Microscale synthesis of isotopically labeled 6R‐N5, N10 methylene‐5, 6, 7, 8‐tetrahydrofolate

Abstract

AbstractA one‐pot chemo‐enzymatic microscale synthesis of isotopically labeled R‐[6‐YH; 11‐XH] N5, N10 methylene‐5, 6, 7, 8‐tetrahydrofolate (CH2H4folate) is presented, where Y=1 or 2 represents protium or deuterium, and X=1, 2 or 3 represents protium, deuterium or tritium, respectively. In this procedure, Thermoanaerobium brockii alcohol dehydrogenase (tbADH) and Escherichia coli dihydrofolate reductase (ecDHFR) were used simultaneously in the reaction mixture. First, tbADH stereospecifically catalyzes a hydride transfer from [2‐YH] iPrOH to the re face of C‐4 NADP+. The ecDHFR then reduced 7, 8‐dihydrofolate (H2folate) to form (6S)‐H4folate. Finally, the enzymatic reactions were followed by chemical trapping with isotopically labeled formaldehyde ([XH]‐HCHO) to form the final product. The preparation of deuterium‐ and tritium‐labeled formaldehyde is also presented. Two reverse phase HPLC methods were developed for analysis and purification of product R‐[6‐YH; 11‐XH] CH2H4folate. This isotopically labeled cofactor can be used to study 1° and 2° kinetic isotope effects (KIEs) with any CH2H4folate dependent enzyme as demonstrated by studies with E. coli thymidylate synthase (TS). Copyright © 2005 John Wiley & Sons, Ltd.