Strategies for Protecting Group Free Glycosidation

by

Melissa Ann Cochran

A thesis submitted in conformity with the requirements for the degree of Master of Sciences Graduate Department of Chemistry

University of Toronto

© Copyright by Melissa Ann Cochran 2011

ii

Strategies for Protecting Group Free Glycosidation

Melissa Ann Cochran

Master of Sciences

Graduate Department of Chemistry University of Toronto

2011

Abstract

The synthesis of glycoconjugates is of interest in biological and medicinal research. There are

numerous approaches to the synthesis of glycosides involving protecting group free methods.

This thesis outlines what has been achieved in the field and two novel approaches for O-

glycosidation. The first approach involves the use of a toluenesulfonohydrazide glycoside with a

purification handle designed for simple glycoside purifications. The butyl 3-O-octyl-D-

glucopryanoside was successfully synthesized but did not have the desired property of yielding

simple-to-purify glycosides as products. The second approach uses a thiouronium glycosyl

donor; a variety of glycosidations using this donor were investigated. The glucosyl thiouronium

salt donor was shown to undergo glycosidation effectively with simple alcohols.

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Acknowledgments

I would like to start by thanking my supervisor, Professor Mark Nitz, for the opportunity to work

in his group on a variety of glycoside synthesis projects and for sharing his wealth of knowledge

on glycosides with me over the past year.

I would also like to say thank you to Dr. Deborah Zamble for reading my thesis.

A very big thank-you to everyone in the group (Pengpeng, Chibba, Joanna, Rullo, Rodolfo,

Xiong, Somnath, Varvara, Yunshan and Landon), you were all exceptionally wonderful people

to work with and get to know over the last year. Joanna – Thank you for answering plenty of

random questions throughout the year and always being around to chat with. Pengpeng – Thank

you for being an awesome friend to share a work-space with and for ordering all the chemicals.

Chibba – Thanks for helping me learn to read sugar proton NMRs and for sometimes being a

(much needed) distraction from my work. Xiong – Thank you for your helpful suggestions on

my project for Lautens’ class in fall term. I wish you all good-luck with your remaining time in

the Nitz lab and in whatever you chose to do afterwards.

I would also like to thank the staff in the NMR lab for all of their help and for what they do to

keep the NMR lab running smoothly for us. Thank-you to Ken, and everyone in ChemStore, for

always having what I needed to get my work done.

Lastly, I would like to thank my friends and family for all of their love and support throughout

the last year. It is without a doubt due to all of you that I made it through this last year and that I

am now writing these acknowledgements, so again, thank-you so very much.

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Table of Contents

Acknowledgments .............................................................................................................................. iii  

Table  of  Contents .................................................................................................................................iv  

List  of  Abbreviations...........................................................................................................................vi  

List  of  Figures..................................................................................................................................... viii  

List  of  Schemes .....................................................................................................................................ix  

1   An  Introduction  to  Glycosides  and  Glycosidations.............................................................1  1.1   Protecting  Group  Free  Glycosidations.......................................................................................... 2  

2   O-­Glycoside  Synthesis...................................................................................................................3  2.1   Fischer-­Type  Glycosidations............................................................................................................ 3  2.2   Remote  Activation ............................................................................................................................... 7  2.3   Novel  Glycosyl  Donors........................................................................................................................ 9  

3   S-­Glycoside  Synthesis ................................................................................................................ 12  3.1   Direct  S-­Glycosidation......................................................................................................................12  3.2   Synthesis  of  1-­thiols  and  their  use  as  S-­glycosidation  precursors....................................13  

4   N-­Glycoside  Synthesis................................................................................................................ 15  4.1   Azide  Formation.................................................................................................................................15  4.2   Hydrazide  Formation .......................................................................................................................18  4.3   Aminooxy  Linked  Carbohydrate  Conjugates ............................................................................22  4.4   N-­Alkyl  Hydroxylamine  Linked  Carbohydrate  Conjugates..................................................24  

5   Project  Objective......................................................................................................................... 31  5.1   Developing  an  Efficient  Synthesis  for  β-­Glycosides  of  Simple  Alcohols ..........................31  5.2   Investigating  Glycosyl-­Thiouronium  Salt  as  a  Novel  Glycosyl  Donor...............................31  

6   Results  and  Discussion.............................................................................................................. 33  6.1   Developing  an  Efficient  Synthesis  for  β-­Glycosides  of  Simple  Alcohols ..........................33  6.2   Investigating  Glycosyl-­Thiouronium  Salt  as  a  Novel  Glycosyl  Donor...............................34  

7   Conclusions ................................................................................................................................... 38  

8   Experimental ................................................................................................................................ 39  

v

Appendices........................................................................................................................................... 42  

References............................................................................................................................................ 59  

vi

List of Abbreviations

Ac acetyl

AcOH acetic acid

Bn benzyl

Bu3P tributylphosphine

cat catalyst

DCM dichloromethane

DMF dimethylformamide

DMSO dimethylsulfoxide

Et ethyl

EtOAc ethyl acetate

eq equivalents

GalNAc N-acetylgalactosamine

1H NMR proton nuclear magnetic resonance

h hour(s)

J coupling constant

Me methyl

MeCN acetonitrile

MeOH methanol

min minute(s)

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mmol millimole

MOP 3-methoxy-2-pyridyloxy

NBS N-bromosuccinimide

OTf triflate

Ph phenyl

rt room temperature

t time

TBAC tetrabutylammonium chloride

TFA trifluoroacetic acid

TLC thin layer chromatography

TsHydrazide p-toluenesulfonyl hydrazide

viii

List of Figures

Figure 1.1 Examples of natural glycosides………………………………………………………. 1

Figure 2.1 Mechanism for remote activation using MOP donors………………………………... 7

Figure 2.2 Furanosides synthesized by Ferrières via MOP donors…………………………….… 9

Figure 3.1 Arylthiols used in the synthesis of S-arylglycosides………………...……………… 14

Figure 4.1 Bishydrazide linkers synthesized by Wei…………………………………………… 20

Figure 4.2 Photoreactive agent with aminooxy side chain for conjugation with a carbohydrate. 23

Figure 4.3 Oligopeptoid used in the synthesis of glycopeptoids using aminooxy linkage

chemistry…………………………………………………………………………….. 27

Figure 4.4 BA-sugar conjugate with N-methyl-hydroxylamine linker…………………………. 28

Figure 4.5 A chlorambucil glycoconjugate……………………………………………………... 28

Figure 4.7 Substrates used in stability studies of N-glycosides………………………………… 30

Figure 6.1 Anomeric peaks of crude octyl-glucopyranoside, rxn 6.4, t = overnight…………… 37

ix

List of Schemes

Scheme 1.1 Mechanism of glycosidation reaction showing oxocarbenium ion intermediate…… 1

Scheme 2.1 A typical Fischer glycosidation reaction……………………………………………. 3

Scheme 2.2 Ultrasonic assisted Fischer glycosidation with H2O-silica………………………….. 5

Scheme 2.3 Hanessian’s synthesis of MOP donors……………………………………………… 7

Scheme 2.4 Synthesis of glycosyl ester via MOP donor………………………………………… 8

Scheme 2.5 Synthesis of aspirin using MOP donor…………………………………….………... 8

Scheme 2.6 Synthesis of bromobutyl glycosyl donor……………………………….………….. 10

Scheme 2.7 Proposed mechanism for glycosidation using bromobutyl glycosyl donor……….. 11

Scheme 2.8 Synthesis of TSH glycosyl donors………………………………………………… 11

Scheme 2.9 Mechanism for glycosidation using TSH donor…………………………………… 12

Scheme 3.1 Synthesis of S-aryl glycosides……………………………………………………... 13

Scheme 3.2 Glycosyl thiol synthesis…………………………………………………………… 14

Scheme 4.1 Azide formation using Appel reaction conditions with sodium azide.……………. 15

Scheme 4.2 Azide formation from TSH glycosyl donor……………………………………….. 17

Scheme 4.3 An example of a hydrazide synthesizded by Flinn………………………………… 20

Scheme 4.4 Synthesis of pyrazole-5-carbohydrazide N-glycosides……………………………. 21

Scheme 4.5 Synthesis of a biotin-terminated poly(acryloyl hydrazide) scaffold………………. 21

Scheme 4.6 Mechanism of glycolipid formation using the aminooxy functionality…………… 24

Scheme 4.7 Synthesis of N-methyl-hydroxylamine carbohydrate derivatives…………………. 25

x

Scheme 4.8 Synthesis of N-methyl-hydroxylamine linked disaccharide……………………….. 26

Scheme 4.9 Synthesis of ribose-calicheamicin derivative……………………………………… 29

Scheme 5.1 An overview of the synthesis of a glycoside with a purification handle…………... 31

Scheme 6.1 Synthesis of butyl-glucoside with octyl chain purification handle………………... 34

Scheme 6.2 Synthesis of glucosyl-thiouronium salt……………………………………………. 34

Scheme 6.3 a-f Various reactions performed with glucosylthiouronium salt…………………... 35

Scheme 6.4 Synthesis of octyl-glucopyranoside………………………………………………... 37

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1 An Introduction to Glycosides and Glycosidations Glycosides are important biological molecules that are involved in a vast number of biological

processes.1 Many glycosides have therapeutic uses such as anti-cancer therapies, anti-

inflammatories, enzyme inhibitors and antibiotics2 (Figure 1.1). Glycosidation reactions are a

means to produce synthetic analogues of these important molecules. Typical glycosidation

reactions are challenging and laborious. Generally, glycosidations require several protection and

deprotection steps; these steps often result in time-consuming reaction sequences with low

overall yields. The protecting groups used can also influence, and change, the stereochemistry of

the reaction. Control of the stereoselectivity of glycosidation reactions is a challenge in itself

due to the mechanism of the typical reaction, which proceeds through an oxocarbenium ion

intermediate, which can produce a mixture of both α- and β-anomers if not carefully controlled

(Scheme 1.1).

Figure 1.1 Examples of natural glycosides. (a) Aesculin (aids in the identification of bacterial

species); (b) Salicin (an anitinflammatory found in willow bark); (c) Apertin (dilates coronary

arteries and blocks calcium channels)

Scheme 1.1 Mechanism of glycosidation reaction showing oxocarbenium ion intermediate

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1.1 Protecting Group Free Glycosidations

Due to the challenges presented when protecting groups are used, synthesis of glycosides without

the use of protecting groups is appealing. Reactions of this type have shorter reaction sequences,

which typically result in higher overall yields. The reactions are more atom economical without

the use of protecting groups – leading to less waste and fewer reagents. The decreased labour

and increased reaction efficiency are also attractive. However, protecting group free reactions

are not without their own set of challenges. The main challenges of these types of reactions are

that they often have difficult purifications, need to be highly selective due to potential competing

reactions and lack the stereocontrol often afforded by protecting groups.

The rest of the introduction will look at advancements made in three specific, important areas of

protecting group free glycoside synthesis: O-glycosides, S-glycosides and N-glycosides, as these

are the focus of the research carried out.

3

2 O-Glycoside Synthesis In the last decade there has been continued progress in the protecting-group free synthesis of O-

glycosides. O-glycosides are the major type of glycoside found in biological systems and thus

have garnered much of the attention of synthetic chemists. Efficient syntheses of O-glycosides

are desired to provide access to these molecules for biological studies and for the discovery of

new therapeutics.

2.1 Fischer-Type Glycosidations

Fischer glycosidations remain the simplest and most effective method for performing simple

glycosidations; they involve the addition of an alcohol to an unprotected sugar in the presence of

an acid catalyst (Scheme 2.1). Research into improving Fischer glycosidations has focused on

evaluating various catalysts, solvents and heating methods to reduce the long reaction times, the

large excesses of alcohol required, the mixtures of products that are formed and the often low

yields. Furthermore, this research has aimed to allow glycosidations with alcohols beyond the

simple low boiling point alcohols, which are used in traditional Fischer glycosidations.2

Scheme 2.1 A typical Fischer glycosidation reaction

Microwave irradiation is one method that has been explored to improve Fischer glycosylations.

Bornaghi and Poulsen3 investigated the effect of microwave irradiation on the reaction to form

the methyl glycoside of N-acetyl-D-glucosamine using anhydrous methanol as a solvent and

Amberlite resin IRN 120H+ as the acid catalyst. They found that optimal results could be

achieved after just 10 min of microwave irradiation at 120 ˚C. After 10 min, the yield of

recovered product was 80% with selectivity at 13.2:1 (α:β); compared to 70% recovered product

and 11.6:1 (α:β) after 8 h of the traditional reflux method. The use of microwave irradiation

improved the yield and reaction time for a variety of alcohols (methanol, ethanol, benzyl alcohol

and allyl alcohol) and other sugars (N-acetyl-D-galactosamine, D-glucose, D-galactose and D-

mannose).

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Roy and Bordoloi4 have also investigated microwave-assisted Fischer glycosylations; their

method uses montmorillonite K-10 as a catalyst. The mixture of D-glucose, montmorillonite K-

10 and dry methanol was allowed to react under microwave irradiation at 90 °C for 10 min.

Optimum selectivity (14.6:1 α:β) and yield (86%) occurred at this time interval. Further

experiments were done to test scope and a variety of monosaccharides and alcohols were found

to give similar yield and good selectivity (8:1 to 15:1).

Though reaction times, yields and selectivity are comparable for both of the above microwave

methods, the montmorillonite K-10 catalyst method can be performed at a lower temperature

with fewer equivalents of catalyst. It is also worth noting that the montmorillonite K-10 catalyst

could be recovered via filtration and reused up to three times without loss of reactivity. Both

methods show that microwave irradiation can be applied to variety of sugar/acceptor

combinations; however, the methods were only tested with monosaccharides and low boiling

alcohols. It would be interesting so see if this method could be applied to more complex sugars

and/or alcohols.

Roy and Mukhopadhyay5 used H2SO4-silica as a catalyst for Fischer glycosidation. The model

reaction involved D-glucose being suspended in propargyl alcohol and stirred at 65 °C. H2SO4-

silica was added and stirred until all the solids had dissolved (~2.5 h), which corresponded to

complete conversion of starting materials; at this point the α:β was 6:1 and the isolated yield was

75%. In the model reaction of D-glucose and propargyl alcohol, the α:β ratio was found to

improve to 10:1 and yield increased to 80% if the reaction mixture was allowed to stir for 3 h at

65 °C after complete dissolution of starting materials. Recovery of the product involved cooling

the reaction mixture to r.t. and transferring it to a silica gel column; excess propargyl alcohol was

eluted with DCM followed by elution of the product with 15:1 DCM/MeOH. Reactions were

then done on a variety of sugars and alcohols to investigate the scope. Overall, Fischer

glycosidations were successful, giving high yields and alpha selectivity using fewer equivalents

of alcohol (5 eq) and in shorter reaction times (2-8 h) than traditional methods, showing that this

method can be used as a general approach to Fischer glycosidations.

Shaikh et al6 looked at a method involving the combination of two already established methods

to improving Fischer glycosidations. The method involves using ultrasonic technology in

combination with H2SO4-silica as a catalyst (Scheme 2.2). The reactions were done on a 1 mmol

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scale by suspending the starting material in 5 equivalents of the acceptor alcohol and stirring in

an ultrasound bath at 40 °C. The yield using propargyl alcohol as the acceptor, in reactions with

various sugars, ranged from 82-98% and selectivity favoured the α-anomer (the only product in

most cases and 10:1 (α:β) otherwise). Reaction times were approximately 30 min with lactose

taking 12 h. The yield for lactose was 98%, which is interesting as dissacharides can be

susceptible to hydrolysis under Fischer glycosidation conditions.

Scheme 2.2 Ultrasonic assisted Fischer glycosidation with H2O-silica

Comparing the two methods that involve the use of H2SO4-silica as a catalyst it is clear that there

is a benefit in using ultrasonic technology in decreasing reaction times as the yields and

selectivity in both methods are comparable.

The use of ionic liquids as solvents for Fischer O-glycosidations is an area that has recently been

explored. Unprotected carbohydrates only dissolve in the most polar organic solvents, which can

be difficult to remove from reaction mixtures and the use of ionic liquids eliminates the need to

use these solvents. Lindhart et al7 looked at room temperature ionic liquids (RTILs) as solvents

for unprotected glycoside synthesis after they proved to be useful solvents for the protection of

carbohydrates. They used RTIL 1-ethyl-3-methylimidazolium benzoate ([emIm][ba]) as the

solvent, in the presence of Amberlite IR-120 (H+) as a catalyst. They successfully produced the

benzyl-α-glycosides of D-glucose, D-mannose and N-acetyl-D-galactosamine. The yields of the

reaction were moderate (27-64%) and the authors suggest that the moderate yields are likely due

to the viscosity of the RTIL, which may prevent adequate mixing of the heterogeneous

suspensions. Various disaccharides were also synthesized by this approach using p-

toluenesulfonic acid (TsOH) as the catalyst, which was also found to decrease the viscosity of

the reaction. The reactions used 2-10 equivalents of a partially protected acceptor, either 2,3,4-

Tri-O-allyl- or 2,3,4-tri-O-benzyl-protected methyl α-D-glucoside. Again, the α-glycosides were

isolated and yields were modest, ranging from 20-41%, possibly due to the difficulty in

recovering the products from the RTIL. The methods described above improve upon Fischer

glycosidation in shortening reaction times and giving good anomeric selectivity; although this

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method does not improve the yield and a large excess of alcohol is required, the methodology is

extendable to dissacharide sugars, a place where typical Fischer glycosidations are not

successful.

Lindhart et al8 have also used the above chemistry to synthesize a variety of alkyl glycosides for

use in protein-carbohydrate interaction studies. The yields were between 12-35% and in many

cases product was not isolated. This is once again due to the viscosity of the RTIL and the

difficulty in recovering the products from the ionic liquid. Even with the low yields, this method

still represents an approach to a range of glycosides that are not achievable under traditional

Fischer glycosidation conditions. Yields may be improved upon discovery of an efficient way to

recover the products.

Auge and Sizun9 have been doing work with ionic liquids as a greener approach to Lewis acid

catalyzed glycosidations. The RTIL used was 1-­‐butyl-­‐3-­‐methylimidazolium  

trifluoromethanesulfonate  ([BMIM][OTf])  and  Sc(OTf)3  was  used  as  the  Lewis  acid  to  do  a  

glycosidation   on   a   variety   of   sugars   with   a   variety   of   alcohols.     Yields   ranged   from   51-­‐

100%,   which   was   a   significant   improvement   compared   to   the   neat   reactions   they  

performed   without   an   ionic   liquid;   yields   in   the   neat   reactions   ranged   from   14-­‐75%.    

Selectivity   also   varied   depending   on   the   sugar   or   alcohol   being   used,   ranging   from   61-­‐

100%  α.    The  importance  of  this  study  is  the  greener  approach  to  the  glycosidations  due to

the lower equivalents of alcohol used (5 eq) compared to other methods that typically use

approximately 20 equivalents; it also uses a minimal amount of catalyst (5 mol %); the reaction

does not involve the use of harsh organic solvents; and lastly, the RTIL can be recovered and

used up to three times without loss of yield and selectivity. The recovery of the RTIL is

achieved by filtering the crude reaction mixture through a silica gel pad eluting with 0-15%

MeOH in EtOAc; giving first the glycoside followed by the elution of the ionic liquid.

Noteworthy is that a model reaction was found to proceed with only 1 equivalent of octanol and

1 mol% of Lewis acid to give the desired product in 58% yield.

A comparison of the reports using RTILs shows that they can provide a greener solvent to

perform Fischer glcosidations and that the reaction can be generalized to a wide range of

alcohols; however, methods are required for easier recovery of products. It is important to note

7

that these reactions require long reaction times; depending on the sugar and acceptor being used,

reaction times average 6 to 24 h.

2.2 Remote Activation

Hanessian2 introduced a method that involves activating the anomeric group by interaction of a

promoter with an atom not attached to the anomeric center (Figure 2.1). The details of this

method are extensively covered in Hanessian’s2 review of glycosyl reactions involving

unprotected glycosyl donors; however, there has been further research in this area since this

publication.

Figure 2.1 Mechanism for remote activation using MOP donors

The Hanessian lab developed the 3-methoxy-2-pyridyloxy (MOP) donors for protecting group

free glycosidation. These substrates are synthesized in one of two ways (Scheme 2.3). In the

first method, silver 3-methoxy-2-pyridoxide is used to couple the MOP acceptor with a

peracetylated glycosyl halide. The reaction is done at 100-110 °C in toluene for a short period of

time and a simple filtration gives the desired 1,2-trans anomeric configuration in good yield.

The second method involves using 3-methoxy-2-(1H)-pyridone in the presence of a phase-

transfer catalyst (either (hexyl)4)-NHSO4 or Bu4NBr) in a mixture of aqueous NaOH and DCM.

Yields for this method ranged between 45-60%. Both methods require the donors to be de-O-

acetylated using NaOMe in MeOH (Scheme 2.3).

Scheme 2.3 Hanessian’s synthesis of MOP donors

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In 2002, Hanessian’s group elaborated the use of the MOP donors for the synthesis of glycosyl-

1-carboxylates.10 The reaction of either α-D-gluco or D-galacto MOP glycosyl donor with an

excess of a carboxylic acid (20 eq for benzoic acid and 2-phenylacetic acid; 200 eq for pivalic

acid) without any promoter and using DMF or MeCN as a solvent gave good to excellent (58-

98%) yields and selectivity favoring the α-glycosyl esters (5:1 to 30:1 α:β) (Scheme 2.4).

Scheme 2.4 Synthesis of glycosyl ester via MOP donor

Improved reaction times and selectivity in the glycosidation were achieved by using 6-O-tert-

butyldiphenylsilyl (OTBDPS) protected donors which allowed solubility in DCM. Promotion of

the reaction with only 1.5 equivalents of carboxylic acid with MeOTf gave 62-70% yield with

impressive stereoselectivity >50:1 (α:β). The MOP donors provide a valuable approach to

synthesize glycosyl-1-carboxylates as these compounds are prone to ester migration when

deprotection steps are required.

The method outlined here has been applied to the synthesis of aspirin pro-drugs. Using aspirin

as the carboxylic acid (Scheme 2.5) the glycosyl ester was produced in 68% yield and following

cleavage of the TBDPS group a completely water-soluble aspirin derivative could be released

within 30 min at pH 1. This discovery hints that MOP glycosyl donors could have future

applications in drug delivery. The paper showed the reaction can be applied to a variety of

different carboxylic acids; however, the scope of the method has yet to be confirmed on a wide

range of MOP donors.

Scheme 2.5 Synthesis of aspirin using MOP donor

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Ferrières et al11,12,13 have used the remote activation approach to synthesize a variety of

furanosyl derivatives (Figure 2.2). However, for the purposes of this report, these will not be

discussed in depth.  

 

Figure  2.2  Furanosides  synthesized  by  Ferrières  via  MOP  donors  

MOP  donors  are  particularly  appealing  substrates  for  glycosidations  due  to  their  versatility.    

As  outlined  above,  many  MOP  donors  have  been  synthesized  that  work  to  varying  degrees  

for  different  types  of  reactions.    They  can  be  used  to  synthesize  a  variety  of  different  types  

of   O-­‐glycosides   such   as   glycosyl-1-carboxylates.     This   perhaps   being   the   most   appealing  

application,   as   these   types   of   glycosides   under   other   conditions   are   susceptible   to  

unwanted   rearrangements   that   are   not   seen   when   using   MOP   donors.     This   specific  

application  has  also  given  light  to  the  idea  that  MOP  glycosyl  donors  may  have  applications  

in   drug   delivery.     Lastly,   they   have   also   been   used   extensively   to   develop   an   array   of  

different  furanosides  that  may  have  applications  in  biology.    

2.3 Novel Glycosyl Donors

Another   area   of   current   interest   in   glycoside   synthesis   is   the   discovery   of   novel  

unprotected  glycosyl  donors.    Glycosyl  donors  often  require  many  steps  in  their  syntheses;  

this   adds   to   the   overall   number   of   steps   required   for   glycosidation   reactions,   thus  

decreasing  overall  yield  from  the  start.    It  is  for  this  reason  that  groups  have  searched  for  

10

novel  unprotected  glycosyl  donors   that   can  be  easily   synthesized   in  high  yield   in  a   small  

number  of  steps.  

Davis   et   al14   have   explored   the   use   of   bromobutyl   unprotected   glycosyl   donors.     The   4-­‐

bromobutyl   tetra-­‐O-­‐   acetyl-­‐a-­‐D-­‐mannopyranoside  was   synthesized   in   three   steps   in   42%  

overall  yield  (Scheme  2.6).    A  direct  Fischer  synthesis  was  also  possible,  however  the  yield  

of  the  final  product  was  only  18%.    A set of glycosidation reactions were explored using either

MeOH (5 eq) or diacetone galactose (1 eq) as acceptors while varying the solvent, reaction time

and activator. Reactions proceeded best using DMF or DCM as the solvent and AgOTf as an

activator. Under these conditions, using the diacetone galactose acceptor, the yield was 66%

with a 9:1 (α:β) ratio. The mechanism of this reaction is believed to involve an activation step

where a soft Lewis base is interacting with a hard Lewis base; however, aspects of the

mechanism are still uncertain (Scheme 2.7). Though the yields are only moderate, the ability to

use unprotected donors reduces the number of overall yield-reducing steps. This paper proves

that bromobutyl unprotected glycosyl donors can function to synthesize simple glycosides and

further investigation should be done to determine the generality of the method.

Scheme 2.6 Synthesis of bromobutyl glycosyl donor

Scheme 2.7 Proposed mechanism for glycosidation using bromobutyl glycosyl donor

11

Our lab has synthesized N’-glycopyranosulfonohydrazides (GSHs) as glycosyl donors for use as

unprotected donors for O-glycosidations.15 N-acetyl-D-glucosamine (GlcNAc) was successfully

transformed into N-acetyl-D-glucosamine sulfonohydrazide on a multigram scale and

disaccharide donors were synthesized on milligram scales in excellent yields (Scheme 2.8). The

synthesis of GSHs has the advantage of not requiring any protection/deprotection steps, making

its synthesis simple and high yielding.

 

Scheme 2.8 Synthesis of TSH glycosyl donors

Donors were reacted with various alcohols (20 eq), in the presence of NBS (2.4 eq), using DMF

as a solvent with good yields (70-87%) and good selectivity (1:6 – 1:10 α/β). These results

show the versatility of the synthesized donors for use in preparing a variety of different

glycosides. A proposed mechanism is that the reaction proceeds through a diazene intermediate.

Elimination of sulfinic acid and nitrogen gas gives the oxocarbenium ion (Scheme 2.9). The

oxocarbenium ion is trapped by the incoming alcohol and the stereochemistry of the attack is

biased by the neighbouring acetamido group. The sulfinic acid that is generated undergoes

oxidation in situ to generate the sulfonyl halide. The main limitation of the method outlined

above is that the reaction does require a large excess of alcohol (20 eq); however, it is a method

to β-glycosides, which is uncommon compared to methods to synthesize α-glycosides.

 

Scheme 2.9 Mechanism for glycosidation using TSH donor

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3 S-Glycoside Synthesis Though there is much more focus on O-glycosidation, S-glycosidation does have an important

place in carbohydrate chemistry. S-glycosidation is important in the synthesis of many

glycoconjugates due to the orthogonal reactions possible with thiol nucleophiles. Current

literature focuses on the synthesis of the aforementioned conjugates from glycosyl-thiols.

3.1 Direct S-Glycosidation

Shoda et al16 have recently done work on synthesizing aryl-thioglycosides from fully unprotected

sugars. The basis for this work was previous work by their group in using 2-chloro-1,3-

dimethylimidaxolinium chloride (DMC) to synthesize 1,6-anhydrosugars from unprotected

sugars. They hypothesized that if the reactions were carried out in the presence of a thiol, the 1-

thiolglycoside would result. They started by screening bases using D-glucose as the model

substrate and determined that 10 equivalents of triethylamine worked most efficiently. Reactions

were carried out on a handful of different sugars and arylthiols using 3 equivalents of DMC, 10

equivalents of Et3N and 3-7 equivalents of aryl thiol depending on the aryl thiol being used

(Scheme 3.1). Reactions were stirred in a mixture of H2O and MeCN for 1 h.

Scheme 3.1 Synthesis of S-aryl glycosides

They were able to use this approach to synthesize five glucosides using five different aryl thiols

(Figure 2.1). Reactions were done at -15°C (1), 0°C (3,2,5) and room temperature (4) using a 1:1

H2O/MeCN mixture. Yields for all five reactions were excellent (90%-quant) and selectivity

was also excellent – exclusively β for 4 and 5 and 6.7:1, 4.5:1 and 10:1 (α/β) for 1, 2 and 3,

respectively, using glucose as the carbohydrate.

13

Figure 3.1 Arylthiols used in the synthesis of S-arylglycosides

They were also used the above method to synthesize the aryl thioglycoside of four other sugars

(cellobiose, lactose, laminaribiose and melibiose) using aryl thiol 5. These reactions were all

done at 0 °C and used a 4:1 H2O/MeCN mixture. The yields for these reactions were

quantitative and gave exclusively the β-product.

Overall the method provides a route to aryl thioglycosides with short reaction times, excellent

yields and β-selectivity. A limitation of the above data is that it does not confirm the scope of

the reaction with a variety of monosaccharides. In the future, it would be of interest to see if this

method can apply to oligosaccharides as well.

3.2 Synthesis of 1-thiols and their use as S-glycosidation precursors

In 2006, Davis et al17 reported the synthesis of glycosyl thiols from fully unprotected sugars.

The developed method was first used on protected sugars and was then extended to fully

unprotected sugars. They used Lawesson’s reagent (1.5 eq) in anhydrous dioxane at 110 °C for

48 h (Scheme 3.2). This was followed by the reaction mixture being cooled and filtered through

Celite and concentrated. The crude product was then dissolved in wet chloroform and methanol;

a solution of tributylphosphine was stirred into the mixture, which was then stirred for 4 h. The

product was purified by column chromatography. The yields were good for the three sugars

tested (glucose, mannose and galactose), ranging from 63-71%. The single step and absence of

protecting groups makes the transformation an efficient route to the synthesis of

glycoconjugates.

14

Scheme 3.2 Glycosyl thiol synthesis

As mentioned previously, once synthesized, glycosyl thiols have many applications in biological

chemistry. Davis et al18,19 used the 1-thiosugars they synthesized in the addition of the 1-

thiosugars to selenylsulfide-activated single cysteine mutant proteins to give complete

conversion to the corresponding glycoproteins. Many other groups have been able to use

glycosyl thiols to produce various types of glycoconjugates that may be useful for biological

studies.20,21,22,23,24,25

15

4 N-Glycoside Synthesis N-glycosidations are an appealing alternative to O-glycosidations as building blocks to many

larger molecules that have potential as therapeutics, as they are often simpler to form and can be

easily elaborated from existing structures. Recently significant advances in the protecting group

free approaches to the synthesis of azido-glycosides, glycosyl hydrazides and alkoxyamine

glycosides have been reported.

4.1 Azide Formation

In constrast to other N-glycosides the formation of glycosyl azides proceeds through an activated

glycosyl donor, but high yields and selectivity can be achieved due to the high nucleophilicity of

the azide.

Gouin and Kovensky26 were successful in combining Appel reaction conditions with sodium

azide as a system to achieve the synthesis of azido glycosides from unprotected sugars.

Optimization of the reaction was done using D-mannose as a model substrate and it was found

that complete disappearance of starting material required 2 equivalents of PPh3, 2 equivalents of

CBr4 and 10 equivalents of NaN3; the reaction was stirred at room temperature for 60 h in DMF

(Scheme 4.1). To simplify the purification and characterization efforts, the crude mixtures were

per-acetylated in Pyr/Ac2O. The D-mannopyranosyl azide was isolated in 41% yield in a 1:1

ratio of anomers.

Scheme 4.1 Azide formation using Appel reaction conditions with sodium azide

The reaction was optimized by increasing the excess of reagents used (4 eq PPh3/CBr4 and 20 eq

NaN3). This had no effect on the yield, which was 42% in this case; but resulted in substitution

at the C6 position as well as at the anomeric position. The selectivity of the reaction also

remained unchanged. Reactions with D-glucose and D-galactose were explored. The yields of the

products for these sugars were 39% and 40%, respectively; and selectivity was 1:4 (α/β) and

pure β, respectively. When the amount of reagent used was doubled to eight equivalents of

16

PPh3/CBr4 the C6 position underwent substitution. The yields were 39% and 20%, respectively;

and selectivity was 1:3 (α/β) and pure β, respectively.

The methodology was extended to di- (maltose) and tri-saccharides (maltotriose). When using 4

eq of PPh3/CBr4 it was the primary alcohol groups that were converted to azide groups. The

yields were 49% for maltose and 26% for maltotriose. Again, the amount of reagent was

increased to 8 eq, which resulted in the azidation of the anomeric position with yields of 49% for

maltose, 26% for maltotriose and 13% for lactose (in the case of 4 eq of reagent the product

could not be separated from a complex mixture of side products). Selectivity was 1:3 (α/β) for

both maltose and maltotriose and pure β for lactose. The yields for this reaction are moderate,

however, it does represent a new and efficient way to carry out azidations without the use of

protecting groups. Of particular interest here is the ability to control which of the carbons

undergo substitution based on the amount of reagent used.

Chambert et al27 developed a similar method for the generation of azido sugars; it involves the

use of hydrazoic acid under Mitsunobu conditions. There reaction involves stirring the sugar in

DMF with PPh3 and DIAD (2 eq each) in the presence of excess HN3 overnight. The HN3 was

produced in one of two ways: the first follows known procedures28 to produce a dry HN3 solution

in toluene, this solution was then used directly in the Mitsunobu reactions; the second method is

the direct treatment of an excess of NaN3 with H2SO4 in DMF, the mixture was dried in sodium

sulphate and then added to the sugar solution to be used in the reactions. Using the HN3 in a

solution of toluene with glucose, only the mono-azide was formed in 88% yield with 5:2 α/β

selectivity. Increasing the number of equivalents gave the diazide with substitution at the

anomeric and C6 positions in 91% yield with 3:1 α/β selectivity. The authors did not extend this

chemistry to any other aldoses, as Nitz15 and Shoda29 had reported on modifications of aldoses

for the synthesis of β-glycosyl azides during the time their work was being completed. When the

reaction was extended to ketoses (fructose and isomatulose), a complex mixture of monoazide

products was obtained. Again, increasing the amount of reagent led to the production to

diazides, however, there were also monoazides present. The method here does not necessarily

present any advantages over the methods presented by Gouin, Nitz and Shoda; however, it does

present new chemistry and gives a different option in producing azido sugars.

17

As mentioned previously in the O-glycosidation section, our group as successfully developed

TSH glycosyl donors.15 It is possible to use TSH donors for the synthesis of glycosyl azides. It

was found that the reaction of the GlcNAc TSH donor with TBAC and lutidine in DMF resulted

in the formation of an oxazoline intermediate (observed in situ) which could then be opened with

NaN3 to give the glycosylazide in 73% yield (Scheme 4.2). It is worth noting that the formation

of the glycosyl azide directly in the glycosylation reaction was not possible due to the azide

source being incompatible with the conditions necessary to activate the TSH donor.

Scheme 4.2 Azide formation from TSH glycosyl donor

Though the above synthesis requires two steps, the synthesis of the TSH donor is quite simple

(as outlined earlier) and thus the above method of azidation is appealing. The yields are far

better than those using the method of Gouin and Kovensky and the reaction is accomplished

overnight, rather than 60 h.

Shoda et al29 extended chemistry they previously developed for the synthesis of S-glycosides to

the synthesis of azido-glycosides. They use 2-chloro-1,3-dimethylimidaxolinium chloride

(DMC) to form β-glycosyl azides in aqueous media. The reactions were carried out in water

using base (the base used depended on the sugar and was either Et3N, DIPEA or 2,6-lutidine;

equivalents of base also varied between 6 and 75), DMC (equivalents varied depending on sugar

and ranged between 3 to 25) and 2.5 M NaN3. The reactions took between 0.5 and 48 h

depending on the complexity of the sugar and were done at either 0 °C or room temperature.

18

Overall isolated yields for these reactions ranged from 49 to 87%; with only the β-anomer

formed in most cases. Noteworthy is that this was applied to a complex decasaccharide and an

isolated yield of 87% was obtained.

This method’s main benefit over the above-mentioned methods is that it has much lower reaction

times for glucose as well as other oligosaccharides (2-7 sugar units). Besides reaction time, an

advantage to this methodology is that it is has been shown to give high isolated yields of high

order sugar units, which previously have not been accessible. Also, this method works

irrespective as to whether there is a hydroxy group or acetamide group at the 2-position; this is

not explored in the above methodologies and it is known that changing the group at the 2-

position can affect whether a given reaction is successful.

4.2 Hydrazide Formation

The formation of glycosyl hydrazides as a method for N-glycosidation has been investigated by

many different groups and has been used for different purposes such as the formation of novel

glycosyl donors15 and as linkers for glycoconjugation reactions. The use of hydrazides for

glycoconjugation reactions is appealing because the cyclic structure of the sugar, which is often

lost when traditional reductive amination methods are used. Feizi et al30 provided an example

showing this advantage when they developed a hydrazide-biotin derivative that could

successfully be coupled to unprotected oligosaccharides. Their approach represented an

advantage to reductive amination because the cyclic structure of the sugar was maintained during

the conjugation reaction; as well, only a small excess of the biotion derivative was required.

There has been much exploration into the use of hydrazides as a method of glycoconjugate

formation in the past decade as well.

Augé and Lubin-Germain31 investigated the reaction between glucose and acylhydrazides to

develop a method of forming long-chain glycosyl derivatives. Long-chain glycosyl derivatives

are of interest due to having many areas of application such as: purification of membrane

proteins, molecular recognition in glycobiology or immunology and, of particular interest to

Augé and Lubin-Germain, as surfactants. They looked to developing long-chain

glycosylhydrazides as a new class of sugar surfactants. Acylhydrazines of various chain lengths

were coupled to glucose in MeOH at 65 ˚C to give the glucosylhydrazides. Reaction yields

ranged from 70 to 98%. The chemistry was extended to maltose to determine if the reaction

19

would proceed with a dissacharide; octanoylhydrazide gave the corresponding glycosylhydrazide

in 55% yield. The major anomer in all cases was the β-anomer. The importance of this

chemistry is that it shows that long chain glycosylhydrazides can be easily synthesized and

purified in high yields.

In 2001, Peluso and Imperiali32 synthesized alanine-β-hydrazide and coupled it to GlcNAc using

the following conditions: 1 M solution of GlcNAc in a sodium acetate buffer (0.1 M, pH 5.6, 5

eq) was added to a solution of the alanine-β-hydrazide (12 µmol) in DMSO (50 µL). After 24 h

there was complete conversion, however there was a mixture of both the cis and trans-β-isomers.

This chemistry represents an early approach to synthesizing glyco-peptide conjugates using

hydrazide linkers and many extensions to this methodology have been made.

Sugar conjugation with other biologically important molecules is an area of great interest,

especially in the discovery of probes and therapeutics. Flinn et al33 developed a one-step method

of producing a sugar with a chemoselective linker for controlled conjugation. The linkage

chemistry they wished to explore relies on hydrazone formation and it is therefore necessary to

introduce a hydrazide moiety. Sugars were reacted with adipic dihydrazide (10 eq) at 80 ˚C for 8

h in a 1:1 mixture of water and MeCN (Scheme 4.3). Glucose gave only a 13% yield, due to

purification issues – the molecular weight of the product and reactants is similar, despite the

reaction being near quantitative. Higher molecular weight sugars gave better results; lactose had

a yield of 73% and LewisY tetrasaccharide (reacted at lower temperature for 6 days in a buffer

solution) had a yield of 45%. The selectivity was estimated to be 90% β in all cases; however,

the α proton signal’s proximity to the water signal made greater accuracy difficult.

Glucosamine, N-acetylglucosamine and glucuronic acid were tested but products were not

isolated. One of the major difficulties in glycosylhydrazide synthesis is the formation of the

acyclic hydrazone product rather than the cyclic pyranose form; this method proved to be useful

in providing the pryanose form. The limitation of this method is that it gives low isolated yields

of lower molecular weight products; however, it is useful for larger saccharides and gives even

tetrasaccharides in moderate yield. Also noteworthy, is that these products can be formed in

mildly acidic to neutral pH allowing modification to achieve optimal results depending on the

sugar moiety being used. The products are stable and are able to be conjugated to amine-

functionalized surfaces.

20

Scheme 4.3 An example of a hydrazide synthesizded by Flinn

Wei et al34 have also developed bifunctional linkers for conjugating sugars to various substrates

using bishydrazides. Two different bishydrazide linkers were synthesized easily from their

corresponding α, ω-diols by base-promoted carboxymethylation with ethyl bromoacetate in

THF, followed by treatment with excess hydrazine in anhydrous MeOH under inert atmosphere.

Adapting conditions previously reported by Shin, sugar-bishydrazide conjugates were formed in

moderate to high yields. The best yields were obtained by treating the sugar in AcOH/NaOAc

buffer (pH 4.2) with 10 equivalents of bishydrazide linker at 70 ˚C for 48 h. 13C NMR

confirmed the pyranoside nature of the sugar, while 1H NMR confirmed that the linkage was the

β- anomer. The yields varied depending on the sugar, ranging from 51-86%. The sugars used

were lactose, a pulmonary trisaccharide and 2’-fucosyllactose. It was found in further studies

that the sugar-bishydrazide conjugates react readily with standard labeling and bioconjugate

reagents. The limitation of this method is similar to the previously discussed linker-conjugate

method: it has not been extended to monosaccharides. The reaction conditions and yields of the

reactions are similar between the two methods; however, the method presented here extends the

chemistry to different linkers. Wei varies chain length of the linker looking at a two different

ester linkages (Figure 4.1), whereas Flinn looks only at an alkyl linker with no variation in

carbon chain length.

Figure 4.1 Bishydrazide linkers synthesized by Wei

Pyrazole derivatives with a sugar moiety have many biological activities and thus their synthesis

has been given a lot of attention. Lian et al35 synthesized pyrazole-5-carbohydrazide N-

glycosides and investigated their anti-tumor properties. A variety of derivatives were

21

synthesized using either D-glucose or D-xylose in a mixture of ethanol and acetic acid over 2-4 h

at reflux (Scheme 4.4). The yields were between 81 and 90% and all products were exclusively

the β-anomer. The chemistry presented here shows that glycosyl hydrazide derivatives of

complex organic molecules can be synthesized high yields using a simple method. This is very

important for the synthesis of sugar-derivatives with potential therapeutic properties as they can

be synthesized efficiently for further biological testing. An area for future exploration would be

to determine if the synthesis of these derivatives is possible with larger saccharide units.

Scheme 4.4 Synthesis of pyrazole-5-carbohydrazide N-glycosides

Another method of reacting sugars with a hydrazide derivatives was looked at by Godula and

Bertozzi.36 The research involved a general strategy for the formation of glycopolymers based

on the ligation of reducing sugars to an acryloyl hydrazide polymer scaffold. They began by

synthesizing a biotin-terminated poly(acryloyl hydrazide) scaffold (Scheme 4.5).

Scheme 4.5 Synthesis of a biotin-terminated poly(acryloyl hydrazide) scaffold

Conjugation of a variety of sugars to this scaffold went smoothly with the following reaction

conditions: 1.1 (or 2.2 eq) of sugar, in acetate buffer (pH 5.5), with 0.5% aniline (cat.) at 50 ˚C

for 24 h. The yields of these reactions ranged from 37 to 93% with a high selectivity for the β-

anomer. The advantage of this method is that they were able to reduce the amount of sugar to a

22

mere 0.6 equivalents and still achieve yields of 17-68%; thus this reaction can be done where

conserving reagents is of concern. The method also eliminates the laborious carbohydrate

prefunctionalization that is typically required for glycopolymer synthesis. The method sees a

reduction in yields of products when N-acetyl sugars are used, these yields are moderate (37-

64%) compared to the higher yields of the other sugars investigated.

In 2007, our group published an important study on the stability of glycosyl-hydrazide

derivatives.37 The stability of 1-glucosyl-2-benzoylhydrazines was investigated in terms of

hydrolysis in aqueous solution. It was found that hydrolysis was rapid under even mildly acidic

conditions and that hydrolysis was somewhat dependent on the electronic properties of the

hydrazide; the hydrazide with the lowest pKa was found to be the most stable. These derivatives

were found to be very stable at neutral pH. This study presents an idea of the types of conditions

that are both useful and limiting to form glycosyl-hydrazides; as well as the ideal conditions that

they can be further used under.

The chemistry presented above shows that hydrazides are an appealing method for sugar

conjugation to other biologically-relevant molecules and that the examples of applications for

using hydrazides for glycoconjugation are numerous. There are many efficient ways of doing

this with unprotected sugars to give good yields of desired products. The products can then be

used in many other biological investigations such as therapeutic discoveries.

4.3 Aminooxy Linked Carbohydrate Conjugates

The aminooxy functionality offers an alternative and significant improvement to the methods

available for forming N-glycosides. The main complication in glycoconjugation reactions using

the aminooxy linker functionality is that they form mainly an acyclic oxime, which is in

equilibrium with the pyranose product. The investigation of whether or not sugars formed cyclic

or acyclic products when reacting with hydroxylamine goes back to the 1930’s when Wolform et

al38 investigated whether various sugar oximes were present as the cyclic or acyclic isomer. In

1975, Finch and Merchant39 investigated the structures for arabinose and glucose oximes. Both

of these groups used the methods of Richsbieth40 to synthesize the desired glycosyl oximes.

In 1996, Dumy et al41 incorporated amino acids containing the aminooxy functionality into

peptide sequences. They were then able to form glycoconjugates with various sugars, under mild

23

conditions, in good yields (ranging from 60 to 70%) in 5-120 h. In a comparison study, they

found that their new method was much faster than the typical reductive amination method. Chait

et al42 also investigated the use of the aminooxy linkage to synthesize peptide glycoconjugates

using procedures previously developed by Rose43 and Canne et al44. In 1998, Dumy et al45

continued to investigate the use of the aminooxy funcationality to synthesize glycopeptide

conjugates. They successfully synthesized L-homocanaline derivatives that contained the

aminooxy side chain and incorporated the derivative into short peptide sequences. One peptide

sequence was reacted with maltose to give the acyclic product in a mixture of isomers in 65%

yield. Although the aminooxy functionality represents an appealing alternative to reductive

amination, it often results in the formation of acyclic glycosides. In most cases, this is

undesirable, due to the fact that the products may not have the same biological activity as the

cyclic derivatives. There has been much exploration into this method over the last decade.

Hatanaka et al46 looked at the synthesis of biotinyl photoprobes from unprotected sugars made

with an aminooxy linker between the sugar and the photoprobe. The photoreactive agent (Figure

4.2) was coupled to N-acetyllactosamine (LacNAc) using the following conditions: NBoc-

protected photoreactive agent was treated with 50% TFA-DCM at 0 ˚C for 30 min, after

evaporation, the residue was dissolved in 80% aq MeCN containing 0.5 eq of sugar. The

mixture was incubated at 37 ˚C for 40 h in the dark at pH 5-6 (adjusted with

diisopropylethylamine). A mixture of products (63%) was formed and separated on HPLC. The

products were identified as the acyclic oxime (both E and Z isomers) and the cyclic pyranoside

in strictly beta confirmation.

Figure 4.2 Photoreactive agent with aminooxy side chain for conjugation with a carbohydrate

They were able to successfully attach the photoreactive reagent to the biologically important

probes Lewis X trisaccharide and sialyl Lewis X tetrasaccharide. Both of these also gave a

mixture of oxime and pyranoside products (78% for the tri- and 71% for the tetrasaccharide).

24

For the purpose of this group’s research, the mixture could be used and labeling still occurred.

However; there are certainly cases where a mixture of products would not be favourable and

when separated, the yields of each component would be low; thus this chemistry is only good for

the specific use it has been presented for.

Keller et al47 synthesized glycolipids for the fuctionalization of liposomes and lipoplexes using

the aminooxy functionality. They began by synthesizing a cholesterol-based lipid that contained

an aminooxy functional group. The functionalized lipid was then coupled to unprotected

monosaccharides and oligosaccharides. Coupling reactions were done in a DMF/aqueous acetic

acid buffer system with pH 4; at this pH the proton concentration is optimal for the reaction to

give the cyclic product via the mechanism described in the scheme 4.6. Reactions took 1 – 7

days at room temperature; with more complex sugars taking longer. Yields were moderate to

high, ranging from 40 to 85%. The selectivity remained consistent with the various sugars,

favouring the β-anomer, 85:15. Though the reaction times are somewhat lengthy, it is

favourable because only pyranose derivatives are formed and in decent yields. It is also

noteworthy that a larger variety of both simple and more complex saccharides were investigated

here.

Scheme 4.6 Mechanism of glycolipid formation using the aminooxy functionality

Andreu et al48 give another example that shows the diversity of the applications of the aminooxy

linker. They successfully used the method to immobilize carbohydrates on a surface for

carbohydrate-lection interaction studies.

4.4 N-Alkyl Hydroxylamine Linked Carbohydrate Conjugates

Dumy et al49 made advancement to this area of investigation when they used an N-

alkylsubstituted hydroxylamine derivative. They were able to synthesize N-glycosides in

25

moderate to excellent yields (30 to 95%) with the formation of only the pyranoside product. The

selective formation of only the cyclic product was a major advantage over previous methods that

involved the formation of a mixture of both cyclic and acyclic products. In most cases, the β-

anomer was produced exclusively, with only mannose preferring the production of the α-

anomer.

In 2002, Peri et al50 reported the synthesis of methyl 6-deoxy-6-methoxyamino-D-

glucopyranoside (Scheme 4.7), which they successfully coupled to glucose, galactose and N-

acetylglucosamine in high yields (80-92%) after 4-6 h (Scheme 4.8). Reactions were carried out

in either DMF/acetic acid (2:1) or aqueous sodium acetate buffer (pH 4.5). The reactions with

glucose and N-acetylglucosamine gave exclusively the β-anomer; there was a small amount of

the α-anomer (7:1 β/α) in the case of galactose. The reaction with mannose did not proceed as

smoothly and only gave 35% yield with a 1:5 (β/α) ratio of anomers. The methodology was

successfully extended to synthesize a trisaccharide in 65% yield and could also be used in SPPS.

The methodology offers some appealing advantages over other SPPS techniques such as that it

can be done in the presence of water, does not require any glycosylation promoter and, in the

case of glucose and N-acetylglucosamine, affords the β-product. Through further studies51 it

was shown that these carbohydrate mimetics have similar conformational behaviour to their

natural counterparts.

Scheme 4.7 Synthesis of N-methyl-hydroxylamine carbohydrate derivatives

26

Scheme 4.8 Synthesis of N-methyl-hydroxylamine linked disaccharide

Methyl-aminooxy chemistry has been used extensively to produce glycopeptides derivatives. In

2002, Carrasco et al52 made advancement to the synthesis of glycopeptides when they combined

the ideas of using a short amino side chains (only 1-2 carbons) and utilizing the N-methyl-

aminooxy functionality to synthesize peptides that could react selectively with sugars and result

in products that more closely resembled natural glycopeptides. They successfully made a

homoserine derivative that could be incorporated into short peptide sequences. Glycosylation of

the peptides proceeded smoothly in aqueous sodium acetate buffer (pH 4.0 or 5.1) at 40-45 ˚C

for 24-48 h. Reactions with glucose and lactose, in various trials, gave yields ranging from 60-

85%. They extended this study further when they reported the synthesis of two other small side-

chain N-methyl-aminooxy peptide derivatives and their reaction with glucose.53 They also

reported the synthesis of an Fmoc N-methyl-aminooxy derivative that was suitable for Fmoc-

chemistry based SPPS;54 thus broadening the applications and accessibility of the method. The

main limitation with the chemistry presented by this group is that the scope of the reaction, in

terms of sugars that can successfully be conjugated, has not been investigated.

Due to the success of the methyl-aminooxy functionality for glycoconjugation reactions it is an

attractive tool for library synthesis. Thorson et al55 successfully synthesized a 78-member

library of digitoxin glycoconjugates using the methyl-aminooxy linker. Using the library they

were able to explore the cytotoxicity of the derivatives on nine cancer cell lines and prove that

the sugar moiety has an affect on the toxic behaviour of the anti-cancerous digitoxin. This

example shows again the broad applications of the methyl-aminooxy functionality - it can be

used to find new, more potent therapies by quickly synthesizing many derivatives of a known

glycoconjugate with therapeutic properties.

The aminooxy linkage chemistry was extended to synthesizing glycopeptoids by Carrasco et al.56

They started by synthesizing a N-methylaminoxy submonomer and then incorporating it into

27

oligopeptoids using standard solid-phase peptoid synthesis procedures; four different model

peptoids were synthesized. A glycosidation reaction was then performed with glucose (50 M

excess) and oligopeptoid (Figure 4.3) (3.8 mM) in 0.1 M NaOAc, pH 4.0, 40 ˚C for 12 h. It was

found that results could be improved using microwave irradiation (10 min) and a 200 molar

excess of glucose.

Figure 4.3 Oligopeptoid used in the synthesis of glycopeptoids using aminooxy linkage

chemistry

The methodology was extended to other sugars with reactions taking 10 min except in the cases

of D-lactose and N-acetyl-D-galactose, which both took 5 h. This method expands known

chemistry to new areas and improves the method by decreasing reaction time by the use of

microwave irradiation; however, it does require a large excess of sugar. It would be an

interesting exploration to determine if other chemistry that has been done with these linkages

could be improved with microwave irradiation.

Goff and Thorson57 expanded the N-methylaminooxy linker chemistry in order to make betulinic

acid (BA) – sugar conjugates (Figure 4.4) in an attempt to enhance the properties of BA. BA and

its reduced form exhibit many biological functions including anti-cancer and anti-HIV

properties. Using the previously developed methodology, they optimized the reaction conditions

for their purposes. The optimized reaction conditions involved dissolving BA and 3 equivalents

of sugar in MeOH/DCM (6:1) and stirring for 48 h at 40 ˚C. With these conditions they were

able to create a 37-member library of BA-sugar conjugates and investigate their properties. The

average isolated yield was 33% and the selectivity was not strongly biased and the ratio of

anomers depended on the sugar used. The advantage of this study is that it expands known

chemistry and shows its use as a tool for natural product glycodiversification. The drawback is

that it shows that the selectivity of the methodology is not general, as seen in Peri’s study where

all of the sugars looked at favoured the β-anomer.

28

Figure 4.4 BA-sugar conjugate with N-methyl-hydroxylamine linker

Goff and Thorson58 were able to extend the methodology discussed above to create another

library of compounds, once again showing how this methodology can be used to glycodiversify

natural compounds. In this study they synthesized a 54-member library of glycoside-

chlorambucil conjugates (Figure 4.5) and investigated the anti-cancer activity of the library.

Once again, the conditions were optimized for the specific reaction. They stirred the

chlorambucil with 2 equivalents of sugar in 1.5 equivalents of AcOH in MeOH for 3-48 h at 40

˚C. The average yields depended on the type of sugar used and were as follows: tetroses (79 ±

10%), deoxy sugars (69% ± 6%), pentoses (62 ± 7%), and hexoses (56 ± 5%). Unlike previous

libraries of similar compounds, a strong 1,2-trans relationship was typically observed in the

products.

Figure 4.5 A chlorambucil glycoconjugate

Recently, Thorson et al59 extended the use of aminooxy functionality to a new area. They

developed glycosyloxyamine neoglycosylation as a way to synthesize calicheamicin derivatives.

Ribose was reacted with calicheamicin (CLM) α or N-acteyl-CLM γ with AcOH (1.5 eq) in

MeOH at 40 ˚C for 20-48 h (Scheme 4.9). The reaction gave primarily the β-anomer in 33-64%

yield. This report shows that the aminooxy functionality can be a useful tool for the synthesis of

complex oligosaccharide containing compounds.

29

Scheme 4.9 Synthesis of ribose-calicheamicin derivative

Our group has done studies on the stability of glycosyl hydroxyl-amine derivatives.60 The paper

discusses finding the optimal conditions for the formation of hydroxyl-amine based

glycoconjugates. Nine different glycogonjugates were synthesized (Figure 4.7). The optimal

conditons were found to be: 0.75 M sugar and hydrazide, at pH 4.5 (2 M NH4OAc), 37 ˚C for 72

h. Isolated yields were 80 to 90% and only the β-anomer was formed in all cases. The

hydrolysis studies revealed that hydrolysis rates are accelerated in acidic conditions and are

affected by the electronic properties of the sugar; electron rich sugars hydrolyze significantly

faster than electron poor sugars. These results are important as they offer information on the best

conditions for the synthesis of these types of molecules; as well, perhaps even more importantly

for biologically active glycoconjugates, it gives information on the conditions that these

molecules remain stable under.

30

Figure 4.7 Substrates used in stability studies of N-glycosides

There has been extensive exploration into the aminooxy linkage chemistry for producing N-

glycosides. This chemistry is an efficient way to product many different types of

glycoconjugates and is advantageous for the synthesis of even very complex glycoconjugates.

31

5 Project Objective

5.1 Developing an Efficient Synthesis for β-Glycosides of Simple Alcohols

The goal of this project was to develop a protecting group free method of generating β-

glycosides of simple alcohols. This is a significant challenge as all of the reported methods for

protecting group free synthesis from simple sugars lead to predominately the α-glycosidic

products (see O-glycosidation chapter).

Preliminary work was done in our lab using glycosyl-tosylhydrazides as an unprotected glycosyl

donor.61 It is on this chemistry that we wish to expand. To facilitate the development of this

new approach to protecting group free glycosidations we sought to explore a model system that

would yield glycosides that could be readily purified and analyzed. The hypothesis was that the

installation of a purification handle (an -OC8H17 group) would allow for easy purification of any

glycoside products by reverse phase C18 chromatography (Scheme 5.1). With this handle, rapid

screening of a variety of glycosidation conditions would be carried out to determine the optimal

conditions for selective glycosidation. In future iterations the glycosylation could be carried out

with an unsubstituted glycosyl donor and the purification protocol optimized.

Scheme 5.1 An overview of the synthesis of a glycoside with a purification handle

5.2 Investigating Glycosyl-Thiouronium Salt as a Novel Glycosyl Donor

The goal of this project was to synthesize a glucosyl-thiouronium salt and to investigate what

types of glycosides it can be used to synthesize. The appeal of this strategy is the simple

synthesis of the glucosyl-thiouronium salt; previously synthesized in our lab by Paul and

Chibba.61 Also, thiourea derivatives have been extensively covered in the literature as

32

organocatalysts and from this literature it may be possible to design glycosyl-thiouronium salts

for specific glycosidations.62,63

33

6 Results and Discussion

6.1 Developing an Efficient Synthesis for β-Glycosides of Simple Alcohols

A glycoside of a simple alcohol was successfully synthesized (Scheme 6.1). The installation of

the octyl-chain was completed by reacting diacetic-D-glucose (6) with iodooctane and sodium

hydrazide in DMF to give the desired product (7) in 85% yield after column chromatography.

Compound 7 was then stirred with 90% TFA in DCM for 90 min at rt; however, 1H NMR data of

the crude product showed that the deprotection step was not complete. Refluxing for 3 h in 80%

AcOH successfully removed the acetal protecting groups; unfortunately side products were

produced in the reaction that could not be removed and lead to complications in the subsequent

synthetic steps. Ultimately it was found that stirring compound 7 with DOWEX® 50WX2-100

ion-exhange resin in MeOH/H2O (5:1) overnight at 43 ˚C, gave compound 8 in 95-100% crude

yield. Previously in the literature, compound 8 was prepared and isolated in approximately 20%

yield.64 Our slightly modified approach gives better yields. The crude product 8 was used in the

next step without complication. Next, using chemistry previously developed in the lab, the 3-

octyloxy-3-deoxy-glucose (8) was reacted with p-toluenesulfonylhydrazide with AcOH as a

catalyst in DMF to give (3-octyloxy-3-deoxy-β-D-glucopyranosyl)-p-toluenesulfono-hydrazide

(9) in 43% isolated yield.

The final step of the sequence was the glycosidation reaction. Compound 9 was reacted with

butanol (10 eq) as a model simple alcohol using NBS (2.4 eq) as an activator in the presence of

lutidine (3 eq) to give the desired glycosylated product (10). TLC of the crude product showed a

mixture of products and it was the hope that these would easily be separated using a C18 column.

The separation of these compounds proved to be difficult on the C18 column. Elution was done

a gradient of H2O and MeOH. 1H NMR analysis showed that at 100% H2O the excess lutidine

was eluted; at 35% MeOH an unidentified tosylated product was eluted; at 60% MeOH butyl

glycoside product (10) as well as tosylated products were eluted; and at 100% MeOH yet another

tosylated product was eluted along with some sugar product. The mixtures of various products

were not readily separated and as such could not easily be identified via 1H NMR. This column

was performed several times, on different batches of compound 10; however, the results were

consistent – a mixture of inseparable products was always eluted.

34

As the goal of the project was to develop a simple purification method for the unprotected

glycosidation products it was determined unsuccessful and other glycosidation reactions were

explored.

Scheme 6.1 Synthesis of butyl-glucoside with octyl chain purification handle

6.2 Investigating Glycosyl-Thiouronium Salt as a Novel Glycosyl Donor

Glucosyl thiouronium salt was successfully synthesized and the types of reactions it could

participate in were investigated. The synthesis of glucosyl thiouronium salt (12) began by

forming the glucosyl-TSH derivative (11) (Scheme 6.2). This was done as has been previously

reported15 by reacting glucose with p-toluenesulfonyl hydrazide in DMF/H2O (5:1), with AcOH

as catalyst, at 37 ˚C overnight, giving compound 11 in 89% isolated yield. Compound 11 was

then treated with TBAC (3 eq), and NBS (2.2 eq) in MeCN, followed by the addition of thiourea

and stirred overnight to give compound 12; which precipitates out of solution and is easily

collected via vacuum filtration (85% yield).

Scheme 6.2 Synthesis of glucosyl-thiouronium salt

35

With compound 12 in hand various types of reactions were explored (Scheme 6.3 a – f). The

reactions were all done on a 4 mM scale in NMR tubes in d6-DMSO, as a means to quickly

determine which reactions were taking place by running a 1H NMR at time zero and again after

letting the reaction run overnight. The first reaction (Scheme 6.3a) was to determine if O-

glycosides could be synthesized from this starting compound. In the first trial, compound 12 was

reacted with D4-MeOH (20 eq) in the presence of DIEA (3 eq) in d6-DMSO. When comparing

the overnight 1H NMR to the one at time zero, it was difficult to determine if there was any

reaction occurring; due to the high equivalents of base used on such a small scale reaction, the

base’s proton signals were swamping out the NMR. The reaction was then done without base

(Scheme 6.3b) to get a better idea as to whether this reaction was taking place. With the lack of

base, the overnight 1H NMR showed that there was in fact conversion to the methyl-glucoside

(13) occurring. Based on integration values (see Appendices, rxn 6.3b, t = overnight) the α/β

ratio was 1:1.7. This is better than the Fischer reaction, which typically has a 1:1 anomeric ratio

of products. This result was promising and would be returned to after the investigation of other

reactions.

Scheme 6.3 a-f Various reactions performed with glucosylthiouronium salt

36

The next reaction investigated was between compound 12 and imidazolidine-2-thione to form

compound 14 (Scheme 6.3c). Our interest in this reaction was due to the possibility of using

other thiourea derivatives as potential catalysts in the glycosidation reaction. An excess (5 eq)

of imidazolidine-2-thione was used and, as in the case of reaction a, the 1H NMR signals were

swamped by exchangeable protons from the imidazolidine-2-thione and the reaction was

inconclusive. To work around this issue, the imidazolidine-2-thione was stirred in D2O, in order

to exchange the protons for deuterium. The exchanged imidazolidine-2-thione was lyophilized

and used to evaluate the thiourea exchange. The reaction was performed again with the

deuterium derivative of the imidazolidine-2-thione (Scheme 6.3d) and it was determined, based

on the overnight 1H NMR that no thiourea exhange was occurring.

Next, a reaction with compound 12 and tetrabutylammonium hydrogen-phosphate was

investigated (Scheme 6.3e). Glycosyl phosphates are typically difficult to synthesize but are

important biosynthetic intermediates in glycobiology. The 1H NMR was inconclusive and we

were unable to determine if compound 16 was produced based on overnight 1H NMR data.

Synthesis of a Glycosyl ester from compound 12 was evaluated (Scheme 6.3f). As discussed in

the O-glycosidation chapter, these types of glycosides are difficult to synthesize due to being

prone to rearrangements to 2-O-esters. The synthesis of compounds like 17 directly from

glycosylthiouronium salts would provide a useful route to glycosyl esters. However, there was

no evidence of reaction after 24 h based on 1H NMR. Evidence of the reaction occurring would

have been new anomeric peaks on the 1H NMR; however, the only thing observed was an

increase in the hydrolysis that was already present on the NMR at t = 0.

Based on the data that the reaction between 12 and deuterated MeOH was successful, we decided

to investigate whether longer chain O-glycosides could be synthesized. We reacted 12 with

octanol (20 eq) and monitored the reaction by 1H NMR (Scheme 6.4). After 24 h, the 1H NMR

showed some hydrolysis and the desired product (Figure 6.1). From here, we planned to do this

reaction on a larger scale and attempt to purify on C18 column.

37

Scheme 6.4 Synthesis of octyl-glucopyranoside.

Figure 6.1 Anomeric peaks of crude octyl-glucopyranoside, rxn 6.4, t = overnight

At this point, it was necessary to synthesize more glucosylthiouronium salt. This proved not to

be reproducible. In attempted reactions a significant amount of hydrolysis was observed.

Evaluating subsequent reactions using the impure thiouronium salt 12 was not possible. Further

work is required to optimize the synthesis of the thiouronium salt before further synthetic studies

are undertaken.

β-glucoside

α-hydrolysis

α-glucoside

38

7 Conclusions Butyl 3-O-octyl-D-glucopryanoside was successfully synthesized. However, purification of this

compound did not turn out to be simple. Using both column chromatography and a reverse

phase C18 column the crude mixture of products remained inseparable. After several attempts, it

was determined that this method of simple purification of glycosides was unsuccessful.

Glucosyl thiouronium salt was successfully synthesized from glucosyl-TSH, but this synthesis

proved difficult to repeat. Several different types of reactions were explored. Promising O-

glycosidations were observed using the glucosyl thiouronium salts as donors. It is possible that

this salt could be a viable glycosyl donor if it can be synthesized without the formation of the

hydrolysis product.

39

8 Experimental All chemicals were purchased from Sigma-Aldrich and used as purchased. All 1H NMR were

run on a Varian 400 MHz NMR or a Bruker 400 MHz NMR spectrometer.

3-O-octyl-1,2:5,6-di-O-isopropylidene-α-D-glucose (7)

Diacetic-D-glucose (2.0 g, 7.68 mmol) was dissolved in DMF (20 mL)

and cooled to 0 ˚C. To this, NaH (0.83 g, 34.56 mmol, 60% dispersion in

mineral oil) was added and the reaction mixture was stirred for 10 min at

which point iodooctane was added (3.69 mL, 15.36 mmol) and the

reaction stirred for 30 min at 0 ˚C. Stirring was continued for 30 min as the reaction warmed to

r.t. The reaction mixture was diluted with 70 mL of water and extracted with DCM (2 x 35 mL).

The organic extracts were washed with brine (20 mL), dried (MgSO4) and concentrated. The

resulting compound was purified using column chromatography (9:1 pentanes:EtOAc) to yield a

colourless oil (2.70 g, 85%). 1H NMR (400 MHz, CDCl3) δ 5.84 (d, J = 3.6 Hz, 1H); 4.49 (d, J

= 4.0 Hz, 1H); 4.29-4.25 (m, 1H); 4.11-4.03 (m, 2H); 3.97-3.94 (m, 1H); 3.82 (d, J = 3.2, 1H);

3.60-3.54 (m, 1H); 3.50-3.45 (m, 1H); 1.53-1.23 (m, 24H); 0.87-0.84 (t, 3H).65

3-O-octyl-D-glucose (8)

Prior to use, DOWEX resin was washed with MeOH. To a mixture of

diacetic-4-octyloxy-D-glucose (0.85 g, 2.05 mmol) in MeOH/H2O (5:1,

140 mL), DOWEX ion exchange resin (27 g) was added. The mixture

was stirred at 50 ˚C overnight. The reaction mixture was then filtered and concentrated. The

crude product was used in the following step without purification.

(3-O-octyl-β-D-glucopyranosyl)-p-toluenesulfonohydrazide (9)

Compound 8 (4.3 g, 14.67 mmol) and p-

toluenesulfonohydrazside (3.8 g, 20.54 mmol) were

dissolved in DMF (30 mL) and AcOH was added (0.55 mL).

After stirring overnight the reaction mixture was concentrated and the crude product was purified

using column chromatography (98:2 EtOAc/Pentanes). The resulting residue was crystallized

from toluene to give a white solid (3.28g, 48%). 1H NMR (400 MHz, CD3OD) δ 7.78 (d, J = 8.0

40

Hz, 2H); 7.38 (d, J = 8.0, 2H); 3.84-3.80 (m, 2H); 3.55 (dd, J = 6.8 Hz, 1H); 3.37 (t, 1H); 3.24-

3.19 (m, 1H); 3.15-3.06 (m, 2H); 2.43 (s, 3H); 2.31 (s, 2H); 1.59 (m, 2H); 1.38-1.30 (m, 10H);

0.90 (t, 3H).

Butyl 3-O-octyl-D-glucopryanoside (10)

All glassware was oven dried and cooled in a dessicator prior to use.

Butanol was dried by bubbling N2 through the liquid. Compound 11

(25 mg, 0.054 mmol) was placed in a vial that was sealed and purged

with N2. To this, MeCN (1 mL), lutidine (18.9 µL, 0.16 mmol), BuOH (49.6 µL, 0.54 mmol)

and NBS (23 mg, 0.13 mmol) were added. After 45 min the reaction mixture was concentrated.

The resulting residue was diluted with water and purification on a C18 column was attempted.

The mixture of products remained inseparable.

Glucosyl-thiouronium Salt (12)

Glassware was oven dried and cooled in a dessicator prior to use.

Glucopyranosulfonohydrazide (11, 0.5 g, 1.43 mmol) was prepared

previously in our lab15 and placed in a flask, which was then purged

with N2. Dry MeCN (25 mL was added via syringe, followed by the addition of

tetrabutylammonium chloride (1.2 g, 4.31 mmol) and NBS (0.56 g, 3.15 mmol). At this point

the solution turns orange and bubbles. Once the production of gas bubbles has subsided thiourea

(0.33 g, 4.31 mmol) was added and the reaction was stirred overnight. A white precipitate is

formed and collected via vacuum filtration (0.29 g, 85%). 1H NMR data reported by Paul.61

Methyl-D-glycopyranoside (13)

In a small vial, compound 12 (4.0 mg, 0.013 mmol) was mixed with D4-

MeOH (0.01 mL, 0.25 mmol) in D6-DMSO (1 mL). The reaction

mixture was placed in an NMR tube and a 1H NMR was taken at time

zero. A second 1H NMR was taken after the reaction had been left overnight at r.t.

41

Octyl-D-glucopyranoside (18)

All glassware was oven dried and cooled in a dessicator. Compound

12 (25 mg, 0.078 mmol) was mixed with octanol (0.25 mL, 1.57

mmol) in anhydrous DMSO (2 mL). After stirring over night, the

mixture was diluted with 20 mL of H2O and washed with EtOAc. Purification was attempted on

a C18 column using a MeOH/H2O gradient but proved to be unsuccessful.

42

Appendices

1H NMR 400 MHz (CDCl3, 25 ˚C)

43

1H NMR 400 MHz (CDCl3, 25 ˚C)

44

1H NMR 400 MHz (CD3OD, 25 ˚C)

45

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.3a, t= 0

46

1HNMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.3a, t=overnight

47

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.3b, t=0

48

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.3b, t=overnight

49

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.3c, t = 0

50

1H NMR 400 MHZ (d6-DMSO, 25 ˚C) Rxn 6.3c, t = overnight

51

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.3d, t = 0

52

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.3d, t = overnight

53

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.3e, t = 0

54

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.3e, t = overnight

55

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.3f, t = 0

56

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.3f, t = overnight

57

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.4, t = 0

58

1H NMR 400 MHz (d6-DMSO, 25 ˚C) Rxn 6.4, t = overnight

59

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