Application of Hypervalent Iodine Reagents for Novel Cysteine Labeling

Selective functionalization of peptides and proteins has always been a valuable tool for the study and manipulation of biological processes. In this context, cysteines are of particular interest. Their intrinsic high nucleophilicity makes these amino acids excellent targets for peptides and proteins modification. As a result, cysteine labeling techniques have been extensively investigated. Nevertheless, while bioconjugation requires high efficiency, selectivity and kinetic under mild and aqueous conditions, most of the reported methodologies exhibit significant limitations. For instance, lack of chemoselectivity, as well as low or uncontrolled reactivity, are frequently reported. Furthermore, many of these methods rely on reagents challenging to prepare, store and use. In this regard, hypervalent iodine reagents are attractive. Heterocyclic λ3-iodanes demonstrated high reactivity and selectivity, together with great stability and low toxicity. Nevertheless, applications to peptides and proteins modification remain rare. Employing ethynylbenziodoxolone reagents (EBXs), we previously described an efficient and chemoselective alkynylation of organosulfur compounds. The process was performed under “open-flask” conditions, at room temperature, without any metal- or additive-assistance. In collaboration with Dr. Adibekian and co-workers, we then employed this methodology to intracellular proteomic profiling of cysteine residues. Nevertheless, only the most reactive cysteines were efficiently labeled. Accordingly, the goal of my thesis was to investigate the reactivity between any cysteine residues and EBX reagents, under mild and aqueous conditions. We started our investigations with treatment of glutathione, a naturally occurring tripeptide, with an azide-containing EBX reagent. Although a complete conversion of the starting material was observed, the corresponding thioalkyne product was not formed. Instead, we observed thiol-yne reactivity and selective thiol trans-addition in β-position of the EBX reagent, leading to vinylbenziodoxolone product (VBX). This efficient cysteine labeling exhibited high selectivity, fast kinetic and great robustness. Various cysteine-containing peptides and proteins, along with diverse EBX reagents, were well tolerated. When an azide-containing EBX reagent was employed, strain-promoted azide-alkyne cycloadditions (SPAAC) were successfully achieved. Alternatively, the hypervalent iodine function was engaged in aqueous Suzuki-Miyaura cross-couplings. Various boronic acids, containing electron-rich and –deficient aryls, heteroaryls and vinyls, were successfully subjected to this palladium-promoted process. Remarkably, both SPAAC and cross-coupling were orthogonal to each other. We then investigated the singular reactivity of the TMS-EBX reagent. Under basic aqueous conditions, we observed fast desilylation of the hypervalent iodine reagent. Upon glutathione treatment, the resulting EBX substrate was engaged in a thiol-yne reaction. Nevertheless, the corresponding VBX product exhibited a low stability and led to alkynylated glutathione. Further optimization furnished an efficient, selective and rapid ethynylation of numerous cysteine-containing peptides.