A green-by-design biocatalytic process for atorvastatin intermediate

Abstract

The development of a green-by-design, two-step, three-enzyme process for the synthesis of a key intermediate in the manufacture of atorvastatin, the active ingredient of the cholesterol lowering drug Lipitor R , is described. The first step involves the biocatalytic reduction of ethyl-4-chloroacetoacetate using a ketoreductase (KRED) in combination with glucose and a NADP-dependent glucose dehydrogenase (GDH) for cofactor regeneration. The (S) ethyl-4-chloro-3-hydroxybutyrate product is obtained in 96% isolated yield and >99.5% e.e. In the second step, a halohydrin dehalogenase (HHDH) is employed to catalyse the replacement of the chloro substituent with cyano by reaction with HCN at neutral pH and ambient temperature. The natural enzymes were highly selective but exhibited productivities that were insufficient for large scale application. Consequently, in vitro enzyme evolution using gene shuffling technologies was employed to optimise their performance according to predefined criteria and process parameters. In the case of the HHDH reaction, this afforded a 2500-fold improvement in the volumetric productivity per biocatalyst loading. This enabled the economical and environmentally attractive production of the key hydroxynitrile intermediate. The overall process has an E factor (kg waste per kg product) of 5.8 when process water is not included, and 18 if included.

Rapid asymmetric reduction of ethyl 4-chloro-3-oxobutanoate using a thermostabilized mutant of ketoreductase ChKRED20

“…In addition, when significant improvement of catalytic properties is demanded, the recombinant enzymes can always be engineered via directed evolution to meet the process requirements. Hence, the application spectrum of the KREDs has been greatly expanded (Gooding et al 2010;Huisman et al 2010;Kosjek et al 2008;Liang et al 2010;Ma et al 2010;Solano et al 2012).…”

Identification of ketone reductase ChKRED20 from the genome of Chryseobacterium sp. CA49 for highly efficient anti-Prelog reduction of 3,5-bis(trifluoromethyl)acetophenone

“…Requirements [2] Table S4) [7][8][9][10][11][12]. In addition, ChKRED20 could use 2-propanol as a cheap co-substrate for cofactor regeneration, and the crude enzyme could easily tolerate 40% (v/v) of 2-propanol in the reaction system.…”

“…In the last decade, successful application of ketone reductases (KRED) either as whole-cells or isolated enzymes has risen rapidly. In particular, several processes using isolated/crude recombinant enzymes coupled with efficient recycling systems have been developed into industrial-scale production with excellent stereoselectivity and economic advantage [7][8][9][10][11][12]. The use of recombinant enzymes greatly facilitates the scale-up with high volumetric productivity and the absence of side reactions [2,13,14].…”

Characterization of a robust anti-Prelog short-chain dehydrogenase/reductase ChKRED20 from Chryseobacterium sp. CA49

“…Both cell-free enzymes and whole microorganisms have been applied to the asymmetric reduction processes. Recent years, in particular, have seen the extensive application of isolated/crude recombinant enzymes coupled with efficient cofactor recycling systems being developed into industrial-scale production [8][9][10][11][12][13]. The use of recombinant enzymes greatly facilitates the scale-up with high volumetric productivity and the absence of side reactions [2,[14][15][16].…”