Stereoselective synthesis of nicotinamide β-riboside and nucleoside analogs

Nicotinamide riboside (β-NAR) is an intermediate in one biosynthetic pathway by which nicotinamide is converted into NAD. In fact, nicotinamide or nicotinamide riboside derived from degradation of NAD can be reused by nicotinamide phosphoribosyl transferase or ribosylnicotinamide kinase, respectively, to form the ribotide NMN that is adenylated to NAD by nicotinamide mononucleotide adenylyltransferase (NMNAT). Human NMNAT is an indispensable enzyme in both denovo and salvage/recycling pathways for NAD biosynthesis, catalyzing the conversion of NMN or its deamidated form NaMN into NAD or NaAD, respectively. On the other hand, it is known that many bacterial NMNATs strongly prefer the deamidated NaMN as a substrate. NAD is a co-factor in numerous enzyme-catalyzed redox reactions in all living organisms and plays a fundamental role in cellular metabolic processes. It is crucial thus that proper level of NAD are regulated and maintained for cellular survival. There is evidence that hNMNAT is weakly expressed in tumor cells, determining thus interest in the enzyme as a target for anticancer drugs. In this respect, some nicotinamide riboside analogs have been described as antitumor agents involving their metabolic conversion to NAD analogs by NMNAT activity.

Kinetic and structural studies of both human and bacterial NMNAT require the β-anomer of nicotinamide monoribotide as a substrate and of nicotinamide riboside as a product of the NAD degradation pathway. The stereospecificity of NAD-mediated reactions is determinant in all living organisms because the pyridine ribotide moiety of NAD reacts only in the β-configuration.

The key elements of our synthesis involved the formation of silylated nicotinamide as starting material, and then its coupling with peracylated sugars in the presence of trimethylsilyl trifluoromethanesulfonate. However, the author found that when a large excess of trimethylsilyl chloride in hexamethyldisilazane was used to silylate the nicotinamide, a poor yield of the desired nucleoside was obtained and the purification of 3-(carbamoyl)-1-(2,3,5-tri-O-acetyl-b-D D-ribofuranosyl) pyridinium triflate was very difficult. In addition, the amount of TMSOTf proved to be crucial in this reaction.

The author obtained a stereoselectivity in the synthesis of NAR by condensation of nicotinamide with both 1,2,3,5-tetra-O-acetyl-b-D D-ribofuranose and 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D D-ribofuranose using carefully controlled conditions. In this procedure the author used 2equiv of TMSCl to silylate the dry nicotinamide under reflux. The intermediate was directly coupled with the protected sugars (1 eqiv) under Vorbruggen’s conditions in the presence of a catalytic amount of TMSOTf in 1,2-dichloroethane, to give corresponding intermediate in high yield. Only β-anomers of the protected N-nucleo-sides were obtained. β-NAR was then obtained by basic hydrolytic deblocking of compounds 9 or 11 at  5℃. The low temperature was required to minimize cleavage of the glycosidic linkage. In the case of corresponding intermediate, for removal of benzoyl groups, two days were required under methanolic ammonia. The nucleoside product was then purified by chromatography on activated charcoal and isolated as a white solid.

In summary, a stereoselective reaction for β-NAR and its analogs via TMSOTf-mediated glycosylation of the silylated nicotinic bases using controlled conditions was shown.




Palmarisa Franchetti, Bioorganic & Medicinal Chemistry Letters 14 (2004) 4655-4658