Chemoenzymatic Preparation of 4′‐Thioribose NAD +

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.