Directed Evolution of a Cytochrome P450 Carbene Transferase for Selective Functionalization of Cyclic Compounds

Transfers of carbene moieties to heterocycles or cyclic alkenes to obtain C(sp^2)–H alkylation or cyclopropane products are valuable transformations for synthesis of pharmacophores and chemical building blocks. Through their readily tunable active-site geometries, hemoprotein “carbene transferases” could provide an alternative to traditional transition metal catalysts by enabling heterocycle functionalizations with high chemo-, regio-, and stereocontrol. However, carbene transferases accepting heterocyclic substrates are scarce; the few enzymes capable of heterocycle or cyclic internal alkene functionalization described to date are characterized by low turnovers or depend on artificially introduced, costly iridium–porphyrin cofactors. We addressed this challenge by evolving a cytochrome P450 for highly efficient carbene transfer to indoles, pyrroles, and cyclic alkenes. We first developed a spectrophotometric high-throughput screening assay based on 1-methylindole C3-alkylation that enabled rapid analysis of thousands of P450 variants and comprehensive directed evolution via random and targeted mutagenesis. This effort yielded a P450 variant with 11 amino acid substitutions and a large deletion of the non-catalytic P450 reductase domain, which chemoselectively C_3-alkylates indoles with up to 470 turnovers per minute and 18 000 total turnovers. We subsequently used this optimized alkylation variant for parallel evolution toward more challenging heterocycle carbene functionalizations, including C_2/C_3 regioselective pyrrole alkylation, enantioselective indole alkylation with ethyl 2-diazopropanoate, and cyclic internal alkene cyclopropanation. The resulting set of efficient biocatalysts showcases the tunability of hemoproteins for highly selective functionalization of cyclic targets and the power of directed evolution to enhance the scope of new-to-nature enzyme catalysts

Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions

The surge in reports of heme-dependent proteins as catalysts for abiotic, synthetically valuable carbene and nitrene transfer reactions dramatically illustrates the evolvability of the protein world and our nascent ability to exploit that for new enzyme chemistry. We highlight the latest additions to the hemoprotein-catalyzed reaction repertoire (including carbene Si–H and C–H insertions, Doyle–Kirmse reactions, aldehyde olefinations, azide-to-aldehyde conversions, and intermolecular nitrene C–H insertion) and show how different hemoprotein scaffolds offer varied reactivity and selectivity. Preparative-scale syntheses of pharmaceutically relevant compounds accomplished with these new catalysts are beginning to demonstrate their biotechnological relevance. Insights into the determinants of enzyme lifetime and product yield are providing generalizable cues for engineering heme-dependent proteins to further broaden the scope and utility of these non-natural activities

Mathematical models: a key to understanding HIV envelope interactions?

The spikes of the human immunodeficiency virus (HIV) mediate viral entry and are the most important targets for neutralizing antibodies. Each spike consists of three identical subunits. The role of the spike's subunits in antibody binding is not fully understood. One experimental approach to analyze trimer function uses assays with mixed envelope trimer expressing cells or viruses. As these experiments do not allow direct observation of subunit functions, mathematical models are required to interpret them. Here we describe a modeling framework to study (i) the interaction of the V1V2 loop with epitopes on the V3 loop and (ii) the composition of quaternary epitopes. In a first step we identify which trimers can form in these assays and how they function under antibody binding. We then derive the behavior of an average trimer. We contrast two experimental reporting systems and list their advantages and disadvantages. In these experiments trimer formation might not be perfectly random and we show how these effects can be tested. As we still lack a potent vaccine against HIV, and this vaccine surely has to stimulate the production of neutralizing antibodies, mixed trimer approaches in combination with mathematical models will help to identify vulnerable sites of the HIV spike

Directed Evolution of a Cytochrome P450 Carbene Transferase for Selective Functionalization of Cyclic Compounds

Transfers of carbene moieties to heterocycles or cyclic alkenes to obtain C(sp^2)–H alkylation or cyclopropane products are valuable transformations for synthesis of pharmacophores and chemical building blocks. Through their readily tunable active-site geometries, hemoprotein “carbene transferases” could provide an alternative to traditional transition metal catalysts by enabling heterocycle functionalizations with high chemo-, regio-, and stereocontrol. However, carbene transferases accepting heterocyclic substrates are scarce; the few enzymes capable of heterocycle or cyclic internal alkene functionalization described to date are characterized by low turnovers or depend on artificially introduced, costly iridium–porphyrin cofactors. We addressed this challenge by evolving a cytochrome P450 for highly efficient carbene transfer to indoles, pyrroles, and cyclic alkenes. We first developed a spectrophotometric high-throughput screening assay based on 1-methylindole C3-alkylation that enabled rapid analysis of thousands of P450 variants and comprehensive directed evolution via random and targeted mutagenesis. This effort yielded a P450 variant with 11 amino acid substitutions and a large deletion of the non-catalytic P450 reductase domain, which chemoselectively C_3-alkylates indoles with up to 470 turnovers per minute and 18 000 total turnovers. We subsequently used this optimized alkylation variant for parallel evolution toward more challenging heterocycle carbene functionalizations, including C_2/C_3 regioselective pyrrole alkylation, enantioselective indole alkylation with ethyl 2-diazopropanoate, and cyclic internal alkene cyclopropanation. The resulting set of efficient biocatalysts showcases the tunability of hemoproteins for highly selective functionalization of cyclic targets and the power of directed evolution to enhance the scope of new-to-nature enzyme catalysts

Stereodivergent cyclopropanation of unactivated alkenes with heme proteins

Cyclopropyl motifs are present in a variety of compounds important to pharmaceutical, agrochemical, and fragrance industries. The asymmetric synthesis of cyclopropanes is often performed under harsh conditions with toxic, precious metal chiral catalysts. In 2013, the first example of biocatalytic alkene cyclopropanation was reported, using an engineered cytochrome P450 enzyme [1]. Since then, several heme proteins were reported to cyclopropanate a variety of styrenyl alkenes [2], but none have been shown to asymmetrically cyclopropanate more challenging substrates such as unactivated, aliphatic alkenes using the native iron-heme cofactor. Here we report that heme proteins can cyclopropanate unactivated alkenes and that stereoselectivity and activity can be tuned by directed evolution. A few rounds of site-saturation mutagenesis and screening yielded four protein variants with high enantio- and diastereoselectivity for complementary isomers, enabling stereodivergent synthesis of aliphatic cyclopropanes. These iron-porphyrin proteins are fully genetically encoded, and the reactions can be performed under mild, aqueous conditions with whole cells or purified protein. The protein enhances the activity of the native iron-heme cofactor, giving access to a broad array of cyclopropanated products. This example showcases the ability to quickly and efficiently engineer proteins for non-natural biocatalytic function. [1] P.S. Coelho, E.M. Brustad, A. Kannan, F.H. Arnold, Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes., Science. 339 (2013) 307–10. [2] O.F. Brandenberg, R. Fasan, F.H. Arnold, Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions, Curr. Opin. Biotechnol. 38 (2017) in press

Alternate heme ligation steers activity and selectivity in engineered cytochrome P450-catalyzed carbene transfer reactions

We report a biocatalytic platform of engineered cytochrome P450 enzymes to carry out carbene-transfer reactions using a lactone-based carbene precursor. By simply altering the heme-ligating residue, we obtained two enzymes that catalyze olefin cyclopropanation (Ser) or S–H bond insertion (Cys). Both enzymes exhibit high catalytic efficiency and stereoselectivity, thus enabling facile access to structurally diverse spiro[2.4]lactones and α-thio-γ-lactones. Computational studies revealed the mechanism of carbene S–H insertion and explain how the axial ligand controls reactivity and selectivity. This work expands the catalytic repertoire of hemeproteins and offers insights into how these enzymes can be tuned for new chemistry

Engineering Chemoselectivity in Hemoprotein-Catalyzed Indole Amidation

Here we report a cytochrome P450 variant that catalyzes C_2-amidation of 1-methylindoles with tosyl azide via nitrene transfer. Before evolutionary optimization, the enzyme exhibited two undesired side reactivities resulting in reduction of the putative iron-nitrenoid intermediate or cycloaddition between the two substrates to form triazole products. We speculated that triazole formation was a promiscuous cycloaddition activity of the P450 heme domain, while sulfonamide formation likely arose from surplus electron transfer from the reductase domain. Directed evolution involving mutagenesis of both the heme and reductase domains delivered an enzyme providing the desired indole amidation products with up to 8400 turnovers, 90% yield, and a shift in chemoselectivity from 2:19:1 to 110:12:1 in favor of nitrene transfer over reduction or triazole formation. This work expands the substrate scope of hemoprotein nitrene transferases to heterocycles and highlights the adaptability of the P450 scaffold to solve challenging chemoselectivity problems in non-natural enzymatic catalysis

Engineering Chemoselectivity in Hemoprotein-Catalyzed Indole Amidation

Here we report a cytochrome P450 variant that catalyzes C_2-amidation of 1-methylindoles with tosyl azide via nitrene transfer. Before evolutionary optimization, the enzyme exhibited two undesired side reactivities resulting in reduction of the putative iron-nitrenoid intermediate or cycloaddition between the two substrates to form triazole products. We speculated that triazole formation was a promiscuous cycloaddition activity of the P450 heme domain, while sulfonamide formation likely arose from surplus electron transfer from the reductase domain. Directed evolution involving mutagenesis of both the heme and reductase domains delivered an enzyme providing the desired indole amidation products with up to 8400 turnovers, 90% yield, and a shift in chemoselectivity from 2:19:1 to 110:12:1 in favor of nitrene transfer over reduction or triazole formation. This work expands the substrate scope of hemoprotein nitrene transferases to heterocycles and highlights the adaptability of the P450 scaffold to solve challenging chemoselectivity problems in non-natural enzymatic catalysis

Stereoselective Enzymatic Synthesis of Heteroatom-Substituted Cyclopropanes

The repurposing of hemoproteins for non-natural carbene transfer activities has generated enzymes for functions previously accessible only to chemical catalysts. With activities constrained to specific substrate classes, however, the synthetic utility of these new biocatalysts has been limited. To expand the capabilities of non-natural carbene transfer biocatalysis, we engineered variants of Cytochrome P450_(BM3) that catalyze the cyclopropanation of heteroatom-bearing alkenes, providing valuable nitrogen-, oxygen-, and sulfur-substituted cyclopropanes. Four or five active-site mutations converted a single parent enzyme into selective catalysts for the synthesis of both cis and trans heteroatom-substituted cyclopropanes, with high diastereoselectivities and enantioselectivities and up to 40 000 total turnovers. This work highlights the ease of tuning hemoproteins by directed evolution for efficient cyclopropanation of new substrate classes and expands the catalytic functions of iron heme proteins