Solid-phase syntheses and studies of conformationally constrained analogues of the calcitonin gene peptide superfamily polypeptides calcitonin (Ct) and islet amyloid polypeptide (IAPP) [Elektronische Ressource] = Festphasensynthesen und Untersuchungen von konformationell-eingeschränkten Analoga der Calcitonin-Gen-Peptid-Superfamilien-Polypeptide Calcitonin (Ct) und Islet-Amyloid-Polypeptid (IAPP) / vorgelegt von Athanasios Kazantzis

De
Solid-phase syntheses and studies of conformationally constrained analogues of the calcitonin gene peptide superfamily polypeptides calcitonin (Ct) and islet amyloid polypeptide (IAPP) Festphasensynthesen und Untersuchungen von konformationell-eingeschränkten Analoga der Calcitonin-Gen-Peptid-Superfamilien-Polypeptide Calcitonin (Ct) und Islet Amyloid Polypeptid (IAPP) DISSERTATION der Fakultät für Chemie und Pharmazie der Eberhard-Karls-Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften 2004 vorgelegt von Athanasios Kazantzis Tag der mündlichen Prüfung: 17.02.2004 Dekan: Prof. Dr. H. Probst 1. Berichterstatter: Prof. Dr. Dr. h. c. mult. W. Voelter 2. Berichterstatter: Priv. Doz. Dr. A. Kapurniotu Die vorliegende Arbeit wurde unter Anleitung von Priv.Doz. Dr. A.
Publié le : jeudi 1 janvier 2004
Lecture(s) : 23
Source : W210.UB.UNI-TUEBINGEN.DE/DBT/VOLLTEXTE/2004/1113/PDF/4_FINAL_FORM.PDF
Nombre de pages : 161
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Solid-phase syntheses and studies of conformationally constrained analogues
of the calcitonin gene peptide superfamily polypeptides calcitonin (Ct)
and islet amyloid polypeptide (IAPP)

Festphasensynthesen und Untersuchungen von konformationell-eingeschränkten
Analoga der Calcitonin-Gen-Peptid-Superfamilien-Polypeptide
Calcitonin (Ct) und Islet Amyloid Polypeptid (IAPP)




DISSERTATION



der Fakultät für Chemie und Pharmazie
der Eberhard-Karls-Universität Tübingen


zur Erlangung des Grades eines Doktors
der Naturwissenschaften



2004

vorgelegt von
Athanasios Kazantzis









































Tag der mündlichen Prüfung: 17.02.2004


Dekan: Prof. Dr. H. Probst
1. Berichterstatter: Prof. Dr. Dr. h. c. mult. W. Voelter
2. Berichterstatter: Priv. Doz. Dr. A. Kapurniotu









































Die vorliegende Arbeit wurde unter Anleitung von Priv.Doz. Dr. A. Kapurniotu
in der Zeit von Oktober 1999 bis Dezember 2003 an der Abteilung für Physikalische
Biochemie des Physiologisch-chemischen Instituts der Eberhard-Karls-Universität Tübingen
(1999-2002) und am Institut für Biochemie des Klinikums der RWTH Aachen (2002-2003)
durchgeführt.
i

Acknowledgments

I would like to express my sincere gratitude and appreciation to my supervisor Priv. Doz. Dr.
Aphrodite Kapurniotu for introducing me to the field of peptide synthesis and for supervising
and guiding me throughout my PhD thesis which provided the motivation and encouragement
for accomplishing this work. Valuable and thoughtful discussions during these years
enlightened my perspectives and provided new insights into my studies. I would also like to
thank her for the design of the peptide analogues and her assistance in the experimental
planning.
I am thankful to Prof. Dr. Dr. h. c. Wolfgang Voelter for the continuous support and interest
in my PhD work.
I am thankful to Prof. Dr. Jürgen Bernhagen for teaching me and supervising the performance
of the bioactivity assays (RIA and ELISA) in his laboratory and also for his continuous
support and interest in my work.
I am thankful to Doris Finkelmeier for assisting me during the bioactivity assays. I would like
to thank Kostas Tenidis, Andreas Buck, Anita Horn, Anke Schmauder, Michaela Waldner,
Jürgen Beck, and Thomas Hirsch for their assistance in the syntheses and HPLC purifications
and for the friendly atmosphere in the Tübingen lab. I would also like to thank Kostas Tenidis
and Anke Schmauder for performing the cytotoxicity assays and FT-IR spectroscopy. I would
also like to thank Gunnar Müllenweg for helpful discussions and Marco Müsken and Heidi
Vasen for the friendly atmosphere in the Aachen laboratory.
I am thankful to Miriam Fecker for helping me with various kind of administrative paperwork
during my studies.
I am also thankful to Gabriele Fahrbüchel for her help and assistance with regard to the
several issues related to my stay in Germany.
I would like to thank Dr. Roland Wacker and all the coworkers in the MALDI-MS lab that
acquired the mass spectra of the peptide samples. Their continuous effort was an invaluable
help to my data and results analysis.
I wish to express my thanks to my coworkers in Aachen: Michael Thiele, Helge Fünfzig,
Manfred Dewor, Marianna Tatarek-Nossol, Hannelore Didden, and Dr. Hongqi Lue for their
friendship and lab assistance.
I am also thankful to my friends in Tübingen: Marcus, Daniel, Florian, Marc, Tina, Florian,
Ilka, Andreas, Celine, and Mafiu for their good friendship and optimism during these years. ii
Finally, I would like to thank my parents and my brother for their patience, support and
encouragement.
It should be mentioned that parts of this thesis have been published in:

1.) A. Kazantzis, M. Waldner, J. W. Taylor, & A. Kapurniotu, Eur. J. Biochem., 269,
780-791, 2002. iiiList of Abbreviations


Ab Absorbance
Ac Acetyl
AC Adenylate cyclase
ACN Acetonitrile
AcOH Acetic acid
AcO anhydride 2
ACS American chemical society
Adoc 1-(1´-adamantyl)-1-methyl-ethoxycarbonyl
Boc t-Butoxycarbonyl
Bom π-Benzyloxymethyl
BOP 1-Benzotriazolyloxy-tris-dimethylamino-phosphonium hexafluorophosphate
BSA Bovine serum albumin
Bzl Benzyl
cAMP cyclo-Adenosine monophosphate
CD Circular dichroism
CNS Central nervous system
Ct Calcitonin
Gradient change over time ∆G
DCC Dicyclohexylcarbodiimide
DCM Dichloromethane
DIC Diisopropylcarbodiimide
DIEA Diisopropylethylamine
DKP Diketopiperazine
DMF Dimethylformamide
DMS Dimethylsulf
DMSO Dimethylsulfoxide
DTT 1,4-Dithio-DL-threitol
DTAB Dodecyltrimethylammonium bromide
DVB Divinylbenzene
EC Effective concentration at half-maximal response 50
EDT 1,2-ethanedithiol
EIA Enzyme immuno assay iv List of Abbreviations
Eq Equivalent
Et Ethyl
EtO Diethylether 2
FAB Fast atom bombardment
Fmoc 9-Fluorenylmethoxycarbonyl
FT-IR Fourier transform infrared spectroscopy
Gdn HCl Guanidine hydrochloride
GPCR G-protein coupled receptor
HATU 2-(1H-9Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexaflurophosphate
HBTU 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
HF Hydrogen fluoride
HFIP 1,1,1,3,3,3-hexafluoro-2-propanol
HPLC High performance liquid chromatography
HOBt 1-Hydroxybenzotriazole
hrs hours
IAPP Islet amyloid polypeptide
IC Inhibition concentartion at half-maximal response 50
iPrOH Isopropyl alcohol
MALDI Matrix assisted laser desorption ionization
MBHA p-Methylbenzhydrylamine
Me Methyl
min minutes
Mob 4-Methoxybenzyl
MS Mass spectroscopy
Mtt 4-Methyltrityl
MTT 3-[4,5-dimethylthiazol-1-yl]-2,5-diphenyltetrazolium bromide
NMP N-methyl pyrrolidinone
NMR Nuclear magnetic resonance
OBt 1-Benzotriazolyl ester
Oc Hx Cyclohexyl ester
OD Optical density
OFm Formyl ester
p.a pro analysi
PBS Phosphate buffer saline v List of Abbreviations
Pip 2-Phenyl isopropyl ester
Pmc 2,2,5,7,8-Pentamethylchroman-6-sufonyl
PyBOP 1-Benzotriazolyloxy-tris-pyrrolidinophosphonium hexafluorophosphate
Rink MBHA 4-(2´, 4´-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy acetamido-norleucyl
RP Reversed phase
RPMI Roswell Park Memorial Institute
RT Room temperature
SAR Structure-activity relationship
SD Standard deviation
SPPS Solid phase peptide synthesis
TAS Thioanisole
TBTU Benzotriazole-1-yl-1,1,3,3,-tetramethyluronium tetrafluoroborate
tBu tert-Butyl
TFA Trifluoroacetic acid
TFE 2,2,2-Trifluoroethanol
TIS Triisopropylsilane
TMB 3,3´, 5, 5´-Tetramethylbenzidine
TMD Trans membrane domain
Trt Trityl
UV Ultraviolet
Z Benzyloxycarbonyl
vi List of Abbreviations
Amino-Acids Abbreviations


[Ala, A] Alanine
[Aib] 2-Aminoisobutyric acid
[Arg, R] Arginine
[Asn, N] Asparagine
[Asp, D] Aspartic acid
[Cys, C] Cysteine
[Dab] 2,4-Diaminobutyric acid
[Dap] 2,3-Diaminopropionic
[Glu, E] Glutamic acid
[Gln, Q] Glutamine
[Gly, G] Glycine
[His, H] Histidine
[Ile, I] Isoleucine
[Leu, L] Leucine
[Lys, K] Lysine
[Met, M] Methionine
[Nle, B] Norleucine
[Orn] Ornithine
[Phe, F] Phenylalanine
[Pro, P] Proline
[Ser, S] Serine
[Thr, T] Threonine
[Trp, W] Tryptophan
[Tyr, Y] Tyrosine
[Val, V] Valine
[Xaa] Unspecified
viiTable of contents

Acknowledgments...................................................................................................................... i
List of Abbreviations...............................................................................................................iii
Table of contents.....................................................................................................................vii
1 Abstract.................................................................................................................................. 1
2 Objective ................................................................................................................................ 5
3 Introduction ........................................................................................................................... 8
3.1 Solid Phase Peptide Synthesis (SPPS) ............................................................................. 8
3.1.1 Diketopiperazine formation..................................................................................... 11
3.1.2 Acidic hydrolysis of N-methylated peptides........................................................... 12
3.2 Constrained peptides...................................................................................................... 13
3.2.1 Rational design of drugs: constrained peptides....................................................... 15
3.3 Calcitonin gene peptide superfamily.............................................................................. 16
3.3.1 Calcitonins............................................................................................................... 16
3.3.2 Islet Amyloid Polypeptide (IAPP) .......................................................................... 19
4 Materials and Methods ....................................................................................................... 23
4.1 Materials ........................................................................................................................ 23
4.2 Methods .......................................................................................................................... 24
4.2.1 SPPS using the Boc-strategy................................................................................... 24
α4.2.1.1 Cleavage of the N -Boc-group of glutamine ................................................... 24
4.2.1.2 Cyclization strategy in synthesis by Boc-chemistry ........................................ 25
4.2.2 SPPS using the Fmoc-strategy ................................................................................ 26
4.2.2.1 Cyclization strategy in Fmoc-chemistry .......................................................... 27
4.2.2.2 Coupling of Cys residues in Fmoc-SPPS......................................................... 28
4.2.3 Coupling of N-methyl residues ............................................................................... 29
4.2.4 Coupling on N-methyl residues 32
4.2.5 Kaiser test................................................................................................................ 36
4.2.6 Attachment of the C-terminal amino acid to the resin ............................................ 37
4.2.6.1 Attachmeinal amino acid for the synthesis of Ct analogues
using Boc-strategy........................................................................................................ 37
4.2.6.2 Attachment of the C-terminal am
using Fmoc-strategy ..................................................................................................... 37
4.2.6.3 Attachmeinal amino acid for the synthesis of IAPP analogues
...................................................................................................................................... 37
4.2.6.4 Determination of the substitution level of resin in Fmoc-strategy................... 38
4.2.7 Final deprotection of side-chains and cleavage from the resin ............................... 38
4.2.7.1 Final deprotection of side chains and cleavage of full length sequences from
the resin in Boc-chemistry............................................................................................ 38
4.2.7.2 Final deprotection of side chains and cleavage of full length sequences from
the resin in Fmoc-chemistry......................................................................................... 39
4.2.7.3 Deprotection of side chains and cleavage of partial peptide sequences from the
resin in Fmoc-chemistry............................................................................................... 40

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