Large-scale chemoenzymic synthesis of calcium (6S)-5-formyl-5,6,7,8-tetrahydrofolate [(–)-leucovorin] using the NADPH recycling method

Chemoenzymic large-scale synthesis of the calcium salt of (6S)-5-formyltetrahydrofolic acid [(–)-leucovorin, (6S)-5] was achieved from folic acid 1via(6S)-tetrahydrofolic acid [(6S)-3] by using dihydrofolate reductase (DHFR) produced by Escherichia coil, harbouring a high-expression plasmid, pTP64–1. On the other hand, for the diastereoselective reduction of 7,8-dihydrofolic acid 2 to tetrahydrofolate (6S)-3, a new NADPH recycling system was constructed by coupling with glucose dehydrogenase from Gluconobacter scieroides. Having these enzymic systems to hand, compound 1 was reduced by zinc powder in alkaline solution to give compound 2 which, without isolation, was reduced enzymatically to afford tetrahydrofolate (6S)-3(94% de). The pH adjustment of the reaction mixture containing dihydrofolate 2 was done with phosphoric acid in order to remove zinc ion which inhibited the following enzymic reduction. The formed tetrahydrofolate (6S)-3 was converted into entirely optically pure N-formyl compound (6S)-5 on a large scale. The specific rotation value of (–)-leucovorin was [α]D20–13.3 (c 1, water). For the comparison of pharmacological effects, a completely optically pure form of (+)-leucovorin [(6R)-5] was also prepared on a preparative scale. Compound (6S)-5 was 300-fold more active compared with the (6R)-diastereoisomer.