"Ghadiri reported the first synthetic peptide nanotubue in 1993, which has triggered extensive studies on peptide-based nanotubes and their potential application in molecular wires, catalysts and novel drug delivery vehicles. Our concerns focus on chromophore-modified cyclic peptides, which open a new way to design and synthesize novel nanoscale electronic or photonic devices, and are expected to provide the highly efficient electron and energy transfer that such devices require. This research concerned the design and synthesis of chiral a-amino acids with specific chromophores, including N-ethyl-3-carbazolylalanine and 9-anthrylalanine, and an 8-mer linear peptide (H-Aib-Car-Aib-Phe-Aib-Bpa-Aib-Phe-OH) and its corresponding cyclic peptide cyclo(Aib-Car-Aib-Phe-Aib-Bpa-Aib-Phe) that incorporate the N-ethyl-3-carbazolylalanine. This thesis describes the relevant background, synthetic strategies, experiments and results in detail. The carbazole derivatives were found to be very labile to strong acid, which might have caused self-condensation. In order to avoid the formation of acid-derived side-products, the Wittig-Horner reaction was used successfully in preparation of N-protected-3-(N'-ethyl-3-carbazolyl)-DL-alanine methyl ester. Dual enzymatic hydrolyses were developed to produce the chiral amino acids with high enantiomeric excess. ChiroCLEC-BL was used to selectively hydrolyze the N-acetyl-L-amino acid methyl ester, while amanoacylase was adopted to remove the acetyl group from the resulting N-acetyl-L-amino acid. Two model peptides were synthesized, a 4-mer peptide (H-Car-D-Ala-Bpa-D-Ala-OH) via the Boc-strategy, and an 8-mer peptide (H-Ala-D-Ala-Npa-D-MeAla-Ala-D-Ala-Bpa-D-Ala-OH) by the Fmoc-strategy. Eventually, the target linear peptide was synthesized via the Fmoc-strategy and then cyclized in solution."

No description available.

This thesis describes NMR studies which probe weak interactions between amino acid side chains in folded peptide structures. Aromatic/aromatic interactions between facing phenylalanine residues have been probed in antiparallel β-sheets, while aromatic/proline interactions have been examined using cyclic peptide disulfides that occur in the venom of marine cone snails. Novel intramolecular hydrogen bonded structures in hybrid peptides containing backbone homologated residues, specifically γ-amino acids, are also described. Chapter 1 provides a brief background to the principles involved in the design of antiparallel β-sheet structures and an introduction to previous studies on aromatic/aromatic and aromatic/proline interactions in influencing peptide conformations. A summary of the NMR methods used is also presented. Chapter 2 discusses the structural characterisation of a designed 14 residue, three stranded β-sheet peptide, Boc-LFVDP-PLFVADP-PLFV-OMe (LFV14). The results described in this Chapter support the presence of multiple conformational states about the χ1 (Cα-Cβ) torsional degree of freedom for the interacting aromatic pairs in solution. Chapter 3 presents the structural characterisation of a designed 19 residue three stranded hybrid β-sheet peptide, Boc-LVβFVDPGLβFVVLDPGLVLβFVV-OMe (BBH19). β-amino acid residues (β-phenylalanine, βPhe) were incorporated at facing positions on antiparallel β-sheets. The BBH19 structure provides an example of interaction between the N and C-terminal strands in a three stranded structure with an α/β hybrid backbone. Chapter 4 focuses on studies of the conformations of the contryphan In936 (GCVDLYPWC*) from Conus inscriptus and the related peptide Lo959 (GCPDWDPWC*) from Conus loroissi. Both peptides possess a macrocyclic 23 membered ring, with multiple accessible conformational states. Chapter 5 describes conformational analysis of a novel 20 membered cyclic peptide disulfide, CIWPWC (Vi804), from Conus virgo. NMR structures were calculated for Vi804 and an analog peptide, CIDWPWC, DW3-Vi804. Chapter 6 explores the solution conformation of hybrid sequences containing α and γ residues. Oligopeptides of the type (αγ)n and (αγγ)n have been studied in solution by NMR methods. Chapter 7 provides a summary of the results described in this thesis and highlights the major conclusions.

HIV-1 has evolved with a great deal of functional complexity contained within a very small genome by encoding small, but critical viral proteins within larger viral genes and dividing the replication cycle into early and late phases to differentially produce all proteins leading to efficient replication and virion release. Early replication is restricted by the host spliceosome that processes HIV-1 vRNA transcripts so only the small intragenomic proteins are produced, one of which is Rev (Regulator of Virion Expression). Rev in turn governs the transition from early to late replication by interacting with a highly structured region of vRNA termed the Rev Response Element (RRE). The binding of Rev to the RRE is believed to cause a change in the vRNA tertiary structure and inhibition of splicing of the vRNA. Once, a Rev:RRE complex is formed, a nuclear export signal within Rev facilitates the export of partially spliced and unspliced vRNA to the cytoplasm. During late replication the partially spliced and unspliced vRNA is translated to polyproteins and is packaged into a budding virion where the viral aspartyl protease (HIV-1 PR) autocatalytically excises itself from the larger polyprotein and processes the remaining polyproteins to release all viral structural and functional proteins to form a mature and infectious virion. Since the vRNA salvaged by Rev is translated to the polyproteins containing HIV-1 PR, the inhibition of Rev function will reduce the amount of HIV-1 PR available and thereby reduce the amount of HIV-1 PR therapeutics required to elicit a clinical effect. Therfore a combination approach to HIV-1 treatment using suitably developed therapeutics that inhibit Rev and HIV-1 PR function represents an attractive synergistic approach to treating HIV-1 infection in vivo. The work of this thesis was divided into two parts, the first part was concerned with HIV-1 PR structural biology and addressing problems encountered with inhibitor design. A bicyclic peptide (based on inhibitors of analogous structure) was co-crystallised with active HIV-1 PR to develop an enzyme-product (E-P) complex and with a catalytically inactive mutant HIV-1 PR to provide an analogy to the enzyme-substrate (E-S) complex. Both structures of the E-P and E-S complexes were solved to 1.6Å resolution and were compared to a hydroxyethylamine isostere enzyme-inhibitor complex (E-I), highlighting the similarity of binding mode for all ligands. The inhibitor in the E-I complex was translated towards the S1 - S3 pockets of the substrate binding cleft relative to the substrate in the E-S complex due to the increased length of the hydroxylethylamine isostere compared to the peptide backbone, although the inhibitor "puckered" the isostere linkage and maintains a binding mode similar to the substrate with very little overall differences in the position of the ligands and surrounding protein. The similarity of the E-S, E-I and E-P complexes was attributed to the macrocyclic ligands ordering the surrounding protein environment, especially the protein -strand "flap" structures that form a roof over the ligands in the active site but were not found to close more tightly in any of the trapped catalytic states. The new structures allowed refinement of details of the mechanism of peptide hydrolysis. The mechanism relies on the optimal nucleophilic attack of a water molecule on the scissile amide bond with concerted acid-base catalysis of the active site aspartyl residues intitiated by D125. The alignment and intrinsic position of the N-terminus of the bicyclic substrate was interpreted as being critical to facilitate efficient electron transfer with the bicyclic substrate. An N-terminal cyclic inhibitor, similar to the N-terminal portion of the bicyclic substrate, was used to address a major problem in HIV-1 PR drug design termed "cooperativity," where the sequential optimisation of an inhibitor (or substrate) to individual pockets of the substrate binding cleft, can negatively impact on adjacent and downfield subsites and thereby alter the binding mode of the "optimised" inhibitor. The technique referred to here as "templating" uses the N-terminal cycle to lock the binding mode into a known conformation, probing the S1' and S2' pockets. The structure activity relationship suggested that by viewing the S1' - S3' pockets as a single trough, bulky aromatic groups attached to an N-alkyl sulfonamide could be directed along the line of the trough without adverse interactions with the tops of the S1' and S3' pockets, providing very potent inhibitors. It was also found that specificity and potency of an inhibitor can be maintained with smaller functionalities that carry their bulk low and close to the inhibitor backbone in the S2' pocket, making the P2 functionalities more substrate-like. The second part of the thesis was concerned with establishing suitable surface plasmon resonance assays for testing potential inhibitors of Rev function. Recombinant Rev and its minimal RNA aptamer target (stem loop II of the RRE termed RBE3), were expressed, purified, and used to develop BIAcore-based assays and test potential inhibitors of their interaction. The system was applied to screening of aminoglycoside antibiotics and other small molecules in a competitive assay, and also to quantitative assay of Neomycin and moderate sized analytes: Rev and three peptidic analogues of the high-affinity binding site of Rev - the native peptide, succinylated form of the peptide and a form incorporating a novel helix-inducing cap. The peptide and protein assay was undertaken to test the proposition that helix induction of the high-affinity binding site of Rev can increase affinity for the biologically important RNA target and thereby form the basis of a new class of inhibitors. The screen of small molecule antagonists found that Neomycin was the best inhibitor of the Rev:RBE3 interaction and that efficacy of other aminoglycosides was due to the neamine-base structure presenting charge to bind to the RNA and blocking interaction with Rev. The quantitative assay was optimised to reduce non-specific interactions of Rev protein to allow reliable studies of the analytes with RBE3 by the sytematic testing of buffers and modifiers. It was found that mutliple analytes bound to the RBE3 aptamer and a comparison of the KD values found that the native and capped peptides had similar affinity for RBE3 RNA (native slightly greater at 21 ± 7nM cf capped 41 ± 10nM) that was greater than the Rev protein (101 ± 19nM), however the succinylated peptide exhibited stronger binding with a KD ≤8nM and Neomycin had the lowest affinity (KD 13 ± 3M). The similarity of the native and capped peptides may be due to the high concentration of salt in the assay buffers and was necessary for the stability of the Rev protein, but is sufficient to influence secondary structure of the peptides. Therefore, it could not be stated that the helix-inducing cap increased the affinity of the native peptide for the biologically important therapeutic target. The work conducted in this thesis firmly establishes foundations for the continued development of inhibitors against both Rev and HIV-1 PR that play key roles in the HIV-1 replication strategy. It is envisaged this work could lead to a novel synergistic therapeutic approach to treating HIV-1 infection.

No description available.