Development of a Biocatalytic Process as an Alternative to the (−)-DIP-Cl-Mediated Asymmetric Reduction of a Key Intermediate of Montelukast

Abstract

A KetoREDuctase (KRED) engineered via directed evolution technologies catalyzed the asymmetric reduction of (E)-methyl 2-(3-(3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)-3-oxopropyl)benzoate to the corresponding (S)-alcohol, a key intermediate in the synthesis of montelukast sodium (Singulair). Through synergistic efforts in process chemistry, molecular biology, bioinformatics and high throughput screening, a KRED with very high enantioselectivity (>99.9% ee) was developed for an economical and simple process that takes advantage of the physical properties of the substrate and product. The evolved KRED is an efficient and robust enzyme for catalyzing the reaction of an essentially water insoluble substrate (c log P ≈ 7) at a 100 g/L loading in the presence of ∼70% organic solvents at 45 °C. The biocatalytic process currently runs at >200 kg scale.

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

“…ChKRED20 was well expressed in E. coli and represented over 60% of the total soluble protein (Fig. 5); therefore, lyophilized powder of crude enzyme was applied as the catalyst according to recent successful industrial applications of ketone reductases [7][8][9]. Product (R)-1b was achieved with excellent enantioselectivity (>99.9% ee) and conversion (>99%) in 24 h (Table 3, entry 1).…”

“…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].…”

“…In a recent work by Ouyang et al [22], whole cells of Leifsonia xyli CCTCC M 2010241 were applied to reduce 200 mM (51.2 g/l) 1a. A conversion rate of 91.8% was achieved in 30 h with a high cell concentration of 300 g/l, and no further conversion could be achieved with prolonged reaction time of up to 50 h. In contrast to most academic practices, a typical industrial-scale biotransformation catalyzed with KREDs requires significant substrate concentration of at least 100 g/l, a substrateto-enzyme ratio of >50 to facilitate post-processing, as well as a high conversion rate of >95% to facilitate product purification [2,7,8]. Therefore, novel KRED enzymes or KRED-producing stains with more robust characteristics are in high demand for the anti-Prelog reduction of 1a.…”

Bioreductive production of enantiopure (S)-duloxetine intermediates catalyzed with ketoreductase ChKRED15

“…The bioreduction serves as a powerful approach for the production of optically active alcohols, which are widely used as fundamental intermediates for pharmaceuticals, agrochemicals and other products with chiral centers [1][2][3][4][5][6]. With the increased availability of recombinant ketoreductases, industrial-scale processes using cell-free enzymes coupled with efficient recycling systems have been developed rapidly in the past decade, which provides high volumetric productivity, excellent stereoselectivity, absence of side reactions and other economic advantages [7][8][9][10][11][12].…”