Anti-Markovnikov alkene oxidation by metal-oxo–mediated enzyme catalysis

Abstract

Catalytic anti-Markovnikov oxidation of alkene feedstocks could simplify synthetic routes to many important molecules and solve a long-standing challenge in chemistry. Here we report the engineering of a cytochrome P450 enzyme by directed evolution to catalyze metal-oxo-mediated anti-Markovnikov oxidation of styrenes with high efficiency. The enzyme uses dioxygen as the terminal oxidant and achieves selectivity for anti-Markovnikov oxidation over the kinetically favored alkene epoxidation by trapping high-energy intermediates and catalyzing an oxo transfer, including an enantioselective 1,2-hydride migration. The anti-Markovnikov oxygenase can be combined with other catalysts in synthetic metabolic pathways to access a variety of challenging anti-Markovnikov functionalization reactions.

Extending Designed Linear Biocatalytic Cascades for Organic Synthesis

“…It is worth to note, that for a certain multi‐step transformations different cascades have been developed like for the formal hydration of alkenes (see Scheme and ). The shorter cascade (Scheme ) was enabled due to recent developments of a new reaction . Nevertheless, comparing the various reviews on cascades, certain transformations pop up several times, like the amination of alcohols„ transformation of alcohol to esters or the transformation of a CH 2 moiety to a carbonyl (mostly ketone).…”

“…Anti‐Markovnikov oxidation of alkenes is a long‐standing challenge in chemistry and identifying suitable biocatalytic methods could simplify synthetic routes. Recently, an engineered cytochrome P450 enzyme from the rhodobacterium Labrenzia aggregata (P450LA1) was developed by directed evolution, which catalyzed an anti‐Markovnikov oxidation of styrenes to the corresponding carbonyl compound (Scheme ) . The final best performing variant displayed eight amino acid exchanges (A103L, M118L, R120H, V123I, I326V, V327M, H385V, and M391L), overall substituting 3 % of the heme domain amino acids.…”

Inside Cover: Directed Evolution: Bringing New Chemistry to Life (Angew. Chem. Int. Ed. 16/2018)

“…In a powerful example of how an enzyme active site can be engineered to promote one reaction pathway over another, postdoctoral fellow Stephan Hammer directed the evolution of an alkene antiMarkovnikov oxygenase (aMOx), which catalyzes the conversion of alkenes into the anti-Markovnikov carbonyl compounds. [12] Intrigued by a report that the cytochrome P450 from Labrenzia aggregata made some phenyacetaldehyde as a side product when it oxidized styrene to the epoxide, Hammer looked more deeply and discovered that this promiscuous reactivity did not involve epoxidation followed by isomerization to the aldehyde, as had been proposed. [13] He correctly surmised that it instead went through a competing, stepwise mechanism involving radical/cation intermediates and a 1,2-hydride migration (Figure 4 A).…”

“…[14] He then exploited this side activity to direct the evolution of by far the most active, and the first enantioselective, direct aMOx catalyst. [12] Using earth-abundant iron, dioxgen, and a recyclable cofactor (NADPH), the laboratory-evolved P450 enzyme catalyzes thousands of turnovers for anti-Markovnikov oxidation of different substituted styrenes, including hindered substrates such as internal and 1,1-disubstituted alkenes.…”

“…[12] This coupled enzyme system yields valuable chiral alcohols with high enantiomeric excess and has the added benefit of recycling the NADPH cofactor. The antiMarkovnikov alkene functionalization is an important step in the production of many valuable chemicals, which are now in principle approachable through the combination of an evolved aMOx with other enzymes or chemical catalysts (Figure 4 C).…”