Abstract
The cyclic five-membered disulfide 1,2-dithiolane, known for its moderate reactivity and ease of preparation, holds significant promise as a recognition unit in probe design and drug development. However, potential limitations in activation specificity—most notably the diminished selectivity toward thioredoxin reductase (TrxR) caused by nonspecific reactions with abundant low-molecular-weight (LMW) thiols such as glutathione (GSH)—raise concerns about its reliability in biological settings. Here we systematically evaluate the activation behavior of 1,2-dithiolane by synthesizing a panel of prodrugs and fluorescent probes incorporating either amine- or hydroxyl-linked cargoes. Our results reveal that TrxR-mediated selective activation of the 1,2-dithiolane unit is achievable when the cargo is incorporated within an amine-based framework. In contrast, hydroxyl-linked conjugates undergo rapid cleavage by physiological GSH levels, resulting in a pronounced loss of TrxR selectivity. Generally, the recognition site 1,2-dithiolane, the linker unit and the leaving group in a cargo coordinate to determine the selectivity activated by TrxR. Overall, this study resolves ambiguities in previous reports, reconciles conflicting observations, and provides new conceptual guidance for the use of the 1,2-dithiolane scaffold in the design of biofunctional molecules.
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The data supporting the findings of this study are available in the main text and Supplementary Information, and from the corresponding author(s) upon request. Source data are provided with this paper.
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Acknowledgements
This research was supported by the China Postdoctoral Science Foundation under Grant Number 2025M783554 (J.Z.).
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J.Z. conceived and led the study, designed and coordinated the overall experimental strategy, synthesized all compounds, performed all spectroscopic and HPLC analyses, processed and interpreted the data, wrote the original draft of the manuscript, and contributed funding support for the research. H.L. performed the TrxR1-knockout–related cellular imaging, cytotoxicity assays, and associated experiments (Figs. 7D and 9 and Supplementary Figs. 11 and 16–18). T.L. conducted the computational docking studies (Supplementary Figs. 6 and 12). J.W. conducted the cytotoxicity assays shown in Supplementary Fig. 9. B.C. provided prodrug Se1-DOX, and T.M. provided probe AFC-SS. M.Y. performed the cellular imaging experiments in Supplementary Fig. 15. S.-H.W. contributed computational docking support. X.L. analyzed the data and contributed to discussion. B.Z. provided instrumentation support and reviewed key data. C.Z. provided the HCT116 cell lines (WT, NC, and TrxR1-KO) and TrxR, and supervised the experimental design, execution, and data analysis of all HCT116-related studies, including the TRi-1 inhibition experiments. J.F. contributed to project conception, supervised the research, provided input on data interpretation, performed manuscript writing and submission as well as revision, and offered funding and instrumentation support. All authors reviewed and approved the final manuscript.
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Zhao, J., Liu, H., Liu, T. et al. Insights into the activation patterns of 1,2-dithiolane unit in biofunctional molecules. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70678-8
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DOI: https://doi.org/10.1038/s41467-026-70678-8
