Synthesis and Use of the Bifunctional Sulfenic Acid Probe BCN-E-BCN for In Vitro and Cell-Based Assays of Protein Oxidation

Abstract

The reversible oxidation of cysteine thiol groups to sulfenic acid by reactive oxygen species (ROS) such as hydrogen peroxide can impact protein function, activity, and localization. As a consequence, ROS have profound effects on cell functions including proliferation, differentiation, and survival. Furthermore, there are clear associations between the effects of ROS on cells and the etiology of several diseases including cancer and neurodegeneration. In spite of the importance of cysteine sulfenylation as a validated post-translational modification, its labile nature impedes efficient and reproducible detection of proteins with cysteine sulfenic acid residues. To overcome this challenge, we developed a novel cell-permeable bifunctional reagent, consisting of two linked bicyclo[6.1.0]nonyne (BCN) moieties coupled with a short ethylenediamine-derived linker (BCN-E-BCN) that enables the detection of sulfenylated proteins in vitro and in intact cells. The two symmetrical BCN groups allow protein sulfenic acids to be selectively tagged with a BCN at one end while allowing for copper-free click chemistry with azide-tagged reagents of the opposite BCN. In this protocol, the synthesis of BCN-E-BCN and its use to detect cysteine sulfenic acids will be detailed. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Copper-mediated cyclopropanation of 1,5-cyclooctadiene. Basic Protocol 2: Synthesis of endo- and exo-bicyclononyne. Basic Protocol 3: Synthesis of endo-BCN-E-BCN. Basic Protocol 4: BCN-E-BCN treatment of wild-type and cysteine-deficient mutant recombinant cofilin protein. Basic Protocol 5: BCN-E-BCN labeling in live cells. Basic Protocol 6: Western blotting and visualization of BCN-E-BCN-labeled samples.