Underpinning Studies of Native Chemical Ligation

Abstract

This thesis details kinetic investigation into some factors involved in native chemical ligation (NCL) and this knowledge can be applied in the field of peptide/protein chemistry. NCL is one of the two principal techniques used in the chemical synthesis of proteins, where an N-terminal cysteine thiolate ligates with a C- terminal thioester that upon rearrangement forms the native amide bond.
Thioester hydrolysis is one of the destructive pathways that can minimise the efficiency of NCL. We therefore monitored the kinetics of such reactions of model amino acid derived thioesters, based on alanine and phenylalanine. 1H NMR spectroscopy and UV-Vis spectrophotometry was utilised to establish the second-order rate constants for deuteroxide, hydroxide and deuterium-catalysed hydrolysis reactions of alanine and phenylalanine based alkyl thioesters (69 and 70, respectively). For the former substrate, kDO and kHO values were 0.121 and 0.207 M-1s-1 and for the latter substrate, the corresponding values were 0.113 and 0.155 M-1s-1, respectively. The similarity of these second-order rate constants for the hydrolysis reactions of alanine and phenylalanine-based thioesters suggest insignificant dependence upon the steric hindrance of C-terminus. In addition, for the similarity of kDO and kHO for each individual substrate demonstrates a small secondary solvent isotope effect, suggesting a concerted mechanism for hydrolysis of these thioesters.
Stability of some of the most common thiol-containing amino acids and catalysts were examined in deuterated phosphate buffer and it was found that they undergo dimerisation (common for thiol-containing species). In another series of experiments, 5-fold excess of TCEP quenched the mentioned side reaction.
Preliminary thiol-thioester exchange kinetic studies with amino acid based thioesters and MESNa indicated the reaction reaching equilibrium over time and a net increase in the forward second-order rate constant as the pD of the reaction increased, owing to higher concentration of the MESNa being in thiolate form (i.e. the active form).