Many syntheses of organic molecules require that certain carbon-hydrogen bonds are targeted for reaction over others with similar reactivity (16). This high selectivity to one specific C–H bond is frequently achieved by a remote activating group in the molecule (known as remote functionalization). A particularly attractive group of synthesis targets in pharmaceutical chemistry are chiral lactams, which are structural motifs in many bioactive compounds. The challenge is to design catalytic processes that can start from easily accessible derivatives of carboxylic acids and provide tunable control over stereoselective installation of the nitrogen at desired positions in the substrate, leading to lactams of varying ring size. On page 575 of this issue, Cho et al. (7) achieve this goal by harnessing the power of directed evolution. The authors engineered a toolbox of enzymes that are not found in nature for the remote C–H functionalization of chemically diverse substrates to yield valuable lactam products.

Cho et al. used cytochrome P450 monooxygenases, a large class of enzymes involved in a wide range of oxidation reactions in nature. P450 monooxygenases contain a characteristic heme prosthetic group and can catalyze highly selective oxidations, such as the hydroxylation of nonactivated C–H bonds, even when substrate flexibility makes selective functionalization more difficult. For example, Manning et al. have recently reported the regio- and enantioselective midchain C–H hydroxylation of simple fatty acid substrates by a native cytochrome P450 enzyme (8).

Hemeproteins, such as P450s, have also been the basis of new-to-nature enzymes, which catalyze reactions that have not been found in metabolic pathways. In earlier work, Arnold and coauthors took advantage of directed evolution technology to engineer cytochrome P450 variants, dubbed P411s, that catalyze the C–H insertion of sulfonyl nitrenes to form C–N bonds (9). Singh et al.have shown that carbonyl-based nitrene donors can also be substrates for intramolecular C–H amination processes (10).

Cho et al. based their study on the recent discovery that a P450 is implicated in the biosynthesis of benzastatins through an acyl-protected hydroxylamine to form the reactive iron nitrenoid species (11). The use of carbonyl-based nitrene donors opens the door to synthetically useful amides, but they are also susceptible to decomposition. Cho et al. used directed evolution to develop P450-derived enzyme mutants that can direct carbonyl nitrenes to enantio- and regioselective C–H amidation rather than the competing decomposition pathways.

Cho et al. selected β-lactams as the initial target products because of their prevalence in pharmaceutical applications. They identified a model substrate containing two sets of reactive C–H bonds: one that could yield β-lactam as the C–H amidation product, and another that could yield the corresponding δ-lactam. They expressed a panel of hemeproteins, including native P450 enzymes and the P411 variants described in earlier work (9), in Escherichia coli cells. One P411 variant generated small amounts of β- and δ-lactams and appeared to bypass any degradation to the corresponding amine by-product. The authors chose this variant as the parent enzyme for directed evolution experiments to create a more effective lactam synthase enzyme.


A route to stereoselective lactam synthesis

Cho et al. have engineered cytochrome P450 enzymes that catalyze the stereoselective functionalization of particular C–H bonds to form a range of lactam products. Ph, phenyl; piv, pivaloyl.




Four rounds of directed evolution later, an enzyme variant (LSsp3) emerged that displayed total turnover numbers (TTNs) as high as 223,000, yields of up to 96%, regioisomeric ratios (r.r.) of up to 25:1, and enantiomeric excesses (ee) as high as 96% for almost exclusively β-lactam products. LSsp3 bears six mutated amino acid residues in close proximity to the heme iron in the active site. The authors screened a diverse selection of substrates against this variant and demonstrated its exceptional selectivity for β-lactam rings over the less strained δ-lactam equivalents. They were able to scale up and purify several lactam products, including one product at gram scale.

Cho et al. used further rounds of directed evolution to generate additional lactam synthase variants, each displaying selectivity for either δ-lactams (LSsp2), β-lactams (LSβ), or δ-lactams (LSδ). In a final example that showcases the tunability of their process, the authors selected a nitrene precursor containing three sets of reactive C(sp3)–H bonds and used the engineered lactam synthase enzymes to each selectively generate β-, γ-, or δ-lactam products (see the figure).

The work reported by Cho et al. represents a crucial step forward in the search for tunable catalysts capable of remote C–H functionalizations. It is particularly impressive for its use of highly flexible substrates. One of the technical restrictions that will need to be overcome is the requirement for strictly anaerobic reaction conditions. However, this work clearly demonstrates the power of directed evolution to develop remote C–H activation strategies with highly tunable chemo-, regio-, and stereoselectivities, accessing a diverse range of chiral lactams from common starting materials.



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