Design, synthesis and structural evaluation of peptidomimetics towards foldamers, PNAs and non covalent inhibitors of the 20S proteasome [Elektronische Ressource] / vorgelegt von Andrea Bordessa
261 Pages
English
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Design, synthesis and structural evaluation of peptidomimetics towards foldamers, PNAs and non covalent inhibitors of the 20S proteasome [Elektronische Ressource] / vorgelegt von Andrea Bordessa

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Design, synthesis and structural evaluation of peptidomimetics
towards foldamers, PAs and non covalent inhibitors of the 20S
proteasome

Dissertation

zur Erlangung des Doktorgrades der Naturwissenschaften
Dr. rer. nat.
an der Fakultät für Chemie und Pharmazie
der Universität Regensburg und der Universität Paris XI (Frankreich)

vorgelegt von

Andrea Bordessa

aus

Germasino (Italy)


Regensburg 2008













This work was supervised by Prof. Dr. Oliver Reiser in Regensburg and
Dr. Sandrine Ongeri and Prof. Dr. Sames Sicsic in Paris

th
Thesis submission on November 17 , 2008

rd
Thesis defence on December 3 , 2008

Examination committee: Prof. Dr. Oliver Reiser
Dr. Sandrine Ongeri
Prof. Dr. Burkhard König
Prof. Dr. Sigurd Elz





The following research work was performed from November 2005 to October 2007 in the
Institute of Organic Chemistry of the University of Regensburg under the supervision of Prof. Dr.
Oliver Reiser and from November 2007 to October 2008 at the University of Paris XI, in the
Laboratoire de Molécules Fluorées et Chimie Médicinale, BioCIS, UMR-CNRS 8076 under the
supervision of Dr. Sandrine Ongeri and Prof. Dr. Sames Sicsic.

























I would like to thank Prof. O. Reiser, Dr. Sandrine Ongeri and Prof. Sames Sicsic for having
given me the opportunity to join their research groups and for their help in these three years.
I also thank the Marie Curie commission for financial support during this PhD programme.
CHAPTER 1 INTROCUTION 1
1.1 Peptide generalities 1
1.2 Primary structure 2
1.3 Secondary structure 2
1.3.1 α-Helix 2
1.3.2 β-sheets 3
1.3.3 turns 4
1.4 Tertiary structure 5
1.5 Quaternary structure 5
1.6 Conformational studies of the secondary structure 6
1.6.1 NMR studies 6
1.6.2 Choice of the solvent 6
1.6.3 2D NMR 6
1.6.4 Hydrogen-deuterium exchange and variation of the
temperature 7
1.6.5 Circular dichroism 7
1.6.6 IR in solution 8
1.6.7 X-ray crystallography 9
1.7 Peptidic coupling 9
1.7.1 Coupling reagents 9
1.7.2 Solution phase synthesis 12
1.7.3 Solid phase peptide synthesis (SPPS) 12
1.7.4 Protecting groups 13
1.8 Peptidomimetics 15
CHAPTER 2 δ-AMINO ACIDS TOWARDS FOLDAMERS AND PNAs 17
2.1 Synthesis of cyclic δ-amino acids 17
2.1.1 Three membered rings 17
2.1.4 Four membered rings 20
2.1.5 Five membered rings 22
2.1.6 Six membered rings 27
2.1.7 Bicyclic δ-amino acid 30
2.2 δ-amino acids in foldamers 39
2.3 δ-amino acids in peptide nucleic acids (PNAs) 44
2.3.1 PNA based on aminoethylglicine 44
2.3.2 Linear analogues of Aeg-PNA 48
2.3.3 Cyclic PNAs 52
2.4 Synthesis of δ-amino acids 59
2.4.1 Aim of this work 59
2.4.2 Cyclopropanation 60
2.4.3 Ozonolysis 61
2.4.4 Sakurai allylation 61
2.4.5 Retroaldol lactonisation 62
2.4.6 Introduction of the nitrogen moiety 63
2.4.7 Lactamisation 64
2.4.8 Boc-protection 65
2.4.9 PMB removal by CAN 66
2.4.10 Double bond oxidation 66
2.5 Synthesis of α,δ-pentapeptide 66
2.6 Conformational analysis of the pentapeptide 261 69
2.6.1 IR in solution 69
2.6.2 CD spectroscopy 69
2.6.3 NMR analysis 70
2.6.4 Temperature scan and measurement of the coupling
constants 71
2.6.5 2D NMR and molecular modelling studies 72
2.7 Synthesis of α,δ-heptapetide 73
2.7.1 IR in solution 74
2.7.2 CD spectroscopy 75
2.7.3 Temperature scan and measurement of the coupling
constants 76
2.7.4 2D NMR and molecular modelling studies 77
2.8 Synthesis of PNAs 78
2.8.1 Fmoc protection 79
2.8.2 Reduction of the lactone 80
2.8.3 Coupling with thymine 80
2.8.4 PMB removal by CAN 81
2.8.5 Coupling with adenine 82
CHAPTER 3 PROTEASOME AND INHIBITORS 85
3.1 Role of 20S proteasome 85
3.2 Mechanism of the ubiquitin-proteasome pathway 85
3.3 Proteasome inhibitors 86
3.3.1 Covalent inhibitors 86
3.3.2 Peptide aldehydes 91
3.3.3 Peptide boronates 92
3.3.4 Lactacystin and its derivatives 93
3.3.5 Peptide vinyl sulfonates 94
3.3.6 Epoxyketones 95
3.3.7 Non covalent proteasome inhibitors 96
3.4 Biological effect of proteasome inhibitors 100
3.5 Molecular modelling 101
3.5.1 Docking 101
3.5.2 Genetic algorithm 102
3.5.3 Free energy function 104
3.5.4 3D grids 104
3.5.5 Hydrogen bonds 105
3.5.6 The torsional term 105
3.6 Previous works in this lab and aim of this work 106 3.6.1 Literature and crystallographic studies 109
3.6.2 Choice of the docking parameters 111
3.6.3 Docking of the lead molecule and virtual screening
of new candidates 116
3.6.4 Synthesis of the fluorinated peptidomimetic 295 120
3.6.5 Solution phase synthesis of the inhibitors 121
3.7 Results and discussion 128
3.8 Conclusions 139
CHAPTER 4 EXPERIMENTAL PART 141
4.1 Instruments and general techniques 141
4.2 Synthesis of the compounds 143
SUMMARY 190
REFERENCES AND NOTES 196
ANNEX I Docking results 208
ANNEX II NMR Data 212
















Abbreviations
Ac = Acyl
AcOEt = Ethyl acetate
Ar = Aryl
Bn = Benzyl
Boc = tert-Butoxycarbonyl
Bu = Butyl
CAN = Cerium Ammonium Nitrate
Cbz = Benzyloxycarbonyl
CD = Circular Dichroism
COSY =Correlation spectroscopy
DIBAL-H =Diisobutylaluminium Hydride
DIPEA = Diisopropylethyl Amine
DMF = Dimethylformamide
DMSO = Dimethylsulfoxide
DNA = Deoxyribonucleic acid
EDC = Ethyl-N,N-dimethyl-3-aminopropylcarbodiimide
ee = Enantiomeric Excess
EI = Electronic impact
Eq. = equivalent
Fmoc = 9-Fluorenylmethoxycarbonyl
h = hours
HBTU = O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
HOBt = Hydroxybenzotriazol
HOAt = 1-hydroxy-7-azabenzotriazole
IR = Infrared spectroscopy
Me = Methyl
MeOH = Methanol
min = Minutes
m.p. = Melting Point
MS = Mass Spectroscopy
NMR = Nuclear Magnetic Resonance
NOE = Nuclear Overhauser Effect
PNA = Peptide Nucleic Acid
PG = Protecting Group
Py = Pyridine
PMB = para-Methoxybenzyl
RMSD = Root Mean Square Deviation
ROESY = Rotating Frame NOE Spectroscopy
RT = Room Temperature
TFE = Trifluoroethanol



















CHAPTER 1 ITRODUCTIO

1.1 Peptide generalities


Peptides (from the Greek πεπτίδια, "small digestibles") are short polymers formed from the
linking, in a defined order, of α-amino acids. The link between one amino acid residue and the
next is known as an amide bond or a peptide bond. Proteins are polypeptide molecules (or consist
of multiple polypeptide subunits). The convention is that peptides are shorter than 50 amino acids
residues and polypeptides/proteins are longer. Natural peptides and proteins are mainly composed
of 20 α-amino acids to which we can add a few other ones which are relatively rare in nature.
Amino acids are organic molecules which possess an amine and a carboxylic acid. α-amino acids
can present different lateral chains leading to molecules with completely different physical
properties. The simplest α-amino acid is the glycine, which is the only achiral amino acid.
Natural α-amino acids have been classified in five categories: acidic, neutral, basic, hydrophobic
and hydrophilic. When the amino acid is not A glycine, the C is a chiral center and natural α
amino acids are all present in the L-configuration in the nomenclature of Fischer. A few ones
extracted from exotic molluscs or in cell walls of some bacterias can be in a D-configuration but
their occurrence in nature is anecdotic in comparison to the supremacy of L-amino acids. (Figure
1)

COOH
R
H N H2
αH N COOH2 R
L-amino acid in Fischer representationα-amino acid
Figure 1

1