Chemoselective bioconjugation to cysteine residues is one of the most convenient protein modification strategies due to its highly nucleophilic properties and relatively low abundance. Although a lot of different chemical methods have been recently applied to modify sulfhydryl groups [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. In a modular fashion, phosphonamidates can be introduced into unprotected peptides via the Staudinger-phosphonite reaction with azides. Solid-phase peptide synthesis allows the installation of aromatic and aliphatic azides, which can be selectively modified after cleavage from the resin. The first chemoselective Staudinger-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 can be 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 peptide-linked, enhanced stability Antibody-Drug-Conjugates (ADCs) as well as functional cell-penetrating peptide-protein conjugates. [4]