The mechanism behind enhanced reactivity of unsaturated phosphorus(v) electrophiles towards thiols.

Vinyl- and ethynyl phosphorus(v) electrophiles are a versatile class of thiol-reactive reagents suitable for cysteine-selective peptide and protein modifications, especially for the generation of antibody conjugates. Herein we investigated the reactivity of various P(v) reagents towards thiol addition. Complementing previous studies, we observed that the heteroatoms X (X = S, O, NH) as well as the vinyl- vs. ethynyl-substituent bound to phosphorus greatly influence the overall reactivity. These experimentally observed trends, as well as the high Z-selectivity for thiol additions to ethynyl derivatives, were further elucidated using DFT calculations. Hyperconjugation was a key means of stabilizing the intermediate generated upon the thiol addition, thus determining both the reactivity and stereoselectivity of unsaturated P(v) electrophiles. Specifically, the energetically low-lying σ antibonding orbital of the P-S bond more readily stabilizes the electron density from the lone pair (LP) of the generated carbanion, rendering the phosphonothiolates more reactive compared to the derivatives bearing oxygen and nitrogen. Our studies provide a detailed mechanistic picture for designing P(v)-based electrophiles with fine-tuned reactivity profiles.

Chemically Induced Vinylphosphonothiolate Electrophiles for Thiol-Thiol Bioconjugations.

Herein we introduce vinylphosphonothiolates as a new class of cysteine-selective electrophiles for protein labeling and the formation of stable protein-protein conjugates. We developed a straightforward synthetic route to convert nucleophilic thiols into electrophilic, thiol-selective vinylphosphonothiolates: In this protocol, intermediately formed disulfides can be chemoselectively substituted with vinylphosphonites under acidic conditions to yield the desired vinylphosphonothiolates. Notably, this reaction sequence enables the installation of vinylphosphonothiolate electrophiles directly on cysteine side chains within peptides and proteins. In addition to labeling the monoclonal antibody trastuzumab with excellent cysteine-selectivity, we applied our protocol for the site-specific conjugation of two proteins with unique cysteine residues yielding a nonhydrolyzable phosphonothiolate-linked diubiquitin and an ubiquitin-α-synuclein conjugate. The latter was recognized as a substrate in a subsequent enzymatic ubiquitination reaction.

Modern Ligation Methods to Access Natural and Modified Proteins.

Proteins and peptides are gaining increasing interest as tools and targets in fundamental research and drug discovery. Growing research applications have prompted the need for methodologies that produce homogenous peptide and protein material. The development of efficient, chemoselective ligation reactions using unprotected peptide fragments presents a key solution for this challenging task. This review outlines modern ligation methods that enable the synthesis of both native, and also labelled or post-translationally modified peptides and proteins. The ligation methods herein discussed focus on the formation of the backbone amide bond.