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-formy ltetrahydrofolic acid [(-)leucovorin, (6S)-5] was achieved from folic acid 1 via (6S) -tetrahydrofolic acid [(6S)-3] by using dihydrofolate reductase (DHFR) produced by Escherichia coil, harbouring a high-expre ssion 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 dehyd rogenase from Gluconobacter scleroides. Having these enzymic systems t o 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 rea ction mixture containing dihydrofolate 2 was done with phosphoric acid in order to remove zinc ion which inhibited the following enzymic red uction. The formed tetrahydrofolate (6S)-3 was converted into entirely optically pure N-formyl compound (6S)-5 on a large scale. The specifi c rotation value of (-)-leucovorin was [alpha](20)(D) -13.3 (c 1, wate r). For the comparison of pharmacological effects, a completely optica lly pure form of (+)-leucovorin [(6R)-5] was also prepared on a prepar ative scale. Compound (6S)-5 was 300-fold more active compared with th e (6R)-diastereoisomer.