Дисертації з теми "Constrained peptides"

In this thesis, constrained 7/8 helix has been developed to enhance the stability of 7/8 helical structure in polar and aqueous solvents for biological application. The synthesis of constrained 7/8 helical peptides has been achieved in two steps. In the first step, a 7/8 helical peptide chain comprising alternating α-L-amino acids and α-D-aminoxy acids was assembled by standard solution phase peptide synthesis protocol. In the second step, a covalent cross-linker as the conformational constraint was incorporated into 7/8 helical peptide at adjacent α-amino acid residues by consecutive intramolecular ring-closing olefin metathesis reaction and catalytic hydrogenation reaction. Conformational properties of constrained 7/8 helical peptides have been explored by applying NMR spectroscopy, theoretical calculation, and circular dichroism spectroscopy to three constrained tetrapeptides 2.2–2.4 and one non-cross-linked reference peptide 2.1. It was discovered that constrained 7/8 helical peptides with a saturated methylene chain as the covalent cross-linker maintained the structural feature of alternating N–O turns and γ-turns, and exhibited increased stability in both organic solvents and aqueous media. No obvious difference was observed for the covalent cross-linker with six, seven, or eight methylene units in improving the stability of 7/8 helix. The constrained 7/8 helix with enhanced structural stability was applied in the design of α-helix mimics. Based on the structure of helix D in the crystal structure of CD81 large extracellular loop, the putative receptor of Hepatitis C Virus envelop 2 (HCV E2) protein, constrained 7/8 helical hexapeptides 4.1 and 4.2 with free N-terminus were designed and synthesized in the form of TFA salt as the candidates of helix D mimics. It was expected that peptides 4.1 and 4.2 could act as the inhibitors of the interaction between helix D and HCV E2 to block HCV entry into cells. Unfortunately, the biological activity test on the ability of peptides 4.1 and 4.2 in HCV inhibition revealed that none of these peptides exhibited detectable inhibitory effect on the entry of HCV into cells and HCV replication at the concentration of 100 μM.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

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 SSβ₆Fc, Fcβ₆SS and SC₁₅β₆Fc. These peptides exhibited very weak dependence on overpotential, characteristic 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 sulfurlinker group were found to be important in determining the rate of electron transfer. Conversely, the equivalent α-peptide SSα₆Fc exhibited electron transfer behaviour characteristic of the less 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 catalystmediated 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 SSβ₆Fc, SSβ₆Et and acetyl-protected SC₁₅β₆Fc) were not suitable for solution phase structural studies; however, molecular modelling 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 ringclosing 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 cyclooctylbased 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.

Les travaux décrits dans ce manuscrit concernent la synthèse d’oligomères de γ-amino acides hétérocycliques contraints, appelés ATCs (acides 4-Amino-(méthyl)-1,3-Thiazole-5-Carboxyliques), leur application en catalyse énamine et leur étude structurale. Les monomères d’ATC sont construits autour d’un noyau thiazole inséré entre les carbones Cα-Cβ, permettant de limiter la valeur de l’angle dièdre ζ à 0°. La présence de deux points de substitution, sur le carbone γ asymétrique et en position 2 du noyau aromatique, permet une large diversification structurale des ATCs. Ainsi, plusieurs séries d’oligomères ont été synthétisées par couplages peptidiques sur support solide. Une étude structurale de ces oligomères par RMN, IR-TF, cristallographie RX et dichroïsme circulaire a démontré qu’ils adoptaient une structure en helice C9, résultant d’un réseau de liaisons hydrogène de type COi---NHi+2 s’établissant tout au long de la séquence. L’objectif du projet présenté ici vise à étudier l’impact de la conformation des architectures développées, à la fois sur la sélectivité et sur l’induction asymétrique dans la réaction de nitro-Michael pour trois réactifs différents. Le dernier axe de ce travail a été de développer une méthode de modélisation sous contraintes RMN spécifique à la génération de modèles tridimentionel d’oligomères d’ATCs
The work described in this manuscript is devoted to the synthesis of heterocyclic constrained γ-amino acids, named ATCs (4-Amino-(methyl)-1,3-Thiazole-5-Carboxylic acids), their application in enamine catalysis and their structural study. ATC monomers are built around a thiazole ring providing a conformational limitation around the Cα and Cβ at 0°. The presence of two diversification points both on the γ asymmetric carbon and on the position 2 of the aromatic ring, allows a large structural diversification of the ATCs. Therefore, several oligomers were synthesized using solid phase peptide synthesis. A structural study of these oligomers, employing NMR, FTIR, circular dichroism and crystallography RX, demonstrated that they adopt a C9-right-handed helix stabilized by a hydrogen bond pattern between COi---NHi+2 along the helix. The objective of the project presented in this manuscript was the design and the structural characterization of molecular edifices with predictable folding properties and the systematic study of structure-function relationships in the nitro-Michael addition reaction, for three different substrates. Eventually, the last part of this work focused on the development of a new methodology, specific to ATC-oligomers, to perform 3D-modelling studies using NMR refinement

The recent upsurge of interest in the peptide-based drug molecules has been accompanied by a great deal of attention to the design of stereochemically defined non-proteinogenic amino acids. As a continuous effort to develop efficient syntheses of χ-constrained amino acids in our group, we recently have developed a practical methodology for the asymmetric synthesis of substituted pyroglutamic acid, glutamic acid and proline analogues, which are of important use in examining the relationships between conformation and bioactivities of biologically important peptides (e.g. DPDPE, α-MSH). The key step in this method is an asymmetric Michael addition reaction between a chiral Ni(II)-complex of the glycine Schiff base (S)-NiGlyBPB, and derivatives of α,β-unsaturated carboxylic acids. This new method is the first highly diastereoselective, room temperature, organic base-catalyzed, asymmetric Michael addition reaction. Excellent chemical yields and diastereoselectivity, along with the simplicity of experimental procedure, renders the present method of immediate use for preparation of various novel beta-substituted pyroglutamic acids, glutamic acids and prolines. Decomposing the resulting addition products in acidic medium, followed by neutralizing with ammonia, gave optically pure substituted pyroglutamic; acids in good yields (>80%). The substituted pyroglutamic acids were converted to the corresponding substituted glutamic acids by hydrolysis in 6N HCl, or to substituted proline analogues by selective reduction of amide carbonyl group to a methylene group. Both novel substituted glutamic acids and prolines are being incorporated into biologically important peptide MT-II analogues for structure-activity studies.

Topographical constraint is the most powerful approach for the design of bioactive peptides to explore the bioactive conformation of crucial side-chain pharmacophores of amino acid residues in peptide-receptor recognition and signal transduction. Novel topographically constrained amino acids β-isopropylphenylalanine and 2',6'-dimethyl-2,3-methanophenylalanine have been designed and synthesized. Incorporation of the four optically pure β-isopropylphenylalanine stereoisomers into deltorphin I produced four peptide analogues of [β-iPrPhe]Deltorphin I with differentiated bioactivities. The most potent and selective analogue, [(2S,3R)-β-iPrPhe]Deltorphin I showed an IC₅₀ nM binding affinity, and a 29000 fold selectivity for the δ-opioid receptor over the μ opioid receptor. Combined molecular modeling and NMR studies indicated that the (2S,3R)-β-iPrPhe³ residue in the analogue favors the trans rotamer, and can induce the linear peptide to form a low-energy folded conformation which was proposed as the bioactive conformation for the δ-opioid receptor. Coupling four optically pure, conformationally constrained β-methyl-2',6'-dimethyltyrosine (TMT) with L-Tic formed four dipeptide analogues of TMT-L-Tic. The most potent and selective analogue, (2S,3R)-TMT-L-Tic showed 9 nM binding affinity and 4000 fold selectivity to the δ vs μ opioid receptor. The lowest-energy conformation of (2S,3R)-TMT-L-Tic was suggested to be the bioactive one in which TMT side chain is trans and Tic side chain is in a gauche (+) conformation. Bicyclic oxytocin antagonist [dPen¹, cyclo(Glu⁴ Lys⁸)]OT (BC-OT) (pA₂ = 8.10) is an excellent template to examine further topographical ideas. Substitution of Tyr² with the topographically constrained para-methoxy-β-methyl-2',6'-dimethyltyrosine (p-MeOTMT) amino acids produced two very potent antagonists [(2S,3S)-p-MeOTMT²]BC-OT (pA₂ = 8.26) and [(2R,3R)-p-MeOTMT²]BC-OT(pA₂ = 7.80), and two inactive analogues [(2S,3R)-p-MeOTMT²]BC-OT and [(2R,3S)-p-MeOTMT²]BC-OT. These interesting results can be attributed to the biased side-chain conformation, gauche(+) and gauche(-) in (2S,3S)-p-MeOTMT and (2R,3R)-p-MeOTMT respectively, and trans in both (2S,3R)-p-MeOTMT and (2R,3S)-p-MeOTMT residues. Rational design of non-peptide mimetics from peptide leads is still elusive. Based on the δ-opioid selective lead [(2S,3R)-TMT¹]DPDPE and SAR of δ-opioid selective ligands, the first generation of non-peptide mimetics have been designed and synthesized. The new lead SL-3111 showed binding affinity IC₅₀ = 8 nM, and over 2000 fold selectivity for the δ-opioid receptor over the μ receptor.

The synthesis of seventeen novel conformationally constrained analogues of the neurohypophyseal peptide hormone oxytocin is described. Synthesis of the peptides was accomplished using solid-phase synthesis techniques on either Merrifield or p-methyl-benzhydrylamine resin. Cleavage of peptides from the solid support and deprotection were carried out by either ammonolysis followed by treatment with sodium in liquid ammonia or anhydrous HF. Disulfide formation was accomplished by treatment of the deprotected peptide with aqueous potassium ferricyanide. Purification of the peptide analogues involved a combination of either partition and/or size exclusion chromatography followed by reverse-phase high-performance liquid chromatography. Several conformationally constrained unnatural amino acids were incorporated into the synthetic peptides. Two were prepared and incorporated as a mixture of isomers and the resulting peptides were separated and purified by HPLC. The types of analogues prepared fall into three categories: analogues incorporating conformational restrictions in positions 1 and 2; bicyclic oxytocin peptides; oxytocin antagonists with changes at the Asn⁵ residue. The peptides with conformational restrictions at position 1 or 2 are: [Tic²]OT, [DTic²]OT, [DTic²,Thr⁴]OT, [β-MePhe²]OT, [ΔPhe²]OT, [Cys(CH₂)₅¹,Phe²,Thr⁴,Orn⁸]OT and [Pen¹,DPhe²,Thr⁴,Orn⁸]OT. Bicyclic peptide analogues and their monocyclic precursors include: [Mpa¹,Lys⁴,Glu⁵]OT, [Mpa¹,Lys⁴,Glu⁵]OT, [Mpa¹,Glu⁴,Lys⁸]OT, and [Mpa¹,Glu⁴,Lys⁸]OT. Antagonists with changes in the Asn⁵ residue are: [Pen¹,DPhe²,Thr⁴,Thr⁵,Orn⁸]OT; [Pen¹,DPhe²,Thr⁴,Leu⁵,Orn⁸]OT; [Pen¹,DPhe²,Thr⁴,Asp⁵,Orn⁸]OT; and [Pen¹,DPhe²,Thr⁴,Tyr⁵,Orn⁸]OT. Biological assays of these analogues for oxytocic activity in the rat uterus model have shown one of the β-MePhe²-containing peptides, [L-threo-β-MePhe²]OT, to be a very potent agonist and one bicyclic, [Mpa¹,Glu⁴,Lys⁸]OT to be an extremely potent oxytocin antagonist. Initial biophysical investigations employing 250 MHz nuclear magnetic resonance spectroscopy were also undertaken in order to determine possible solution conformations of these peptide analogues.

Activator Protein-1 (AP-1) is an oncogenic transcription factor that is dysregulated in numerous human cancers, making it an attractive therapeutic target. AP-1 forms via interaction of cJun and cFos proteins, which intertwine to generate a ‘coiled coil’ (CC) structure. Thus, the cJun/cFos α-helical CC domains responsible for dimerisation are appealing targets for inhibiting AP-1 formation and activity. Helical peptide antagonists that sequester cJun can be derived from the cFos CC domain by selection of more optimal amino acids for increased binding affinity. Peptides can then be downsized and modified to improve therapeutic potential. Two approaches aimed to identify novel short peptides against cJun. The first was to covalently cyclise amino acid side chains in existing cFos-derived peptide “FosW”, with the aim of constraining FosW into a stable helix to allow downsizing without significant loss of binding structure and affinity. Using circular dichroism spectroscopy and isothermal titration calorimetry, a series of helix constrained peptides were characterised, from which a peptide was identified that retained 88 % of FosW binding affinity whilst being 22 % shorter, and which entered breast cancer cells in vitro, with preliminary data suggesting potential ability to inhibit AP-1 in cellulo. The second approach was to combine two existing high-throughput peptide selection systems, with the aim of benefitting from overlap in their strengths and weaknesses. Combination of in vitro CIS display and in cellulo Protein-fragment Complementation Assay successfully isolated a high affinity peptide from a hugely diverse library, and future refinements to further exploit this approach, particularly for short peptide selection, were formulated. Thus, molecules and techniques derived here may expedite the future development of therapies for cancers featuring AP-1 dysregulation.

Le but de cette thèse a concerné le développement de deux types de structures oligomériques contraintes, basées soit sur des dérivés non naturels d’acides aminés cycliques, soit sur des dimères cycliques de Peptide Nucleic Acids (PNA), en vue d’analyser leur tendance à adopter des conformations pré-organisées, imitant les conformations actives des protéines ou des acides nucléiques. La première partie de cette thèse a trait à l’élaboration de synthons clefs dérivant d’acides α-aminés cycliques non naturels (acides 4-oxopipécoliques 6-substitués), à leur utilisation potentielle dans la synthèse de peptidomimétiques contraints, homogènes et hétérogènes, ainsi qu’à leurs limites dans cette utilisation. Dans le cadre de ce travail, une nouvelle voie de synthèse permettant d’accéder à des résidus N-protégés d'acides 4-oxo-pipécoliques 6substitués a été mise au point. La deuxième partie de cette thèse concerne l’élaboration de dimères d’α-PNA (di-α-PNA) cycliques, dans lesquels les chaînes latérales de deux monomères d’α-PNA consécutifs sont «agrafées» via un pont lactame. Une stratégie de synthèse a tout d'abord été développée en phase liquide, puis appliquée à la synthèse en phase solide de di-α-PNA «agrafés» modèles, incorporant des bases nucléiques thymine
The purpose of this thesis concerned the development of two kinds of constrained oligomeric structures, based either on unnatural cyclic α-amino acids derivatives or on cyclic Peptide Nucleic Acid (PNA) dimers, in view of analyzing their propensity to adopt pre-organized conformations mimicking the active conformations of proteins or nucleic acids. The first part of this thesis reports on the elaboration of new cyclic αamino-acids (6-substituted 4-oxopipecolic acids) building blocks, their potential use in the synthesis of constrained homogenous and heterogeneous peptidomimetics as well as their limitations in this use. As part of this work, a new methodology has been developed for the synthesis of N-protected 6-substituted 4-oxo-pipecolic acids residues. The second part of this thesis reports on the elaboration of constrained α-PNA dimers (di-α-PNA), in which the side-chains of two consecutive α-PNA monomers are “stapled” via a lactam bridge. A synthetic orthogonal strategy has been first developed in liquid-phase then applied to the solid-phase synthesis of models “stapled” di-α PNA incorporating thymine nucleobases

Chemistry
Ph.D.
In the study of structurally restricted cyclic β-amino acids and peptides, methanopyrrolidine-5-carboxylic acids (MetPyr-5-acids), or 5-syn-carboxy-2-azabicyclo[2.1.1] hexanes, and derivatives were investigated. MetPyr-5-acids are a series of highly conformationally constrained β-proline derivatives, which belong to a novel category of β-amino acids utilized as building blocks for the synthesis of β-peptides. These β-peptides lack the backbone hydrogen bonds necessary for folding in the usual manner. Substituents and functional groups in this ring system were envisioned to impact the folding properties and functionalities of the corresponding β-peptides. In the present study, the analogues of MetPyr-5-acids with C6- substitutions were prepared, and the folding properties of their peptides were explored. To introduce different functionalities at C6 in MetPyr-5-acids, 6-syn-hydroxymethyl substituted derivatives were synthesized and were used as key intermediates. In the synthesis of this core structure, the major steps in their preparation included the Michael addition of benzyloxymethyl allyl amine to 3-butynone, followed by UV light irradiation of the diene to afford 5-acetyl-6-benzyloxymethyl-2-azabicyclo[2.1.1]hexane. Haloform (Br2/NaOH) oxidation of the acetyl group leads to the 6-substituted MetPyr-5-acid. Resolution of the racemate was achieved either by resolving (±)-6-syn-benzyloxymethyl-MetPyr-5-acid via a classical crystallization resolution method using (S)-(-)-α-methylbenzylamine, or by chiral preparative HPLC separation of (±)-6-syn-benzyloxymethyl-MetPyr-5-acid methyl ester. The absolute stereochemistry was confirmed by X-ray crystallography of a derivative. Novel analogs with a range of functionalities incorporated at the C6 position in MetPyr-5-acid were synthesized from 6-syn-hydroxymethyl-MetPyr-5-acid methyl ester, and include hydrophilic groups such as hydroxyl, amino, methyl ether, and hydrophobic groups, such as substituted phenyl groups and triazole. From the protected C6-substituted analogs of MetPyr-5-acids, peptide oligomers of C6-benzyloxymethyl-2,4-methanopyrrolidine-b-amino acid were prepared up to the length of octomer in high yields. This series of oligomers were characterized by circular dichroism (CD) and indicated enhanced order of folding uniformity for the tetramer and up, with increasing ordered folding for longer oligomers. The octomer exhibited minimal solvent effects, and was stable with increasing temperature up to 80 °C. Analysis by NMR of the iso-butyric amide capped monomer indicated a mixture of cis/trans conformation favoring the cis conformation. This was slightly different from the C6 unsubstituted iso-butyric amide derivative, which favored the trans conformation. For the dipeptide, the C6-benzyloxymethyl substitution increased the percentage of cis conformation of the dipeptide amide bond, but the major peptide had the trans conformation. This demonstrated that C6 substitutions could shift the cis/trans equilibrium towards the cis conformation. Longer oligomers showed ordered secondary folding structure as demonstrated by the increase in ellipticity per amino acid unit, but was too complicated to be determined by NMR analysis. Both the CD patterns and molecular model calculation predicted that the longer oligomers (tetramer and above) favor the trans conformation. This preference was driven by the backbone dipole effect. II. SYNTHESIS OF 2,2-DISUBSTITUTED PYRROLIDINE-3-CARBOXYLIC ACIDS Due to the perceived steric influence of 2,2-disubstitution in the pyrrolidine-3-carboxylic acid, it is believed that the adjacent amide/peptide bonds should result in a trans amide bond conformation. Because of the difficulty in introducing disubstitution at the hindered C2 position, the synthesis of such derivatives has not been successful. For this reason a new method was introduced to prepare novel derivatives, at the N- and C- termini of protected 2,2-dimethyl pyrrolidine-3-carboxylic acid, i.e., benzyloxycarbonyl protected 2,2-dimethylpyrrolidine-3-carboxylate. This procedure included the Michael addition of 2-nitropropane to dimethyl fumarate, followed by ring closure of the amino ester derived from reduction of the nitro ester providing the pyrrolidinone. Reduction of the pyrrolidinone to the pyrrolidine with borane finished 2,2-dimethylpyrrolidine-3-carboxylate in moderate overall yield. A preliminary set of two amides, iso-butyric amide and 3,5-dichlorobenzamide of this 2,2-dimethylpyrrolidine-3-carboxylate, were also prepared. NMR analysis of this pyrrolidine derivative suggested the amide bonds adopted the trans conformation. It was concluded that steric bulk of the 2,2-disubstitution favorably influenced the trans amide conformation. This demonstrated that trans amide conformation control of a β-proline amide was possible.
Temple University--Theses

A systematic practical method to prepare highly chi (χ)-constrained amino acids has been developed. It was found that increasing the size of R¹ (see figure) from H to Me to Et to n-Pr led to decreased reactivity of the starting complexes. In the case of R¹ as i-Pr, no alkylation was observed. With an increase of the size of R² from H to Me to Et to i-Bu, the reactivities of the alkyl bromides decreased. The starting Schiff bases had more effective stereocontrol at the α-carbon center than at the β-carbon center. The starting Schiff bases showed differential reactivity toward the racemic electrophile (kinetic resolution). Satisfactory differentiations were obtained at room temperature which makes this method synthetically useful. In the case of R¹ as H (NiGlyBPB), the thermodynamically-controlled stereoselectivity of alkylation was as high as 30:1. [DIAGRAM OMITTED]* A series of dipeptide analogues (TMT-Tic and DMT-Tic) were designed and synthesized to mimic the potent and highly selective delta-opioid receptor pentapeptide ligand-[(2S,3R)TMT¹]-DPDPE and thus to explore the topographical requirements for recognition of ligands at the opioid receptor through bioassays and NMR studies to facilitate the design of non-peptide compounds to be used as therapeutic agents for pain. (2S,3R)-TMT-L-Tic was found to have best binding affinities at the δ-opioid receptor in TMT-Tic series. In preliminary NMR studies, it was found that these designed peptide ligands have their own distinct conformations in the aqueous media. Meanwhile some modified non-peptide analogues of SL-3111 were prepared to continue our efforts to find effective non-peptide ligands for the δ-opioid receptor. More systematic studies are still ongoing using NMR and computational methods. *Please refer to dissertation for diagram.

Aberrant protein-protein interactions often result in disease, and as such, effective protein-protein interaction inhibitors are needed to mitigate the disease state. These interaction interfaces often involve secondary structural motifs, for example, an α-helix or β-sheet. Small molecule drugs are not well suited to inhibit protein-protein interactions however constrained peptides, have shown to have great therapeutic potential. Short peptides display little secondary structure in aqueous solution and as such, peptide sequences derived from a protein-protein interaction interface for use as a protein-protein interaction inhibitor, must be constrained into the native secondary structure. This can be achieved by installing a linker between the side-chains of two appropriately spaced amino-acids in the sequence. Many different linker chemistries have been designed and implemented with good biological results. However, these constrained peptide therapeutics are still restricted by traditional small molecule drug hurdles including cell permeability, protease degradation and the ability to visualise and track a molecule intracellularly. Linkers such as the all-hydrocarbon metathesis linker have shown great promise in reducing protease degradation and increasing cell permeability, however a fluorescent tag is still necessary to visualise a drug candidate. Here, a bimane linker is proposed as a new peptide linker to help overcome these limitations. Dibromobimane is reacted with thiol-containing amino-acid side chains to introduce a new fluorescent constraint in a series of model peptides. The reaction conditions with dibromobimane are optimised in solution to reveal that a buffered system is required for the cyclisation to occur efficiently. Optimal reaction conditions, determined by monitoring the increase of the fluorescent product, were 0.5 mg/ml peptide in 10 mM PBS with one equivalent of dibromobimane. The reaction was shown to be facile and versatile; in this thesis an array of peptides with varied sequence length, constraint length and amino-acid composition were cyclised under the same conditions, all reaching reaction completion in under 30 minutes. Additionally, these same conditions were applied successfully to react monobromobimane with series of short peptides. Cyclisation on reaction with dibromobimane, was also demonstrated on-resin with similar efficiency. The fluorescent properties of the resultant peptides were then explored to reveal that pH does not affect the observed fluorescence however a longer peptide length resulted in greater fluorescence intensity. Furthermore, acyclic mono-bimane-functionalised peptides displayed lower fluorescence intensity than the bimane-cyclised counterparts. The fluorescence of the bimane cyclised peptide could be detected as low as 10 nM on a plate reader, which is expected to further improve on a more sensitive instrument. The secondary structure of a series of tri- and penta-peptides were investigated through CD and NMR techniques. It was deduced that the bimane linker can induce β-strand like structure in an i-i+2 constrained peptide; in contrast an i-i+4 constrained pentapeptide with homocysteine in the 1 and 5 positions results in a 3₁₀ helical like structure. β-alanine containing analogues of these peptides were also synthesised and showed minimal structure.This work outlines the synthesis of macrocyclic peptides containing a peptide constraint, in the form of a fluorescent bimane, both in solution and on-resin to produce cyclised peptides. The fluorescent properties of the resultant peptides have been shown to be biologically compatible with great fluorescence sensitivity. Furthermore, different secondary structure can be introduced by simply alterations of the constraint length from i-i+2 to i-i+4. This work provides a foundation on which to design new fluorescent bimane-cyclised peptide-based protein-protein interaction inhibitors.
Thesis (M.Phil.) -- University of Adelaide, School of Physical Sciences, 2017.

A limited number of drug targets can be exploited by conventional drug-like compounds as the vast majority of disease-associated targets are involved in protein-protein interactions (PPI). PPI targets possess binding surfaces that lack a well-defined hydrophobic pocket amenable for binding to small drug-like compounds. A new class of therapeutics that has shown great potential at modulating PPI are macrocyclic peptides, particularly for their ability to bind to large and topologically complex protein surfaces as well as their potential to access intracellular targets. However, the efficiency of macrocyclic peptides at mediating PPIs and permeating cell membranes is conformation dependent. Here, I describe the role of peptide conformation on target recognition using three clinically relevant PPI targets: the Kelch like ECH Associated Protein-1 (KEAP1), (Chapter Two and Chapter Three); the RET receptor tyrosine kinase (Chapter Four); and β-catenin (Chapter Five). Guided by published X-ray crystal structures, peptides derived from PPI epitopes were designed and structurally constrained to mimic the conformation of the natural PPI recognition motif. In Chapter Two, I report the development of a cyclic heptapeptide derived from the transcription factor Nuclear Factor (Erythroid-derived 2)-Like 2 (Nrf2) with similar affinity for KEAP1 as native Nrf2 through conformational optimization of a linear Nrf2-derived heptapeptide. Efforts to improve the potency and physicochemical properties of the cyclic heptapeptide are discussed in Chapter Three. In Chapter Four, I describe the design of dimeric peptides as tool compounds to investigate the mechanism by which the interaction between glial cell-line derived neurotrophic factor family ligands (GFLs) and GPI-linked co-receptors, GFRα, induce RET signaling. These peptides were derived from the β-sheet regions of GFLs, GDNF and ART, that interact with GFRα1 and GFRα3, respectively. Peptide cyclization and the introduction of a β-turn promoting motif yielded GFL mimetic peptides with stronger affinity for GFRα. Lastly, Chapter Five focuses on exploring the scope of i, i+4 carbamate and amino-staples as a novel peptide stapling system to stabilize α-helical peptides. An axin-derived α-helical peptide that disrupts the β-catenin/TCF4 interaction was used as a model to determine the effect of peptide α-helical stabilization on binding affinity for β-catenin.
2023-07-07T00:00:00Z

博士
國立東華大學
化學系
102
Synthesis of a naturally occurring cyclic tetrapeptide cyclo(Gly-L-Ser-L-Pro-L-Glu) [cyclo(GSPE)] was performed and crystal structure of it was obtained. X-ray crystal structure of cyclo(GSPE) showed a cis-trans-cis-trans (two transoid amide bonds between Gly-Ser, Pro-Glu and two cisoid amide bonds between Ser-Pro, Glu-Gly) peptide bond sequence. The conformation of synthesized cyclo(GSPE) fixes the coordination to lead ion in a 1:1 ratio. This cyclo(GSPE)-Pb complex was constructed as an asymmetric 3D network in the crystalline state. The polymerization of a rigid asymmetric cyclic tetrapeptide in the presence of a heavy metal ion represents the first example of a new class of macrocyclic complexes. Cyclo(GHPE), an analogue of the natural product, was also synthesized. A few unprotected α-cyclic tripeptides (CtPs) have been previously reported. Four new CtPs, cyclo(L-Ser-L-Ser-L-Ser)OBn, cyclo(L-Glu-L-Glu-L-Glu), cyclo(L-Ser-L-Pro-L-Glu), cyclo(L-Ser-L-Pro-L-Ser), cyclo(L-Ser-L-Pro-Gly) that consist of either protected glutamate (E(OBn)), protected serine (S(Bn)), or proline (P) were prepared from corresponding linear tripeptides. We were able to synthesize cyclo(E-E-E) and cyclo(S-P-E) by regioselective enzymatic hydrolysis of α-methyl or benzyl ester on glutamate of corresponding linear precursors Boc-E(OBn)-E(OBn)-E(OBn)(OMe) and Boc-S(Bn)-P-E(OBn)2. An attempt was also made to synthesize CtPs that contained unprotected glycine (G). Although cyclo(S-P-G) was obtained, all the attempts to synthesize the reported natural product cyclo(G-P-E) failed. Since NMR patterns of the natural product are similar to that of synthesized cyclic hexapeptide cyclo(G-P-E)2, we propose that the reported natural product may be a cyclic hexapeptide. Four water-soluble C3-symmetric cages containing α-cyclic tripeptide were efficiently synthesized. Two of the cages are 1,2,3-triazole containing big cages. The key steps include a click reaction to incorporate L-glutamic or L-aspartic acids to a tripodal linker and a unique one-pot cyclotrimerization. Other two cages were amide linked small cages. Glutamyl small cage showed intense blue color, which changes with pH. Aspartyl small cage was able to encapsulate three molecules of D2O or water. It also showed five times selectivity towards D2O molecule.

Structural studies of peptides are of great importance in developing novel and effective biomaterials ranging from drugs and vaccines to nano materials with industrial applications. In addition, they provide model systems to study and mimic the protein conformations. The ability to generate folded intramolecularly hydrogen bonded structures in short peptides is essential for peptide design strategies, which rely on the use of folding nuclei in the construction of secondary structure modules like helices and β-hairpins. In these approaches, conformational choices at selected positions are biased, using local stereochemical constraints, that limit the range of accessible backbone torsion angles. X-ray crystallographic studies of designed peptides provide definitive proof of the success of a design strategy, and provide essential structural information that can be utilized in the future design of biologically and structurally important polypeptides. Recent trends in peptide research focus on the incorporation of β-, γ- and higher homologs of the α-amino acid residues in designed peptides as they confer more proteolytic stability to the polypeptides. X-ray crystallographic studies of such modified peptides containing non-protein residues are essential, since information on the geometric and stereochemical properties of modified amino acids can only be gathered from the systematic structural studies of synthetic peptides incorporating them. This thesis reports a systematic study of the structures and conformations of amino acid derivatives and designed peptides containing stereochemically constrained α-, β- and γ-amino acid residues and the structural studies of polymorphic peptide helices. The structures described in thesis contain the Cα,α-dialkyalted α-residues α-aminoisobutyric acid (Aib) and 1-aminocyclohexane-1-carboxylic acid (Ac6c), the β-amino acid residue 1-aminocyclohexane acetic acid (β3,3Ac6c) and the γ-amino acid residue 1-aminomethylcyclohexaneacetic acid (gabapentin, Gpn). The crystal structure determination of peptides incorporating conformationally constrained α-, β- and γ- amino acid residues permitted the characterization of new types of hydrogen bonded turns and polymorphs. The studies enabled the precise determination of conformational and geometric parameters of two ω-amino acid residues, gabapentin and β 3,3Ac6c and provided detailed information about the conformational excursions possible for peptide molecules. This thesis is divided into 10 chapters. Chapter 1 gives a general introduction to the stereochemistry of the polypeptide chain, description of backbone torsion angles of α- and ω- amino acid residues and the major secondary structures of α-peptides, β-peptides, γ-peptides and hybrid peptides. A brief introduction to polymorphism and weak interactions, in particular aromatic interactions, is also provided, followed by a discussion on X-ray diffraction and solution to the phase problem. Chapter 2 describes the crystal structures of gabapentin zwitterion and its eight derivatives (Ananda, Aravinda, Vasudev et al., 2003). The crystal structure of the gabapentin zwitterions determined in this study is identical to that previously reported (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643). Eight of the nine achiral compounds crystallized in centrosymmetric space groups P21/c, C2/c or Pbca, while one derivative (Tos-Gpn-OH) crystallized in non-centrosymmetric space group Pna21 with four independent molecules in the asymmetric unit.The structural studies presented in this chapter reveal that the geminal substituents on the Cβ atom limits the values of dihedral angles θ1 and θ2 to ±60°, resulting in folded backbone conformations in all the examples. Intramolecular hydrogen bonds with 7-atoms in the hydrogen bond turn (C7) are observed in three derivatives, gabapentin hydrochloride (GPNCL), Boc-Gpn-OH (BGPNH) and Piv-Gpn-OH (PIVGPN), while a 9-atom hydrogen bonded turn (C9) is observed in Ac-Gpn-OH (ACGPH). Unique structural features, such as an unusual anti conformation of the COOH group (in ACGPH) and positional disorder of the cyclohexane ring (in BGPNN), indicating the co-existence of both the interconvertible chair conformations, are revealed by the crystal structure analyses. Chapter 3 describes the structural characterization of novel hydrogen bonded conformations of homo oligomers of Gpn. The crystal structures of three peptides, Boc-Gpn-Gpn-NHMe (GPN2), Boc-Gpn-Gpn-Leu-OMe (GPN2L) and Boc-Gpn-Gpn-Gpn-Gpn-NHMe (GPN4) provide the first crystallographic characterization of two new families of polypeptide structures, the C9 helices and C9 ribbons (Vasudev et al., 2005, 2007), in which the molecular conformations are stabilized by contiguous C9 turns formed by the hydrogen bonding between the CO group of residue (i) and the NH group of residue (i+2). The C9 hydrogen bond is characterized by a specific combination of the four torsion angles for the Gpn backbone, with the torsion angles θ1 and θ2 adopting g+/g+ or g /g- conformations. The structural analysis also permits precise determination of hydrogen bond geometry for the C9 structures, which is highly linear in contrast to the analogous γ-turn hydrogen bonds in α-peptides. A comparison of the backbone conformations in the three peptides reveals two classes of C9 hydrogen bonded secondary structures, namely C9 helices and C9 ribbons. The packing arrangement in these γ-peptides follows the same patterns as the helix packing in crystals of α-peptides. Chapter 4 describes ten crystal structures of short hybrid peptides containing the Gpn residue (Vasudev et al., 2007). In addition to the C7 and C9 hydrogen bonded turns which are defined by the backbone conformations at the Gpn residue, hybrid turns defined by a combination of backbone conformations at the α and γ-residues or at the β and γ-residues have been determined. Peptides Boc-Ac6c-Gpn-OH (ACGPH), Piv-Pro-Gpn-Val-OMe (PPGPV) and Boc-Val-Pro-Gpn-OH (VPGPH) reveal molecular conformation stabilized by intramolecular C9 hydrogen bonds, while Boc-Ac6c-Gpn-OMe (ACGPO) and Boc-Gpn-Aib-OH (GPUH) are stabilized by a C7 hydrogen bonded turn at the Gpn residue. An αγ hybrid turn with 12 atoms in the intramolecular hydrogen bonded rings (C12 turns) has been observed in the tripeptide Boc-Ac6c-Gpn-Ac6c-OMe (ACGP3), while βγ hybrid turns with 13 atoms in the hydrogen bonded ring (C13 turns) have been characterized in the tripeptides Boc-βLeu-Gpn-Val-OMe (BLGPV) and Boc- βPhe-Gpn-Phe-OMe (BFGPF). The two βγ C13 turns belong to two different categories and are characterized by different sets of backbone torsion angles for the β and γ residues. A γα C10 hydrogen bond, which is formed in the N→C direction (NHi ••• COi+2), as opposed to the regular hydrogen bonded helices of α-peptides, has also been observed in BFGPF. The Chapter provides a comparison of the backbone torsion angles of the Gpn residue in various hydrogen bonded turns and a brief comparison of the observed hydrogen bonded turns with those of the α-peptides. Chapter 5 describes the crystal structures of three αγ hybrid peptides which show C12/C10 mixed hydrogen bond patterns (Vasudev et al., 2007, 2008a; Chatterjee, Vasudev et al.,2008a). The insertion of gabapentin in the predominantly α-amino acid sequences in Boc-Ala-Aib-Gpn-Aib-Ala-OMe (AUGP5) and Boc-Leu-Gpn-Aib-Leu-Gpn-Aib-OMe results in the observation of helices stabilized by αα C10 (310-turn) and αγ C12 turns. The tetrapeptide Boc-Leu-Gpn-Leu-Aib-OMe reveals a novel conformation, stabilized by C12 (αγ) and C10 (γα) hydrogen bonds of opposite hydrogen bond directionalities. The conformations observed in crystals have been extended to generate C12 helix and C12/C10 helix with alternating hydrogen bond polarities in ( αγ)n sequences. The structure determination of three crystals, providing five molecular conformations, presented in this chapter provides the first crystallographic characterization of two types of helices predicted for the regular αγ hybrid peptides from theoretical calculations. The crystal structure of Boc-Ala-Aib-Gpn-Aib-Ala-OMe also provides an example for the co-existence of left-handed and right-handed helix in the asymmetric unit. Chapter 6 describes the structural studies of αγ hybrid peptides containing Aib and Gpn residues, and is divided into two parts. The first part presents the crystal structure analysis of peptides of sequence length 2 to 4, with alternating Aib and Gpn residues, and illustrates the conformational variability in αγ hybrid sequences as evidenced by the observation of conformational polymorphs (Chatterjee, Vasudev et al., 2008b; Vasudev et al., 2007; Ananda, Vasudev et al., 2005). The peptide Boc-Gpn-Aib-NHMe (GUN), Boc-Aib-Gpn-Aib-OMe (UGU), Boc-Gpn-Aib-Gpn-Aib-OMe (GU4O), Boc-Aib-Gpn-Aib-Gpn-OMe (UG4O) and Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), all of which are potential candidates for exhibiting αγ C12 hydrogen bonds, reveal molecular conformations stabilized by diverse hydrogen bonded turns such as C7, C9, C12 and C17 in crystals. The conformational heterogeneity in this class of hybrid peptides is further evidenced by the observation of three polymorphs in the monoclinic space group P21/c for the tetrapeptide Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), providing four independent peptide molecules adopting two distinct backbone conformations. In one polymorph, C12 helices terminated with an unusual three residue ( γαγ) C17 turn is observed, while the unfolding of helical conformation by solvent insertion into the backbone is observed in the other two polymorphs. The studies indicate the possible utility of Gpn residue in stabilizing locally folded conformations in the folding pathway, thus permitting their crystallographic characterization in multiple crystal forms. A discussion of the structural and conformational features of Gpn residues determined from all the crystal structures is presented in the Chapter, along with a φ-ψ plot for the Gpn residue. Part 2 of Chapter 6 describes the crystal structures of two octapeptides, Boc-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-OMe (GU8) and Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe (LFVUG8), featuring C12 turns at the Aib-Gpn segments (Chatterjee, Vasudev et al., 2009). GU8 folds into a C12 helix flanked by C9 hydrogen bonds at both the termini, while LFVUG8 adopts β-hairpin conformation with a chain-reversing C12 turn at the central Aib-Gpn segment. A remarkable feature of the Aib-Gpn turn in the β-hairpin structure is the anti conformation about the Cβ-Cα (θ2) bond, which is the only example of a Gpn residue not adopting gauche conformation for both θ1 and θ2. The crystal structures of the two peptides, mimicking the two major secondary structural elements of α-peptides in hybrid polypeptides, permits a comparative study of the mode of molecular packing in crystals of α-peptides and hybrid peptides. The chapter also discusses theoretical calculations on αγ hybrid sequences, which reveal new types of C12 hydrogen bonded turns. Chapter 7 describes the crystal structures of conformationally biased tert-butyl derivatives of Gpn. The crystallographic characterization of the E (trans) and Z (cis) isomers of the residue,three protected derivatives and a tripeptide provides examples of C7 and C9 hydrogen bonded conformations, suggesting that the C7 and C9 hydrogen bonds can be formed by Gpn residues with both the chair conformations of the cyclohexane ring. Chapter 8 describes the systematic structural studies of the derivatives and peptides of the stereochemically constrained β- amino acid residue, β3,3Ac6c (Vasudev et al., 2008c). The backbone torsion angles φ and θ adopt gauche conformation in majority of the examples, owing to the presence of a cyclohexane ring on the Cβ atom. In contrast to Gpn, β3,3Ac6c does not show strong preference for adopting intramolecularly hydrogen bonded conformations. Of the 16 crystal structures determined, intramolecular hydrogen bonds involving the β-residue are observed only in 4 cases. The amino acid zwitterion (BAC6C), the hydrochloride (BACHCL) and the dipeptide Boc-β3,3Ac6c-β3,3Ac6c-NHMe (BAC62N) form N-H•••O hydrogen bonds with 6-atoms in the hydrogen bond ring (C6 turns). An αβ hybrid C11 hydrogen bonded turn is characterized in the dipeptide Piv-Pro-β3,3Ac6c-NHMe, which is distinctly different from the C11 hydrogen bonds observed in αβ hybrid peptide helices. Several unique structural features such as a dynamic disorder of the hydrogen atom of the carboxylic acid group (in BBAC) and cis geometry of the urethane bond (in BBAC, BAC62N and BPBAC) have been observed in this study. A comparison of the backbone conformations of β3,3Ac6c with other β- amino acid residues is also provided. Chapter 9 describes the crystallographic characterization of a new polymorph of gabapentin monohydrate and crystal structures of the zwitterions of E and Z isomers of tert-butylgabapentin and its hydrochloride and hydrobromide (Vasudev et al., 2009). A comparison of the crystal structures of the monoclinic form (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643) of gabapentin monohydrate and the newly characterized orthorhombic form reveals identical molecular conformations and intermolecular hydrogen bond patterns in both the polymorphs. The two polymorphs show differences in the orientation of molecules constituting a layer of hydrophobic interactions between the cyclohexyl side chains. A comparison of the packing arrangements of the zwitterionic amino acid molecules in the crystal structures of gabapentin monohydrate, the tert-butyl derivatives and other co-crystals of gabapentin that had been characterized so far, is provided which would facilitate prediction of new polymorphs of the widely used drug molecule, Gpn. Chapter 10 describes the crystallization of α-peptide helices in multiple crystal forms (Vasudev et al., 2008b). Crystal structures of two peptides, Boc-Leu-Aib-Phe-Phe-Leu-Aib-Ala-Ala-Leu-Aib-OMe (LFF), Boc-Leu-Aib-Phe-Ala-Leu-Ala-Leu-Aib-OMe (D1) in two crystal forms and the crystal structure of a related sequence, Boc-Leu-Aib-Phe-Ala-Phe-Aib-Leu-Ala-Leu-Aib-OMe (D10) permit an analysis of the molecular conformation and packing patterns of peptide helices in crystals. The two polymorphs of LFF, crystallized in the space groups P21 and P22121, reveal very similar molecular conformation (α/310-helix) in both the polymorphic crystals; the two forms differ significantly in the pattern of solvation. The crystal structure determination of a monoclinic (P21) and an orthorhombic polymorph (P21212) of D1 provides five different peptide conformations, four of which are α-helical and one is a mixed 310/α-helix. The crystal structure determination of the three peptides provide an opportunity to compare the nature and role of aromatic interactions in stabilizing molecular conformation and packing and its significance in the observation of polymorphism. An analysis of the Cambridge Structural Database and a model for nucleation of crystals in hydrophobic peptide helices are also discussed.

This thesis discusses the design and production of peptides with side chain linkers that are intended to bind to the F3 domain of talin. The talin F3 domain was targeted as it is involved in the activation of integrin membrane proteins present in platelets. The over activation of these integrins can result in clotting within the blood vessels causing heart disease, however, current medication targeting integrin have negative side effects. The design and synthesis of short peptides based on the sequence of the β3 integrin tail that binds to the F3 domain of talin is presented. The binding affinity of peptides to the talin F3 domain was tested using NMR titrations to reveal the ideal location for the linker in the production of potential therapeutics which target integrin activation. Side chain linked peptides with high helical content have previously been shown to improve binding affinity. This drove investigation of side chain constrained peptides to increase their helical content, and thus, their binding affinity to talin F3 domain and cellular uptake. It is demonstrated that side chain linkers are effective in stabilising the helical structure of the short peptides. When incorporated in the β3 integrin sequence in specific locations, lactam linkers improved binding affinity of these peptides to the talin F3 domain. Additionally, all-hydrocarbon and triazole linkers enhanced the peptide’s cellular uptake when compared to the native peptide of this sequence. The position and type of side chain linkers were investigated. The result of which showed that the position of the linker had a significant impact on the binding affinity to talin. The lactam linker between residues in positions 725 and 729 created a peptide (7) with the highest binding affinity. The cell penetration of peptides with different linker types was tested using NIH 3T3 mouse cells, and HEK298 cells. A number of side chain linkers were tested with the triazole linker producing the most α-helical peptide, and the all-hydrocarbon linker producing peptides with the greatest cellular uptake.
Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2018

Chapter One introduces the concept of peptide 'secondary structure' with an emphasis on β-strand geometry in macrocycles. This structural design is crucial for targeting different proteases. The significance of the macrocylic β-strand ‘bioactive’ conformation is discussed in detail. In particular the exploitation of the conformationally constrained peptidomimetic macrocylic backbone, which is constrained by a number of synthetic approaches to lock the ‘bioactive’ conformation in place. Chapter Two describes simple and scalable methodology for the preparation of N-Cbz protected amino acids by reaction with Cbz-Cl which uses a mixture of aqueous sodium carbonate and sodium bicarbonate to maintain the appropriate pH. This method proceeds without the formation of by-products. The method is extended to large scale preparation of an intermediate zofenopril, an ACE inhibitor. Chapter Three describes new peptidic templates constrained into a β-strand geometry by linking acetylene and azide containing P₁ and P₃ residues of a tripeptide by Huisgen cycloaddition. The conformations of the macrocycles are defined by NMR studies and those that best define a β-strand are shown to be potent inhibitors of the protease calpain. The β-strand templates presented and defined here are prepared under optimized conditions and should be suitable for targeting a range of proteases and other applications requiring such geometry. Chapter four describes a new approach to non-covalent peptide-based nanotubular or rodlike structures, whereby the monomeric units are preorganised into a β-strand geometry that templates the formation of an extended and unusual parallel β-sheet rod-like structure. The conformational constraint is introduced by Huisgen cycloaddition to give a triazolebased macrocycle, with the resulting self-assembled structures stabilized by a well-defined series of intermolecular hydrogen bonds. Chapter Five the 26S proteasome has emerged over the past decade as an attractive therapeutic target in the treatment of cancers. Here, we report new tripeptide aldehydes that are highly specific for the chymotrypsin-like catalytic activity of the proteasome. These new CT-L specific proteasome inhibitors demonstrated high potency and specificity for cancer cells, with therapeutic windows superior to those observed for benchmark proteasome inhibitors, MG132 and Bortezomib. Constraining the peptide backbone into the β-strand geometry was associated with decreased activity in vitro and reduced anticancer activity, suggesting that the proteasome prefers to bind a conformationally flexible ligand. Using these new proteasome inhibitors, we show that the presence of an intact p53 pathway significantly enhances cytotoxic activity, thus suggesting that this tumor suppressor is a critical downstream mediator of cell death following proteasomal inhibition. Chapter Six peptide derived protease inhibitors represent an important class of compounds with the potential to treat a wide range of serious medical conditions. Herein we describe the synthesis of a series of triazole containing macrocylic protease inhibitors preorganised in a β-strand conformation and evaluate their selectivity and potency against a panel of protease inhibitors. A series of acyclic azido-alkyne-based aldehydes is also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards Calpain II, Cathepsin L and S and the 26S proteasome chymotrypsin-like activity. Importantly, the first examples of potent and selective inhibitors of Cathepsin S were identified and shown to adopt a well-defined β-strand geometry by NMR, X-ray and molecular docking studies. Chapter Seven describes simple and efficient methodology for the selective acylation and alkylation of biotin at its 3′-nitrogen. This methodology is used to prepare of other biotin derivatives.
Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, 2012

Research undertaken in this thesis focuses on electron transfer in peptides constrained into either a 3₁₀-helical or a β-strand conformation in order to progress the field of molecular electronics. Chapter One: Natural proteins have evolved to promote electron transfer in many biological processes. However, their complex conformational nature inhibits a thorough investigation, so in order to study electron transfer in proteins, simple peptide models containing redox active moieties present as ideal candidates. Chapter One introduces the importance of secondary structure characteristic to proteins/peptides, and its relevance to electron transfer. The proposed mechanisms responsible for such electron transfer are discussed, along with the various approaches used to further constrain the peptides into their geometric conformations. The methods used to characterize the conformation of all peptides synthesized throughout this thesis are outlined, as are details of the electrochemical techniques used to investigate their electronic properties. A literature review describing several factors that have been shown to influence electron transfer in peptides, and a brief summary of molecular electronics follows. Chapter Two: Two 3₁₀-helical peptides were synthesized, one constrained via a covalent side-chain staple using Huisgencycloaddition, and the other a linear analogue. Both peptides contain a redox active terminal ferrocene moiety, and were separately attached to a single walled carbon nanotube (SWCNT)/gold electrode array for electrochemical analysis. The effect of backbone rigidity imparted by the side-bridge constraint was revealed, which was shown to restrict the necessary torsional motions that lead to facile intramolecular electron transfer along the peptide backbone. High level calculations were used to support the electrochemical observations. Chapter Three: A series of peptides constrained into either a 3₁₀-helix or β-strand conformation were synthesized, each containing a varied number of electron rich alkene side chains. The ability of the alkene(s) to facilitate electron transfer through the peptides by exploiting a hopping mechanism, and thus act as a “stepping stone” was investigated. Ring closing metathesis was used to further rigidify the backbones of a helical and a β-strand peptide via side chain tethers. The ensuing saturated and unsaturated compounds were electrochemically interrogated in order to explore any possible interplay between the effects of the alkene side-chains and backbone rigidity. High level calculations were conducted to verify the observed electrochemical data. Chapter Four: Two β-strand peptides were synthesized, one constrained via a covalent side-chain staple using Huisgen cycloaddition, and the other a linear analogue. Both peptides contain a redox active terminal ferrocene moiety, and were separately attached to a SWCNT/gold electrode array for electrochemical analysis. The charge transfer pathway was determined to be intramolecular by measuring the electron transfer rate at various concentrations of the constrained peptidebound to the electrode. This pathway is analogous to charge transfer through a molecular junction involving a single peptide. Theoretical conductance simulations were then undertaken using two peptide analogues in order to establish a link between the electrochemical observations and conductance measurements through a molecular junction. Chapter Five: Two macrocyclic peptides were synthesized, one constrained into a 3₁₀-helical conformation by linking its i to i+3 residues to form a lactam bridge, and the other constrained into a β- strand geometry via a lactam-bridge tether, linking its i to i+2 residues. These peptides were chosen in order to define the role of the amide bond in a lactam bridge constraint. Direct linear analogues of each were used to establish the effect on electron transfer from a terminal amide bond located in an untethered side-chain. High level calculations were also conducted in order to elucidate the mechanism(s) responsible for electron transfer in each of the linear and macrocyclic helical peptides.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Physical Sciences, 2015.

This thesis entitled “Design and Synthesis of Peptidomimics Constrained in Helical and Sheet Conformations Using a Novel Covalent Surrogate for the Peptide Main Chain Hydrogen Bond” is divided into six chapters. Chapter 1: Introduction to Ordered Conformations of Peptides and Strategies for Constraining Short Peptides in Ordered Conformations. The first chapter describes the different types of protein secondary structures and introduces the various prominent strategies developed thus far to constrain short peptides in ordered secondary structure-like conformations, with specific emphasis on helical and parallel β-sheet folds. Chapter 2: Design of Structure and General Methodology for the synthesis of Novel H-Bond Surrogate Constrained Cyclic α-Helical Mimics Here we develop the first design of the propyl linker as a covalent surrogate for the peptide H-bond. The first synthetic methodology is described for the synthesis of constraining shortest peptide sequences (tripeptides) in α-helix-like conformations. The Macrolactamization strategy proved to work best as the final step for cyclization. All residues of the turn are completely retained in the constrained sequence, unlike any other earlier method. More importantly, there are no metal involved as catalysts in any of the synthetic transformations, hence removing the problem of metal-bound cyclic structures – which have otherwise rendered these structures non-usable as drug leads in the earlier models. Gly-rich peptides have been constrained as extreme cases of highest chain entropy and least helix propensity. Both secondary and tertiary amide containing peptides have been synthesized using this protocol. Note that the macrolactamization was found to be better than the Fukuyama-Mitsunobu N-alkylation protocol for the final cyclization step. Chapter 3: Synthesis of C-terminal Extended HBS-Constrained Helical Turn Mimics – Validation of the Versatility of Current synthetic protocol The developed cyclization protocol is extended towards the synthesis of C-terminal extended α-helical turn mimics using a solution phase peptide synthesis procedure. Peptides which extend belong the helical turn by a high entropy Gly-residue at the C-terminal are synthesized. The versatility of the synthetic methodology to accommodate sterically constrained amino acid residues – in the form of phenylalanine residue – at any of the positions i+1, i+2 or i+3 of the constrained helical turn is demonstrated. The synthesized are easily isolated without need for column chromatography, in high purity and good yields – this is due to the presence of the N-terminal amino group, salts of which are easily triturated to remove all other organic impurities. Chapter 4: Synthesis and CD conformational analyses of HBS constrained α-Helical turn mimics containing residues with improved helical propensities Alanine residue has the highest helix propensity among all other natural α-amino acid residues. Its enthalpic contribution to the helical conformation is 1 kcal/mol more than that for the Gly residue, which has the least propensity. Incorporation of Ala residue in the Gly-rich cyclic sequences in either the middle of constrained tripeptide or as the C-terminal extended residue has been accomplished. Comparison of the CD spectra of the synthesized cyclic α-helical turn peptides reveals that a tertiary amide linkage is essential for the propyl linker at the C-terminal amino appendage, for helicity to be observed. Helicity improves upon introduction of the first extended residue. The constrained and C-terminal extended α-helical turn mimics show consistently high helicity irrespective of the helix propensities of the component residues showing that the covalent propyl linker surrogate for the H-bond overwhelms the natural propensities of individual amino acid residues towards enabling stabilization of the helical turn and offer far better structural organization to this cause. Chapter 5: Synthesis of shortest HBS-constrained 310 and - helical peptide analogues The unique versatility of the novel covalent propyl linker surrogate for the peptide H-bond is exhibited by its ability to constrain dipeptides in 310-helix like structures. This is the first and the only HBS model that can achieve this synthetic target as the synthetic protocol allows the conservation of both the residues as is in the constrained helical turn. Similarly, the trapping of a pentapeptide in a C-terminal extended rare and unstable -helix like cyclic structure using the current HBS linker is achieved. Considering the high entropic cost for cyclizing such a long 16-membered chain into a constrained structure, this again exhibits the versatility of the currently developed HBS design and the currently developed synthetic methodology. Chapter 6: First design and synthesis of novel H-bond surrogate constrained parallel β-sheet mimics H-bonding interactions stabilize another prevalently observed secondary structure, other than helical structures, namely the -sheets. The parallel -sheets that almost qualify for super secondary structures due to the high contact orders in them are thought to mimic in models, unlike the easier antiparallel -sheets. Here we replace the inter-strand peptide H-bond between parallel -strands to create excised templates as parallel -sheet nucleators. The propyl linker acts as a dynamic linker in these models and the two amino groups are protected with bulky sulphonamides, in order to provide Thorpe-Ingold effect to the peptide chain. The protocol for synthesizing these models has been described and the different analogues that are synthesized thus have been described. This is the first instance of synthesis of parallel -sheet mimics using the covalent surrogates for the peptide H-bond.

Conformationally constrained ligands and their flexible analogues were prepared as inhibitors of the Grb2 SH2 domain in order to study the structural and energetic effects of ligand preorganization in protein-ligand interactions. The compounds were prepared by using trans-cyclopropane-containing amino acid mimics, macrocyclization, or [alpha,alpha]-disubstituted amino acid residues. All trans-cyclopropane containing peptides were more potent than their corresponding succinate containing analogues due to an enthalpic advantage. Surprisingly, the binding of constrained peptides to the domain was entropically disfavored relative to their flexible controls. Effects of proton transfer and desolvation as being the source of the unprecedented entropic penalty for the constrained ligands relative to their respective controls were precluded, and X-ray crystallographic studies revealed that the binding conformations for the respective cyclopropane and succinate containing ligands were similar. This led us to believe that differential changes in protein dynamics may occur upon binding of the constrained and flexible ligands, which could contribute to the observed binding energetics. Two 23-membered macrocyclic ligands were slightly more potent than their corresponding linear controls. The amino acids used to link the N- and C-termini of the linear peptides to form the macrocycles were found to affect the energetics of binding. In one case, the 23-membered macrocycle was more potent than its control due to an entropic advantage, whereas the other 23-membered macrocycle was more potent than its control because it benefited from an enthalpic advantage. [alpha,alpha]-Disubstituted and [alpha]-monosubstituted residues that varied in hydrophobic character were incorporated into Grb2 SH2 domain binding tripeptides, and binding became more favorable as nonpolar surface area increased only for the set of tripeptides possessing cyclic [alpha,alpha]-disubstituted residues. The increase in affinity was due to an increasing enthalplic term, whereas the entropy of binding became less favorable. A total synthesis of (±)-lycopladine A was achieved in five steps from known compounds. The tricyclic core of the natural product was prepared utilizing a novel two-step sequence comprising a conjugate addition of a metalated picoline derivative followed by an intramolecular enolate arylation. It was demonstrated that the natural product existed in a solvent dependent equilibrium with its isomeric lactol.
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