Synthesis of R and S tritiated reduced beta-nicotinamide adenine dinucleotide 2' phosphate |
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Authors: | McCracken Jocelyn A Wang Lin Kohen Amnon |
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Institution: | Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA. |
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Abstract: | Nicotinamides are ubiquitous cofactors used by many biological systems as redox agents. Stereospecifically labeled cofactors are useful in many studies of nicotinamide-dependent enzymes. Enzyme-directed synthesis of these cofactors is rather common but their stability imposes significant challenges on yield, purity, and preservation. This paper presents the stereospecific synthesis of reduced R- and S-4-3H] beta-nicotinamide adenine dinucleotide 2' phosphate (NADPH). The method of Valera et al. Biochem. Biophys. Res. Commun. 148 (1987) 515] was modified to a synthetic procedure that produces both isotopic diastereomers within 2h with an improved yield of 75-90% after purification and lyophilization. In the synthesis, 4-3H]NADP+ was generated as an intermediate (which can be isolated if desired). The specific radioactivities reported here are 2.7 and 1.1 Ci/mmol for the S and R diastereomers, respectively. Specific radioactivities ranging from carrier-free to trace labeling can be achieved with a minor change to the procedure. |
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Keywords: | Tritium Labeled nicotinamide Alcohol dehydrogenase Glucose dehydrogenase Reverse-phase HPLC Tritiated nicotinamide Labeled nicotinamide |
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