A chemical synthesis of nicotinamide adenine dinucleotide (NAD⁺)

Abstract

A practical synthesis of nicotinamide mononucleotide (β-NMN) and a high yield coupling with AMP-morpholidate that also provides NAD⁺ in an efficient manner are reported.

References

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8. Chromatography on Dowex resin (two successive columns, 1X2 formate, 50WX8 H⁺ form) followed by subsequent lyophilization of the aqueous fractions provided β-NMN in 80% yield, 97% purity (120 g scale). The two resin column isolation process proved to be operationally simple and the Dowex 50WX8 resin (H⁺ form) served the dual purpose of purification and, more importantly, pH adjustment to provide the desired inner salt form of β-NMN.
9. The crude β-NMN was initially purified by a resin-free isolation [i.e. precipitation followed by treatment with activated carbon (Ecorsob-C, P-502-H)]. Although the process is simple, the yield for the next coupling reaction with AMP-morpholidate under the optimized condition was only moderate (it still required pH adjustment using H⁺ cation resin). Alternatively, a direct resin chromatography using Dowex (50W X 8-100) resin column was developed (97% purity, eluted with 5% formic acid in water). However, this procedure required high resin loads with the product eluting in a large volume of water.
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17. For a successful high yielding coupling reaction, reagents and the solvents were dried. A MnCl₂ solution was prepared by dissolution of the commercial tetrahydrate in formamide to give a 0.2 M stock solution. The solution was dried over 4 Å molecular sieves for several days prior to use. Typically the solution had 0.5 M H₂O content and was used for the reaction successfully. Use of commercial anhydrous MnCl₂ proved inferior.
18. Purification of NAD⁺ using resin chromatography at room temperature resulted in partial decomposition of purified NAD⁺(5% purity loss). Therefore, the chromatography was carried out at 5 °C (110 g scale, > 99% purity).