Triphenylphosphonium-Derived Protein Sulfenic Acid Trapping Agents: Synthesis, Reactivity, and Effect on Mitochondrial Function

Abstract

Redox-mediated protein modifications control numerous processes in both normal and disease metabolism. Protein sulfenic acids, formed from the oxidation of protein cysteine residues, play a critical role in thiol-based redox signaling. The reactivity of protein sulfenic acids requires their identification through chemical trapping, and this paper describes the use of the triphenylphosphonium (TPP) ion to direct known sulfenic acid traps to the mitochondria, a verified source of cellular reactive oxygen species. Coupling of the TPP group with the 2,4-(dioxocyclohexyl)propoxy (DCP) unit and the bicyclo[6.1.0]nonyne (BCN) group produces two new probes, DCP-TPP and BCN-TPP. DCP-TPP and BCN-TPP react with C165A AhpC-SOH, a model protein sulfenic acid, to form the expected adducts with second-order rate constants of k = 1.1 M^-1 s^-1 and k = 5.99 M^-1 s^-1, respectively, as determined by electrospray ionization time-of-flight mass spectrometry. The TPP group does not alter the rate of DCP-TPP reaction with protein sulfenic acid compared to dimedone but slows the rate of BCN-TPP reaction compared to a non-TPP-containing BCN-OH control by 4.6-fold. The hydrophobic TPP group may interact with the protein, preventing an optimal reaction orientation for BCN-TPP. Unlike BCN-OH, BCN-TPP does not react with the protein persulfide, C165A AhpC-SSH. Extracellular flux measurements using A549 cells show that DCP-TPP and BCN-TPP influence mitochondrial energetics, with BCN-TPP producing a drastic decrease in basal respiration, perhaps due to its faster reaction kinetics with sulfenylated proteins. Further control experiments with BCN-OH, TPP-COOH, and dimedone provide strong evidence for mitochondrial localization and accumulation of DCP-TPP and BCN-TPP. These results reveal the compatibility of the TPP group with reactive sulfenic acid probes as a mitochondrial director and support the use of the TPP group in the design of sulfenic acid traps.

Scheme 1.

Synthesis of DCP-TPP and BCN-TPP

Scheme 2.

Reactions of DCP-TPP (1), BCN-TPP (2), and BCN-OH with C165A AhpC-SOH and AhpC-SSH

Figure 1.

Reaction kinetics of DCP-TPP (A) and BCN-TPP (B) probes with C165A AhpC-SOH. Oxidized protein was reacted with each probe, and at set time points samples were taken and desalted and their species abundance determined using ESI-TOF-MS. To account for differences in batches of AhpC-SOH, the plateau of each reaction is normalized to a value of 1, and all data points are expressed relative to the plateau.

Figure 2.

Effects of mitochondria-targeted dimedone and BCN derivatives (DCP-TPP and BCN-TPP) on mitochondrial respiration. (A) Seahorse Mito Stress test for DCP-TPP. Pre-treatment with the compound was started 1 h before the analysis. (B) Seahorse Mito Stress test for BCN-TPP. Pre-treatment with the compound was started 1 h before the analysis. (IC) Quantification of DCP-TPP and BCN-TPP effects on the mitochondrial basal respiration; values are presented relative to the basal respiration before compound exposure. (D) Quantification of DCP-TPP effects on ATP production and proton leak. Error bars represent the standard error of mean.

Figure 3.

Control studies to evaluate the effects of dimedone, BCN-OH, and TPP-COOH on mitochondrial respiration. (A) Seahorse Mito Stress test for dimedone, BCN-OH, and TPP-COOH. (B) Quantification of effects on mitochondrial basal respiration; values are presented relative to the basal respiration before compound exposure. (C) Quantification of effects on ATP production, proton leak, and respiratory capacity. Error bars represent the standard error of mean.

Chart 1.

Structures of Mitochondrial-Directed Sulfenic Acid Probes