Chemo-enzymatic synthesis of optically pure l-leucovorin, an augmentor of 5-fluorouracil cytotoxicity against cancer

Abstract Optically pure l -leucovorin was synthesized on a large scale by the combination of chemical and enzymatic processes. After reduction of folate with zinc, dihydrofolate was reduced asymmetrically to (6)-tetrahydrofolate by use of dihydrofolate reductase from E. coli C600 pTP600 , with simultaneous NADPH cofactor recycling using glucose dehydrogenase from Gluconobacter scleroideus KY3613. Calcium l -leucovorin·4H 2 O (113g) was obtained from (6S)-tetrahydrofolate via 5,10-methenyltetrahydrofolate by formylation, reflux, addition of calcium ions and floricil column chromatography, with an overall yield of 50% based on folate. The l -leucovorin showed optical purity of 99.9% de as (6S)-form.