title: Microscale synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate as a cofactor for thymidylate synthase.
abstract: A one-pot synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4F) is presented, where x=1, 2, or 3 represents hydrogen, deuterium, or tritium, respectively. The current procedure offers high-yield, high-purity, and microscale-quantity synthesis. In this procedure, two enzymes were used simultaneously in the reaction mixture. The first was Thermoanaerobium brockii alcohol dehydrogenase, which stereospecifically catalyzed a hydride transfer from C-2-labeled isopropanol to the re face of oxidized nicotinamide adenine dinucleotide phosphate to form R-[4-xH]-labeled reduced nicotinamide adenine dinucleotide phosphate. The second enzyme, Escherichia coli dihydrofolate reductase, used the xH to reduce 7,8-dihydrofolate (H2F) to form S-[6-xH]5,6,7,8-tetrahydrofolate (S-[6-xH]H4F). The enzymatic reactions were followed by chemical trapping of S-[6-xH]H4F with formaldehyde to form the final product. Product purification was carried out in a single step by reverse phase high-pressure liquid chromatography separation followed by lyophilization. Two analytical methods were developed to follow the reaction progress. Finally, the utility of the labeled cofactor in mechanistic studies of thymidylate synthase is demonstrated by measuring the tritium kinetic isotope effect on the enzyme's second order rate constant.
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Nitish
authors.initials: N
authors.lastname: Agrawal
authors.forename: Cornelia
authors.initials: C
authors.lastname: Mihai
authors.forename: Amnon
authors.initials: A
authors.lastname: Kohen
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes / Q000138:chemical synthesis* / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D013763:Tetrahydrofolates / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D013940:Thymidylate Synthase / Q000378:metabolism*; D014316:Tritium / Q000737:chemistry
chemical_list: D003067:Coenzymes; D013763:Tetrahydrofolates; D014316:Tritium; D006859:Hydrogen; D003903:Deuterium; D013940:Thymidylate Synthase