Chemoselective synthesis of boronopeptides and their biological applications

Abstract

Non-natural late-stage modifications to peptide chains have been in effect since the 1950s to improve stability, binding affinity, and targeting selectivity. Recent research focuses on merging synthetic organic chemistry and chemical biology to develop robust methods for chemical modifications to peptide chains that do not interfere with their native biological functions but rather serve as upgrades. In this regard, boronic acids (BA) pose as an exciting module for the diversification of peptide function due to their extensive bonding nature with endogenous nucleophiles. In the thesis, we have established two methods to install BA derivatives on cysteine residues in peptides and proteins. The first method employs the well-known thiol-ene click chemistry where an alkene handle appended BA molecule was effectively installed on cysteine residue(s) in the presence of a photoinitiator and 365 nm light. The strategy yielded clean to quantitative conversion with eight different BA derivatives spanning over eight biologically active peptides. The BA derivatives were handpicked based on the diverse reaction genres offered by them, which in turn were demonstrated by four chemical modules – cyclization, functional group conversion, disulfide formation and α-nucleophile conjugation. The established methodology was diligently used to install BA derivatives, known for their superior sugar binding ability, on programmed cysteine residues placed on wheat germ agglutinin (WGA) peptide which reportedly binds selectively to sialyl-glycans. Currently, the overexpression of sialyl-glycan antigens has been considered as the hallmarks of cancer progression and the in-use clinical techniques involve the use of lectins which are costly and difficult to handle. We hypothesized that a synergistic binding exerted by the designed boronopeptides will prove to be as effective as a lectin, thereby providing a cheaper alternative. NMR and cell imaging studies proved the binding selectivity of the boronopeptides while flow cytometer and EC50 studies showed that Wulff boronate conjoined peptides are pretty effective in recognising the glycans. In a separate investigation, we endeavoured to confront the imminent challenge presented by antimicrobial resistance (AMR) by affixing boronic acid probes to ubiquicidine, a widely recognized antimicrobial peptide. The designed peptides show increased salt tolerance and the ability to differentiate sterile inflammation from infection. Radiolabelling with 99mTc and 68Ga enabled superior imaging techniques to be applied to in vivo studies with mice. In our second method, we employed practically simple and benign SN2 reaction to install methyl boronic acid on cysteine residues in peptides and proteins. A quantitative conversion can be achieved within 10 min in water at physiological pH with only 1.5-2 equivalent of the reagent, providing a green technology to modify sensitive biomolecules. This opens up avenues to create multivalent peptides which provide enhanced affinity and specificity.