It is of ongoing interest to develop new approaches for efficient and selective modification of cysteine residues on biomolecules. Here we present a comprehensive study on a newly developed isoxazolinium-mediated cysteine modification of peptides and proteins. Using a stoichiometric amount of isoxazolinium reagents generated in situ from a catalytic amount of silver salts, cysteine-containing peptides can be efficiently modified to afford products in nearly complete conversions. With the optimized conditions, free cysteine containing proteins HSA and BSA, as well as a site-directed mutated therapeutic protein (BCArg) can be efficiently and selectively labelled using small amounts of the isoxazolinium reagents. We find that the phenylacyl thioether linkage bearing an alkyne moiety can be rapidly cleaved under irradiation of UV-A light, giving the formation of a thioaldehyde moiety, which can be converted back to cysteine by reduction.

Selective modification of the N-terminus of peptides and proteins is a promising strategy for single site modification methods. Here we report N-terminal selective modification of peptides and proteins by using 2-ethynylbenzaldehydes (2-EBA) for the production of well-defined bioconjugates. After reaction screening with a series of 2-EBA, excellent N-terminal selectivity is achieved by the reaction in slightly acidic phosphate-buffered saline using 2-EBA with electron-donating substituents. Selective modification of a library of peptides XSKFR (X = either one of 20 natural amino acids) by 2-ethynyl-4-hydroxy-5-methoxybenzaldehyde (2d) results in good-to-excellent N-terminal selectivity in peptides (up to >99:1). Lysozyme, ribonuclease A and a therapeutic recombinant Bacillus caldovelox arginase mutant (BCArg mutant) are N-terminally modified using alkyne- and fluorescein-linked 2-EBA. Alkyne-linked BCArg mutant is further modified by rhodamine azide via copper(I)-catalyzed [3 + 2] cycloaddition indicating that the reaction has high functional group compatibility. Moreover, the BCArg mutant modified by 2-ethynyl-5-methoxybenzaldehyde (2b) exhibits comparable activity in enzymatic and cytotoxic assays with the unmodified one.

Isatin-based small molecules targeting bacterial peptidoglycan glycosyltransferase are potent antimicrobial agents against S. aureus, E. coli and methicillin-resistant Staphylococcus aureus strains.

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