Boc Solid-Phase Synthesis of Conjugable Cyclic Depsipeptide Cotransin for Use as a Biological Probe
Creators: name: Warren, Thomas
Publisher: name: University of Surrey
Year: 2023

description: Cyclic depsipeptides (peptides containing an ester bond) have demonstrated a range of useful bioactive properties and have applications as drugs and biological tools for drug development. Cotransin, a cyclic depsipeptide inhibitor of the protein translocation channel Sec61 of the endoplasmic reticulum, has been identified as a useful probe molecule for investigating the molecular function of protein-conducting channels in living cells. An analogue of cotransin capable of conjugation with a fluorophore for use as a probe in a biological assay (BRET assay) to establish target binding was required. It was also recognised that a generalised and robust synthetic methodology to obtain other biologically active cyclic depsipeptides capable of fluorophore conjugation would be useful. This thesis aimed to use the side chain amine of lysine for fluorophore conjugation as well as side chain attachment of lysine to a solid support, which enables convenient solid phase cyclisation of the peptide. Solid phase peptide synthesis (SPPS) to obtain cyclic depsipeptides using basic conditions (‘Fmoc SPPS’) has resulted in problems due to unwanted cyclisation (diketopiperazine formation) or hydrolysis of the ester bond. This project instead used the acidic conditions of ‘Boc SPPS’ to avoid these problems. Thus, a Fmoc-derived linker was used to allow cleavage of the final peptide under mild basic conditions, instead of the hazardous hydrofluoric acid conditions which are usually required for Boc SPPS. In this thesis, the use of this Boc SPPS methodology for the synthesis of a series of lysine substituted cotransin analogues is reported.<br>
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description: N-[(9-Hydroxymethyl)-2-fluorenyl]succinamic acid (HMFS) was synthesised in 5 steps in 48% overall yield, which was attached to aminomethyl polystyrene (AMPS) in quantitative yield, after optimisation, to form HMFS-AMPS. Conversion of HMFS-AMPS into the Fmoc-OSu linker was improved to 60% using DSC/aq. HCl, as determined through an investigation using solution phase modelling. The remaining HMFS-AMPS hydroxyl sites were selectively acetylated using an acetic anhydride/pyridine method that was developed. Synthesis strategies were developed for the orthogonally protected lysine derivatives required for synthesis of the Lys-cotransin analogues. Boc-Lys-OAll was synthesised from Boc-Lys(Fmoc)-OH in two steps in 72% yield. Z-Lys-OAll was synthesised from Z-Lys(Boc)-OH in two steps in 62% yield, and HO-D-Lys-OAll was synthesised from D-Lys(Z)-OH in three steps in 78% yield. Initial attempts to synthesise the Lys1,6-cotransin peptides using the Fmoc-OSu linker were unsuccessful and resulted in C-terminal synthesised linear Lys1,6-cotransin peptides missing the lysine residue. Alternative Fmoc linkers were investigated and the Fmoc-ONPh linker appeared to show quantitative side chain attachment of lysine. Thus, Lys1-cotransin, Lys6-cotransin, Lys2-cotransin, and Lys4-cotransin were synthesised using the Fmoc-ONPh linker. Lys6-cotransin (0.055 g crude yield) was lost during the attempted purification and Lys1-cotransin (0.104 g crude yield, 5.9 mg final yield), Lys2-cotransin (0.070 g crude yield, 0.7 mg final yield), and Lys4-cotransin (0.082 g crude yield, 0.9 mg final yield) were obtained in low yields. The C-terminal synthesised by-product peptides were still detected, but in a decreased extent compared to when the Fmoc-OSu linker was used. Furthermore, incomplete cyclisation of Lys2-cotransin and Lys4-cotransin occurred, contributing to the low yields obtained.<br>