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Stereoselective amino acid synthesis by photobiocatalytic oxidative coupling

Nature volume 629, pages 98–104 (2024)Cite this article

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Abstract

Photobiocatalysis—where light is used to expand the reactivity of an enzyme—has recently emerged as a powerful strategy to develop chemistries that are new to nature. These systems have shown potential in asymmetric radical reactions that have long eluded small-molecule catalysts1. So far, unnatural photobiocatalytic reactions are limited to overall reductive and redox-neutral processes2,3,4,5,6,7,8,9. Here we report photobiocatalytic asymmetric sp3–sp3 oxidative cross-coupling between organoboron reagents and amino acids. This reaction requires the cooperative use of engineered pyridoxal biocatalysts, photoredox catalysts and an oxidizing agent. We repurpose a family of pyridoxal-5′-phosphate-dependent enzymes, threonine aldolases10,11,12, for the α-C–H functionalization of glycine and α-branched amino acid substrates by a radical mechanism, giving rise to a range of α-tri- and tetrasubstituted non-canonical amino acids 13,14,15 possessing up to two contiguous stereocentres. Directed evolution of pyridoxal radical enzymes allowed primary and secondary radical precursors, including benzyl, allyl and alkylboron reagents, to be coupled in an enantio- and diastereocontrolled fashion. Cooperative photoredox–pyridoxal biocatalysis provides a platform for sp3–sp3 oxidative coupling16, permitting the stereoselective, intermolecular free-radical transformations that are unknown to chemistry or biology.

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Fig. 1: Photobiocatalytic asymmetric sp3–sp3 oxidative cross-coupling via cooperative triple catalytic cycles.
Fig. 2: Discovery and optimization of photobiocatalytic asymmetric sp3–sp3 oxidative cross-coupling.
Fig. 3: Photobiocatalytic asymmetric sp3–sp3 oxidative coupling for ncAA synthesis.
Fig. 4: Enantio- and diastereoselective synthesis of β-methyl ncAAs and enantioselective synthesis of α-tetrasubstituted ncAAs.

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Data availability

All data are available in the main text and the Supplementary Information. Plasmids encoding PLP radical enzymes reported in this study are available for research purposes from Y.Y. under a material transfer agreement with the University of California Santa Barbara.

Change history

  • 17 May 2024

    In the version of the article initially published, the Supplementary Data files (NMR spectra and X-ray data) were missing and are now available in the HTML version of the article.

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Acknowledgements

This research is supported by the NIH (R35GM147387 to Y.Y. and R35GM128779 to P.L.) and the Packard Foundation (Y.Y.). PLP enzyme mining is supported by the Herman Frasch Foundation (947-HF22 to Y.Y.). We acknowledge the NSF BioPACIFIC MIP (DMR-1933487) and NSF MRSEC at UCSB (DMR-2308708) for access to instrumentation. Computational studies were carried out at the University of Pittsburgh Center for Research Computing and the Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) programme, supported by NSF award numbers OAC-2117681, OAC-1928147 and OAC-1928224. We are grateful to Y. Wang (University of Pittsburgh) for critical reading of this manuscript as well as P. C. Ford (University of California Santa Barbara) and Q. Zhu (University of Utah) for helpful discussions on photo- and electrochemistry.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, CA, USA

    Tian-Ci Wang, Zheng Zhang, Zhiyu Bo, Jiedong Li & Yang Yang

  2. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA

    Binh Khanh Mai & Peng Liu

  3. Biomolecular Science and Engineering (BMSE) Program, University of California Santa Barbara, Santa Barbara, CA, USA

    Yang Yang

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Contributions

Y.Y. conceived and directed the project. T.-C.W. discovered and optimized the oxidative photobiocatalytic process. T.-C.W. and Z.Z. performed the directed evolution and carried out the substrate scope studies. Z.B. and J.L. prepared the organoboron substrates. B.K.M. carried out the computational studies, with P.L. and Y.Y. providing guidance. Y.Y., P.L. and B.K.M. wrote the manuscript with the input of all other authors.

Corresponding authors

Correspondence to Peng Liu or Yang Yang.

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Competing interests

Y.Y., T.-C.W. and Z.Z. are inventors on a patent application submitted by the University of California Santa Barbara that covers compositions, methods and applications of biocatalytic non-canonical amino acid synthesis. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Mechanistic studies.

a, TEMPO trapping studies. Reaction conditions: 1a’ (1.0 equiv, 3.0 mM), 2a (10 equiv, 30.0 mM), 1 mol% TmPLPα1 (30 μM), 10 mol% PLP (300 μM), 2 mol% (fac)-Ir(ppy)3 (60 μM), Co(NH3)6Cl3 (2.0 equiv, 6.0 mM), TEMPO (3.0 equiv, 9.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h. b, Radical generation studies. Reaction conditions: 1a’ (1.0 equiv, 3.0 mM), 2 mol% (fac)-Ir(ppy)3 (60 μM), Co(NH3)6Cl3 (2.0 equiv, 6.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h.

Extended Data Fig. 2 Computational studies on threonine aldolase-catalysed oxidative cross-coupling.

a, Computed energy profile using a theozyme model at the (U)ωB97X-D/6-311+G(2d,2p)-SDD(Ir)/SMD(PhCl)//(U)B3LYP-D3/6-31G(d)-SDD(Ir) level of theory. Except those in external aldimine 13, active-site residues are omitted for clarity. Enthalpy values (ΔH) are with respect to 13. b, Activation enthalpies for radical additions to quinonoid species computed using theozyme and a cofactor-only model. Enthalpies are relative to the van der Waals complex (15). c, Optimized structures of regio- and enantioselectivity-determining radical addition transition states.

Extended Data Fig. 3 UV-vis spectroscopic analysis of threonine aldolase variants upon the introduction of D-alanine, L-alanine and glycine.

a, TmTA W86N (TmPLPα1) at pH 8. b, TmTA W86N (TmPLPα1) at pH 9.

Supplementary information

Supplementary Information

Experimental procedures, methods, supplementary figures and tables, and characterization data.

Reporting Summary

Supplementary Data 1

NMR spectra.

Supplementary Data 2

X-ray data.

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Wang, TC., Mai, B.K., Zhang, Z. et al. Stereoselective amino acid synthesis by photobiocatalytic oxidative coupling. Nature 629, 98–104 (2024). https://doi.org/10.1038/s41586-024-07284-5

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