Microscale synthesis of 2-tritiated isopropanol and 4R-tritiated reduced nicotinamide adenine dinucleotide phosphate.
An improved microscale synthesis of 2-tritiated isopropanol ([2-3H]iPrOH) and R-tritiated reduced beta-nicotinamide adenine dinucleotide 2(')-phosphate (R-[4-3H]NADPH) is presented. The current procedure offers high yield, high purity, and small-quantity synthesis and is shorter than previous procedures. [2-3H]iPrOH was prepared by reduction of acetone with [3H]NaBH(4) under reflux conditions. [2-3H]iPrOH was used to reduce NADP(+) to R-[4-3H]NADPH with alcohol dehydrogenase from Thermoanaerobium brockii at 40 degrees C. This equilibrium reaction was drawn toward products by trapping the acetone with an excess of semicarbazide. This improvement enables a better control of the reaction time, as the enzymatic reduction became rate determining. Greater than 75% of the product was identified as reduced cofactor by reverse-phase (RP) HPLC. Longer reaction led to decomposition of the product and lower yield. Purification was carried out by semipreparative RP HPLC followed by lyophilization and yielded a compound of high purity that was preserved at -80 degrees C. Applications of the new procedure to a wide variety of specific radioactivities ranging from carrier-free to a few Ci/mmol are discussed. The intriguing formation of radioactive NADP(+) by-product (the major product in some reported procedures), was also studied and minimized in the procedure described below. Finally, the usage of the labeled cofactor is demonstrated with the enzymes dihydrofolate reductase and thymidylate synthase.
Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
Nitish
N
Agrawal
Amnon
A
Kohen
D019840:2-Propanol / Q000138:chemical synthesis*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D001421:Bacteria, Anaerobic / Q000201:enzymology; D001671:Biochemistry / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005612:Freeze Drying; D009249:NADP / Q000138:chemical synthesis*; D013237:Stereoisomerism; D014316:Tritium / Q000737:chemistry
D014316:Tritium; D009249:NADP; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+); D019840:2-Propanol