The Synthesis and Surface Studies of β-Amino Acids & β-Peptides

307 Pages·2007·6.06 MB·English

The Synthesis and Surface Studies
of β-Amino Acids & β-Peptides

A thesis submitted
in the partial fulfilment
of the requirements for the degree
of
Doctor of Philosophy in Chemistry
by
Kelly Helen Anderson

University of Canterbury
2007
Work in this thesis has appeared in the following
publications:

Brooksby, P. A.; Anderson, K. H.; Downard, A. J.; Abell, A. D., Langmuir 2006, 22, (22), 9304-
9312.

Gardiner, J.; Anderson, K. H.; Downard, A.; Abell, A. D., Journal of Organic Chemistry 2004,
69, (10), 3375-3382.
Table of Contents i
Table of Contents

Abstract ......................................................................................................................iii
Abbreviations......................................................................................................................vi
Acknowledgements.............................................................................................................ix
Chapter 1 Introduction.................................................................................................1
1.1 Electron Transfer in Proteins.................................................................................2
1.2 Conformation of Peptides......................................................................................4
1.3 Research Described in This Thesis......................................................................15
1.4 References...........................................................................................................16
Chapter 2 Electron Transfer and Surface Studies of Immobilised β-Peptides......18
2.1 Introduction.........................................................................................................19
2.2 Preparation and Electrochemical Analysis of β-peptide SAMs..........................35
2.3 Discussion of Electrochemical Analysis of β-Peptide SAMs.............................58
2.4 Conclusions and Future Work.............................................................................67
2.5 References...........................................................................................................69
Chapter 3 Cross Metathesis on Gold.........................................................................71
3.1 Introduction.........................................................................................................72
3.2 Synthesis of Olefins.............................................................................................77
3.3 Cross Metathesis Reactions on a Gold Electrode................................................81
3.4 Electrochemical Analysis....................................................................................83
3.5 Conclusions and Future Work.............................................................................90
3.6 References...........................................................................................................91
Chapter 4 Synthesis of Compounds Suitable for Surface Immobilisation.............93
4.1 Functionalised β-peptides....................................................................................94
4.2 Peptide Design and Retrosynthetic Analysis.....................................................103
4.3 Synthesis of Model Compounds........................................................................105
4.4 Synthesis of β-Peptides.....................................................................................116
4.5 Elucidation of Secondary Structure...................................................................130
4.6 Conclusions and Future Work...........................................................................143
4.7 References.........................................................................................................145
Chapter 5 Conformationally Constrained β-Amino Acids....................................147
5.1 Introduction.......................................................................................................148
5.2 Synthesis of Fluorinated β-Amino Acids..........................................................154
5.3 Synthesis of Cyclic β-Amino Acids using Organozinc Reagents.....................162
5.4 Synthesis of Cyclic β-Amino Acids from Allylglycine....................................170
5.5 Conclusion and Future Work............................................................................180
5.6 References for Chapter Five..............................................................................181
Chapter 6 Experimental............................................................................................184
6.1 General Methods and Procedures......................................................................185
6.2 General Synthetic Methods...............................................................................191
6.3 Experimental Work Described in Chapter 2.....................................................194
6.4 Experimental Work Described in Chapter 3.....................................................195
6.5 Experimental Work Described in Chapter 4.....................................................201
6.6 Experimental Work Described in Chapter 5.....................................................246
6.7 References for Chapter Six................................................................................267
Appendix A Electroanalytical Methods......................................................................269
Table of Contents ii
A.1 The Potentiostat.................................................................................................269
A.2 Electron Transfer Mechanisms at the Electrode................................................270
A.3 The Cyclic Voltammetry Technique.................................................................271
A.4 Cyclic Voltammetry Theory..............................................................................274
A.5 The Chronoamperometry Technique.................................................................277
A.6 Chronoamperometry Theory.............................................................................278
Appendix B ...................................................................................................................281
Appendix C Crystallographic Data.............................................................................282
C.1 Crystallographic data for cycloheptene-based β-amino acid 5.63.....................282
C.2 Crystallographic data for conjugate addition/fluorination product 5.43a..........287
C.3 References for the Appendix.............................................................................296
Abstract iii

Abstract

This thesis examines the synthesis of conformationally constrained β-amino acids and β-
peptides, and the electron transfer properties of the latter when immobilised on gold.
Additionally, cross metathesis on gold was investigated as a method for surface
functionalisation.

Chapter One introduces the concepts of electron transfer in nature, how it is facilitated by the
secondary structure in α-peptides, and why β-peptides might be useful for studying electron
transfer. This is followed by a discussion of the properties of β-peptides, including the enhanced
stability and variety of helical secondary structures and the greater potential for functionalisation
of the peptide backbone when compared to α-peptides. Finally, the conformational constraints
of ring-systems on cyclic amino acids is discussed, with reference to the stabilising effect of
these compounds on peptide secondary structures.

Chapter Two describes the electrochemical analysis of β-hexapeptides immobilised on gold. The
chapter is prefaced by a discussion of the important electron transfer mechanisms for peptides,
the fabrication of peptide-gold self-assembled monolayer (SAM) interfaces, and the electron
transfer in helical α-peptides. β-Peptides containing an electroactive ferrocene moeity were
immobilised on gold and studied using cyclic voltammetry and chronoamperometry. The latter
method was used to examine the dependence of the electron transfer rate on overpotential,
thereby determining the likely mode of electron transfer through the β-peptides SSβ Fc, Fcβ SS
6 6
and SC β Fc. These peptides exhibited very weak dependence on overpotential, characteristic
15 6
of electron transfer behaviour of an electron hopping mechanism (which is also thought to occur
in helical α-peptides). Both the dipole moment of the peptides and the structure of the sulfur-
linker group were found to be important in determining the rate of electron transfer. Conversely,
the equivalent α-peptide SSα Fc exhibited electron transfer behaviour characteristic of the less
6
efficient tunnelling mechanism, which is thought to operate in strand-like peptides.

Chapter Three examines the application of cross metathesis, using a Grubbs’ second generation
catalyst, as a means to functionalise olefin-terminated self-assembled monolayers on gold.
Abstract iv
Firstly, an introduction into the limited published research on cross metathesis on both planar
surfaces and nanoparticles is given. Olefin-terminated thiol 3.18, suitable for immobilisation on
gold, and solution phase olefin-terminated ferrocene 3.10 were synthesised as reactants for cross
metathesis studies. An analytical methodology was developed involving the cross metathesis of
surface-immobilised 3.18 with ferrocene 3.10 in dichloromethane, whereby the concentration of
electroactive cross metathesis product 3.22 was monitored electrochemically as a function of
time. The concentration of surface-immobilised product 3.22 was determined by integration of
the oxidation peak area and found to be highly dependent on both the concentration of
immobilised olefin reactant 3.18 and reaction time. Furthermore, the surface concentration of
ferrocenyl model disulfide 3.21 and thiol 2.18 decayed markedly upon addition of Grubb’s
catalyst, as revealed by the decrease in the oxidation peak area, which suggested that catalyst-
mediated desorption was occurring.

Chapter Four details the solution-phase synthesis of ferrocene- and thiol-functionalised β-
hexapeptides used in both the electron transfer studies described in chapter two, and in the
determination of secondary structure using circular dichroism and NMR techniques. The
synthesis of simple model compounds 4.14, 4.16 and 4.18 established the incompatibility of the
deprotection of methyl and benzyl ester protecting groups with protected-thiol and disulfide
linkers, leading to the use of N-hydroxysuccinmide-activated sulfur-linkers 4.20 and 4.22 in
further synthesis. A number of β-hexapeptides were synthesised by amide coupling of β-
tripeptides functionalised at the N- and C-termini. Structural studies of the methanol soluble β-
hexapeptide 4.60 suggested that the covalent attachment of ferrocene moeity to the C-terminus
of a β-peptide did not disrupt the formation of a 14-helix in solution. β-peptides containing
functionality at both the C- and N-termini (such as SSβ Fc, SSβ Et and acetyl-protected
6 6
SC β Fc) were not suitable for solution phase structural studies; however, molecular modelling
15 6
suggested that helical conformations are the most stable these β-peptides in solution phase.

Chapter Five outlines the synthesis of novel cyclic β-amino acids by two different general
synthetic routes. The first uses an efficient conjugate addition/fluorination reaction of α,β-
unsaturated esters with lithiated chiral secondary amines to prepare the novel cyclopentyl- and
cyclohexyl-based fluorinated β-amino acids 2.43a and 2.43b. The high diastereoselectivity of
this reaction, which introduces two stereocentres into the achiral unsaturated esters, is directed
by the configuration of the attacking amine. The second methodology utilizes the versatile ring-
closing metathesis reaction in the synthesis of novel cyclic β-amino acids. A stereoselective
Abstract v
trans-alkylation of olefinic β-amino acids gave the required β-dienes 5.62 and 5.77. Optimised
cyclisation yields were achieved with a Grubb’s 2nd generation catalyst for diene 5.62 and
Grubb’s 1st generation catalyst for diene 5.77, to give the trans-cycloheptyl- and cyclooctyl-
based β-amino acids 5.63 and 5.78, respectively. The attempted synthesis of cyclononyl-based
β-amino acid 5.87 using both catalysts yielded only cyclic dimer products 5.88 and 5.89. The
trans configuration of the 5.62 diene was confirmed by x-ray crystallography.

Chapter Six is an experimental chapter and outlines the electrochemical setup and analysis, and
the synthesis, purification and characterisation of compounds described in this thesis.
Abbreviations vi
Abbreviations

α (1) designates an amino acid of the general structure
NH CR CO H
2 2 2
(2) the activity coefficient
β (1) designates an amino acid of the general structure
NH CR CR CO H
2 2 2 2
(2) tunnelling parameter (Å-1)
η overpotential
Γ surface coverage
δ NMR chemical shift
Ac O
acetyl
Ala alanine
Bn benzyl
Boc tert-butoxy
br s broad singlet (in NMR)
CA chronoamperometry
CD circular dichroism
CM cross metathesis
CV cyclic voltammetry
d doublet (in NMR)
DCC N,N'-dicyclohexylcarbodiimide
DCM dichloromethane
DIPEA N,N-diisopropylethylamine
DMAP 4-(dimethylamino)pyridine
DMSO Dimethylsulfoxide
EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
Abbreviations vii
ET electron transfer
Et N triethylamine
3
EtOAc ethyl acetate
f F/RT where F, R, and T are the Faraday and gas constants, and
absolute temperature, respectively
Fc ferrocene
HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate
HOBT N-hydroxybenzotriazole
HRMS high resolution mass spectrometry
Hz hertz (in NMR)
J coupling constant (in NMR)
i initial current
0
k electron transfer rate (s-1)
et
k 0 standard electron transfer rate (s-1)
et
k kinetic prefactor
0
Leu leucine
m multiplet (in NMR)
min minute(s)
mp melting point
n the number of electrons transferred
NHS N-hydroxysuccinimide
NMR nuclear magnetic resonance
Ph phenyl
ppm parts per million
q quartet (in NMR)
R distance between hopping sites
1
RCM ring closing metathesis
Abbreviations viii
R donor-acceptor centre-to-centre distance
DA
ROESY rotational nuclear overhauser effect spectroscopy
rt room temperature
s singlet (in NMR)
SAM(s) self-assembled monolayer(s)
SCE saturated calomel electrode
t time
t triplet (in NMR)
TFA trifluoroacetic acid
TFE 2,2,2-trifluoroethanol
THF tetrahydrofuran
TLC thin layer chromotography
Val valine