Parallel evaluation of nucleophilic and electrophilic chemical probes for sulfenic acid: Reactivity, selectivity and biocompatibility

Abstract

Cysteine sulfenic acids (Cys-SOH) are pivotal modifications in thiol-based redox signaling and central intermediates en route to disulfide and sulfinic acid states. A core mission in our lab is to develop bioorthogonal chemical tools with the potential to answer mechanistic questions involving cysteine oxidation. Our group, among others, has contributed to the development of nucleophilic chemical probes for detecting sulfenic acids in living cells. Recently, another class of Cys-SOH probes based on strained alkene and alkyne electrophiles has emerged. However, the use of different models of sulfenic acid and methodologies, has confounded clear comparison of these probes with respect to chemical reactivity, kinetics, and selectivity. Here, we perform a parallel evaluation of nucleophilic and electrophilic chemical probes for Cys-SOH. Among the key findings, we demonstrate that a probe for Cys-SOH based on the norbornene scaffold does not react with any of the validated sulfenic acid models in this study. Furthermore, we show that purported cross-reactivity of dimedone-like probes with electrophiles, like aldehydes and cyclic sulfenamides, is a not meaningful in a biological setting. In summary, nucleophilic probes remain the most viable tools for bioorthogonal detection of Cys-SOH.

Reaction-based fluorogenic probes for selective detection of cysteinyl oxidation in living cells

“…Kinetic and fluorescence characterization of phenaline-1,3-dione derivatives. We first sought to assess the reactivity of phenaline-1,3-dione (1) and its analogs with an established small-molecule model for cysteine sulfenic acid, known as CSA 19,20 (Fig. 2a).…”

“…3). This Gpx3 variant has one redox-sensitive cysteine, C36 that is readily oxidized to sulfenic acid using stoichiometric amounts of hydrogen peroxide (H2O2) and has been well-validated as a model for the study of protein sulfenic acid reactivity 19,20 . Intact mass spectrometry (MS) analysis demonstrated that Gpx3 C36 sulfenic acid (Gpx3-SOH) formed the anticipated adduct with CysOx probes in high yield, while reduced Gpx3 (Gpx3-SH) was not modified (Fig.…”

Defining and refining the cysteine redoxome with sulfur chemical biology

“…While this model, characterized in a handful of cases, can provide convenient answers to nagging questions surrounding target selectivity for a diffusible second messenger like H2O2 an equally troubling new set of selectivity issues arise, which cannot be satisfactorily addressed in target identification experiments that employ so-called "trap mutants" in which the peroxidase lacks the resolving cysteine. Furthermore, positing the redox relay as the exclusive mechanism of cysteine OxiPTM contradicts the proteome-wide discovery 28 of cysteine sulfenic and sulfinic acids OxiPTMs, known respectively as S-sulfenylation and S-sulfinylation, using reaction-based probes whose chemoselectivity has been shown in numerous reports 15,29 .…”

Sirtuin Oxidative Post-translational Modifications

“…Sulfenylation is a reversible oxidative modification where cysteine thiol is converted to a sulfenic acid (RS-OH) by reaction with two electron oxidants such as the peroxides H 2 O 2 or ONOO – ( Table 3 ; Alvarez and Radi, 2003 ; Yang et al, 2016 ); cysteine sulfenylation is reducible by common intracellular reductants such as reduced glutathione ( Reddie and Carroll, 2008 ; Conte and Carroll, 2013 ; Gupta and Carroll, 2014 ). In recent years, protein sulfenylation has been described in numerous systems due to the advent of dimedone-based and other sulfenylation-selective probes ( Holmila et al, 2018 ; Shi and Carroll, 2020 , 2021 ). In in vitro studies of recombinantly purified Sirt1, Sirt2, Sirt3, and Sirt5, sulfenylation was not detected by the DYn-2 dimedone-based probe following treatment with 100 μM H 2 O 2 ( Kalous et al, 2016 , 2020 ).…”