Chemoenzymatic Preparation of 4'-Thioribose NAD.

Abstract

This chemoenzymatic procedure describes a strategy for the preparation of 4'-thioribose nicotinamide adenine dinucleotide (S-NAD⁺ ), including chemical synthesis of nicotinamide 4'-riboside (S-NR), recombinant expression and purification of two NAD⁺ biosynthesis enzymes nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), and enzymatic synthesis of S-NAD⁺ . The first basic protocol describes the procedures for introduction of nicotinamide onto 4'-thioribose and subsequent deprotection to generate S-NR as the key intermediate for enzymatically synthesizing S-NAD⁺ . In the second basic protocol, experimental methods are detailed for the production of recombinant human NRK1 and NMNAT1 to catalyze conversion of S-NR to S-NAD⁺ . The third basic protocol presents the enzymatic approach for the generation of S-NAD⁺ from S-NR precursor. © 2019 by John Wiley & Sons, Inc.