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Development of a versatile and efficient C–N lyase platform for asymmetric hydroamination via computational enzyme redesign

Abstract

Although C–N bonds are ubiquitous in natural products, pharmaceuticals and agrochemicals, biocatalysts forging these bonds with high atom-efficiency and enantioselectivity have been limited to a few select enzymes. In particular, ammonia lyases have emerged as powerful catalysts to access C–N bond formation via hydroamination. However, the use of ammonia lyases is rather restricted due to their narrow synthetic scope. Herein, we report the computational redesign of aspartase, a highly specific ammonia lyase, to yield C–N lyases with cross-compatibility of non-native nucleophiles and electrophiles. A wide range of non-canonical amino acids (ncAAs) are afforded with excellent conversion (up to 99%), regioselectivity >99% and enantioselectivity >99%. The process is scalable under industrially relevant protocols (exemplified in kilogram-scale synthesis) and can be facilely integrated in cascade reactions (demonstrated in the synthesis of β-lactams with N-1 and C-4 substitutions). This versatile and efficient C–N lyase platform supports the preparation of ncAAs and their derivatives, and will present opportunities in synthetic biology.

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Fig. 1: Biosynthetic strategy for producing ncAAs through hydroamination reactions.
Fig. 2: A flowchart for computational active site redesign of AspB.
Fig. 3: Computationally redesigned AspB-catalysed amine (a–n) addition to unsaturated acids (1–9).
Fig. 4: Synthesis of ncAAs at preparative scale.
Fig. 5: Chemoenzymatic synthesis of β-lactam compounds and modification of the alkyne tag in β-lactam heterocycles via click reaction.

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The findings of this study are available within the paper and its Supplementary Information. All data are available from the corresponding author upon reasonable request.

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Acknowledgements

This work is supported by the National Key R&D Programme of China (grant no. 2018YFA0901600), the National Natural Science Foundation of China (grant nos. 31822002, 31870055), the Biological Resources Programme (grant no. KFJ-BRP-017-58) and the Key Research Program of Frontier Sciences (grant no. ZDBS-LY-SM014) from the Chinese Academy of Sciences.

Author information

Authors and Affiliations

Authors

Contributions

B.W. initiated the project. Y.C. performed the computational work. Y.W., W.T., T.L., X.C. and R.L. performed biocatalytic experiments. Y.B. and T.Z. performed preparative-scale synthesis of amino acids and derivatives. Y.C. and B.W. provided supervision and input on experimental design and wrote the manuscript, which was revised and approved by all authors. Y.C., Y.W. and W.T. contributed equally to this work.

Corresponding author

Correspondence to Bian Wu.

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The authors declare no competing interests.

Additional information

Peer review information Nature Catalysis thanks Csaba Paizs, Christopher K Prier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Tables 1–3, Figs. 1–62 and Notes 1–7.

Reporting Summary

Supplementary Data PDB 1

Rosetta-predicted model of BA15.

Supplementary Data PDB 2

Rosetta-predicted model of AA15.

Supplementary Data PDB 3

Rosetta-predicted model of PA15.

Supplementary Data PDB 4

Rosetta-predicted model of FA15.

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Cui, Y., Wang, Y., Tian, W. et al. Development of a versatile and efficient C–N lyase platform for asymmetric hydroamination via computational enzyme redesign. Nat Catal 4, 364–373 (2021). https://doi.org/10.1038/s41929-021-00604-2

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