Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes

Abstract

Terminal‐selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio‐ and enantioselectivity. A sensitive colorimetric high‐throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative saturation mutagenesis, sextuple‐mutant P450pyrSM1 was created for the hydroxylation of n‐octane (1) to give (S)‐2‐octanol (2) with 98 % ee and >99 % subterminal selectivity. The engineered P450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the CH activation and functionalization of alkanes and the preparation of enantiopure alcohols. Molecular modeling provided structure‐based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple‐mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene (3) to afford (S)‐1‐phenyl‐2‐propanol (4) with 95 % ee and 98 % subterminal selectivity.

Enhancing cofactor recycling in the bioconversion of racemic alcohols to chiral amines with alcohol dehydrogenase and amine dehydrogenase by coupling cells and cell‐free system

“…Although the alcohol substrates 1a–b are easily available, amines ( R )‐ 3a–b are important intermediates for the syntheses of ( R , R )‐formoterol, tamsulosin, and antihypertensive dilevalol (Ye et al, ). As no nonenantioselective ADH is available, S ‐selective CpsADH and R ‐selective PfODH (Yang, Liu, & Li, ) were chosen for the oxidation of the racemic alcohols 1a–b to ketones 2a–b for their high activity and complementary enantioselectivity. A NOX from Bacillus sp.…”

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

“…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. [5,6] However, methods for the enantioselective allylic C À H oxidation of simple linear compounds remain a challenge as asymmetry-inducing factors are missing. [1] Extensive research toward the enantioselective synthesis of these moieties has resulted in multistep approaches comprising prefunctionalization and protection group chemistry as well as functional group interconversions and oxidation state manipulations.…”

“…Chiral (wÀ2)-hydroxy-w-alkenoic acids as well as their esters, vinyl lactones, and protected alcohols are pivotal structural elements abundantly present in diverse natural and biologically active compounds including decanolides, oxylipins, antibiotics, and pheromones. [5,6] However, methods for the enantioselective allylic C À H oxidation of simple linear compounds remain a challenge as asymmetry-inducing factors are missing. [2] A chemoenzymatic route with an alcohol dehydrogenase catalyzed selective reduction as the key step was reported by our group and represents arguably the most efficient access nowadays (2 or 3 steps, 73-77 % overall yield, > 99 % ee, both enantiomers accessible).…”

“…[4] In this context, significant success in the highly topical field of CÀH activation was achieved by Agudo et al and Li et al regard-ing the enzymatic oxidation of cyclic and linear molecules, respectively. [5,6] However, methods for the enantioselective allylic C À H oxidation of simple linear compounds remain a challenge as asymmetry-inducing factors are missing. [7] The enantioselective allylic acetoxylation of terminal olefins using a combination of a Pd II /bis(sulfoxide) system with benzoquinone as the terminal oxidant and Cr III (salen) as a chiral Lewis acid cocatalyst reported by White and co-workers in 2008 was a milestone and ee values up to 63 % were achieved; the desired products were obtained in 69-89 % yield and 50-57 % ee as roughly 5:1 mixtures with the corresponding linear alcohols.…”

Enantioselektive allylische Hydroxylierung von ω‐Alkensäuren und ‐estern mittels der P450‐BM3‐Monooxygenase

“…[4] In diesem Zusammenhang wurden hervorragende Ergebnisse auf dem hochaktuellen Gebiet der C-HAktivierung hinsichtlich der enzymatischen Oxidation cyclischer und linearer Moleküle durch Agudo et al und Li et al erreicht. [5,6] Dennoch bleibt die enantioselektive allylische C-H-Oxidation von einfachen linearen Verbindungen aufgrund fehlender Faktoren für die Induktion von Asymmetrie eine Herausforderung. [7] [9,10] Als native Funktion von P450 BM3 wird die subterminale Hydroxylierung von Fettsäuren durch Insertion eines O-Atoms aus molekularem O 2 in C-H-Bindungen vermutet; darüber hinaus wurde die Vielseitigkeit und Wandelbarkeit dieser Monooxygenase durch diverse Protein-Engineering-Studien aufgezeigt.…”