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:

Biocatalytic strategy for the construction of sp3-rich polycyclic compounds from directed evolution and computational modelling

Nature Chemistry (2024)Cite this article

Subjects

Abstract

Catalysis with engineered enzymes has provided more efficient routes for the production of active pharmaceutical agents. However, the potential of biocatalysis to assist in early-stage drug discovery campaigns remains largely untapped. In this study, we have developed a biocatalytic strategy for the construction of sp3-rich polycyclic compounds via the intramolecular cyclopropanation of benzothiophenes and related heterocycles. Two carbene transferases with complementary regioisomer selectivity were evolved to catalyse the stereoselective cyclization of benzothiophene substrates bearing diazo ester groups at the C2 or C3 position of the heterocycle. The detailed mechanisms of these reactions were elucidated by a combination of crystallographic and computational analyses. Leveraging these insights, the substrate scope of one of the biocatalysts could be expanded to include previously unreactive substrates, highlighting the value of integrating evolutionary and rational strategies to develop enzymes for new-to-nature transformations. The molecular scaffolds accessed here feature a combination of three-dimensional and stereochemical complexity with ‘rule-of-three’ properties, which should make them highly valuable for fragment-based drug discovery campaigns.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: sp2- versus sp3-rich molecular ‘fragments’ for applications in FBDD.
Fig. 2: Biocatalytic intramolecular cyclopropanation of benzothiophene substrates.
Fig. 3: Intramolecular cyclopropanation reactions promoted by Mb biocatalysts.
Fig. 4: Crystal structures of MbBTIC-C2 and MbBTIC-C3 and the corresponding stereochemical models.
Fig. 5: Analysis of the MbBTIC-C2-, MbBTIC-C3- and MbBTIC-C3+-catalysed cyclization reactions by MD simulations.

Similar content being viewed by others

Data availability

Protein crystal structure coordinates have been deposited with the Protein Data Bank (PDB) under accession numbers 7SLH (MbBTIC-C3) and 7SLI (MbBTIC-C2). Crystallographic data for the structures reported in this Article have been deposited at the Cambridge Crystallographic Data Centre under CCDC deposition numbers 2157009 (2a), 2157011 (2d), 2157007 (4a), 2157010 (4f) and 2157008 (4k). Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/.

References

  1. Bornsceuer, U. T. et al. Engineering the third wave of biocatalysis. Nature 485, 185–194 (2012).

    Article  ADS  Google Scholar 

  2. Devine, P. N. et al. Extending the application of biocatalysis to meet the challenges of drug development. Nat. Rev. Chem. 2, 409–421 (2018).

    Article  Google Scholar 

  3. Slagman, S. & Fessner, W. D. Biocatalytic routes to anti-viral agents and their synthetic intermediates. Chem. Soc. Rev. 50, 1968–2009 (2021).

    Article  CAS  PubMed  Google Scholar 

  4. Savile, C. K. et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309 (2010).

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Huffman, M. A. et al. Design of an in vitro biocatalytic cascade for the manufacture of islatravir. Science 366, 1255–1259 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  6. McIntosh, J. A. et al. Engineered ribosyl-1-kinase enables concise synthesis of molnupiravir, an antiviral for COVID-19. ACS Cent. Sci. 7, 1980–1985 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Brandenberg, O. F., Fasan, R. & Arnold, F. H. Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions. Curr. Opin. Biotechnol. 47, 102–111 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Schwizer, F. et al. Artificial metalloenzymes: reaction scope and optimization strategies. Chem. Rev. 118, 142–231 (2018).

    Article  CAS  PubMed  Google Scholar 

  9. Ren, X. & Fasan, R. Engineered and artificial metalloenzymes for selective C–H functionalization. Curr. Opin. Green Sustain. Chem. 31, 100494 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Liu, Z. & Arnold, F. H. New-to-nature chemistry from old protein machinery: carbene and nitrene transferases. Curr. Opin. Biotechnol. 69, 43–51 (2021).

    Article  CAS  PubMed  Google Scholar 

  11. Ramsden, J. I., Cosgrove, S. C. & Turner, N. J. Is it time for biocatalysis in fragment-based drug discovery? Chem. Sci. 11, 11104–11112 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Erlanson, D. A., Fesik, S. W., Hubbard, R. E., Jahnke, W. & Jhoti, H. Twenty years on: the impact of fragments on drug discovery. Nat. Rev. Drug Discov. 15, 605–619 (2016).

    Article  CAS  PubMed  Google Scholar 

  13. Erlanson, D. A., Davis, B. J. & Jahnke, W. Fragment-based drug discovery: advancing fragments in the absence of crystal structures. Cell Chem. Biol. 26, 9–15 (2019).

    Article  CAS  PubMed  Google Scholar 

  14. Jhoti, H., Williams, G., Rees, D. C. & Murray, C. W. The ‘rule of three’ for fragment-based drug discovery: where are we now? Nat. Rev. Drug Discov. 12, 644 (2013).

    Article  CAS  PubMed  Google Scholar 

  15. Murray, C. W. & Rees, D. C. Opportunity knocks: organic chemistry for fragment-based drug discovery (FBDD). Angew. Chem. Int. Ed. 55, 488–492 (2016).

    Article  CAS  Google Scholar 

  16. Morley, A. D. et al. Fragment-based hit identification: thinking in 3D. Drug Discov. Today 18, 1221–1227 (2013).

    Article  PubMed  Google Scholar 

  17. Over, B. et al. Natural-product-derived fragments for fragment-based ligand discovery. Nat. Chem. 5, 21–28 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Yu, B., Zheng, Y. C., Shi, X. J., Qi, P. P. & Liu, H. M. Natural product-derived spirooxindole fragments serve as privileged substructures for discovery of new anticancer agents. Anticancer Agents Med. Chem. 16, 1315–1324 (2016).

    Article  CAS  PubMed  Google Scholar 

  19. Coelho, P. S., Brustad, E. M., Kannan, A. & Arnold, F. H. Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339, 307–310 (2013).

    Article  ADS  CAS  PubMed  Google Scholar 

  20. Bordeaux, M., Tyagi, V. & Fasan, R. Highly diastereoselective and enantioselective olefin cyclopropanation using engineered myoglobin-based catalysts. Angew. Chem. Int. Ed. 54, 1744–1748 (2015).

    Article  CAS  Google Scholar 

  21. Tinoco, A., Steck, V., Tyagi, V. & Fasan, R. Highly diastereo- and enantioselective synthesis of trifluoromethyl-substituted cyclopropanes via myoglobin-catalyzed transfer of trifluoromethylcarbene. J. Am. Chem. Soc. 139, 5293–5296 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Key, H. M. et al. Beyond iron: iridium-containing P450 enzymes for selective cyclopropanations of structurally diverse alkenes. ACS Cent. Sci. 3, 302–308 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chandgude, A. L. & Fasan, R. Highly diastereo- and enantioselective synthesis of nitrile-substituted cyclopropanes by myoglobin-mediated carbene transfer catalysis. Angew. Chem. Int. Ed. 57, 15852–15856 (2018).

    Article  CAS  Google Scholar 

  24. Knight, A. M. et al. Diverse engineered heme proteins enable stereodivergent cyclopropanation of unactivated alkenes. ACS Cent. Sci. 4, 372–377 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wittmann, B. J. et al. Diversity-oriented enzymatic synthesis of cyclopropane building blocks. ACS Catal. 10, 7112–7116 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Nam, D., Steck, V., Potenzino, R. J. & Fasan, R. A diverse library of chiral cyclopropane scaffolds via chemoenzymatic assembly and diversification of cyclopropyl ketones. J. Am. Chem. Soc. 143, 2221–2231 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Vargas, D. A., Tinoco, A., Tyagi, V. & Fasan, R. Myoglobin-catalyzed C–H functionalization of unprotected indoles. Angew. Chem. Int. Ed. 57, 9911–9915 (2018).

    Article  CAS  Google Scholar 

  28. Brandenberg, O. F., Chen, K. & Arnold, F. H. Directed evolution of a cytochrome P450 carbene transferase for selective functionalization of cyclic compounds. J. Am. Chem. Soc. 141, 8989–8995 (2019).

    Article  CAS  PubMed  Google Scholar 

  29. Srivastava, P., Yang, H., Ellis-Guardiola, K. & Lewis, J. C. Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation. Nat. Commun. 6, 7789 (2015).

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Sreenilayam, G., Moore, E. J., Steck, V. & Fasan, R. Metal substitution modulates the reactivity and extends the reaction scope of myoglobin carbene transfer catalysts. Adv. Synth. Catal. 359, 2076–2089 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Dydio, P. et al. An artificial metalloenzyme with the kinetics of native enzymes. Science 354, 102–106 (2016).

    Article  ADS  CAS  PubMed  Google Scholar 

  32. Ohora, K. et al. Catalytic cyclopropanation by myoglobin reconstituted with iron porphycene: acceleration of catalysis due to rapid formation of the carbene species. J. Am. Chem. Soc. 139, 17265–17268 (2017).

    Article  Google Scholar 

  33. Villarino, L. et al. An artificial heme enzyme for cyclopropanation reactions. Angew. Chem. Int. Ed. 57, 7785–7789 (2018).

    Article  Google Scholar 

  34. Zhao, J. M., Bachmann, D. G., Lenz, M., Gillingham, D. G. & Ward, T. R. An artificial metalloenzyme for carbene transfer based on a biotinylated dirhodium anchored within streptavidin. Catal. Sci. Technol. 8, 2294–2298 (2018).

    Article  CAS  Google Scholar 

  35. Carminati, D. M. & Fasan, R. Stereoselective cyclopropanation of electron-deficient olefins with a cofactor redesigned carbene transferase featuring radical reactivity. ACS Catal. 9, 9683–9697 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Stenner, R., Steventon, J. W., Seddon, A. & Anderson, J. L. R. A de novo peroxidase is also a promiscuous yet stereoselective carbene transferase. Proc. Natl Acad. Sci. USA 117, 1419–1428 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chandgude, A. L., Ren, X. & Fasan, R. Stereodivergent intramolecular cyclopropanation enabled by engineered carbene transferases. J. Am. Chem. Soc. 141, 9145–9150 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ren, X. K., Liu, N. Y., Chandgude, A. L. & Fasan, R. An enzymatic platform for the highly enantioselective and stereodivergent construction of cyclopropyl-δ-lactones. Angew. Chem. Int. Ed. 59, 21634–21639 (2020).

    Article  CAS  Google Scholar 

  39. Yong, K., Salim, M. & Capretta, A. Intramolecular carbenoid insertions: reactions of α-diazo ketones derived from furanyl-, thienyl-, (benzofuranyl)-, and (benzothienyl)acetic acids with rhodium(II) acetate. J. Org. Chem. 63, 9828–9833 (1998).

    Article  CAS  Google Scholar 

  40. Padwa, A., Wisnieff, T. J. & Walsh, E. J. Synthesis of cycloalkenones via the intramolecular cyclopropanation of furanyl diazo ketones. J. Org. Chem. 51, 5036–5038 (1986).

    Article  CAS  Google Scholar 

  41. Zhang, R. K. et al. Enzymatic assembly of carbon–carbon bonds via iron-catalysed sp3 C–H functionalization. Nature 565, 67–72 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  42. Wei, Y., Tinoco, A., Steck, V., Fasan, R. & Zhang, Y. Cyclopropanations via heme carbenes: basic mechanism and effects of carbene substituent, protein axial ligand, and porphyrin substitution. J. Am. Chem. Soc. 140, 1649–1662 (2018).

    Article  CAS  PubMed  Google Scholar 

  43. de Visser, S. P., Ogliaro, F., Harris, N. & Shaik, S. Multi-state epoxidation of ethene by cytochrome P450: a quantum chemical study. J. Am. Chem. Soc. 123, 3037–3047 (2001).

    Article  PubMed  Google Scholar 

  44. Schroder, D., Shaik, S. & Schwarz, H. Two-state reactivity as a new concept in organometallic chemistry. Acc. Chem. Res. 33, 139–145 (2000).

    Article  CAS  PubMed  Google Scholar 

  45. Sreenilayam, G. & Fasan, R. Myoglobin-catalyzed intermolecular carbene N–H insertion with arylamine substrates. Chem. Commun. 51, 1532–1534 (2015).

    Article  CAS  Google Scholar 

  46. Kondrashov, D. A., Zhang, W., Aranda, R. T., Stec, B. & Phillips, G. N. Jr. Sampling of the native conformational ensemble of myoglobin via structures in different crystalline environments. Proteins 70, 353–362 (2008).

    Article  CAS  PubMed  Google Scholar 

  47. Blomberg, M. R. A., Borowski, T., Himo, F., Liao, R. Z. & Siegbahn, P. E. M. Quantum chemical studies of mechanisms for metalloenzymes. Chem. Rev. 114, 3601–3658 (2014).

    Article  CAS  PubMed  Google Scholar 

  48. Himo, F. Recent trends in quantum chemical modeling of enzymatic reactions. J. Am. Chem. Soc. 139, 6780–6786 (2017).

    Article  CAS  PubMed  Google Scholar 

  49. Lind, M. E. S. & Himo, F. Quantum chemistry as a tool in asymmetric biocatalysis: limonene epoxide hydrolase test case. Angew. Chem. Int. Ed. 52, 4563–4567 (2013).

    Article  CAS  Google Scholar 

  50. Liao, R. Z. & Thiel, W. On the effect of varying constraints in the quantum mechanics only modeling of enzymatic reactions: the case of acetylene hydratase. J. Phys. Chem. B 117, 3954–3961 (2013).

    Article  CAS  PubMed  Google Scholar 

  51. Ryan, J. et al. Transaminase triggered aza-Michael approach for the enantioselective synthesis of piperidine scaffolds. J. Am. Chem. Soc. 138, 15798–15800 (2016).

    Article  CAS  PubMed  Google Scholar 

  52. Zawodny, W. et al. Chemoenzymatic synthesis of substituted azepanes by sequential biocatalytic reduction and organolithium-mediated rearrangement. J. Am. Chem. Soc. 140, 17872–17877 (2018).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the US National Institutes of Health (grant no. GM098628, R.F.). R.F. acknowledges support from the Cancer Prevention and Research Institute of Texas (CPRIT RR230018) and Robert A. Welch Foundation (Chair, AT-0051). D.A.V. acknowledges support from the National Science Foundation Graduate Fellowship Program. K.N.H. and A.S. acknowledge support from the National Science Foundation (CHE-1764328). M.G.-B. acknowledges support from the Spanish Ministerio de Ciencia e Innovación (MICINN; project PID2019-111300GA-I00) and the Ramón y Cajal programme via the RYC 2020-028628-I fellowship. The authors are grateful to W. Brennessel and J. Jenkins (University of Rochester) for assistance with crystallographic analyses. MS and X-ray instrumentation at the University of Rochester are supported by the US National Science Foundation (grant nos. CHE-0946653 and CHE-1725028) and the US National Institutes of Health (grant no. S10OD030302).

Author information

Author notes

  1. These authors contributed equally: David A. Vargas, Xinkun Ren, Arkajyoti Sengupta.

Authors and Affiliations

  1. Process Research and Development, Merck, Rahway, NJ, USA

    David A. Vargas

  2. College of Engineering and Applied Sciences, Nanjing University, Nanjing, China

    Xinkun Ren

  3. Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA

    Arkajyoti Sengupta & K. N. Houk

  4. Environment Research Institute, Shandong University, Qingdao, People’s Republic of China

    Ledong Zhu

  5. Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, TX, USA

    Satyajit Roy & Rudi Fasan

  6. Institut de Química Computacional i Catàlisi (IQCC), Departament de Química, Universitat de Girona, Girona, Spain

    Marc Garcia-Borràs

Authors
  1. David A. Vargas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xinkun Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arkajyoti Sengupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ledong Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Satyajit Roy
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marc Garcia-Borràs
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. K. N. Houk
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rudi Fasan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.A.V., X.R., S.R. and R.F. conceived the project, designed the experiments and analysed all the experiments with guidance from R.F. K.N.H. and M.G.-B. mentored L.Z. and A.S. for MD and quantum mechanics calculations, and contributed to the writing of the mechanistic parts of the paper. D.A.V. and R.F. wrote the paper with input from all of the authors. All authors discussed the results and contributed to the final paper.

Corresponding authors

Correspondence to K. N. Houk or Rudi Fasan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Chemistry thanks Adrian Mulholland and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Extended data

Extended Data Fig. 1 Calculated reaction pathways for C2- and C3-benzothienyl diazo ester substrates.

DFT analysis of the reaction mechanism for intramolecular cyclopropanation of C2- and C3-benzothiophenyl-methyl-diazoacetate catalyzed by a truncated iron porphyrin with an axial 4-methylimidazole ligand as a simplified model called Fe-Porphyrin. The reaction proceeds via a (1) iron−carbene formation followed by (2) cyclopropanation (2 C–C bonds). ΔG values are calculated at the B3LYP-D3BJ/def2TZVP (SMD, ε=4) // B3LYP-D3BJ/6-31G(d)+SDD (Fe) level. For each stationary point, the Gibbs free energy is provided for its lowest energy spin state. Detailed free energy profiles and additional data are provided in Supplementary Fig. 8 and Supplementary Table 5 in Supporting Information.

Supplementary information

Supplementary Information

Supplementary Information

Reporting Summary

Supplementary Data 1

Compound 2a—crystallographic information file.

Supplementary Data 2

Compound 2a—checkCIF report.

Supplementary Data 3

Compound 2d—crystallographic information file.

Supplementary Data 4

Compound 2d—checkCIF report.

Supplementary Data 5

Compound 4a—crystallographic information file.

Supplementary Data 6

Compound 4a—checkCIF report.

Supplementary Data 7

Compound 4f—crystallographic information file.

Supplementary Data 8

Compound 4f—checkCIF report.

Supplementary Data 9

Compound 4k—crystallographic information file.

Supplementary Data 10

Compound 4k—checkCIF report.

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

Vargas, D.A., Ren, X., Sengupta, A. et al. Biocatalytic strategy for the construction of sp3-rich polycyclic compounds from directed evolution and computational modelling. Nat. Chem. (2024). https://doi.org/10.1038/s41557-023-01435-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41557-023-01435-3

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