Chemoselective bioconjugation to cysteine residues is one of the most convenient site-selective protein modification strategies due to its unique nucleophilic properties and relatively low abundance. Although a lot of different chemical strategies have been recently applied to modify Cys-residues[1], a technique that combines high selectivity, stable linkage and ease of introduction of the thiol reactive moiety is still rarely found in the literature.
In our group, we have developed a series of P(III) compounds including phosphites and phosphonites that react chemoselectively with azides to form stable P(V)-conjugates such as phosphor- or phosphonamidates.[2] These Staudinger-type reactions were applied successfully to small molecules, dyes, polymers as well as unprotected peptides and proteins in the presence of several functional groups. Furthermore, we showed that alkyne-containing phosphonites can be used for a formal and modular conjugation of two azido-containing molecules.[3]
In the current presentation, we extend the use of such phosphorous reagents by the introduction of unsaturated phosphonamidates for the selective modification of Cys- residues, named P5-labeling. In a modular fashion, phosphonamidates can be introduced into functional molecules and unprotected peptides via the Staudinger-phosphonite reaction with azides. This first chemoselective reaction induces reactivity for the subsequent conjugation to thiols, which can be performed in aqueous buffers and proceeds with a unique selectivity. The resulting conjugates were obtained in high yields and we observed a remarkable stability towards exchange with external thiols. Building upon this outstanding performance we were able to apply our method to the generation of novel enhanced stability Antibody-Drug-Conjugates (ADCs) as well as functional protein-peptide-conjugates.[4]