10] Preparation and purification of nicotinamide mononucleotide analogs

Abstract

This chapter discusses the methods for the preparation and purification of nicotinamide mononucleotide analogs. Many nicotinamide mononucleotide derivatives have been prepared chemically as intermediary products in different syntheses of NAD + and NAD + analogs. The preparative methods for the chemical synthesis of NMN molecules become complicated when labile pyridine derivatives, such as thionicotinamide and selenonicotinamide, are used as starting material. It has been shown that thionicotinamide cannot be directly N 1 -alkylated and that selenonicotinamide is stable in aqueous solutions for only a few hours. A simple enzymic method has been developed, for obtaining nicotinamide mononucleotide (NMN) analogs, with alterations to the pyridine moiety of the molecule, starting with the available NAD(P) + analogs. In this method, the crude nucleotide pyrophosphatase from Crotalus venom was used. No attempt was made to purify the pyrophosphatase, by separating the contaminating enzymes, notably the 5′-nucleotidase. The pyrophosphate linkages of NAD(P) + and the NAD(P) + analogs investigated are hydrolyzed, except in 6-AN-ADP + and INH-ADP + , because of low substrate specificity of this nucleotide pyrophosphatase. It was found that only NMN, either obtained commercially or prepared from NADP + , is condensed enzymically with adenosine triphosphate (ATP). Under the conditions of the standard assay selected, none of the NMN analogs (thio-NMN, 3-AcPyr-MN, or 3-OHPyr-MN) reacts with ATP.