Molecular Modification Of An Alcohol Dehydrogenase For The Preparation Of A Ticagrelor Intermediate

Short-chain alcohol dehydrogenases(E.C.1.1.1.x;x=1 or 2,ADHs)from the SDR family,also known as ketoreductases(KREDs)or carbonyl reductases(CRs),catalyze the interconversion of alcohols to aldehydes or ketones by the transfer of protons on C4 of the NAD(P)H nicotinamide ring.Based on their high stereoselectivity under mild conditions,ADHs have been widely studied and uesed in the asymmetric synthesis of chiral alcohols.The purpose of this paper is to investigate the preparation of the ticagrelor intermediate(S)-2-chloro-1-(3,4-difluorophenyl)ethanol by asymmetric reduction of the substrate 2-chloro-l-(3,4-difluorophenyl)ethanol catalyzed by ADHs.The alcohol dehydrogenase ADH-E03 from Leifsonia naganoensis was found to have high catalytic activity in the asymmetric synthesis of(S)-2-chloro-1-(3,4difluorophenyl)ethanol via gene mining and experimental comparison.However,the catalytic activity of wild-type ADH-E03 for 2-chloro-l-(3,4-difluorophenyl)ethanol still needs to be improved,as does its tolerant to high concentrations of isopropanol.Directed evolution and semi-rational design strategies were used to modify wild-type ADH-E03 and four three-point combinatorial mutants with superior performance including T100K/L207I/A7S,T100K/L207I/S105P,T100K/L207I/D107R and T100K/L207I/S148Lwere obtained.Their specific enzyme activities were increased to 7.4,7.6,8.0 and 9.6-fold of the wild type,respectively.The best mutant T100K/L207I/S148L showed the most excellent thermal stability,with a 5.7-fold increase in residual enzyme activity after 30 min treatment at 55℃ compared to the wild type,and it showed the highest catalytic efficiency when applied to 500 g/L substrate conversion.The best mutant was selected as a catalyst to further optimize the reaction conditions for the asymmetric reduction of the substrate 2-chloro-1-(3,4-difluorophenyl)ethanone to synthesize(S)-2-chloro-1-(3,4-difluorophenyl)ethanol.The biotransformation reaction of 500 g/L substrate ketone to the product chiral alcohol could achieve more than 99.9%conversion ratio with the ee value over 99%in 8 h by adding 40 g/L wet cells and 2 equiv isopropanol at 50℃,pH 6.0 and a one-time feeding mode without the addition of coenzyme NAD+.