Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Harnessing transaminases to construct azacyclic non-canonical amino acids

Nature Synthesis volume 3, pages 662–669 (2024)Cite this article

Subjects

Abstract

Non-canonical amino acids (ncAAs) are prized building blocks in the synthesis of natural products, designer peptides and drug molecules. Despite their general utility, the structural complexity of these molecules still presents an enormous challenge for chemical synthesis. Here we develop a one-pot chemoenzymatic approach for the construction of azacyclic ncAAs with multiple substitutions and various ring sizes. A promiscuous transaminase was identified to convert a wide range of diketoacids to the corresponding α-amino acids. A spontaneous cyclic imine formation was followed by a stereocontrolled chemical reduction to generate the corresponding products in one pot with high stereoselectivity. More than 25 azacyclic ncAAs were successfully prepared using this approach. This work demonstrates the value of developing hybrid biocatalytic–chemocatalytic approaches to prepare privileged small molecule motifs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Importance and prevalence of azacyclic ncAAs.
Fig. 2: Preparation of 2,n-diketocarboxylic acid substrates.
Fig. 3: Substrate scope exploration of one-pot ncAA synthesis with MtIlvE and hydrogenation.
Fig. 4: Synthetic logic for accessing branched azacyclic ncAAs with our chemoenzymatic strategy.

Similar content being viewed by others

Data availability

Full experimental details, Supplementary Fig. 1 and Supplementary Tables 1 and 2 are available in the Supplementary Information. Crystallographic data for compound 16l-cis-trans reported in this Article have been deposited at the Cambridge Crystallographic Data Centre under deposition number CCDC 2278016. Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/.

References

  1. Walsh, C. T., Brien, R. V. O. & Khosla, C. Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds. Angew. Chem. Int. Ed. 52, 7098–7124 (2013).

    Article  CAS  Google Scholar 

  2. Budisa, N. Expanded genetic code for the engineering of ribosomally synthetized and post-translationally modified peptide natural products (RiPPs). Curr. Opin. Biotechnol. 24, 591–598 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Du, Y. et al. Incorporation of non-canonical amino acids into antimicrobial peptides: advances, challenges, and perspectives. Appl. Environ. Microbiol. 88, e01617–e01622 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Hoesl, M. G. & Budisa, N. In vivo incorporation of multiple noncanonical amino acids into proteins. Angew. Chem. Int. Ed. 50, 2896–2902 (2011).

    Article  CAS  Google Scholar 

  5. Ngo, J. T. & Tirrell, D. A. Noncanonical amino acids in the interrogation of cellular protein synthesis. Acc. Chem. Res. 44, 677–685 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Agostini, F. et al. Biocatalysis with unnatural amino acids: enzymology meets xenobiology. Angew. Chem. Int. Ed. 56, 9680–9703 (2017).

    Article  CAS  Google Scholar 

  7. Katoh, T. & Suga, H. In vitro genetic code reprogramming for the expansion of usable noncanonical amino acids. Annu. Rev. Biochem. 91, 221–243 (2022).

    Article  CAS  PubMed  Google Scholar 

  8. Sun, S. B., Schultz, P. G. & Hyuk, C. Therapeutic applications of an expanded genetic code. ChemBioChem 15, 1721–1729 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wals, K. & Ovaa, H. Unnatural amino acid incorporation in E. coli: current and future applications in the design of therapeutic proteins. Front. Chem. 2, 15 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Rezhdo, A., Islam, M., Huang, M. & Van Deventer, J. A. Future prospects for noncanonical amino acids in biological therapeutics. Curr. Opin. Biotechnol. 60, 168–178 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Inokuma, T. Synthesis of non-canonical amino acids and peptide containing them for establishment of the template for drug discovery. Chem. Pharm. Bull. 69, 303–313 (2021).

    Article  CAS  Google Scholar 

  12. Hickey, J. L., Sindhikara, D., Zultanski, S. L. & Schultz, D. M. Beyond 20 in the 21st century: prospects and challenges of non-canonical amino acids in peptide drug discovery. ACS Med. Chem. Lett. 14, 557–565 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gentilucci, L., De Marco, R. & Cerisoli, L. Chemical modifications designed to improve peptide stability: incorporation of non-natural amino acids, pseudo-peptide bonds, and cyclization. Curr. Pharm. Des. 16, 3185–3203 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Kiss, L., Mándity, I. M. & Fülöp, F. Highly functionalized cyclic β-amino acid moieties as promising scaffolds in peptide research and drug design. Amino Acids 49, 1441–1455 (2017).

    Article  CAS  PubMed  Google Scholar 

  15. Rovero, P. et al. Agonist activity at the kinin B1 receptor: structural requirements of the central tetrapeptide. J. Med. Chem. 44, 274–278 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Carney, D. W., Schmitz, K. R., Truong, J. V., Sauer, R. T. & Sello, J. K. Restriction of the conformational dynamics of the cyclic acyldepsipeptide antibiotics improves their antibacterial activity. J. Am. Chem. Soc. 136, 1922–1929 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Cummings, A. E. et al. β-Branched bmino acids stabilize specific conformations of cyclic hexapeptides. Biophys. J. 116, 433–444 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Matzov, D. et al. Structural insights of lincosamides targeting the ribosome of Staphylococcus aureus. Nucleic Acids Res. 45, 10284–10292 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Owen, D. R. et al. An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19. Science 374, 1586–1593 (2021).

    Article  CAS  PubMed  Google Scholar 

  20. Link, J. O. The discovery of velpatasvir (GS-5816): the potent pan-genotypic once-daily oral HCV NS5A inhibitor in the single-tablet regimens Epclusa® and Vosevi®. Top. Med. Chem. 32, 81–110 (2019).

    Article  CAS  Google Scholar 

  21. Armishaw, C. et al. Rational design of α-conotoxin analogues targeting α7-nicotinic acetylcholine receptors. J. Biol. Chem. 284, 9498–9512 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Garrigou, M. et al. Accelerated identification of cell active KRAS inhibitory macrocyclic peptides using mixture libraries and automated ligand identification system (ALIS) technology. J. Med. Chem. 65, 8961–8974 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kreuzfeld, H. J., Döbler, C., Schmidt, U. & Krause, H. W. Synthesis of non-proteinogenic (d)- or (l)-amino acids by asymmetric hydrogenation. Amino Acids 11, 269–282 (1996).

    Article  CAS  PubMed  Google Scholar 

  24. Gross, M. F., Martinez, P. & Carolina, N. Asymmetric catalytic synthesis of β-branched amino acids via highly enantioselective hydrogenation of α-enamides. J. Am. Chem. Soc. 117, 9375–9376 (1995).

    Article  Google Scholar 

  25. Roff, G. J., Lloyd, R. C., Turner, N. J., Park, S. & Road, M. A versatile chemo-enzymatic route to enantiomerically pure β-branched α-amino acids. J. Am. Chem. Soc. 126, 4098–4099 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Ji, J. et al. Highly enantioselective synthesis of non-natural aliphatic α-amino acids via asymmetric hydrogenation. Org. Biomol. Chem. 13, 7624–7627 (2015).

    Article  CAS  PubMed  Google Scholar 

  27. Zuend, S. J., Coughlin, M. P., Lalonde, M. P. & Jacobsen, E. N. Scaleable catalytic asymmetric Strecker syntheses of unnatural α-amino acids. Nature 461, 968–970 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Yu, F. et al. Regioselective α-cyanation of unprotected alicyclic amines. Org. Lett. 24, 6364–6368 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mazaleyrat, J., Rage, I., Xie, J., Saw-da, J. & Wakselman, M. Synthesis of a potential ‘suicide substrate’ of the HIV-1 protease, incorporating a 3-acetoxy-Δ-4,5-(l)-pipecolic acid as proline substitute. Tetrahedron Lett. 33, 4453–4456 (1992).

    Article  CAS  Google Scholar 

  30. Hattori, K. & Grossman, R. B. Functionalized cis- and trans-fused bicyclic α-amino acids via stereoselective double annulation and dequaternization reactions. J. Org. Chem. 68, 1409–1417 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. Klein, C. & Hüttel, W. A simple procedure for selective hydroxylation of l-proline and l-pipecolic acid with recombinantly expressed proline hydroxylases. Adv. Synth. Catal. 353, 1375–1383 (2011).

    Article  CAS  Google Scholar 

  32. Glucides, H. Stereoselective synthesis of C-6 substituted pipecolic acid derivatives. formal synthesis of (+)-indolizidine 167B and (+)-indolizidine 209D. Heterocycles 57, 1807–1830 (2002).

    Article  Google Scholar 

  33. Davis, F. A., Zhang, H., Lee, S. H., V, T. U. & Pennsyl, V. Masked oxo sulfinimines (N-sulfinyl imines) in the asymmetric synthesis of proline and pipecolic acid derivatives. Org. Lett. 3, 759–762 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Miyazaki, M., Naito, H., Sugimoto, Y., Yoshida, K. & Kawato, H. Synthesis and evaluation of novel orally active p53–MDM2 interaction inhibitors. Bioorg. Med. Chem. 21, 4319–4331 (2013).

    Article  CAS  PubMed  Google Scholar 

  35. Castro, A. J. et al. Enantioselective synthesis of α-amino acetals and α-amino acids by nucleophilic 1,2-addition to diethoxyacetaldehyde SAMP hydrazone. Angew. Chem. Int. Ed. 32, 418–421 (1993).

    Article  Google Scholar 

  36. Zhang, S., Li, Q., He, G., Nack, W. A. & Chen, G. Stereoselective synthesis of β-alkylated α-amino acids via palladium-catalyzed alkylation of unactivated methylene C(sp3)–H bonds with primary alkyl halides. J. Am. Chem. Soc. 135, 12135–12141 (2013).

    Article  CAS  PubMed  Google Scholar 

  37. Chen, G. et al. Ligand-enabled β-C−H arylation of α-amino acids using a simple and practical auxiliary. J. Am. Chem. Soc. 137, 3338–3351 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hauer, B. Embracing nature’s catalysts: a viewpoint on the future of biocatalysis. ACS Catal. 10, 8418–8427 (2020).

    Article  CAS  Google Scholar 

  39. Wu, S., Snajdrova, R., Moore, J. C., Baldenius, K. & Bornscheuer, U. T. Biocatalysis: enzymatic synthesis for industrial applications. Angew. Chem. Int. Ed. 60, 88–119 (2021).

    Article  CAS  Google Scholar 

  40. Winkler, C. K., Schrittwieser, J. H. & Kroutil, W. Power of biocatalysis for organic synthesis. ACS Cent. Sci. 7, 55–71 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Hussain, S. et al. An (R)-imine reductase biocatalyst for the asymmetric reduction of cyclic imines. ChemCatChem 7, 579–583 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Payer, S. E., Schrittwieser, J. H., Grischek, B., Simon, R. C. & Kroutil, W. Regio‐ and stereoselective biocatalytic monoamination of a triketone enables asymmetric synthesis of both enantiomers of the pyrrolizidine alkaloid xenovenine employing transaminases. Adv. Synth. Catal. 358, 444–451 (2016).

    Article  CAS  Google Scholar 

  43. France, S. P. et al. One-pot cascade synthesis of mono- and disubstituted piperidines and pyrrolidines using carboxylic acid reductase (CAR), ω-transaminase (ω-TA), and imine reductase (IRED) biocatalysts. ACS Catal. 6, 3753–3759 (2016).

    Article  CAS  Google Scholar 

  44. Costa, B. Z. et al. Synthesis of 2,5-disubstituted pyrrolidine alkaloids via a one-pot cascade using transaminase and reductive aminase biocatalysts. ChemCatChem 10, 4733–4738 (2018).

    Article  CAS  Google Scholar 

  45. Taday, F., Ryan, J., O’Sullivan, R. & O’Reilly, E. Transaminase-mediated amine borrowing via shuttle biocatalysis. Org. Lett. 24, 74–79 (2022).

    Article  CAS  PubMed  Google Scholar 

  46. Petermeier, P. et al. Asymmetric synthesis of trisubstituted piperidines via biocatalytic transamination and diastereoselective enamine or imine reduction. Adv. Synth. Catal. 365, 2188–2202 (2023).

    Article  CAS  Google Scholar 

  47. Hepworth, L. J. et al. Enzyme cascades in whole cells for the synthesis of chiral cyclic amines. ACS Catal. 7, 2920–2925 (2017).

    Article  CAS  Google Scholar 

  48. Chen, M., Liu, C. & Tang, Y. Discovery and biocatalytic application of a PLP-dependent amino acid γ-substitution enzyme that catalyzes C–C bond formation. J. Am. Chem. Soc. 142, 10506–10515 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Watkins-Dulaney, E. J. et al. Asymmetric alkylation of ketones catalyzed by engineered TrpB. Angew. Chem. Int. Ed. 60, 21412–21417 (2021).

    Article  CAS  Google Scholar 

  50. Li, F., Yang, L. C., Zhang, J., Chen, J. S. & Renata, H. Stereoselective synthesis of β-branched aromatic α-amino acids by biocatalytic dynamic kinetic resolution. Angew. Chem. Int. Ed. 60, 17680–17685 (2021).

    Article  CAS  Google Scholar 

  51. Amorim Franco, T. M., Hegde, S. & Blanchard, J. S. Chemical mechanism of the branched-chain aminotransferase IlvE from Mycobacterium tuberculosis. Biochemistry 55, 6295–6303 (2016).

    Article  CAS  PubMed  Google Scholar 

  52. Smith, D. R. et al. Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics. J. Bacteriol. 179, 7135–7155 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Nelson, K. E. et al. Evidence for lateral gene transfer between Archaea and Bacteria from genome sequence of Thermotoga maritima. Nature 399, 323–329 (1999).

    Article  CAS  PubMed  Google Scholar 

  54. Matsui, I. et al. The molecular structure of hyperthermostable aromatic aminotransferase with novel substrate specificity from Pyrococcus horikoshii. J. Biol. Chem. 275, 4871–4879 (2000).

    Article  CAS  PubMed  Google Scholar 

  55. Okazaki, N. et al. Cloning and nucleotide sequencing of phenylalanine dehydrogenase gene of Bacillus sphaericus. Gene 63, 337–341 (1988).

    Article  CAS  PubMed  Google Scholar 

  56. Cini, E., Bifulco, G., Menchi, G., Rodriquez, M. & Taddei, M. Synthesis of enantiopure 7-substituted azepane-2-carboxylic acids as templates for conformationally constrained peptidomimetics. Eur. J. Org. Chem. 2012, 2133–2141 (2012).

    Article  CAS  Google Scholar 

  57. Romney, D. K., Sarai, N. S. & Arnold, F. H. Nitroalkanes as versatile nucleophiles for enzymatic synthesis of noncanonical amino acids. ACS Catal. 9, 8726–8730 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Page, P., Barrage, S., Baldock, L. & Bradley, M. The synthesis of symmetrical spermine conjugates using solid-phase chemistry. Bioorganic Med. Chem. Lett. 8, 1751–1756 (1998).

    Article  CAS  Google Scholar 

  59. Kalisiak, J. et al. Identification of a new endogenous metabolite and the characterization of its protein interactions through an immobilization approach. J. Am. Chem. Soc. 131, 378–386 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Martin, J., Eisoldt, L. & Skerra, A. Fixation of gaseous CO2 by reversing a decarboxylase for the biocatalytic synthesis of the essential amino acid l-methionine. Nat. Catal. 1, 555–561 (2018).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Funding for this work is provided, in part, by the ACS Green Chemistry Institute Pharmaceutical Roundtable grant and the Welch Foundation (grant number C2159). T.-H.C. acknowledges fellowship support from the Ministry of Education, Taiwan. We thank M. Gantcher (Rice University) for his assistance in some of the experiments described in this work. We are grateful to the Shared Equipment Authority at Rice University for access to their instrumentation.

Author information

Authors and Affiliations

  1. Department of Chemistry, BioScience Research Collaborative, Rice University, Houston, TX, USA

    Tsung-Han Chao, Xiangyu Wu, Yu Fu & Hans Renata

  2. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Licheng Yang

Authors
  1. Tsung-Han Chao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiangyu Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yu Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Licheng Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hans Renata
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.-H.C., L.Y. and H.R. conceived the work. T.-H.C., X.W., Y.F., L.Y. and H.R. designed all the experiments described in the manuscript. T.-H.C., X.W. and H.R. wrote the manuscript.

Corresponding author

Correspondence to Hans Renata.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Synthesis thanks Andrew Buller, Sabine Flitsch and Elaine O’Reilly for their contribution to the peer review of this work. Primary Handling Editor: Peter Seavill, in collaboration with the Nature Synthesis team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Experimental details, Supplementary Figs. 1 and 2, and Tables 1 and 2.

Reporting Summary

Supplementary Data 1

X-ray crystallographic data for compound 16l-cis–trans, CCDC 2278016.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chao, TH., Wu, X., Fu, Y. et al. Harnessing transaminases to construct azacyclic non-canonical amino acids. Nat. Synth 3, 662–669 (2024). https://doi.org/10.1038/s44160-024-00514-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s44160-024-00514-8

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing