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Microscale synthesis and kinetic isotope effect analysis of (4R)-[Ad-14C,4-2H] NADPH and (4R)-[Ad-3H,4-2H] NADPH

✍ Scribed by Atsushi Yahashiri; Arundhuti Sen; Amnon Kohen

Book ID
102367481
Publisher
John Wiley and Sons
Year
2009
Tongue
French
Weight
171 KB
Volume
52
Category
Article
ISSN
0022-2135

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✦ Synopsis

Abstract

We present a one‐pot chemo‐enzymatic microscale synthesis of NADPH with two different patterns of isotopic labels: (4__R__)‐[Ad‐^14^C,4‐^2^H] NADPH and (4__R__)‐[Ad‐^3^H,4‐^2^H] 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‐^14^C] NAD^+^ and [Ad‐^3^H] NAD^+^ were phosphorylated by NAD^+^ kinase to produce [Ad‐^14^C] NADP^+^ and [Ad‐^3^H] NADP^+^, respectively. Thermoanaerobium brockii alcohol dehydrogenase was then used to stereospecifically transfer deuterium from C2 of isopropanol to the re face of C4 of NADP^+^. After purification by HPLC, NMR analysis indicated that 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. Copyright Β© 2009 John Wiley & Sons, Ltd.

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