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

Abstract

Chemoenzymic large-scale synthesis of the calcium salt of (6s) -5-formyltetrahydrofolic acid [ (-)leucovorin, (6S)-5] was achieved from folic acid 1 via (6s)-tetrahydrofolic acid [(6S)-3] by using dihydrofolate reductase (DH FR) produced by Zscherichia coli, 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 scleroides. 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) -

Multi‐Enzymatic Cascade Reactions: Overview and Perspectives

“…A field where applications of reactor engineering are particularly useful is coenzyme regeneration. A (native enzyme)/(native coenzyme)/(batch reactor) system was used for chemo‐enzymatic synthesis with NADPH recycling;149,150 while an (immobilized enzyme)/(native coenzyme)/(batch reactor) system was used for 12‐ketochenodeoxycholic acid synthesis 151. Membrane reactors were also used in different ways, such as (native enzyme)/(macromolecular coenzyme)/(uncharged membrane reactor)152 and (native enzyme)/(native coenzyme)/(charged membrane reactor) 153…”