Enantioselective Allylic Hydroxylation of ω‐Alkenoic Acids and Esters by P450 BM3 Monooxygenase

Abstract

Chiral allylic alcohols of ω-alkenoic acids and derivatives thereof are highly important building blocks for the synthesis of biologically active compounds. The direct enantioselective C-H oxidation of linear terminal olefins offers the shortest route toward these compounds, but known synthetic methods are limited and suffer from low selectivities. Described herein is an enzymatic approach using the P450 BM3 monooxygenase mutant A74G/L188Q, which catalyzes allylic hydroxylation with high to excellent chemo- and enantioselectivities providing the desirable secondary alcohols.

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…The high activity for unsaturated fatty acids and branched fatty acids led to speculations whether these compounds constitute its natural (Tables 6, 7) [104,105]. Interestingly, whereas ω-C=C bonds increased the regioselectivity towards ω-2, probably due to the activated allylic location of hydroxylation (Table 6, entries 2, 3, and 9), a terminal alkyne-group was demonstrated to deactivate the enzyme by forming an adduct with the heme (Table 6, entry 10) [102,106]. The self-sufficiency of BM3 and its various reactions inspired the characterization of numerous additional CYP102 members, such as CYP102D1 from Streptomyces avermitiliswhich is the only characterized CYP102 outside the Bacillus family [107], CYP102A7 from Bacillus licheniformis (Table 5, entries 11, 30, and 40, Table 6, entries 8 and 13 and Table 7, entries 5 and 10) [78], and CYP102A2 and CYP102A3, both from Bacillus subtilis [108].…”

“…The double-variant A7G/L188Q gave the highest activity for the tested esters, however, the corresponding short chain ω-alkenoic acids were almost not converted. Only the longest tested carboxylic acid, 11-undecenoic acid, was accepted by the variant and in an upscaling (10 mM) (S)-9-hydroxy-undec-10-enoic acid was isolated with 80% yield (Table 6, entry 2) [106].…”

P450 BM3‐Catalyzed Regio‐ and Stereoselective Hydroxylation Aiming at the Synthesis of Phthalides and Isocoumarins

“…In the next step of our study, we screened our in‐house CYP BM3 mutant library, encompassing 121 mutants and the wild‐type enzyme, to investigate the regio‐ and enantioselectivity of the hydroxylation of methyl 2‐ethylbenzoate ( 3 a ) in order to find a more selective BM3 catalyst. The BM3 library is based on the mutated active‐site residues R47, Y51, A74, F87 and L188 . GC analysis of samples was performed with regard to formation and ee of 3‐methylisobenzofuran‐1(3 H )‐one ( 4 a ).…”

“…The F87 library was constructed by site‐saturation mutagenesis, with substitutions to Ala, Gly, Val, Leu, Ile, Ser, Cys, Tyr, His, Asp, Asn and Arg . P450 BM3 variants F87A and F87V were obtained as described previously, whereas the other mutants were generated by QuikChange PCR (see Section S2 in the Supporting Information) . A standard PCR approach was used for the A328 library generation with primers encoding the amino acids Gly, Val, Leu, Ile, Phe, Tyr, His, Asp, Arg and Ser.…”

“…Our laboratory has engineered P450 BM3 for the benzylic hydroxylation of a variety of toluene derivatives, and the double mutant F87A L188C showed remarkably increased turnovers relative to the wild‐type enzyme . In addition, we also pointed out the potential of the P450 BM3 double mutant A74G L188Q for the allylic hydroxylation of ω‐alkenoic acids and esters on preparative scale with high to excellent chemo‐ and enantioselectivities, providing S ‐configured allylic alcohols …”