Systematic kinetic assay of phenylsulfonyl pyridine derivatives with free thiol for site-specific modification of proteins

Abstract

Site-specific modification of proteins with functional groups is a powerful way of understanding the dynamics and functions of proteins. Recently reported phenylsulfonyl pyridine is a thiol-specific moiety and is of great interest for the use of high resolution nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) in structural biology. However, the reactivity of phenylsulfonyl pyridines to free thiols was barely understood, especially considering the effect of the substituent groups in pyridine on the kinetic activity. In this work, we used a number of phenylsulfonyl pyridine derivatives and systematically evaluated the kinetic properties of these compounds with free cysteine at different pHs by means of NMR and UV spectroscopy. The experimental results show that the reactivity of phenylsulfonyl pyridines with free thiols can be tuned by substitution of pyridine or metal ion coordination, with a range of reaction rates from one to five orders of magnitude. These kinetic parameters provide valuable insights into the protein site-specific modification by the phenylsulfonyl pyridine moiety, as demonstrated in the present study.