| Introduction |
Carbon-hydrogen (C-H) bonds, while prevalent in organic molecules, are typically unreactive and present a challenge for chemical manipulation. Recent advancements in C-H functionalisation technology are changing this perspective, highlighting the critical need for selectively installing sp3 carbon-alkyl groups within hydrocarbon frameworks. This study introduces the initial iron-based catalysts capable of enantio-, regio-, and chemo-selective intermolecular alkylation of sp3 C-H bonds through carbene C-H insertion.
These catalysts, derived from a cytochrome P450 enzyme (specifically, a "cytochrome P411" variant with a cysteine-to-serine axial ligand substitution), are fully genetically encoded and produced within bacteria. Through directed evolution, their activity and selectivity can be precisely modulated. The utilisation of iron, the most abundant transition metal, in this demanding chemistry presents a valuable alternative to noble metal catalysts, which have traditionally dominated the C-H functionalisation field. The enzymes, refined in the laboratory, effectively functionalise a range of substrates containing benzylic, allylic, or α-amino C-H bonds, exhibiting high turnover and exceptional selectivity. Furthermore, these highly efficient enzymes facilitate the creation of streamlined synthetic pathways to various natural products. The demonstration that these enzymes mediate sp3 C-H alkylation using their intrinsic iron-haem cofactor broadens the utility of natural haem protein diversity for this abiological transformation, fostering the advancement of novel enzymatic C-H functionalisation reactions in chemistry and synthetic biology.
Biological systems employ a restricted set of chemical strategies for carbon-carbon (C-C) bond formation in organic molecule construction. While many methods rely on functional group manipulation, certain enzymes, such as radical S-adenosylmethionine (SAM) family members, can perform sp3 C-H bond alkylation. This approach has proven highly versatile for structural diversification, playing a crucial role in the biosynthesis of diverse natural products and cofactors. However, existing biological mechanisms for this transformation are confined to enzymes that transfer a methyl group or conjugate an activated radical acceptor substrate to specific molecules, with methylation being a common mode for sp3 C-alkyl installation by radical SAM enzymes.
This research aimed to establish a new enzymatic strategy for sp3 C-H bond alkylation, drawing inspiration from the widespread biological C-H functionalisation process of C-H oxygenation. Cytochrome P450 enzymes achieve C-H oxygenation using a haem cofactor, activating molecular oxygen to generate a high-energy iron-oxo intermediate capable of selective insertion into a substrate C-H bond. Consequently, the combination of a haem protein and a diazo compound was hypothesised to generate a protein-confined iron-carbene species, which could then engage in a selective C-H insertion reaction with a second substrate. While haem proteins have been shown to conduct carbene transfer processes such as cyclopropanation and heteroatom-hydrogen bond insertions, their sp3 C-H bond functionalisation remained challenging. Small-molecule metal-carbene sp3 C-H insertion, particularly intermolecular and stereoselective variants, typically relies on transition metal complexes of rhodium, iridium, and others. Artificial metalloproteins for carbene C-H insertion have been engineered by incorporating iridium-porphyrin into apo haem protein variants. Although uncommon, some instances of iron-carbene sp3 C-H insertion exist; these iron-catalysed reactions often necessitate elevated temperatures, are stoichiometric, or are limited to intramolecular processes, indicating a high activation energy barrier for C-H insertion with an iron-carbene. However, given that the protein scaffold of an enzyme can significantly enhance reaction rates and even confer activity to an otherwise unreactive cofactor, it was anticipated that directed evolution could reconfigure a haem protein to overcome the barrier for the iron-carbene C-H insertion reaction and acquire this new function.
Initial investigations involved screening a panel of seventy-eight haem proteins, including variants of cytochromes P450, cytochromes c, and globin homologues. These haem proteins, within whole Escherichia coli (E. coli) cells, were reacted with p-methoxybenzyl methyl ether (1a) and ethyl diazoacetate (2) at room temperature under anaerobic conditions. The reactions were subsequently analysed for the formation of the C-H alkylation product 3a. Haem proteins from two superfamilies exhibited low levels of this promiscuous activity, establishing the feasibility of creating C-H alkylation enzymes with diverse protein architectures. An engineered variant of cytochrome P450BM3 from Bacillus megaterium, featuring an axial cysteine-to-serine mutation (cytochrome "P411"), P-4 A82L, yielded 3a with 13 total turnovers (TTN). Additionally, nitric oxide dioxygenase from Rhodothermus marinus with the Y32G mutation (Rma NOD Y32G) catalysed the reaction with 7 TTN. A second alkane substrate, 4-ethylanisole (1i), was also processed by these nascent C-H alkylation enzymes, albeit with lower turnover numbers. The haem cofactor alone (iron protoporphyrin IX) or in the presence of bovine serum albumin showed no activity.
Using P411 P-4 A82L as the initial template, successive rounds of site-saturation mutagenesis and screening in whole E. coli cells were conducted to identify biocatalysts with enhanced activity and enantioselectivity for C-H alkylation. Amino acid residues targeted for mutagenesis included those lining the active site pocket, residing on loops and other flexible protein regions, or possessing a nucleophilic side chain. Improved variants were then evaluated in reactions using clarified E. coli lysate with p-methoxybenzyl methyl ether (1a) and 4-ethylanisole (1i). Five rounds of mutagenesis and screening led to variant P411-gen6, which provided product 3a with 60 TTN. Unlike the native monooxygenase activity, the C-H alkylation process does not require reducing equivalents from the FAD and FMN domains of the enzyme. Given that these domains might be dispensable for C-H alkylation, systematic truncations of P411-gen6 were performed to ascertain the minimal domain(s) required for catalytic activity. Intriguingly, removing the FAD domain, which accounts for 37% of the full-length protein's amino acids, resulted in an enzyme with higher C-H alkylation activity: P411ΔFAD-gen6 produced 3a with 100 TTN, representing a 1.7-fold increase compared to P411-gen6. This suggests that the FAD domain might exert negative allosteric effects on C-H alkylation activity. Further investigations with these truncated enzymes confirmed their usability in whole E. coli cells, clarified E. coli cell lysate, and as purified proteins.
Eight additional rounds of mutagenesis and screening resulted in P411-CHF (P411ΔFAD C-H Functionalisation enzyme). P411-CHF exhibits a 140-fold improvement in activity over P-4 A82L, yielding 3a with excellent stereoselectivity (2020 TTN, 96.7:3.3 e.r. using clarified E. coli lysate). Subsequent studies indicate that stereoselectivity could be further improved by conducting the reaction at lower temperatures (e.g., 4 °C) without significant changes in TTN. Enzymatic C-H alkylation can be performed on a millimole scale: with 1.0 mmol of substrate 1a, E. coli containing P411-CHF at 4 °C provided 3a in 82% isolated yield, 1060 TTN, and 98.0:2.0 e.r.
Preliminary mechanistic investigations were conducted to understand the nature of the C-H insertion step. Independent initial rates for reactions involving substrate 1a or deuterated substrate 1a-d2 revealed a normal kinetic isotope effect (KIE) of 5.1 for C-H alkylation catalysed by P411-CHF, indicating that C-H insertion is rate-determining. Using E. coli containing P411-CHF, a variety of benzylic substrates were evaluated for coupling with ethyl diazoacetate. Both electron-rich and electron-deficient functionalities on the aromatic ring were well-tolerated, and cyclic substrates also served as suitable coupling partners. Alkyl benzenes successfully underwent functionalisation at secondary benzylic sp3 C-H bonds. Notably, in the biotransformation of substrate 1l, which possesses both tertiary and secondary benzylic C-H bonds, P411-CHF preferentially functionalised the secondary position despite its higher C-H bond dissociation energy (BDE). The carbene intermediate generated from ethyl diazoacetate belongs to the acceptor-only class. Unlike the more commonly utilised donor/acceptor carbenes, acceptor-only intermediates are more electrophilic, rendering selective reactions with this carbene class a significant challenge for small-molecule catalysts. Our findings illustrate that P411-CHF can manage this highly reactive intermediate to provide the desired sp3 C-H alkylation products with high enantioselectivity.
Enzymes can achieve exceptional reaction selectivity due to their capacity to form multiple interactions with substrates and intermediates throughout a reaction cycle. It was hypothesised that the protein scaffold could be modified to generate complementary enzymes that access different reaction outcomes for a given substrate. When P411-CHF was challenged with 4-allylanisole (1m), a substrate capable of both C-H alkylation and cyclopropanation, C-H alkylation product 3m was observed to dominate, with a selectivity greater than 25:1. In contrast, a related full-length P411 variant, P-I263F, containing thirteen mutations in the haem domain relative to P411-CHF, exclusively catalysed the formation of cyclopropane product 3m''. Furthermore, despite the known reactivity of silanes with iron-carbene, P411-CHF generated C-H alkylation product 3h when substrate 1h was employed in the reaction (though Si-CH insertion product 3h'' was also observed, its formation may not have been catalysed by P411-CHF). Conversely, reaction with P-I263F yielded only the Si-CH insertion product. These examples highlight a remarkable characteristic of macromolecular enzymes: different products can be obtained simply by altering the amino acid sequence of the protein catalyst.
Enzymatic C-H alkylation extends beyond the functionalisation of benzylic C-H bonds. Structurally distinct molecules containing allylic or propargylic C-H bonds are excellent substrates for this chemistry. Unlike compounds 1a-1m, which feature a rigid benzene ring, compounds 4a-4c and 4e possess flexible linear alkyl chains. Their successful enantioselective alkylation suggests that the enzyme active site can accommodate substrate conformational flexibility while maintaining a preferred substrate orientation relative to the carbene intermediate. To illustrate the utility of this biotransformation, the methodology was applied to the formal synthesis of lyngbic acid. Marine cyanobacteria incorporate this versatile biomolecule into the malyngamide family of natural products, and total synthesis approaches often utilise lyngbic acid as a strategic intermediate. Using E. coli containing P411-CHF, intermediate 5a was produced on a 2.4 mmol scale in 86% isolated yield, 2810 TTN, and 94.7:5.3 e.r. Subsequent hydrogenation and hydrolysis quantitatively yielded (R)-(+)-6, which can be further processed into (R)-(+)-lyngbic acid via decarboxylative alkenylation.
As part of the substrate scope investigations, P411-CHF was applied to alkyl amine compounds. Such compounds typically pose difficulties for C-H functionalisation methods because the amine functionality can coordinate to and inhibit the catalyst or lead to undesirable side reactions (e.g., ylide formation and associated rearrangements). With substrates 7a or 7b, which contain both benzylic C-H bonds and α-amino C-H bonds, P411-CHF yielded the corresponding α-amino ester product with high efficiency. Notably, benzylic C-H insertion was not observed (with 7a) or significantly reduced (with 7b), despite the generally lower bond dissociation energies (BDEs) of benzylic C-H bonds compared to α-amino C-H bonds. Additionally, N-aryl pyrrolidines (7c-7e) proved to be excellent substrates, undergoing selective alkylation at the α-amino sp3 position. With P411-CHF, the sp3 C-H alkylation of 7c surpassed a Friedel-Crafts type reaction on the aryl ring, a process often favoured by other carbene-transfer systems. Furthermore, alkylation product 8d offers a plausible route for the synthesis of β-homoproline, a structural motif relevant to medicinal chemistry applications.
Given that P411-CHF alkylates both primary and secondary α-amino C-H bonds, the enzyme's selectivity for these positions was examined. Employing N-methyl tetrahydroquinoline 7f as the alkane substrate, P411-CHF delivered α-amino ester products with 1050 TTN and a 9:1 ratio of regioisomers (C2:C1), with 73.0:27.0 e.r. for 8f. As the tetrahydroquinoline ring is a privileged structural motif in natural products and bioactive molecules, its selective functionalisation could offer a concise strategy for alkaloid synthesis. To enhance the selectivity for alkylation of 7f, variants along the evolutionary pathway from P-4 A82L to P411-CHF were assessed. P411-gen5 exhibited even greater regioselectivity and furnished the product with the inverse stereo-preference. In a 3.0 mmol scale reaction, E. coli containing P411-gen5 yielded 8f in 85% yield with excellent selectivity (1310 TTN, >50:1 r.r., 8.9:91.1 e.r.). In just a few steps, the enzymatic product was successfully converted to the alkaloid (R)-(+)-cuspareine.
Finally, the incorporation of different alkyl groups was investigated. Utilising various diazo reagents, enzymatic C-H alkylation can diversify one alkane substrate, such as 7a, into multiple products. The scope of diazo substrates extends beyond ester-based reagents; Weinreb amide diazo compound 9c and diazoketone 9d were observed to participate in enzymatic C-H alkylation, yielding products 10c and 10d, respectively. However, additional substitution at the α-position of the carbene is generally not well-tolerated by P411-CHF and current related enzymes. With the exception of 10b, reactions involving disubstituted carbene reagents did not provide significant quantities of the desired products.
This study validates that a cytochrome P450 can attain the capacity to construct C-C bonds from sp3 C-H bonds, and that both activity and selectivity can be significantly improved through directed evolution. Nature presents a vast array of potential alternative starting points for further expanding the reaction's scope and achieving other selectivities. The cytochrome P450 superfamily can process an immense range of organic molecules for its native oxygenation chemistry; it is envisioned that P411-derived enzymes and the broader natural haem protein diversity can be harnessed to generate families of C-H alkylation enzymes that replicate the scope and selectivity characteristic of nature's C-H oxygenation catalysts. |
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