Y. Kuge et al., LARGE-SCALE CHEMOENZYMIC SYNTHESIS OF CALCIUM (6S)-5-FORMYL-5,6,7,8-TETRAHYDROFOLATE [(-)-LEUCOVORIN] USING THE NADPH RECYCLING METHOD, Journal of the Chemical Society. Perkin transactions. I, (11), 1994, pp. 1427-1431
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.