Janne Mauritz Weisell Structure and Function of Charge...

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences

Publications of the University of Eastern Finland

Dissertations in Forestry and Natural Sciences

isbn 978-�952-�61-�0856-�8

Janne Mauritz Weisell

Structure and Function ofCharge Deficient PolyaminesThe Latest Generation of Isosteric Charge Deficient Spermine Analogues

Charge is the fundamental property

that enables polyamines to interact

through reversible ionic interactions

with many different biomolecules.

Modifications in the structure of

polyamine analogues can alter their

conformation without causing any

changes in the acidity of the amino

groups in the polyamine backbone.

This thesis focuses on analogues

that have reduced basicity of their

amino groups and therefore are not

fully protonated at physiological pH.

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Janne Mauritz WeisellStructure and Function of

Charge Deficient PolyaminesThe Latest Generation of Isosteric Charge

Deficient Spermine Analogues

JANNE MAURITZ WEISELL

Structure and function of

charge deficient polyamines

The latest generation of isosteric charge deficient spermine analogues


No 78

Academic Dissertation To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for

public examination in the Auditorium L22 in Snellmania Building of the University of Eastern Finland, Kuopio, on August 16th 2012, at 12 o’clock noon

School of Pharmacy

Kopijyvä Oy Kuopio, 2012

Editors: Prof. Pertti Pasanen

Prof. Pekka Kilpeläinen, Prof. Matti Vornanen Distribution:

Eastern Finland University Library / Sales of publications [email protected]

www.uef.fi/kirjasto

ISBN: 978-952-61-0856-8 (printed) ISSN: 1798-5668

ISSNL: 1798-5668 ISBN: 978-952-61-0857-5 (PDF)

ISSN: 1798-5676 (PDF)

Author’s address: School of Pharmacy

University of Eastern Finland P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected]

Supervisors: Professor Jouko Vepsäläinen, Ph.D.

School of Pharmacy University of Eastern Finland P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected] Tuomo Keinänen, Ph.D. School of Pharmacy University of Eastern Finland P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected] Ale Närvänen, Ph.D. School of Pharmacy University of Eastern Finland P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected] Professor Seppo Auriola, Ph.D. School of Pharmacy University of Eastern Finland P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected]

Reviewers: Professor Francisca Sánchez-Jiménez, Ph.D Department of Molecular Biology and Biochemistry University of Malaga MALAGA SPAIN

Jari Ratilainen, Ph.D Kuurna-Kulhontie 93 KULHO FINLAND

Opponent: Professor Alexander Gabibov, Ph.D., Dr.Sc. (Chemistry)

Laboratory of Biocatalysis Institute of Bioorganic Chemistry Russian Academy of Sciences MOSCOW RUSSIA

ABSTRACT: Charge is the fundamental property that enables polyamines to interact through reversible ionic interactions with many different biomolecules. Modifications in the structure of polyamine analogues can alter their conformation without causing any changes in the acidity of the amino groups in the polyamine backbone. This thesis focuses on analogues that have reduced basicity of their amino groups and therefore are not fully protonated at physiological pH.

The aim of this study was to conduct a systematic biological characterization of the newest isosteric charge deficient analogue of Spm, 1,12-diamino-3,6,9-triazadodecane (SpmTrien), which is capable of chelating Cu2+. In addition, the corresponding Spd analogue, 1,8-diamino-3,6-diazaoctane (Trien), was systematically characterized for its effects in polyamine metabolic route. Furthermore, the protonation and pKa values of SpmTrien were determined in order to gain a deeper understanding of the biochemical features of this molecule.

The determined pKa values of SpmTrien (3.3, 6.3, 8.5, 9.5 and 10.3) are extremely low for amino groups and the net charge of the molecule is three (+3) at pH 7.4. Thus this novel compound is a functional chemical tool with which to study the role of charged amines in the regulation of polyamine-dependent biological processes. Both Trien and SpmTrien displayed polyamine like properties to some extent. With respect to their biological properties, Trien and SpmTrien were found to be substrates of the polyamine acetylating enzyme spermidine/spermine N1-acetyltransferace (SSAT). Altough Trien or its acetylated metabolites were not substrates of either spermine oxidaze (SMO) or N1-acetyl polyamine oxidase (APAO), N12-Ac-SpmTrien was a good substrate of APAO resulting Trien as the oxidation product. Furthermore, the concentration of SpmTrien was found to be as high inside cells as the concentration of natural polyamines even though the molecule did not compete efficiently for transportation inside DU145 cells, suggesting that some other mechanism is available for the entry of the molecule into the cells. Trien has already been used in various different studies, ranging from Cu2+ chelation to cancer treatment, thus proving its potential. Since SpmTrien is a bioactive precursor of Trien, it should also have great potential as a chemical research tool; this study provides a firm foundation for future research.

National Library of Medical Classification: QU 61; QU 120 Medical Subject Headings: Polyamines; Biogenic Polyamines/metabolism; Spermine/analogs & derivatives*; Magnetic Resonance Spectroscopy; Mass Spectrometry; Cell Proliferation/drug effects; Spermine/chemistry

Preface The present study was started in the University of Kuopio, Department of Biosciences 2008 and finished in the University of Eastern Finland, School of Pharmacy 2012. The names changed but the location did not. The enzyme, cell and animal experiments were conducted in the A.I. Virtanen Institute, University of Eastern Finland. The departments are acknowledged for providing the facilities for this work and the Academy of Finland, Graduate School of Organic Chemistry and Chemical Biology and Aleksanteri Mikkonen Foundation are thanked for their financial support. My deepest and most sincere thanks go to my principal supervisor Professor Jouko Vepsäläinen for his excellent guidance throughout the process. I also want to thank my other supervisors: Tuomo Keinänen was a very important driving force into the field of polyamines; Ale Närvänen showed me how to complete through many projects with huge enthusiasm; Seppo Auriola contributed me important knowledge about mass spectrometry. Furthermore, I wish to express gratitude to the reviewers, Professor Francisca Sánchez-Jiménez and Dr. Jari Ratilainen, for their constructive work. There can not be thanks enough to express my gratitude to my coauthors. You are all greatly appreciated expecially Dr. Mervi Hyvönen, Dr. Mikael Peräkylä, Dr. Pasi Soininen and Prof. Alex Khomutov. Without you, this dissertation would not ever have seen the light of day. Without the expert technical assistance of Ms. Maritta Salminkoski I would still be in the laboratory trying to start the first synthesis. Ms. Anne Karppinen and Ms. Tuula Reponen are also acknowledged for their expert technical assistance, which helped so much in the biochemistry related works. When I started my academic career I was introduced to the great atmosphere of the old “Kemian Laitos”. Millions of thanks to the people from there, expecially the coffee room philosophy group. All of my friends are greatly acknowledged for the extra curricula activity that kept me sane. Mother and Father are greatly appreciated for their most important part of the process, making and growing me. My wife Tiina, son Eemeli and the still unborn baby, you are the light of my life and give me happiness by your very existence. Cheers! Janne

LIST OF ABBREVIATIONS

2,2,3,3-F4-Put 2,2-3,3-tetrafluoroputrescine, 2,2,3,3-

tetrafluorobutane-1,4-diamine 2,2-F2-Put 2,2- difluoroputrescine, 2,2 -difluorobutane-1,4-

diamine 2,2-F2-Spd 2,2-difluorospermidine 2-2-2-2 1,11-diamino-3,6,9-triazaundecane 2-F-Put 2-fluoroputrescine, 2- fluorobutane-1,4-diamine 3-3-3 norspermine, 1,11-diamino-4,8-diazaundecane 6,6-F2-Spd 6,6- difluoro-spermidine, 6,6-difluoro-1,8-diamino-4-

azaoctane 6,6-F2-Spm 6,6-diflurospermine, 6,6-difluoro-1,12-diamino-4,9-

diazadodecane 6-F-Spd 6-fluoro-spermidine, 6-fluoro-1,8-diamino-4-

azaoctane 7,7-F2-Spd 7,7- difluoro-spermidine, 7,7-difluoro-1,8-diamino-4-

azaoctane 7-F-Spd 7-fluoro-spermidine, 7-fluoro-1,8-diamino-4-

azaoctane AdoMetDC S-adenosylmethionine decarboxylase AOE-Put N-(2-aminooxyethyl)-1,4-diaminobutane APA 1-aminooxy-3-aminopropane APAO N1-acetyl polyamine oxidase AP-APA 1-aminooxy-3-N-(3-aminopropyl)-aminopropane AZ antizyme BE-3-3-3 N1,N11-diethylnorspermine, 1,11-bis(ethylamino)-4,8-

diazaundecane BE-3-3-3-3 1,15-di(ethylamino)-4,8,12-triazapentadecane BESpm N1,N12-diethylspermine, 1,12-bis(ethylamino)-4,9-

diazaundecane BSA bovine serum albumin DAT N1,N8-Ac2-Trien, N1,N8-diacetyl-1,8-diamino-3,6-

diazaoctane dcSAM decarboxylated S-adenosylmethionine DFMO α-Difluoromethyl ornithine, 2,5-Diamino-2-

(difluoromethyl)pentanoic acid FBESpm N1,N12-ditrifluoroethyl-spermine, 1,12-

bis(trifluoroethylamino)-4,9-diazaundecane GAPA γ-guanidinooxypropylamine

MAT N1-Ac-Trien, N1-acetyl-1,8-diamino-3,6-diazaoctane NMR nuclear magnetic resonance ODC ornithine decarboxylase Orn ornithine, (2S)-2,5-diaminopentanoic acid oxa-Spm 1,12-diamino-4,9-diaza-5-oxadodecane Put putrescine, 1,4-diaminobutane SMO spermine oxidase Spd spermidine, 1,8-diamino-4-azaoctane SpdSy spermidine synthase Spm spermine, 1,12-diamino-4,9-diazadodecane SpmSy spermine synthase SpmTrien 1,12-diamino-3,6,9-triazadodecane SSAT spermidine/spermine N1-acetyltransferase Trien triethylenetetramine, 1,8-diamino-3,6-diazaoctane

LIST OF THE ORIGINAL PUBLICATIONS

This dissertation is based on the following original articles:

I Weisell J, Hyvönen MT, Vepsäläinen J, Alhonen L, Keinänen TA,

Khomutov AR, Soininen P. Novel isosteric charge-deficient

spermine analogue—1,12-diamino-3,6,9-triazadodecane: synthesis,

pKa measurement and biological activity.

Amino Acids 38: 501-507, 2010

II Weisell J,* Hyvönen MT,* Häkkinen MR, Grigorenko NA, Pietilä M,

Lampinen A, Kochetkov SN, Alhonen L, Vepsäläinen J, Keinänen TA,

Khomutov AR. Synthesis and Biological Characterization of Novel

Charge-Deficient Spermine Analogues

Journal of Medicinal Chemistry 53: 5738-5748, 2010

*Equal contribution

III Cerrada-Gimenez M,* Weisell J,* Hyvönen MT, Park MH, Alhonen L,

Vepsäläinen J, Keinänen TA. Complex N-Acetylation of

Triethylenetetramine

Drug Metabolism and Disposition 39: 2242-2249, 2011

*Equal contribution

IV Weisell J, Vepsäläinen J, Peräkylä M. Microstates of the charged

species of 1,12-diamino-3,6,9-triazadodecane (SpmTrien) studied

with atomistic computer simulations and cluster expansions.

(Submitted)

The publications have been reproduced with the permission of the copyright

owners.

AUTHOR´S CONTRIBUTION

All of the publications of this dissertation are a result from a long and fruitfull collaboration between the research groups from Kuopio and Moscow. The author has been privlidged to be a part of the dynamic team behind the publications.

For publication I, the author developed a novel synthesis route for SpmTrien and characterized the synthesis products. Furthermore, the author measured and interpreted the 2D spectra needed to study the protonation of SpmTrien and determined the macroscopic pKa values. The author participated also in the planning of the experiments.

For publication II, the author was a part of the synthesis team and synthesized a part of the compounds. In addition, the author characterized all of the compounds with NMR and mass spectrometry. The author performed also the in vitro tests with recombinant proteins. The author participated in the planning of the experiments.

For publication III, the author characterized a novel type of intramolecular acetyl migration reaction. The author synthesized and characterized the compounds of the publication. The author participated also in the planning of the experiments.

For manuscript IV, the author synthesized the compound and measured the needed NMR spectra. The author participated also in the planning of the experiments.

In addition to the above mentioned contributions, the author contributed also to the writing process of all previously mentioned papers. In addition the author contributed to the interpretation of the results.

Contents

1 Introduction ............................................................................................................... 15 2 Review of the literature ........................................................................................... 17 2.1 Polyamines .............................................................................................................. 17 2.1.1 A brief introduction to the polyamines ................................................................... 17 2.1.2 Metabolism of natural polyamines ......................................................................... 18 2.2 Overview on the Acidity ....................................................................................... 21 2.2.1 Macroscopic and microscopic pKa values of polyprotic acids or bases .................... 22 2.2.2 Short overview to the NMR methods used for the pKa determination of polyamines and their analogues ...................................................................................... 24 2.3 Introduction to charge deficient polyamine analogues .................................... 26 2.4 Fluorinated analogues of the natural polyamines ............................................. 26 2.4.1 Chemistry of the fluorinated polyamine analogues ................................................ 28 2.4.2 Analogues in the polyamine metabolizing pathway. .............................................. 29 2.4.3 Analogues and post-translational modifications .................................................... 30 2.4.4 Fluorinated analogues of N,N-di-ethyl-spermine .................................................. 30 2.4.5 Fluorine containing charge deficient ornithine analogues. .................................... 30 2.5 Aminooxyanalogues of natural polyamines ....................................................... 32 2.5.1 Chemistry of the aminooxy and –oxa analogues .................................................... 32 2.5.2 Biochemical features of the aminooxy analogues of putrescine .............................. 33 2.5.3 Biochemical features of the isosteric aminooxy analogues of Spd and Spm ........... 34 2.6 Polyamine analogues with altered carbon chain length and number of amino groups............................................................................................................................. 35 2.6.1 The chemistry of the polyamine analogues with altered carbon chain lengths. ...... 35 2.6.2 The biochemistry of the charge deficient polyamine analogues with altered chain length. .............................................................................................................................. 37 2.6.3 Trien and SpmTrien in the polyamine catabolizing pathway ................................ 38 2.6.4 Biochemical properties of the N,N´-di-ethylated charge deficient analogues with altered chain lengths ........................................................................................................ 38 2.6.5 Charge deficient polyamine conjugates with altered chain length ......................... 39 2.7 The charge deficient polyamines bearing a cyclic ring in their backbone ...... 40 3 References .................................................................................................................. 41 4 Aims of the study...................................................................................................... 49 5 General methods ....................................................................................................... 51 4.1 Synthetic methods .................................................................................................. 51 4.2 NMR spectroscopy ................................................................................................. 51 4.3 Mass spectrometry.................................................................................................. 52

4.4 Computational methods ........................................................................................ 52 4.5 Recombinant protein methods ............................................................................. 52 4.6 Cell culture methods .............................................................................................. 52 4.7 in vivo methods ...................................................................................................... 53 6 PAPER I ...................................................................................................................... 55 7 PAPER II ..................................................................................................................... 63 8 PAPER III ................................................................................................................... 75 9 PAPER IV ................................................................................................................... 85 10 Summary ................................................................................................................ 101 11 Future prospects .................................................................................................... 103

15

1 Introduction

Naturally occurring polyamines spermidine (1,8-diamino-4-azaoctane, Spd) and Spermine (1,12-diamino-4,9-diazadodecane, Spm) and their diamine precursor putrescine (1,4-diaminobutane, Put) are an absolute requirement for mammalian cell growth. Their concentrations can reach millimolar levels in rapidly proliferating cells and changes in the homeostasis of the natural polyamines are often linked to various pathological conditions, such as acute pancreatitis, diabetes and cancer.1, 2 The positive charge of the natural polyamines and their analogues at physiological pH is their most important feature, ensuring their molecular recognition by different cellular constituents and subsequently enabling the regulation of important cellular functions, such as proliferation, differentiation and functioning of different ion channels. Individual polyamines have been shown to interact in a specific manner with DNA and also with different proteins. This property separates the polyamines from all other positively charged cations found inside cells.3, 4 Various different families of polyamine analogues have been synthesized not only for research purposes but also for potential therapeutic use, but only a very small number of these families have the total charge decreased at physiological pH as compared to their natural counterparts. Analogues that contain amino groups that are not protonated at physiological pH will be called charge-deficient in this thesis. The only charge-deficient families of analogues that have been systematically biochemically characterized before the beginning of this work were the difluorinated-5, 6 and aminooxy7-10 analogues of Put, Spd and Spm. In addition, the protonation of difluorospermines and –spermidines has been examined in detail with NMR to differentiate the amounts of all microscopic populations of different charges of the molecules.11

Even though the polyamines have been used in plethora of different studies for over forty years now, their biochemistry still has not been fully resolved. Recently, a novel mechanism for the regulation of antizyme (AZ) has been proposed through ionic binding of Spm and Spd.12 Alternatively charged polyamine analogues could be used in interaction studies to illustrate the importance of the localization of the charge. The main goal of this work was to conduct a comprehensive chemical and biochemical characterization of the novel family of spermine analogues. One of its subsequent aims which was needed to achieve the satisfactory characterization of these analogues was to develop an 1H-15N 2D NMR method to allow the determination of protonation of polyamine analogue, 1,12-diamino-3,6,9-triazadodecane (SpmTrien), which has largely overlapping signals in both 1H and 13C spectra.(Paper I)

17

2 Review of the literature

2.1 POLYAMINES

2.1.1 A brief introduction to the polyamines Polyamines are a class of compounds that contain many amino groups, which are connected to each other via a hydrophobic carbon chain. In general, polyamines can be either linear or branched but biochemically the term polyamines is used to denote the naturally occurring polyamines, Spd and Spm and their diamine precursor Put (hereafter all will be considered as natural polyamines), that are linear positively charged molecules at physiological pH (figure 1).

Figure 1. The chemical structures and the pKa values13 of the naturally occurring polyamines.

The natural polyamines can be found in all eukaryotic cells and the concentrations of polyamines within cells can reach mM levels. Most of the polyamines are bound to various different cellular constituents. This is due to one key feature of these molecules, i.e. their the positive charge, that enables them to interact with negatively charged components of the cell such as DNA, RNA, anionic proteins and phospholipids.3,14,15 However, it is not simply the charge but rather the correct distribution of the charge(s) along the polyamine backbone that give the unique features of polyamines and this differentiates them from other important cations such as Mg2+ or Zn2+ cations.4 The charge enables Spd and Spm to link the major and minor grooves of DNA both inter— or intramolecularly, with a preference for interacting with the single DNA strand.3, 16 The stability of the polyamine-DNA-complexes follows the number of positive charges, as the complexes made with Spm are the most stable and those with Put being the least stable. Moreover, norspermine (1,11-diamino-4,8-diazaundecane, 3-3-3) and the N1,N11-diethylnorspermine (1,11-bis(ethylamino)-4,8-diazaundecane, BE-3-3-3) were reported to have a much lower binding affinity for DNA as well as a different binding site when compared to the natural polyamines.17 Polyamine-tRNA interaction studies have revealed that the preferred

18

binding sites of Spd, Spm and Put differ slightly from their complexes with DNA and at low concentrations, both Spd and Spm bind more effectively to tRNA than DNA.18 In addition, the polyamine-protein interactions show charge dependency. For example, when the interactions of Spd, Spm and diethyl analogues, BE-3-3-3 and 1,15-(ethylamino)4,8,12-triazapentadecane (BE-3-3-3-3) with bovine serum albumin (BSA) were studied, the stability of the BSA-Spm complex was found to be higher than that of the BSA-Spd complex, and likewise, the BSA-BE-3-3-3-3 complex was more stable than BSA-BE-3-3-3.19 Furthermore, the BSA complexes of Spd and Spm were found to be more stable than the complexes with the diethylated analogues. Recent results have indicated that polyamines can also modulate protein-protein interactions and therefore control the functions of proteins.14 Specific binding sites for the polyamines in the CYP11A1 electron transfer system were found. These could modulate interactions between protein components and thus affect the biosynthesis of steroid hormones. Positively charged polyamines can also modulate ion-channel functions.20

The homeostasis of natural polyamines is strictly regulated and the changes in their concentration are linked to various different diseases.1, 2 There are high concentrations of polyamines in rapidly proliferating cells and polyamine metabolism has been a target of cancer chemotherapy and the development of inhibitors of polyamine metabolism has been of keen interest to many different research groups.21 It is also good to remember that not only a high concentration of polyamines but also a low level can create pathological conditions. Acute pancreatitis was found to be cured with polyamine analogues that could fulfill the tasks of the lacking natural polyamines.22

The facts that the polyamines are bound to various different macromolecules within cells through reversible ionic interactions and the changes in their homeostasis create pathological states to cells give the base for this work as the modulation of charge can create new and important knowledge about the interactions of different macromolecules with polyamines and their analogues.

2.1.2 Metabolism of natural polyamines The natural polyamines Put, Spd and Spm are synthesized from the amino acids ornithine and methionine.3, 23 The biosynthesis of the molecules as well as the catabolism is tightly regulated and there is also an active transport system for polyamines (figure 2).

19

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Ornithine decarboxylase (ODC, EC 4.1.1.17) is the first rate limiting enzyme of polyamine metabolism, producing Put by decarboxylating the amino acid, ornithine (Orn). S-adenosylmethionine is transformed by S-adenosylmethionine decarboxylase (AdoMetDC, EC 4.1.1.50) into decarboxylated S-adenosylmethionine (dcSAM), which is used as a substrate for the aminopropyltransferases, spermidine synthase (SpdSy, EC 2.5.1.16) and spermine synthase (SpmSy, EC 2.5.1.22), which are constitutively expressed and active as homodimers. Both ODC and AdoMetDC have very short half-lives that change during the cell cycle.25 The aminopropyltransferases catalyze the synthesis of Spd from Put and subsequently Spm from Spd. Both Spd and Spm are N1-acetylated by the highly inducible polyamine catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT, EC 2.3.1.57).26-28 SSAT has a short half-life and it utilizes acetyl coenzyme A for the acetylation reaction. The acetylation of the polyamines Spd and Spm converts the molecules into substrates of constitutively expressed N1-acetyl polyamine oxidase (APAO, EC 1.5.3.13), which catabolizes acetylated Spm to Spd and acetylated Spd to Put29, 30. The acetylated forms of Spd and —Spm can also be exported from the cell. The most recently discovered polyamine catabolizing enzyme is spermine oxidase (SMO, EC 1.5.3.16), which uses Spm as a substrate producing Spd, 3-aminopropanal and H2O2.31-33 Furthermore, SMO is inducible and a major source of reactive oxygen species in various pathogenic states.1, 34 Even though the biosynthesis of the polyamines is tightly regulated, the active polyamine transport can also regulate the polyamine concentration and it is often upregulated in pathological states in the cell.35 The polyamine transport system has been characterized in E. Coli and yeast (for review see36), but the eukaryotic polyamine transport system is complicated. Recently, some convincing data has been published36-38 that has facilitated the creation of models for the mammalian polyamine uptake system24. The polyamine transporter is regulated by a small short living protein, antizyme, which controls the transport by feedback-inhibition. Antizyme has high affinity for ODC and the binding leads to the dissociation of the ODC dimer into monomers and ultimately to the proteosomal degradation of ODC.39

The polyamines can also be terminally deaminated by other enzymes that are not involved in the polyamine catabolizing route.40, 41 For example, deamination can be achieved by many different enzymes such as monoamine oxidase, diamine oxidase and serum amine oxidase.

21

2.2 OVERVIEW ON THE ACIDITY

Proton transfers are common phenomena in both chemistry and biochemistry and ionic interactions are crucial in the molecular recognition of different biomolecules. Since acids and bases can exist in a veriety of structures a fundamental knowledge about acidity (and basicity) is important. It is also useful to appreciate what factors affect the acidity of compounds. The simplest examples of an acid or a base are the easily recognizable functional groups. Often the acidity or basicity of a compound correlates with its reactivity and almost all sorts of reactions can be explained as acid-base reactions. For example, when acetic acid is added to water it undergoes a reaction forming a negatively charged ion while protonating water (scheme 1). This phenomenon is called the dissociation of acetic acid.

Scheme 1. The dissociation of acetic acid in water

The thermodynamics of this reaction can be expressed as a Ka value, which can be used to compare the acidity to water (Equation 1).

Eq 1.

The Ka values can vary extensively between strong acids and bases and therefore the acidity scale, which is called pKa, the negative logarithm of Ka, is used to compare the strengths of acids. Since the pH affects the concentration of H3O+ molecules in solution, thus altering also Ka and pKa values, the protonation of acetic acid can be examined with the Henderson-Hasselbalch equation (equation 2)

Eq 2.

As mentioned above, different functional groups have different pKa values based on their acidities and some representative examples are given in figure 3. In general, the pKa value indicates the bond strength and/or ability to stabilize the formed charge in the X—H system; a weak bond has a low pKa value and a strong bond has a high pKa value.

Figure 3. The pKa values of various different molecules

22

The pKa values of the above compounds are explained mainly due to their different electronegativities, e.g. the CF3 group is more electronegative than CH3, or due to resonance properties, e.g. CH3CO2- has two resonance structures but RO- has only one.

The elecronegativity affects the acidity in two ways, the first via the electronegativity of the atom, which the proton is directly attached and the other way is caused by electron withdrawing groups near the acidic site of the molecule. This phenomenon is called an inductive effect and one of many examples is the change in acidity caused by a neighboring electronegative group. The electronegativity of atoms follows the number of protons in the nucleus; this can be seen in the periodic table where the electronegativity increases when one moves from left to right and from bottom to top. Other internal factors that affect the acidity of compounds are electrostatic effects, hybridization, aromaticity and solvation

The external factors such as exchange of the solvent from H2O to DMSO can cause dramatic changes in the pKa values of molecules. The pKa value can vary considerably if the molecule binds to an active site of an enzyme, which corresponds to a completely different environment as compared to a solution. The differences in the pKa values of free amino acid residues solely or in a protein backbone might even explain some of the differences in the specificities of the interactions encountered with cationic polyamines.

2.2.1 Macroscopic and microscopic pKa values of polyprotic acids or bases As mentioned above, the pKa value describes the particular pH value in which half of the population of an acid or a base is in an ionic form. With molecules that contain only one acidic or basic group, the pKa value also describes how the charge is localized in the molecule while the pH changes. This is not the case with polyamines, as their name already reveals, because there are more functional groups that can be ionized and all of these groups have their own distinct pKa values. The macroscopic pKa values of these molecules contain only the information about the populations of different charge states in each pH but not how the charge is localized in the structure. These charge populations can be calculated using the Henderson-Hasselbalch equation42, 43, which gives a good approximation of the net charge of a molecule at the calculated pH. The localization of the charge is important for the recognition of the polyamines therefore providing more accurate information about the interactions of cationic polyamines. The constants that describe the protonation of the individual amino groups are called microscopic pKa values and they also contain the information needed to assign the localization of the charge into the polyamine backbone.44,

45 For example, Spd has three pKa values but the charge of mono- and diprotonated states can be represented in three different isomers and 12 microscopic pKa values are needed to describe the protonation of Spd in detail (scheme 2).

23

Scheme 2. A) Macroscopic protonation Spd. L represents Spd and H the charge; B) Microscopic protonation of Spd. Each arrow

represents a distinct microscopic pKa value.

The three most common methods used to determine pKa values of molecules are titrations using potentiometry, spectrophotometry and NMR with potentiometric titration being the most often used in the pKa determination of polyamines. In contrast, the spectrophotometric method cannot be used because polyamines lack chromophores in their structure.46 The potentiometric titration is an automated method and it can be uset to determine the macroscopic pKa values of the polyamines from a small amount of sample. The other option is the NMR-pH titrations that allow the estimations of both macro- and microscopic pKa values.44, 45, 47 The NMR measurements can also be used to extract structural data about the molecule. The selection of the strategy for the calculation of microscopic pKa

values of polyamines and their analogues depend on the amount of amino groups, the pKa

values, and the general structure of polyamine. It is known that all of the microscopic pKa values of polyamines, or any polyprotic acid, with more than three non-identical ionizable groups cannot be determined without making special assumptions or having additional information.48 Thus, cluster expansion methods need to be used for the molecules that do not conform to the above criteria. With these computational methods, the microconstants can be calculated from even complex molecules, with many ionizable groups, using only macroscopic pKa values.49

24

2.2.2 Short overview to the NMR methods used for the pKa determination of polyamines and their analogues NMR-pH titrations are commonly used for the determination of the acid dissociation constants of polyamines (figure 4). The method provides molecular insights into the protonation thus providing detailed information about the microspecies at different pH values. The main disadvantage of the method is the higher amount of the compound needed as compared to the traditional potentiometric titration, even though the compound of interest can be retained from the sample(s) in either basic or salt form. D2O is used as a solvent for the most of the NMR—pH titrations to avoid an excess H2O peak. A correction has to be added to the pD values in order to obtain pH values.50, 51 NMR-pH titrations can also be done from impure mixtures if the compounds in the mixture do not affect each other´s protonation as well as in various different solvent systems including biological fluids.

Figure 4. An example of 1H-NMR-pH titrations; pH vs. chemical shift of aminooxy neighboring CH2-group of 1-aminooxy-3-

aminopropane (APA) around the pKa of the aminooxy group (4.2).52

The pKa values and protonation of metal chelating polyamines with ethylenic chain between amino groups were studied using 1H NMR-pH titrations already in 1964.53 The macroscopic pKa values and the protonation of Spd have been determined with 13C—54 and 15N NMR55 methods with comparable accuracy to those obtained with the potentiometric titration. Furthermore, the 15N NMR method displayed advantages over the 1H— and 13C NMR methods since it had higher sensitivity and more straightforward spectral analysis. However, the low abundance of 15N (0.368 %) makes the method less favorable. Later, a 1H—13C coupled 2D NMR method was used to determine the protonation and macroscopic pKa values of spermine.44 1H— and 13C NMR data was combined for simultaneous analysis

25

giving the most precise results and 2D NMR was shown to be superior in the determination of overlapping 1H signals of Spm. The 13C NMR method has also been used for the detection of pKa values of polyamine-containing wasp toxins with 1H-13C NMR being used to obtain 1H NMR shifts.56 In this case, the proton chemical shifts of Spm part of PhTX-343 did not change sufficiently during ionization to allow non-linear analysis illustrating the superiority of the use of 13C NMR chemical shifts for the detection of protonation. 13C NMR chemical shifts were used also for the determination of protonation constants of fluorinated polyamine analogues.11 Recently, the 1H-15N NMR method with indirect detection of 15N NMR shifts was applied to obtain the protonation map of the next generation charge deficient Spm analogue (figure 5) (Paper I). This method was chosen to overcome the spectral analysis problems caused by the highly overlapping 1H and 13C data.

Figure 5. Overlay of 1H-15N correlated 2D NMR spectra in pH range 2 to 11 (From left to right) of 1,12-diamino-3,6,9-triazadodecane

(SpmTrien). Each of the five nitrogens is represented with a different color.57

26

2.3 INTRODUCTION TO CHARGE DEFICIENT POLYAMINE ANALOGUES

Only a few different rational methods have been used to reduce the basicity of the amino groups in the polyamine backbone and thus to decrease the net charge of polyamine analogues. The ideal way to alter the basicity of any polyamine analogue would be one that causes as little steric disturbance as possible so that the analogue would still resemble the natural polyamines as much as possible. Currently, there are a few different ways available to reduce the basicity of the amino groups in the polyamine backbone. The four most widely used methods are: 1) the introduction of one or two fluorines in the polyamine backbone; 2) exchange of NH2CH2- for NH2O-; 3) reduction of the length of carbon chain to two between the amino groups; 4) the use of conformationally restrained cyclic amines instead of an aliphatic chain. In addition to these classes of analogues, also compounds that have their secondary amino groups exchanged to ether or thioether functionality have been synthesized but an analysis of their properties has been intentionally omitted from this thesis. Charge deficient polyamine analogues represent valuable tools for biochemists trying to elucidate the importance of the charge in polyamine recognition and binding.

2.4 FLUORINATED ANALOGUES OF THE NATURAL POLYAMINES

The substitution of hydrogen atoms to the most electronegative element fluorine (Pauling electronegativity 4) in the polyamine backbone was one of the first strategies used to influence the pKa values of neighbouring amino groups (table 1). Fluorine is the smallest feasible element which can replace hydrogen, even though its size is nearly isosteric to O, but often the C—H bond can be replaced with C—F bond with minimal steric disturbances.58 On the other hand, the C—F bond can mimic C=O bond since it has the same kind of charge distribution. Moreover, fluorine forms hydrogen bonds, where it acts as an acceptor, similarly to C=O group. The NMR sensitivity of the naturally occurring stable isotope 19F is comparable to the sensitivity of 1H in NMR studies providing the opportunity to use 19F NMR spectroscopy for the monitoring of the fluorinated compounds in vitro and in vivo, while the radioactive isotope of fluorine (18F) can be used for positron emission topography (PET) experiments. Fluorinated putrescine analogues have been used in both PET and NMR experiments in vivo. 59, 60

27

Table 1. The structures and the pKa values of the fluorinated polyamine analogues.

Abbr. Compound pKa Net

Charge Ref. 1 2 3 4

Put

10.72 9.44 1.99 13

2-F-Put

9.75 8.15 1.84 6

2,2-F2-Put

9.45 6.80 1.19 6

2,2-3,3-F4-Put

6.2 4.6 0.06 61

Spm

10.98 10.09 8.83 7.94 3.74 11

6-F-Spm

10.7 9.7 8.04 6.62 2.95 11

6,6-F2-Spm

10.7 9.8 7.74 5.11 2.69 11

Spd

10.49 9.81 8.34 2.89 11

6-F-Spd

10.49* 10.40#

9.0* 9.55#

6.91* 7.18#

2.22* 2.37#

11* 5#

6,6-F2-Spd

10.2* 10.34#

9.76* 9.29#

5.16* 5.70#

2.00* 2.01#

11* 5#

7-F-Spd

10.28 9.00 7.80 2.69 5

7,7-F2-Spd

10.25 8.24 6.64 2.02 5

2,2-F2-Spd

10.30 7.30 5.50 1.45 5

DESpm

11.20 10.77 9.42 8.25 3.87 62

FDESpm

10.48 9.41 4.81 4.09 1.99 62

The pKa values were determined with *NMR— or #potentiometric titration. The net charge at pH 7.4 has been calculated from the published pKa values using the Henderson-Hasselbach equation.

28

2.4.1 Chemistry of the fluorinated polyamine analogues

The first fluorinated polyamine analogues were synthesized over 20 years ago when the

synthesis of the radioactive 2-18F-putrescine was described60. In addition, non radioactive 2-

fluoroputrescine (2-F-Put) and 2,2-3,3-tetrafuoroputrescine (2,2,3,3-F4-Put) were synthesized

at the same time63 (scheme 3). The introduction of one fluorine into the 2-position of the

putrescine backbone slightly lowered the net charge at pH 7.4 but it still remained mostly

doubly positively charged, while the net charge of 2,2- difluoroputrescine (2,2-F2-Put) was

predominantly +1 and both of the amino groups in 2,2-3,3-F4-Put were in the base form

(table 1).

a) DAST, CH2Cl2, -78ºC; b) PhtNK, DMF; c) Conc HCl

Scheme 3. The synthetic routes to 2-F-Put and 2,2,3,3-F4-Put.

The syntheses of 2,2-F2-Put and the higher spermidine and spermine derivates have been

carried out starting from 2,2-difluoro-1,4-butanediol but the only available references to

these compounds are in patents64, 65, and though the authors have claimed that they intend

to publish these syntheses in the peer reviewed scientific literature, they are still not

available in the published literature (scheme 4).

Scheme 4. The synthesis of the gem-difluoro-polyamines.

While the 2-F-Put still resembles Put at physiological pH, the effect of the attachment of a

single fluorine into the polyamine backbone of Spd or Spm causes a more extensive

decrease in the net charge of the molecules and the net charge of the gem-difluoro-

analogues of Spd and Spm is drastically lower as compared to the parent polyamines (table

1). The pKa values of 6-fluoro-spermidine (6-F-Spd) and 6,6- difluoro-spermidine (6,6-F2-

Spd) reported by Frassineti et al.11 determined by 13C-NMR differ from those reported by

29

Baillon et al.5 who used potentiometric titration. Interestingly, there is no major difference in the net charge, calculated at pH 7.4 for the published pKa values of both methods, of 6-F-Spd but the pKa value of the primary amine of aminobutyl end reveals a 0.55 unit difference between the methods. Similarly the estimation of the net charge of 6,6-F2-Spd is nearly identical with both methods but the pKa of the primary amine of the aminobutyl end shows a marked increase as determined with the NMR method in comparison to the pKa value determined with potentiometric titration. All of the mono fluorinated analogues discussed later are racemic mixtures of both enantiomers and one must keep in mind the fact that at least the enantiomers of the α-methylated analogues of Spd and Spm have different biochemical properties.66

2.4.2 Analogues in the polyamine metabolizing pathway. Both 2-F-Put and 2,2-F2-Put are substrates of SpdSy and metabolized into 6-F-Spd and 6,6-F2-Spd, respectively67, but neither 7-fluoro-spermidine (7-F-Spd) nor 7,7- difluoro-spermidine (7,7-F2-Spd) were detected6, 67. Furthermore, the accumulation of 2-F-Put was twice as high as the accumulation of 2,2-F2-Put in tumors and even higher in normal cells, which is not surprising, as 2-F-Put possesses almost the same net charge as Put. The affinity of 2-F-Put is high (Km 12 μM) for SpdSy and 2,2-F2-Put has a similar affinity to the natural substrate Put, but it showed mixed type inhibition at concentrations above 25 μM. The mono- and difluorospermidines, except for 2,2-difluorospermidine (2,2-F2-Spd), were found to be substrates of SpmSywith different substrate capabilities.5 This was one of the first pieces of evidence for the role of charge in polyamine biochemistry. The affinity of 6-F-Spd is identical to that of Spd for SpmSy and 6,6-F2-Spd have almost as good affinity whereas the affinity of 7-F-Spd is elevated eight fold and that of 7,7,-F2-Spd is increased by over 100 fold. Interestingly, 7-F-Spd and 7,7-F2-Spd show substrate inhibition of the enzymes with reversed Ki values compared to the Km values; 7-F-Spd had much higher Ki than 7,7-F2-Spd.

While both 2-F-Put and 2,2-F2-Put were able to substitute for Put in the biosynthetic pathway, 2-F-Put did not support cell growth68, even though there are also reports that 2,2-F2-Put on the other hand did seem to be able to support normal, but slightly retarded, growth of chick embryos after administration of α-difluoromethyl ornithine (DFMO)67. Put is a known activator of eukaryotic SAM-decarboxylase and the modulation of the charge in both 2-F-Put and 2,2-F2-Put decreased the affinity of the enzyme for these analogues.69

The fact that 2-F-Put behaves nearly identically to Put in vitro and in vivo, even though it is dehalogenated by diamine oxidase60, made the following polyamine metabolic flux possible using the ornithine analogue 4-fluoro-ornithine, which is metabolized by ODC to 2-F-Put.68,

70 These studies also confirmed that 6-F-Spd and 6-F-Spm were substrates of SSAT. While there are also some findings indicating that 6,6-F2-Spd may also be a substrate of SSAT no convincing data has been presented for this speculation.71 The gem-difluoro spermidines were tested to examine if they were substrates of hepatic acetyltransferase activity. In experiments conducted with the desalted supernatant fraction of rat liver it was observed that both 7,7-F2-Spd and 2,2-F2-Spd were 1.8 times better substrates than Spd; 6,6-F2-Spd also showed good substrate properties (1.4 times better).7 It is worth noting that the

30

acetylating enzyme of the hepatic fraction is unknown and Vmax for each of the acetylation was under 100 pmol/min /mg soluble liver protein. Furthermore, 7,7-F2-Spd was converted to two acetylated metabolites, whereas only one metabolite was observed with the other two analogues

2.4.3 Analogues and post-translational modifications Due to the decrease in the pKa values of 6,6-difluorospermine (6,6-F2-Spm) the affinity of the analogue for calf thymus DNA was greatly reduced when compared to Spm.72 6-F-Spd and 6,6-F2-Spd were shown to be inhibitors of deoxyhypusine synthase (IC50 48μM and 14 μM respectively) whereas 7,7-F2-Spd caused no inhibition suggesting that both of the primary amines need to be protonated to permit the recognition by the enzyme.73 Deoxyhypusine synthase catalyzes also a reverse reaction where the end product is homospermidine 6-F-Spd, 6,6-F2-Spd nor 7,7-F2-Spd were not donors of the diaminobutyl chain to the main product deoxyhypusine or homospermidine.74 Different substrate profiles were also observed for gem-difluorospermidines with glutathionylspermidine synthetase of E.coli where 2,2-F2-Spd was found to be a superior substrate of the enzyme when compared to the natural substrate Spd or other fluorinated Spd analogues.75 These findings provided some mechanistic insights into the way that this enzyme functions e.g. substrate preferences.

2.4.4 Fluorinated analogues of N,N-di-ethyl-spermine The charge deficient analogue of N1,N12-diethylspermine (1,12-bis(ethylamino)-4,9-diazaundecane, BESpm) N1,N12-ditrifluoroethyl-spermine (1,12-bis(trifluoroethylamino)-4,9-diazaundecane, FBESpm) was synthesized to allow investigation of the role of charge in the polyamine transport system. This analogue competed only weakly with Spd for uptake and did not have any growth retarding features of BESpm in L1210 cells.76 Furthermore, BESpm and FBESpm can be considered as being nearly identical, although with different total charges at pH 7.4, because their Nα-Nα´ and Nα-Nβ distances are reported to be nearly identical. However FBESpm could not modulate NMDA receptor activity nor did it exert any effect on the polyamine biosynthetic enzymes and polyamine pools.62, 76 Another fluorinated analogue of BESpm N1-N12-diethyl-6,6-gem-difluoro-spermine has been synthesized starting from 1,3-propanediol using the Mitsunobu reaction with low yields but its chemical and biochemical properties have not been clarified.77

2.4.5 Fluorine containing charge deficient ornithine analogues. DFMO (figure 6) is one of the most widely used inhibitors of polyamine metabolism in studies of polyamine biochemistry. It is a rationally designed suicide inhibitor of pyridoxal-5′-phosphate (PLP) dependent ODC. The introduction of the difluoromethyl group into the α-position of ornithine caused a reduction in the pKa values of the molecule, but the reduction was not responsible for the inhibitory effect of the molecule. The ability of DFMO to inhibit ODC activity is believed to be based on F- to function as a leaving group58 and the mechanism by which DMFO inhibits ODC is now well characterized78. DFMO is used as a racemic mixture of both D- and L-enantiomers and both of the enantiomers form the covalent enzyme-inhibitor complex with ODC, latter posesses 20-times higher affinity for

31

the enzyme.79 Interestingly, absorption of DFMO showed enantioselectivity in the rat with the L-form being less well absorbed.80

Figure 6. The structure and the pKa values of DFMO and Orn

The decrease in the pKa value of the α-amino group of DFMO as compared to Orn did not affect the molecular recognition by ODC but the molecule posessed other rather interesting chemical features. When DFMO was reacted with di-tert-butyl dicarbonate, only the mono Boc- derivate of DFMO was formed, thus indicating that the alpha-amino-group of the analogue was nonreactive.81 Furthermore, the monoprotected DFMO was used in peptide coupling reaction and did not form any polymers. This is rather surprising as other α-difluoro amino acids have been successfully incorporated into the middle of the peptide chain and also into the C-terminal.82 Furthermore, the steric effect of α-difluoromethyl phenylalanine should be at least as strong as those found in DFMO. This highlights the possibility that there might be alternative explanations for the non-reactivity of DFMO other than simply the steric effect.

Other fluorinated analogues of Orn have also been synthesized with higher inhibitory potencies than DFMO, e.g. α-fluoromethyl ornithine and its methyl- and ethyl esters83 and α-fluoromethyl-dehydroornithine and its ethyl ester84, 85.

32

2.5 AMINOOXYANALOGUES OF NATURAL POLYAMINES

Another family of charge deficient analogues of natural polyamines was introduced in the middle of the 1980s when a charge deficient isosteric analogue of putrescine, 1-aminooxy-3-aminopropane (APA), was used to inhibit ODC.9 The introduction of the aminooxy or —oxa group into the polyamine backbone caused a minimal steric disturbance compared to the original NH2CH2 moiety making the family of analogues very similar in their spatial characteristics to the corresponding polyamines. The pKa value of aminooxy group is around five, which is roughly five orders of magnitude lower compared to the amino group, therefore meaning that these molecules are charge deficient at physiological pH. This causes a drop of one unit in the total net charge of the analogues at physiological pH when compared to their parent polyamines. The main disadvantage of the exchange of amino groups to their aminooxy equivalents in the analogues of Put and Spd is their high reactivity with carbonyl containing molecules, this can also be considered as an advantage, the aminooxy containing molecules are suitable for use in click chemistry86.

2.5.1 Chemistry of the aminooxy and –oxa analogues There are few feasible synthetic routes for the preparation of APA and the other aminooxy analogues of natural polyamines.87-90 All of the synthetic strategies rely on the use of N-protected hydroxylamines, which react readily with electrophiles i.e. for the synthesis of APA, ethyl N-hydroxyacetimidate, a good starting material for any of these molecules, was first reacted with 1-bromo-3-chloropropane and the reaction intermediate was then converted into APA (scheme 5).87 For the synthesis of 3H-labeled APA 3-aminoxypropionitrile was reduced with catalytic tritylation into the tritylated product.91 In addition, the backbone modified analogues of APA have been synthesized with a similar methodology.90 The charge of APA and other aminooxy analogues of Put is one (+1) in physiological pH because of the low pKa value of the aminooxy group.

Scheme 5. One of the synthetic routes for the synthesis of APA87

The synthesis of Spd analogues started from a protected aminooxy precursor (ethyl N-hydroxyacetimidate), which was first converted to a suitable synthon and then extended with diamine to 1-aminooxy-3-N-(3-aminopropyl)-aminopropane (AP-APA) or N-(2-aminooxyethyl)-1,4-diaminobutane (AOE-Put). The 3H- labeled derivatives of AP-APA and AOE-PU have also been synthesized.89 The aminooxy Spm analogues were synthesized with similar reactions using the orthogonal protective group strategy.92 The synthesis of the monoacetylated derivates of AOE-Put, AP-APA89 and AOSpm92 has also been described. Furthermore, oxa-analogue of Spm (1,12-diamino-4,9-diaza-5-oxadodecane, oxa-Spm) was

33

prepared using this same synthetic strategy.93 In addition, the synthesis of the oxa-analogue of Spd and bisoxaanalogue of Spm have been described.94 The pKa values for the aminooxy or –oxa polyamine analogues have not been fully characterized and only the pKa values of the aminooxy or –oxa groups are known (figure 7).

Figure 7. The structures of the isosteric amino-oxy and –oxa analogues of natural polyamines and the pKa values of the aminooxy and

–oxa group.52

2.5.2 Biochemical features of the aminooxy analogues of putrescine APA, the isosteric analogue of Put, is a very effective competitive inhibitor of mammalian ODC (Ki 3.2nM); it irreversibly inhibits liver AdoMetDC and is also a competitive inhibitor of bovine brain SpdSy9 and it is not metabolized to longer analogues. Interestingly, APA did not show any inhibition of AdoMetDC or SpdSy from E. coli, although it could block ODC of bacteria with Ki of 1 nM.95 Furthermore, APA did not compete with natural polyamines for transport even in situations where the polyamines had been depleted.96 The bisaminooxy analogue of Put was not an inhibitor of ODC9 whereas the introduction of hydroxyl— or fluorine into the 2- position in the backbone of APA retained the ODC inhibitory efficacy (table 2)90. Disturbances of Golgi apparatus were observed, when BHK cells were treated with APA.97 In all of the studied cases, APA passively accumulated inside cells. However, in the case of L.donovani it proved possible to convert the compound into an actively transported inhibitor, γ-guanidinooxypropylamine (GAPA, pKa 6.71)98, which itself inhibited L.donovani ODC with a Ki value of 60 μM, while IC50 towards amastigotes was already 9.0 μM99. Moreover, GAPA, in contrast to APA was found to be effective even against sodium antimony gluconate-resistant forms of the parasite.100

34

Table 2. Inhibition of mammalian ODC by hydroxylamine-containing analogues of putrescine

Compound Abbreviation. Source of ODC IC50, M Ref.

APA

Mouse liver

Rat liver

2 10-8

1,5 10-8

9

90

Bis-APA Mouse liver > 10-6 9

2-OH-APA Rat liver 3,8 10-8 90

R-2-OH-APA Rat liver 4,9 10-8 90

S-2-OH-APA Rat liver 3,3 10-8 90

2-F-APA Rat liver 1,4 10-8 90

The table adapted from 101

2.5.3 Biochemical features of the isosteric aminooxy analogues of Spd and Spm

The Spd analogues AP-APA and AOE-Put are both moderate inhibitors of ODC and good

inhibitors of AdoMetDC and SpmSy.7 The affinity of the analogues was dependent on the

localization of the charge as AOE-Put had an order of magnitude higher affinity than Spd

while the affinity of the AP-APA was an order of magnitude lower. Both of the Spd

analogues have been postulated to be substrates of SSAT but no data has been presented to

verify this hypothesis.89 Like the parent Spd, AOE-Put was not a substrate of APAO

whereas the acetylated AOE-Put was shown to be a weak substrate (kcat 0.54 s-1).102 Both AP-

APA and AOE-Put, exhibited moderate inhibition of cell proliferation in a dose dependent

manner.103 Both of the isosteric Spd analogues had different effects on the rat hippocampal

NMDA receptor complex, AP-APA was found to be an antagonist whereas AOE-Put was a

partial agonist.104

The Spm analogue, AOSpm, did not show any inhibitory activity towards ODC or

AdoMetDC, therefore differentiating it from the aminooxy analogues of Put or Spd.10 It was

demonstrated to be a substrate of SSAT and to effectively compete for uptake with all three

natural polyamines. Thus, it seems to be actively transported through polyamine transport

system and it was postulated that AOSpm might be recognized similarly as Spm.

Interestingly, AOSpm did not have any impact on polyamine levels or on the proliferation

of Caco-2 cells10 or BHK cells while it was shown to moderately inhibit the growth of the

rapidly proliferating L1210 cells8. The aminooxa analogue of Spm, oxa-Spm, was designed

35

to act as an inhibitor of SMO93 but its biochemistry, as well as the biochemical properties of other oxa analogues of Spd and Spm94, is still unclear.

Only AP-APA and AOSpm, where the two positive charges were separated with trimethylene moiety, were able to induce DNA aggregation when the interactions of the polyamine analogues AP-APA, AOE-Put and AOSpm with DNA were studied.105 Furthermore, AOSpm was found to induce DNA condensation at physiological pH whereas the Spd analogues showed similar properties only at low pH values. AOSpm was also shown to protect DNA from oxidative damage and in that way was similar to Spm.10 AP-APA was not an inhibitor of deoxyhypusine synthase, thus resembling to another charge deficient Spd isostere, 7,7-F2-Spd.73

2.6 POLYAMINE ANALOGUES WITH ALTERED CARBON CHAIN LENGTH AND NUMBER OF AMINO GROUPS

While the introduction of fluorine or the aminooxy group into the polyamine backbone decreases the charge of the molecules with minimal steric disturbances, the third family of charge deficient analogues changes display in the location and the number of the amino groups. When the length of the carbon chain between amino groups is shortened, then the total charge of the analogues is decreased and some of the pKa values of the molecules can be very low. Most of the biochemical studies with these kinds of analogues have concentrated on what features of polyamines are needed to support cell growth, polyamine transport and DNA condensation. Some, but not all, of the analogues belonging to this family are isosteric to the natural polyamines.

2.6.1 The chemistry of the polyamine analogues with altered carbon chain lengths. There are various different synthetic strategies which can be exploited to form a C—N bond and this topic has been widely reviewed106, 107 The same synthetic methodology can be followed for the synthesis of the analogues with altered chain lengths as for the natural polyamines and their metabolites. The synthesis of the symmetrical analogues of the family is straightforward108, 109 whereas seven synthetic steps are needed with orthogonal protection for the synthesis of 1,12-diamino-3,6,9-triazadodecane110 (SpmTrien, 3-2-2-2) (scheme 6). Recently, a large scale synthesis for SpmTrien was devised starting from triethylenetetraamine (1,8-diamino-3,6-diazaoctane, Trien) with a simple Michael addition of acrylonitrile followed by a catalytic hydrogenation, but purification of the nitrile intermediate was rather challenging (Paper I). The acetylated metabolites of SpmTrien (N1— and N12-mono-Acetyl SpmTrien) and Trien (N1-Ac-Trien (MAT) and N1,N8-Ac2-Trien (DAT)) have also been synthesized (Paper II).

36

a(a) H2N(CH2)2NH(CH2)2OH; (b) CbzCl, (c) MsCl, Et3N; NaHCO3; (d) H2N(CH2)2NH2; (e) o-C6H4(OH)CHO/THF; (f) MeONH2; (g) H2/Pd, AcOH,

MeOH

Scheme 6. A) The synthesis of the polyamine analogue 3-2-3 using Michael Addition reaction followed by hydrogenation108; B) the

synthesis of SpmTrien110

The reduction of the carbon chain length between amino groups from three or four to two creates a significant increase in the electrostatic repulsion between the amino groups.13 The electrostatic repulsion between amino groups decreases when the chain is elongated to propylenic and it becomes irrelevant with butylenic or a longer carbon spacer. This phenomenon can be seen from the studies of the pKa values of diamines with different lengths of the aliphatic carbon chain between the amino groups.111 Compounds with only a methylenic spacer between the amino groups are unstable. Thus, there is a large decline in the pKa values of the polyamine analogues of this family, which contain the short ethylenic spacer between amino groups (table 3). All of the pKa values of the analogues from this family have been determined with potentiometric methods and only the protonation of the analogue SpmTrien was determined with NMR (Paper I). The protonation of SpmTrien is interesting as the protonation does not occur in the classical way and an amphoteric character of nitrogens is observed from the 15N shifts. Thus, the protons seem to drift between secondary amines of the molecule at the physiological pH, which is a unique feature of the polyamine analogue. It is important to note that the pKa values of other polyamine analogues have not been studied with 1H-15N coupled 2D NMR method, which revealed the drifting proton(s) of SpmTrien.

37

Table 3. The structures and the pKa values of the polyamine analogues with altered chain length. The net charge in pH 7.4 is

calculated from the published pKa values using the Henderson-Hasselbach equation.

Compound Abbr.

pKa Net

Charge* Ref

1 2 3 4 5

2-3 10.21 9.17 6.10 2.03 13

2-3-2 10.08 9.26 6.88 5.45 2.23 13

3-2-3 10.53 9.77 8.30 5.59 2.90 13

Trien 9.74 9.07 6.59 3.27 1.97 13

SpmTrien 10.3 9.5 8.5 6.3 3.3 2.99 112

3-2-2-3 10.72 10.50 8.94 7.89 3.96 3.73 13

2-2-2-2 9.70 9.14 8.05 4.70 2.92 2.80 13

2.6.2 The biochemistry of the charge deficient polyamine analogues with altered chain

length.

Most of the members of this family have been studied for their ability to replace the

functions of natural polyamines; restore growth after DFMO induced growth arrest of cells;

the uptake competition and cell migration (table 4). Even though all of the analogues can

mimic at least some functions of the natural polyamines109 and also interact with DNA in

vitro72, 113, this group of analogues has not been systematically evaluated for their effects in

the polyamine metabolizing pathway with the exception of the newest analogue in this

family, SpmTrien, which is isosteric to Spm, as well as the molecule from which SpmTrien

was derived, Trien, which is isosteric to Spd. The ability of the analogues to cause a B-to-Z

transition in the DNA conformation correlated with their ability to overcome the DFMO

induced growth arrest.113 Interestingly, Trien was found to restore cell growth after DFMO

induced growth arrest in DU145 cells (Paper I) while it was ineffective in IEC-6 cells109.

Furthermore, Trien and SpmTrien were both found to compete with [14C]-Put for transport

but the competition with 14C-labelled Spd and Spm was weak, suggesting that they had low

affinity toward polyamine transport (Paper II). Nevertheless, the concentration of SpmTrien

was found to be as high inside cells as the concentration of natural polyamines, ponting to

38

another mechanism to account for the entry of the molecule into the cells. In addition, a copper chelating isosteric charge deficient analogue of norspermine, 1,11-diamino-3,6,9-triazaundecane (2-2-2-2), has been synthesized but it has not been viewed as a polyamine even though it has been used in stem cell differentiation experiments and clearly affected CD34 and CD38 expression levels.114 2-2-2-2 has been undergoing phase I/II clinical trials for transplantation of umbilical cord blood derived hematopoietic stem cells grown ex vivo. 115

Table 4. The ability of the charge deficient analogues with the altered chain length to replace the functions of natural polyamines.

Abbreviation Uptakea Cell growthb Cell migrationb Ref.

2-3 No Yes Yes 109

2-3-2 No Yes Yes 109

3-2-3 No Yes Yes 109, 113

Trien No No(IEC-6 Cells)

Yes(DU145 cells) Yes 109, 112, 116

SpmTrien No Yes ND. 112, 116

3-2-2-3 ND. Yes ND. 113

a Competition with [14C]Spd, bGrowth recovery after PA depletion with DFMO; ND. Not determined

2.6.3 Trien and SpmTrien in the polyamine catabolizing pathway Both Trien and SpmTrien were found to be substrates of SSAT with Vmax values around 1.2 (μmol/min)/mg of enzyme for both of the analogues and Km values of 144 and 106 μM, respectively (Paper II). In addition, Trien was a substrate of mouse SSAT in vivo, but also another enzyme, recombinant human thialysine acetyltransferase, was identified as being able to acetylate Trien in vitro (Paper III). While Trien or its acetylated metabolites were not substrates of either of the FAD-dependent polyamine oxidases, SMO and APAO, N12-Ac-SpmTrien proved to be a good substrate of APAO, resulting Trien as the oxidation product. This provided additional information about the differences between substrate recognition of these polyamine oxidases. SMO seems to need adequate protonation of the central part of the substrate polyamines while APAO is not so demanding about the charge state of the substrate molecule(s), which is not surprising because SMO has narrower substrate specificity than APAO. It was found that SpmTrien downregulated ODC and AdoMetDC activity and also moderately induced SSAT activity while Trien did not evoke any changes in the enzyme activities (Papers I, II).

2.6.4 Biochemical properties of the N,N´-di-ethylated charge deficient analogues with altered chain lengths The isosteric charge deficient analogue of di-ethyl-Spm, BE-SpmTrien, is the only diethylated isosteric charge deficient analogue of BE-Spm, which has been systematically tested with the polyamine catabolizing enzymes as well as in vitro (Paper II). Another di-ethylated analogue from this family, BE-3-2-3 has also been tested for its ability to induce changes in the conformation of DNA and for its ability to substitute for some of the

39

functions of the natural polyamines.113 BE-3-2-3 could not overcome DFMO induced growth arrest. Interestingly, treatment of 9L cells with BE-3-2-3 caused a significant elevation in the Put and Spd concentrations over 3 days. In addition, BE-SpmTrien possessed no ability to overcome DFMO induced growth arrest and it did not have any biological activity towards regulatory enzymes of polyamine metabolism (Paper II).

2.6.5 Charge deficient polyamine conjugates with altered chain length The natural polyamines and their analogues with reduced charges and chain lengths have been conjugated with different lipids to create novel agents for non-viral gene delivery.117, 118 Polyamines can interact with DNA very efficiently and they can cause DNA condensation. Therefore they are very interesting molecules to be used in experiments attempting transfection in vitro and in vivo. The chemistry and biological properties of polyamine-steroid conjugates have been reviewed by Blagbrough et al.119 Long polyamines with linear and branched polyethyleneimine (—NCH2CH2N—) chain have also been investigated in non-viral gene therapy systems.120

40

2.7 THE CHARGE DEFICIENT POLYAMINES BEARING A CYCLIC RING IN THEIR BACKBONE

The last family of the charge deficient polyamine analogues has a more rigid structure in their backbone due to the replacement of the aliphatic carbon chain between amino groups with a cyclic ring. This family was synthesized to learn more about the features of the polyamine transport system76 as well as to investigate the regulation of the NMDA-receptor62. The charge deficient analogues of this family have an aromatic pyridine ring at their carbon chain and have total charge of two at the physiological pH (table 5). The properties of the pyridine bearing polyamines were compared with polyamines bearing a non-aromatic piperidyl ring system in their backbone. While these analogues provided interesting insights into the role of charge in polyamine transport system as well as interactions of SSAT, ODC and AdoMetDC with their substrates, they have not subsequently been extensively utilized in the studies clarifying the biochemistry of either polyamines or the NMDA-receptor except for the longest pyridine containing analogue, which was a very efficient partial agonist of the NMDA-receptor with EC50 values in the nM range62.

Table 5. The charge deficient polyamine analogues bearing cyclic ring at their backbone and their fully protonated counterparts. The net charge in pH 7.4 is calculated from the published pKa values62,76 using the Henderson-Hasselbach equation.

Compound pKa Net

charge 1 2 3 4

11.18 10.67 8.88 7.17 2.97

9.55 9.18 <2a <2a 1.98

10.93 10.69 9.43 8.45 3.91

9.23 7.64 4.43 3.77 1.62

10.93 10.82 10.01 9.10 3.98

9.81 8.85 5.69 4.93 1.98

apKa value was approximated to be 2 in the calculations of the net charge

41

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104. Berger, M. L.; Khomutov, A. R.; Rebernik, P. Aminooxy-analogues of spermidine: new partial agonists and antagonists at the polyamine site of the rat hippocampal NMDA receptor complex. Neurosci. Lett. 1996, 203, 25-28.

105. Ramirez, F. J.; Thomas, T. J.; Antony, T.; Ruiz-Chica, J.; Thomas, T. Effects of aminooxy analogues of biogenic polyamines on aggregation and stability of calf thymus DNA. Biopolymers 2002, 65, 148-157.

106. Bergeron, R. J. Methods for the selective modification of spermidine and its homologs Acc. Chem. Res. 1986, 19, 105-113.

107. Kuksa, V.; Buchan, R.; Lin, P. K. T. Synthesis of polyamines, their derivatives, analogues and conjugates. Synthesis 2000, 1189-1207.

108. Israel, M.; Rosenfield, J. S.; Modest, E. J. Analogs of Spermine and Spermidine. I. Synthesis of Polymethylenepolyamines by Reduction of Cyanoethylated α,ι-Alkylenediamines1,2. J. Med. Chem. 1964, 7, 710 -716.

109. McCormack, S. A.; Zimmerman, B. J.; Israel, M.; Blanner, P.; Johnson, L. R. Structural requirements of analogues of polyamines for migration and growth of IEC-6 cells. Molecular Pharmacology 1995, 48, 724-729.

110. Khomutov, A. R.; Grigorenko, N. A.; Demin, A. V.; Vepsäläinen, J.; Casero, R. A.; Woster, P. M. A charge-deficient analogue of spermine with chelating properties. Bioorg. Khim. 2005, 31, 303-311.

111. Bertsch, C. R.; Fernelius, W. C.; Block, B. P. A Thermodynamic Study of Some Complexes of Metal Ions with Polyamines. J. Phys. Chem. 1958, 62, 444-450.

112. Weisell, J.; Hyvonen, M. T.; Vepsalainen, J.; Alhonen, L.; Keinanen, T. A.; Khomutov, A. R.; Soininen, P. Novel isosteric charge-deficient spermine analogue--1,12-diamino-3,6,9-triazadodecane: synthesis, pKa measurement and biological activity Amino Acids 2010, 38, 501-507.

113. Basu, H. S.; Feuerstein, B. G.; Deen, D. F.; Lubich, W. P.; Bergeron, R. J.; Samejima, K.; Marton, L. J. Correlation between the Effects of Polyamine Analogues on DNA Conformation and Cell Growth. Cancer Research 1989, 49, 5591-5597.

114. Peled, T.; Landau, E.; Mandel, J.; Glukhman, E.; Goudsmid, N. R.; Nagler, A.; Fibach, E. Linear polyamine copper chelator tetraethylenepentamine augments long-term ex vivo expansion of cord blood-derived CD34+ cells and increases their engraftment potential in NOD/SCID mice. Exp. Hematol. 2004, 32, 547-555.

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115. de Lima, M.; McMannis, J.; Gee, A.; Komanduri, K.; Couriel, D.; Andersson, B. S.; Hosing, C.; Khouri, I.; Jones, R.; Champlin, R.; Karandish, S.; Sadeghi, T.; Peled, T.; Grynspan, F.; Daniely, Y.; Nagler, A.; Shpall, E. J. Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I/II clinical trial Bone Marrow Transplant. 2008, 41, 771-778.

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4 Aims of the study

The correct localization of charges in the backbone of Spm, Spd and all of their analogues is one of the key descriptors determining the distinct biological effects of these molecules. Thus, one could predict that even small changes to the total charge and the distribution of the charges along the polyamine backbone would create compound with very interesting biochemical properties and which could help to clarify the complex biochemistry of polyamines and their analogues. The aims of this thesis were to examine the chemical and biochemical properties of SpmTrien, the latest charge deficient Spm analogue, and the corresponding Spd analogue, Trien. The more specific aims were:

i) To determine the acid dissociation constants and the protonation of the novel charge deficient Spm analogue, SpmTrien (papers I and IV)

ii) To examine whether SpmTrien, Trien and their acetylated analogues were substrates of polyamine the catabolizing enzymes (paper II)

iii) To determine to what extent SpmTrien could mimic the natural polyamines (papers I and II)

iv) To characterize the metabolism of SpmTrien and Trien in vitro and in vivo (papers II and III)

v) To clarify the chemical features of the acetylated metabolites of SpmTrien and Trien (paper III)

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5 General methods

The general methods that were used in this thesis are briefly described in this chapter. The more detailed methodologies can be found in the papers cited after each general procedure.

4.1 SYNTHETIC METHODS

The synthesis of the novel polyamine analogues and their acetylated derivates relied on the classical strategy of using orthogonally protected precursors (Paper II). Typically, the required polyamine derivatives were prepared from commercially available starting materials by using Cbz-group protection followed by chain elongation with the required carbon chain moiety containing a suitable leaving group (e.g. mesylate). In the last step, the protecting groups were removed with catalytic hydrogenation and the target molecules were crystallized as HCl salts. This type of synthetic methodology provides high purity analogues and each intermediate can be readily purified with conventional chromatography. A large scale synthesis was also deviced for the synthesis of SpmTrien (Paper I). A simple Michael addition reaction of acrylonitrile with Trien was followed by purification on aluminium oxide. After catalytic hydrogenation, SpmTrien was crystallized as the HCl salt. Methanol and aqueous ammonia mixture proved to be best for complex purification of the nitrile intermediate when neutral alumina was used as the column material.

4.2 NMR SPECTROSCOPY

In the characterization of the synthetic products, 1H and 13C NMR spectra were measured on Bruker Avance 500 DRX (Germany) using tetramethylsilane (TMS) in CDCl3 or sodium 3-(trimethylsilyl)-1-propanesulfonate (TSP) in D2O as the internal standards. Chemical shifts are given in ppm, and the letter "J" indicates normal 3JHH сouplings and all J values are given in Hz (Papers I, II, III). 2D NMR spectra were measured using standard pulse programs. A double-tube system to avoid mixing of H2O and D2O was used to reveal the protonation order of SpmTrien and for the follow up of the intramolecular acyl migration reaction (Papers I, III, IV). The sample preparation for each experiment is described in detail in the appropriate publication.

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4.3 MASS SPECTROMETRY

All mass spectra were obtained on an Applied Biosystems/MDS Sciex QSTAR XL spectrometer using the ESI technique. The operation and the spectral processing were performed on the manufacturer´s software. The high resolution mass spectra were recorded using direct infusion (Papers I, II). The intramolecular acetyl migration reaction products were separated using reversed phase chromatography and heptafluorobutyric acid as the ion pairing agent prior to mass spectrometric detection (Paper III).

4.4 COMPUTATIONAL METHODS

SCC-DFTB/MM MD simulations, QM calculations and Cluster expansion were used to obtain the necessary information about the charge tautomers of SpmTrien (Paper IV).

4.5 RECOMBINANT PROTEIN METHODS

The recombinant proteins were produced in E. Coli and purified with His-tag affinity chromatography (Papers II, III). Substrate and kinetic experiments with APAO and SMO were analysed with HPLC using post-column o-phthaldialdehyde-derivatization (Paper II). The inhibition experiments for both oxidizing enzymes were studied using Ampliflu red that was converted into a fluorescent compound (resorufin) by horseradish peroxidase when H2O2 was released in the enzyme oxidation reaction (Paper II).

In the determination of the acetylating activity of the acetyltransferase enzymes, cation binding cellulose discs were used to separate the [14C]- acetylated products from the [14C] labeled acetyl-CoA substrate (Papers II, III).

4.6 CELL CULTURE METHODS

Typically, cellular experiments were performed with DU145 cells (Papers I, II). The samples for the polyamine measurements were taken and mixed 9:1 with 50 % sulphosalicylic acid containing 100 M diaminoheptane. The polyamine concentrations were quantified with HPLC using post-column o-phthaldialdehyde-derivatization. The supernatant fraction from the cell lysate was used for enzymatic assays of polyamine metabolic enzymes (Papers I and II: AdoMetDC, ODC, SSAT. Paper II; SMO and APAO). [14C]-labelled Put, Spd or Spm were used in the competitive uptake experiments and cycloheximide was used in the experiments where antizyme was depleted (Paper II).

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4.7 IN VIVO METHODS

The animal experiments on Trien metabolism were performed with three different mouse lines, SSAT overexpressing, SSAT knockout and syngenic mice (Paper III). The animal experiments were approved by the Animal Care and Use Committee at the Provincial Government of Southern Finland and carried out in accordance with the EU regulations (Paper III).

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10 Summary

The macroscopic pKa values of SpmTrien (3.3, 6.3, 8.5, 9.5 and 10.3), which were determined with potentiometric titration, are comparable to those of similar altough symmetric molecules (Paper I). Based on the 15N NMR—pH titration results, the protonation of SpmTrien occured first at both ends of the molecule. Interestingly, there was equilibrium in the protonation of the secondary amino groups of SpmTrien when the pH value was between 7 and 8. This caused a ‘‘wrong way’’ evolution in the 15N chemical shifts, which can be explained by a proton migration from the central N-III amino group to either NII or N-IV amino groups when the third proton had been introduced into the molecule. This phenomenom was further studied with two separate computational methods in order to obtain the accurate resolution of the microstates of each population of charges. The computational results showed that the equilibrium in the protonation of the secondary amino groups did not favor the protonation of the N-II amino group (Paper IV). This is not surprising because the electrostatic repulsion between charges at the backbone of SpmTrien should be smaller when either N-III or N-IV is first protonated. Furthermore, the protonation of SpmTrien was different from that in the previously known similar compounds with symmetry, providing some explanation for the unique biochemical properties of this compound. It has to be noted that the symmetrical analogues are still lacking a systematic biochemical characterization. The comparison of the computational methods showed that there was no difference between the computational method that was based on 15N NMR chemical shifts and the cluster expansion method, based solely on macroscopic pKa values. The cluster expansion method provided reliable results without the need for time consuming NMR measurements and computation time and thus it can be confidently used to calculate different microstate populations of polyamines and their analogues with accuracy. It is necessary to observe that the selection of the descriptors has to be made with care to the analogue of choice and only similar molecules can be used when being compared to previous data.

Both of the isosteric charge deficient analogues studied in this thesis, Trien and SpmTrien, showed polyamine-like properties to some extent. Trien was able to restore cell growth after DFMO induced growth arrest in DU145 cells (PAPER I and II). Furthermore, Trien and SpmTrien both were found to compete with [14C]-Put for transport to some extent but the competition with 14C-labelled Spd and Spm was low, suggesting that they possessed low affinity for the polyamine transporter (PAPER II). However, the concentration of SpmTrien was found to be as high inside cells as the concentration of the natural polyamines, suggesting another mechanism for the entry of the molecule into the cell. Both Trien and SpmTrien were found to be substrates of SSAT. While Trien or its acetylated metabolites were not substrates of either SMO or APAO, N12-Ac-SpmTrien was a good substrate of APAO, resulting in the formation of Trien as the oxidation product (PAPER II).

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This provided additional information about the differences between the substrate recognition properties of these polyamine oxidases. SMO seems to need adequate protonation of the central part of the substrate polyamine while APAO is not so fussy about the charge state of the substrate molecule(s). This is not surprising because SMO has narrower substrate specificity than APAO. SpmTrien downregulated ODC and AdoMetDC activity and also moderately induced SSAT activity while Trien did not evoke any changes in the enzyme activities. In addition, Trien was shown to be a substrate of mouse SSAT in vivo, but the enzyme was not the sole acetylator of the molecule and another enzyme, recombinant human thialysine acetyltransferase, was identified as being able to acetylate Trien in vitro (Paper III). As yet, the metabolism of SpmTrien has not studied in experimental animals and these experiments will need to be conducted to understand the complex metabolism of these polyamine analogues. Both of the recongnized metabolites of Trien, MAT and DAT, were observed to undergo intramolecular acetyl migration reaction in the acidified HPLC sample matrix (Paper III). This phenomenon may hamper also the determination of the metabolism of SpmTrien with the conventional HPLC method. Thus an LC-MS based method, relying on the use of deuterated molecules as internal standards, will need to be developed before one can clarify the in vivo metabolism of this Spm analogue.

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11 Future prospects

The isosteric charge deficient analogues of Spm and Spd, i.e. SpmTrien and Trien, have

shown interesting properties both chemically and biochemically. The studies into the

protonation of SpmTrien showed that microstate populations of different charges of

SpmTrien can be calculated with sufficient accuracy without NMR data simply by using the

cluster expansion method. Microstate populations of other analogues can also be

determined with the same methodology, providing an opportunity to extract more

chemical knowledge from pKa values to explain the differences in the biochemical

properties of different novel analogues. Our goal in the near future is to determine the pKa

values and protonation of differently backbone substituted Spd and Spm analogues in

order to elucidate whether the changes in the conformation of these analogues give rise to

internal factors which alter the pKa values in addition to the obvious steric changes.

Trien has been shown to be an interesting compound with many different potential drug

uses. Based on our current knowledge about the properties of this compound it seems to

have also other mechanisms of action in addition to known Cu2+ chelation. The metabolism

of this compound will be further studied to provide novel insights into the different

signaling routes that may be influenced after the administration of Trien. Furthermore, the

uptake of SpmTrien is higher than that of Trien and the possible drug use of this compound

will hopefully be conducted in the future. Both of the compounds share a similar backbone

with the norspermine isostere, 2-2-2-2, which would also be an interesting compound for

examination in a systematic characterization of the polyamine metabolizing pathway.


Publications of the University of Eastern Finland

Dissertations in Forestry and Natural Sciences

isbn 978-�952-�61-�0856-�8

Janne Mauritz Weisell

Structure and Function ofCharge Deficient PolyaminesThe Latest Generation of Isosteric Charge Deficient Spermine Analogues

Charge is the fundamental property

that enables polyamines to interact

through reversible ionic interactions

with many different biomolecules.

Modifications in the structure of

polyamine analogues can alter their

conformation without causing any

changes in the acidity of the amino

groups in the polyamine backbone.

This thesis focuses on analogues

that have reduced basicity of their

amino groups and therefore are not

fully protonated at physiological pH.

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Janne Mauritz WeisellStructure and Function of

Charge Deficient PolyaminesThe Latest Generation of Isosteric Charge

Deficient Spermine Analogues

Janne Mauritz Weisell Structure and Function of Charge...

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