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Nucleophilic catalysis of acylhydrazone equilibration for protein-directed dynamic covalent chemistry

Abstract

Dynamic covalent chemistry uses reversible chemical reactions to set up an equilibrating network of molecules at thermodynamic equilibrium, which can adjust its composition in response to any agent capable of altering the free energy of the system. When the target is a biological macromolecule, such as a protein, the process corresponds to the protein directing the synthesis of its own best ligand. Here, we demonstrate that reversible acylhydrazone formation is an effective chemistry for biological dynamic combinatorial library formation. In the presence of aniline as a nucleophilic catalyst, dynamic combinatorial libraries equilibrate rapidly at pH 6.2, are fully reversible, and may be switched on or off by means of a change in pH. We have interfaced these hydrazone dynamic combinatorial libraries with two isozymes from the glutathione S-transferase class of enzyme, and observed divergent amplification effects, where each protein selects the best-fitting hydrazone for the hydrophobic region of its active site.

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Figure 1: Transimination reactions for DCC.
Figure 2: Aniline-catalysed acylhydrazone formation.
Figure 3: Structure of GST illustrating H- and G-sites.
Figure 4: GST-templated DCLs.
Figure 5: GST-templated DCLs of GSH conjugates.
Figure 6: GST ligands.
Figure 7: Molecular modelling of amplified DCL components with the GST active site.

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Acknowledgements

The authors would like to thank EastChem for the award of a studentship to V.T.B. and the Marie Curie Early Stage Training Network (Syn4chembio) and School of Chemistry at Edinburgh for awarding a studentship to A.M.C. R.B. is supported by an EC Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 223461. M.F.G. is an Engineering and Physical Sciences Research Council (EPSRC) Leadership Fellow. The authors thank A. Cooper (University of Glasgow) for ITC measurements and helpful discussions. N. Petitjean is thanked for the synthesis of hydrazone–GSH conjugates.

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V.T.B., A.M.C., D.J.C. and M.F.G. conceived and designed the experiments, V.T.B. and A.M.C. performed the experiments, and T.L., R.B. and A.M.C. carried out molecular modelling. All authors discussed the results and co-wrote the manuscript.

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Correspondence to Dominic J. Campopiano or Michael F. Greaney.

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Bhat, V., Caniard, A., Luksch, T. et al. Nucleophilic catalysis of acylhydrazone equilibration for protein-directed dynamic covalent chemistry. Nature Chem 2, 490–497 (2010). https://doi.org/10.1038/nchem.658

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