Navigating the Unnatural Reaction Space: Directed Evolution of Heme Proteins for Selective Carbene and Nitrene Transfer

Abstract

Despite the astonishing diversity of naturally occurring biocatalytic processes, enzymes do not catalyze many of the transformations favored by synthetic chemists. Either nature does not care about the specific products, or if she does, she has adopted a different synthetic strategy. In many cases, the appropriate reagents used by synthetic chemists are not readily accessible to biological systems. Here, we discuss our efforts to expand the catalytic repertoire of enzymes to encompass powerful reactions previously known only in small-molecule catalysis: formation and transfer of reactive carbene and nitrene intermediates leading to a broad range of products, including products with bonds not known in biology. In light of the structural similarity of iron carbene (Fe═C(R¹)(R²)) and iron nitrene (Fe═NR) to the iron oxo (Fe═O) intermediate involved in cytochrome P450-catalyzed oxidation, we have used synthetic carbene and nitrene precursors that biological systems have not encountered and repurposed P450s to catalyze reactions that are not known in the natural world. The resulting protein catalysts are fully genetically encoded and function in intact microbial cells or cell-free lysates, where their performance can be improved and optimized by directed evolution. By leveraging the catalytic promiscuity of P450 enzymes, we evolved a range of carbene and nitrene transferases exhibiting excellent activity toward these new-to-nature reactions. Since our initial report in 2012, a number of other heme proteins including myoglobins, protoglobins, and cytochromes c have also been found and engineered to promote unnatural carbene and nitrene transfer. Due to the altered active-site environments, these heme proteins often displayed complementary activities and selectivities to P450s.Using wild-type and engineered heme proteins, we and others have described a range of selective carbene transfer reactions, including cyclopropanation, cyclopropenation, Si-H insertion, B-H insertion, and C-H insertion. Similarly, a variety of asymmetric nitrene transfer processes including aziridination, sulfide imidation, C-H amidation, and, most recently, C-H amination have been demonstrated. The scopes of these biocatalytic carbene and nitrene transfer reactions are often complementary to the state-of-the-art processes based on small-molecule transition-metal catalysts, making engineered biocatalysts a valuable addition to the synthetic chemist's toolbox. Moreover, enabled by the exquisite regio- and stereocontrol imposed by the enzyme catalyst, this biocatalytic platform provides an exciting opportunity to address challenging problems in modern synthetic chemistry and selective catalysis, including ones that have eluded synthetic chemists for decades.

Scheme 1. Native and Engineered Activity: P450BM3-Catalyzed C–H Hydroxylation

Scheme 2. Structural Similarity of Fe Oxene, Carbene, and Nitrene

Scheme 3. P450-Catalyzed Diastereo- and Enantioselective Cyclopropanation of Styrenes (2012)

TTN = total turnover number. Conditions: 30 mM styrene, 10 mM EDA, 0.2 mol % catalyst, and 10 mM Na₂S₂O₄.

Scheme 4. Rationally Engineered “P411” Catalyst for in Vivo Cyclopropanation (2013)

Reaction carried out with neat substrates (170 mM EDA) for 24 h. All other reactions were carried out with 8.5 mM EDA.

Scheme 5. Axial Ligand Effect in the Biocatalytic Cyclopropanation in Vivo and the Development of P450BM3 Hstar (2014)

Scheme 6. Development of P450BM3 Hstar

Scheme 7. Substrate Scope of P450BM3 Hstar

Scheme 8. Diastereodivergent Biocatalytic Synthesis of Heteroatom-Substituted Cyclopropanes (2018)

Reaction carried out with P411-VAC_cis V87T. Reaction carried out with P411-VAC_cis V87I. Reaction carried out with P411-VAC_cis A328N. Reaction carried out with P411-VAC_cis V87F.

Scheme 9. Stereodivergent Biocatalytic Synthesis of Disubstituted Cyclopropanes: Access to All Four Stereoisomers (2018)

Scheme 10. Biocatalytic Stereoselective Synthesis of Cyclopropane Cores of Medicinal Agents (2014 and 2016)

Scheme 11. Directed Evolution of Enantiodivergent P411 Catalysts for Cyclopropenation (2018)

Scheme 12. P411-Catalyzed Cyclopropenation of Terminal Aliphatic Alkynes and Internal Alkynes (2018 and 2020)

Scheme 13. P411-Catalyzed Bicyclobutanation of Aromatic Alkynes (2018)

Scheme 14. Directed Evolution of Rma cyt c for Enantioselective Si–H Insertion (2016)

Scheme 15. Engineered Rma cyt c-Catalyzed Enantioselective Si–H Insertion (2016)

Scheme 16. Directed Evolution of Rma cyt c for Enantioselective B–H Insertion (2017)

Scheme 17. Engineered Rma cyt c-Catalyzed Enantioselective B–H Insertion (2017–2019)

Reaction carried out with Rma cyt c V75R M100D M103D. Reaction carried out with Rma cyt c V75R M100D M103F. Reaction carried out with Rma cyt c Y71C V75P M89C M99C M100D. Reaction carried out with Rma cyt c Y44I V75S M99A M100L M103D (BOR-CF₃). Reaction carried out with Rma cyt c V75R M99Q M100D T101Y M103Y (BOR^LAC). The absolute stereochemistry of these products were not determined.

Scheme 18. Directed Evolution of P411_CHF for Enantioselective Intermolecular C–H Insertion (2019)

Scheme 19. P411_CHF-Catalyzed Enantioselective Intermolecular C–H Insertion (2019)

Variant P411-IY(T327I) was used.

Scheme 20. P411-PFA-Catalyzed Enantioselective Intermolecular C–H Insertion (2019)

Scheme 21. Truncated P411-Catalyzed Selective C–H Functionalization of Indoles and Pyrroles (2019)

Reaction carried out with P411-HF M263A. Reaction carried out with P411-HF C87A M263E A268G T327P A328Y L437 M.

Scheme 22. P450-Catalyzed Enantioselective C–H Amidation for Sultam Synthesis (2013)

Reaction carried out using P411 in intact E. coli cells.

Scheme 23. Enzyme-Controlled, Regiodivergent C–H Amidation (2014)

Scheme 24. Directed Evolution of Intermolecular C–H Amidase (2017)

Scheme 25. P411-Catalyzed Intermolecular Benzylic C–H Amidation (2017)

Scheme 26. P411-Catalyzed Asymmetric Amidation of Primary, Secondary, and Tertiary C(sp³)–H Bonds (2019)

Scheme 27. P411-Catalyzed Asymmetric Imidation of Sulfides (2014 and 2016)

Scheme 28. P411-Catalyzed Asymmetric Aziridination (2015)

Scheme 29. Bez-Catalyzed Nitrene Transfer in the Biosynthesis of Benzastatins (2018)

Scheme 30. Comparison between Fe=O and Fe=NH

Scheme 31. Engineered Rma Cytochrome c-Catalyzed Asymmetric Aminohydroxylation (2019)

Scheme 32. P411-Catalyzed Asymmetric Amination of Benzylic and Allylic C(sp³)–H Bonds (2020)

Figure 1

Evolutionary trajectory of P450 carbene and nitrene transferases.

Figure 2

Key active-site residues in P450BM3 for the engineering of carbene and nitrene transferases.