title: Microscale synthesis and kinetic isotope effect analysis of (4R)-[Ad-14C, 4-2H] NADPH and (4R)-[Ad-3H,4-2H] NADPH.
abstract: We present a one-pot chemo-enzymatic microscale synthesis of NADPH with two different patterns of isotopic labels: (4R)-[Ad-14C,4-2H] NADPH and (4R)-[Ad-3H,4-2H] NADPH. These co-factors are required by an enormous range of enzymes, and isotopically labeled nicotinamides are consequently of significant interest to researchers. In the current procedure, [Ad-14C] NAD+and [Ad-3H] NAD+were phosphorylated by NAD+kinase to produce [Ad-14C] NADP+and [Ad-3H] NADP+, respectively.Thermoanaerobium brockiialcohol dehydrogenase was then used to stereospecifically transfer deuterium from C2 of isopropanol to thereface of C4 of NADP+. After purification by HPLC, NMR analysis indicated the deuterium content at the 4R position is more than 99.7 %. The labeled cofactors were then used to successfully and sensitively measure kinetic isotope effects for R67 dihydrofolate reductase, providing strong evidence for the utility of this synthetic methodology.
authors.affiliation: Department of Chemistry, The University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yahashiri
authors.affiliation: Department of Chemistry, The University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Arundhuti
authors.initials: A
authors.lastname: Sen
authors.affiliation: Department of Chemistry, The University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Amnon
authors.initials: A
authors.lastname: Kohen
keywords: Thermoanaerobium brockii alcohol dehydrogenase; dihydrofolate reductase; isotopic labeling; kinetic isotope effect; labeled NADPH; nicotinamide adenine dinucleotide 2′ phosphate