Development of New Synthetic Approaches to Biologically-Active Peptides

Synthetic peptides are widely used in drug research due to their high selectivity and efficaciousness, as well as in chemical biology as tools for modulating protein-protein interactions. While there are a large number of naturally occurring peptides, which can be used for the development of new pharmaceuticals, there is also an increasing interest in rationally designed synthesised and optimised peptides. In the field of chemical biology peptides are used to understand complex biological processes, where gaining knowledge and understanding can lead to improved human health and the development of novel drug targeting strategies. Since the synthesis of the first peptide back in 1882, there have been major changes to the peptide synthesis protocol, leading to robust Fmoc-solid phase peptide synthesis that is used in most laboratories today. However, the peptide purification protocols have been lacking behind, meaning peptide purification is still a major bottleneck in fast, efficient, environmentally friendly peptide synthesis. In this thesis, a new peptide synthesis and purification method has been developed and optimised making use of an acrylamide-scavenging approach. Different strategies to introduce an N-terminal acrylamide cap have been explored, including the use of acryloyl chloride and acrylic anhydride, where the latter was suited for automated SPPS. It was found that replacement of piperidine by DABCO for Fmoc-deprotections was equally efficient and suitable for automated SPPS. The scavenging of acrylamide-tagged deletions was performed making use of a thiol-resin, of which the rate was determined by LCMS in peptide model systems. Upon optimisation of acrylamide-tagging and Fmoc-deprotection conditions, the new purification method was compatible with both manual and automated SPPS, requiring only minor adjustments from standard synthesis protocols. This allowed for the synthesis and purification of the diabetes peptide exenatide (39 amino acids), without making use of preparative-HPLC. An attempt was made to extend this methodology to the synthesis of cyclic thioether peptides; however, this did not provide conclusive results. An alternative approach to cyclising peptides was explored, making use of disulfide crosslinking with hexafluorobenzene in biologically active p53 peptides. Residues of the biologically active pDI peptide that had been shown to be tolerant to substitution were replaced with cysteine (analogues), including D-cysteine, homocysteine and penicillamine. It was found that replacement of L-cysteine by any of the analogues resulted in altered binding affinity and selectivity. Finally, the use of fluorine as a reporter for 19F NMR analysis of biological molecules was investigated. Fluorinated prolines were used to explore protein-protein interactions and dynamics, specifically to study the peptidyl-prolyl isomerase cyclophilin D (CypD). First, short substrates were synthesised, including a section of Bcl2 (8 amino acids) and F0F1 ATPase subunit B (9 amino acids), which formed a proof of principle. Significant line broadening was observed in the 19F NMR spectrum with increased protein concentration, indicating substrate binding. These results were more apparent for the Subunit B substrate, indicating it is a better binder to CypD than the Bcl2 peptide. The synthesis of another CypD substrate, a part of the poly proline motif in p53, was optimised for the incorporation of 4S-fluoroprolines. A very significant finding was that each proline residue had a distinct chemical shift. The native cis-trans ratios of each peptide was determined, after which some changes in the cis-trans ratios were observed in the presence of CypD. For some of the fluorinated proline residues line broadening was observed upon incubation with CypD, indicating the occurrence of protein-protein interactions

Fluorinated Prolines as Conformational Tools and Reporters for Peptide and Protein Chemistry

Amide bonds at the proline nitrogen are particularly susceptible to rotation, affording cis and trans isomers. Installation of a stereochemically defined electron-withdrawing fluorine atom or fluorinated groups has the power to influence the cis–trans conformational preferences of the amide bond in X–(F)Pro (where X = any other amino acid). Advantageously, this also provides a sensitive reporter for 19F nuclear magnetic resonance (NMR) studies of protein conformation, interactions, and dynamics. We deliberately use the term “fluorinated prolines” as an all-encompassing term to describe proline analogues containing one or more fluorine atoms and to avoid confusion with the more well-known 4-fluoroprolines. This review presents a critical discussion of the growing repertoire of fluorinated prolines that have been described and, importantly, provides a comparison of their uses and relative influence on amide-bond conformation and discusses the significant potential of using 19F NMR as a tool to probe conformational changes in polypeptides

Tuning the binding affinity and selectivity of perfluoroaryl-stapled peptides by cysteine-editing.

A growing number of approaches to 'staple' α-helical peptides into a bioactive conformation using cysteine cross-linking are emerging. Here we explore the replacement of L-cysteine with 'cysteine analogues' in combinations of different stereochemistry, side chain length and beta-carbon substitution, to examine the influence that the thiol-containing residue(s) has on target protein-binding affinity in a well explored model system, p53-MDM2/MDMX. In some cases, replacement of one or more L-cysteine residues afforded significant changes in the measured binding affinity and target selectivity of the peptide. Computationally constructed homology models indicate that some modifications, such as incorporating two D-cysteines favourably alter the positions of key functional amino acid side chains, which is likely to cause changes in binding affinity, in agreement with measured SPR data

Development of Brain Targeting Peptide Based MMP-9 Inhibiting Nanoparticles for the Treatment of Brain Diseases with Elevated MMP-9 Activity.

Latent and active levels of cerebral matrix metalloproteinase 9 (MMP-9) are elevated in neurological diseases and brain injuries, contributing to neurological damage and poor clinical outcomes. This study aimed developing peptide-based nanoparticles with ability to cross the blood-brain-barrier (BBB) and inhibit MMP-9. Three amphiphilic peptides were synthesised containing brain-targeting ligands (HAIYPRH or CKAPETALC) conjugated with MMP-9 inhibiting peptide (CTTHWGFTLC) linked by glycine (spacer) at the N-terminus, and the peptide sequences were conjugated at the N- terminus to cholesterol. 19F NMR assay was developed to measure MMP-9 inhibition. Cell toxicity was evaluated by the LDH assay, and dialysis studies were conducted with/without fetal bovine serum. An in vitro model was employed to evaluate the ability of nanoparticles crossing the BBB. The amphiphilic peptide (Cholesterol-GGGCTTHWGFTLCHAIYPRH) formed nanoparticles (average size of 202.8 nm) with ability to cross the BBB model. MMP-9 inhibiting nanoparticles were non-toxic to cells, and reduced MMP-9 activity from kobs of 4.5 × 10-6s-1 to complete inhibition. Dialysis studies showed that nanoparticles did not disassemble by extreme dilution (40 folds), but gradually hydrolysed by serum enzymes. In conclusion, the MMP-9 inhibiting nanoparticles reduced the activity of MMP-9, with acceptable serum stability, minimal cell toxicity and ability to cross the in vitro BBB model

Affinity and kinetics study of anthranilic acids as HCA(2) receptor agonists

Medicinal Chemistr