Synthesis and analysis of hypusine-containing dipeptides and eukaryotic translation initiatiation factor 5A peptide fragments

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Material Information

Title:
Synthesis and analysis of hypusine-containing dipeptides and eukaryotic translation initiatiation factor 5A peptide fragments additional tools to study the amino acid hypusine.
Physical Description:
xv, 194 leaves : ill. ; 29 cm.
Language:
English
Creator:
Della Vecchia, Matthew J., 1975-
Publication Date:

Subjects

Subjects / Keywords:
Research   ( mesh )
Dipeptides -- analysis   ( mesh )
Peptide Initiation Factors -- analysis   ( mesh )
Dipeptides -- chemical synthesis   ( mesh )
Peptide Initiation Factors -- chemical synthesis   ( mesh )
Department of Medicinal Chemistry thesis Ph.D   ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Medicinal Chemistry -- UF   ( mesh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 2002.
Bibliography:
Bibliography: leaves 182-193.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Matthew J. Della Vecchia.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 028914134
oclc - 51887876
System ID:
AA00022671:00001

Table of Contents
    Title Page
        Page i
        Page ii
    Dedication
        Page iii
    Acknowledgement
        Page iv
        Page v
    Table of Contents
        Page vi
        Page vii
    List of Tables
        Page viii
        Page ix
        Page x
    List of abbreviations
        Page xi
        Page xii
        Page xiii
    Abstract
        Page xiv
        Page xv
    Chapter 1. Introduction
        Page 1
        Page 2
        Page 3
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        Page 5
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    Chapter 2. Synthesis I-solution phase
        Page 41
        Page 42
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    Chapter 3. Synthesis II-solid phase
        Page 57
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    Chapter 4. Experimental-synthesis I and II
        Page 78
        Page 79
        Page 80
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        Page 99
        Page 100
        Page 101
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    Chapter 5. Analytical-HPLC, tissue extractions, and HPLC-MS
        Page 103
        Page 104
        Page 105
        Page 106
        Page 107
        Page 108
        Page 109
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        Page 128
        Page 129
        Page 130
        Page 131
        Page 132
        Page 133
        Page 134
    Chapter 6. NMR-additional analyses of EIF-5A peptide fragments
        Page 135
        Page 136
        Page 137
        Page 138
        Page 139
        Page 140
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    Chapter 7. Summary and conclusions
        Page 176
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    List of references
        Page 182
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        Page 188
        Page 189
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        Page 193
    Biographical sketch
        Page 194
        Page 195
        Page 196
Full Text










SYNTHESIS AND ANALYSIS OF HYPUSINE-CONTAINING DIPEPTIDES AND EUKARYOTIC TRANSLATION INITIATION FACTOR 5A PEPTIDE FRAGMENTS:
ADDITIONAL TOOLS TO STUDY THE AMINO ACID HYPUSINE
















By

MATTHEW J. DELLA VECCHIA












A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2002



























Copyright 2002

by

Matthew J. Della Vecchia

























This dissertation is dedicated to my beautiful wife, Stephanie.















ACKNOWLEDGMENTS

I sincerely thank Dr. Raymond J. Bergeron for his cooperation, support, and

patience throughout the course of my graduate studies and research here at the University of Florida. He has provided me with a unique opportunity to explore numerous aspects of scientific research. Completing this dissertation under his direction has allowed me to become a well-rounded scientist with a broad foundation upon which to build a career in science.

I also thank the members of my committee for their input into this dissertation. Dr. Margaret James, Dr. John Perrin, Dr. Richard Streiff, and Dr. Charles Allen have provided much appreciated advice that has helped to make this work a success.

I am also grateful to the many members of Dr. Bergeron's research group, both past and present. All have contributed to this work in some way by helping with the design, execution, and/or interpretation of various experiments. Dr. W. Weimar, Dr. J. McManis, Dr. R. Milefer, Dr. E. Eyler-McManis, Mr. R. Smith, Mr. B. McCosar, Mr. S.

Algee, Mr. C. Zimmerman, Mrs. E. Nelson, Mr. T. Vinson, Mrs. H. Yao, Dr. M. G. Xin,

Dr. J. Bussenius, Mr. N. J. Nguyen. Dr. G. Huang, Ms. J. Weigand, Ms. T. Fannin, Ms.

T. Lindstrom, Mrs. K. Ratliffe-Thompson, Mr. M. Slusher, and Mrs. R. Smith have all provided expert advice to me during my career as a graduate student. Additional thanks go to Dr. Carrie Haskell-Luevano's research group as well for valuable discussions and assistance pertaining to solid phase peptide chemistry techniques. Perhaps even more valuable are the many friendships that have developed while working here.


iv









Special thanks go to Mr. Jim Rocca of the UF McKnight Brain Institute (AMRIS) for his guidance with high field NMR studies. His great patience and wonderful teaching ability have allowed me to explore and exploit a very powerful analytical tool. I would also like to thank Mr. D. Plant, Dr. A. Edison, and Dr. C. Zachariah for their help with the NMR instruments as well.

Thanks are also due to Dr. J. V. Johnson of the UF Chemistry Department for his generous assistance with the HPLC-MS experiments. I would also like to thank Mr. S. McClung and Mr. S. McMillan of the UF ICBR Protein Chemistry Core Facility for their help with protein sequencing and MALDITOF-MS.

My parents, Lucio and Carmen Della Vecchia, have also given me much

encouragement and support throughout my graduate studies and my entire life. Their constant prayers and encouragement for me have truly been a blessing. My in-laws, the Honorable Judge Hale and Rebecca Stancil, have also stood behind me and supported me in my studies. Their care and acceptance of me into their fiunily are greatly appreciated. Thanks are also due to my brother, Peter, and sister, Bethany, and my many brothers and sisters in-law for being true friends.

Finally, I wish to thank my wife Stephanie for her love, encouragement, and

patience throughout this learning experience. She has always believed in me, knowing that I would be successful in completing this research project and writing this dissertation. She has inspired me to do my very best and to not give up despite difficult circumstances. I thank God that He has blessed me with such a wonderful wife to join me on life's journey.






V














TABLE OF CONTENTS

p~ge

ACKNOW LEDGMENTS ......................................................................................... iv

LIST OF FIGURES ...................................................................................................... viii

LIST OF ABBREVIATIONS ..................................................................................... xi

ABSTRACT ................................................................................................................. xiv

CHAPTER

1 INTRODUCTION ....................................................................................................... 1

Polyamines .................................................................................................................. I
Hypusine and Eukaryotic Initiation Factor 5A ...................................................... 14
Hypusine and eIF-5A: Functions and Roles ........................................................ 22
Isolation and Identification of Hypusine Dipeptides .............................................. 26
Proposal .................................................................................................................... 29

2 SYNTHESIS I-SOLUTION PHASE .................................................................... 41

Previous Synthetic M ethods for Hypusine ............................................................ 41
Preparation of Hypusine Reagent .......................................................................... 42
Synthesis of Hypusine and Hypusine-containing Dipeptides .................................. 45
Synthesis of 13C-labeled and 3H-labeled Hypusine Reagent .................................. 47
Preparation of Deoxyhypusine Reagent ................................................................. 49

3 SYNTHESIS 1-SOLID PHASE ......................................................................... 57

Solid Phase Peptide Chemistry (SPPS)-General .................................................... 58
Synthesis of eIF-5A Peptide Fragments ................................................................. 67

4 EXPERIMENTAL-SYNTHESIS I AND II ............................................................... 78

General ...................................................................................................................... 78
Synthesis of a-(P-alanyl)- and a-(y-aminobutyryl)-Hypusine Precursors ............... 80
Synthesis of a-(3-alanyl)- and at-(y-aminobutyryl)-Hypusine ................................ 84
Synthesis of 3H-Hypusine ..................................................................................... 88
Solid Phase Peptide Synthesis-Use of Hypusine and Deoxyhypusine Reagents ......... 89


vi








5 ANALYTICAL-HPLC, TISSUE EXTRACTIONS, AND HPLC-MS ..................... 103

HPLC Fluorescence Assay for Hypusine and Hypusine Dipeptides ......................... 103
Use of 3H-Hypusine as a Tracer for Tissue Extraction Method Development ........... 108
Extraction and HPLC Analysis of Rat Brain Tissue for Hypusine and Dipeptides .... 112 HPLC-MS Analysis of Hypusine, Hypusine Dipeptides, and Samples from the Extracts of the Rat Brain Homogenate .............................................................................. 114

6 NMR-ADDITIONAL ANALYSES OF EIF-5A PEPTIDE FRAGMENTS ............. 135

G eneral .................................................................................................................... 135
Analysis of Hypusine Dipeptides ............................................................................. 137
Analysis ofelF-5A Peptide Fragments .................................................................... 139
pH Titration NMR Study of eIF-5A (Hypusineso and 13C Hypusine) 20mers ........... 142
2D Analysis of eF-5A Peptide Fragments ............................................................... 146
D iscussion ............................................................................................................... 151

7 SUMMARY AND CONCLUSIONS ....................................................................... 176

LIST OF REFEREN CES ............................................................................................. 182

BIOGRAPHICAL SKETCH ....................................................................................... 194




























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LIST OF FIGURES



Fige Page

1-1 The Polyam ines ....................................................................................................... 34

1-2 Polyamine Biosynthetic Pathway I-Access to Putrescine.........................................35

1-3 Polyamine Biosynthetic Pathway II-A) dcAdoMet and B) Spermidine and Spermine 36 1-4 Post-Translational Modification of Lysine to Hypusine-A) Hypusine and B)
Hypusine Biosynthesis on elF-5A...................................................................37

1-5 Eukaryotic Initiation Factor 5A Amino Acid Sequence (No Hypusine Modification) 38 1-6 Deoxyhypusine Synthase (DOHS)-Modification of Human elF-5A (Lys5o) .............39

1-7 Hypusine and Hypusine-Dipeptides.........................................................................40

2-1 Synthetic Scheme #1-Synthesis of Hypusine Reagent.............................................51

2-2 Synthetic Scheme #2-Synthesis of Hypusine Dipeptides.........................................52

2-3 Synthetic Scheme #3-Synthesis of 13C Hypusine Reagent.......................................53

2-4 Synthetic Scheme #4-Synthesis of 3H-Hypusine .....................................................54

2-5 Analysis of Compounds During Synthesis-A) TLC for Hypusine Dipeptides and B)
Scintillation Counts for 3H-Hypusine ..............................................................55

2-6 Synthetic Scheme #5-Synthesis of Deoxyhypusine Reagent....................................56

3-1 General FMOC Solid Phase Peptide Synthesis and Reaction Vessel........................70

3-2 FMOC-Leuss5-WANG RESIN..................................................................................71

3-3 FMOC Synthesis-A) Deprotection and B) Activation of FMOC Amino Acids for
SPP S ............................................................................................................... 72

3-4 Amino Acid Coupling in SPPS................................................................................73

3-5 Structures of Coupling Reagents used in SPPS ........................................................74

viii









3-6 Structures of Standard FMOC-protected Amino Acids used in SPPS.......................75

3-7 elF-5A 5-, 8-, and 14-Amino Acid Peptide Fragments.............................................76

3-8 elF-5A 20 and 21 Amino Acid Peptide Fragments with Lysso, DHyps5o, Hyps5o, and
S3C H yps5o ........................................................................................................ 77

5-1 OPA/NAC Derivatization of Primary Amines .........................................................120

5-2 Pre-Column Derivatization of Hypusine, 1-Ala-Hypusine, and GABA-HypusineA) Sample HPLC Chromatogram and B) Standard Curve ...............................121

5-3 Comparing the Reagents-A) OPA/2ME vs. OPA/NAC and B) Time Course of
OPA/N AC Reagent.........................................................................................122

5-4 Elution of 3H-Hypusine through G-25 Sephadex in Relation to A) Blue Dextran and
Phenol Red and B) Excess Protein from Tissue Extract...................................123

5-5 HPLC Chromatogram of Rat Brain Extract--"Suspect Region" ................................. 124

5-6 HPLC Chromatogram of Rat Brain Extract Mixed with Standards (a.k.a "Spike
Experim ent") ..................................................................................................125

5-7 HPLC-MS-UV Chromatogram of OPA/NAC Di-Derivatized Hypusine, O-AlaHypusine, and GABA-Hypusine ..................................................................... 126

5-8 HPLC-MS-(+)ESI-MS of Hypusine after OPA/NAC Precolumn Derivatization.......127

5-9 HPLC-MS--(+)ESI-CID-MS/MS of the [M+H]+ ions of the Hypusine and
D ipeptide Standards ........................................................................................ 128

5-10 Proposed Structures of OPA/NAC Di-Derivatized Compounds after LCMS............129

5-11 Possible Fragmentation Pathway for Di-Derivatized GABA-Hypusine....................130

5-12 Comparison of HPLC-MS Data of A) Hypusine Standard and B) Rat Brain Extract
from Experiment #1 Containing Hypusine "Suspect"......................................131

5-13 Comparison of HPLC-MS Data of A) GABA-Hypusine Standard and B) Rat Brain
Extract from Experiment #1 Containing GABA-Hypusine "Suspect"..............132

5-14 HPLC-MS of Rat Brain Extract Mixed with A) Hypusine and B) GABA-Hypusine
Standards........................................................................................................ 133

5-15 HPLC-MS of Rat Brain Extract with Hypusine Standard.........................................134

6-1 1D 'H NMR of2S, 9R Hypusine (300 MHz)...........................................................159

6-2 1iD 'H NMR (300 MHz) of Hypusine Dipeptides-A) a-(P-alanyl)-Hypusine and B)
a-(y-aminobutyryl)-Hypusine..........................................................................160

ix








6-3 ID 'H NMR (500 MHz, 7 oC) ofelF-5A 5, 8, 14, and 20mer (Hypso) Peptides-A)
Amide (-NH) and Histidine Aromatic Region (9.0-7.0 ppm) and B) a-'H
Region (4.8-3.5 ppm ) ..................................................................................... 161

6-4 ID IH NMR (500 MHz, 7 oC) ofelF-5A 5, 8, 14, and 20mer (Hypso) Peptides-A)
His 3-CH2 and Lys e-CH2 region (3.60-2.80 ppm) and B) M43 and E42 -CH2 Y
(2.85-2.20 ppm )..............................................................................................162

6-5 ID IH NMR (500 MHz, 7 oC) of elF-5A 5, 8, 14, and 20mer (Hypso) Peptides-A)
Aliphatic Region (2.2-1.1 ppm) and B) Terminal Methyl Groups (1.1-0.5
ppm ) ...............................................................................................................163

6-6 ID 'H NMR (500 MHz, 7 oC) of elF-5A 20mer (Hypso)-pH Titration from 2.63 to
7.60 ..................................................................................................................164

6-7 1ID 'H NMR (500 MHz) of elF-5A 20mer (Hypso)-pH Titration Expansions A)
2.75-2.10 ppm and B) Amide/Aromatic Region 8.9-6.8 .................................165

6-8 3C NMR (27 oC) of labeled Hyps5o -CH2 on C11 of Hypusine Side Chain-A) pH
Titration from 2.95-7.10 and B) Coupled 13C Spectrum of C11, pH 2.95 .........166

6-9 13C NMR Spectra Expansions of elF-5A 20mer for (13CHypso), A) 182-170 ppm,
B) 170-160 ppm, C) 140-125ppm, D) 125-110Oppm.......................................167

6-10 13C NMR Spectra Expansions for elF-5A 20mer (Hypso), A) 72-49 ppm, B) 49-30
ppm, C) 30-10, D) 182-10 ppm ......................................................................168

6-11 TOCSY NMR Spectra (500 MHz, 7 oC), Aliphatic and a-'H to Amide Region-A)
5mer (L58ss-A54), B) 8mer (L58ss-Hs51), C) 14mer (L58-K50so-T45).............................169

6-12 TOCSY NMR Spectra (500 MHz, 7 oC), Amide Region-A) 5mer (L58ss-A54), B)
8mer (L58ss-H51), C) 14mer (L58-K50so-T45)...........................................................170

6-13 TOCSY NMR Spectra (500 MHz, 7 oC), Aliphatic and a-'H to Amide Region-A)
20mer (Kso), B) 20mer (DHyps5o), C) 20mer (Hypso) .......................................171

6-14 HMQC NMR Spectra Comparison (500 MHz, 7 oC)-A) and B) 20mer (Lys5o); C)
and D ) 20m er (13CH ypso) ................................................................................. 172

6-15 Comparison of ID 'H NMR Spectra for elF-5A 20mer (Hypso) at 500 MHz and 750
MHz-A) Amide/Aromatic Region 9.2-7.2 and B) a-'H Region 4.9-3.7.........173

6-16 elF-5A 20mer (Hypso)-(A) TOCSY vs. (B) NOESY, 750 MHz, 7 oC ......................174

6-17 Superimposed TOCSY and NOESY for elF-5A 20mer with Hypso (750 MHz,
70C)-Sequence Assignment from NMR Data-a-'H to Amide Region.............175




x














LIST OF ABBREVIATIONS


ADC arginine decarboxylase (EC 4.1.1.19)
[a]25D specific optical rotation at 25 'C for D (sodium) line
AdoMet S-adenosylmethionine
AdoMetDC S-adenosylmethionine decarboxylase (EC 4.1.1.50)
AIDS acquired immunodeficiency syndrome
AIH agmatine iminohydrolase (EC 3.5.3.12)
A1203 alumina, aluminum oxide
aq aqueous
ARD AIDS related diarrhea
Ar argon
P3-ALA 3-alanine
BBO broad-band probe, NMR
BOC tert-butoxycarbonyl
BOP benzotriazol-1 yl-oxy-tris(dimethylamino)phosphonium
hexafluorophosphate
c concentration (g/100 mL), for optical rotation
oC centigrade degree; Celsius degree
C6H6 hexanes
CBz carbobenzoxy
CD3OD deuterated methanol
CH2C12 methylene chloride
CH3CN acetonitrile
CH3OH methanol
CHCl3 chloroform
CHO chinese hamster ovary cells
CO2 carbon dioxide
COOH carboxylic acid functional group
CPM counts per minute
8 (delta) chemical shift
D20 deuterated water
DAO diamine oxidase
dcAdoMet decarboxylated S-adenosylmethionine
DFMO a-difluoromethylornithine
DHyp deoxyhypusine
DIEA or DIPEA diisopropyl ethylamine
DMF dimethylformamide
DNA deoxyribonucleic acid
DOHH deoxyhypusine hydroxylase
DOHS deoxyhypusine synthase

xi








DPM disintegrations per minute
e.g. (exempli gratia) for example
eIF-5A eukaryotic translation initiation factor 5A
EtOAc ethyl acetate
FAD flavin adenine dinucleotide
FMOC 9-fluorenylmethoxycarbonyl
GABA y-aminobutyric acid
h hour
H2 hydrogen
H20 water
HBr hydrogen bromide
HBTU 2-(1H-benzotriazol- 1-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate
HCI hydrogen chloride
HIV human immunodeficiency virus
HMQC heteronuclear multiple quantum correlation
spectroscopy
HOAc acetic acid
HOBt 1-hydroxybenzotriazole
HPLC High performance liquid chromatography
Hyp hypusine
i.e. (id est) that is
KCN potassium cyanide
X (lambda) wavelength
LCMS liquid chromatography mass spectrometry
LiAIH4 lithium aluminum hydride
M molar concentration: moles per liter
MALDITOF-MS matrix assisted laser desorptive time of flight mass
spectrometry
MAO monoamine oxidase
mCi milliCurie
MeOH methanol
2-ME 2-mercaptoethanol
MGBG methylglyoxal bis(guanylhydrazone)
MHz megahertz
mm minute
ml milliliter (cubic centimeter)
mmol millimole (10-3 moles)
mRNA messenger ribonucleic acid
MS (HRMS) mass spectrometry (high resolution mass spectrometry)
N normal (equivalent per liter)
N2 nitrogen
Na2SO4 sodium sulfate
NaH sodium hydride
NaHCO3/Na2CO3 sodium bicarbonate/sodium carbonate
NaOCH3 sodium methoxide
NaOH sodium hydroxide

xii









NCPA N-carbamoylputrescineamidase (EC 3.5.1.53)
(NJI2)2C(NH2) guanidine
NH3 ammonia
NH40H ammonium hydroxide
NMDA N-methyl-D-aspartate
NIMR nuclear magnetic resonance
NOESY nuclear overhauser effect spectroscopy
OD optical density (absorbance)
GDC omithine decarboxylase
GNSu N-hydroxysuccinimide
OPA/NAC o-phthaldialdehyde/N-acetyl-L-cysteine
PAG polyarnine oxidase (EC 1.5.3.11)
PAPT putrescine aminopropyltransferase; sperinidine synthase
(EC 2.5.1.16)
Pd-C palladium carbon
pH acid-base scale: log of reciprocal of hydrogen ion
concentration.
PLP pyridoxal 5'-phosphate
ppm parts per million
PtG2 platinum dioxide
Put putrescine; 1 ,4-butanediamine
PyBOP c0 benzotriazolyloxy-tris[pyrroldino]-phosphonium
hexafluorophosphate
(R) rectus (right) stereodescriptor
Rev viral regulatory protein for FHV
Rf TLC compound migration distance indicator
RRE rev response element, arginine rich domain of rev
protein
rpm revolutions per minute
(5) sinister (left) stereodescriptor
SAG serum amine oxidase
SAPT spermine amninopropyltransferase, spermine synthase
(EC 2.5.1.22)
Spd spermidine; N' -(3-aminopropyl)-1,4-butanediamine
Spin spermine; N', M'-bis(3 -aminopropyl)- 1,4-butanediamine
SPPS solid phase peptide synthesis
SSAT spermidine/spermine N'-acetyltransferase (EC 2.3.1.57)
TFA trifluoroacetic acid
TUiF tetrahydrofuran
TLIP tetrahydropyran
TIS triisopropylsilane
TLC thin-layer chromatography
TMS tetramethylsilane
TOCSY total correlation spectroscopy
TRNA transfer ribonucleic acid
TSP (NaTSP) 3-trimethylsilyipropionate, (sodium salt)
UV ultraviolet


xiii














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND ANALYSIS OF HYPUSINE-CONTAINING DIPEPTIDES AND EUKARYOTIC TRANSLATION INITIATION FACTOR 5A PEPTIDE FRAGMENTS:
ADDITIONAL TOOLS TO STUDY THE AMINO ACID HYPUSINE By

Matthew J. Della Vecchia

August 2002


Chairman: Dr. Raymond J. Bergeron
Major Department: Medicinal Chemistry

Hypusine is an amino acid originally isolated from the extracts of bovine brain in 1971. Currently, this amino acid has been located only on a single protein, eIF-5A. This 154 amino acid protein (human) undergoes a post-translational modification that transforms a specific lysine residue (Lys50, human) to hypusine. Two enzymes, deoxyhypusine synthase and hydroxylase, and the polyamine spermidine are required for this highly conserved modification to occur. Despite its categorization as an initiation factor for protein synthesis, the exact function of eIF-5A is still unclear. However, the lysine to hypusine modification is essential for the protein's full function. Particularly interesting are recent experiments that have implicated eIF-5A's involvement in the progression of HIV. Inhibition of the hypusine modification on eIF-5A causes HIV replication to be reduced, thus providing a potential for a therapeutic outcome.





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In order to understand and determine the functions of hypusine, elF-5A, and its two reported dipeptides, a-(y-aminobutyryl)-hypusine and ao-(P-alanyl)-hypusine, it is imperative that reliable and efficient techniques be developed to synthesize and analyze these compounds. The proper tools to carry out these experiments are lacking as hypusine and deoxyhypusine reagents, hypusine dipeptides, and elF-5A peptide fragments are not commercially available.

This dissertation describes the multi-step synthesis and analysis of hypusine dipeptides and 3H-labeled hypusine as well as the incorporation of hypusine, deoxyhypusine, and 13C-labeled hypusine reagents into eIF-5A peptide fragments using solid phase peptide synthetic techniques. The radiolabeled compound has enabled the development of a new tissue extraction method for the analysis of free hypusine and hypusine dipeptides from rat brain. Extracts were analyzed for the presence of these compounds via precolumn derivatization on HPLC and HPLC-MS systems. EIF-5A peptide fragments containing lysine, deoxyhypusine, hypusine, and 13C-labeled hypusine at position 50 in human eIF-5A have been characterized by ID and 2D high field homonuclear and heteronuclear NMR experiments. The data collected here provides a basis for the future analysis of hypusine and eIF-5A peptides and their interactions with other components of various viral proteins.













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CHAPTER 1
INTRODUCTION

Polyamines

Polyamines are organic compounds containing two or more amine moieties

separated by aliphatic carbon chains of varying length. The three most common naturally occurring polyamines are putrescine (Put; 1,4-butanediamine), spermidine (Spd; N'-(3aminopropyl)-1,4-butanediamine), and spermine (Spm; N, N4-bis(3-aminopropyl)- 1,4butanediamine) (Fig. 1-1). These polyamines are considered ubiquitous since at least one of these compounds has been detected in almost all cells studied, both prokaryotic and eukaryotic, plant and animal (Jainne et al., 1991; Thomas & Thomas, 2001). These low molecular weight bases have been detected in the millimolar concentration range in proliferating mammalian cells (Cohen, 1971; Thomas & Thomas, 2001). Other polyamines, such as cadaverine (1,5-pentanediamine) and 1,3 propanediamine, occur in nature as well and have been identified as polyamine metabolites in human and rat urine (Muskiet et al., 1995).

Polyamine History

Polyamines are considered essential for cell growth and differentiation, and the biosynthetic pathways for the formation of the major polyamines in vivo are well established (Shantz & Pegg, 1999). Despite this fact, most biochemistry courses and textbooks typically offer only minor discussions on this group of compounds. Much less is said regarding their initial discovery. In actuality, there is a vast history to the study of




I





2

polyamines that dates back over three hundred years. The historical summary given below is taken from Cohen (1971).

One of the first scientists to report data related to polyamines was Antoni van Leeuwenhoek. In his letter to the Royal Society of London in 1678, Leeuwenhoek described the gradual formation of colorless crystals in samples of seminal fluid. The French chemist Nicolas Vauquelin also described a similar observation in 1791, apparently unaware of Leeuwenhoek's discovery some 113 years earlier. Nearly a century later in 1878, Schreiner reported that these crystals were a phosphate salt of an organic base. The name spermine was eventually given to this organic base in 1888 but it was not until Rosenheim synthesized spermine and the related polyamine, spermidine, that the correct structure of this compound was finalized in 1926 (Cohen, 1971).

The discovery of putrescine is attributed to Brieger who isolated this compound

from animal tissues in 1885. Ladenburg synthesized putrescine a year later, confirmed its structure, and verified Brieger's work (Cohen, 1971). In the years that followed, more reliable syntheses of the polyamines were developed and studies to determine the exact biological functions and roles of these compounds began. A majority of the research to date has focused on the polyamines as they occur in cells of bacteria, yeast, and mammals. More recently, the role of polyamines in cells of higher plants has also been investigated (Bagni & Tassoni, 2001).

Putrescine, spermidine, and spermine are di-, tri-, and tetraamines respectively. The nature of these compounds, multiple amines separated by carbon backbones, has led to an abbreviated method to denote their structure. The number of carbons between each amine is used to describe each compound. Thus, putrescine can be abbreviated as "4,"





3

while spermidine and spermine are denoted as "3-4" and "3-4-3" respectively (Fig. 1-1). At physiological pH the polyamines are protonated making the compounds di-, tri-, and tetracations respectively as well. This unique polyionic nature is thought to be a main reason why polyamines tend to exhibit a wide range of effects in nature (Janne et al., 1991). Since the 1970's, numerous studies have been published regarding the functions and roles of these organic compounds. Still it is generally considered that their exact functions and roles are yet unknown. Future study in this field is indeed warranted as the research completed to date has shown promise in terms of the potential development of treatments for a variety of debilitating human diseases such as cancer (Marton & Pegg, 1995).

Polyamine Biosynthesis

As stated, the polyamines are necessary for eukaryotic cell growth. Severe depletion of these compounds reduces growth in mammalian cells (Tome & Gerner, 1997). On the other hand, elevated polyamine levels within a cell can be toxic (Marton & Pegg, 1995; Thomas & Thomas, 2001). Cells maintain a balance among the three major polyamines and interconversion can occur in order to maintain this balance. Thus, eukaryotic cells possess a multilevel polyamine regulation system by which the synthesis, catabolism, uptake and excretion of putrescine, spermidine, and spermine can be controlled (Pegg et al., 1995).

The primary carbon and nitrogen sources for putrescine, spermidine, and

spermine are the amino acids L-methionine, L-arginine, and L-ornithine (Jdnne et al., 1991). Several key enzymes work in conjunction with these amino acids to create a polyamine biosynthetic pathway responsible for the production of polyamines within a





4

cell. These enzymes are (1) arginase (EC 3.5.3.1), (2) ornithine decarboxylase (ODC, EC

4.1.1.17), (3) arginine decarboxylase (ADC, EC 4.1.1.19), (4) agmatinase (EC 3.5.3.11),

(5) agmatine iminohydrolase (AIH, EC 3.5.3.12), (6) N-carbamoylputrescine amidase (NCPA, EC 3.5.1.53), (7) S-adenosylmethionine decarboxylase (AdoMetDC, EC 4.1.1.50), (8) spermidine synthase (PAPT, putrescine aminopropyltransferase, EC

2.5.1.16), (9) spermine synthase (SAPT, spermidine aminopropyltransferase, EC

2.5.1.22), (10) spermidine/spermine Ni-acetyltransferase (SSAT, EC 2.3.1.57), and (11) polyamine oxidase (PAO, EC 1.5.3.11) (Morgan, 1998; Bagni & Tassoni, 2001). Though each of these enzymes is important for the overall synthesis ofpolyamines, ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase, spermine synthase, and spermidine/spermine N'-acetyltransferase are considered key. A generalized pathway for the biosynthesis of polyamines is shown in Figures 1-2 and 13a,b (Marton & Pegg, 1995). The pathways for the synthesis of each polyamine and the enzymes that promote these syntheses are discussed below. Formation of Putrescine

In eukaryotes, ornithine decarboxylase (ODC) forms putrescine directly by

removing the -COOH functional group from L-ornithine, releasing CO2 (Fig. 1-2). ODC is a unique enzyme in that compared to most mammalian enzymes whose half-lives are measured in days, it has a relatively short half-life of 10 to 60 minutes depending on species (Russell & Snyder, 1969; Morgan, 1998). ODC, regardless of source, is a dimer of identical subunits and absolutely requires pyridoxal 5'-phosphate (PLP), an electrophilic catalyst, for activity. PLP dependent enzymes form covalent Schiff base intermediates with their substrates, which help to labilize functional groups like -COOH





5

in the case of ODC. The amino acid sequence of this enzyme displays over 90% identity among mammalian species (McCann & Pegg, 1992). This suggests that this enzyme's structure and function are also quite similar among mammalian species. The enzyme's substrate, ornithine, is present in human plasma and is also a product of the urea cycle (Morgan, 1998). Ornithine used for the synthesis of putrescine via ODC may be drawn from these sources.

Mammalian cells and fungi that lack a complete urea cycle may utilize the

enzyme arginase to yield putrescine via a two-step process. Arginase first cleaves the guanidine group from L-arginine to form L-omithine and urea (Fig. 1-2). ODC then converts ornithine to putrescine as described above. In animals, the decarboxylation of omithine is the only possible synthetic route for putrescine, whereas in bacteria and higher plants, both arginase and ODC pathways are involved in the production of putrescine (Bagni & Tossoni, 2001).

Putrescine can also be synthesized from arginine via enzymes different from arginase. These reactions typically occur in bacteria and higher plants, but not mammalian cells (Bagni & Tossoni, 2001). Initially, ADC removes the -COOH functional group from L-arginine to form agmatine (Fig. 1-2). Then, agmatine is hydrolyzed by agmatinase to form putrescine and urea.

In plants, an additional route to putrescine also exists. After agmatine is derived from arginine as described above, it is converted to N-carbamoylputrescine and NH3 by AIH. N-carbamoylputrescine is then hydrolyzed by N-carbamoylputrescine amidase to yield NH3, CO2, and putrescine (Morgan, 1998). Clearly, the synthesis of putrescine follows a rather elaborate scheme that is not limited to a single route. Multiple routes to





6

the formation of this polyamine attest to its requirement in normal cell function among a variety of species.

Formation of Spermidine and Spermine

S-adenosylmethionine (AdoMet) normally serves as a substrate involved in methylation reactions in the biosynthesis of amino acids and in capping procedures during eukaryotic transcription (Stryer, 1995). S-adenosylmethionine can also serve as a substrate for S-adenosylmnethionine decarboxylase (AdoMetDC). AdoMetDC utilizes a covalently bound pyruvate as its prosthetic group instead of PLP (as in the case of ornithine decarboxylase). This enzyme removes the terminal -COOH group from AdoMet to form decarboxylated S-adenosylmethionine (dcAdoMet) (Fig. 1-3a). Once Sadenosylmethionine is decarboxylated, it is committed to a role in polyamine biosynthesis, unable to participate as a substrate in methylation reactions (Shantz & Pegg, 1999). Instead, dcAdoMet becomes the substrate for the next enzyme in the polyamine biosynthetic pathway, putrescine aminopropyltransferase (PAPT), also called spermidine synthase.

The active form of PAPT is a dimer of two identical subunits. This enzyme has also been purified from various microbial, plant and mammalian sources. Unlike ODC and AdoMetDC, spermidine synthase does not require an enzyme cofactor (Pegg et al., 1995). Spermidine synthase is responsible for transferring the aminopropyl group from dcAdoMet to putrescine (Fig. 1-3b). In this manner, spermidine is formed along with 5'methylthioadenosine as a side product.

Finally, spermine synthase (SAPT) transfers an aminopropyl group from an

additional molecule of dcAdoMet to spermidine resulting in the formation of spermine.






7

Like spermidine synthase, spermine synthase also consists of two identical subunits of equal size. Though they catalyze nearly identical reactions and are similar in size, PAPT and SAPT are quite specific in that they require putrescine and spermidine as substrates respectively (Pegg et al., 1995).

Polyamine Catabolism

As stated earlier, cells must maintain a careful balance of each polyamine in order to function properly. Each reaction described above for the formation of each polyamine is essentially irreversible. Thus, an additional set of enzymes is required to convert any of the polyamines back to its precursor(s) in order to maintain proper concentrations. Both spermidine and spermine can be acetylated by spermidine/spermine Aacetyltransferase (SSAT). This enzyme appears to be ubiquitous in mammalian tissues as well (Casero & Pegg, 1993). The nucleotide sequences for this enzyme from human, hamster, and mouse are more than 95% homologous. This homology as well as conservation among various species may indicate the potential importance of this protein in cellular function (Morgan, 1998). Similarly, this homology may indicate a necessary amino acid sequence required for the structure and functionality of this enzyme. SSAT is the rate-limiting step of the polyamine catabolic pathway. Intracellular activity of SSAT increases in response to elevated levels of polyamines within cells (Casero & Pegg, 1993). Induction of SSAT reduces the concentrations of spermidine and spermine within a cell, thus preventing the cytotoxic levels of polyamines from accumulating (Shantz & Pegg, 1999). An acetyl group from acetyl-coenzyme A is transferred to an aminopropyl group of either spermidine or spermine by SSAT (Fig. 1-3b). The acetylated polyamines are then modified further as described below.





8

In 1977, H61ttl used the term "polyamine oxidase" to describe various enzymes that complete the back conversion to spermidine and putrescine by oxidizing N'acetylspermidine or N'-acetylspermidine. Typically, oxidases are enzymes that catalyze oxidation reactions in which molecular oxygen is the electron acceptor but oxygen atoms do not appear in the oxidized product. Seiler designates the oxidases as follows: (1) polyamine oxidase (PAO), (2) diamine oxidase (DAO), (3) serum amine oxidases (SAO),

(4) monoamine oxidase (MAO) (Seiler, 2000). As the acetylated polyamines are cleaved at secondary amino nitrogens, spermidine or putrescine and 3-acetamidoproponal are released (Fig. 1-3b). Polyamine oxidase is a flavin adenine dinucleotide (FAD) dependent enzyme whereas the diamine oxidases utilize Cu(II) as a cofactor (Thomas & Thomas, 2001). The polyamine oxidases primarily act on the N'-acetylated polyamines and spermine. The diamine oxidases catabolize diamines such as putrescine, cadaverine and histamine as well as Spd and Spm. Serum amine oxidase also contains copper and catalyzes the deamination of Spd and Spm. The product of spermidine and spermine catabolism, 3-acetamidoproponal, can be further metabolized to both 3-alanine and yaminobutyric acid (Seiler, 2000).

Polyamines-Functions and Roles

It is quite clear that the path to synthesizing the major polyamines in vivo is

intricate and detailed. An equally intricate pathway exists, complete with its own set of enzymes, in order to catabolize and maintain the required amounts of these polyamines within a cell. Undoubtedly, the polyamines must be required for some unambiguous, definitive function. However, this exact unambiguous function is yet unknown. In





9


addition to being regarded as essential for normal cell growth and proliferation, polyamines have been shown to be involved in numerous biological processes.

Spermidine and spermine are tri- and tetracations respectively at physiological pH. With a net positive charge of three and four, these two polyamines are considered the most cationic small molecules within a cell (Davis et al., 1992). As such, these compounds interact with a variety of anionic molecules and cellular structures. These polyamines bind to polyanionic molecules such as DNA, RNA, and phospholipids (Igarashi et al., 1982). Polyamines have a distributed charge due to their carbon backbones. This spacing may allow polyamines to stabilize the conformation of DNA and guard against denaturation (Marton & Morris, 1987). Putrescine, spermidine and spermine can increase the melting temperature of DNA by as much as 40 'C in low salt buffers (Tabor, 1962; Thomas & Bloomfield, 1984). Polyamines appear to also have roles in embryonic development, the cell cycle, cancer, neurochemistry, and pulmonary and immune system functions (Thomas & Thomas, 2001). Enzyme Inhibition

Researchers have attempted to elucidate the functions of polyamines in a variety of manners, the first of which is through the inhibition of the enzymes involved in the polyamine biosynthetic pathway. Inhibition of the synthetic enzymes leads to decreased concentrations of Put, Spd, and Spm. Inhibition of the enzymes involved in catabolizing the polyamines leads to over accumulation of the polyamines within cells. Developing inhibitors specific for each enzyme in the pathway has proved challenging and has resulted in numerous publications regarding potential polyamine function. Some





10

inhibitors of the polyamine biosynthetic pathway are discussed below and are reviewed elsewhere (Marton & Pegg, 1995).

The most targeted of the enzymes in the pathway, from an antineoplastic

viewpoint, is omithine decarboxylase. The most widely used inhibitor developed to target ODC is a-difluoromethylornithine (DFMO). Inhibition of ODC by DFMO leads to a major reduction in the amounts of putrescine and spermidine, causing a significant reduction in cell growth once spermidine is depleted (Marton & Pegg, 1995). The effects of DFMO are considered cytostatic rather than cytotoxic and clinical studies of this compound on human tumors have proved disappointing (Schecter et al., 1987). This apparent failure of DFMO for the treatment of human tumors is attributed to the residual amounts of spermine that remain in cells despite treatment with DFMO (Marton & Pegg, 1995). Also, due to the short half-life of ODC, cancer-like cells can overcome an attempt to block its activity very quickly (Bergeron et al., 1995a). Polyamine Analogues

Targeting the enzymes involved in the synthesis of polyamines has proved

problematic. Another approach to targeting the polyamine biosynthetic network in hopes of a therapeutic outcome is to develop polyamine analogues. In theory, synthetically prepared polyamine-like compounds could be incorporated into and distributed throughout a cell. If appearing enough like the endogenous polyamines, the synthetic analogues would mimic the natural polyamines within a cell. A cell's production of the natural polyamines would decrease and perhaps shut down completely, in effect killing a tumor. Numerous compounds have been synthesized and tested for their impact on polyamine pools and their effect on the polyamine regulatory enzymes (Bergeron et al.,





11


1995a, 1997a). Extensive studies, including molecular modeling, have also been carried out to determine the relationship between biological activities and structural factors of the polyamines and their analogues. For example, varying the length of the carbon backbone in polyamines changes the distance between nitrogen atoms. In turn, this affects charge distribution within the compound altering its interaction with various anionic compounds in the cell. Furthermore, analogues are also prepared by altering the terminal alkylating groups placed on the polyamines. Terminally dialkylated analogues and homologues of spermine, which exhibit antineoplastic activity against a number of tumor cells lines, both murine and human, have been synthesized in Bergeron's laboratories (Bergeron et al., 1994, 1995a, 1997a). The structural requirement of spermidine in supporting cell proliferation has been well documented. Spermidine homologues with a greater than two-carbon extension compared with spermidine exhibited decreased ability to rescue cells from DFMO treatment (Porter & Bergeron, 1983).

Polyamine analogues have also proved to be potent antidiarrheal compounds

(Sato et al., 1991). In the course of human immunodeficiency virus (HV), many patients experience serious diarrhea (AIDS related diarrhea, ARD). Current treatments involve the application of various antirnotility agents and even hormonal therapy. Polyamines have been demonstrated to have a profound effect on the gastrointestinal tract (Tansy et al., 1982). By optimizing the length of methylene backbones to obtain ideal separation between charged centers and by incorporating additional functional groups to allow additional routes for metabolism, polyamine analogues with reduced toxicity have been developed for use against ARD (Bergeron et al., 1996, 2001).





12

Early studies on the effects of polyamines on whole animals models reported changes in blood pressure and slowing of pulse and respiration depending on the dose and route of administration of the polyamines (Wrede, 1924; Tabor & Tabor, 1956). In addition to studying polyamine analogues as antidiarrheals, Bergeron's group has also assessed polyamine analogues for their effectiveness as antiarrhythmics (Bergeron et al., 1998c). The compounds tested included both polyamine antimetabolites and putrescine mimics. The most active analogues observed were dicationic, tetraamines with pyridine ring substitution on the terminal nitrogens-these compounds were able to reverse the progression of an arrhythmic event induced by isoproterenol in a rat model (Bergeron et al., 1998c).

Polyamines may also play an important role in the central nervous system (CNS). Polyamines can cross the blood-brain barrier in limited quantities but the CNS depends on de novo synthesis of Spd and Spm as well (Seiler, 1997). The brain contains the highest activity of spermine synthase. Spm is attributed to the functions of neurons whereas Spd plays a role in the conservation and formation of myelin-rich layers. Unusual concentrations of polyamines in the brain are found during the course of several diseases such as Alzheimer's and schizophrenia. Gilad et al. (1997) have observed increased levels of polyamines in the brains of rats exposed to external stressors. Several N,N' terminal dialkylated homologues of the tetraamine spermine have been shown to exhibit activity at the N-methyl-D-aspartate (NMDA) receptor-channel complex, a ligandgated ion channel involved in excitatory neurotransmission in the mammalian CNS (Bergeron et al., 1995b). Though limited, information indicating the specific





13

involvement of polyamines in neuropsychiatric disorders exists and further attention to the role ofpolyamines in the CNS is warranted.

The roles of polyamines are indeed numerous and diverse. In some cases, their exact function remains ambiguous. In others, their role is quite well understood. Clearly much insight into the functions and roles of polyamines has been gained over the last few decades. The potential for the development of therapeutic outcomes based on the polyamine biosynthetic network is great and clinical trials testing the effectiveness of polyamine analogues are ongoing (Bergeron et al., 2000). An Unambiguous Role for the Polyamines

Much has been written on the role ofpolyamines and their interactions with DNA, RNA, and protein biosynthesis. A severe depletion in polyamine levels in mammalian cells leads to reduced cell growth and even cell death. This may be due in part to the involvement of polyamines in the posttranslational modification of eukaryotic translation initiation factor 5A (eIF-5A). More specifically, it has been shown that the polyamine spermidine is the sole aminobutyl donor required to modify a specific lysine residue at a single position (Lys5o, human eIF-5A) to the rare amino acid known as hypusine (Fig. 14a). Two enzymes affect this modification: deoxyhypusine synthase and deoxyhypusine hydroxylase. Hypusine is an essential component of the eIF-5A protein and the enzymes involved in the biosynthesis of hypusine have become targets for growth inhibition as well. The remainder of this paper will focus on this unique amino acid hypusine, the only known protein on which this modification occurs (elF-5A), the enzymes that facilitate the modification, and the methods developed in this laboratory to study this biological system more efficiently and effectively.





14

Hypusine and Eukaryotic Initiation Factor 5A Hypusine, a basic amino acid, was first isolated from the homogenates of bovine brain tissue via extraction with trichloroacetic acid followed by extensive ion-exchange chromatography (Shiba et al., 1971). From 200 fresh bovine brains weighing 82.0 kg (obtained from a slaughterhouse), approximately 137 mg of hypusine as the dihydrochloride salt were obtained. The brains were first homogenized in 5-10% trichloroacetic acid in a Waring blender. The homogenates were diluted and centrifuged. Supernatants were filtered through celite and then purified through several ion-exchange columns to isolate basic compounds. Eluates were evaporated to dryness and analyzed by high-voltage paper electrophoresis with ninhydrin development. During this process, an unidentified amino acid was observed. Fractions containing this compound were combined and evaporated to dryness in vacuo. The dried residue was dissolved in 90% methanol and 6 mL of ether. Cooling this solution overnight yielded 105 mg of crystalline material. The mother liquor yielded an additional crop of 32 mg of hypusine (Shiba et al., 1971).

Shiba's group proceeded to analyze this unidentified compound by nuclear

magnetic resonance (NMR), mass spectrometry (MS), chemical degradation experiments, thin layer chromatography (TLC), and amino acid analysis (Shiba et al., 1971). The structure of this compound was then determined and confirmed to be N6-(4-amino-2hydroxy-butyl)-2,6-diaminohexanoic acid. It was given the common name of hypusine because it was composed of both bldroxyputrescine and lysine moieties (Fig. 1-4a). The stereochemistry of this molecule was determined to be S at the C-2 position and R at the C-9 position (Park et al., 1982a; Shiba et al., 1982). The formal nomenclature of hypusine was later revised to (2S, 9R)-2,1 1-diamino-9-hydroxy-7-azaundecanoic acid.






15


Other reports refer to hypusine more simply as N-(4-amino-2-hydroxybutyl)lysine (Park et al., 1981).

Once isolated, the distribution of this amino acid was studied in various organs of rats and in the brains of rabbits and oxen (Nakajima et al., 1971). Hypusine was determined in the brain, liver, kidney, muscle, spleen, heart, intestine, uterus, lung, blood, and skin of 6-month old Wistar rats. Approximately 1-8 nmol/g of hypusine were detected in the various organs. A similar concentration range for hypusine was observed in the central nervous systems of rabbits and oxen. The renal clearance of hypusine in human urine was also reported to be over 100 mL/min (Nakajima et al., 1971). Eukaryotic Translation Initiation Factor (elF) 5A

Protein synthesis requires the coordinated interaction of greater than 100 macromolecules such as tRNA, mRNA, and ribosomes (Stryer, 1995). In general, protein synthesis consists of three stages-initiation, elongation, and termination. Initiation describes the reactions that precede the formation of the peptide bond between the first two amino acids of a protein within a cell. Initiator tRNA binds to the start signal, AUG in eukaryotes, nearest the 5' end of mRNA. Ribosome subunits join together and begin moving along the mRNA in a step-wise manner. Elongation includes the formation of the first peptide bond between the first two amino acids in the protein sequence followed by subsequent peptide bond formations that synthesize the protein in the 5' (N-terminus) to 3' (C-terminus) direction. Termination of protein sequence elongation occurs when a stop signal is recognized along the mRNA. The completed peptide chain is then released from the ribosome.





16

Initiation is considered the rate-determining step for protein synthesis. Eukaryotic initiation factors (elFs) regulate the initiation process and assist with the joining of the 60S and 40S ribosome subunits to form an 80S particle along mRNA. Numerous initiation factors are required during the first stage of protein synthesis. When one or more initiation factors are absent or inhibited, initiation of protein synthesis may not occur or will occur to a much lesser extent.

Eukaryotic translation initiation factor (elF) 5A is an initiation factor that has the unique distinction of undergoing a post-translational modification that is required for its own activity and for cell proliferation. This modification involves the formation of a unique amino acid known as hypusine exclusively at the Lysso residue in human elF-5A precursor (Fig. 1-4b). The polyamine spermidine is required for this conversion of lysine to hypusine. An enzyme known as deoxyhypusine synthase catalyzes the coupling of the 4-aminobutyl group from spermidine to the s-amino group of the Lysso residue to form a deoxyhypusine residue. This is followed by hydroxylation of the deoxyhypusine residue by deoxyhypusine hydroxylase to form the fully functional elF-5A. Discovery of elF-5A

Eukaryotic initiation factor 5A is an 18 kDa protein. This initiation factor has been found in both eukaryotic cells and archaea, but not in eubacteria (Chen & Jao, 1999). The amino acid sequence of this protein is highly conserved from yeast to humans. The 12 amino acids closest to the hypusine modification site are highly conserved among various species as well: Ser-Thr-Ser-Lys-Thr-Gly-Hypusine-His-GlyHis-Ala-Lys (Park et al., 1997). The human version of this protein has 154 amino acids in its sequence (Fig. 1-5). First isolated from rabbit reticulocyte proteins, elF-5A was





17


categorized as initiation factor M2Ba (Kemper et al., 1976). In 1983, this protein was rediscovered and renamed eIF-4D. It was found to be identical to IF-M2Ba (Cooper et al., 1983). The human cDNA for this protein was cloned in 1989 and the protein was again renamed eIF-5A (Smit-McBride et al., 1989).

In 1981, the metabolic labeling of an 18 kDa protein by radioactive spermidine or putrescine was observed (Park et al., 1981; Chen & Liu, 1981). Researchers incubated Chinese hamster ovary (CHO) cells with 3H-spermidine or [terminal methylenes3H]spermidine and found that the radioactivity was incorporated into one cellular protein (Park et al., 1981). It was determined that this labeling occurred as a result of spermidine acting as an aminobutyl donor in the post-translational modification of a particular protein, which converted a single lysine to the unique amino acid hypusine. This protein, called Hy+ (indicating the presence of the amino acid hypusine), was also isolated and purified from human lymphocytes and Chinese hamster ovary fibroblasts. It was found that the Hy" protein exhibited electrophoretic properties identical to those of eIF-4D purified from rabbit reticulocytes (Cooper et al., 1983). The Biosynthesis of Hypusine Requires Two Key Enzymes: DOHS and DOHH

As stated, the post-translational modification of a single lysine residue to

hypusine requires the function of two key enzymes: deoxyhypusine synthase (DOHS, E.C. 1.5.1.-) and deoxyhypusine hydroxylase (DOHH, E.C. 1.14.99.29). Park's group

first described the biosynthesis of hypusine as a sequential process (Park et al., 1982a). CHO cells were incubated with [4,5-3H]lysine. After acid hydrolysis of these cells, a small portion of the radioactivity of the cellular protein faction chromatographed to the same position as unlabeled hypusine. This indicated that lysine was a precursor for





18

hypusine formation. A similar experiment was carried out in which CHO cells were incubated with either tritium labeled lysine or [terminal methylenes-3H]spermidine and a metal chelator, a, a-dipyridyl. After digestion of these cells, the label was found to be incorporated into a protein-bound material with chromatographic properties different than those of hypusine. This material was determined to be the unhydroxylated form of hypusine or deoxyhypusine, NV-(4-aminobutyl)lysine (Park et al., 1982b). Analysis of the deoxyhypusine-containing protein by 2D-electrophoresis provided evidence that this protein was identical to that of the hypusine-containing protein. Park used this experimental evidence to propose that the formation of hypusine occurred in the following manner: peptide-bound lysine + spermidine -) peptide-bound deoxyhypusine

- peptide-bound hypusine. Park's group also postulated that a metal-requiring enzyme catalyzed the hydroxylation of deoxyhypusine since hypusine formation was inhibited by the presence of a chelating agent and the effects of this chelating agent were reversed upon addition of ferrous sulfate (Park et al., 1982b). Deoxyhypusine Synthase

Soon after the above scheme for hypusine formation on a single protein was

proposed, the enzymes responsible for this post-translational modification were studied. Deoxyhypusine synthase (DOHS) is the enzyme responsible for the first step in hypusine biosynthesis. Approximately 40-43 kDa in size depending on species, DOHS is an NAD+/NADH dependent enzyme (Chen & Dou, 1988). This enzyme has been purified to homogeneity by substrate elution affinity chromatography from Neurospora crassa (Chen & Tao, 1995). DOHS has also been purified and characterized from rat testis and HeLa cells by ammonium sulfate fractionation and ion exchange chromatography (Wolff





19

et al., 1990; Klier et al., 1995). The enzyme is a homotetramer and its amino acid sequence appears to be highly conserved, especially towards the C-terminal region (Chen & Jao, 1999).

The reaction by which deoxyhypusine synthase converts lysine on precursor eIF5A to deoxyhypusine can be expressed in four steps (Fig. 1-6). First, the enzyme and its NAD+ cofactor catalyze the dehydrogenation and oxidative cleavage of spermidine (Wolff et al., 1990). This is followed by the formation of an enzyme-imine intermediate and 1,3-diaminopropane as a side product. The iminobutyl group is then transferred from the enzyme-imine intermediate to the lysine residue on eIF-5A. Lastly, the imino group is reduced to form the deoxyhypusine residue on eIF-5A. As Lys50 in the human elF-5A protein has been identified as the sole residue to undergo this modification to date, Lys329 in the human deoxyhypusine synthase enzyme has been identified as the key residue involved in the enzyme-substrate intermediate discussed above (Joe et al., 1997). When Lys329 of the enzyme was substituted with alanine or arginine, the enzyme-substrate intermediate formation and DOHS activity was completely eliminated. Mutations at nine other conserved lysine residues within the enzyme (Lys 141, 156, 205,212, 226, 251 and 338) did not affect enzyme activity in the same manner as mutations at Lys329. It was determined that Lys329 is located at the active center of the enzyme near the NAD-binding site and in the middle of the predicted spermidine-binding pocket thus enabling it to accept the aminobutyl group (Joe et al., 1997). Recently, the crystal structure of the human form of deoxyhypusine synthase with bound NAD cofactor has been reported (Liao et al., 1998). The crystal structure observed in this study shows that the entrance to the active site is blocked by a two-turn a-helix. This suggests that the reaction steps





20

described above are accompanied by significant conformational changes in the enzyme (Liao et al., 1998). Undoubtedly, the crystal form of the enzyme is likely to be in quite a different conformation than it would normally be in solution or in vivo. A significant conformation change may occur immediately upon solvation of this enzyme prior to exposure to immature elF-5A or NAD+. The establishment of a basis of knowledge on the elF-5A protein in its solution phase is warranted and therefore pursued in this study. Deoxyhypusine Hydroxylase

Deoxyhypusine hydroxylase (DOHH) is the second enzyme involved in the

biosynthesis of hypusine on eIF-5A. It is responsible for adding a hydroxyl functional group in the R configuration to the C-9 position of the deoxyhypusine residue on the elF5A intermediate. To date, this enzyme has not been purified to homogeneity. In fact, the study of this particular enzyme has proved to be much more problematic than that of DOHS. The main difficulty in the development of a purification method for this enzyme is the fact that a convenient assay to monitor the hydroxylation reaction is lacking (Chen & Jao, 1999). DOHH has only been partially purified from the homogenates of rat testis (Abbruzzese et al., 1986). Adding to the problem is the fact that the partially purified DOHH does not act on the free amino acid, deoxyhypusine. So, there is currently no synthetic substrate other than deoxyhypusine-containing eIF-5A for this enzyme to work on.

Abbruzzese et al. have proposed several indirect methods for the monitoring of DOHH activity. One method relies on the measurement of tritiated water formed as the deoxyhypusine hydroxylase enzyme displaces a tritium at the C-9 position of deoxyhypusine. Another method requires a dual-labeled protein substrate-both 14C and





21

H are incorporated into the side chain of deoxyhypusine by incubating CHO cells with both 1,4-14C putrescine and 2,3-3H putrescine. This is a rather creative method. Theoretically, as DOHH acts upon the dual-labeled substrate, one fourth of the tritium is released whereas all of the 14C is retained. The degree of hydroxylation can be estimated from the change in the ratio of 3H to 14C in the protein fraction (Abbruzzese et al., 1988). Still, these methods do not lend themselves to convenience and reliability. A dually labeled compound may also create cross contamination problems and 3H:14C ratios may not be accurate.

More recently, an assay for deoxyhypusine hydroxylase was developed using HeLa cells incubated with either 3H-spermidine or 14C-spermidine and a metal chelator (Csonga et al., 1996). Metal chelators prevented DOHH from completing the hydroxylation of deoxyhypusine on eIF-5A and in the process provided deoxyhypusinecontaining eIF-5A, the natural substrate to form hypusine-containing eIF-5A. In the process, the dependency of this enzyme on a metal ion, Fe3+, was determined. Hypusinecontaining eIF-5A can be oxidized by periodate to yield lysine-containing-elF-5A (elF5A precursor), formaldehyde and P-aminopropionaldehyde. These aldehydes can then be extracted and measured for their radioactivity. Oxidation will theoretically only occur in the presence of the hypusine containing eIF-5A (-OH at C-9) and therefore the radiolabeled byproducts of this oxidation will be proportional to the activity of DOHH. Others are attempting to refine this technique in order to fully purify deoxyhypusine hydroxylase (Chen & Jao, 1999). At the time of this writing, no further progress has been reported regarding the full purification and characterization of deoxyhypusine hydroxylase.





22

Hypusine and elF-5A: Functions and Roles How important is hypusine? What role does eIF-5A play? Clearly these are important questions. It should be obvious that this unique amino acid, protein, and the enzymes that synthesize them play a key role in nature. Two enzymes have been devoted to the process of producing this unique amino acid on a single residue of a single protein. The highly conserved nature of the amino acid sequence in the protein also suggests a vital cellular role. Like the polyamines, the exact role for free and protein-bound hypusine and its parent protein are as of yet unknown. Also like the polyamines, numerous studies have been conducted to elucidate the functions of these biological molecules. The main pathway taken to determine the functions of hypusine and eIF-5A has been that of developing inhibitors of the two key enzymes involved in the formation of hypusine-DOHS and DOHH. In this manner, much insight has been gained into the functions and roles of hypusine and eIF-5A. In the same manner, these same studies have made solving the puzzle of hypusine and eIF-5A more difficult as well.

Hypusine and the hypusine-containing eIF-5A protein are essential in cell

proliferation (Park et al., 1993, 1997). Early on, a correlation was observed between hypusine synthesis and cell growth. Hypusine was shown to be essential for the in vitro activity of eIF-5A in the stimulation of methionyl-puromycin synthesis (Kemper et al., 1976). This in vitro study mimics the formation of the first peptide bond in protein synthesis. However, another study showed that protein synthesis in yeast continued even after introducing a gene variant that depleted eIF-5A (Kang et al., 1993). Studies have shown that preventing the synthesis of mature, hypusine-containing eIF-5A by blocking the synthesis of its aminobutyl donor spermidine also leads to the arrest of growth in eukaryotic cells (Park et al., 1997). Hypusine synthesis is suppressed in cells where





23

spermidine has been depleted and, upon replenishment of spermidine or a suitable spermidine analogue, hypusine synthesis is dependent upon the amount of spermidine or analogue that has been supplemented (Marton & Pegg, 1995; Tome & Gemer, 1997).

Despite the fact that the exact cellular function(s) of eIF-5A are still unknown, it is clear that cellular growth is adversely affected in its absence and in the absence of its hypusine residue. Studies involving S. cerevisiae show that total protein synthesis is only reduced to 30% of normal levels in eIF-5A deficient cells (Kang & Hershey, 1994). This evidence suggests that eIF-5A and hypusine are not required solely for protein synthesis. In any case, there is an undeniable link between polyamines, eIF-5A, and hypusine and hypusine is absolutely required for eIF-5A to be completely functional, whatever function that may be.

Several inhibitors of the enzymes, as well as analogues of the substrates, involved in the biosynthesis of hypusine on eIF-5A have been employed in order to gather more information on the function of these molecules. A number of spermidine analogues have been synthesized as described above in hopes of discovering an effective antineoplastic drug. Several inhibitors of the polyamine biosynthetic pathway have also been proposed (e.g. DFMO). It is possible that these analogues and inhibitors function as antiproliferatives due to the fact that by depleting the natural source of spermidine, the aminobutyl donor required for hypusine synthesis on eIF-5A is also depleted.

The most potent spermidine analogue reported thus far is N'-guanyl-l,7diaminoheptane (GC7), which causes growth arrest of mammalian and archaea cells (Jakus et al., 1993; Jansson et al., 2000). It is thought that this compound competes with spermidine for DOHS. Metal chelators have also been used as inhibitors of





24

deoxyhypusine hydroxylase. Compounds like mimosine (3-(N-(3-hydroxypyridin-4one))-2[S]-aminopropionic acid) reportedly cause an arrest in cell proliferation at the Gl/S boundary of the cell cycle (Hanauske-Abel et al., 1995). Blocking S-phase entry interferes with the onset of DNA replication and therefore hints that eIF-5A may play a role in this manner. Studies such as these are complicated by the fact that chelators such as mimosine have exhibited other cellular functions such as interfering with DNA synthesis by inhibiting deoxyribonucleotide metabolism (Gilbert et al., 1995). Methylglyoxal bis(guanylhydrazone) (MGBG) is also a reported inhibitor of AdoMetDc (Marton & Pegg, 1995). This inhibitor prevents the decarboxylation of Sadenosylmethionine therefore cutting off the supply of dcAdoMet required for the synthesis of spermidine. Elimination of spermidine can prevent the formation of hypusine by removing the aminobutyl donor source.

It should be noted that the inhibitors used to study the hypusine modification on eIF-5A lack specificity, like mimosine, and exhibit general toxicity as well, like DFMO, MGBG, and GC7. Caution should be taken when interpreting the results obtained to date, as when examined individually they may not tell the complete story of eIF-5A and hypusine. Undoubtedly, better tools are required to study this biological system. The crystal structure of DOHS obtained by Liao's group in 1998 should provide a template for the development of more specific inhibitors of this enzyme. Tools developed in Bergeron's laboratory, to be discussed later in this paper, should also aid in the elucidation of the functions and roles of hypusine and eIF-5A.

The decrease or shutdown of cellular proliferation as a result of the prevention of eIF-5A/hypusine formation has encouraged further study of this biological system as a





25

target for antineoplastic development. Other studies have linked hypusine and eIF-5A function to human immunodeficiency virus type 1 (HIV-1). EIF-5A has been shown to interact with Rev, a viral regulatory protein essential for HIV-1I replication (Ruhl et al., 1993). Rev functions by activating the nuclear export of unspliced (intron-containing) viral mRNAs (Pollard & Malim, 1998). Rev is a 13 kDa RNA binding protein. It contains at least three functional domains: an arginine-rich domain, a nuclear export signal (NES), and a homomultimerization domain (Hope, 1999). The arginine-rich domain of the Rev protein allows it to bind specifically to a 240-base region of complex RNA secondary structure called the Rev response element (RRE). RNA gel mobility shift assays have indicated the interaction between RRE RNA and both deoxyhypusineand hypusine-containing eIF-5A protein (Liu et al., 1997). No interaction is observed with the unmodified, lysine-containing eIF-5A protein. From this observation, it is clear that eIF-5A may function as a cellular cofactor involved in the recognition and nuclear export of certain mRNA in addition to its potential role in protein synthesis (Park et al., 1997). From this observation, a potential antiviral therapeutic window also arises. If the interaction of mature eIF-5A, Rev, and RRE can be prevented, it is conceivable that HIV replication can be halted. Indeed, mutant forms of eIF-5A that form a complex with Rev and RRE were reported to inhibit HIV- 1 replication in human lymphocyte cell lines (Bevec et al., 1996). Several studies have characterized the interactions between Rev and RRE by NMR (Peterson, et al., 1994, 1996; Battiste, et al., 1995, 1996). To date, no studies have examined the interactions between Rev and RRE with elF-5A by NMR. Future study regarding eIF-5A and hypusine function is warranted as it offers a potential antiviral therapeutic outcome.





26

Isolation and Identification of Hypusine Dipeptides

GABA or y-aminobutyric acid is an abundant constituent of the brain. It is the

major inhibitory neurotransmitter in the mammalian central nervous system (Siegal et al., 1994). GABA meets the five classical criteria for assignment as a neurotransmitter: (1) it is present in the nerve terminal, (2) it is released from electrically stimulated neurons,

(3) there is a mechanism for reuptake of the released neurotransmitter, (4) its application to target neurons mimics the action of inhibitory nerve stimulation, and (5) specific receptors for GABA exist (Siegal et al., 1994). The effects of this neurotransmitter are widespread. A variety of evidence suggests a role for altered GABA function in several neurological and psychiatric disorders of humans such as epilepsy, schizophrenia, sleep disorders, and Parkinson's disease. Also, recall that GABA and P-alanine are terminal catabolites of polyamine metabolism as well (Morgan et al., 1998). a-(y-aminobutyryl)-hypusine

In light of the fact that both GABA and hypusine are abundant in the brain, there should be ample opportunity (and enzymes no doubt) to couple the two compounds together to form a dipeptide. A novel GABA-containing peptide was isolated from bovine brain and determined to be a-(y-aminobutyryl)-hypusine, or GABA-hypusine (Fig. 1-7) (Sano et al., 1986). Bovine brains (1.2 kg) obtained from slaughterhouses were dissected, boiled in water and then homogenized and extracted with trichloroacetic acid. Extracts were centrifuged and supernatants were filtered through a series of ion exchange columns in order to isolate basic aliphatic amino acids. High-voltage paper electrophoresis, TLC, and HPLC techniques were used to examine column eluates. Recall that the amino acid hypusine was isolated and identified in a similar manner by





27

this same research group. Fractions collected ater hypusine was detected yielded an unknown compound. Two ninhydrin positive substances were observed when the acid hydrolysate of this unknown was examined by electrophoresis and TLC. The TLC Rf values in three different solvent systems were the same as those for hypusine and GABA. In a similar manner, migration distances in high-voltage electrophoresis matched those for hypusine and GABA as well. Analysis of the acid hydrolysate of the unknown compound by HPLC indicated approximately equimolar amounts of GABA and hypusine. It was determined by HPLC that approximately 0.9 pmol of a-(yaminobutyryl)-hypusine was isolated from 1.2 kg (0.75 nmol/g) of bovine brain. The structure of the isolated dipeptide was verified by comparison with a synthetic version of GABA-hypusine. Accounting for loses due to purification, the authors estimated the actual concentration of GABA-hypusine in bovine brain to be > 1 nmol/g.

Further study by this same group determined that GABA-hypusine has a

distribution restricted to the brain (Sano et al., 1987). The peptide was detected in the brain tissues of the rat, rabbit, ox, and monkey. The dipeptide was not detected in the brain of the dog or in a specimen of cat brain. Concentration of the dipeptide in the various brain tissues ranged from 1.3 to 9.5 nmol/g wet weight (Sano et al., 1987). a-(13-alanyl)-hypusine

-alanine has also been isolated in the form of a hypusine dipeptide: a-(P3alanyl)-hypusine (Fig. 1-7) (Ueno et al., 1991). The same group responsible for the isolation of hypusine and GABA-hypusine accomplished this work using a procedure similar to that described above. Approximately 1 pimol of this dipeptide was isolated from 1090g of bovine brain. Two ninhydrin positive substances were also produced upon





28

acid hydrolysis of this compound. The two substances were determined to be P-alanine and hypusine by TLC, HPLC, and high-voltage electrophoresis analysis.

Several dipeptides containing an )-amino acid and a basic amino acid have been identified and divided into two groups: those that contain GABA and a basic amino acid and those that contain P-alanine and a basic amino acid. In addition to GABA-hypusine discussed above, the following GABA containing dipeptides have been found in mammalian brain: homocarnosine (or GABA-histidine), homoanserine (or GABA-1methylhistidine), GABA-lysine and GABA-cystathionine. In addition to a-(1-alanyl)hypusine discussed above, the following 1-alanine containing dipeptides have been found in mammalian brain: carnosine (a-(P-alanyl)histidine), anserine (a-(P-alanyl)-lmethylhistidine), and a-(P-alanyl)lysine.

The function of these dipeptides is not known. All that is known is that these compounds are found in excitatory tissues such as nervous system and muscle. Carnosine is rich in olfactory neurons and is considered to have a neurotransmitter role in the olfactory system (Sano et al., 1986). An enzyme known as carnosine synthetase was shown to catalyze the synthesis of dipeptides containing both e0-amino acids and basic amino acids (Sano et al., 1987).

The isolation and identification of the two hypusine dipeptides discussed above has currently only been accomplished by a single group of researchers. Their last published report regarding these compounds was published over 10 years ago (Ueno et al., 1991). There is the distinct possibility that many more of these hypusine-containing peptides have yet to be isolated. There also remains the possibility that these hypusine dipeptides discovered were actually artifacts of the extraction process used. In other





29

words, it is possible that GABA- and P-ALA-hypusine are dipeptide sequences from yet another hypusine-containing protein. Clearly, more work in this area is merited and better synthetic and analytical tools are required to accomplish this task.

Proposal

In order to understand and determine the functions of hypusine, eIF-5A, and the novel dipeptides, a-(y-aminobutyryl)-hypusine and ca-(P-alanyl)-hypusine, it is imperative that reliable and efficient techniques be developed to synthesize and analyze these compounds. The proper tools to carry out these experiments are lacking. Hypusine, deoxyhypusine, hypusine dipeptides, and eIF-5A peptide fragments cannot be purchased from commercial sources. Additional syntheses and analyses of these compounds are required. In this study, the synthesis and analysis of hypusine dipeptides, 3H-hypusine, and the incorporation of hypusine, deoxyhypusine, and 3C labeled hypusine reagents into elF-5A peptide fragments has been explored. A new tissue extraction method for the analysis of free hypusine and hypusine dipeptides from rat brain has also been developed. Finally the complete characterization of elF-5A peptide fragments containing lysine, deoxyhypusine, hypusine, and 13C labeled hypusine (position 50 in human elF-5A) by both ID and 2D 'H and 13C NMR experiments were completed in order to establish a basis of NMR data for the 20 amino acid region of elF5A immediately surrounding the lysine to hypusine modification site.

A synthetic scheme reported by Bergeron for the preparation of a hypusine

reagent has been used as the basis for the synthesis of the hypusine-containing dipeptides described in this study (Bergeron et al., 1997b, 1998a). This synthetic scheme is quite versatile due to the presence of orthogonal protecting groups. Thus, the fully protected





30

dipeptide was achieved via the selective removal of a BOC protecting group from the hypusine reagent (Gibson et al., 1994) followed by the coupling of BOC-GABA or BOC1-alanine to the free N' amine. Protecting groups were removed with TFA and TIS and the compounds were purified via ion exchange column. The compounds a-(yamniinobutyryl)-hypusine and a-(P-alanyl)-hypusine have been synthesized and characterized by TLC, MS, optical rotation and NMR. To date, only a brief mention of a synthetic scheme to access GABA-hypusine in low yields (5%) has been reported (Sano et al., 1986, 1987). In addition, no synthetic scheme for the synthesis of a-(13-alanyl)hypusine has been described prior to this. The synthetic scheme described in Chapter 2 allows access to the GABA- and a-(-alanyl)-hypusine dipeptides from Bergeron's hypusine reagent with a 46-48% overall yield. In addition to increased yields of these compounds, the natural stereochemistry of the hypusine moiety (2S, 9R) is maintained throughout the synthesis as a result of steps to prevent racemization from occurring (Bergeron et al., 1998a).

Another hindrance to the study of hypusine and hypusine containing dipeptides is the lack of a routine pre-column derivatization HPLC assay to detect these compounds. Synthetic routes to these compounds provide standards to use for the development of such an assay. The use of OPA/NAC as a precolumn derivatization agent for the routine separation of hypusine, o-(P3-alanyl)-hypusine, and y-(aminobutyryl)-hypusine has been explored and compounds can be routinely detected below 0.5 nmol (,ex = 340 nm, )em = 440 nm). The successful development of an HPLC analytical technique to detect these standards can then be adapted for the routine analysis of these compounds in animal





31

tissues, such as rat brain. The precolumn derivatization of hypusine and its dipeptides has also been successfully adapted to an HPLC-MS system (Chapter 5).

To be able to routinely assay for hypusine and its dipeptides in the free form (nonprotein bound) in animal tissues such as rat brain, an efficient tissue extraction method must also be developed. It is important to subject the tissues to conditions that will only extract the free form of the compounds and not hydrolyze them from their parent proteins (i.e. hypusine from eIF-5A). Key to this method is the availability of a tracer compound that can be used to develop and gauge the efficiency of the extraction method. Again the versatility of the hypusine reagent synthetic scheme described in Chapter 2 lends itself to the incorporation of a tritium label into the hypusine molecule. The 31-2 gas reduction of a terminal nitrile will incorporate a tritium label into the hypusine molecule at the C-I 1 position. The use of this novel 3H-hypusine as a tracer for the development of an efficient tissue extraction method for free hypusine and hypusine dipeptides has been explored (Chapter 5). Tissues were homogenized and extracted with 0.1 M NaHCO3/Na2CO3 buffer (pH 10.7) followed by centrifugation and neutralization of the supernatant. The extract was then lyophilized followed by purification through G-25 sephadex before HPLC analysis. Combining the extraction method developed using the 3H-hypusine tracer compound with the OPA/NAC IPLC assay has allowed for the detection of compounds in rat brain tissue that coelute with both hypusine and GABAhypusine standards. HPLC-MS analysis was able to elucidate the identity of one of the suspect peaks as hypusine.

In addition, the same synthetic scheme used to access the dipeptides and 3Hhypusine is also versatile enough to allow the incorporation of a 13C label into the





32

hypusine reagent at the C- 11 position (Chapters 2 & 4). These hypusine reagents were then used in solid phase peptide synthesis (SPPS). Prior to this dissertation, hypusine reagents have been successfully incorporated into hexapeptides (Bergeron et al., 1997b, 1998a). Using FMOC solid phase peptide chemistry synthetic techniques, hypusine and deoxyhypusine reagents, as well as a 13C labeled hypusine reagent were successfully incorporated into 20 and 21 amino acid peptide sequences matching the hypusinecontaining region of the human eIF-5A protein, Cys3s-Leu5s (Chapter 3). Furthermore, these peptides have been characterized by HPLC, MALDITOF-MS, and amino acid sequencing. Each peptide synthesized has a purity ranging from 85-99% and no deletion sequences were detected.

Both ID and 2D NMR studies ('H and 13C) on eIF-5A peptide fragments have been completed as well (Chapter 6). The integrity of the 13C labeled reagent was assessed via 13C NMR experiments. The presence of a '3C labeled carbon within a large peptide was easily identified in the NMR spectrum. Other NMR techniques such as TOCSY (Total Correlation Spectroscopy), NOESY (Nuclear Overhauser Effect Spectroscopy), and HMQC (Heteronuclear Multiple Quantum Correlation spectroscopy) were used to help assign the NMR spectrum of each eIF-5A peptide fragment as fully as possible. The synthesis and analysis of these eIF-5A peptide fragments may provide a more detailed understanding of the amino acid region surrounding the hypusine modification site in the eIF-5A protein. Prior to this dissertation, no NMR data for hypusine-containing eIF-5A peptides up to 20 amino acids in length has been acquired.

The hypusine reagent scheme described by Bergeron is indeed a powerful

synthetic tool. This study has capitalized on that versatility in order to access previously





33

unavailable synthetic compounds and develop new analytical techniques for their analysis. The ability to synthesize, analyze, and detect hypusine containing compounds will indeed provide additional, necessary tools that will aid the complete understanding of the functions and roles of the unique amino acid hypusine, its dipeptides, and the single protein on which hypusine is formed via a post-translational modification, eIF-5A.





34







H2N NH2
putrescine 1,4-butanediarnine 11411





H2N---- N __, NH2
H

spennidine Nl-(3-aminopropyl)-1,4-butanediamine t'341#

H
H2N N
H

spermine N', N4-bis(3-aminopropyl)-1,4-butanediamine 1134-311




HX' NH2 H2N'-' NH2
1,3-propanediamine cadaverine
1,5-pentanediamine



Figure 1-1. The Polyamines






35







NH

H2N 'K N OH
H
NH2
L-arginine


ARGINNE DECARBOXYLASE ARGEVASE
(ADQ

C02 urea



NH 0

H2N ) N ^., NH2 H2N OH
H NH2


agmatine L-omithine



A GAM TINE ORIVITHINE
IM17VOHYDROLASE (AIH) AGAMTINASE DECARBOXYL4SE (ODC)





NH, \urea f C02

H2N NH2

putrescine

0

H2N ) N
H

N-carbamoylputrescine i -, A C02 + NH3
N-CARBAWYLPUTRESUVE AMDASE (NCPA)



Figure 1-2. Polyarnine Biosynthetic Pathway I-Access to Putrescine







36





0
s OH Mt

NH2 N N 0
s .112
+ OH
Nf* N
ATP
A S-adenosybnethionine s
Pi + ppi H (AdoMet) N +
OH On
Dwaftxytated S-adenosylmethiDnine H (doMoMet
OH 00





H2N N H2 0 0
H'k N'k
putrescine Fig. 1-2 H
B P04- 3-Acetamidopropanal
-*-Mkk- oxtd=
,S) (PAM:
W2 drAdM*t H

v HA" N N
H
N Nl-acetylspermidine 0


0 ;0 s )e-oMmfe-w
T-methylthioadenine NH2 AceO4 Co-A

HX' N
H 3-Acetamidopropanal
spermidine
P04
ddm
SyW- (,VAP n. H H
&AdvA*i H2N--' N N-_ N
H
5'-methylthioadenine Nl-acetylspermine 0



Sp-mdbW4-mhw H rmy),
H2N"" N &eoiCo-A
H

spermine



Figure 1-3. Polyarnine Biosynthetic Pathway H-A) dcAdoMet and B) Spermidine and Spermine







37







NH2
12
H2N 7 5 3 2 1 OH
N
H
A
6H 0



""; drox ytUtre sdne IyNINE





NH2


B

H2N, HN


DEOXYHYPUSINE NH-HI!S5j GLYS2--, SYNTHAS")OHS) N' A N
H2N .. ..... ............. N.#*,,,, NH2 9'
e-NH2 Lysint5o H DEOXYHYPUSINE,
(human cIF-5A precursor) (umnature cIF-5A)
SPERMIDINE (amino butyl donor)




NH2


HO DEOXYHYPUSINE
HYDROXYLASE (DOHH)
HN




'THR4&-GLY49 N" NH-HISSI-GLY52
H

HYPUSENE,
(biologic&W active form of cW-5A)




Figure 1-4. Post-Translational Modification of Lysine to Hypusine-A) Hypusine and B) Hypusine Biosynthesis on eIF-5A








38


















-W m .0 10, v le, -,n" -0,



tA tn -C,< Z W -C L4
IN.



CA A W Od Q a 000 Q CP x Q Q 0 LA M -C A za
= hf n u u ..4,4 - > > > :>
ca CAM a 00 00 >. .11 -C &4 Oc .4 -CC



W L9 z
a 94 U W rd a a a u
C. 0A 0 m x = IH x 0


x Ic > > cr ":a
NN m -Musa 44 XXMM-;bl>&bWXW%- to;* t. i. Q w Ed
0
> > > -.Z.4 X X 26
.4.4 06 .4
b4 Do -tafA ;01
P t-;g W. 54 .00aaWw
0 0 U bc U W W uw
o
Ic Oc b, W 61 A 0 0 0 fa .0

ra bA a a cl a w %4 C4
>>> .......... 3.3 tk. IL -:P- -'-t
-CjUfAfA(JGXUUZ 00 -C -C -C -C:A w -.4 r 0 (D
ld 9- -C 001 X0A.
W ad le 0 .4014 al: Cw COW M -Ut-49
sc -C 0.4 M .3 X=
mc 3c W Id ca X bo
ie lu
0 04

Mtn W !* Vl 4
a W SC

. . . . . .
to CA -C cn m f. Z fe. f. k. C4 ck P i. tr)

vc $Cj Ok 0
0 X x cp 0,E- I- C) C) = I% M E 0
z m N la = = ; -3 =14 Cc
nip E> lb. 26 ak 3c m

bhm
n r
ri tw (Lip 94 ad aeifrX X






a a K ri
tn w r-a ul w Ic x a a W to le w .)
A. A . . . 0
a Ozf oot coin

rl4A- u -ijacli at v 4a 0 u v t4 .j
c -iQ
bqvqmjjf I tf)
u 1) 0) X L$ td ; v
64 >. N Q
'cl tA
Cn Ki

to






39






H2N' H H2N" N4'
., NH2 NHI

DOHS-NADH
spermidine (2) \ HA".' NH2


Deoxyhypusine Synthase (DOHS)
NAD if 1, 3 diaminopropane

DOHS-Lys329-N4' NHI
NADH



H (3)
el[MA-Lys5o
(4) DOHS-NADH

eIF-5A-Lys5o--eN, NF







( NH2
eIF-5A-LyS50-N
H
Deoxyhypusine eIF-5A intermediate I NAD+ cofactor catalyzes the dehydrogenation and oxidative cleavage of spermidine
2 Formation of an enzyme-imine intermediate and 1,3-diaminopropane side product 3 Transfer of iminobutyl group from the intermediate to the lysine residue on eIF-5A
4 Reduction to form the deoxyhypusine residue on elF-5A (Joe et al., 1997)



Figure 1-6. Deoxyhypusine Synthase (DOHS)-Modification of Human eIF-5A (Lys5o)






40










NH2
H2N H
H C jl WN 0
OH 0 WK i? 2b.31

Hypusine

0; NH2 HN
7 C H NO
H2N OH Wi. Wi.1384.39
6 H--H 0

a-(P-alanyl)-hypusine


NH2


HN c 0
7 41H
H2N OH it.! 3 t8.41
6 H^*
H 0

a-(y-aminobutyryl)-hypusine


Figure 1-7. Hypusine and Hypusine-Dipeptides












CHAPTER 2
SYNTHESIS I-SOLUTION PHASE

Previous Synthetic Methods for Hypusine The importance of obtaining a synthetic route to hypusine in order to study this unique amino acid more carefully has been established in the previous chapter. One initial reported synthesis of this amino acid was based on the coupling of L-lysine and 4amino-1-bromo-2-butanol with the secondary alcohol component being derived from malic acid (Shiba et al., 1982). Both isomers of hypusine, (2S, 9R) and (2S, 9S), were prepared as crystalline dihydrochloride salts in this manner. It was determined that (2S, 9R)-2,11-diamino-9-hydroxy-7-azaundecanoic acid, or (2S, 9R) hypusine, was identical with naturally occurring hypusine in all respects (Shiba et al., 1982).

In response to the growing interest in hypusine and its origin, Tice and Ganem

(1983a) reported the preparation of functionalized hydroxyputrescine-aldehyde adducts to use in the synthesis of hypusine. Furthermore, Tice and Ganem (1983b) also reported an additional synthesis of hypusine after retrosynthetic analysis of the compound suggested that the coupling of the N7-C8 bond might be prepared via reductive amination of a cyclic aldehyde with a protected form of lysine. Also, the use of dibenzyltriazones as amino protecting groups and a D-asparagine amino derivative were demonstrated to be useful in the syntheses of hypusine, deoxyhypusine, spermidine, and spermidine analogues (Knapp et al., 1992).

Hypusine was obtained in low yields by Shiba's pathway (Shiba et al., 1982).

Ganem's method required the cumbersome separation of diastereomers (Tice & Ganem,


41





42

1983b). These hindrances prompted Bergeron's group to explore alternative methodologies that would allow easier access to hypusine and related compounds. An initial strategy focused on the design of an electrophilic 4-amino-2-hydroxybutane synthetic reagent that would allow the necessary four carbon extension on the lysine residue while simultaneously providing a hydroxyl group and a terminal amino group (i.e. hydroxyputrescine moiety) (Bergeron et al., 1993). Commercially available (R) and

(S) epichlorohydrins were used as chiral epicyanohydrin precursors in this study. Reaction of the epichlorohydrins with an Ne-benzyl-Na-carbobenzoxy-L-lysine benzyl ester yielded the (2S, 9S) chlorohydrin and oxirane in 75% and 8% yield respectively. The chloride ion of the chlorohydrin compound was then displaced by cyanide. This was followed by the simultaneous reduction of the nitrile and removal of the benzyl- and carbobenzyloxy-protecting groups via hydrogenation with PtO2 (catalyst) in acetic acid. Neutralization gave the hypusine freebase and reacidification yielded (2S, 9R)-(+)hypusine dihydrochloride for an overall 53% yield (Bergeron et al., 1993).

Preparation of Hypusine Reagent

In order to study hypusine in a peptide environment, particularly within an elF-5A peptide sequence surrounding the Lyss5o-Hypso (human elF-5A) modification site, a useful "hypusine reagent" is required. This reagent would also have orthogonal protecting groups, which would allow the amino acid to be coupled to adjacent residues in a peptide sequence and at the same time protect the hypusine side chain from undergoing modification during the coupling process. These protecting groups should also be readily removed under conditions that will result in the maximum yield of the unprotected peptide while avoiding undesirable cleavage of the peptide bonds. Additionally, the ideal





43

hypusine reagent would be suitable for both traditional solution phase peptide chemistry as well as more recent solid phase peptide synthetic methodologies, including both manual and automated techniques.

Successful preparation of hypusine with relatively good yields led to the

development of both hypusine and deoxyhypusine reagents that could be incorporated into peptide syntheses (Bergeron et al., 1997a, 1998a). The synthesis described below (Bergeron et al., 1998a) and displayed in Figure 2-1 is the basis for the preparation of additional hypusine and deoxyhypusine containing peptides to be discussed in Chapter 3. More detailed synthetic data (i.e. reagent amounts, yields, temperature conditions, etc.) for the precursors prepared in this study via the schemes presented by Bergeron are given in Chapter 4.

The first step in the synthesis of the hypusine reagent was the protection of the Na of commercially available Ne-CBZ-L-lysine tert-butyl ester (BACHEM Bioscience Inc.) with di-tert-butyl dicarbonate in aqueous NaHCO3 and chloroform (Fig. 2-1). The fully orthogonally protected lysine reagent (1) was prepared from this biphasic reaction. The BOC, CBz, and O-t-butyl protecting groups now present on this molecule can be selectively removed under distinctive conditions without affecting the integrity of the other protecting groups. The Ne-CBZ group was then removed via hydrogenation with Pd-C catalyst (2) followed by the coupling of the now free N.- of the lysine compound with (S)-(+)-epichlorohydrin in cyclohexane. The precipitated product was filtered and purified to obtain 3. Benzyl chloroformate was then condensed with the chlorohydrin (3) to yield the N -CBZ protected compound (4). The carbamate linkage at this point in the synthesis prevents azetidinium formation (which can cause racemization at C-9) that was





44

observed during the initial scale-up of the hypusine reagent synthesis (Bergeron et al., 1998a). Potassium cyanide in the presence of 18-crown-6 was used to incorporate an additional carbon and nitrogen into the compound (5). The 18-crown-6 reagent forms a complex with KCN allowing the cyano group to act as a nucleophile and displace the chlorine atom on the chlorohydrin compound (Cook et al., 1974). Though not shown in the scheme presented, compound 5 can be converted to a Mosher ester in order to verify chiral integrity at the C-9 position (Dale & Mosher, 1973). Resonances for the 9R and 9S methoxy groups resulting from the Mosher reaction are easily observed and distinguishable via 'H NMR (Bergeron et al., 1998a).

The nitrile (5, non-Mosher ester) was then reduced with H2 (g) and a mixed

catalyst of Pd-C and PtO2 to give 6. Since hydrogenation also removes CBZ protection on N-7 and creates a free terminal amine on N-12, both amines were reprotected with N(benzyloxycarbonyloxy)succinimide (CBZ-ONSu) to yield 7. The addition of trifluoroacetic acid (TFA) and triethylsilane (as a scavenger) removed both the BOC and t-butyl ester protecting groups leaving the di-CBZ amino acid, 8. A tetrahydropyran (THP) ester was formed at the C-9 position via addition of 3,4-dihydro-2H-pyran to the secondary alcohol (9). This was done in order to prevent the nucleophilic reactivity of the hydroxyl group during the coupling of the hypusine reagent during solution or solid phase peptide synthesis. Finally, the Na of the reagent was protected with a 9fluorenylmethoxycarbonyl (FMOC) group by reacting 9 with a solution of 9fluorenylmethyl N-succinimidyl carbonate in DMF and 9% Na2CO3 (10). This fully protected hypusine reagent (10) was then utilized in the solid phase peptide synthesis of hypusine-containing eIF-5A peptide fragments discussed in Chapter 3.





45

Synthesis of Hypusine and Hypusine-containing Dipeptides

Due to the fact that in recent studies hypusine has been routinely isolated using

methods such as acid hydrolysis of proteins (Bergeron et al., 1998b), there is concern that its original isolation from bovine brains using trichloroacetic acid extracts may not have yielded free hypusine at all, but simply hypusine cleaved from elF-5A. Complicating the matter further, the same group that performed the original isolation of hypusine has also reported the isolation of two hypusine-containing dipeptides: a-(y-aminobutyryl)hypusine and a-(P-alanyl)-hypusine (Sano et al., 1986, 1987; Ueno et al., 1991).

In order to explore this matter further, certain tools are required. The main tool required is a hypusine standard. A reliable, reproducible synthetic scheme is essential to access such a standard. The versatility of the hypusine reagent scheme developed by Bergeron, and described in detail above, was capitalized upon in order to prepare hypusine and the two hypusine-containing dipeptides--a-(P-alanyl)-hypusine and a-(yaminobutyryl)-hypusine (Fig. 2-2).

Hypusine. (2S, 9R) Hypusine was prepared according to the scheme described for the preparation of the hypusine reagent in Figure 2-1. Compounds 1-7 were synthesized according to the method of Bergeron (1998a) described above. Once the nitrile was reduced to give compound 6, TFA and triisopropylsilane (TIS) were added to remove the BOC and t-butyl ester protecting groups. The compound was then purified by ion exchange chromatography to yield hypusine as the dihydrochloride salt.

The Dipeptides. Compounds 1-7 were synthesized according to the method of

Bergeron (1998a) described above. Using a method modified from a procedure described by Gibson (1994), compound 12 was prepared by the selective removal of a BOC





46

protecting group in the presence of CBZ and t-butyl ester protecting groups (Fig. 2-2). First, hydrochloride gas was carefully bubbled into dry ethyl acetate for approximately 30 minutes followed by dilution with additional dry ethyl acetate to produce approximately I M HCI in ethyl acetate. (A freshly opened bottle of analytical grade ethyl acetate was used; ethyl acetate was further dried by eluting through a small column of alumina directly into the flask where HC1 gas was introduced). The di-CBZ, BOC, O-t-butyl protected hypusine reagent (7) was then dissolved in the freshly prepared 1 M HCI in ethyl acetate and stirred at room temperature under nitrogen. The appearance of the free amine was monitored by TLC (both UV and ninhydrin development, 9:1 CHC3/MeOH). Compound 12 was obtained in 64% yield. Approximately 80% yields were obtained when compounds 13 and 14 were synthesized by coupling BOC-3-alanine-OH and BOCy-aminobutyric acid to compound 12 using the PyBOP coupling reagent (Fig. 3-5) in methylene chloride (Coste et al., 1990). The coupling reaction was also monitored by TLC for the disappearance of starting materials and the emergence of fully protected dipeptide (1:5 C6HdEtOAc). PyBOP was utilized due to its description as a "safe, nontoxic alternative" to the widely used BOP reagent (Coste et al., 1990). Compounds 13 and 14 were individually deprotected via the addition of 30% HBr/HOAc and trifluoroacetic acid in the presence of pentamethylbenzene and phenol as cation scavengers. Extraction of the scavengers with methyl-t-butyl ether followed by concentration of the aqueous acetic acid layer yielded 15 and 16 (crude) as a mixture of trifluoroacetic acid and acetic acid salts. These compounds were converted to hydrochloride salts by eluting through an ion-exchange column with 0.1 to 6 N HC1. The collected fractions were then pooled and concentrated to yield 15 and 16 as hydrochloride





47

salts (clear, light yellow oils, >90% yields). ('H and 13C NMR spectra, optical rotation, HRMS data were obtained for compounds 12-16). Rf values on TLC (Fig. 2-5a) agreed quite well with those presented in previous reports (Sano et al., 1986, 1987; Ueno et al., 1991). NMR data for the dipeptides will be presented and discussed in Chapters 4 and 6. These dipeptides, along with their hypusine precursor, were then utilized as standards for the development of an HPLC and LCMS analytical assay (Chapter 5).

Synthesis of 13C-labeled and 3H-labeled Hypusine Reagent

The hypusine reagent synthetic scheme is versatile enough to also allow various labels to be incorporated into the hypusine molecule. This is of great importance as the presence of a label would allow a researcher to follow the hypusine compound in various tracer and metabolism studies as well as enhanced NMR experiments.
13C-labeled Hypusine Reagent. The formation of the hypusine reagent requires the addition of KCN and 18-crown-6 in order to integrate an additional carbon and terminal amine to the hypusine backbone (compound 5, Fig. 2-1). Quite simply, commercially available K13CN (Sigma-Aldrich) can be substituted into this synthesis (Fig. 2-3) and the 13C-labeled hypusine reagent can be prepared as described above (5a10a). The uniform incorporation of this label can be easily assessed by 13C-NMR. These NMR experiments will be discussed in greater detail in Chapter 6. A portion of the 13Clabeled hypusine reagent described here was utilized in the solid phase peptide synthesis of 13C hypusine-containing eIF-5A peptide fragments discussed below (Chapter 3).

3H-labeled Hypusine. A radioactive label can also be incorporated into the hypusine reagent synthetic scheme. The incorporation of nitrile functionality into the hypusine molecule was subsequently followed by a reduction with 112 (g) in the presence of mixed catalysts Pd-C and PtO2 in acetic acid to give 6 (Fig. 2-1). Alternatively, 3H2





48

gas can be used to reduce the nitrile to a terminal amine and in the process incorporate two non-exchangeable tritium atoms at the C-II position (Fig. 2-4).

A portion of compound 5 was reduced with 3H2 (g) in the presence of mixed catalysts, Pd-C and PtO2 in acetic acid, at Moravek Biochemicals (Brea, CA). As a result, compound 6b with a 3H label on C-II was obtained (approximately 5 mL of 1.0 mCi/mL; 649.2 mg/mL in ethanol). Radiochemical purity as determined by TLC (1:4:4 NH4OH/CH2CI2/MeOH) was 98% and specific activity was 600 mCi/mmol. In Bergeron's laboratories, compound 6 was prepared by reduction with non-radioactive H2

(g) and mixed catalysts PtO2 and Pd-C in acetic acid (slightly yellow oil, -68 % yield).

Due to the elevated level of radioactivity of the hypusine precursor, 50 mg of the unlabeled compound 6 was added to 1.5 mL of the radiolabeled 6b. This "cold dilution"l process resulted in a specific activity of 16 mCi/mmol (almost a 38 fold dilution from the stock solution). The solution containing both radiolabeled and unlabeled compound 6/6b was concentrated via rotary evaporation followed by azeotropic removal of excess ethanol with methylene chloride. Then, additional methylene chloride was added and the reaction flask was cooled in an ice bath. TFA was added to the flask and the mixture was allowed to stir under N2 (g) atmosphere for approximately one hour at room temperature. The mixture was again concentrated to dryness and then cooled in an ice bath. TFA was again added and the solution was allowed to stir at room temperature under a N2 (g) atmosphere for approximately three hours. TLC analysis of the reaction mixture was used to determine when all starting material had been fully deprotected (2:1:1 MeOHICH2C12/NH4OH). Again, the contents of the flask were concentrated to dryness followed by methylene chloride washes. The radioactive residue was then carefully





49

purified via flash chromatography (15 x 2.5 cm, flash silica, 2:1:1 methanol, methylene chloride, ammonium hydroxide).

As compound 18 eluted from the column, fractions were collected and analyzed by thin layer chromatography and scintillation counting. In this manner, the radiolabeled product was observed to co-elute with the unlabeled product. Figure 2-5b depicts how the scintillation counts complimented TLC analysis of the fractions collected. The strongest levels of radioactivity (CPM) were detected in the same fractions containing the compound with an Rf identical to that of the hypusine standard. Fractions 16-45 were transferred to a tared flask and concentrated to dryness followed by three 15 mL additions of methylene chloride to remove any excess methanol and ammonia. The product, a colorless oil, was then acidified via microliter additions of 0.1 N HCI (aq), transferred in

1 mL portions to small, brown glass vials, and stored in the freezer. A 3H-labeled hypusine reagent of the same form as compound 10 has not yet been prepared. However, it is clear that the syntheses described here could easily be adapted to obtain a radiolabeled hypusine reagent. The 3H-labeled hypusine compound was used as a tracer compound for the development of tissue extraction assays described in Chapter 5.

Preparation of Deoxyhypusine Reagent
The preparation of the deoxyhypusine reagent by Bergeron et al. (1998a) followed a scheme analogous to that discussed above for the hypusine reagent. Of course, the only difference in the structure of the final reagent is the lack of the hydroxyl group at C-9. First, following the method of Sunimoto, the Na-BOC-tert-butyl ester of L-lysine is reacted with 3-cyanopropanal in benzene over molecular sieves as shown in Figure 2-6 (Sumimoto & Kobayashi, 1966). This condensation reaction was followed by a reduction





50

with H2 (g) and PtO2 catalyst to obtain the deoxyhypusine backbone. An additional reduction using H2 (g) and a mixed catalyst (Pd-C/PtO2) in acetic acid was required to convert the terminal nitrile to a terminal amine. The N-7 and N-12 amines were then protected via reaction with CBZ-ONSu, as for the hypusine reagent, to form the di-CBZ, BOC, O-t-butyl protected deoxyhypusine reagent. To allow this reagent to be incorporated into solid phase peptide synthesis, TFA and TIS were added to remove the BOC and t-butyl protecting groups. This was followed by the addition of FMOC-ONSu to protect the alpha nitrogen atom. A portion of the deoxyhypusine reagent (19) described here was utilized in the solid phase peptide synthesis of deoxyhypusinecontaining eIF-5A peptide fragments discussed in Chapter 3.







51






tM2 HA

CBZ-HN t-Bu
0
Ne-CBZ-L4ysine t-butyl ester hydrochloride


a

NH-BOC NH-80C
If C
R-HN 0-t-Bu 11- 0---i"'N 0-t-Bu
0 OH 0

b R = CBZ, I d R = K 3:(9S)
F-R=H-HCI,2 R = CBZ, 4: (9S)

e


NH-8OC NH-BOC
f
R-HN 0-t-Bu NC--r- N 0-t-BU

OH R 0 OH CBZ 0


C R = H 2 HOAc, 6: (9R)
R = CBZ, 7: (9R) 5:(9R)




h


NH2 NH-R
CBZ-HN,_,,,,_,-,, OH CBZ-HN,_,,^,,,,-, OH

H 6BZ 0 THpO CBZ 0
8:(9R)
j R = K 9:(9R)
E R = FMOC, 10: (9R)






Reagents: (a) (BOQ20, NaHC03; (b) H2, Pd-C; (c) (S)-(+)- epichlorohydrin; (d) benzyl chloroformate,
triethylamine; (e) KCN, 18-aown-6; (1) H2, Pd-C, PtO2; (g) CBZ-ONSU, KHC03; (h) TFA, triethYlSdarr.
CH2Cl2; (i) 3,44hydro-2H-pyran; 0) FMOC-ONSu, Na2CO3
Bergeron et al., 1998a



Figure 2-1. Synthetic Scheme #I-Synthesis of Hypusine Reagent






52








NH-BOC
7
CBZ-HN,_,-N,'^ 'N --., 04-BU 7: 9R

OH CBZ 0





NH2
CBZ-HN 0-t-Bu 12..9R

OH CBZ 0


b
0
if HN R

CBZ-HN R = -CH2C"2NH-BOC, 13
R = -CH2CH2CH2NH-BOC, 14 OH CBZ 0


c


0 0

HN) NH2 HN- 1 141-12
OH
N N
H H
15H 0 OH 0

15: a-(P-alanyl)-hypusine 3 HCI 16: a+-arninobutyryl)-hypusine 3 HCI




Reagents: (a) IM HCI in ethyl acetate; (b) PyBOP, BOC-04anine or BOC-GABA, diisopropylethylamine, CH2C'2; (C) 30*/* HBr/Acetic acid, trifluoroacetic acid, phenol, pentarnethylbenzene followed by ion exchange w/ 0. 1 to 6 N HCI


Figure 2-2. Synthetic Scheme #2-Synthesis of Hypusine Dipeptides






53












NH-BOC
CI~N wtOt u 4:(9S)
OH CBZ O0





K- 13CN, 18-crown-6






UH-BOC
O-t-Bu5a: (9R), OH CBZ 0C C1



Scheme 1 (Fig. 2-1)



NH-FMOC
CBZ-HN,13 yOH 108: (9R)

THP6 CBZ 0


13 C labeled potassiumn cyanide is used to obtain the 13 C hypusine reagent Figure 2-3. Synthetic Scheme #3-Synthesis of 13 C Hypusine Reagent






54






!UH-BOC

O-t-Bu 4: (9S)
OH CBZ



KCN, 18-crown-6





NH-BOC

N N 0t-BU S:(9R)
OH 6BZO


3HPd-C, PtO2,HOAC




NH-BOO 6b: (9R), RHNY .N O-t-Bu R =H -2 HOAc
3H2 OH 0



TFA


NIH2
H2N OH
3H2 5OH 0 18:(9R), 3H@C11


3 2gas is used for the mixed catalyst reduction of the nitrite Figure 2-4. Synthetic Scheme #4-Synthesis of 3 H-Jlypusine






55






Citric acid 2:21
HCUNaOK/H20 CH2CIAMOWNH40H

I Ikk








pp

LOK K,91 0
to # 1TIALM,
Rf ;.36 0.30 0.28
Rt 0.23 0.20 0.19

4 .........

H Hoy H y Hoy

H: Hypusine, 0: P-Ala-hypusine, y: GABA-hypusine, Hoy: co-spot



Monkoring 3"pUSIne in Flash (SHica) Column Fmctions
8.00E+07 1211 M9OH/CN2CIVWH40Hj
7.OOE+07 FrwdowV CPM Toed EMin!m
16 686 1.098 Vwdon
20 4268 6,829x 107
6.OOE+07 25 4398 T021.107
30 39% 6 _-M x 107
M 5.WE+07 35 1414 2Xa x IC
IL 40 544 8.704.lLt
IM 4.OOE+07 45 31 62ODx le

3.OOE+07 2.OOE+07 1.OOE+07 O.OOE+00




Figure 2-5. Analysis of Cornpounds During Synthesis-A) TLC for Hypusine Dipeptides and B) Scintillation Counts for 3 H-Hypusine






56






NH2 HCI
NC,-,--Y H CBZ-HN -t-Bu

0 0
3-cyanoproponal N-CBZ-L-lysine t-butyl ester hydrochloride


ab


NH-BOC
0-t-Bu

6BZ 0





1 tIH-BOC
R-HN 04 Bu R=H- HOAc
N d R=CBZ
R 0






NH2
CBZ-HN OH

CBZ 0





NH-FMOC CBZ-HN,_,--_,--, N OH 19

6BZ 0





Reagents: (a) benzene, molecular sieves; (b) H2, PtO2, THF; (c) H2, Pd-C, Pt02; (d) CBZ-ONSU, KHC03; (e)
TFA, triethytsilane, CH2CI2; (1) FMOC-ONSu, Na2CO3
Bergeron et at, 1998a

Figure 2-6. Synthetic Scheme #5-Synthesis of Deoxyhypusine Reagent












CHAPTER 3
SYNTHESIS HI-SOLID PHASE

In order to more completely understand the mechanisms by which hypusine is formed on elF-5A and the roles this amino acid and protein play in nature, it is key to develop routine access to these compounds synthetically. The synthesis of hypusine, hypusine dipeptides, and various labeled hypusine compounds has been described in Chapter 2. This chapter focuses on solid phase peptide synthetic techniques and the incorporation of the hypusine, deoxyhypusine, and 13C-hypusine reagents into peptide fragments matching the sequence of the elF-5A protein surrounding the Lysso-Hyp5o modification site (Figures 1-5, 3-7, and 3-8).

The synthesis of hypusine-containing peptides up to 21 amino acids in length is described below. The successful preparation of such peptides demonstrates the utility of the various hypusine reagents. To date, the hypusine and deoxyhypusine reagents have only been incorporated into hexapeptides (Bergeron et al., 1998a). The "3C-hypusine reagent was previously untested. The successful incorporation of these reagents into larger peptides via solid phase peptide synthesis described below displays the potential for preparing the entire elF-5A protein synthetically in its three naturally occurring stages: Lysineso (elF-5A precursor), Deoxyhypusines5o (elF-5A intermediate), and Hypusine50 (mature, fully functional elF-5A). The availability of such proteins would provide the necessary substrates for which to study the two key enzymes known to be responsible for the post-translation modification of elF-5A: deoxyhypusine synthase (DOHS) and deoxyhypusine hydroxylase (DOHH). Furthermore, analysis of the 20


57





58

amino acid peptide fragments by various techniques such as high field NMR may help to define the hypusine modification site more fully and provide an NMR fingerprint of this post-translational modification site as well. This information is advantageous for future experiments that would attempt to observe the interactions of these peptides with other viral proteins such as Rev and the Rev Response Element in HIV.

Solid Phase Peptide Chemistry (SPPS)-General In 1963, Merrifield introduced solid phase peptide synthesis (SPPS). His idea was to develop methodologies that would enhance the field of peptide chemistry and facilitate the synthesis of previously unattainable compounds. SPPS demonstrated the first practical use of an insoluble polymer with bound reagents in organic synthesis (Stewart & Young, 1984). Merrifield linked the C-terminal residue of the peptide to be synthesized to an insoluble polymer. Through a series of deprotection and coupling steps, the peptide was synthesized one residue at a time from the C- to N-terminus. Numerous papers have been published using SPPS as the means to synthesize a variety of peptides of varying lengths. Solid phase peptide synthesis has been reviewed extensively elsewhere (Stewart & Young, 1984; Fields & Noble, 1990; Chan & White, 2000; Pennington & Dunn, 1994). This paper will focus on the use of Fmoc SPPS only.

Fmoc SPPS refers to the use of Na-Fmoc protected amino acids during the

synthesis of the peptide. The Fmoc group is labile to mildly basic conditions (i.e. 20% piperidine in DMF) whereas the side-chain protecting groups are typically labile to acidic conditions (i.e. TFA) as is the resin linker. The hypusine and deoxyhypusine reagents discussed earlier are well suited for use in Fmoc SPPS and their incorporation into eIF5A peptide fragments is discussed below.






59




Reaction Vessel

The reaction vessel used for SPPS is a glass cylinder that contains a glass frit at the bottom leading to a three-way valve (Fig. 3 -1). This valve allows for the introduction of an inert gas such as N2 from beneath the frit to allow gentle mixing of the resins and reagents when in the vessel. When reactions are being stirred by the gas flow, they are said to be "bubbling". When reactions have gone to completion, excess reagents and solvents are drained through the frit into a waste container leaving the peptide bound to the resin inside the reaction vessel. The unique design allows SPPS to be a "one-pot" method.

Prior to the synthesis of the various peptides described below, the reaction vessels were acid washed and silanized. This is a necessary step prior to SPPS because many peptides, especially those that exhibit basic and hydrophobic characteristics, adhere to the surfaces of the glassware. Silanization of the glass reaction vessel prevents this from occurring and facilitates the SPPS procedure (Stewart &Young, 1984).

Silanization. First, concentrated 112S04 was added to the reaction vessel and allowed to "bubble" with N2 for at least one hour. The acid was then drained and the reaction vessel was rinsed with copious amounts of distilled water. The vessel was then dried in an oven. After drying and returning to room temperature, 10% dichlorodimethylsilane in dry toluene was added to the reaction vessel and allowed to bubble with N2 for one hour. After draining the silanization solution, the reaction vessel was immediately rinsed three times with excess dry toluene. Following this, the vessel was filled with dry methanol and allowed to bubble with N2 for 15 minutes. The





60

methanol was then drained and the vessel was rinsed with copious amounts of methanol (three washes) followed by acetone (three washes). The reaction vessel was then thoroughly dried in an oven overnight (personal communication, Dr. Haskell-Luevano, University of Florida).

FMOC Chemistry and Wang Resin

The hypusine reagents were designed for use in standard Fmoc solid phase

peptide synthesis. Thus, it was necessary to choose an Fmoc-amino acid substituted resin suitable for this type of chemistry so that these novel reagents could be incorporated into larger peptides. Wang resin, considered one of the standard supports used in Fmoc SPPS, was developed by Wang in 1973 and was chosen for the syntheses of all peptide acids described in this dissertation (Fig. 3-2). The resin consists of 1% divinylbenzene beads, 100-200 mesh, onto which an acid-labile linker, p-hydroxybenzyl alcohol has been attached (Wang, 1973). Wang resin was originally prepared by allowing Merrifield's resin to react with either methyl-4-hydroxybenzoate and NaOCH3 followed by LiAI4 reduction or 4-hydroxybenzyl alcohol and NaOCH3 to form p-hydroxybenzyl alcohol substituted resin. The p-hydroxybenzyl alcohol linker portion of the resin can then be attached to the first M'-Fmoc protected amino acid in the desired peptide sequence (Wang, 1973).

Fmoc-Leu-Wang resin. In order to synthesize peptides containing the amino acid sequence surrounding the Lyss0 residue for post-translational modification to hypusine in eIF-5A, Fmoc-Leu-Wang Resin (Leu5s in the human eIF-5A peptide sequence) with a 0.36 mmol/g substitution (1.0 g resin = 0.127 g FMOC-Leu + 0.873 g resin) was purchased from Peptides International (Fig. 3-2). Beginning with Leu5g





61

allowed the syntheses of peptides containing eight and up to 12 amino acids respectively on either side of the Lys5o modification site. Higher resin efficiency was available for purchase but was avoided due to earlier experiences of peptide aggregation, difficult couplings, and cross-linking known to occur during solid phase peptide synthesis at higher resin substitutions (Pennington & Dunn, 1994).

The powder-like FMOC-Leu-Wang resin (1.0 g) was placed into the reaction

vessel and solvated or "swelled" in 20 mL of dry distilled DMF with N2 bubbling for at least three hours prior to the first deprotection and coupling cycle. A worksheet was prepared so as to standardize each step and minimize errors in the SPPS method (Stewart & Young, 1984; Haskell-Luevano, personal communication). FMOC-deprotection

The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group developed by Carpino and Han (1970, 1972) was designed to mask primary amines, rendering them inactive as nucleophiles. This protecting group is desirable in a multi-step synthesis as well as SPPS because it exhibits acid stability. Other protecting groups such as BOC and benzyl-based groups can be cleaved in its presence thus adding Fmoc to the list of orthogonal protecting groups. Only under basic conditions can the Fmoc be cleaved to reveal a free, terminal amine (Greene & Wuts, 1999).

The fluorene ring system of the Fmoc protecting group exhibits an electron withdrawing effect on the hydrogen on the 3-carbon. This causes the hydrogen to be acidic and therefore readily removed by a base such a piperidine (pKa 11.22) (Carpino & Han, 1970). The products of this reaction are a DMF-soluble dibenzofulvene (which may form an adduct with piperidine), carbon dioxide, and the free amine (Fig. 3-3a). The





62

absence of acid in this reaction ensures that the resultant amine is indeed free (Bodansky, 1993). Other deprotection methods, such as the use of trifluoroacetic acid for the removal of BOC protecting groups, generate a protonated amine. This protonated amine must then be converted to the free amine prior to coupling. Deprotection of the Fmoc group with a base such as piperidine, and followed by simply rinsing the resin with excess DMF, generates the free amine directly making it immediately available for peptide coupling.

In this laboratory, the Fmoc protecting group was removed from the amino terminus of the resin-bound peptide in the following manner. After the resin was properly swelled in DMF, a solution of 20-30% piperidine in DMF was added to the resin first for two minutes with N2 agitation. The solution was drained and the procedure was repeated with fresh 20-30% piperidine in DMF for 25 min. The t1/2 for the deprotection of Fmoc-Valine-OH with 20% Piperidine in DMF is six seconds-excess reaction times ensured complete removal of the Fmoc protecting group (Greene & Wutz, 1999). The resin was then washed four times with DMF to remove any traces of piperidine. A small sample of the resin was then removed from the batch and subjected to the Kaiser (Ninhydrin) test (discussed later in this chapter). This test determines if the Fmoc protecting group has been removed to reveal a terminal -NH2. This free amine is available to act as a nucleophile in the coupling of the next amino acid in the peptide sequence.

Amino Acid Activation and Coupling

The mechanism for the activation and coupling of amino acids during solid phase peptide synthesis is detailed in Figure 3-3b and Figure 3-4. Fmoc SPPS typically





63

requires the formation of benzotriazole esters on the amino acids to be coupled (Fields & Noble, 1990). Activation agents used for this procedure are (1) benzotriazol-1-yloxy tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (2) benzotriazol-1yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), (3) N-[(1HbenzotriazoI- 1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), and (4) 1-hydroxybenzotriazole (HOBt) (Fig. 35).

After the resin is properly solvated in DMF and the terminal Fmoc protecting

group is removed, the next Fmoc-protected amino acid is dissolved in a solution of DMF and activating agents to produce a benzotriazole ester on its carboxyl terminus (Fmoc amino acid reagents are shown in Fig. 3-6). This solution is then added to the deprotected peptide-resin in the reaction vessel and allowed to bubble with N2. Reactions times are typically 2-3 hours. Some couplings required the filtration and re-addition of coupling reagents and occasionally overnight reactions times to obtain complete couplings. After the reaction is determined to be complete by the Kaiser test, excess reagents are drained away leaving the now elongated, resin-bound, terminally Fmoc protected peptide in the reaction vessel. Coupling reactions are monitored as described below. It should be noted that nucleophilic sites on the side chains of all amino acids used during the peptide synthesis are blocked with protecting groups that are stable to the conditions of the deprotection and coupling cycle. Kaiser (Ninhydrin) Test

It is necessary to monitor the SPPS reaction at each coupling step in order to

avoid incomplete peptide chains, or deletion sequences, from developing throughout the






64

synthesis. Kaiser et al. first reported a method for the determination of the completeness of amino acid coupling during SPPS in 1970. Peptide chemists routinely use this method today. Based on the ninhydrin reagent described by Troll and Canan (1953), the ninhydrin reagent of the Kaiser test reacts with free Na-amino groups to form a deep blue/violet color known as Ruhemann's purple (X. 570 nmn).

Kaiser test reagent. Three solutions were prepared for the Kaiser test as follows:

(1) 2 mL of 0.001 M KCN (6.5 mg KCN in 100 mL distilled, deionized H20) diluted to 100 mL with pyridine; (2) 50 mg ninhydrin (Aldrich) in 1 ml ethanol; (3) 80 g phenol in 20 mL ethanol. After a coupling reaction or Fmoc deprotection, a small sample of the resin was removed from the reaction vessel on a pipette tip and placed in a glass test tube. Approximately 20-30 IiL of each reagent were added to the resin. The solution was mixed via mild shaking or vortexing for five seconds and then heated in a heating block (1 10 'C) for four minutes. A clear, golden yellow solution color identical to that of ninhydrin in ethanol was indicative of a negative result (i.e. no free amines) or complete coupling. In strong contrast, the presence of free Na-amines after Fmoc deprotection or an incomplete coupling reaction caused the solution to turn deep blue/violet.

It should be noted that the Kaiser test, as described above, is only semiquantitative. The amount of free amine present is estimated visually based on the intensity of the blue/violet color observed in the test solution. Stated more simply, the darker the blue/violet color, the more free amine is considered to be present (Sarin et al., 1981). Kaiser described peptide couplings (verified by titration) ranging from 76% to 99.4% completion as exhibiting "dark blue to trace blue solution" colors respectively (Kaiser et al., 1970). As coupling proceeds to completion, the terminal Na-amines





65

become amides in the peptide backbone and therefore are unavailable to react with the ninhydrin reagent. When only a clear golden yellow color is observed, the coupling reaction is said to be complete.

Despite the Kaiser test's visual indication that a coupling is complete, it is

possible that a small percentage of free amine may still be present on the resin. This minor amount of Na-amine may not cause a color change significant enough to be observed by the naked eye. Unprotected, this amine is available to react in subsequent coupling cycles. Over the course of a lengthy peptide synthesis, this small percentage of uncoupled amnine can lead to a significant amount of deletion peptide. A more accurate quantitative method for the monitoring of SPPS by the ninhydrin reaction was proposed by Sarin et al. (1981) and has more recently been standardized by various automated peptide synthesis equipment manufacturers. This method involves monitoring the ninhydrin chromnophore (Ruhemann's purple) in solution at 570 nim. However, this method may be considered a slightly tedious and unnecessary measure to the manual synthetic peptide chemist. Since acylat ions/couplings are considered high yield reactions, very often these highly quantitative measures determine that only fractions of a percent of Na -amines remain unreacted (Fields & Noble, 1990). Therefore, these methods are more practical when incorporated into an automated peptide synthesizer where the chemist cannot personally sample the beads from the reaction vessel after each deprotection and coupling cycle. For manual SPPS the practice of "capping", even when the visual Kaiser test indicates complete coupling, eliminates any unreacted anmines that may remain on the resin. Capping, described below, is more commonly and easily





66

employed during manual SPPS and was used throughout the synthesis of the peptides described in this paper.

In summary, the Kaiser test was utilized to monitor the status of terminal amines on the resin-bound eIF-5A peptide fragments throughout each step of SPPS described in this dissertation. A coupling was determined to be incomplete if any color other than that of the ninhydrin reagent itself was observed after heating the resin in the presence of the ninhydrin reagents. Occasionally, when applying the Kaiser test to assess the extent of FMOC deprotection (prior to coupling), even the sampled resin beads were stained. This staining of the beads has been attributed to the blue/violet chromophore being sonically bound to the resin bearing a positive charge (Sarin et al., 1981). Capping

Deletion peptides that differ from the desired peptide sequence by only one or two residues may not be easily separated from the desired peptide by HPLC or other purification techniques. For example, their affinity for the HPLC column is nearly identical causing them to coelute. Further, mass spectrometry and Edman degradation (sequence) analysis of what appears to be a single, pure compound by HPLC usually reveals that there is indeed more than a single peptide sequence present. Therefore, terminating the peptide chain by "capping" any unreacted Na-amines prior to the next coupling substitutes a terminated peptide for a potential deletion peptide. Theoretically, terminated peptides will then differ from the desired peptide significantly enough, for example in both molecular weight and N-terminal functionality (N-acetyl-peptide vs. NH2-peptide), that separation by HPLC methods should be relatively simple (Fields & Noble, 1990; Dunn & Pennington, 1994).





67

Capping reagent. During the synthesis of the peptides described in this paper, any uncoupled amines were blocked using acetic anhydride in the presence of a tertiary amine such as DIEA in CH2C12 (Stewart & Young, 1984; Pennington & Dunn, 1994). This reactive agent effectively acetylated any primary amines still available on the peptide-resin. Specifically, a 2:1:2 ratio of acetic anhydride/DIEA/CH2C2 was prepared. After each coupling was deemed complete by the traditional Kaiser test, 10-20 mL of this "capping" solution were added to the resin and allowed to bubble with N2 for 15-30 minutes. The solution was then drained and the resin was rinsed with excess CH2C12 three times, followed by five rinses with DMF. All steps were recorded on the peptide worksheet as described earlier. In this manner, the amino termini of any peptide chains where coupling may have been incomplete were covalently blocked (Fields & Noble, 1990). Facilitating the purification process by terminating unwanted peptide chains prior to cleaving the peptide from the resin enhanced the final yields of the desired peptide sequences.

Synthesis of eIF-5A Peptide Fragments

The sequences and expanded structure of the peptides synthesized in this study using the Fmoc SPPS methods described above are shown in Figures 3-7 and 3-8. Peptides of 5, 8, 14, and 20 amino acid residues of the human eIF-5A sequence have been synthesized from Leu5s toward Lys39. These peptides contain a lysine residue at position 50 and represent the unmodified form of eIF-5A. In addition, 20-residue sequences containing deoxyhypusine, hypusine, and '3C-labeled hypusine at position 50 have also been synthesized using the reagents described in the previous chapter. A 21-residue sequence, Leu58 to Cys39, with hypusine at position 50 was also prepared. To determine their purity and sequence integrity, each peptide was carefully characterized by HPLC,





68

MALDITOF-MS, and amino acid sequencing. The peptides have also been analyzed by nuclear magnetic resonance at various temperatures, pHs, and field strengths (Chapter 6).

A typical peptide synthesis followed a series of deprotection and coupling steps as depicted in Figure 3-1. Prior to each reaction, the resin was swelled for 1-2 minutes in DMF and rinsed with excess DMF. The terminal Fmoc was removed via a two minute then 25 minute reaction with 20-30% piperidine in DMF. This was followed by four washes with excess DMF. A portion of the resin was sampled and subjected to the Kaiser test. If a positive result was obtained, the next amino acid in the peptide sequence and the appropriate activating agents (i.e. HBTU, HOBT, etc.) were dissolved in DMF and added to the reaction vessel with DIEA. Typical coupling times were 2-3 hours. Occasionally a reaction was observed to be incomplete after this time period. Reagents were then drained from the reaction vessel and fresh reagents were added and allowed to react overnight. Upon completion of coupling (as determined by the Kaiser test), the resin was washed with excess DMF and CH2C12 followed by the addition of "capping" reagents to terminate any unreacted peptide chains that might remain on the resin. After capping, the resin was again washed with excess DMF and the cycle of deprotection and coupling was repeated.

Final Deprotection and Cleavage from Resin

After the successful coupling of the final amino acid in the peptide sequence, the terminal Fmoc was removed as described above. The peptide was then subjected to chemical conditions that simultaneously removed all side chain protecting groups and cleaved the peptide from the solid support. A cleavage "cocktail" was prepared with 95% trifluoroacetic acid, 2.5% H20, and 2.5% triisopropylsilane (as scavenger). An





69

excess of the cleavage cocktail was added to the resin and mixed with a gentle N2 gas flow. After 2.5 hours, the solution was drained and collected. The resin was washed three times with excess cleavage cocktail. All washes were collected and then partitioned into centrifuge tubes. Cold ether was added to each tube in order to precipitate the peptide and extract scavengers and side chain protecting groups. The tubes were sealed and centrifuged for 15 min at 2500 rpm, 10 *C. The ether was carefully decanted leaving a white residue in the centrifuge tubes. A total of three cold ether extractions were carried out. The peptide residues were allowed to dry in a desiccator under vacuum overnight.

Peptides prepared with Hypusine reagents. Peptides containing the

deoxyhypusine, hypusine, and 13C-labeled hypusine reagents required the use of a slightly different cleavage cocktail due to the presence of CBZ protecting groups that were not labile to trifluoroacetic acid cleavage conditions. For these peptides, the resinbound peptide was first transferred from the reaction vessel to a round bottom flask and then cooled in an ice bath. A cleavage cocktail consisting of 10 mL TFA, 500 pL of TIS, phenol, and pentamethylbenzene (also cooled) was then added. The mixture was stirred for five minutes under N2 followed by the addition of 400 VL of 30% HBr in acetic acid under N2 for 15 minutes. The flask was allowed to warm to room temperature. After 1.5 hours, the resin beads were filtered away and the solution was concentrated to near dryness via rotary evaporation. The residue was then treated with 10% acetic acid (aq) and extracted with cold diethylether. The aqueous layer was then lyophilized. All crude peptides were purified via preparative HPLC (Chapter 4).







70








FMOC-HN-Ami no Ai-20-30% piperidine in DMF H2N-Amnifo Ad--- Wash resin, Kaiser Test





FMOC-activated amino acid, coupling reagents FMOC-HN--Amliflo Addn-Amino Add1 Q







Deprotection & Cleavage Workup, HPLC



















Figure 3-1. General FMOC Solid Phase Peptide Synthesis and Reaction Vessel






71












p-AlkoxylbenzyWcohol (WANG RESIN) 0 70FMOC-prolecting group Leucine58 p-Alkoxylbenzylakohol (WANG RESIN)

Figure 3-2. FMOC-Leu5g-WANG RESIN









72







piperidincinDMF


H

0 0
0

0

d




2 30 min RT











0 /
C02 W +
1, Ol H2N

0
Dib ffWvmw--"bk in DMF, rinwd away
Wmq wi& pip-dime
Deprotection of FMOC-LeU58-Wang Rmin with 20-30% Pipffidine in DNff





H457

H (HC"
G, 0 N(rHh f*CKA
0 -KCH3)2 I M I
N N
N 'N
N 0 ACH36 FMW' 0 MCH h
N
'N' N N 11 N
0
Tit HUTU Oat
Tn Tlt





HiW
MCK


(H,,r fil' MCH.). Fmw 0- N N Fmw RCHh B
N
Tlt N'
-N, N N'-Fm*c-NH.Hhip,-OBt Tit Ad"W Amino Acid Activation of FMOC-His,57(Trt)-OH with HBTU/DIEA/DNE






Figure 3-3. FMOC Synthesis-A) Deprotection and B) Activation of FMOC Amino Acids for SPPS








73













FmO&- H oat 0
-H2N
HisS7 7 - --FmocNH-is-Ot NNH2 Leucmne 58 Wang Resin Activated Amino Acid


Til DMF
N2 bobbling







H \OBt 0 0

Fmoc0
N
H

-N

N
Trt









H + N
N0
Fmoc' N" N
H I
o0
N
-OBt regenerated, can N activate additional amino acid
Til Fmoc-His57(Tht)-Leu5g-Wang Resin for oupling

Coupling of Activated FMOC-His57{Trt)-OBt to NH2-Leuss-Wang Resin








Figure 3-4. Amino Acid Coupling in SPPS






74





N(CH3)2




(H3C)2N3)
N N(CH3)2 2 P








OZN OPF6

PBO




/_uIIJII N NCHV 13 1

Fiur 35.Stucurs foulNg eget used H SP








75













0
H
Fn= "OH
Fmoc.
Frwc OH N
H
,eeIO4
141 t 619.71 0 M 3939
0
TO

FnDOJAUCMD.O" Fmoo-Hishibra(Trt)-OH Fmoc-Vidiw4M
9-FkwerAmOlbO"CAftwA-LIAmw W-9-Fluorawbnetboxycubonyl-N -Trh*L4iisudme



0
H H
N J ,
Fmoc OH H 0
2 H
Fmcc OH F. JH OH
N N
2MP49. 54 H wlOh9.35 H
0 :29731

NH
BOC

Fmoc Lysin"w)-OH Fnwo-Alanme-OH H20 Fmm-Glyane-OH
N'-%Fh-enyJnwd-y-b-y 9-Fhwrwytw&dioxyauborrJ-L-Aknim H20 9-FhxxurAwAthoxycarbonyl-Lr*ane




0
0 t43u -0 -t-BU H H
Ffficic OH
Fmoc, F. OH
N' W 74n17,46 W CgHOO, C2()H2lNO4S
H H 38144 Mol. WL 371.45
0 0 S


Fmoc-Threontnm(&Bu)-(W Fnwo%erme(o4uWM F131004401131mina-M
9-RumawhoWmycubon)4-0-t-BuWt-LThmmm 94'lu--ykntd"carb-yk 44W-L-Senne 9-FkwruWkmdwwcaftnyl-L-WAtbwmne




0 0-443u 0

N,
Fmoc Om

Fmoc F. J' OH
OH ww'10647 N"' "" 11 W COII.S,71
H L91Y, 313.41
H r I
0 0 Trt


Fmoi;-G AcuK04-Bii) OH FnKie-Waxwe-OH Fnxm Imne Crrt).OH
9-FhwrmirAmOdlOxyCvboqy L-(Hvww Acid 9-Fkwrwoimcghoryoubonyt-L4solmmam 9-FhbmarAnwd*owycubDayI.&TritykC"m
044krA Eaw




Figure 3-6. Structures of Standard FMOC-protected Amino Acids used in SPPS







76










H ,H
NJ jj"
H2N 20
I-r "
0 0 N 0

L?6H
'I.
NH2 t&. 69





H2N OH
H 0 0 H 0 H 0 21
N
N N
N LON98091.02
H NH2



H2N


OH OH
H
H2NJN O'XNf H
'jN- y" N 22
H 0 H 0 H 0 0 0 H 0 H 0
-"OH N

gH yO
WN W.
N 71
NH2 H NH2 N


THR45-SER46-LYS47-THR4$-GLY49-LYSso-HIS51-GLY52-HIS53-ALA54-LY855-VALw-HM,57-LEUm

1 5 mer
8 mer

14 nvr





Figure 3-7. eIF-5A 5-, 8-, and 14-Amino Acid Peptide Fragments







77










HS 0 OH XSO
OH OH OH 0 H 40
hN N HN 4 N NO N ? NI, H, 5 HN HN 'OH
H 0 H H o H o H o H o H H o H o H

N N N"
NH2 NH2 H H NH2 H

(CYS3g)-LYS3rlLE40,VAt4 I-GW42-WT4,-SER4-4-Tfflt45-SERWLYS47-THR4rGLy4T&-HLI-GLY5rMS5rALA54-LYS55-VALWMSsrLEL s



Xso = Lysine NF 23
C95Hi62N30027S Mol. Wt.: 2188.55


H
*50 = Deoxyhypusine N," 24
Nl$
C99HQIN3 027S Mol. Wt.: 2 59.68

QH
X50 Hypusine 25

C99H171N 0 S NF
MOL Wc IM'68

OH
X50 13C-Hyposine 13 26

C9813CH171N31028S
Mol. Wu 2276.68

OH
X50 Hypusine, (Cysteine 38) 27

C H N 0 S aol'W2358%


Figure 3-8. elF-5A 20 and 21 Amino Acid Peptide Fragments with Lys5o, DHyp5o, Hyp5o, and "C Hyp5o












CHAPTER 4
EXPERIMENTAL-SYNTHESIS I AND II General

Reagents were purchased from various chemical companies such as Aldrich,

Fluka, or Sigma and were used without further purification. No-CBZ-L-lysine tert-butyl ester hydrochloride was obtained from BACHEM Bioscience Inc. BOC-j3-alanine-OH and BOC-y-aminobutyric acid were purchased from NovaBiochem and PyBOP coupling reagent was purchased from Acros. Fisher Optima-grade solvents were used routinely and organic extracts were dried with anhydrous Na2SO4. Acetonitrile used for the KCN/18-crown-6 reaction was distilled from Nail. HCI in ethyl acetate (1 M) was prepared by bubbling HCI (g) into dry ethyl acetate and then diluting to I M with additional dry ethyl acetate. Ethyl acetate was dried by elution through A1203 into a clean, dry flask under N2 atmosphere. KCN was finely and carefully ground and dried for 20 h at 145 C. Silica gel 32-60 (40 gm "flash") from Selecto, Inc. (Kennesaw, GA) was used for flash column chromatography. Molecular biology grade AG 50W-X8 Resin (200-400 mesh, Na' form) from BioRad was used for ion exchange chromatography. 'H NMR and 13C NMR spectra were recorded on a 300 MHz (Varian) for hypusine and hypusine dipeptide precursor compounds. The 500 and 750 MHz (Avance Bruker, 5mm TXI or BBO probes) instruments at the UF McKnight Brain Institute Advanced Magnetic Resonance and Imaging Spectroscopy (AMRIS) Facility were used to analyze the eIF-5A peptide fragment compounds. Samples were analyzed at room temperature (- 25 oC) and pH was not adjusted unless otherwise stated (as for pH titration analyses described in 78






79

Chapter 6). Solvent and reference standard conditions are stated for each compound analyzed. Chemical shifts for proton NMR are given in parts per million downfield from an internal reference standard such as tetramethylsilane (TMS), or external reference standard such as sodium 3-trimethylsilylpropionate (TSP) in D20. '3C NMR chemical shifts in CD3OD are calibrated on the CD3OD septuplet peak at 49.0 ppm unless otherwise stated. 13C NMR chemical shifts for eIF-5A peptides are referenced to external TSP in D20. Coupling constants (J) are in hertz. Melting points were determined on a Fisher-Johns melting point apparatus and are uncorrected. FAB mass spectra were run in a 3-nitrobenzylalcohol matrix. Elemental analyses were run at Atlantic Microlabs (Norcross, GA). Optical rotations were obtained at 589 nm (the Na D-line) on a Perkin Elmer 141 Polarimeter with c expressed as grams of compound per 100 mL. 3H2 gas hydrogenation was performed at Moravek Biochemicals Inc. (Brea, CA). A Beckman LS 5000 TD was utilized for scintillation counting.

The hypusine reagents (10, 10a, and 19) were prepared as previously described.

All other Fmoc-amino acids, reagents and reaction vessels for SPPS were purchased from Peptides International. Crude peptides were routinely purified by HPLC on a Vydac C18 "semi-prep" reverse phase column (25cm x 1 cm). The Gilson HPLC system consisted of two model 306 pumps, a model 805 manometric module, a model 811 C dynamic mixer, a model 119 UV/Vis detector, a 401 dilutor, and a model 231 sample injector. The flow rate was set to 5.00 mL/min with a 30 minute solvent gradient (10% CH3CN, 0.1% TFA in H20 to 100% CH3CN). The detector was set to X = 220 nm. An Isco "Foxy" fraction collector was used to collect purified peptides.





80

MALDITOF-MS was performed on a Perseptive Biosystems Voyager DE PRO Biospectrometry Workstation (Framingham, MA) at the UF ICBR Protein Chemistry Core Facility. Samples were run in an a-cyano-4-hydroxycinnamic acid matrix (10 mg a-cyano-4-hydroxycinnamic acid dissolved in 1 mL 1:1 0.1% TFA in H20 and CH3CN). Amino acid sequencing using standard Edman degradation chemistry was also performed at the UF ICBR Protein Chemistry Core Facility on an Applied Biosystems 494 HT Procise Protein Sequencer (Foster City, CA). Samples for amino acid analysis (AAA) were hydrolyzed with 6 N HCI and run on an Applied Biosystems 420 Amino Acid Analyzer (Milford, MA) with a Water Associates PicoTag Workstation for hydrolysis at the UF ICBR Protein Chemistry Core as well. Precolumn PTC-derivatization was employed with this method.

Synthesis of a-(i-alanyl)- and a-(y-aminobutyryl)-Hypusine Precursors

Na-BOC-Ne-CBZ-L-lysine tert-Butyl Ester (1). Sodium hydrogen carbonate (2.81 g, 33.47 mmol) in water (75 mL) was added to A-CBZ-L-lysine tert-butyl ester hydrochloride (12.00 g, 32.18 mmol) in 100 mL chloroform. The mixture was stirred at room temperature for 5 min under an N2 atmosphere. Di-tert-butyl dicarbonate (7.02 g, 32.18 mmol) in chloroform (50 mL) was added and the mixture was refluxed for 2.0 h and then allowed to cool to room temperature. The layers were separated, the aqueous layer was extracted with chloroform (3 x 100 mL), and the combined organic layers were dried. The organic layers were concentrated to dryness and purified by flash chromatography (20% ethyl acetate/hexane) to yield 1 (14.04 g, 99.9%) as a colorless oil: 'H NMR (CD3OD, TMS): 61.30-1.82 (mn, 6 H), 1.43 (s, 9 H), 1.45 (s, 9 H), 3.11 (t, 2 H, J= 6.7), 3.93 (dd, I H, J= 8.2, 5.2), 5.06 (s, 2 H), 7.24-7.38 (m, 5 H); 3C NMR: 624.0,





81

28.3, 28.7, 30.4, 32.4, 41.4, 55.8, 67.3, 80.4, 82.5, 128.8, 128.9, 129.4, 138.4, 158.1,

158.9, 173.8; [a]21D +4.3* (c 1.75, CHC13).

Na-BOC-L-lysine tert-Butyl Ester Hydrochloride (2). Compound 1 (14.01 g, 32.09 mmol) was dissolved in ethanol (122 mL) and 1 N HCI (36 mL). A catalyst, 10% Pd-C (1.20 g), was added and H2 gas was introduced. After 20.5 h the black suspension was filtered through Celite and washed with ethanol. The filtrate was concentrated, and the residue was dried in vacuo to give 2 as the hydrochloride salt (11.89 g, 100%): 'H NMR (CD3OD, TMS): 81.40-1.82 (m, 6 H), 1.44 (s, 9 H), 1.46 (s, 9 H), 2.92 (t, 2 H, J = 7.6), 3.95 (dd, 1 H, J= 8.9, 4.9); '3C NMR: 523.9, 28.0, 28.3, 28.7, 32.1, 40.5, 55.5,

80.5, 82.7, 158.2, 173.5; [a]23D -20.0* (c 0.86, CH3OH).

(2S,9S)-2-[(tert-Butoxycarbonyl)aminol-10O-chloro-9-hydroxy-7-azadecanoic Acid tert-Butyl Ester (3). A solution of 2 (10.87 g, 32.09 mnmol) in chloroform (200 mL) was extracted with saturated NaHCO3 solution (2 x 150 mL) to neutralize the HCI salt. The aqueous layer was extracted with chloroform (4 x 100 mL). The organic layer was dried, concentrated, and further dried under vacuum. The resulting oil was dissolved in cyclohexane (60 mL). (S)-(+)-epichlorohydrin (3.25 g, 35.12 mmol) was added under an Ar atmosphere. After 35 h, the precipitated product was filtered, washed with cold cyclohexane, and dried under vacuum to give 3 (4.89 g, 39%) as a fine, white powder: The procedure described was repeated with an additional 3.35 g of 2 and yielded 1.28 g (33% yield) of the white powder. mp 86-88 oC; 'H NMR (CD3OD, TMS): 51.30-1.82 (m, 6 H), 1.43 (s, 9 H), 1.45 (s, 9 H), 2.58-2.65 (m, 3 H), 2.76 (dd, 1 H, J= 12.2, 3.8),

3.51 (dd, 1 H, J= 11.2, 5.7), 3.56 (dd, 1 H, J= 11.1, 5.0), 3.84-3.91 (m, 1 H), 3.94 (dd, 1





82


H, J= 9.1, 5.6); "3C NMR: 824.6, 28.3, 28.7, 30.0, 32.6,48.2, 50.3, 53.5, 55.8, 71.1,

80.4, 82.5, 158.1, 173.8; [a]23D -24.9* (c 1.00, CH3OH).

(2S,9S)-2-[(tert-Butoxycarbonyl)aminol-7-(carbobenzyloxy)-10-chloro-9hydroxy-7-azadecanoic Acid tert-Butyl Ester (4). A solution of benzyl chloroformate (3.67 g, 21.50 mmol) in chloroform (30 mL) was added over a period of 15 min to an icecold solution of 3 (6.05 g, 15.31 mmol) in chloroform (40 mL) under an Ar atmosphere. Triethylamine (3.09 g, 30.62 mmol) in chloroform (30 mL) was then added dropwise and the reaction mixture was stirred for 4.5 h at room temperature. The reaction mixture was extracted with I N HCI (160 mL) and water (160 mL). The aqueous extract was dried, concentrated and purified by flash chromatography (33% ethyl acetate/hexane) to yield 4 (6.89 g, 85%) as a colorless oil: 'H NMR (CD3OD, TMS): 6 1.30-1.82 (min, 6 H), 1.44 (s,

9 H), 1.45 (s, 9 H), 3.21-3.55 (min, 6 H), 3.93 (dd, 1 H, J= 8.6, 5.5), 4.0 (mi, 1 H), 5.12 (s, 2 H), 7.30-7.37 (m, 5 H); '3C NMR: 824.1, 28.3, 28.8, 32.5, 49.4, 52.2, 55.7, 68.4, 71.1,

80.4, 82.5, 129.0, 129.1, 129.6, 138.0, 157.5-158.4 (br), 158.1, 173.7; [a]23D -21.2' (c

1.00, CH3OH).

(2S,9R)-2-[(tert-Butoxycarbonyl)amino]-7-(carbobenzyloxy)-10-cyano-9hydroxy-7-azadecanoic Acid tert-Butyl Ester (5). A mixture of dry KCN (9.24 g, 141.89 mmol), 18-crown-6 (1.021g, 3.86 mmol), and 4 (6.81 g, 12.87 mmol) in dry acetonitrile (300 mL) was stirred at 60 oC for 20 h under an Ar atmosphere. After the reaction mixture was cooled to rt it was filtered through Celite and concentrated to dryness. The residue was purified by flash chromatography (33% ethyl acetate/hexane, then 50% ethyl acetate/hexane) to give 5 (4.69 g, 70%) as a colorless oil: 'H NMR (CD3OD, TMS): 81.28-1.80 (in, 6 H), 1.44 (s, 9 H), 1.45 (s, 9 H), 2.42-2.72 (m, 2 H),





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3.22-3.47 (m, 4 H), 3.92 (mn, 1 H, J= 8.4, 5.5), 4.08 (mn, 1 H), 5.13 (s, 2 H), 7.20-7.38 (m,

5 H); 3C NMR: 924.0,24.2,28.3,28.7, 32.4, 38.4, 55.7, 67.1, 68.4, 80.4, 82.5, 118.9,

129.1,129.2, 129.6, 137.9, 157.5-159.0 (br), 158.1, 173.8. [a]22D -18.1* (c 0.96,

CH3OH).

(2S,9R)-2-[(tert-Butoxycarbonyl)amino]-11-amino-9-hydroxy-7azaundecanoic Acid tert-Butyl Ester, Diacetate Salt (6). A mixed catalyst of 10% PdC (0.50 g) and PtO2 (0.93 g) was added to a solution of 5 (4.66 g, 8.97 mmol) in glacial acetic acid (112 mL). 1H2 gas was introduced and after 24 h the catalysts were removed via filtration through Celite. The filtrate was concentrated in vacuo and excess acetic acid was removed via addition of toluene to form an azeotrope. This provided 6 as a colorless oil (4.57 g, 100%): 'H NMR (D20, external TSP): 81.44 (s, 9 H), 1.47 (s, 9 H), 1.50-1.80 (mn, 8 H), 2.04 (s, 6 H), 3.01-3.24 (m, 6 H), 3.97 (dd, 1 H, J= 9.1, 5.2), 4.06

(tt, 1 H, J= 9.6, 3.3); 13C NMR (D20, internal standard CH3OH = 49.5 ppm): 622.7, 24.2, 26.7, 28.3, 28.8, 29.3, 32.1, 33.2, 38.2, 53.9, 56.1, 66.4, 81.1, 84.5, 158.9, 175.0,

178.8; [a]25D -17.1* (c 1.00, CH3OH).

(2S,9R)-11-[(Benzyloxycarbonyl)aminol-2-[(tert-butoxycarbonyl)aminoj-9hydroxy-7-(carbobenzyloxy)-7-azaundecanoic Acid tert-Butyl Ester (7). Compound 6 (4.57 g, 8.98 mmol) was dissolved in water (150 mL) and diethyl ether (150 mL). This biphasic solution was vigorously stirred and cooled to 0 C under an Ar atmosphere. KHCO3 (14.02 g, 140.0 mmol) was added to the solution. Then N(benzyloxycarbonyloxy)-succinimide (CBZ-ONSu, 8.06 g, 32.33 mmol) was added in several portions over 20 min. The reaction mixture was allowed to warm to room temperature and stir for an additional 5 h. The layers were then separated and the





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aqueous layer was extracted with diethyl ether (3 x 200 mL). Ether layers were combined, dried and concentrated. Purification of the residue by flash chromatography (50% ethyl acetate/hexane) gave 7 (3.83 mg, 65%) as a colorless oil: 'H NMR (CD3OD, TMS): 31.30-1.80 (mi, 8 H), 1.43 (s, 9 H), 1.44 (s, 9 H), 3.11-3.44 (m, 6 H), 3.82 (m, 1 H), 3.92 (m, 1 H), 5.06 (s, 2 H), 5.10 (s, 2 H), 7.24 -7.38 (m, 10 H); 13C NMR: 624.1, 28.3, 28.8, 32.5, 35.9, 38.6, 54.0, 54.7, 55.8, 67.4, 68.3, 69.0, 80.4, 82.5, 128.8, 128.9,

129.1, 129.5, 129.6, 138.1, 138.4, 158.1, 158.4, 158.9, 173.8; [a]22D -12.1* (c 1.00,

CH3OH).

Synthesis of a-(3-alanyl)- and a-(y-aminobutyryl)-Hypusine

(2S,9R)-11-[(Benzyloxycarbonyl)amino]-2-amino-9-hydroxy-7(carbobenzyloxy)-7-azaundecanoic Acid tert-Butyl Ester (12). The BOC protecting group was selectively removed from 7 (3.83 g, 5.82 mmol) via the addition of 5 mL of dry I M HCI in ethyl acetate (chilled) to six-638 mg fractions (approximately I mmol each) of the di-CBZ, Boc, O-t-Butyl protected hypusine reagent. The reaction mixtures were allowed to stir at room temperature under an Ar atmosphere and were monitored by TLC (9:1 CHCl3I/MeOH) until all starting material was consumed (i.e. converted to free amine). Typical reaction times were 3 to 4 h. The solutions were then concentrated to remove excess HCl/EtOAc and then dried under vacuum. The glass-like materials were then dissolved in ethyl acetate (45 mL), combined, and washed with saturated NaHCO3 solution (3 x 50 mL). The aqueous and organic layers were separated followed by extraction of the aqueous layer with ethyl acetate (3 x 100 mL). The organic layers were combined and dried over Na2SO4, filtered, and concentrated to dryness. Purification by flash chromatography (90% MeOH:CHC13) yielded the free amine, a clear oil 12 (2.05g,





85


64%): 'H NMR (CD3OD, TMS): 81.28-1.76 (m, 8 H), 1.45 (s, 9 H), 3.11-3.45 (mn, 7 H), 3.82 (m, 1 H), 5.06 (s, 2 H), 5.10 (s, 2 H), 7.29-7.34 (m, 10 H); "3C NMR: 523.3, 23.7, 29.2, 35.5, 35.9, 38.6, 54.1, 54.8, 55.5, 67.4, 68.3, 68.9, 82.2, 128.8, 129.0, 129.1, 129.5,

129.6, 138.1, 138.4, 158.4, 158.9, 175.9; [a]22D +4.20 (c 1.00, CH3011). HRMS m/z called for C30H44N307 558.3179, found 558.3181; Anal. (C301143N307) C, H, N calcd: C 64.61, H 7.77, N 7.53, found C 63.59, H 7.67, N 7.26.

BOC-i-Alanyl-(2S,9R)-Hypusine-[N7,NI2-di-CBZ]-9-hydroxy-O-t-Bu (13). A

solution of 12 (249 mg, 0.45 mmol) and BOC-13-alanine-OH (110 nmg, 0.58 mmol) in 5 mL of methylene chloride was cooled to 0 oC under an Ar atmosphere, and allowed to stir for 10 min. PyBOP (348 mg, 0.67 mmol) coupling reagent was added followed by diisopropylethyl amine (101 mg, 0.78 mmol) 15 min later. The reaction mixture was allowed to stir for 10 min before warming to room temperature. After 1.5 hrs, the reaction mixture was diluted with saturated NaCl solution and extracted with CHCl3 (3 x 20 mL). The combined organic layers were successively washed with ice-cold 10% citric acid/brine (1:1, 20 mL), brine (20 mL), saturated NaHCO3 solution/brine solution (1:1, 20 mL), and brine (20 mL). The organic layer was dried with Na2SO4 and concentrated in vacuo. Purification via flash chromatography (17% hexane/ethyl acetate) yielded a colorless oil 13 (266 mg, 82%): 'H NMR (CD3OD, TMS): 8 1.30-1.80 (mn, 8 H), 1.42 (s, 9H), 1.44 (s, 9 H), 2.40 (t, 2 H,J= 6.6) 3.11-3.48 (m, 8 H), 3.81 (s, br, 1 H), 4.22 (s, br, 1 H) 5.08 (s, 2 H), 5.11 (s, 2 H), 7.24-7.38 (m, 10 H); 13C NMR: 624.1,28.3,28.8,32.2, 35.9, 37.0, 38.0, 38.6, 54.1, 54.4, 54.6, 67.4, 68.3, 68.9, 80.2, 82.8, 128.8, 128.9, 129.1,

129.5, 129.6, 138.1,138.4, 158.1, 158.4, 159.0, 173.1,174.0; [a]9"D-10.5 (c 1.00,