

3CUr1Es-z-cholestane; the more active amphiphile in the epimeric pair of 3CUr-z-

cholestane is tenfold more potent than that of the 3CUr1Es-z-cholestane against MRSA.

For the rest of the microorganisms, the active concentrations of all homologs within these

series are above 210 μM.

V.3.2 The Most Active Amphiphiles within Each Homologous Series

From Table V.1, the most active amphiphile within a homologous series can be

identified. Plots of log MIC vs chain length in the 3CUrn series (Figure V.1) clearly

show parabolic figures, which simply means that cut-off effects are reached against E.

coli, M. smegmatis, C. albicans, and C. neoformans. Amphiphiles 3CUr20 and 3CUr18

are the most active against E. coli and M. smegmatis, respectively (Figure V.1, left), and

3CUr16 is the most active against both C. albicans, and C. neoformans (Figure V.1,

Figure V.1 Relationship between log MIC of the 3CUrn series vs chain length for E. coli, M. smegmatis (left) and C. albicans, C. neoformans, and S. cerevisiae (right). Error bars (not shown for clarity) are ± 0.3. Lines connecting points are eye guides

-4.8

-4.3

-3.8

-3.3

12 14 16 18 20 22

chain length

log

[MIC

] (M

)

E. coli M. smegmatis

-5-4.5

-4-3.5

-3-2.5

-2

12 14 16 18 20 22chain length

log

[MIC

] (M

)

C. albican C. neoformansS.cerevisiae

176

right). Against S. cerevisiae, the amphiphiles with longer hydrocarbon chain display

better activity (Figure V.1, right). Within this series, the cut-off effect has not yet been

reached.

In the two-tailed series against C. neoformans, plots of log MIC vs chain length display

two different patterns (Figure V.2). A cut-off effect has been reached in the 3CUr(n)2

series, but not in the 3CUr1(n)2 series (Figure V.2, left). Amphiphile 3CUr(10)2 appears

to be the most active amphiphile. Within the chain length tested, the longest homolog of

3CUr1(n)2 is the most active amphiphile. Amphiphile 3CUr1(12)2 is even more active

than the most active homolog in the 3CUrn series; 3CUr1(12)2 is sevenfold more active

than 3CUr16. An ideal cut-off effect is shown on these two-tailed series against C.

albicans (Figure V.2, right), where the activity of the amphiphiles increases up to a point,

then the activity ceased within the highest concentrated tested.

Figure V.2 Relationship between log MIC of the 3CUr(n)2 and 3CUr1(n)2 series vs chain length for C. neoformans (left) and C. albicans (right). Error bars (not shown for clarity) are ± 0.3. Lines connecting points are eye guides

-5.5

-5

-4.5

-4

-3.5

-3

-2.5

12 14 16 18 20 22 24

chain length

log

[MIC

] (M

)

3CUr(n)2 3CUr1(n)2

-4

-3.5

-3

-2.5

-2

-1.5

12 14 16 18 20 22 24

chain length

log

[MIC

] (M

)

3CUr(n)2 3CUr1(n)2

177

As the last two series contains a pair of epimeric compounds in each series, there

is no cut-off effect for this discussion. However, a particular epimer is found to be more

active than the other. Amphiphiles 3CUr-β-cholestane and 3CUr1Es-α-cholestane are

more active against MRSA compared to their epimeric pairs.

V.3.3 Various Specificities Patterns of Antimicrobial Activity

V.3.3.1 One- and Two-tailed Amphiphiles

C. neoformans was unique among the microorganisms tested by virtue of its

susceptibility to members of both series of two-tailed amphiphiles and the one-tailed tri-

carboxylato dendritic amphiphiles (Table V.1). M. smegmatis, E. coli, K. pneumoniae,

and S. cerevisiae were distinguished from the other microorganisms by their

susceptibility to all five members of the 3CUrn series tested. (Although the three

bacteria have an outer membrane, S. cerevisiae and the remaining microorganisms lack

an outer membrane, so that is apparently not the common structure responsible for

susceptibility.)

These results with long chains contrasted with those studies6,7 that have shown

that short chain free fatty acids (C8–C12) exhibit highest antibiotic activity against a

variety of mycobacteria, including M. smegmatis. C. neoformans was unique by its

susceptibility to all five members of the 3CUr1(n)2 series (Table V.1). With the

exception of C. neoformans (e.g., 3CUr(10)2), the panel of microorganisms, including

the other pathogenic yeast C. albicans, were resistant to the 3CUr(n)2 series (Table V.1).

Amphiphile-series specificity is shown when a particular class of

amphiphile⎯3CUrn, 3CUr(n)2 or 3CUr1(n)2⎯is specifically active against a tested

microorganism. Amphiphile-series-specific antimicrobial activities were displayed by

178

the 3CUrn series against E. coli, K. pneumoniae, M. smegmatis, and Saccharomyces

cerevisiae (Table V.1). Members of the 3CUrn series were significantly more active

against those microorganisms than the corresponding members of the 3CUr(n)2 and

3CUr1(n)2 series. The one exception to that statement was activity against S. cerevisiae

by 3CUr(8)2 (MIC = 380 µM) and 3CUr16 (MIC = 550 µM); 3CUr(8)2 showed

incomplete inhibition but 3CUr16 showed complete inhibition.

Species specificity is displayed when a particular microbial species is uniquely

susceptible to a particular amphiphile. The 3CUrn homologs are active against the two

Gram-negative bacteria, a mycobacterium, and S. cerevisiae; they are not active against

Gram-positive bacteria, the pathogenic yeast, and a filamentous fungus (Table V.1).

Two-tailed amphiphiles were only active against the yeast, but not the bacteria,

mycobacterium, or filamentous fungus. For the yeasts tested, some homologs of the

3CUr(n)2 series had antimicrobial activity against both C. albicans and C. neoformans,

while all but one of the 3CUr1(n)2 amphiphile series had strong antimicrobial activity

against C. neoformans.

As only particular members of the homologous series with specific chain length

show distinct activities, these amphiphiles also display another type of specificity,

namely chain-length specificity. Chain-length specificity is displayed when amphiphiles

in a homologous series with a particular chain length are exceptionally active compared

to those with other chain lengths. Chain-length specificity was displayed by members of

the 3CUrn series against M. smegmatis, S. cerevisiae, C. albicans, and C. neoformans,

by those of the 3CUr(n)2 series against C. albicans and C. neoformans, and by those of

the 3CUr1(n)2 series against C. neoformans (Table V.1).

179

In the 3CUrn series, the MIC of 3CUr16 against both C. albicans and C.

neoformans is 47 μM, the MIC of 3CUr18 against M. smegmatis is 33 μM, and the MIC

of 3CUr22 against S. cerevisiae was 32 μM. This chain-length specificity was not found

in the 3CUrn series against the rest of the microorganisms tested and the homologs of

3CUrn were not particularly active against those microorganisms. Among the two-tailed

amphiphiles, the three homologs 3CUr1(10)2, 3CUr1(11)2, and 3CUr1(12)2 showed

unique and promising MICs of 15 µM, 7.2 µM, and 6.9 µM, respectively, against C.

neoformans.

Chain-length specificity was particularly demonstrated by 3CUr(8)2 against C.

albicans (MIC = 190 μM) and 3CUr(10)2 against C. neoformans (MIC = 62 μM) (Table

V.1). As the strain of C. albicans was grown at 37 °C, it had low cell surface

hydrophobicity,8 which likely contributed to relative resistance of the cells to

amphiphiles.

V.3.3.2 Cholestane-based Amphiphiles

As seen in the previous series of amphiphiles, there was no general pattern

observed in the cholestane-based amphiphiles; the amphiphiles behaved differently

against the tested microorganisms. The only common pattern observed was that these

cholestane-based amphiphiles were not active against yeast; none of the yeasts tested

responded to the cholestane-based amphiphiles to any degree under given concentration.

Epimer specificity was displayed by both series against S. aureus and MRSA, and

by 3CUr-z-cholestane series against M. luteus. Epimer specificity was not displayed

against both Gram-negative K. pneumoniae and E. coli (3CUr-z-cholestane) for both

Gram-positive L. plantarum and M. luteus (for 3CUr1Es-z-cholestane), M. smegmatis,

180

and filamentous A. niger.

V.3.4 Comparing Hydrophobicity and Antimicrobial Activity

It was our intention next to explore how amphiphiles with similar hydrophobicity,

but different tail topology will affect their antimicrobial activity. The amphiphiles

maintain common head groups and linker. However, the tails were arranged in different

geometry. As the number of carbon on the tail(s) is maintained equal, amphiphiles with

the same number of carbon in their tails will have similar hydrophobicity.

V.3.4.1 One- and Two-tailed Amphiphiles

Partitioning of these amphiphiles into both outer and cytoplasmic membranes

should be a major contributor to their antimicrobial activity. Based on simple

approximations of hydrophobicity, the distribution coefficient increases as the chain

length increases. Calculations9 of logD at pH 7.4 give values that range from –5.1 for

3CUr14 to –1.9 for 3CUr22 in increments of 0.8 for each additional –CH2–CH2– unit,

from –5.2 for 3CUr(7)2 to –2.0 for 3CUr(11)2 in increments of 0.8 for each additional –

CH2– in each chain, and from –5.0 for 3CUr1(7)2 to –1.0 for 3CUr1(12)2 in increments

of 0.8 for an additional –CH2– in each chain. Because of the multiple ionization states of

the headgroup, logD is the appropriate choice. These calculations suggest that the tri-

headed amphiphiles are substantially more likely to favor being in a hydrophilic phase

than in a hydrophobic phase.

Comparing logD vs MIC for each member of the three series reveals the

similarities with respect to the antimicrobial activity against M. smegmatis (Figure V.1,

left) and C. albicans (Figure V.1, right). For M. smegmatis, all series show a parabolic

181

dependence on logD; the 3CUrn series gives the best activities. The 3CUr(n)2 and

3CUr1(n)2 series overlap each other in activity against M. smegmatis such that a logD of

–2 to –3 predicts the best activity. All series show similar pattern of activity against C.

albicans, a microorganism that shows chain-length specificity. The one-tailed

amphiphile 3CUr16 (logD = –4.3) has better activity than 3CUr(8)2 (logD = –4.4) and

3CUr1(8)2 (logD = –4.2).

Figure V.3 Relationship between log MIC of one- and two-tailed amphiphiles vs logD for M. smegmatis (left) and C. albicans (right). Error bars (not shown for clarity) are ± 0.3

Comparing logD vs MIC for each member of the three series reveals the

differences with respect to the antimicrobial activity against C. neoformans (Figure V.2,

left) and S. cerevisiae (Figure V.2, right). For C. neoformans, there is no similarity in the

patterns among the three series. Amphiphiles 3CUr16 (logD = –4.3), 3CUr(10)2 (logD =

–2.8), and 3CUr1(12)2 (logD = –1.0) are the most active members of the respective

series. Clearly, the 3CUr1(n)2 series is the best among these three series. For S.

cerevisiae, the 3CUrn and 3CUr1(n)2 series show similar patterns of improving activity

with increasing hydrophobicity, although the 3CUrn series is significantly more active.

-5

-4.5

-4

-3.5

-3

-2.5

-2

-6 -4 -2 0

logD

log

[MIC

] (M

)

3CUrn 3CUr(n)23CUr1(n)2

-5

-4

-3

-2

-1

-6 -4 -2 0

logD

log

[MIC

] (M

)


182

The 3CUr(n)2 series has a parabolic dependence with the best activity at 3CUr(8)2 (logD

= –4.4).

Figure V.4 Relationship between log MIC and logD for C. neoformans (left) and S. cerevisiae (right). Error bars (not shown for clarity) are ± 0.3

V.3.4.2 Cholestane-based Amphiphiles

The values9 of logD at pH 7.4 for 3CUr-z-cholestane and 3CUr1Es-z-

cholestane were –2.62 and –3.68, respectively. Based on simple approximations of

hydrophobicity, addition of a spacer –CH2COO− simply decreased the distribution

coefficient by 1.06.

In each pair of epimeric amphiphiles, the distribution coefficients were exactly the

same, yet the activity, represented by MICs, against several organisms were different.

This evidence suggests that geometry of the molecules affect their biological activities.

V.4 Comparison with Prior Work

A number of compounds containing a ureido group, where both nitrogen atoms

are monosubstituted, have been synthesized. In those studies, a nitrogen is attached to a

-5.5

-5-4.5

-4-3.5

-3-2.5

-2

-6 -4 -2 0

logD

log

[MIC

] (M

)


-5

-4.5

-4

-3.5

-3

-2.5

-2

-6 -4 -2 0

logDlo

g [M

IC] (

M)


183

long single alkyl chain while the other nitrogen is attached to a various group, which can

be classified as depsipeptide,10 pyrimidine nucleoside,11 glucosamine,12 crown ether,13

and penicillin14 substituents. All the compounds above have displayed antibacterial10,12-14

and antifungal11,12 activity.

Only one study15 evidences that urea derivatives where one of the nitrogen on the

ureido functional group is attached to two symmetrical n-alkyl chain have shown utility

as agricultural fungicides. On the other hand, many compounds containing two

symmetrical alkyl chains bonded to a nitrogen atom, not necessarily associated with a

ureido functional group, have been synthesized and studied for their antibacterial and

antifungal activity. To our knowledge, none of these compounds have anionic

headgroups as reported in this study. In these previous studies, the compounds are in the

neutral16,17 or charged form.18-21 The charged forms involve quarternary ammonium

with chloride counterions,18-20 or intercalation complexes21 with montmorillonite or

saponite. In a study20 of antibacterial properties against S. aureus, several homologous

tertiary amines are employed to quarternize crosslinked chloromethyl polystyerenes;

antibacterial efficiency increases as the alkyl chain length increases.

To the best of our knowledge, no urea derivative, where one of the nitrogen on the

ureido functional group attached to a swallowtail, is found to show antibacterial or

antifungal properties in the open literature. However, there are several compounds

containing a nitrogen atom bonded to a swallowtail, a long fatty chain bonded through

the middle carbon atom, have been synthesized. Unlike compounds containing a nitrogen

atom directly bonded to two symmetrical fatty chains, there are only few studies on

compounds containing a nitrogen atom bonded to a swallowtail. These compounds have

184

been investigated for antibacterial22,23 and antifungal23 activity. In the studies, these

compounds have cationic22,23 and neutral23 headgroups.

Several compounds where cholestane moiety is attached to a ureido linker have

been synthesized. No study of antifungal or antibacterial properties in such compounds

was found. Nevertheless, two studies pointed out that these compounds in their neutral

forms were actually display anticancer activity.24,25 Apart from that, a number of

compounds containing cholestane moiety indeed have been synthesized26-28 or even

isolated from natural product;29-32 these compounds exhibit antibacterial26-31 and

antifungal26,30-32 properties. To the best of our knowledge, no compound with the general

structure of linker-spacer-hydrophobic moiety, such as that in 3CUr1Es-z-cholestane,

were found in the open literature. However, compounds containing a cholestane moiety

bonded to an ethanoate moiety have been synthesized and investigated for their

antibacterial,33 antimycobacterial,34 and antifungal33,35 properties.

V.5 Conclusions

Overall, we concluded that the antimicrobial activity displayed is modest with a

couple of promising leads. However, we could say that the one-tailed series is more

active than the others. In general, the five series of dendritic tri-carboxylato amphiphiles

did not display a uniform pattern of activity against the broad spectrum of

microorganisms tested, but displayed amphiphile-series-, species-, chain-length-, or

epimer-specific patterns. It would be interesting to further the investigation on the

antimicrobial activity where the cut-off effect has not been reached.

185

V.6 Experimental Procedures V.6.1 Preparation of Tri-carboxylato Amphiphiles Solutions in Aqueous Triethanolamine

An aqueous triethanolamine was prepared by the following manner.

Triethanolamine (4.900 g) was added to a 100-mL volumetric flask, and then diluted with

water until the mark. The stock (12,500 mg/L) solutions for all homologues were easily

prepared by simply vortexing the tricarboxylic acid in the aqueous triethanolamine

solution. Each tri-headed amphiphile⎯3CUrn, 3CUr(n)2, 3CUr1(n)2, 3CUr-z-

cholestane, and 3CUr1Es-z-cholestane⎯(25 mg) was dissolved into aqueous

triethanolamine (2 mL). An aliquot (200 μL) of the resulting solution was diluted with

aqueous triethanolamine (2 mL) to give a more dilute solution (568 mg/L). These

original and the diluted solutions were called the stock and trial solutions, respectively.

V.6.2 Microbial Strains, Culture Conditions, and Preparations of Inocula for Susceptibility Testing

Strains of E. coli strain C (ATCC strain 13706), K. pneumoniae (ATCC strain

4352), L. plantarum (ATCC strain 14917), S. aureus (ATCC strain 6538), and M.

smegmatis (ATCC strain 607) were obtained from the American Type Culture Collection.

A methicillin-resistant isolate of S. aureus (MRSA) was obtained from the Microbiology

Laboratory, Danville Community Hospital (Virginia) and S. cerevisiae, C. albicans, C.

neoformans, A. niger, and M. luteus strains were obtained from the Virginia Tech

Microbiology teaching culture collection.

Colonies of E. coli, K. pneumoniae, S. aureus, MRSA, M. luteus, S. cerevisiae, C.

albicans, and C. neoformans were grown on 1/10-strength Brain Heart Infusion Broth

(BBL Microbiology Systems, Cockeysville, MD) containing 0.2% (wt/vol) sucrose

186

(BHIB+S) and 1.5% (wt/vol) agar. L. plantarum was grown on ¼-strength Tryptic Soy

Broth (TSB, BBL Microbiology Systems, Cockeysville, MD) containing 0.2% glucose

(TSB+G) and 1.5% (wt/vol) agar. M. smegmatis was grown on Middlebrook 7H10 agar

(BBL Microbiology Systems, Cockeysville, MD) and A. niger on Potato Dextrose Agar

(PDA, BBL Microbiology Systems, Cockeysville, MD). Streaked plates were incubated

at 37 °C for 3–7 days, except for that of A. niger, which was incubated in the dark at 30

°C. A single colony for each microbe except A. niger was used to inoculate 5 mL of

1/10-strength BHIB+S (E. coli, K. pneumoniae, M. luteus, S. aureus, and MRSA), full

strength TSB (L. plantarum), Middlebrook 7H9 broth (M. smegmatis), or Yeast Extract

Peptone Maltose broth (S. cerevisiae, C. albicans and C. neoformans) and incubated at

37°C (S. cerevisiae and M. luteus at 30 °C) for 4–7 days. After growth, the resulting

broth cultures were diluted with buffered saline gelatin [BSG, gelatin (0.1 g/L), NaCl (8.5

g/L), KH2PO4 (0.3 g/L), Na2HPO4 (0.6 g/L)] to equal the turbidity of a No. 1 McFarland

Standard. Spores of A. niger were scraped from the surface of PDA and suspended in 5

mL of 1/10-strength BHIB+S and that suspension transferred to a sterile test tube. The

turbidity was adjusted to a No. 1 McFarland Standard by dilution with BSG.

To check for viability and contamination, broth cultures of all organisms, except

A. niger, were streaked on Plate Count Agar (BBL Microbiology Systems, Cockeysville,

MD), and the plates incubated at 37 °C for 3–4 days, except those for M. luteus and S.

cerevisiae grown at 30 °C. To check for viability and contamination on A. niger, the

spore suspensions were streaked on PDA and incubated at 37 °C for 3–4 days.

For the work reported here, all cultures and suspensions that were used as inocula

were uncontaminated and the colonies had the expected morphologies. All viable,

187

uncontaminated inocula were stored up to 14 days at 4 °C until used without any

differences in susceptibility to antimicrobial compounds.

V.6.3 Measurement of MICs

MICs of compounds dissolved in aqueous triethanolamine were measured by

broth microdilution in 96-well microtitre plates.2 Preliminary experiments demonstrated

that 5% (wt/vol) triethanolamine/water did not inhibit the growth of any microorganism

tested. A two-fold dilution series of tri-headed amphiphiles⎯3CUrn, 3CUr(n)2,

3CUr1(n)2, 3CUr-z-cholestane, and 3CUr1Es-z-cholestane⎯was prepared in 96-well

microtitre plates (96 wells: rows A–H, columns 1–12) in the following manner. Aliquots

(50 μL) of 1/10-strength BHIB+S (pH 7.4) were placed in all the wells except for those in

column 1. Stock solutions (100 μM of 25 mg amphiphile 5% triethanolamine/water),

were placed in column 1. An aliquot (50 μL) was removed from a well in column 1 and

mixed with the BHIB+S in the well of column 2. Then, an aliquot (50 μL) of this mixture

was removed and mixed with BHIB+S in column 3. This process was repeated through

column 11, at which point an aliquot (50 μL) was removed and discarded. Column 12

was the blank (positive growth) control, BHIB+S only, for each row. An aliquot (50 μL)

of the suspension of the organism adjusted to No.1 McFarland Standard was added to all

wells in a row. Aqueous triethanolamine without any amphiphile was also tested for

antimicrobial activity by using the same protocol; no antimicrobial activity was found.

For E. coli and K. pneumoniae, the volumes of the medium and the inoculum were

doubled. The resulting inoculated dilution series were incubated at 30 °C (37 °C for E.

coli and K. pneumoniae) and growth, as turbidity, scored visually and recorded on the

188

fourth day (the seventh day for E. coli and K. pneumoniae). The MIC of each compound

was measured in triplicate and was defined as the lowest concentration of drug resulting

in a prominent visible decrease in turbidity (incomplete) or an absence of visible turbidity

(complete) compared to the drug-free control. In many cases (identified in Table V.1 by

superscript a), inhibition was incomplete.

References for Chapter V 1 Cain, C. C.; Henry, A. T.; Waldo III, R. H.; Casida Jr., L. J.; Falkinham III, J. O.

Identification and characteristics of a novel Burkholderia strain with broad-spectrum antimicrobial activity. Appl. Environ. Microbiol. 2000, 66, 4139-4141.

2 Williams, A. A.; Sugandhi, E. W.; Macri, R. V.; Falkinham III, J. O.; Gandour, R.

D. Antimicrobial activity of long-chain, water-soluble, dendritic tri-carboxylato amphiphiles. J. Antimicrob. Chemother. 2007, in press.

3 Vorum, H.; Brodersen, R.; Kragh-Hansen, U.; Pedersen, A. O. Solubility of long-

chain fatty acids in phosphate buffer at pH 7.4. Biochim. Biophys. Acta 1992, 1126, 135-142.

4 Kaneko, D.; Olsson, U.; Sakamoto, K. Self-assembly in some N-lauroyl-L-

glutamate/water systems. Langmuir 2002, 18, 4699-4703. 5 Boris, S.; Barbes, C. Role played by lactobacilli in controlling the population of

vaginal pathogens. Microbes Infect. 2000, 2, 543. 6 Seidel, V.; Taylor, P. W. In vitro activity of extract and constituents of

Pelagonium against rapidly growing mycobacteria. Int. J. Antimicrob. Agents 2004, 23, 613.

7 Kondo, E.; Kanai, K. Lethal effect of long-chain fatty acids on mycobacteria. Jpn.

J. Med. Sci. Biol. 1972, 25, 1-13. 8 Masuoka, J.; Hazen, K. C. Differences in the acid-labile component of Candida

albicans mannan from hydrophobic and hydrophilic yeast cells. Glycobiology 1999, 9, 1281-1286.

189

9 Czimadia, F. logP and logD calculator, http://intro.bio.umb.edu/111-112/OLLM/111F98/newclogp.html; November 15 2006.

10 Leese, R. A.; Curran, W. V.; Borders, D. B. Lipodepsipeptide antibiotics and

methods of preparation. U.S. Pat. Appl. Publ. 2003, US 2003224475 A1, 14pp. 11 Kiyoto, T.; Yotsuji, A.; Kajita, T.; Takashima, K.; Miyao, N.; Nomura, N.

Preparation of 5-deoxy-5-ureido-β-D-allofuranosyluronic acid pyrimidine derivatives as antifungal agents and chitin synthetase inhibitor. Jpn. Kokai Tokkyo Koho 1999, JP 11029590 A2, 45pp.

12 Kurita, H.; Imanishi, Y.; Ishida, A.; Onta, T.; Ohashi, M. Preparation of

glucosamine derivative having leukocyte-increasing and infection-preventing (antibacterial or antifungal) activity. Can. Pat. Appl. 1995, CA 2135536, 55pp.

13 Oros, G.; Szogyi, M.; Cserhati, T. Effect of some new crown ethers on plant-

related bacteria and their possible mode of action. Acta Microbiologica Hungarica 1986, 33, 117-123.

14 Koenig, H. B.; Metzger, K. G.; Offe, H. A.; Schroeck, W. Antibacterial activity

and structural constants of acylureidomethylpenicillins. Special Publication - Chemical Society 1977, 28(Recent Adv. Chem. β-Lactam Antibiot.), 78-91.

15 Yamada, Y.; Saito, J.; Gotoh, T.; Katsumata, O.; Sakawa, S. Trichloroacryloyl

urea derivatives, process for their preparation and their use as fungicides in agriculture and horticulture. Eur. Pat. Appl. 1984, EP 118093 A1, 42pp.

16 Genco, C. A.; Maloy, W. L.; Kari, U. P.; Motley, M. Antimicrobial activity of

magainin analogues against anaerobic oral pathogens. Int. J. Antimicrob. Agents 2003, 21, 75-78.

17 Quinn, F. R.; Driscoll, J. S.; Hansch, C. Structure-activity correlations among

Rifamycin B amides and hydrazides. J. Med. Chem. 1975, 18, 332-339. 18 Jiang, S., L; Wang, L.; Yu, H.; Chen, Y. Preparation of crosslinked polystyrenes

with quaternary ammonium and their antibacterial behavior. React. Funct. Polym. 2005, 62, 209-213.

19 Langsrud, S.; Sundheim, G.; Borgmann-Strahsen, R. Intrinsic and acquired

resistance to quaternary ammonium compounds in food-related Pseudomonas spp. J. Appl. Microbiol. 2003, 95, 874-882.

190

20 Walsh, S. E.; Maillard, J.-Y.; Russel, A. D.; Catrenich, C. E.; Charbonneau, D. L.; Bartolo, R. G. Activity and mechanisms of action of selected biocidal agents on Gram-positive and -negative bacteria. J. Appl. Microbiol. 2003, 94, 240-247.

21 Oya, A.; Funato, Y.; Sugiyama, K. Antimicrobial and antifungal agents derived

from clay minerals. Part V. Montmorillonite and saponite containing some antimicrobial ammonium ions. J. Mater. Sci. 1994, 29, 11-14.

22 Grier, N.; Dybas, R. A.; Strelitz, R. A.; Witzel, B. E.; Dulaney, E. L. Antibacterial

N-[ω,ω'-bis(alicyclic and aryl)-sec-alkyl]-1,3-diamino-2-propanol dihydrochloride salts. J. Pharm. Sci. 1982, 71, 365-367.

23 Cox, J. M.; Marsden, J. H. E.; Elmore, N.; Shephard, M. C.; Burrell, R. A.

Thienopyrimidines.1979, US 4146716, 17pp. 24 Kim, J. C.; Choi, S. K.; Cho, I. S.; Yu, D. S.; Ryu, S. H.; Moon, K. H. Synthesis

and antitumor evaluation of N-alkyl-N-nitrosocarbamoyl-3α -amino-and 3β -amino-5α -cholestane derivatives. 1985, 29, 62-69.

25 Johnston, T. P.; McCaleb, G. S.; Opliger, P. S.; Montgomery, J. A. Synthesis of

potential anticancer agents. XXXVI. N-nitrosoureas. 2. Haloalkyl derivatives. J. Med. Chem. 1966, 9, 892-910.

26 Elmegeed, G. A.; Wardakhan, W. W.; Younis, M.; Louca, N. A. Synthesis and

antimicrobial evaluation of some novel cholestane heterocyclic derivatives. Arch. Pharm. 2004, 337, 140-147.

27 Todorova, D.; Tupova, M.; Zapreva, V.; Milkova, T.; Kujumdjiev, A.

Transformation of amino steroids into pharmacologically active amides of phenolic acids. Biosci. 1999, 54, 65-69.

28 Krieg, R.; Wyrwa, R.; Mollmann, U.; Gorls, H.; Schonecker, B. Novel (N-

ferrocenylmethyl)amines and (N-ferrocenylmethylene)imines derived from vicinal steroid amino alcohols and amines: synthesis, molecular structure, and biological activity. Steroids 1998, 63, 531-541.

29 Kamenarska, Z.; Gasic, M. J.; Zlatovic, M.; Rasovic, A.; Sladic, D.; Kljajic, Z.;

Stefanov, K.; Seizova, K.; Najdenski, H.; Kujumgiev, A.; Tsvetkova, I.; Popov, S. Chemical composition of the brown alga Padina pavonia (L.) Gaill. from the Adriatic Sea. Botanica Marina 2002, 45, 339-345.

30 Rao, M. N.; Shinnar, A. E.; Noecker, L. A.; Chao, T. L.; Feibush, B.; Snyder, B.;

Sharkansky, I.; A, S.; Zhang, X.; Jones, S. R.; Kinney, W. A.; Zasloff, M. Aminosterols from the dogfish shark Squalus acanthias. J. Nat. Prod. 2000, 63, 631-635.

191

31 Wehrli, S. L.; Moore, K. S.; Roder, H.; Durell, S.; Zasloff, M. Structure of the novel steroidal antibiotic squalamine determined by two-dimensional NMR spectroscopy. Steroids 1993, 58, 370-378.

32 D'Auria, M. V.; Fontana, A.; Minale, L.; Riccio, R. Starfish saponins. Part XLII.

Isolation of twelve steroidal glycosides from the Pacific Ocean starfish Henricia laeviuscola. Gazz. Chim. Ital. 1990, 120, 155-163.

33 Madureira, A. M.; Ascenso, J. R.; Valdeira, L.; Duarte, A.; Frade, J. P.; Freitas,

G.; Ferreira, M. J. U. Evaluation of the antiviral and antimicrobial activities of triterpenes isolated from Euphorbia segetalis. Nat. Prod. Res. 2003, 17, 375-380.

34 Cantrell, C. L.; Lu, T.; Fronczek, F. R.; Fischer, N. H.; Adams, L. B.; Franzblau,

S. G. Antimycobacterial Cycloartanes from Borrichia frutescens. J. Nat. Prod. 1996, 59, 1131-1136.

35 Anke, T.; Werle, A.; Zapf, S.; Velten, R.; Steglich, W. Favolon, a new antifungal

triterpenoid from a Favolaschia species. J. Antibiot. 1995, 48, 735-736.

192

Chapter VI Intrinsic Activity of the 3CUrn Homologus Series Against Mycobacterium smegmatis

It had been discussed (Chapter I), that our goal is to design amphiphiles that have

good antimicrobial activity and does not involve detergency as the mechanism of action.

In order to fulfill the goal, we needed to explore solubility and CMC. In a homologous

series, antimicrobial activity can show a cut-off effect,1 known earlier as the parabolic

case,2 where antimicrobial activity increases up to an optimal point as chain length

increases; after that, the activity decreases. To conclude that the cut-off effect is due to

the intrinsic activities3,4 (i.e., the maximal response that a compound induces) or the

decreased solubility of the homologous amphiphiles, the amphiphiles must fully dissolve

in aqueous media.

We had designed amphiphiles that are water-soluble. This should allow the

biological assays of all amphiphiles to take place without encountering any solubility

problem. Following this, all homologous tri-carboxylato amphiphiles were screened for

the antimicrobial activity, which was represented as the MIC. As the solubility was no

longer an issue, it could be eliminated as the cause of a cut-off effect. Consequently, we

intended to explore if a cut-off effect was due to the intrinsic activity. How would we do

this?

In the previous biological assays (Chapter V), the suspension of the

microorganisms used was adjusted to equal the turbidity of a No. 1 McFarland Standard,

which has a cell density of 3 × 108 colony forming unit (CFU). For this number, it was

possibly too many of microorganisms added to the amphiphiles. Under this condition,

there might not be enough amphiphiles to inhibit the microorganisms. In other words, we

could not determine if the amphiphiles gave their maximum response. As we observed

193

some amphiphiles showed complete inhibition, and some others showed incomplete

inhibition. In order to overcome this problem, we intended to explore this antimicrobial

activity in more detail, where the MICs were measured based on the maximum response

of the amphiphiles. Under this condition, we should only observe the complete

inhibition, where we had to be certain that the amphiphiles should saturate the

microorganims. One way to satisfy this was to control the cell density of the

microorganisms used, that it should be less than that of a No. 1 McFarland Standard.

In this new protocol, MICs of the series were measured at several different initial

cell densities, expressed as colony-forming units (CFU/mL). We will vary the density of

the suspension of the microorganism by ten-fold dilution starting with supposedly that

where the turbidity has been adjusted to equal the turbidity of a No. 1 McFarland

Standard. In this dilution series, the more diluted suspension contains fewer

microorganisms. The least concentrated suspension should allow us to count the number

of the colony grown in term of CFU. This dilution series are used in the biological assays

following the previous protocol (Chapter V); both amphiphile concentration and cell

density of the suspension of the microorganism are varied. Under a certain concentration

(of suspension of the microorganism) and below, we expect they become saturated with

the amphiphile. In other words, the observed MIC should not change, instead, it should

be a constant; this is the MIC threshold, which is caused by the intrinsic activity.

In a homologous series, if the cut-off effect is observed under this protocol, we

can conclude that such a phenomenon is due to intrinsic activity. Following this, we

should compare these MICs to the CMC to determine if detergency is involved in the

mechanism of action of the antimicrobial activity. In this chapter, we would only address

194

the intrinsic activity of the 3CUrn homologous series against M. smegmatis.

VI.1 The “inoculum effect”

In studies of antimicrobial activity, the MIC is often observed to increase as initial

cell density increases. This phenomenon is referred to as the “inoculum effect”.5-7 Thus

patients with higher microbial loads often require higher drug doses.6 Gehrt et al.5 have

proposed that the “inoculum effect” reflects the increased demand for a given drug as the

number of cells and targets increases. From a bioorganic mechanistic perspective, the

“inoculum effect” may provide information about a change in the mechanism of action.

A mathematical model (Equation 1) of the “inoculum effect”, developed by Li

and Ma8 from a study of four aminoglycosides against Escherichia coli and

Staphylococcus aureus, shows the dependence of MIC on initial cell density (i.e.,

inoculum). Equation 1 describes the independence of log MIC at low inoculum by

log MIC = log MIC0 + (e − 1) (1)k (log I - log Itr ).

defining log MIC0 as the intrinsic activity, log I as the inoculum, log Itr as the inoculum at

the threshold immediately before the rise in MIC, and k is a constant that decribes the rate

of increase in MIC. Equation 1 is flat at low inoculum and the rises exponentially after

Itr, which varies by drug (see Figure VI.1 below).

VI.2 Intrinsic Activity of Homologous Amphiphiles

Our study addresses Gruber’s thesis that “many [reported] chain length profiles of

apparent potencies…produce an unclear, or misleading impression of the role [of] alkyl

chain length”.4 Gruber suggests that increasing the number of measurements by varying

195

the drug–cell ratios provides at least a qualitative measure of how membrane partitioning

affects activity. Gruber’s suggestion should lead to measurements where the amphiphiles

saturate the cells; hence the activity of the amphiphiles should be at an optimal value.

Our studies of measuring MICs as a function of initial cell density follow Gruber’s

suggestion. The results provide measurements of the intrinsic activity, which is equal to

log MIC0 in Equation 1.

Scheme VI.1 Simplified kinetic model of inhibition of cell growth

n drug + cell drugn . cell

k grow k inhibit

Keq

In our study, the MIC to prevent bacterial growth within a given time can be

described as the minimal concentration of drug needed to achieve the following

equality—kinhibit[drugn·cell] = kgrow[cell]—as described in Scheme VI.1. In this simplified

model, the drug diffuses into or onto a cell. Undoubtedly, more than one drug molecule

is needed to inhibit the cell. Hence, n indicates the number of drug molecules needed to

inhibit the cell. This n is probably not a specific number, but some average number,

especially, because the sizes of the cells vary. This means that Keq is actually a product

of several equilibria as successive drug molecules are absorbed by the cell. In addition to

the partitioning equilibria, there is the added complication of the microbe developing

drug-destruction and drug-export pathways,9 which drive the equilibrium to the left.

Further, kinhibit is also a composite of many different rate constants and equilibrium

constants.

The results of these experiments—measuring MIC as a function of initial cell

196

density—enable identification of the intrinsic activities. Varying the initial cell density

also provides a measure of how changing the number of cells (and as a result the amount

of cell wall, membranes, and proteins) affects MIC. If partitioning of these amphiphiles

into both outer and cytoplasmic mycobacterial membranes10 is a significant component of

the activity, then estimates of the partitioning coefficients would be useful in measuring

intrinsic activities. At a very low cell density, the cells are so few that we presume that

they are saturated with amphiphile. At the highest cell density, saturation might depend

on the partition coefficient of the amphiphile. For a homologous series, partition

coefficients increase as the chain length increases. For compounds with ionizable groups,

the distribution coefficient, which is comprised of the partition coefficient and the pKa’s,

provides a better estimate of the partitioning between octan-1-ol and water at given pH.

Calculations11 of log D at pH 7.4 give values that range from –5.1 for 3CUr14 to

–1.9 for 3CUr22 in increments of 0.8 for each additional –CH2–CH2– unit. Calculations

of log D at pH 7.4 for natural saturated fatty acids give values that range from 2.4 for

tetradecanoic acid to 5.6 for docosanoic acid. These calculations suggest that the 3CUrn

series substantially favors the hydrophilic phase so that low initial cell densities are

needed to saturate the cell with amphiphile.

To probe the intrinsic activities of these amphiphiles, we measured the MICs of

3Ur14–3Ur22 at several different initial cell densities, expressed as colony-forming units

(CFU)/mL, of M. smegmatis. Experiments were performed in duplicate at two

overlapping ranges of initial inocula—32 to 3.2 × 107 CFU/mL and 500 to 5.0 × 108

CFU/mL. The MIC was defined as the lowest concentration at which an amphiphile

completely inhibited the growth after four days at 37 °C by successive twofold dilution in

197

96-well microtiter plates.

VI.3 Results and Discussions VI.3.1 MICs of the 3CUrn Series

All amphiphiles show an inoculum effect and follow Equation 1. The

antimycobacterial activity of these amphiphiles is constant as the initial cell density

increases until a threshold inoculum (Figure VI.1). These plateaus equal log MIC0, the

intrinsic activity. For these amphiphiles, 3CUr16 has the lowest intrinsic activity. The

log Itr, inoculum at the threshold immediately before the rise in MIC, varies among the

amphiphiles. The lowest log Itr (4.7) is for 3CUr16; the highest log Itr (6.7) occurs for

both 3CUr14 and 3CUr20. If partitioning into a hydrophobic part of the cell were a

dominant mechanism, the log Itr should be lowest for 3CUr14. After the threshold, the

increases in log MICs vs log initial cell densities are approximately the same for all the

amphiphiles.

Figure VI.1 Effect of initial cell density on inhibition of growth of M. smegmatis for the 3CUrn series. Combined data of two separate assays⎯3.2 to 3.2 × 107 CFU/mL and 500 to 5.0 × 108 CFU/mL. Error bars (not shown for clarity) are ± 0.3

-5

-4.5

-4

-3.5

-3

-2.5

0 1 2 3 4 5 6 7 8 9 10log initial cell density (CFU/mL)

log

[MIC

] (M

)

3CUr14 3CUr16 3CUr20

-4.5

-4

-3.5

-3

-2.5

0 1 2 3 4 5 6 7 8 9 10log initial cell density (CFU/mL)

log

[MIC

] (M

)

3CUr18 3CUr22

198

Figure VI.2 shows plots of log MIC vs. chain length at four different initial cell

densities to better illustrate the effect of chain length on activity. Figure VI.2 also

illustrates Gruber’s thesis that at cell densities above the lowest threshold, one is

measuring apparent values and can be misled as to which chain length gives the best

intrinsic activity. At the initial cell densities of 3.2 × 104 CFU/mL and lower, the profile

is the same and the intrinsic activity is revealed (Figure VI.2 left, circles). The most

active compound is 3CUr16. One might have reached a similar conclusion at an initial

cell density of 5 × 108 CFU/mL (Figure VI.2 left, diamonds). The data at that inoculum

show almost no variation among the amphiphiles, except that the MIC for 3CUr16 is

only one dilution lower than the rest. On the other hand, at initial cell densities of 5 × 106

and 3.2 × 105 (Figure IV.2, right), one would conclude that both 3CUr16 and 3CUr18

are the most active compounds.

Figure VI.2 Effect of chain length on inhibition of growth of M. smegmatis of the 3CUrn series as function of the initial cell density. Error bars (not shown for clarity) are ± 0.3

-5

-4.5

-4

-3.5

-3

-2.5

-2

12 14 16 18 20 22 24

chain length

log

[MIC

] (M

)

5.0E+08 3.20E+04

-4.5

-4

-3.5

-3

-2.5

12 14 16 18 20 22 24

chain length

log

[MIC

] (M

)

5.0E+06 3.2E+05

199

VI.3.2 Comparison with Previous Work

Although the mechanism of action of these amphiphiles remains to be determined,

we expect that these amphiphiles exert their antimycobacterial activity by interacting

with the mycobacterial membrane. Excluding detergency (disrupting cell walls and

solubilizing membranes), these amphiphiles can inactivate microorganisms by several

mechanisms: (1) as monomers, changing fluidity and porosity of membranes; (2) as

monomers, altering membrane-associated pathways; (3) as monomers, changing the flow

of constituents and nutrients between a cell and a medium; and (4) as monomers,

inhibiting specific membrane-associated enzymes. The discovery that the homolog with

the hexadecyl chain gives the best activity contrasts with the results for natural saturated

fatty acids. In an early study12 of several mycobacteria, tetradecanoic acid is slightly

more active than dodecanoic acid in inhibiting the growth of M. smegmatis. In a very

recent study13 dodecanoic acid is the most active of the even-numbered natural saturated

fatty acids up to twenty-six carbons for several mycobacteria, including M. smegmatis.

VI.3.3 Comparison of MICs and CMCs

The MICs at the lowest and highest initial cell densities with the CMCs are

compared (Figure VI.3). The values of MIC at the lowest initial cell density illustrate the

intrinsic activity of the series. As all MICs of the series are lower than the CMCs, we

conclude that detergency is not the likely mechanism of action. In this comparison, the

MIC of 3CUr16 is 100-fold smaller than the CMC, the MIC of 3CUr22, 8-fold smaller

than the CMC. The data at the highest initial cell density illustrate conditions where the

amphiphiles may not be saturating the cells. In this comparison, the MIC of 3CUr16 at

the highest initial cell density is 5-fold smaller than the CMC, the MIC of 3CUr22 at the

200

Figure VI.3 Comparison of log CMC and log MIC of the 3CUrn series at the highest and lowest initial cell densities of M. smegmatis

highest initial cell density equals the CMC. We tentatively conclude that detergency is

the likely mechanism of action at the highest initial cell density for 3CUr22 and 3CUr20.

For the other members of the series detergency is likely a significant contributor to the

mechanism of action.

We initiated this project to design long chain amphiphiles with high CMCs. The

underlying thesis is that the higher the CMC, the less likely detergency will contribute to

the mechanism of action. Even though these amphiphiles do not have MICs low enough

to be considered as leads for drug development, one should always remember that the

therapeutic index (ratio of cytotoxicity to potency) is the ultimate predictor for potential

applications. In this regard, a potential application of these amphiphiles is as affordable

topical anti-infectives. If cytotoxicity is only related to detergency (hence CMC), then a

therapeutic index of 100 looks more promising. Although it is unlikely that cytotoxicity

-5

-4.5

-4

-3.5

-3

-2.5

-2

12 14 16 18 20 22 24

chain length

log

MIC

or C

MC

(M)

high inoculum low inoculum CMCs

201

will be that simple, the strategy of designing amphiphiles with high CMCs is essential to

developing non-detergent amphiphilic microbicides.

VI.4 Conclusions

Water-soluble amphiphiles that have ultra-long chains can be made readily from

first-generation, Newkome-type dendrons. As tris(triethanolammonium) salts, these

amphiphiles show excellent solubility in water. This homologous series of alkyl chains

that extends to docosyl reveals that the cut-off for antimycobacterial activity is the

hexadecyl chain. As the intrinsic activity is well below the CMC, a mechanism of action

that does not involve detergency is suggested.intrinsic activity of the series.

VI.5 Experimental Procedures

To probe the intrinsic activities of these amphiphiles, we measured the MICs of

3Ur14–3Ur22 at several different initial cell densities, expressed as colony-forming units

(CFU)/mL, of M. smegmatis. Experiments were performed in duplicate at two

overlapping ranges of initial inocula—32 to 3.2 × 107 CFU/mL and 500 to 5.0 × 108

CFU/mL. The MIC was defined as the lowest concentration at which an amphiphile

completely inhibited the growth after four days at 37 °C by successive twofold dilution in

96-well microtiter plates.

VI.5.1 Microbial Strain, Culture Conditions, and Preparations of Inoculum for Susceptibility Testing

Mycobacterium smegmatis was obtained from the Virginia Tech Microbiology

culture collection. Two separate assays with initial cell densities ranging from 32 to

3.2 × 107 CFU/mL and 500 to 5.0 × 108 CFU/mL were performed. The different initial

202

cell densities were prepared in the following manner. A single isolated colony of M.

smegmatis on M7H10 agar was used to inoculate sterile M7H9 broth (2 mL) in a screw

capped tube (16 ×125 mm). After incubation without shaking at 37 °C for 5 d, 2 mL was

used to inoculate M7H9 broth (18 mL) in a 125-mL flask and then incubated with

aeration (120 rpm) at 30 °C for 5 d. The culture was transferred to a sterile 50-mL screw

capped centrifuge tube; centrifugation (5,000 × g) for 20 min was performed. The

supernatant-spent medium was discarded, and the cells were suspended in 1/10-strength

BHIB medium (2 mL). A tenfold dilution series was prepared in 1/10-strength BHIB by

transferring the concd cell suspension (0.5 mL) into 4.5 mL of 1/10-strength BHIB to

yield a 10-fold to 107-fold dilution series. The concd cell suspension, measured in

CFU/mL, was measured by spreading different dilutions (0.1 mL each) on M7H10 agar.

The agar plates were incubated at 37 °C and colonies were counted after 4 d. The average

of the colony counts was calculated. MIC measurements were performed within 24 h

after the dilutions were performed.

VI.5.2 Measurement of MICs

Measurement of MICs was carried out in a similar manner is the measurement of

MICs for the preliminary screening (Chapter V). Instead of a suspension of the organism

adjusted to McFarland standard No.1, suspensions of M. smegmatis in different cell

density prepared as above were employed. An aliquot (50 μL) of the microbial

inoculum, M. smegmatis, at a given cell density was added to all wells in a row. Aqueous

triethanolamine without amphiphile 3CUrn was also tested for antimicrobial activity by

using the same protocol; no antimicrobial activity was found. The concentration of the

3CUrn series ranged from 6.25 mg/mL to 5.5 μg/mL. After the plates were incubated at

203

37 °C for 4 d, MICs results were read by comparing the turbidity (due to growth of

microbes) of each test well to the positive control wells. MIC was defined as the lowest

concentration completely inhibiting the growth. The experiments were run in duplicate

for the initial cell densities of 32 to 3.2 × 107 CFU/mL and in single for the initial cell

densities of 500 to 5.0 × 108 CFU/mL.

References for Chapter VI 1 Balgavý, P.; Devínsky, F. Cut-off effects in biological activities of surfactants.

Adv. Coll. Interface Sci. 1996, 66, 23-63. 2 Hansch, C.; Clayton, J. M. Lipophilic character and biological activity of drugs.

II. The parabolic case. J. Pharm. Sci. 1973, 62, 1-21. 3 Gruber, H. J.; Low, P. S. Interaction of amphiphiles with integral membrane

proteins. I. Structural destabilization of the anion transport protein of the erythrocyte membrane by fatty acids, fatty alcohols, and fatty amines. Biochim. Biophys. Acta 1988, 944, 414-424.

4 Gruber, H. J. Interaction of amphiphiles with integral membrane proteins. II. A

simple, minimal model for the nonspecific interaction of amphiphiles with the anion exchanger of the erythrocyte membrane. Biochim. Biophys. Acta 1988, 944, 425-436.

5 Gehrt, A.; Peter, J.; Pizzo, P. A.; Walsh, T. J. Effect of increasing inoculum sizes

of pathogenic filamentous fungi on MICs of antifungal agents by broth microdilution method. J. Clin. Microbiol. 1995, 33, 1302-1307.

6 Soriano, F. Optimal dosage of β-lactam antibiotics: time above the MIC and

inoculum effect. J. Antimicrob. Chemother. 1992, 30, 566-569. 7 Brook, I. Inoculum effect. Rev. Infect. Dis. 1989, 11, 361-368. 8 Li, R. C.; Ma, H. H. Parameterization of inoculum effect via mathematical

modeling: aminoglycosides against Staphylococcus aureus and Escherichia coli. J. Chemother. 1998, 10, 203-207.

204

9 Thrupp, L. D. Antibiotics in laboratory medicine; 2nd ed.; Williams & Wilkins: Baltimore, MD, 1986.

10 Brennan, P. J.; Nikaido, H. The envelope of mycobacteria. Annu. Rev. Biochem.

1995, 64, 29-63. 11 Csizmadia, F. logP and logD calculator, http://intro.bio.umb.edu/111-

112/OLLM/111F98/newclogp.html; September 26 2006. 12 Kondo, E.; Kanai, K. Lethal effect of long-chain fatty acids on mycobacteria. Jpn.

J. Med. Sci. Biol. 1972, 25, 1-13. 13 Seidel, V.; Taylor, P. W. In vitro activity of extracts and constituents of

Pelagonium against rapidly growing mycobacteria. Int. J. Antimicrob. Agents 2004, 23, 613-619.

205

Chapter VII Accomplishments, Conclusions, and Future Work

VII.1 Accomplishments

We have initiated a project designing amphiphiles with antimicrobial properties

that do not involve detergency as the mechanism of action. We have established a route

to synthesize homologous, water-soluble amphiphiles with very hydrophobic tails

utilizing the first-generation, tri-carboxylato Newkome-type dendron attached to a ureido

linker. With this route, three homologous series containing mobile hydrophobic moieties

and two series of epimers containing a rigid cholestane moiety have been generated

easily within a relatively short period of time. The tri-carboxylato amphiphiles have been

obtained in moderate to excellent yields.

Ten new compounds, which were tri-tert-butyl esters and triacids, were generated

in the synthesis of one-tailed, tri-carboxylato amphiphiles. All these new compounds

were fully characterized. A new synthetic route to long-chain alkan-1-amines (n = 20,

22), which are rarely found in the literature, was established. Beginning with low-cost

commercially available alcohols, the three-step synthesis afforded long-chain amines in

moderate yields.

Twenty-two new compounds, which are tri-tert-butyl esters and triacids, were

generated in the synthesis of two-tailed, tri-carboxylato amphiphiles. A new synthetic

route to amines attached to a “swallowtail” was established. Most documented

procedures for the preparation of such amines, which begins with costly ketones, required

extreme conditions. Beginning with relatively low-cost commercially available

bromoalkanes and ethyl formate, this newly established four-step synthesis could be

easily performed with standard laboratory glassware. Six new secondary azides were

206

generated during this process. All compounds⎯eleven tri-tert-butyl esters, eleven

triacids, and six azidoalkanes with a swallowtail⎯were fully characterized.

Eight new compounds, which are epimeric pairs of tri-tert-butyl esters and

triacids, were generated in the synthesis of tri-carboxylato amphiphiles containing the

rigid cholestane moiety. Two new 5α-cholestan-3-yl azidoethanoates, which are a pair of

epimers, had been synthesized during this process. All the ten new compounds are fully

characterized.

We screened all these amphiphiles against a broad spectrum of eleven

microorganisms. During this screening process, more than one hundred biological assays

were performed.

VII.2 Conclusions

The Newkome-type dendron provides a unique headgroup for amphiphiles with a

very hydrophobic moieties. As tris(triethanolammonium) salts, these amphiphiles show

excellent solubility in water. The solubilities of the first three series in aqueous solution

are 19,500 to 25,700 µM depending on the formula weight of the homolog, while those

of the last two are 18,900 and 17,400 μM.

We screened these amphiphiles against a broad spectrum of microorganisms by

broth microdilution in 96-well microtiter plates, where the antimicrobial activity is

represented by MICs. Plots of log MIC vs chain length show the cut-off effect is

observed in the first three series (3CUrn, 3CUr(n)2, and 3CUr1(n)2) against most of the

microorganisms tested. As all amphiphiles are soluble, solubility issues can be ruled out

as the cause of the cut-off effect. Based on the plots of log MIC vs hydrophobicity, we

cannot make the generalization that hydrophobicity is the only factor affecting

207

antimicrobial activity; both hydrophobicity and tail topology affect the antimicrobial

activity.

In general, the five series of dendritic tri-carboxylato amphiphiles show modest

antimicrobial properties with a couple promising leads, and they do not display a uniform

pattern of activity against the broad spectrum of microorganisms tested, but display

amphiphile-series-, species-, chain-length-, or epimer-specific patterns.

Finally, to determine if detergency is the mechanism of action in the antimicrobial

activity, we have compared the MICs to CMCs. For this intention, we have developed a

more detail biological assays to investigate the intrinsic activity (MIC0) of the 3CUrn

series against Mycobacterium smegmatis. As the resulting MIC0’s are below the CMCs,

we conclude that detergency is not the mechanism of action. Amphiphile 3CUr16 is the

most active with the MIC0 100-fold smaller than the CMC.

VII.3 Future Work

As two of the amphiphile series have not reached the cut-off effects, we are

interested in generating longer amphiphiles of the corresponding series to explore the

more active amphiphile. Especially against Cryptococcus neoformans, a longer

3CUr1(n)2 is worth synthesizing and exploring for antimicrobial activity.

A very recent study1 documented that ultra-long chain unsaturated fatty acids⎯2-

hexadecynoic and 2-octadecynoic acids⎯posses antimycobacterial against M.

smegmatis. Thus, synthesis of the tri-headed amphiphiles containing ultra-long,

unsaturated, linear alkyl chains, followed by investigation on antimycobacterial activity is

encouraged.

208

References for Chapter VII 1 Morbidoni, H. R.; Vilcheze, C.; Kremer, L.; Bittman, R.; Sacchettini, J. C.;

Jacobs, W. R. Dual inhibition of mycobacterial fatty acid biosynthesis and degradation by 2-alkynoic acids. Chem. Biol. 2006, 13, 297-307.

209

APPENDICES

210

APPENDIX A: 1H and 13C NMR spectra

211

1H NMR spectrum of 3EUr14

NH

NH

O

O O

3

212


NH

NH

O

O O

3

213


NH

NH

O

O O

3

214


NH

NH

O

O O

3

215


NH

NH

O

O O

3

216

1H NMR spectrum of 3EUr(7)2

N

NO O

O

3H

217


N

NO O

O

3H

218


N

NO O

O

3H

219


N

NO O

O

3H

220


N

NO O

O

3H

221

NH

ONH

O

3

222

1H NMR spectrum of 3EUr1(8)2

NH

ONH

O

3

223


NH

ONH

O

3

224


NH

ONH

O

3

225


NH

ONH

O

3

226


NH

ONH

O

3

227

1H NMR spectrum of 3EUr-α-cholestane

NH

NH

O

O

O

3

228

1H NMR spectrum of 3EUr-β-cholestane

NH

NH

O

O

O

3

229

1H NMR of 3EUr-β-cholestane (expansion)

230

1H NMR spectrum of 3EUr1Es-α-cholestane

ONH

NH

O

O

O

O

3

231

1H NMR spectrum of 3EUr1Es-β-cholestane

ONH

NH

O

O

O

O

3

232

1H NMR spectrum of 3CUr14

NH

O

NH

OH

O 3

233


NH

O

NH

OH

O 3

234

1H NMR spectrum of 3CUr16 (expansion)

235


NH

O

NH

OH

O 3

236


NH

O

NH

OH

O 3

237

1H NMR spectrum of 3CUr20 (expansion)

238


NH

O

NH

OH

O 3

239

1H NMR spectrum of 3CUr(7)2

N

NO OH

O

3H

240

1H NMR of 3CUr(8)2

N

NO OH

O

3H

241


N

NO OH

O

3H

242


N

NO OH

O

3H

243


N

NO OH

O

3H

244

1H NMR spectrum of 3CUr1(7)2

NH

ONH

H

O

3

245


NH

ONH

H

O

3

246


NH

ONH

H

O

3

247


NH

ONH

H

O

3

248


NH

ONH

O

H 3

249


NH

ONH

O

H 3

250

1H NMR spectrum of 3CUr-α-cholestane

NH

NH

O

O

O

H

3

251

1H NMR spectrum of 3CUr-β-cholestane

NH

NH

O

O

O

H

3

252

1H NMR spectrum of 3CUr1Es-α-cholestane

ONH

NH

O

O

O

O

H

3

253

1H NMR spectrum of 3CUr1Es-β-cholestane O

NH

NH

O

O

O

O

H

3

254

1H NMR spectrum of 8-azidopentadecane

N3

255

1H NMR spectrum of 9-azidoheptadecane

N3

256

1H NMR spectrum of 10-azidononadecane

N3

257

1H NMR spectrum of 11-azidohenicosane

N3

258

1H NMR spectrum of 12-azidotricosane

N3

259

1H NMR spectrum of 13-azidopentacosane

N3

260

1H NMR spectrum of 5α-cholestan-3α-yl azidoethanoate

O

ON3

261

1H NMR spectrum of 5α-cholestan-3β-yl azidoethanoate

O

ON3

262

1H NMR spectrum of 5α-cholestan-3β-yl azidoethanoate

263

13C NMR spectrum of 3EUr14

NH

NH

O

O O

3

264


NH

NH

O

O O

3

265


NH

NH

O

O O

3

266


NH

NH

O

O O

3

267


NH

NH

O

O O

3

268

13C NMR spectrum of 3EUr(7)2

N

NO O

O

3H

269


N

NO O

O

3H

270


N

NO O

O

3H

271


N

NO O

O

3H

272


N

NO O

O

3H

273


N

NO O

O

3H

274

13C NMR spectrum of 3EUr1(7)2

NH

ONH

O

3

275


NH

ONH

O

3

276


NH

ONH

O

3

277


NH

ONH

O

3

278


NH

ONH

O

3

279


NH

ONH

O

3

280

13C NMR spectrum of 3EUr-α-cholestane

NH

NH

O

O

O

3

281

13C NMR spectrum of 3EUr-β-cholestane

NH

NH

O

O

O

3

282

13C NMR spectrum of 3EUr1Es-1-cholestane

ONH

NH

O

O

O

O

3

283

13C NMR spectrum of 3EUr1Es-β-cholestane

ONH

NH

O

O

O

O

3

284

13C NMR spectrum of 3CUr14

NH

O

NH

OH

O 3

285


NH

O

NH

OH

O 3

286


NH

O

NH

OH

O 3

287


NH

O

NH

OH

O 3

288


NH

O

NH

OH

O 3

289

13C NMR spectrum of 3CUr(7)2

N

NO OH

O

3H

290


N

NO OH

O

3H

291


N

NO OH

O

3H

292


N

NO OH

O

3H

293


N

NO OH

O

3H

294

13C NMR spectrum of 3CUr1(7)2

NH

ONH

OH

O

3

295


NH

ONH

OH

O

3

296


NH

ONH

OH

O

3

297


NH

ONH

OH

O

3

298


NH

ONH

O

OH 3

299


NH

ONH

O

OH 3

300

13C NMR spectrum of 3CUr-α-cholestane

NH

NH

O

O

O

H

3

301

13C NMR spectrum of 3CUr-α-cholestane (expansion)

302

13C NMR spectrum of 3CUr-β-cholestane

NH

NH

O

O

O

H

3

303

13C NMR spectrum of 3CUr1Es-α-cholestane

ONH

NH

O

O

O

O

H

3

304

13C NMR spectrum of 3CUr1Es-β-cholestane

ONH

NH

O

O

O

O

H

3

305

13C NMR spectrum of 8-azidopentadecane

N3

306

13C NMR spectrum of 9-azidoheptadecane

N3

307

13C NMR spectrum of 10-azidononadecane

N3

308

13C NMR spectrum of 11-azidohenicosane

N3

309

13C NMR spectrum of 12-azidotricosane

N3

310

13C NMR spectrum of 13-azidopentacosane

N3

311

13C NMR spectrum of 5α-cholestan-3α-yl azidoethanoate

O

ON3

312

13C NMR spectrum of 5α-cholestan-3β-yl azidoethanoate

O

ON3

313

APPENDIX B: X-ray tables

314

Table I.1 Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) of 3EUr16.

U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

________________________________________________________________________________

x y z U(eq)

________________________________________________________________________________ O(1) 5406(2) 8565(2) 2493(1) 25(1)

O(2) 3665(2) 13670(2) 2412(1) 25(1)

O(3) 5437(2) 12118(2) 2578(1) 35(1)

O(4) 2010(2) 11480(1) 454(1) 21(1)

O(5) 901(2) 12338(2) 1018(1) 32(1)

O(6) 6393(2) 6243(1) 1283(1) 22(1)

O(7) 6191(2) 7741(2) 832(1) 32(1)

N(1) 3535(2) 9478(2) 2145(1) 19(1)

N(2) 3604(2) 8429(2) 2749(1) 23(1)

C(1) 3999(2) 10021(2) 1801(1) 17(1)

C(2) 4249(2) 8814(2) 2464(1) 19(1)

C(3) 4245(2) 7680(2) 3099(1) 24(1)

C(4) 4457(3) 8335(2) 3460(1) 28(1)

C(5) 5025(2) 7533(2) 3838(1) 29(1)

C(6) 6375(3) 6655(2) 3790(1) 31(1)

C(7) 6908(2) 5963(2) 4188(1) 28(1)

C(8) 8257(3) 5090(2) 4164(1) 32(1)

C(9) 8756(2) 4427(2) 4572(1) 26(1)

C(10) 10132(2) 3582(2) 4567(1) 28(1)

C(11) 10564(2) 2918(2) 4978(1) 25(1)

C(12) 11942(2) 2082(2) 5000(1) 24(1)

C(13) 12294(2) 1420(2) 5413(1) 25(1)

C(14) 13669(2) 579(2) 5455(1) 24(1)

C(15) 13966(2) -75(2) 5873(1) 26(1)

C(16) 15357(2) -845(2) 5943(1) 27(1)

C(17) 15615(2) -1517(2) 6356(1) 32(1)

C(18) 17019(3) -2196(3) 6447(1) 45(1)

C(19) 4525(2) 10922(2) 1956(1) 19(1)

C(20) 3690(2) 11798(2) 2262(1) 29(1)

C(21) 4365(3) 12529(2) 2435(1) 26(1)

C(22) 4152(2) 14561(2) 2553(1) 24(1)

315

C(23) 4425(3) 14388(2) 3010(1) 33(1)

C(24) 5301(3) 14505(3) 2304(1) 36(1)

C(25) 3038(3) 15704(2) 2458(1) 33(1)

C(26) 2830(2) 10610(2) 1519(1) 19(1)

C(27) 3081(2) 11027(2) 1093(1) 19(1)

C(28) 1863(2) 11689(2) 860(1) 21(1)

C(29) 965(2) 12062(2) 154(1) 22(1)

C(30) -179(2) 11757(3) 263(1) 34(1)

C(31) 660(2) 13374(2) 145(1) 26(1)

C(32) 1535(2) 11535(2) -252(1) 29(1)

C(33) 5084(2) 9091(2) 1571(1) 20(1)

C(34) 4782(2) 8079(2) 1405(1) 22(1)

C(35) 5852(2) 7355(2) 1137(1) 21(1)

C(36) 7510(2) 5392(2) 1076(1) 24(1)

C(37) 8616(2) 5802(2) 1098(1) 30(1)

C(38) 7765(3) 4286(2) 1337(1) 33(1)

C(39) 7181(2) 5226(2) 634(1) 31(1)

O(8) 8919(2) 1015(2) 7560(1) 27(1)

O(9) 8526(2) 3776(1) 8685(1) 22(1)

O(10) 8580(2) 2298(2) 9127(1) 37(1)

O(11) 13029(2) -1452(1) 9506(1) 22(1)

O(12) 14212(2) -2112(2) 8935(1) 38(1)

O(13) 11297(2) -3903(2) 7580(1) 31(1)

O(14) 9383(2) -2552(2) 7655(1) 101(1)

N(3) 10962(2) 406(2) 7807(1) 21(1)

N(4) 10457(2) 1275(2) 7163(1) 28(1)

C(40) 10773(2) -139(2) 8199(1) 17(1)

C(41) 10045(2) 897(2) 7511(1) 21(1)

C(42) 9608(2) 1810(2) 6815(1) 25(1)

C(43) 10244(2) 2344(2) 6510(1) 28(1)

C(44) 9465(2) 2836(2) 6119(1) 28(1)

C(45) 8179(2) 3815(2) 6182(1) 26(1)

C(46) 7533(2) 4379(2) 5780(1) 26(1)

C(47) 6214(2) 5299(2) 5814(1) 25(1)

C(48) 5698(2) 5915(2) 5403(1) 26(1)

C(49) 4340(2) 6803(2) 5406(1) 25(1)

316

C(50) 3946(2) 7470(2) 4995(1) 25(1)

C(51) 2580(2) 8320(2) 4961(1) 24(1)

C(52) 2286(2) 8983(2) 4547(1) 24(1)

C(53) 917(2) 9802(2) 4478(1) 25(1)

C(54) 705(2) 10437(2) 4056(1) 25(1)

C(55) -672(2) 11099(2) 3939(1) 31(1)

C(56) -858(3) 11738(3) 3521(1) 37(1)

C(57) -2245(3) 12373(3) 3400(1) 50(1)

C(58) 9741(2) 781(2) 8453(1) 18(1)

C(59) 10003(2) 1870(2) 8554(1) 23(1)

C(60) 8963(2) 2654(2) 8822(1) 21(1)

C(61) 7462(2) 4684(2) 8895(1) 23(1)

C(62) 7311(2) 5793(2) 8644(1) 29(1)

C(63) 6283(2) 4388(2) 8866(1) 28(1)

C(64) 7822(2) 4785(2) 9342(1) 31(1)

C(65) 12059(2) -582(2) 8431(1) 18(1)

C(66) 11964(2) -978(2) 8878(1) 20(1)

C(67) 13212(2) -1573(2) 9100(1) 22(1)

C(68) 14046(2) -2026(2) 9817(1) 21(1)

C(69) 15174(3) -1686(3) 9739(1) 44(1)

C(70) 14386(3) -3342(2) 9806(1) 37(1)

C(71) 13409(3) -1565(3) 10222(1) 33(1)

C(72) 10323(2) -1144(2) 8115(1) 20(1)

C(73) 11156(2) -2097(2) 7834(1) 24(1)

C(74) 10492(3) -2865(2) 7691(1) 31(1)

C(75) 10866(3) -4810(2) 7419(1) 33(1)

C(76A) 12077(4) -5897(4) 7437(3) 51(2)

C(77A) 9906(7) -5009(6) 7728(2) 40(2)

C(78A) 10332(8) -4435(7) 7008(2) 49(2)

C(76B) 12019(12) -5525(14) 7132(6) 43(5)

C(77B) 10644(18) -5523(15) 7733(4) 36(4)

C(78B) 9680(19) -4225(17) 7099(6) 37(4)

O(15) 3144(2) 10294(2) 7221(1) 36(1)

________________________________________________________________________________

317

Table I.2 Bond lengths [Å] and angles [°] of 3EUr16.

_____________________________________________________

O(1)-C(2) 1.236(3)

O(2)-C(21) 1.324(3)

O(2)-C(22) 1.487(3)

O(3)-C(21) 1.216(3)

O(4)-C(28) 1.343(3)

O(4)-C(29) 1.480(3)

O(5)-C(28) 1.205(3)

O(6)-C(35) 1.340(3)

O(6)-C(36) 1.478(3)

O(7)-C(35) 1.205(3)

N(1)-C(2) 1.360(3)

N(1)-C(1) 1.473(3)

N(2)-C(2) 1.352(3)

N(2)-C(3) 1.449(3)

C(1)-C(33) 1.535(3)

C(1)-C(26) 1.539(3)

C(1)-C(19) 1.541(3)

C(3)-C(4) 1.509(4)

C(4)-C(5) 1.528(3)

C(5)-C(6) 1.516(4)

C(6)-C(7) 1.516(3)

C(7)-C(8) 1.506(4)

C(8)-C(9) 1.521(4)

C(9)-C(10) 1.516(4)

C(10)-C(11) 1.519(3)

C(11)-C(12) 1.515(3)

C(12)-C(13) 1.515(3)

C(13)-C(14) 1.517(3)

C(14)-C(15) 1.523(3)

C(15)-C(16) 1.519(3)

C(16)-C(17) 1.518(4)

C(17)-C(18) 1.520(4)

C(19)-C(20) 1.523(3)

C(20)-C(21) 1.507(4)

C(22)-C(23) 1.503(4)

C(22)-C(24) 1.509(4)

C(22)-C(25) 1.518(3)

C(26)-C(27) 1.516(3)

C(27)-C(28) 1.507(3)

C(29)-C(32) 1.513(3)

C(29)-C(31) 1.515(3)

C(29)-C(30) 1.518(4)

C(33)-C(34) 1.523(3)

C(34)-C(35) 1.495(3)

C(36)-C(38) 1.508(4)

C(36)-C(39) 1.518(4)

C(36)-C(37) 1.523(4)

O(8)-C(41) 1.243(3)

O(9)-C(60) 1.338(3)

O(9)-C(61) 1.479(3)

O(10)-C(60) 1.211(3)

O(11)-C(67) 1.334(3)

O(11)-C(68) 1.483(3)

O(12)-C(67) 1.208(3)

O(13)-C(74) 1.317(3)

O(13)-C(75) 1.486(3)

O(14)-C(74) 1.176(3)

N(3)-C(41) 1.365(3)

N(3)-C(40) 1.468(3)

N(4)-C(41) 1.351(3)

N(4)-C(42) 1.454(3)

C(40)-C(72) 1.537(3)

C(40)-C(58) 1.539(3)

C(40)-C(65) 1.543(3)

C(42)-C(43) 1.496(3)

C(43)-C(44) 1.519(3)

C(44)-C(45) 1.518(3)

C(45)-C(46) 1.512(3)

318

C(46)-C(47) 1.504(3)

C(47)-C(48) 1.514(3)

C(48)-C(49) 1.517(3)

C(49)-C(50) 1.517(3)

C(50)-C(51) 1.510(3)

C(51)-C(52) 1.518(3)

C(52)-C(53) 1.513(3)

C(53)-C(54) 1.523(3)

C(54)-C(55) 1.510(3)

C(55)-C(56) 1.517(4)

C(56)-C(57) 1.518(4)

C(58)-C(59) 1.521(3)

C(59)-C(60) 1.501(3)

C(61)-C(62) 1.516(4)

C(61)-C(63) 1.519(3)

C(61)-C(64) 1.526(4)

C(65)-C(66) 1.528(3)

C(66)-C(67) 1.502(3)

C(68)-C(69) 1.508(4)

C(68)-C(71) 1.510(3)

C(68)-C(70) 1.516(4)

C(72)-C(73) 1.519(3)

C(73)-C(74) 1.498(4)

C(75)-C(77B) 1.403(13)

C(75)-C(78A) 1.452(8)

C(75)-C(76A) 1.513(5)

C(75)-C(77A) 1.554(6)

C(75)-C(76B) 1.586(12)

C(75)-C(78B) 1.625(19)

C(21)-O(2)-C(22) 121.8(2)

C(28)-O(4)-C(29) 121.49(19)

C(35)-O(6)-C(36) 120.59(19)

C(2)-N(1)-C(1) 125.9(2)

C(2)-N(2)-C(3) 121.5(2)

N(1)-C(1)-C(33) 111.03(19)

N(1)-C(1)-C(26) 104.45(18)

C(33)-C(1)-C(26) 111.4(2)

N(1)-C(1)-C(19) 111.56(19)

C(33)-C(1)-C(19) 106.92(18)

C(26)-C(1)-C(19) 111.6(2)

O(1)-C(2)-N(2) 122.0(2)

O(1)-C(2)-N(1) 122.8(2)

N(2)-C(2)-N(1) 115.2(2)

N(2)-C(3)-C(4) 114.2(2)

C(3)-C(4)-C(5) 112.9(2)

C(6)-C(5)-C(4) 115.7(2)

C(5)-C(6)-C(7) 112.5(2)

C(8)-C(7)-C(6) 115.2(2)

C(7)-C(8)-C(9) 113.3(2)

C(10)-C(9)-C(8) 115.2(2)

C(9)-C(10)-C(11) 112.8(2)

C(12)-C(11)-C(10) 115.7(2)

C(13)-C(12)-C(11) 112.8(2)

C(12)-C(13)-C(14) 115.2(2)

C(13)-C(14)-C(15) 113.0(2)

C(16)-C(15)-C(14) 114.7(2)

C(17)-C(16)-C(15) 113.4(2)

C(16)-C(17)-C(18) 113.8(2)

C(20)-C(19)-C(1) 115.81(19)

C(21)-C(20)-C(19) 111.3(2)

O(3)-C(21)-O(2) 123.9(2)

O(3)-C(21)-C(20) 123.7(2)

O(2)-C(21)-C(20) 112.4(2)

O(2)-C(22)-C(23) 109.8(2)

O(2)-C(22)-C(24) 110.2(2)

C(23)-C(22)-C(24) 112.6(2)

O(2)-C(22)-C(25) 102.0(2)

C(23)-C(22)-C(25) 111.2(2)

C(24)-C(22)-C(25) 110.5(2)

C(27)-C(26)-C(1) 116.22(19)

C(28)-C(27)-C(26) 111.6(2)

319

O(5)-C(28)-O(4) 125.0(2)

O(5)-C(28)-C(27) 124.1(2)

O(4)-C(28)-C(27) 110.8(2)

O(4)-C(29)-C(32) 102.99(19)

O(4)-C(29)-C(31) 108.9(2)

C(32)-C(29)-C(31) 110.9(2)

O(4)-C(29)-C(30) 110.3(2)

C(32)-C(29)-C(30) 110.7(2)

C(31)-C(29)-C(30) 112.7(2)

C(34)-C(33)-C(1) 115.69(19)

C(35)-C(34)-C(33) 109.1(2)

O(7)-C(35)-O(6) 124.7(2)

O(7)-C(35)-C(34) 123.5(2)

O(6)-C(35)-C(34) 111.8(2)

O(6)-C(36)-C(38) 102.0(2)

O(6)-C(36)-C(39) 110.61(19)

C(38)-C(36)-C(39) 110.8(2)

O(6)-C(36)-C(37) 109.4(2)

C(38)-C(36)-C(37) 111.2(2)

C(39)-C(36)-C(37) 112.3(2)

C(60)-O(9)-C(61) 120.78(19)

C(67)-O(11)-C(68) 123.16(19)

C(74)-O(13)-C(75) 122.2(2)

C(41)-N(3)-C(40) 124.8(2)

C(41)-N(4)-C(42) 121.6(2)

N(3)-C(40)-C(72) 109.91(19)

N(3)-C(40)-C(58) 109.55(19)

C(72)-C(40)-C(58) 108.18(19)

N(3)-C(40)-C(65) 106.60(18)

C(72)-C(40)-C(65) 112.0(2)

C(58)-C(40)-C(65) 110.59(19)

O(8)-C(41)-N(4) 122.4(2)

O(8)-C(41)-N(3) 123.1(2)

N(4)-C(41)-N(3) 114.4(2)

N(4)-C(42)-C(43) 109.9(2)

C(42)-C(43)-C(44) 113.4(2)

C(45)-C(44)-C(43) 115.6(2)

C(46)-C(45)-C(44) 112.8(2)

C(47)-C(46)-C(45) 116.1(2)

C(46)-C(47)-C(48) 112.6(2)

C(47)-C(48)-C(49) 115.9(2)

C(50)-C(49)-C(48) 112.4(2)

C(51)-C(50)-C(49) 116.0(2)

C(50)-C(51)-C(52) 112.4(2)

C(53)-C(52)-C(51) 115.8(2)

C(52)-C(53)-C(54) 112.4(2)

C(55)-C(54)-C(53) 114.8(2)

C(54)-C(55)-C(56) 113.8(2)

C(55)-C(56)-C(57) 113.4(2)

C(59)-C(58)-C(40) 115.6(2)

C(60)-C(59)-C(58) 109.9(2)

O(10)-C(60)-O(9) 124.7(2)

O(10)-C(60)-C(59) 123.1(2)

O(9)-C(60)-C(59) 112.2(2)

O(9)-C(61)-C(62) 102.6(2)

O(9)-C(61)-C(63) 109.1(2)

C(62)-C(61)-C(63) 111.5(2)

O(9)-C(61)-C(64) 110.40(19)

C(62)-C(61)-C(64) 110.6(2)

C(63)-C(61)-C(64) 112.2(2)

C(66)-C(65)-C(40) 112.96(19)

C(67)-C(66)-C(65) 115.2(2)

O(12)-C(67)-O(11) 125.7(2)

O(12)-C(67)-C(66) 125.0(2)

O(11)-C(67)-C(66) 109.3(2)

O(11)-C(68)-C(69) 111.0(2)

O(11)-C(68)-C(71) 103.27(19)

C(69)-C(68)-C(71) 111.5(2)

O(11)-C(68)-C(70) 107.9(2)

C(69)-C(68)-C(70) 112.7(2)

C(71)-C(68)-C(70) 110.0(2)

C(73)-C(72)-C(40) 116.5(2)

320

C(74)-C(73)-C(72) 112.0(2)

O(14)-C(74)-O(13) 123.7(3)

O(14)-C(74)-C(73) 124.0(3)

O(13)-C(74)-C(73) 112.2(2)

C(77B)-C(75)-C(78A) 133.2(7)

C(77B)-C(75)-O(13) 113.0(6)

C(78A)-C(75)-O(13) 108.7(4)

C(77B)-C(75)-C(76A) 75.9(7)

C(78A)-C(75)-C(76A) 115.3(4)

O(13)-C(75)-C(76A) 101.9(2)

C(77B)-C(75)-C(77A) 32.4(6)

C(78A)-C(75)-C(77A) 113.0(4)

O(13)-C(75)-C(77A) 109.2(3)

C(76A)-C(75)-C(77A) 108.0(3)

C(77B)-C(75)-C(76B) 112.3(8)

C(78A)-C(75)-C(76B) 78.0(7)

O(13)-C(75)-C(76B) 101.7(5)

C(76A)-C(75)-C(76B) 40.2(6)

C(77A)-C(75)-C(76B) 140.6(6)

C(77B)-C(75)-C(78B) 113.8(8)

C(78A)-C(75)-C(78B) 27.4(6)

O(13)-C(75)-C(78B) 111.0(7)

C(76A)-C(75)-C(78B) 136.6(7)

C(77A)-C(75)-C(78B) 87.3(6)

C(76B)-C(75)-C(78B) 104.0(8)

_____________________________________

321

Table I.3 Anisotropic displacement parameters (Å2x 103) of 3EUr16. The anisotropic

displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

______________________________________________________________________________

U11 U22 U33 U23 U13 U12

______________________________________________________________________________

O(1) 17(1) 31(1) 26(1) 5(1) -5(1) -9(1)

O(2) 23(1) 22(1) 30(1) -5(1) -3(1) -8(1)

O(3) 29(1) 26(1) 45(1) -6(1) -15(1) -5(1)

O(4) 21(1) 27(1) 12(1) 1(1) -3(1) -6(1)

O(5) 22(1) 40(1) 23(1) -1(1) 1(1) 1(1)

O(6) 23(1) 20(1) 20(1) -3(1) 1(1) -4(1)

O(7) 35(1) 29(1) 23(1) 5(1) 6(1) -5(1)

N(1) 16(1) 21(1) 18(1) 6(1) -2(1) -5(1)

N(2) 18(1) 29(1) 21(1) 9(1) -4(1) -9(1)

C(1) 16(1) 20(1) 15(1) 2(1) 0(1) -7(1)

C(2) 22(2) 18(1) 16(1) 1(1) -2(1) -7(1)

C(3) 26(2) 23(2) 23(2) 5(1) -3(1) -10(1)

C(4) 34(2) 22(2) 27(2) 2(1) -4(1) -8(1)

C(5) 38(2) 27(2) 22(2) 4(1) -9(1) -14(1)

C(6) 39(2) 29(2) 26(2) 3(1) -9(1) -13(1)

C(7) 33(2) 26(2) 25(2) 5(1) -9(1) -11(1)

C(8) 38(2) 33(2) 23(2) 1(1) -9(1) -10(1)

C(9) 31(2) 26(2) 24(2) 6(1) -8(1) -12(1)

C(10) 36(2) 31(2) 20(2) 1(1) -7(1) -15(1)

C(11) 27(2) 26(2) 24(2) 4(1) -3(1) -13(1)

C(12) 27(2) 24(2) 24(2) 1(1) -3(1) -13(1)

C(13) 24(2) 26(2) 27(2) 0(1) -1(1) -11(1)

C(14) 24(2) 26(2) 23(2) 0(1) 0(1) -13(1)

C(15) 25(2) 24(2) 30(2) 2(1) -2(1) -11(1)

C(16) 24(2) 28(2) 32(2) 3(1) 0(1) -12(1)

C(17) 28(2) 25(2) 41(2) 5(1) -2(1) -10(1)

C(18) 34(2) 44(2) 55(2) 18(2) -7(2) -15(2)

C(19) 17(1) 21(1) 18(1) 1(1) -1(1) -7(1)

C(20) 23(2) 26(2) 37(2) -8(1) 0(1) -9(1)

C(21) 27(2) 26(2) 25(2) -7(1) 2(1) -9(1)

C(22) 29(2) 25(2) 21(2) -2(1) -3(1) -13(1)

322

C(23) 40(2) 29(2) 28(2) -6(1) -5(1) -11(1)

C(24) 35(2) 42(2) 36(2) -6(2) 3(1) -20(2)

C(25) 37(2) 23(2) 38(2) -4(1) -5(1) -9(1)

C(26) 20(1) 19(1) 18(1) 1(1) -1(1) -8(1)

C(27) 18(1) 23(1) 15(1) -1(1) -2(1) -8(1)

C(28) 21(2) 23(1) 19(2) 1(1) 1(1) -8(1)

C(29) 21(1) 24(2) 18(2) 3(1) -8(1) -7(1)

C(30) 32(2) 44(2) 30(2) 7(1) -9(1) -20(2)

C(31) 29(2) 24(2) 22(2) 4(1) -7(1) -7(1)

C(32) 32(2) 33(2) 17(2) -1(1) -7(1) -6(1)

C(33) 15(1) 24(1) 18(1) 2(1) -1(1) -6(1)

C(34) 22(1) 21(1) 24(2) -3(1) 2(1) -8(1)

C(35) 20(1) 23(2) 19(2) -2(1) -3(1) -8(1)

C(36) 16(1) 25(2) 26(2) -7(1) 2(1) -2(1)

C(37) 21(2) 32(2) 35(2) 0(1) -2(1) -7(1)

C(38) 29(2) 23(2) 42(2) -1(1) 2(1) -3(1)

C(39) 24(2) 35(2) 32(2) -14(1) 4(1) -9(1)

O(8) 17(1) 39(1) 23(1) 6(1) -3(1) -9(1)

O(9) 22(1) 17(1) 23(1) -3(1) 2(1) -4(1)

O(10) 43(1) 28(1) 32(1) 1(1) 16(1) -7(1)

O(11) 20(1) 29(1) 11(1) 0(1) -3(1) -3(1)

O(12) 21(1) 60(1) 19(1) -2(1) 1(1) 0(1)

O(13) 22(1) 26(1) 46(1) -14(1) 4(1) -8(1)

O(14) 24(1) 82(2) 200(3) -96(2) 5(2) -15(1)

N(3) 15(1) 29(1) 18(1) 5(1) -2(1) -9(1)

N(4) 19(1) 41(1) 22(1) 10(1) -3(1) -11(1)

C(40) 16(1) 21(1) 14(1) 4(1) -1(1) -6(1)

C(41) 22(2) 21(1) 19(2) 0(1) -2(1) -7(1)

C(42) 25(2) 28(2) 20(2) 3(1) -5(1) -8(1)

C(43) 23(2) 36(2) 22(2) 6(1) -3(1) -8(1)

C(44) 30(2) 31(2) 22(2) 8(1) -1(1) -10(1)

C(45) 27(2) 28(2) 23(2) 4(1) -3(1) -10(1)

C(46) 28(2) 23(2) 25(2) 5(1) -6(1) -10(1)

C(47) 30(2) 23(2) 22(2) 6(1) -6(1) -10(1)

C(48) 29(2) 26(2) 24(2) 5(1) -6(1) -11(1)

C(49) 28(2) 26(2) 24(2) 6(1) -6(1) -13(1)

323

C(50) 30(2) 23(2) 21(2) 7(1) -5(1) -10(1)

C(51) 28(2) 25(2) 21(2) 3(1) -3(1) -14(1)

C(52) 28(2) 24(2) 21(2) 3(1) -2(1) -12(1)

C(53) 26(2) 23(2) 26(2) 4(1) -4(1) -11(1)

C(54) 23(2) 23(2) 27(2) 3(1) -2(1) -8(1)

C(55) 29(2) 34(2) 31(2) 9(1) -5(1) -12(1)

C(56) 31(2) 39(2) 35(2) 11(2) -7(1) -7(1)

C(57) 43(2) 65(2) 36(2) 19(2) -11(2) -15(2)

C(58) 19(1) 21(1) 13(1) 1(1) -1(1) -7(1)

C(59) 20(1) 21(1) 25(2) -2(1) 4(1) -5(1)

C(60) 22(2) 21(2) 21(2) -1(1) -2(1) -8(1)

C(61) 18(1) 21(1) 26(2) -9(1) 1(1) -4(1)

C(62) 29(2) 20(2) 34(2) -2(1) 0(1) -5(1)

C(63) 22(2) 31(2) 31(2) -7(1) 2(1) -9(1)

C(64) 25(2) 38(2) 30(2) -14(1) 3(1) -11(1)

C(65) 18(1) 23(1) 13(1) -1(1) 0(1) -7(1)

C(66) 17(1) 26(2) 16(1) 1(1) -1(1) -6(1)

C(67) 22(2) 26(2) 16(2) 2(1) 0(1) -9(1)

C(68) 22(1) 27(2) 16(1) 4(1) -7(1) -9(1)

C(69) 42(2) 73(2) 31(2) 12(2) -9(2) -38(2)

C(70) 45(2) 33(2) 26(2) 1(1) -13(1) -5(1)

C(71) 34(2) 45(2) 16(2) -1(1) -3(1) -10(1)

C(72) 20(1) 24(1) 16(1) 0(1) -2(1) -7(1)

C(73) 21(1) 24(2) 26(2) -3(1) 0(1) -6(1)

C(74) 22(2) 33(2) 37(2) -17(1) 3(1) -8(1)

C(75) 28(2) 28(2) 43(2) -10(2) -2(1) -11(1)

C(76A) 38(3) 29(3) 82(5) -20(3) 1(3) -9(2)

C(77A) 42(4) 43(4) 41(3) 0(3) -4(3) -24(3)

C(78A) 71(6) 39(3) 40(4) -10(3) -6(4) -26(4)

C(76B) 37(7) 48(9) 57(12) -36(8) 18(8) -27(7)

C(77B) 36(10) 38(9) 32(7) 0(7) 5(7) -12(7)

C(78B) 56(12) 45(10) 22(9) -4(7) -10(8) -32(11)

O(15) 23(1) 32(1) 51(1) 1(1) -9(1) -9(1)

______________________________________________________________________________

324

Table I.4 Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3) of 3EUr16.

________________________________________________________________________________

x y z U(eq)

________________________________________________________________________________

H(1A) 2721 9594 2143 23

H(2A) 2777 8638 2720 27

H(3A) 5079 7115 3007 28

H(3B) 3736 7221 3194 28

H(4A) 3636 8948 3538 34

H(4B) 5035 8733 3374 34

H(5A) 5004 8032 4075 34

H(5B) 4475 7093 3909 34

H(6A) 6923 7080 3700 38

H(6B) 6393 6102 3572 38

H(7A) 6863 6524 4407 34

H(7B) 6359 5535 4275 34

H(8A) 8812 5512 4076 39

H(8B) 8307 4517 3950 39

H(9A) 8658 5007 4788 32

H(9B) 8221 3979 4651 32

H(10A) 10678 4027 4499 34

H(10B) 10243 3009 4347 34

H(11A) 10416 3499 5197 30

H(11B) 10023 2464 5041 30

H(12A) 12106 1509 4778 29

H(12B) 12493 2534 4951 29

H(13A) 11742 969 5459 30

H(13B) 12110 2000 5634 30

H(14A) 14228 1027 5415 28

H(14B) 13863 -1 5234 28

H(15A) 13692 509 6094 31

H(15B) 13458 -574 5901 31

H(16A) 15643 -1413 5718 33

H(16B) 15866 -344 5927 33

H(17A) 15175 -2076 6360 38

325

H(17B) 15249 -955 6579 38

H(18A) 17399 -2731 6222 67

H(18B) 17113 -2651 6708 67

H(18C) 17448 -1643 6470 67

H(19A) 5362 10486 2088 22

H(19B) 4669 11367 1713 22

H(20A) 2898 12321 2121 34

H(20B) 3455 11367 2491 34

H(23A) 3663 14399 3157 49

H(23B) 4665 15023 3107 49

H(23C) 5123 13630 3064 49

H(24A) 6035 13807 2392 54

H(24B) 5481 15212 2349 54

H(24C) 5131 14461 2010 54

H(25A) 2833 15760 2162 49

H(25B) 3265 16369 2530 49

H(25C) 2299 15721 2619 49

H(26A) 2409 10045 1484 23

H(26B) 2223 11294 1662 23

H(27A) 3563 11545 1122 23

H(27B) 3609 10340 934 23

H(30A) 94 10904 307 51

H(30B) -798 12019 35 51

H(30C) -571 12152 516 51

H(31A) 321 13691 414 39

H(31B) 26 13758 -71 39

H(31C) 1435 13521 87 39

H(32A) 2309 11687 -306 44

H(32B) 920 11891 -476 44

H(32C) 1743 10684 -237 44

H(33A) 5342 9487 1336 23

H(33B) 5822 8760 1761 23

H(34A) 4668 7586 1638 27

H(34B) 3983 8392 1242 27

H(37A) 8451 6490 911 45

H(37B) 9400 5167 1014 45

326

H(37C) 8710 6014 1382 45

H(38A) 7848 4447 1628 50

H(38B) 8554 3671 1246 50

H(38C) 7058 4022 1306 50

H(39A) 6377 5104 631 46

H(39B) 7858 4538 523 46

H(39C) 7097 5927 464 46

H(3C) 11720 416 7758 25

H(4C) 11259 1195 7146 33

H(42A) 8818 2425 6916 31

H(42B) 9376 1207 6680 31

H(43A) 11072 1739 6433 33

H(43B) 10416 2984 6643 33

H(44A) 9967 3136 5928 34

H(44B) 9328 2185 5982 34

H(45A) 7624 3492 6337 31

H(45B) 8294 4425 6352 31

H(46A) 8072 4742 5636 31

H(46B) 7492 3751 5603 31

H(47A) 6226 5889 6013 30

H(47B) 5639 4923 5924 30

H(48A) 5754 5311 5201 31

H(48B) 6255 6323 5304 31

H(49A) 3762 6389 5473 30

H(49B) 4252 7371 5626 30

H(50A) 4503 7912 4939 30

H(50B) 4103 6890 4775 30

H(51A) 2404 8891 5185 28

H(51B) 2013 7881 5001 28

H(52A) 2828 9449 4517 29

H(52B) 2526 8402 4325 29

H(53A) 666 10391 4697 29

H(53B) 365 9344 4502 29

H(54A) 1141 10999 4053 30

H(54B) 1108 9852 3843 30

H(55A) -1081 11679 4153 38

327

H(55B) -1108 10537 3937 38

H(56A) -430 11162 3307 44

H(56B) -444 12316 3525 44

H(57A) -2654 11802 3383 75

H(57B) -2302 12779 3131 75

H(57C) -2675 12949 3609 75

H(58A) 8928 1035 8298 22

H(58B) 9627 398 8716 22

H(59A) 10829 1633 8700 28

H(59B) 10051 2302 8295 28

H(62A) 8071 5978 8678 44

H(62B) 6564 6447 8741 44

H(62C) 7200 5673 8351 44

H(63A) 6177 4189 8582 43

H(63B) 5536 5069 8946 43

H(63C) 6376 3716 9053 43

H(64A) 7866 4081 9503 46

H(64B) 7178 5482 9461 46

H(64C) 8651 4858 9348 46

H(65A) 12674 -1248 8281 22

H(65B) 12391 57 8429 22

H(66A) 11442 -282 9035 24

H(66B) 11510 -1525 8880 24

H(69A) 14883 -830 9701 66

H(69B) 15616 -2064 9490 66

H(69C) 15757 -1945 9977 66

H(70A) 14996 -3736 10028 56

H(70B) 14764 -3628 9538 56

H(70C) 13616 -3513 9845 56

H(71A) 13178 -712 10224 50

H(71B) 13994 -1941 10452 50

H(71C) 12641 -1743 10252 50

H(72A) 10247 -1522 8383 25

H(72B) 9462 -798 7990 25

H(73A) 11403 -1724 7590 29

H(73B) 11942 -2588 7985 29

328

H(76A) 12340 -6107 7726 76

H(76B) 12748 -5738 7285 76

H(76C) 11929 -6549 7311 76

H(77A) 9789 -5735 7663 59

H(77B) 9089 -4343 7704 59

H(77C) 10236 -5073 8010 59

H(78A) 10068 -5039 6895 73

H(78B) 10973 -4318 6827 73

H(78C) 9593 -3694 7024 73

H(76D) 12804 -5831 7296 65

H(76E) 12107 -5003 6908 65

H(76F) 11859 -6181 7014 65

H(77D) 10439 -6147 7609 54

H(77E) 9932 -5045 7904 54

H(77F) 11408 -5876 7906 54

H(78D) 9522 -4848 6956 55

H(78E) 9885 -3715 6896 55

H(78F) 8919 -3759 7252 55

H(15C) 3590(30) 9556(17) 7246(12) 90(10)

H(15D) 3530(30) 10730(30) 7307(11) 90(10)

________________________________________________________________________________

329

Table I.5 Torsion angles [°] of 3EUr16.

________________________________________________________________

C(2)-N(1)-C(1)-C(33) 57.0(3)

C(2)-N(1)-C(1)-C(26) 177.2(2)

C(2)-N(1)-C(1)-C(19) -62.1(3)

C(3)-N(2)-C(2)-O(1) -1.3(4)

C(3)-N(2)-C(2)-N(1) 177.9(2)

C(1)-N(1)-C(2)-O(1) -2.1(4)

C(1)-N(1)-C(2)-N(2) 178.7(2)

C(2)-N(2)-C(3)-C(4) 83.3(3)

N(2)-C(3)-C(4)-C(5) 174.8(2)

C(3)-C(4)-C(5)-C(6) 67.4(3)

C(4)-C(5)-C(6)-C(7) 175.3(2)

C(5)-C(6)-C(7)-C(8) -178.9(2)

C(6)-C(7)-C(8)-C(9) 179.4(2)

C(7)-C(8)-C(9)-C(10) -177.1(2)

C(8)-C(9)-C(10)-C(11) -178.2(2)

C(9)-C(10)-C(11)-C(12) -178.3(2)

C(10)-C(11)-C(12)-C(13) -178.0(2)

C(11)-C(12)-C(13)-C(14) -179.3(2)

C(12)-C(13)-C(14)-C(15) -179.2(2)

C(13)-C(14)-C(15)-C(16) -174.8(2)

C(14)-C(15)-C(16)-C(17) -178.0(2)

C(15)-C(16)-C(17)-C(18) -174.3(2)

N(1)-C(1)-C(19)-C(20) -47.4(3)

C(33)-C(1)-C(19)-C(20) -168.9(2)

C(26)-C(1)-C(19)-C(20) 69.0(3)

C(1)-C(19)-C(20)-C(21) 172.6(2)

C(22)-O(2)-C(21)-O(3) 1.2(4)

C(22)-O(2)-C(21)-C(20) -178.3(2)

C(19)-C(20)-C(21)-O(3) -49.4(4)

C(19)-C(20)-C(21)-O(2) 130.1(2)

C(21)-O(2)-C(22)-C(23) -62.9(3)

C(21)-O(2)-C(22)-C(24) 61.6(3)

C(21)-O(2)-C(22)-C(25) 179.0(2)

N(1)-C(1)-C(26)-C(27) -167.7(2)

C(33)-C(1)-C(26)-C(27) -47.8(3)

C(19)-C(1)-C(26)-C(27) 71.6(3)

C(1)-C(26)-C(27)-C(28) -174.6(2)

C(29)-O(4)-C(28)-O(5) 0.6(4)

C(29)-O(4)-C(28)-C(27) -177.68(19)

C(26)-C(27)-C(28)-O(5) 36.8(3)

C(26)-C(27)-C(28)-O(4) -144.8(2)

C(28)-O(4)-C(29)-C(32) -177.8(2)

C(28)-O(4)-C(29)-C(31) 64.5(3)

C(28)-O(4)-C(29)-C(30) -59.7(3)

N(1)-C(1)-C(33)-C(34) 55.7(3)

C(26)-C(1)-C(33)-C(34) -60.2(3)

C(19)-C(1)-C(33)-C(34) 177.6(2)

C(1)-C(33)-C(34)-C(35) 171.6(2)

C(36)-O(6)-C(35)-O(7) 1.7(4)

C(36)-O(6)-C(35)-C(34) -177.09(19)

C(33)-C(34)-C(35)-O(7) -59.6(3)

C(33)-C(34)-C(35)-O(6) 119.3(2)

C(35)-O(6)-C(36)-C(38) -179.0(2)

C(35)-O(6)-C(36)-C(39) -61.1(3)

C(35)-O(6)-C(36)-C(37) 63.1(3)

C(41)-N(3)-C(40)-C(72) 57.8(3)

C(41)-N(3)-C(40)-C(58) -60.9(3)

C(41)-N(3)-C(40)-C(65) 179.4(2)

C(42)-N(4)-C(41)-O(8) -2.1(4)

C(42)-N(4)-C(41)-N(3) 179.4(2)

C(40)-N(3)-C(41)-O(8) 5.8(4)

C(40)-N(3)-C(41)-N(4) -175.7(2)

C(41)-N(4)-C(42)-C(43) 169.4(2)

N(4)-C(42)-C(43)-C(44) 175.3(2)

C(42)-C(43)-C(44)-C(45) 60.9(3)

C(43)-C(44)-C(45)-C(46) 172.1(2)

C(44)-C(45)-C(46)-C(47) 175.8(2)

C(45)-C(46)-C(47)-C(48) 173.4(2)

330

C(46)-C(47)-C(48)-C(49) 176.3(2)

C(47)-C(48)-C(49)-C(50) 174.0(2)

C(48)-C(49)-C(50)-C(51) 176.3(2)

C(49)-C(50)-C(51)-C(52) 177.6(2)

C(50)-C(51)-C(52)-C(53) 176.6(2)

C(51)-C(52)-C(53)-C(54) 179.7(2)

C(52)-C(53)-C(54)-C(55) 169.7(2)

C(53)-C(54)-C(55)-C(56) 179.2(2)

C(54)-C(55)-C(56)-C(57) 178.4(3)

N(3)-C(40)-C(58)-C(59) -58.2(3)

C(72)-C(40)-C(58)-C(59) -178.0(2)

C(65)-C(40)-C(58)-C(59) 59.0(3)

C(40)-C(58)-C(59)-C(60) -176.7(2)

C(61)-O(9)-C(60)-O(10) -3.0(4)

C(61)-O(9)-C(60)-C(59) 177.42(19)

C(58)-C(59)-C(60)-O(10) 48.4(3)

C(58)-C(59)-C(60)-O(9) -132.0(2)

C(60)-O(9)-C(61)-C(62) 179.1(2)

C(60)-O(9)-C(61)-C(63) -62.5(3)

C(60)-O(9)-C(61)-C(64) 61.3(3)

N(3)-C(40)-C(65)-C(66) 169.8(2)

C(72)-C(40)-C(65)-C(66) -69.9(3)

C(58)-C(40)-C(65)-C(66) 50.8(3)

C(40)-C(65)-C(66)-C(67) 171.8(2)

C(68)-O(11)-C(67)-O(12) -2.9(4)

C(68)-O(11)-C(67)-C(66) 174.5(2)

C(65)-C(66)-C(67)-O(12) -27.3(4)

C(65)-C(66)-C(67)-O(11) 155.3(2)

C(67)-O(11)-C(68)-C(69) 56.7(3)

C(67)-O(11)-C(68)-C(71) 176.3(2)

C(67)-O(11)-C(68)-C(70) -67.2(3)

N(3)-C(40)-C(72)-C(73) 55.1(3)

C(58)-C(40)-C(72)-C(73) 174.6(2)

C(65)-C(40)-C(72)-C(73) -63.2(3)

C(40)-C(72)-C(73)-C(74) -166.4(2)

C(75)-O(13)-C(74)-O(14) -3.4(5)

C(75)-O(13)-C(74)-C(73) -178.7(2)

C(72)-C(73)-C(74)-O(14) 27.7(4)

C(72)-C(73)-C(74)-O(13) -157.1(2)

C(74)-O(13)-C(75)-C(77B) -87.3(9)

C(74)-O(13)-C(75)-C(78A) 70.9(5)

C(74)-O(13)-C(75)-C(76A) -166.9(4)

C(74)-O(13)-C(75)-C(77A) -52.7(4)

C(74)-O(13)-C(75)-C(76B) 152.1(9)

C(74)-O(13)-C(75)-C(78B) 41.9(8)

________________________________________

331

Table I.6 Hydrogen bonds [Å and °] of 3EUr16.

___________________________________________

D-H...A d(D-H) d(H...A) d(D...A) <(DHA)

____________________________________________________________________________

N(1)-H(1A)...O(8)#1 0.88 2.44 3.201(3) 145.6

N(2)-H(2A)...O(8)#1 0.88 2.02 2.863(3) 159.1

N(3)-H(3C)...O(15)#2 0.88 2.33 3.074(3) 141.6

N(4)-H(4C)...O(15)#2 0.88 2.02 2.832(3) 153.4

O(15)-H(15C)...O(3)#3 0.857(2) 2.00(2) 2.843(4) 169(4)

O(15)-H(15D)...O(1)#3 0.863(2) 1.864(2) 2.718(4) 170(4)

____________________________________________________________________________

Symmetry transformations used to generate equivalent atoms:

#1 -x+1,-y+1,-z+1 #2 x+1,y-1,z #3 -x+1,-y+2,-z+1

332

Table II.1 Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) of 3EUr-α-cholestane. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ________________________________________________________________________________ x y z U(eq) ________________________________________________________________________________ O(1) 4343(4) 7655(4) 1830(1) 50(1)

O(2) -620(4) 9402(4) 1567(1) 60(1)

O(3) -294(4) 10571(3) 1122(1) 44(1)

O(4) 3705(8) 6352(6) 399(2) 123(3)

O(5) 3589(5) 8131(4) 384(1) 64(2)

O(6) 2572(5) 11008(4) 2079(2) 72(2)

O(7) 2610(4) 10050(4) 2614(1) 48(1)

O(8) 9386(4) 5986(4) 1773(1) 51(1)

O(9) 7457(5) 2761(4) 2168(1) 63(2)

O(10) 7683(4) 3979(3) 2635(1) 41(1)

O(11) 4396(4) 4184(4) 1538(2) 66(2)

O(12) 4730(4) 3064(4) 1079(1) 50(1)

O(13) 9020(9) 7089(7) 437(2) 177(5)

O(14) 8637(4) 5388(4) 364(1) 65(2)

N(1) 2519(5) 7219(4) 1651(1) 41(1)

N(2) 3587(5) 6300(4) 2123(1) 41(1)

N(3) 7549(5) 6421(4) 1614(1) 38(1)

N(4) 8654(4) 7267(4) 2102(1) 36(1)

C(1) 2181(5) 8135(5) 1412(2) 38(2)

C(2) 3547(6) 7078(5) 1869(2) 38(2)

C(3) 4633(5) 6006(5) 2370(2) 41(2)

C(4) 4529(6) 4876(5) 2507(2) 45(2)

C(5) 3742(5) 4775(5) 2801(2) 40(2)

C(6) 4045(5) 5495(5) 3147(2) 37(2)

C(7) 3146(5) 5468(4) 3405(2) 33(1)

C(8) 2955(6) 4404(5) 3576(2) 40(2)

C(9) 2010(5) 4369(5) 3816(2) 34(1)

C(10) 2142(5) 5196(5) 4132(2) 37(1)

C(11) 1105(5) 5450(5) 4309(2) 36(2)

C(12) 1317(6) 6572(5) 4461(2) 47(2)

C(13) 2196(6) 7045(5) 4246(2) 44(2)

C(14) 2298(5) 6246(5) 3937(2) 37(2)

333

C(15) 3302(5) 6297(5) 3719(2) 36(2)

C(16) 3439(6) 7397(5) 3563(2) 45(2)

C(17) 4316(5) 7423(5) 3305(2) 47(2)

C(18) 4094(5) 6616(5) 2990(2) 37(2)

C(19) 4911(6) 6748(5) 2704(2) 43(2)

C(20) 5184(5) 5156(6) 3366(2) 44(2)

C(21) 3137(5) 4928(5) 4443(2) 44(2)

C(22) 693(5) 4736(5) 4609(2) 37(1)

C(23) 653(5) 3592(5) 4490(2) 41(2)

C(24) -488(5) 5095(5) 4663(2) 42(2)

C(25) -882(6) 4733(5) 5033(2) 46(2)

C(26) -578(6) 5472(5) 5360(2) 47(2)

C(27) -934(6) 5192(5) 5735(2) 46(2)

C(28) -2208(6) 5174(7) 5705(2) 61(2)

C(29) -441(7) 5942(6) 6040(2) 62(2)

C(30) 905(6) 7969(5) 1287(2) 46(2)

C(31) 307(6) 8856(5) 1054(2) 43(2)

C(32) -258(6) 9626(5) 1283(2) 43(2)

C(33) -875(6) 11457(6) 1286(2) 54(2)

C(34) -296(9) 11684(8) 1690(2) 93(3)

C(35) -2103(6) 11220(7) 1254(3) 73(3)

C(36) -663(7) 12334(6) 1021(3) 76(3)

C(37) 2808(6) 8169(5) 1072(2) 47(2)

C(38) 2643(8) 7246(6) 810(2) 66(2)

C(39) 3400(9) 7198(8) 518(2) 86(3)

C(40) 4261(7) 8274(9) 71(2) 77(3)

C(41) 5411(9) 7779(11) 197(3) 121(5)

C(42) 3660(7) 7772(7) -289(2) 71(2)

C(43) 4287(7) 9459(8) 40(2) 79(3)

C(44) 2433(5) 9162(5) 1637(2) 38(2)

C(45) 2110(6) 9183(5) 2038(2) 43(2)

C(46) 2447(5) 10198(6) 2235(2) 42(2)

C(47) 2866(6) 10954(6) 2872(2) 52(2)

C(48) 3957(7) 11426(7) 2824(2) 72(2)

C(49) 1940(8) 11731(9) 2799(4) 131(5)

C(50) 2972(9) 10412(8) 3259(2) 99(4)

334

C(51) 7172(5) 5491(5) 1390(2) 38(2)

C(52) 8596(6) 6522(5) 1829(2) 36(2)

C(53) 9725(5) 7573(5) 2326(2) 38(2)

C(54) 10123(5) 6803(5) 2640(2) 38(2)

C(55) 9367(5) 6783(5) 2950(2) 36(1)

C(56) 9214(5) 7854(5) 3125(2) 33(1)

C(57) 8298(5) 7797(5) 3382(2) 34(1)

C(58) 8505(5) 6959(5) 3693(2) 38(2)

C(59) 7595(5) 6919(5) 3942(2) 35(1)

C(60) 7416(5) 7974(4) 4127(2) 33(1)

C(61) 6320(5) 8118(5) 4305(2) 39(2)

C(62) 6151(6) 9302(5) 4299(2) 55(2)

C(63) 6783(7) 9743(5) 3981(2) 60(2)

C(64) 7159(6) 8765(5) 3793(2) 43(2)

C(65) 8065(6) 8877(5) 3549(2) 44(2)

C(66) 7747(7) 9651(5) 3232(2) 62(2)

C(67) 8566(7) 9716(5) 2944(2) 55(2)

C(68) 8785(5) 8614(5) 2794(2) 38(2)

C(69) 9568(5) 8650(5) 2498(2) 40(2)

C(70) 10323(5) 8217(6) 3351(2) 46(2)

C(71) 8434(5) 8260(6) 4412(2) 49(2)

C(72) 6289(5) 7618(5) 4700(2) 37(2)

C(73) 6407(6) 6434(5) 4701(2) 51(2)

C(74) 5184(5) 7950(5) 4832(2) 39(2)

C(75) 4965(5) 7467(5) 5198(2) 42(2)

C(76) 3916(6) 7920(5) 5326(2) 45(2)

C(77) 3550(5) 7355(5) 5665(2) 41(2)

C(78) 4441(6) 7395(7) 6023(2) 62(2)

C(79) 2452(6) 7790(7) 5746(2) 68(2)

C(80) 7405(6) 4483(5) 1633(2) 42(2)

C(81) 7000(6) 4556(5) 2017(2) 44(2)

C(82) 7403(6) 3650(5) 2271(2) 42(2)

C(83) 8062(6) 3243(5) 2948(2) 44(2)

C(84) 9094(6) 2682(6) 2878(2) 64(2)

C(85) 7097(6) 2501(6) 2992(2) 61(2)

C(86) 8295(7) 3970(6) 3287(2) 57(2)

335

C(87) 5927(5) 5680(5) 1263(2) 44(2)

C(88) 5291(6) 4815(5) 1025(2) 46(2)

C(89) 4770(6) 3997(6) 1248(2) 49(2)

C(90) 4205(7) 2144(6) 1232(2) 60(2)

C(91) 4798(7) 1891(7) 1629(2) 77(3)

C(92) 2965(6) 2345(7) 1206(2) 70(2)

C(93) 4405(8) 1296(6) 958(3) 74(3)

C(94) 7814(6) 5380(5) 1050(2) 43(2)

C(95) 7737(8) 6292(6) 789(2) 68(2)

C(96) 8540(10) 6312(8) 511(2) 83(3)

C(97) 9413(8) 5200(10) 82(2) 85(3)

C(98) 10556(11) 5511(19) 236(5) 236(12)

C(99) 8956(14) 5804(8) -275(3) 146(6)

C(100) 9309(8) 4087(8) 4(2) 82(3)

O(15) 1441(4) 5337(5) 1994(2) 64(2)

C(101) 1426(8) 4399(7) 1789(3) 88(3)

C(102) 1356(8) 4709(8) 1335(3) 97(3)

O(16) 6458(4) 8155(4) 2006(2) 61(1)

C(103) 6421(8) 9171(7) 1856(5) 142(6)

C(104) 5911(13) 9833(11) 2240(5) 172(6) ________________________________________________________________________________

336

Table II.2 Bond lengths [Å] and angles [°] of 3EUr-α-cholestane. _____________________________________________________

O(1)-C(2) 1.240(8)

O(2)-C(32) 1.200(7)

O(3)-C(32) 1.337(8)

O(3)-C(33) 1.500(8)

O(4)-C(39) 1.241(10)

O(5)-C(39) 1.320(11)

O(5)-C(40) 1.493(9)

O(6)-C(46) 1.197(8)

O(7)-C(46) 1.346(7)

O(7)-C(47) 1.478(8)

O(8)-C(52) 1.220(7)

O(9)-C(82) 1.201(8)

O(10)-C(82) 1.353(8)

O(10)-C(83) 1.476(7)

O(11)-C(89) 1.215(8)

O(12)-C(89) 1.335(8)

O(12)-C(90) 1.483(8)

O(13)-C(96) 1.203(10)

O(14)-C(96) 1.308(10)

O(14)-C(97) 1.499(9)

N(1)-C(2) 1.370(8)

N(1)-C(1) 1.469(8)

N(2)-C(2) 1.341(8)

N(2)-C(3) 1.471(8)

N(3)-C(52) 1.378(8)

N(3)-C(51) 1.466(8)

N(4)-C(52) 1.357(8)

N(4)-C(53) 1.465(8)

C(1)-C(37) 1.530(8)

C(1)-C(44) 1.544(9)

C(1)-C(30) 1.554(9)

C(3)-C(19) 1.519(9)

C(3)-C(4) 1.540(9)

C(4)-C(5) 1.529(8)

C(5)-C(6) 1.534(9)

C(6)-C(7) 1.533(8)

C(6)-C(20) 1.538(9)

C(6)-C(18) 1.545(9)

C(7)-C(8) 1.526(8)

C(7)-C(15) 1.533(8)

C(8)-C(9) 1.534(8)

C(9)-C(10) 1.534(8)

C(10)-C(11) 1.528(8)

C(10)-C(14) 1.538(8)

C(10)-C(21) 1.540(9)

C(11)-C(12) 1.544(9)

C(11)-C(22) 1.549(8)

C(12)-C(13) 1.532(9)

C(13)-C(14) 1.522(8)

C(14)-C(15) 1.545(8)

C(15)-C(16) 1.533(8)

C(16)-C(17) 1.513(9)

C(17)-C(18) 1.516(9)

C(18)-C(19) 1.538(8)

C(22)-C(23) 1.523(9)

C(22)-C(24) 1.545(8)

C(24)-C(25) 1.542(8)

C(25)-C(26) 1.498(9)

C(26)-C(27) 1.511(9)

C(27)-C(29) 1.500(10)

C(27)-C(28) 1.531(9)

C(30)-C(31) 1.522(9)

C(31)-C(32) 1.510(9)

C(33)-C(35) 1.505(11)

C(33)-C(36) 1.514(11)

C(33)-C(34) 1.526(11)

C(37)-C(38) 1.499(9)

C(38)-C(39) 1.494(11)

337

C(40)-C(42) 1.511(12)

C(40)-C(43) 1.522(14)

C(40)-C(41) 1.532(12)

C(44)-C(45) 1.539(8)

C(45)-C(46) 1.503(9)

C(47)-C(48) 1.489(10)

C(47)-C(49) 1.492(12)

C(47)-C(50) 1.530(11)

C(51)-C(87) 1.524(9)

C(51)-C(94) 1.548(8)

C(51)-C(80) 1.553(9)

C(53)-C(54) 1.509(9)

C(53)-C(69) 1.532(8)

C(54)-C(55) 1.543(8)

C(55)-C(56) 1.529(8)

C(56)-C(70) 1.527(9)

C(56)-C(57) 1.548(8)

C(56)-C(68) 1.551(8)

C(57)-C(58) 1.535(8)

C(57)-C(65) 1.550(8)

C(58)-C(59) 1.520(8)

C(59)-C(60) 1.533(8)

C(60)-C(71) 1.512(8)

C(60)-C(64) 1.555(9)

C(60)-C(61) 1.572(8)

C(61)-C(62) 1.529(9)

C(61)-C(72) 1.550(8)

C(62)-C(63) 1.570(9)

C(63)-C(64) 1.526(9)

C(64)-C(65) 1.511(8)

C(65)-C(66) 1.505(9)

C(66)-C(67) 1.539(8)

C(67)-C(68) 1.548(9)

C(68)-C(69) 1.527(8)

C(72)-C(73) 1.524(9)

C(72)-C(74) 1.547(8)

C(74)-C(75) 1.504(8)

C(75)-C(76) 1.531(9)

C(76)-C(77) 1.533(8)

C(77)-C(79) 1.512(9)

C(77)-C(78) 1.536(9)

C(80)-C(81) 1.528(8)

C(81)-C(82) 1.504(9)

C(83)-C(84) 1.497(9)

C(83)-C(86) 1.517(9)

C(83)-C(85) 1.534(10)

C(87)-C(88) 1.528(9)

C(88)-C(89) 1.513(9)

C(90)-C(93) 1.504(11)

C(90)-C(92) 1.514(10)

C(90)-C(91) 1.516(11)

C(94)-C(95) 1.486(10)

C(95)-C(96) 1.494(11)

C(97)-C(100) 1.455(14)

C(97)-C(98) 1.461(14)

C(97)-C(99) 1.517(15)

O(15)-C(101) 1.404(10)

C(101)-C(102) 1.652(14)

O(16)-C(103) 1.404(10)

C(103)-C(104) 1.80(2)

C(32)-O(3)-C(33) 120.3(5)

C(39)-O(5)-C(40) 121.9(6)

C(46)-O(7)-C(47) 119.6(5)

C(82)-O(10)-C(83) 121.6(5)

C(89)-O(12)-C(90) 122.3(5)

C(96)-O(14)-C(97) 121.3(7)

C(2)-N(1)-C(1) 125.3(5)

C(2)-N(2)-C(3) 121.8(6)

C(52)-N(3)-C(51) 123.9(5)

C(52)-N(4)-C(53) 121.3(5)

N(1)-C(1)-C(37) 110.6(5)

338

N(1)-C(1)-C(44) 111.5(5)

C(37)-C(1)-C(44) 107.7(5)

N(1)-C(1)-C(30) 103.3(5)

C(37)-C(1)-C(30) 111.9(5)

C(44)-C(1)-C(30) 111.8(5)

O(1)-C(2)-N(2) 125.0(6)

O(1)-C(2)-N(1) 120.7(6)

N(2)-C(2)-N(1) 114.3(6)

N(2)-C(3)-C(19) 111.8(5)

N(2)-C(3)-C(4) 108.9(6)

C(19)-C(3)-C(4) 110.9(5)

C(5)-C(4)-C(3) 112.9(5)

C(4)-C(5)-C(6) 113.7(5)

C(7)-C(6)-C(5) 111.4(5)

C(7)-C(6)-C(20) 110.8(5)

C(5)-C(6)-C(20) 108.6(5)

C(7)-C(6)-C(18) 108.0(5)

C(5)-C(6)-C(18) 106.7(5)

C(20)-C(6)-C(18) 111.2(5)

C(8)-C(7)-C(15) 109.6(5)

C(8)-C(7)-C(6) 115.3(5)

C(15)-C(7)-C(6) 113.9(5)

C(7)-C(8)-C(9) 115.0(5)

C(8)-C(9)-C(10) 112.8(5)

C(11)-C(10)-C(9) 116.9(5)

C(11)-C(10)-C(14) 99.7(5)

C(9)-C(10)-C(14) 106.2(5)

C(11)-C(10)-C(21) 110.7(5)

C(9)-C(10)-C(21) 110.6(5)

C(14)-C(10)-C(21) 112.3(5)

C(10)-C(11)-C(12) 103.9(5)

C(10)-C(11)-C(22) 122.4(5)

C(12)-C(11)-C(22) 111.1(5)

C(13)-C(12)-C(11) 106.4(5)

C(14)-C(13)-C(12) 103.8(5)

C(13)-C(14)-C(10) 103.4(5)

C(13)-C(14)-C(15) 119.7(5)

C(10)-C(14)-C(15) 115.3(5)

C(7)-C(15)-C(16) 112.3(5)

C(7)-C(15)-C(14) 108.8(5)

C(16)-C(15)-C(14) 111.1(5)

C(17)-C(16)-C(15) 111.6(5)

C(16)-C(17)-C(18) 112.0(5)

C(17)-C(18)-C(19) 110.9(5)

C(17)-C(18)-C(6) 112.4(5)

C(19)-C(18)-C(6) 114.0(5)

C(3)-C(19)-C(18) 111.4(5)

C(23)-C(22)-C(24) 109.4(5)

C(23)-C(22)-C(11) 112.0(5)

C(24)-C(22)-C(11) 108.5(5)

C(25)-C(24)-C(22) 115.8(5)

C(26)-C(25)-C(24) 113.7(5)

C(25)-C(26)-C(27) 117.8(6)

C(29)-C(27)-C(26) 110.5(6)

C(29)-C(27)-C(28) 110.0(6)

C(26)-C(27)-C(28) 111.6(6)

C(31)-C(30)-C(1) 114.9(5)

C(32)-C(31)-C(30) 114.2(5)

O(2)-C(32)-O(3) 125.8(6)

O(2)-C(32)-C(31) 123.9(6)

O(3)-C(32)-C(31) 110.4(5)

O(3)-C(33)-C(35) 109.9(6)

O(3)-C(33)-C(36) 100.5(5)

C(35)-C(33)-C(36) 111.6(7)

O(3)-C(33)-C(34) 109.6(6)

C(35)-C(33)-C(34) 114.2(7)

C(36)-C(33)-C(34) 110.3(8)

C(38)-C(37)-C(1) 115.8(6)

C(39)-C(38)-C(37) 115.4(8)

O(4)-C(39)-O(5) 126.0(8)

O(4)-C(39)-C(38) 121.5(9)

O(5)-C(39)-C(38) 112.1(7)

339

O(5)-C(40)-C(42) 109.4(7)

O(5)-C(40)-C(43) 101.3(7)

C(42)-C(40)-C(43) 112.1(7)

O(5)-C(40)-C(41) 108.0(7)

C(42)-C(40)-C(41) 111.4(8)

C(43)-C(40)-C(41) 113.9(10)

C(45)-C(44)-C(1) 115.9(5)

C(46)-C(45)-C(44) 111.0(5)

O(6)-C(46)-O(7) 125.3(6)

O(6)-C(46)-C(45) 125.3(6)

O(7)-C(46)-C(45) 109.4(6)

O(7)-C(47)-C(48) 110.6(5)

O(7)-C(47)-C(49) 109.7(6)

C(48)-C(47)-C(49) 111.5(8)

O(7)-C(47)-C(50) 100.5(6)

C(48)-C(47)-C(50) 109.7(7)

C(49)-C(47)-C(50) 114.2(8)

N(3)-C(51)-C(87) 103.9(5)

N(3)-C(51)-C(94) 110.4(5)

C(87)-C(51)-C(94) 112.3(5)

N(3)-C(51)-C(80) 111.1(5)

C(87)-C(51)-C(80) 112.5(5)

C(94)-C(51)-C(80) 106.8(5)

O(8)-C(52)-N(4) 124.0(6)

O(8)-C(52)-N(3) 122.4(6)

N(4)-C(52)-N(3) 113.7(5)

N(4)-C(53)-C(54) 112.2(5)

N(4)-C(53)-C(69) 107.6(5)

C(54)-C(53)-C(69) 109.9(5)

C(53)-C(54)-C(55) 112.6(5)

C(56)-C(55)-C(54) 113.6(5)

C(70)-C(56)-C(55) 109.5(5)

C(70)-C(56)-C(57) 110.9(5)

C(55)-C(56)-C(57) 110.1(5)

C(70)-C(56)-C(68) 111.6(5)

C(55)-C(56)-C(68) 107.6(5)

C(57)-C(56)-C(68) 107.0(5)

C(58)-C(57)-C(56) 114.2(5)

C(58)-C(57)-C(65) 111.5(5)

C(56)-C(57)-C(65) 112.2(5)

C(59)-C(58)-C(57) 113.2(5)

C(58)-C(59)-C(60) 112.7(5)

C(71)-C(60)-C(59) 110.2(5)

C(71)-C(60)-C(64) 113.4(5)

C(59)-C(60)-C(64) 105.7(4)

C(71)-C(60)-C(61) 110.6(5)

C(59)-C(60)-C(61) 117.6(5)

C(64)-C(60)-C(61) 98.8(5)

C(62)-C(61)-C(72) 113.5(5)

C(62)-C(61)-C(60) 103.4(5)

C(72)-C(61)-C(60) 117.5(5)

C(61)-C(62)-C(63) 106.7(5)

C(64)-C(63)-C(62) 103.6(5)

C(65)-C(64)-C(63) 118.0(5)

C(65)-C(64)-C(60) 115.2(5)

C(63)-C(64)-C(60) 103.7(5)

C(66)-C(65)-C(64) 111.7(5)

C(66)-C(65)-C(57) 109.9(5)

C(64)-C(65)-C(57) 109.2(5)

C(65)-C(66)-C(67) 114.8(6)

C(66)-C(67)-C(68) 110.2(5)

C(69)-C(68)-C(67) 111.8(5)

C(69)-C(68)-C(56) 111.9(5)

C(67)-C(68)-C(56) 111.4(5)

C(68)-C(69)-C(53) 112.2(5)

C(73)-C(72)-C(74) 111.1(5)

C(73)-C(72)-C(61) 113.4(5)

C(74)-C(72)-C(61) 107.9(5)

C(75)-C(74)-C(72) 115.4(5)

C(74)-C(75)-C(76) 111.8(5)

C(75)-C(76)-C(77) 114.5(5)

C(79)-C(77)-C(76) 110.6(6)

340

C(79)-C(77)-C(78) 110.6(6)

C(76)-C(77)-C(78) 112.7(5)

C(81)-C(80)-C(51) 113.0(5)

C(82)-C(81)-C(80) 111.7(5)

O(9)-C(82)-O(10) 124.7(6)

O(9)-C(82)-C(81) 125.3(6)

O(10)-C(82)-C(81) 110.0(5)

O(10)-C(83)-C(84) 110.5(5)

O(10)-C(83)-C(86) 101.9(5)

C(84)-C(83)-C(86) 111.9(6)

O(10)-C(83)-C(85) 108.8(5)

C(84)-C(83)-C(85) 113.0(6)

C(86)-C(83)-C(85) 110.3(6)

C(51)-C(87)-C(88) 116.0(5)

C(89)-C(88)-C(87) 115.3(6)

O(11)-C(89)-O(12) 124.5(7)

O(11)-C(89)-C(88) 123.6(7)

O(12)-C(89)-C(88) 111.9(5)

O(12)-C(90)-C(93) 102.3(5)

O(12)-C(90)-C(92) 108.9(6)

C(93)-C(90)-C(92) 110.2(7)

O(12)-C(90)-C(91) 110.1(6)

C(93)-C(90)-C(91) 110.4(7)

C(92)-C(90)-C(91) 114.2(6)

C(95)-C(94)-C(51) 115.3(6)

C(94)-C(95)-C(96) 116.4(7)

O(13)-C(96)-O(14) 125.7(9)

O(13)-C(96)-C(95) 123.2(9)

O(14)-C(96)-C(95) 111.0(7)

C(100)-C(97)-C(98) 112.8(13)

C(100)-C(97)-O(14) 103.9(7)

C(98)-C(97)-O(14) 111.4(8)

C(100)-C(97)-C(99) 109.3(8)

C(98)-C(97)-C(99) 111.9(12)

O(14)-C(97)-C(99) 107.1(9)

O(15)-C(101)-C(102) 107.1(8)

O(16)-C(103)-C(104) 98.3(10)

_______________________________________________________________

341

Table II.3 Anisotropic displacement parameters (Å2x 103) of 3EUr-α-cholestane. The anisotropic

displacement factor exponent takes the form: -2�2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

______________________________________________________________________________

U11 U22 U33 U23 U13 U12

______________________________________________________________________________

O(1) 53(3) 58(3) 38(3) 10(2) 8(2) -11(2)

O(2) 70(3) 69(3) 47(3) 8(3) 28(3) 1(3)

O(3) 59(3) 29(2) 46(3) -1(2) 20(2) 4(2)

O(4) 226(9) 94(5) 59(4) 22(4) 56(5) 88(5)

O(5) 96(4) 71(4) 31(3) 5(3) 26(3) 30(3)

O(6) 117(5) 43(3) 49(3) 2(3) -5(3) -23(3)

O(7) 61(3) 53(3) 35(3) -16(2) 21(2) -18(2)

O(8) 49(3) 66(3) 41(3) -7(2) 12(2) 20(3)

O(9) 110(4) 40(3) 41(3) -1(2) 19(3) 20(3)

O(10) 55(3) 36(2) 34(2) 6(2) 14(2) 12(2)

O(11) 73(4) 77(4) 56(3) -12(3) 31(3) -5(3)

O(12) 58(3) 50(3) 48(3) 0(2) 24(2) -4(2)

O(13) 327(13) 145(7) 89(5) -82(5) 128(7) -156(9)

O(14) 85(4) 82(4) 31(3) 0(3) 22(3) -26(3)

N(1) 57(4) 37(3) 28(3) 2(2) 5(3) -1(3)

N(2) 53(3) 44(3) 29(3) 9(3) 15(3) 11(3)

N(3) 54(4) 29(3) 30(3) 3(2) 8(3) 13(2)

N(4) 44(3) 37(3) 31(3) -1(2) 17(2) 7(2)

C(1) 52(4) 35(3) 25(3) 4(3) 5(3) -1(3)

C(2) 55(4) 38(4) 24(3) -3(3) 11(3) 9(3)

C(3) 37(4) 48(4) 39(4) 10(3) 12(3) 8(3)

C(4) 55(4) 46(4) 40(4) 11(3) 24(3) 13(3)

C(5) 47(4) 37(4) 39(4) 5(3) 13(3) 10(3)

C(6) 41(4) 36(4) 35(3) 3(3) 11(3) 1(3)

C(7) 37(3) 32(3) 31(3) 0(3) 7(3) 3(3)

C(8) 61(4) 29(3) 35(4) 1(3) 24(3) 9(3)

C(9) 41(3) 29(3) 36(3) 2(3) 13(3) 4(3)

C(10) 46(4) 33(3) 33(3) -2(3) 12(3) -1(3)

C(11) 49(4) 34(4) 27(3) -5(3) 8(3) 6(3)

C(12) 60(4) 44(4) 39(4) -2(3) 18(3) -5(3)

C(13) 63(4) 39(4) 29(3) -7(3) 9(3) 0(3)

342

C(14) 47(4) 36(4) 29(3) 4(3) 12(3) -1(3)

C(15) 44(4) 39(4) 25(3) 1(3) 5(3) -3(3)

C(16) 57(4) 39(4) 42(4) -5(3) 16(3) -12(3)

C(17) 55(4) 44(4) 44(4) 9(3) 11(3) 1(3)

C(18) 41(4) 39(4) 32(3) 0(3) 10(3) 0(3)

C(19) 53(4) 45(4) 32(3) 13(3) 13(3) 6(3)

C(20) 44(4) 53(4) 38(4) 13(3) 14(3) 13(3)

C(21) 47(4) 56(4) 30(3) 5(3) 8(3) 2(3)

C(22) 46(4) 39(4) 28(3) 0(3) 12(3) 2(3)

C(23) 42(4) 41(4) 42(4) 6(3) 13(3) 6(3)

C(24) 54(4) 40(4) 34(3) 12(3) 14(3) 12(3)

C(25) 59(4) 38(4) 46(4) 11(3) 20(3) 6(3)

C(26) 59(4) 36(4) 50(4) 8(3) 17(4) 3(3)

C(27) 62(4) 41(4) 41(4) 8(3) 28(3) 7(3)

C(28) 74(5) 69(5) 43(4) 3(4) 21(4) 13(4)

C(29) 96(6) 46(4) 50(5) 3(4) 26(4) 14(4)

C(30) 61(4) 34(4) 40(4) -5(3) 0(3) -10(3)

C(31) 57(4) 41(4) 33(4) 0(3) 7(3) 1(3)

C(32) 46(4) 43(4) 41(4) -1(3) 13(3) -5(3)

C(33) 58(5) 43(4) 65(5) 4(4) 20(4) 18(4)

C(34) 128(8) 79(7) 65(6) -41(5) -5(6) 22(6)

C(35) 61(5) 77(6) 86(7) 13(5) 29(5) 16(4)

C(36) 78(6) 50(5) 108(7) 14(5) 38(5) 17(4)

C(37) 68(5) 46(4) 31(4) 2(3) 16(3) 20(4)

C(38) 117(7) 52(5) 32(4) -5(3) 24(4) 12(5)

C(39) 152(9) 85(7) 26(4) 9(4) 31(5) 52(7)

C(40) 73(6) 132(9) 30(4) 14(5) 18(4) 32(6)

C(41) 104(8) 196(13) 66(6) 4(8) 22(6) 73(9)

C(42) 90(6) 88(6) 33(4) -7(4) 8(4) 24(5)

C(43) 80(6) 110(8) 56(5) -13(5) 35(5) -5(6)

C(44) 48(4) 37(4) 31(3) 4(3) 12(3) -6(3)

C(45) 58(4) 37(4) 36(4) -2(3) 16(3) -9(3)

C(46) 46(4) 47(4) 35(4) -2(3) 13(3) -6(3)

C(47) 58(4) 54(4) 47(4) -22(4) 17(3) -23(4)

C(48) 65(5) 97(7) 56(5) -29(5) 11(4) -27(5)

C(49) 71(6) 127(9) 188(12) -129(9) -2(7) 11(6)

343

C(50) 162(10) 96(7) 43(5) -28(5) 32(6) -49(7)

C(51) 53(4) 34(4) 28(3) -1(3) 12(3) 12(3)

C(52) 48(4) 36(4) 29(3) 1(3) 18(3) 12(3)

C(53) 40(4) 41(4) 37(4) -12(3) 19(3) -3(3)

C(54) 40(4) 43(4) 33(3) -5(3) 12(3) 4(3)

C(55) 46(4) 35(3) 27(3) -4(3) 11(3) 9(3)

C(56) 41(4) 35(3) 27(3) -4(3) 16(3) 3(3)

C(57) 38(3) 30(3) 35(3) -6(3) 9(3) -2(3)

C(58) 48(4) 39(4) 28(3) 0(3) 9(3) 4(3)

C(59) 42(3) 34(3) 29(3) -2(3) 6(3) 3(3)

C(60) 36(3) 33(3) 31(3) -9(3) 12(3) -6(3)

C(61) 47(4) 44(4) 27(3) 5(3) 12(3) 0(3)

C(62) 73(5) 51(4) 51(4) 0(4) 37(4) 14(4)

C(63) 86(5) 42(4) 64(5) 6(4) 51(4) 9(4)

C(64) 57(4) 36(4) 41(4) -2(3) 25(3) 4(3)

C(65) 54(4) 36(4) 51(4) -1(3) 32(4) 1(3)

C(66) 103(6) 28(4) 73(5) 12(3) 65(5) 17(4)

C(67) 85(5) 35(4) 55(4) 6(3) 43(4) 6(4)

C(68) 47(4) 36(4) 36(4) 2(3) 22(3) 4(3)

C(69) 48(4) 39(4) 38(4) 2(3) 16(3) -7(3)

C(70) 41(4) 56(4) 43(4) -7(4) 12(3) -5(3)

C(71) 45(4) 65(5) 40(4) -19(4) 17(3) -11(4)

C(72) 40(3) 44(4) 30(3) 2(3) 14(3) -6(3)

C(73) 65(5) 49(4) 45(4) 10(3) 26(4) -2(4)

C(74) 44(4) 42(4) 34(3) -3(3) 14(3) -5(3)

C(75) 43(4) 46(4) 37(3) -3(3) 11(3) -6(3)

C(76) 57(4) 36(4) 43(4) 2(3) 15(3) -2(3)

C(77) 49(4) 47(4) 32(3) -4(3) 18(3) -14(3)

C(78) 77(5) 81(6) 28(4) -9(4) 11(4) -22(5)

C(79) 71(5) 81(6) 64(5) 17(4) 49(4) 6(4)

C(80) 61(4) 39(4) 30(4) 2(3) 16(3) 13(3)

C(81) 58(4) 47(4) 33(4) 9(3) 22(3) 16(3)

C(82) 55(4) 45(4) 31(4) -2(3) 15(3) 5(3)

C(83) 67(4) 38(4) 27(3) 13(3) 10(3) 15(3)

C(84) 67(5) 59(5) 68(5) 15(4) 15(4) 26(4)

C(85) 80(5) 53(5) 52(4) 8(4) 20(4) 3(4)

344

C(86) 85(5) 46(4) 40(4) 6(3) 5(4) 2(4)

C(87) 56(4) 43(4) 33(4) -2(3) 7(3) 1(3)

C(88) 58(4) 44(4) 39(4) 6(3) 12(3) 4(3)

C(89) 58(5) 50(5) 41(4) -2(4) 17(4) 5(4)

C(90) 67(5) 56(5) 60(5) 22(4) 25(4) -4(4)

C(91) 85(6) 74(6) 76(6) 32(5) 27(5) -10(5)

C(92) 62(5) 75(6) 81(6) -6(5) 37(5) -10(4)

C(93) 88(6) 46(5) 94(7) 2(5) 39(5) -12(4)

C(94) 57(4) 49(4) 26(3) -3(3) 12(3) -3(3)

C(95) 100(6) 69(6) 37(4) 9(4) 20(4) 0(5)

C(96) 154(9) 69(6) 32(4) -13(4) 34(5) -46(6)

C(97) 85(6) 136(9) 42(5) -33(6) 32(4) -57(6)

C(98) 101(10) 420(30) 201(17) -190(20) 54(10) -93(15)

C(99) 330(19) 72(7) 58(7) 7(5) 103(10) -23(9)

C(100) 98(7) 109(8) 47(5) 8(5) 36(5) 16(6)

O(15) 39(3) 87(4) 69(4) -24(3) 12(3) 10(3)

C(101) 63(5) 73(6) 135(9) -18(6) 36(6) 20(5)

C(102) 82(6) 77(6) 136(10) -32(7) 27(6) 8(5)

O(16) 46(3) 59(3) 84(4) 13(3) 26(3) 6(2)

C(103) 51(5) 59(6) 320(20) 88(10) 55(8) 19(5)

C(104) 191(15) 104(11) 197(16) -2(11) -39(13) 20(11)

______________________________________________________________________________

345

Table II.4 Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3) of 3EUr-α-cholestane.

________________________________________________________________________________

x y z U(eq)

________________________________________________________________________________

H(1A) 2022 6720 1654 49

H(2A) 2971 5955 2142 49

H(3B) 7080 6946 1612 45

H(4C) 8037 7573 2145 44

H(3A) 5253 6035 2215 49

H(4A) 4252 4428 2286 54

H(4B) 5279 4620 2621 54

H(5A) 3752 4043 2890 48

H(5B) 2972 4936 2676 48

H(7A) 2429 5648 3237 39

H(8A) 3657 4182 3738 48

H(8B) 2787 3891 3366 48

H(9A) 1993 3669 3932 41

H(9B) 1287 4478 3647 41

H(11A) 471 5499 4092 44

H(12A) 619 6986 4415 56

H(12B) 1596 6561 4738 56

H(13A) 1945 7728 4134 52

H(13B) 2918 7138 4417 52

H(14A) 1618 6339 3741 44

H(15A) 3994 6123 3901 43

H(16A) 2715 7634 3419 54

H(16B) 3654 7885 3778 54

H(17A) 5060 7293 3458 56

H(17B) 4329 8126 3190 56

H(18A) 3333 6770 2847 44

H(19A) 4877 7476 2610 51

H(19B) 5682 6612 2834 51

H(20A) 5128 4445 3463 66

H(20B) 5407 5633 3580 66

H(20C) 5744 5175 3196 66

346

H(21A) 3795 4774 4324 66

H(21B) 2952 4317 4586 66

H(21C) 3298 5523 4616 66

H(22A) 1210 4810 4856 45

H(23A) 1405 3359 4460 61

H(23B) 150 3513 4247 61

H(23C) 379 3169 4685 61

H(24A) -509 5867 4655 50

H(24B) -1029 4837 4445 50

H(25A) -1704 4648 4984 55

H(25B) -549 4042 5106 55

H(26A) -902 6162 5281 57

H(26B) 245 5553 5404 57

H(27A) -644 4478 5809 55

H(28A) -2409 4990 5953 91

H(28B) -2521 4655 5515 91

H(28C) -2510 5865 5628 91

H(29A) 375 5941 6060 94

H(29B) -650 5730 6284 94

H(29C) -727 6646 5975 94

H(30A) 790 7319 1136 55

H(30B) 555 7871 1518 55

H(31A) 855 9237 926 52

H(31B) -263 8555 852 52

H(34A) 494 11841 1687 139

H(34B) -356 11073 1851 139

H(34C) -653 12285 1791 139

H(35A) -2416 11080 988 109

H(35B) -2486 11821 1346 109

H(35C) -2206 10606 1410 109

H(36A) -1036 12177 762 114

H(36B) 143 12404 1022 114

H(36C) -958 12988 1107 114

H(37A) 2573 8804 921 57

H(37B) 3616 8238 1169 57

H(38A) 2749 6602 966 79

347

H(38B) 1860 7249 677 79

H(41A) 5330 7020 213 182

H(41B) 5736 8054 447 182

H(41C) 5901 7947 11 182

H(42A) 3644 7014 -255 106

H(42B) 4053 7939 -502 106

H(42C) 2894 8039 -343 106

H(43A) 3525 9722 -37 119

H(43B) 4749 9661 -150 119

H(43C) 4603 9757 288 119

H(44A) 3244 9305 1660 45

H(44B) 2035 9737 1486 45

H(45A) 1292 9089 2018 51

H(45B) 2482 8599 2190 51

H(48A) 3886 11771 2576 108

H(48B) 4527 10879 2840 108

H(48C) 4177 11941 3026 108

H(49A) 1934 12051 2549 197

H(49B) 2053 12272 2996 197

H(49C) 1225 11379 2805 197

H(50A) 3607 9929 3288 148

H(50B) 2285 10024 3275 148

H(50C) 3092 10938 3462 148

H(53A) 10299 7626 2154 46

H(54A) 10893 6987 2758 46

H(54B) 10143 6096 2528 46

H(55A) 8625 6506 2838 43

H(55B) 9694 6299 3155 43

H(57A) 7594 7591 3212 41

H(58A) 8556 6269 3572 46

H(58B) 9231 7098 3856 46

H(59A) 7798 6390 4144 42

H(59B) 6886 6696 3785 42

H(61A) 5691 7809 4123 46

H(62A) 6460 9607 4550 66

H(62B) 5345 9473 4242 66

348

H(63A) 6278 10172 3796 72

H(63B) 7432 10173 4093 72

H(64A) 6485 8492 3622 52

H(65A) 8762 9126 3713 53

H(66A) 7687 10351 3345 75

H(66B) 6998 9464 3094 75

H(67A) 9279 10028 3068 66

H(67B) 8249 10170 2729 66

H(68A) 8052 8337 2664 45

H(69A) 10304 8922 2618 49

H(69B) 9259 9137 2291 49

H(70A) 10890 8243 3183 69

H(70B) 10562 7728 3560 69

H(70C) 10231 8914 3455 69

H(71A) 9108 8162 4296 73

H(71B) 8471 7813 4637 73

H(71C) 8380 8992 4487 73

H(72A) 6923 7911 4884 44

H(73A) 7121 6244 4623 77

H(73B) 5792 6129 4522 77

H(73C) 6386 6166 4957 77

H(74A) 5188 8719 4861 47

H(74B) 4557 7770 4628 47

H(75A) 5617 7589 5399 50

H(75B) 4873 6703 5163 50

H(76A) 3295 7894 5109 54

H(76B) 4056 8663 5394 54

H(77A) 3425 6605 5593 50

H(78A) 5145 7111 5965 93

H(78B) 4192 6979 6225 93

H(78C) 4554 8120 6109 93

H(79A) 1896 7762 5514 101

H(79B) 2558 8517 5831 101

H(79C) 2192 7375 5946 101

H(80A) 7031 3886 1488 51

H(80B) 8219 4344 1676 51

349

H(81A) 7274 5215 2144 53

H(81B) 6173 4573 1976 53

H(84A) 8909 2217 2658 96

H(84B) 9393 2270 3103 96

H(84C) 9655 3191 2827 96

H(85A) 6953 2038 2770 91

H(85B) 6424 2909 3010 91

H(85C) 7298 2083 3223 91

H(86A) 8919 4433 3257 86

H(86B) 8490 3557 3521 86

H(86C) 7628 4388 3304 86

H(87A) 5838 6335 1113 53

H(87B) 5575 5789 1492 53

H(88A) 4692 5139 840 56

H(88B) 5811 4463 877 56

H(91A) 5592 1767 1622 115

H(91B) 4467 1263 1723 115

H(91C) 4718 2478 1799 115

H(92A) 2640 2495 942 105

H(92B) 2848 2943 1367 105

H(92C) 2604 1726 1294 105

H(93A) 5209 1163 981 110

H(93B) 4114 1517 697 110

H(93C) 4024 657 1017 110

H(94A) 8612 5252 1151 52

H(94B) 7528 4757 901 52

H(95A) 7848 6934 945 81

H(95B) 6969 6317 644 81

H(98A) 10836 5078 458 354

H(98B) 10565 6246 313 354

H(98C) 11034 5417 41 354

H(99A) 8207 5547 -377 218

H(99B) 9450 5709 -465 218

H(99C) 8915 6548 -213 218

H(10A) 9664 3695 227 123

H(10B) 9676 3917 -215 123

350

H(10C) 8516 3899 -54 123

H(15B) 788 5572 1976 97

H(10D) 770 3973 1826 106

H(10E) 2112 3990 1877 106

H(10F) 1344 4071 1183 146

H(10G) 2009 5130 1302 146

H(10H) 673 5111 1252 146

H(16C) 5851 7851 1929 91

H(10I) 5897 9228 1614 170

H(10J) 7169 9424 1821 170

H(10K) 5879 10585 2190 257

H(10L) 6415 9698 2479 257

H(10M) 5161 9576 2260 257

________________________________________________________________________________

Table II.5 Torsion angles [°] of 3EUr-α-cholestane.

________________________________________________________________

C(2)-N(1)-C(1)-C(37) 69.1(7)

C(2)-N(1)-C(1)-C(44) -50.8(7)

C(2)-N(1)-C(1)-C(30) -171.0(5)

C(3)-N(2)-C(2)-O(1) -4.0(9)

C(3)-N(2)-C(2)-N(1) 177.3(5)

C(1)-N(1)-C(2)-O(1) -12.3(9)

C(1)-N(1)-C(2)-N(2) 166.4(5)

C(2)-N(2)-C(3)-C(19) 78.8(7)

C(2)-N(2)-C(3)-C(4) -158.2(5)

N(2)-C(3)-C(4)-C(5) -72.1(7)

C(19)-C(3)-C(4)-C(5) 51.4(8)

C(3)-C(4)-C(5)-C(6) -54.8(8)

C(4)-C(5)-C(6)-C(7) 172.5(5)

C(4)-C(5)-C(6)-C(20) -65.1(7)

C(4)-C(5)-C(6)-C(18) 54.8(7)

C(5)-C(6)-C(7)-C(8) 61.3(7)

C(20)-C(6)-C(7)-C(8) -59.8(7)

C(18)-C(6)-C(7)-C(8) 178.2(5)

C(5)-C(6)-C(7)-C(15) -170.7(5)

C(20)-C(6)-C(7)-C(15) 68.2(7)

C(18)-C(6)-C(7)-C(15) -53.8(7)

C(15)-C(7)-C(8)-C(9) 53.2(7)

C(6)-C(7)-C(8)-C(9) -176.7(5)

C(7)-C(8)-C(9)-C(10) -53.9(7)

C(8)-C(9)-C(10)-C(11) 162.5(5)

C(8)-C(9)-C(10)-C(14) 52.4(7)

C(8)-C(9)-C(10)-C(21) -69.7(6)

C(9)-C(10)-C(11)-C(12) -154.9(5)

C(14)-C(10)-C(11)-C(12) -41.1(6)

C(21)-C(10)-C(11)-C(12) 77.4(6)

C(9)-C(10)-C(11)-C(22) 78.5(7)

C(14)-C(10)-C(11)-C(22) -167.7(5)

C(21)-C(10)-C(11)-C(22) -49.3(8)

C(10)-C(11)-C(12)-C(13) 20.4(7)

C(22)-C(11)-C(12)-C(13) 153.8(5)

C(11)-C(12)-C(13)-C(14) 9.1(7)

351

C(12)-C(13)-C(14)-C(10) -35.2(6)

C(12)-C(13)-C(14)-C(15) -165.1(6)

C(11)-C(10)-C(14)-C(13) 47.5(6)

C(9)-C(10)-C(14)-C(13) 169.4(5)

C(21)-C(10)-C(14)-C(13) -69.7(6)

C(11)-C(10)-C(14)-C(15) -179.9(5)

C(9)-C(10)-C(14)-C(15) -58.1(7)

C(21)-C(10)-C(14)-C(15) 62.9(7)

C(8)-C(7)-C(15)-C(16) -177.1(5)

C(6)-C(7)-C(15)-C(16) 52.1(7)

C(8)-C(7)-C(15)-C(14) -53.7(6)

C(6)-C(7)-C(15)-C(14) 175.5(5)

C(13)-C(14)-C(15)-C(7) -175.2(5)

C(10)-C(14)-C(15)-C(7) 60.4(7)

C(13)-C(14)-C(15)-C(16) -51.1(7)

C(10)-C(14)-C(15)-C(16) -175.5(5)

C(7)-C(15)-C(16)-C(17) -50.2(7)

C(14)-C(15)-C(16)-C(17) -172.2(5)

C(15)-C(16)-C(17)-C(18) 53.5(7)

C(16)-C(17)-C(18)-C(19) 173.0(5)

C(16)-C(17)-C(18)-C(6) -58.1(7)

C(7)-C(6)-C(18)-C(17) 56.5(7)

C(5)-C(6)-C(18)-C(17) 176.4(5)

C(20)-C(6)-C(18)-C(17) -65.3(6)

C(7)-C(6)-C(18)-C(19) -176.3(5)

C(5)-C(6)-C(18)-C(19) -56.4(7)

C(20)-C(6)-C(18)-C(19) 62.0(7)

N(2)-C(3)-C(19)-C(18) 70.0(7)

C(4)-C(3)-C(19)-C(18) -51.8(7)

C(17)-C(18)-C(19)-C(3) -175.0(5)

C(6)-C(18)-C(19)-C(3) 57.0(7)

C(10)-C(11)-C(22)-C(23) -47.6(8)

C(12)-C(11)-C(22)-C(23) -171.0(5)

C(10)-C(11)-C(22)-C(24) -168.4(6)

C(12)-C(11)-C(22)-C(24) 68.2(7)

C(23)-C(22)-C(24)-C(25) 76.7(7)

C(11)-C(22)-C(24)-C(25) -160.9(5)

C(22)-C(24)-C(25)-C(26) 89.4(7)

C(24)-C(25)-C(26)-C(27) 179.6(6)

C(25)-C(26)-C(27)-C(29) 173.0(6)

C(25)-C(26)-C(27)-C(28) -64.3(8)

N(1)-C(1)-C(30)-C(31) 176.0(5)

C(37)-C(1)-C(30)-C(31) -64.9(7)

C(44)-C(1)-C(30)-C(31) 56.0(7)

C(1)-C(30)-C(31)-C(32) -96.9(7)

C(33)-O(3)-C(32)-O(2) -3.3(10)

C(33)-O(3)-C(32)-C(31) 176.5(6)

C(30)-C(31)-C(32)-O(2) -29.3(10)

C(30)-C(31)-C(32)-O(3) 150.9(6)

C(32)-O(3)-C(33)-C(35) -63.9(8)

C(32)-O(3)-C(33)-C(36) 178.4(6)

C(32)-O(3)-C(33)-C(34) 62.3(9)

N(1)-C(1)-C(37)-C(38) 59.5(8)

C(44)-C(1)-C(37)-C(38) -178.4(6)

C(30)-C(1)-C(37)-C(38) -55.1(8)

C(1)-C(37)-C(38)-C(39) -170.1(6)

C(40)-O(5)-C(39)-O(4) -2.7(16)

C(40)-O(5)-C(39)-C(38) -176.0(7)

C(37)-C(38)-C(39)-O(4) 148.9(9)

C(37)-C(38)-C(39)-O(5) -37.5(11)

C(39)-O(5)-C(40)-C(42) 64.7(10)

C(39)-O(5)-C(40)-C(43) -176.8(8)

C(39)-O(5)-C(40)-C(41) -56.8(12)

N(1)-C(1)-C(44)-C(45) -44.8(8)

C(37)-C(1)-C(44)-C(45) -166.4(6)

C(30)-C(1)-C(44)-C(45) 70.3(7)

C(1)-C(44)-C(45)-C(46) 177.2(6)

C(47)-O(7)-C(46)-O(6) 5.9(10)

C(47)-O(7)-C(46)-C(45) -175.4(5)

C(44)-C(45)-C(46)-O(6) 24.9(10)

C(44)-C(45)-C(46)-O(7) -153.8(5)

C(46)-O(7)-C(47)-C(48) -64.3(8)

352

C(46)-O(7)-C(47)-C(49) 59.1(9)

C(46)-O(7)-C(47)-C(50) 179.8(6)

C(52)-N(3)-C(51)-C(87) 170.9(5)

C(52)-N(3)-C(51)-C(94) -68.5(7)

C(52)-N(3)-C(51)-C(80) 49.7(7)

C(53)-N(4)-C(52)-O(8) 7.3(9)

C(53)-N(4)-C(52)-N(3) -172.4(5)

C(51)-N(3)-C(52)-O(8) 20.2(9)

C(51)-N(3)-C(52)-N(4) -160.0(5)

C(52)-N(4)-C(53)-C(54) -78.8(6)

C(52)-N(4)-C(53)-C(69) 160.2(5)

N(4)-C(53)-C(54)-C(55) -66.9(6)

C(69)-C(53)-C(54)-C(55) 52.7(7)

C(53)-C(54)-C(55)-C(56) -55.0(7)

C(54)-C(55)-C(56)-C(70) -67.2(6)

C(54)-C(55)-C(56)-C(57) 170.6(5)

C(54)-C(55)-C(56)-C(68) 54.3(7)

C(70)-C(56)-C(57)-C(58) -66.3(7)

C(55)-C(56)-C(57)-C(58) 55.0(7)

C(68)-C(56)-C(57)-C(58) 171.7(5)

C(70)-C(56)-C(57)-C(65) 61.7(7)

C(55)-C(56)-C(57)-C(65) -177.0(5)

C(68)-C(56)-C(57)-C(65) -60.3(7)

C(56)-C(57)-C(58)-C(59) -180.0(5)

C(65)-C(57)-C(58)-C(59) 51.6(7)

C(57)-C(58)-C(59)-C(60) -55.9(7)

C(58)-C(59)-C(60)-C(71) -66.8(6)

C(58)-C(59)-C(60)-C(64) 56.1(6)

C(58)-C(59)-C(60)-C(61) 165.2(5)

C(71)-C(60)-C(61)-C(62) 76.0(6)

C(59)-C(60)-C(61)-C(62) -156.1(6)

C(64)-C(60)-C(61)-C(62) -43.2(6)

C(71)-C(60)-C(61)-C(72) -49.9(7)

C(59)-C(60)-C(61)-C(72) 78.0(7)

C(64)-C(60)-C(61)-C(72) -169.1(6)

C(72)-C(61)-C(62)-C(63) 151.7(6)

C(60)-C(61)-C(62)-C(63) 23.3(7)

C(61)-C(62)-C(63)-C(64) 6.5(8)

C(62)-C(63)-C(64)-C(65) -163.0(6)

C(62)-C(63)-C(64)-C(60) -34.3(7)

C(71)-C(60)-C(64)-C(65) 61.5(7)

C(59)-C(60)-C(64)-C(65) -59.4(7)

C(61)-C(60)-C(64)-C(65) 178.5(6)

C(71)-C(60)-C(64)-C(63) -69.0(6)

C(59)-C(60)-C(64)-C(63) 170.1(5)

C(61)-C(60)-C(64)-C(63) 48.1(6)

C(63)-C(64)-C(65)-C(66) -57.2(9)

C(60)-C(64)-C(65)-C(66) 179.7(6)

C(63)-C(64)-C(65)-C(57) -179.0(6)

C(60)-C(64)-C(65)-C(57) 57.9(8)

C(58)-C(57)-C(65)-C(66) -174.0(6)

C(56)-C(57)-C(65)-C(66) 56.5(7)

C(58)-C(57)-C(65)-C(64) -51.1(7)

C(56)-C(57)-C(65)-C(64) 179.4(5)

C(64)-C(65)-C(66)-C(67) -173.3(6)

C(57)-C(65)-C(66)-C(67) -51.8(9)

C(65)-C(66)-C(67)-C(68) 52.0(9)

C(66)-C(67)-C(68)-C(69) 178.2(6)

C(66)-C(67)-C(68)-C(56) -55.7(8)

C(70)-C(56)-C(68)-C(69) 64.2(6)

C(55)-C(56)-C(68)-C(69) -56.0(7)

C(57)-C(56)-C(68)-C(69) -174.3(5)

C(70)-C(56)-C(68)-C(67) -61.7(6)

C(55)-C(56)-C(68)-C(67) 178.1(5)

C(57)-C(56)-C(68)-C(67) 59.8(7)

C(67)-C(68)-C(69)-C(53) -176.0(6)

C(56)-C(68)-C(69)-C(53) 58.3(7)

N(4)-C(53)-C(69)-C(68) 67.4(7)

C(54)-C(53)-C(69)-C(68) -55.1(7)

C(62)-C(61)-C(72)-C(73) 177.5(6)

C(60)-C(61)-C(72)-C(73) -61.7(8)

C(62)-C(61)-C(72)-C(74) 54.1(7)

353

C(60)-C(61)-C(72)-C(74) 174.8(5)

C(73)-C(72)-C(74)-C(75) 50.5(7)

C(61)-C(72)-C(74)-C(75) 175.4(5)

C(72)-C(74)-C(75)-C(76) 174.4(5)

C(74)-C(75)-C(76)-C(77) 171.7(5)

C(75)-C(76)-C(77)-C(79) -174.6(6)

C(75)-C(76)-C(77)-C(78) 61.0(8)

N(3)-C(51)-C(80)-C(81) 49.3(8)

C(87)-C(51)-C(80)-C(81) -66.7(7)

C(94)-C(51)-C(80)-C(81) 169.7(6)

C(51)-C(80)-C(81)-C(82) -170.5(6)

C(83)-O(10)-C(82)-O(9) -1.7(10)

C(83)-O(10)-C(82)-C(81) 177.4(5)

C(80)-C(81)-C(82)-O(9) -39.5(10)

C(80)-C(81)-C(82)-O(10) 141.4(6)

C(82)-O(10)-C(83)-C(84) 59.7(8)

C(82)-O(10)-C(83)-C(86) 178.7(5)

C(82)-O(10)-C(83)-C(85) -64.9(7)

N(3)-C(51)-C(87)-C(88) -179.1(5)

C(94)-C(51)-C(87)-C(88) 61.7(7)

C(80)-C(51)-C(87)-C(88) -58.8(7)

C(51)-C(87)-C(88)-C(89) 91.6(7)

C(90)-O(12)-C(89)-O(11) 0.4(11)

C(90)-O(12)-C(89)-C(88) -176.8(6)

C(87)-C(88)-C(89)-O(11) 33.7(10)

C(87)-C(88)-C(89)-O(12) -149.0(6)

C(89)-O(12)-C(90)-C(93) -178.0(7)

C(89)-O(12)-C(90)-C(92) 65.3(9)

C(89)-O(12)-C(90)-C(91) -60.6(9)

N(3)-C(51)-C(94)-C(95) -56.8(8)

C(87)-C(51)-C(94)-C(95) 58.7(8)

C(80)-C(51)-C(94)-C(95) -177.6(6)

C(51)-C(94)-C(95)-C(96) 166.8(7)

C(97)-O(14)-C(96)-O(13) 1.5(17)

C(97)-O(14)-C(96)-C(95) -178.7(7)

C(94)-C(95)-C(96)-O(13) -138.2(12)

C(94)-C(95)-C(96)-O(14) 42.0(11)

C(96)-O(14)-C(97)-C(100) 177.6(8)

C(96)-O(14)-C(97)-C(98) 55.9(16)

C(96)-O(14)-C(97)-C(99) -66.8(10)

______________________________________

Table II.6 Hydrogen bonds [Å and °] of 3EUr-α-cholestane.

____________________________________________________________________________

D-H...A d(D-H) d(H...A) d(D...A) <(DHA)

____________________________________________________________________________

O(16)-H(16C)...O(1) 0.84 1.82 2.621(7) 158.1

O(15)-H(15B)...O(8)#1 0.84 1.82 2.625(6) 161.0

N(4)-H(4C)...O(16) 0.88 2.04 2.865(7) 155.7

N(3)-H(3B)...O(16) 0.88 2.30 3.035(7) 141.6

N(2)-H(2A)...O(15) 0.88 2.01 2.849(8) 159.6

N(1)-H(1A)...O(15) 0.88 2.32 3.084(8) 146.0

____________________________________________________________________________

Symmetry transformations used to generate equivalent atoms:

#1 x-1,y,z

354

VITA

Eko Winny Sugandhi was born in Jepara, Jawa Tengah, Indonesia on Oct 8, 1973, to

Sugandhi and Henny Susanti. She attended Gadjah Mada University in Yogyakarta,

where she earned her B.S. equivalent in chemistry in 1996. She came to the US in 1997

to pursue her M.S. degree in chemistry with Dr. Arthur S. Howard at Eastern Michigan

University, Ypsilanti MI. This was the very first time she enjoyed teaching, and

eventually decided to pursue her career in academia. Upon completion of her M.S.

degree, she entered the chemistry graduate program at Virginia Polytechnic Institute and

State University in 2000. She finally obtained her M.S. degree in chemistry in 2002. At

Virginia Polytechnic Institute and State University, she joined the research group of Dr.

Richard D. Gandour. She received her Doctorate degree in chemistry in 2007. She has

been a wife of Ernest Lada since 2002 and a mother of Stanley Lada since 2006.

Synthesis, Characterization, and Antimicrobial Activity of Water … · Synthesis,...

Documents

Transcript of Synthesis, Characterization, and Antimicrobial Activity of Water … · Synthesis,...