Efficient production of an ezetimibe intermediate using carbonyl reductase coupled with glucose dehydrogenase

Abstract

Ezetimibe is a top-selling hypolipidemic drug for the treatment of cardiovascular diseases. Biosynthesis of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one ((S)-ET-5) using carbonyl reductase has shown advantages including high catalytic efficiency, excellent stereoselectivity, mild reaction conditions, and environmental friendness, and was considered as the key step for ezetimibe production. The regeneration efficiency of the cofactor, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is one of the main restricted factor. Recombinant Escherichia coli strain (smCR125) coexpressing carbonyl reductase (CR125) and glucose dehydrogenase were successfully constructed and applied for the production of (S)-ET-5 for the first time. Without extra addition of the coenzyme NADPH, the yield of 99.8% and the enantiomeric excess (e.e.) of 99.9% were achieved under ET-4 concentration of 200 g/L. Using a substrate fed-batch strategy, under the optimal conditions, the substrate ET-4 concentration was increased to 250 g/L with the yield of 98.9% and the e.e. of 99.9% after 12 hr reaction. The space-time yield of 494.5 g L −1 d −1 and the space-time yield per gram biocatalyst of 24.7 g L −1 d −1 g −1 DCW were achieved, which were higher than ever reported for the biosynthesis of the ezetimibe intermediate.

Shortening Synthetic Routes to Small Molecule Active Pharmaceutical Ingredients Employing Biocatalytic Methods

“…With a fed-batch strategy, a 250 g·L –1 substrate concentration was achieved. The reduction yielded 98.9% alcohol with 99% e.e., resulting in a space-time yield of 494.5 g·L –1 · d –1 and a space-time yield per gram DCW (dry cell weight) biocatalyst of 24.7 g·L –1 · d –1 . , …”

“…The reduction yielded 98.9% alcohol with 99% e.e., resulting in a space-time yield of 494.5 g•L −1 • d −1 and a space-time yield per gram DCW (dry cell weight) biocatalyst of 24.7 g•L −1 • d −1 . 120,121 Several chemical total syntheses of ezetimibe were published. 115,122 The challenge is the installation of the three stereocenters with the correct absolute configuration.…”