Maleimide–thiol adducts stabilized through stretching

Abstract

Maleimide–thiol reactions are widely used to produce protein–polymer conjugates for therapeutics. However, maleimide–thiol adducts are unstable in vivo or in the presence of thiol-containing compounds because of the elimination of the thiosuccinimide linkage through a retro-Michael reaction or thiol exchange. Here, using single-molecule force spectroscopy, we show that applying an appropriate stretching force to the thiosuccinimide linkage can considerably stabilize the maleimide–thiol adducts, in effect using conventional mechanochemistry of force-accelerated bond dissociation to unconventionally stabilize an adjacent bond. Single-molecule kinetic analysis and bulk structural characterizations suggest that hydrolysis of the succinimide ring is dominant over the retro-Michael reaction through a force-dependent kinetic control mechanism, and this leads to a product that is resistant to elimination. This unconventional mechanochemical approach enabled us to produce stable polymer–protein conjugates by simply applying a mechanical force to the maleimide–thiol adducts through mild ultrasonication. Our results demonstrate the great potential of mechanical force for stimulating important productive chemical transformations.

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Fig. 1: Single-molecule force spectroscopy of maleimide–thiol adducts.
Fig. 2: Pre-stretching experiment of maleimide–thiol conjugates.
Fig. 3: Chemical characterization of the force-induced hydrolysis of the maleimide–thiol conjugates.
Fig. 4: Ultrasonication increases the stability of maleimide–thiol-based antibody–PEG conjugates.

Code availability

Igor procedures for single-molecule experiment analyses of this study are available from the corresponding author upon request.

Data availability

All data generated and analysed during this study are included in this article and its Supplementary Information, and are also available from the authors upon reasonable request.

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Acknowledgements

This research is supported mainly by the National Natural Science Foundation of China (grants nos. 21522402, 11674153, 11374148, 21774057 and 11334004) and the Fundamental Research Funds for the Central Universities (grant no. 020414380080). The authors thank Y. Li for discussions.

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Yi.C., W.H. and X.G. conceived the project and designed the experiments. W.H., X.G. and H.L. performed the single-molecule experiments and analysed the data. W.H., Y.Y., Z.Z., Y.L.C. and Y.S. performed the ultrasound experiments and analysed the data. W.H., X.W. and Y.Y. performed the antibody stability experiments. Yi.C., W.W. and M.Q. supervised the project. W.H. and Yi.C. wrote the paper with contributions from all authors.

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Correspondence to Wei Wang or Yi Cao.

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Supplementary Methods, Supplementary Notes, Supplementary Figures 1–27, Supplementary Tables 1 and 2.

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Huang, W., Wu, X., Gao, X. et al. Maleimide–thiol adducts stabilized through stretching. Nat. Chem. 11, 310–319 (2019). https://doi.org/10.1038/s41557-018-0209-2

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