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An NAD⁺ analogue enables assembly of structurally diverse artificial photoenzymes for enantiodivergent [2 + 2] cycloadditions

Nature Catalysis volume 8, pages 822–832 (2025)Cite this article

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Abstract

Although artificial enzymes have significantly expanded the scope of enzyme-catalysed transformations, their construction typically relies on the irreversible incorporation of non-natural active sites. Inspired by natural cofactors, here we show a reversible binding strategy for artificial enzyme design using benzophenone adenine dinucleotide (BpAD), a photoactive NAD⁺ analogue that can integrate seamlessly into a broad range of NAD⁺-dependent protein scaffolds. The resulting artificial photoenzymes catalyse both inter- and intramolecular [2 + 2] cycloaddition reactions with excellent enantioselectivity, broad substrate compatibility and notable enantiodivergence. Computational studies confirm the precise binding mode of BpAD and reveal a key exo-attack pathway in the stepwise C–C bond formation mechanism. Notably, BpAD-catalysed reactions are highly orthogonal to those mediated by NAD⁺, allowing simultaneous use without interference. This work introduces a versatile and generalizable approach to artificial enzyme development, leveraging the inherent diversity of NAD⁺-dependent proteins for tailored catalytic applications.

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Fig. 1: Strategies for artificial enzyme design and development of the BpAD cofactor.
Fig. 2: Protein scaffold screening and engineering.
Fig. 3: Investigation of the substrate scope.
Fig. 4: Orthogonality of the NAD+- and BpAD-catalysed reactions.
Fig. 5: Mechanistic studies.

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Data availability

Data relating to the materials and methods, experimental procedures, mechanistic studies and computational calculations, HPLC spectra and NMR spectra are available in the Supplementary Information or from the authors on reasonable request. The configurations of MD simulations are available via GitHub at https://github.com/3dwdw58rds/7WBC-2DKN-research.

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Acknowledgements

We thank the National Key Research and Development Program of China (2022YFA1505600 to H.-J.P.), the National Natural Science Foundation of China (22277052 to H.-J.P. and 22121001 to B.W.), the Natural Science Foundation of Jiangsu Province (BK20232016 and BK20220762 to H.-J.P.) and the High-Level Innovation and Entrepreneurial Research Team Program in Jiangsu (JSSCTD202309 to H.-J.P.) for financial support. We also thank X. Lou from Westlake University for her valuable assistance with the transient absorption spectroscopy measurements.

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  1. These authors contributed equally: Ping Du, Jinsi Li, Tai-Ping Zhou.

Authors and Affiliations

  1. State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBic), ChemBioMed Interdisciplinary Research Center at Nanjing University, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China

    Ping Du, Jinsi Li, Jun Wang, Wenhao Hu, Haoyu Li & Hui-Jie Pan

  2. State Key Laboratory of Physical Chemistry of Solid Surfaces, Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China

    Tai-Ping Zhou & Binju Wang

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Contributions

H.-J.P. conceived and and coordinated the project. P.D. conducted most of the protein scaffold screening, protein evolution and reaction optimization experiments. J.L. designed and synthesized the BpAD cofactor, substrates and standard products. H.L. and J.W. conducted part of the protein evolution work. T.-P.Z. performed the computational studies under the supervision of B.W. W.H. measured the Kd values using ITC. H.-J.P., P.D., J.L., T.-P.Z. and B.W. wrote the paper with input from all authors.

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Correspondence to Binju Wang or Hui-Jie Pan.

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Nature Catalysis thanks Gui-Juan Cheng and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Du, P., Li, J., Zhou, TP. et al. An NAD⁺ analogue enables assembly of structurally diverse artificial photoenzymes for enantiodivergent [2 + 2] cycloadditions. Nat Catal 8, 822–832 (2025). https://doi.org/10.1038/s41929-025-01390-x

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