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Wittig reagents for chemoselective sulfenic acid ligation enables global site stoichiometry analysis and redox-controlled mitochondrial targeting

Nature Chemistry volume 13, pages 1140–1150 (2021)Cite this article

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Abstract

Triphenylphosphonium ylides, known as Wittig reagents, are one of the most commonly used tools in synthetic chemistry. Despite their considerable versatility, Wittig reagents have not yet been explored for their utility in biological applications. Here we introduce a chemoselective ligation reaction that harnesses the reactivity of Wittig reagents and the unique chemical properties of sulfenic acid, a pivotal post-translational cysteine modification in redox biology. The reaction, which generates a covalent bond between the ylide nucleophilic α-carbon and electrophilic γ-sulfur, is highly selective, rapid and affords robust labelling under a range of biocompatible reaction conditions, which includes in living cells. We highlight the broad utility of this conjugation method to enable site-specific proteome-wide stoichiometry analysis of S-sulfenylation and to visualize redox-dependent changes in mitochondrial cysteine oxidation and redox-triggered triphenylphosphonium generation for the controlled delivery of small molecules to mitochondria.

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Fig. 1: Repurposing TPP ylides as probes for electrophilic sulfur in proteins.
Fig. 2: WYne-probe reactivity with sulfenic acid in complex biological settings.
Fig. 3: Profiling S-sulfenylation dynamics with WYneN in cells.
Fig. 4: Proteome-wide analysis of cysteine sulfenic acid site stoichiometry.
Fig. 5: Redox-triggered in situ TPP generation for mitochondrial cargo delivery.

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Data availability

The MS proteomics data have been deposited at the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository52 with the dataset identifier PXD025630. All other data associated with this study are available in the published article and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank L. Sun and T. Zhang from the Beijing Qinglian Biotech Co., Ltd, for their help and technical support. This work was supported by the US National Institutes of Health (R01 GM102187 and R01 CA174864 to K.S.C.) and the National Natural Science Foundation of China (21922702), the National Key R&D Program of China (2016YFA0501303) and the State Key Laboratory of Proteomics (SKLP-K201703 and SKLP-K201804) to J.Y.

Author information

Authors and Affiliations

  1. Department of Chemistry, The Scripps Research Institute, Jupiter, FL, USA

    Yunlong Shi & Kate S. Carroll

  2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing, China

    Ling Fu & Jing Yang

  3. National Center for Protein Sciences—Beijing, Beijing, China

    Ling Fu & Jing Yang

  4. Beijing Institute of Lifeomics, Beijing, China

    Ling Fu & Jing Yang

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  1. Yunlong Shi
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Contributions

Y.S., J.Y. and K.S.C. conceived the project, designed experiments and analysed data. Y.S. synthesized and characterized the compounds. Y.S. and L.F. performed intact MS and quantitative proteomic experiments and data analysis. Y.S. performed the probe validation and cell-based experiments.

Corresponding authors

Correspondence to Jing Yang or Kate S. Carroll.

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Peer review information Nature Chemistry thanks Megan Matthews and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Surveying Wittig reagent reactivity with sulfenic acid.

Wittig reagents 1-7 were screened for reaction with the dipeptide-SOH model compound. Rate constants were obtained in acetonitrile(ACN):25 mM NaOAc (1:2 v/v) pH = 4.9. Isolation yields, literature (in parentheses, reported in DMSO39) and experimental pKa values (in ACN-H2O, see Supplementary Figs. 3-4) are listed if available.

Extended Data Fig. 2 Imaging redox-dependent changes in mitochondrial cysteine oxidation.

a, Amide derivative of Wittig reagents exists predominantly in protonated form, setting stage for enrichment and detecting S-sulfenylation in mitochondria. b, Structure of the mitochondrial targeting sulfenic acid probe WYneN10 with enhanced lipophilicity. c, Live HeLa cells were incubated with BDP-WYneN10 (500 nM) and MitoTrackerTM Deep Red FM (100 nM) in DPBS. After 10 min, confocal images were taken. A scale bar of 20 µm is shown. R, Pearson’s correlation coefficient. d, BDP-WYneN10 tagged S-sulfenylated proteins with fluorescence inside mitochondria. e, BDP-WYneN10 fluorescence responded to external oxidative stress (0-5 mM H2O2) in live A549 cells (n = 4 areas from one representative experiment). f, WYneN10 disrupted mitochondrial respiration in A549 cells to a greater extent than other WYne probes (50 µM) (n = 12 biological replicates). OCR, oxygen consumption rate. Data in e-f are presented as box plots (maximum, 75%, median, 25%, minimum). P values were calculated using a two-tailed t-test. ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–26, Methods and NMR spectra.

Reporting Summary

Supplementary Table 1

Applications of WYne probes for mapping cysteine sulfenic acids in A549 cell lysates.

Supplementary Table 2

Application of WYneN for mapping cysteine sulfenic acids in intact A549 cells.

Supplementary Table 3

WYneN-based in situ S-sulfenylome analysis.

Supplementary Table 4

Proteome-wide analysis of cysteine sulfenic acid site stoichiometry in A549 cells.

Supplementary Data 1

Statistical Source Data for Supplementary Figures.

Source data

Source Data Fig. 2

Statistical Source Data.

Source Data Fig. 2

Unprocessed gel scans.

Source Data Fig. 3

Statistical Source Data.

Source Data Fig. 3

Unprocessed gel scans.

Source Data Fig. 4

Statistical Source Data.

Source Data Fig. 5

Statistical Source Data.

Source Data Extended Data Fig. 2

Statistical Source Data.

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Shi, Y., Fu, L., Yang, J. et al. Wittig reagents for chemoselective sulfenic acid ligation enables global site stoichiometry analysis and redox-controlled mitochondrial targeting. Nat. Chem. 13, 1140–1150 (2021). https://doi.org/10.1038/s41557-021-00767-2

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