Episulfonium chemistry for versatile protein modifications and crosslinking

Proteins mediate intracellular processes through sequence-encoded three-dimensional structures and are dynamically regulated by post-translational modifications. Facile access to well-defined protein conjugates through chemical protein modification for investigation of protein functions is challenging because chemical reactions on biomacromolecules must proceed rapidly in aqueous buffer under biocompatible conditions and provide high site selectivity. Cysteine-selective protein modification strategies offer an attractive solution due to low native cysteine abundance and high thiol nucleophilicity, but many established approaches require synthesis of prefunctionalized electrophiles, multi-step procedures with isolation of intermediates, as well as incorporation of long or unstable linkers. A recently developed vinylthianthrenium-mediated bioconjugation enables fast cysteine-selective protein modification and incorporation of different payloads via a short and stable ethylene linkage. The first part of this work describes the exploration of the reaction scope for versatile protein and peptide bioconjugation with vinylthianthrenium reagents, as well as mechanistic studies on the bioconjugation. The proposed mechanism involves umpolung of nucleophilic cysteinyl thiols via formation of electrophilic episulfonium intermediates, followed by rapid trapping by neighboring nucleophiles, thereby suggesting a fundamental difference from previously established multi-step protocols. The rest of the work is dedicated to the investigation of the episulfonium chemistry for protein labeling and crosslinking in cellulo. Introduction of covalent crosslinks between native amino acids in proteins enables intracellular protein structure probing and characterization of protein–protein interactions based on the identity of the crosslinked residues. However, crosslinking with commonly used bifunctional reagents occurs in two separate steps and can thereby potentially capture unfavorable conformations, whereas crosslinking via irradiation of photoreactive amino acids relies on unnatural amino acid incorporation and activation with UV light. Single-step crosslinking via episulfonium formation provides an alternative to both methods and enables incorporation of short and stable crosslinks. In this work, formation of episulfonium intermediates on protein surfaces for intracellular protein labeling and crosslinking in different cell types was established. The elucidated reactivity profile of vinylthianthrenium-mediated crosslinking suggests versatile reactivity of episulfonium intermediates with all nucleophilic amino acids and formation of short-distance restraints for reliable protein structure probing. Finally, a method for inter-protein crosslinking using a new thianthrenium reagent that enables enrichment of modified products and facilitates identification of protein–protein interactions in cells is presented in the last part of the work. The combined data demonstrate broad applicability of the site-selective episulfonium generation strategy for synthesis of well-defined protein conjugates, covalent disulfide rebridging, and versatile protein crosslinking.