Synthesis of β‐Nicotinamide Riboside Using an Efficient Two‐Step Methodology

Ning Zhang1, Anthony A. Sauve1

1 Department of Pharmacology, Weill Cornell Medical College, New York
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 14.14
DOI:  10.1002/cpnc.43
Online Posting Date:  December, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


A two‐step chemical method for the synthesis of β‐nicotinamide riboside (NR) is described. NR has achieved wide use as an NAD+ precursor (vitamin B3) and can significantly increase central metabolite NAD+ concentrations in mammalian cells. β‐NR can be prepared with an efficient two‐step procedure. The synthesis is initiated via coupling of commercially available 1,2,3,5‐tetra‐O‐acetyl‐β‐D‐ribofuranose with ethyl nicotinate in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf). 1H NMR showed that the product was formed with complete stereoselectivity to produce only the β‐isomer in high yield (>90% versus starting sugar). The clean stereochemical result suggests that the coupling proceeds via a cationic cis‐1,2‐acyloxonium‐sugar intermediate, which controls addition by nucleophiles to generate predominantly β‐stereochemistry. The subsequent deprotection of esters in methanolic ammonia generates the desired product in 85% overall yield versus sugar. © 2017 by John Wiley & Sons, Inc.

Keywords: nicotinamide riboside; nucleoside synthesis; stereoselective; NAD+; two‐step methodology

PDF or HTML at Wiley Online Library

Table of Contents

  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1:

  • 1,2,3,5‐Tetra‐O‐acetyl‐β‐D‐ribofuranose, 98% (Sigma‐Aldrich)
  • Ethyl nicotinate, 99% (Sigma‐Aldrich)
  • Trimethylsilyl trifluoromethanesulfonate (TMSOTf), ≥98.0% (Sigma‐Aldrich; used fresh when received or stored in a desiccator containing Drierite)
  • Anhydrous dichloromethane (DCM), ≥99.8% (contains 40 to 150 ppm amylene as stabilizer; Sigma‐Aldrich)
  • Anhydrous ethyl acetate (EtOAc), 99.8% (Sigma‐Aldrich)
  • Anhydrous methanol (MeOH), 99.8% (Sigma‐Aldrich)
  • Anhydrous triethylamine, ≥99% (Sigma‐Aldrich)
  • Anhydrous hexane, 95% (Sigma‐Aldrich)
  • Sulfuric acid (H 2SO 4), 99.999% (Sigma‐Aldrich)
  • Ammonia solution, 7 N in methanol (Sigma‐Aldrich)
  • Distilled water (Millipore Water Systems)
  • Argon (Tech Air)
  • 0.1 M sodium hydroxide
  • Octadecyl‐functionalized silica gel, 16% to 18% carbon loading, 200 to 400 mesh (Sigma‐Aldrich)
  • Deuterium oxide (D 2O), 99.9 atom % D (Sigma‐Aldrich)
  • 0.1% trifluoroacetic acid (TFA; mobile phase on HPLC)
  • 100 mL single‐neck round‐bottom flasks (VWR Scientific)
  • 100 mL three‐neck round‐bottom flasks (VWR Scientific)
  • Glass condenser, 110 mm (VWR Scientific)
  • Magnetic stirrer with hot plate and magnetic stir bars (VWR Scientific)
  • Oil bath (VWR Scientific)
  • Thermometer, −10° to 110°C (VWR Scientific)
  • High vacuum oil pump (VWR Scientific)
  • TLC plates (VWR Scientific)
  • Separatory funnel (VWR Scientific)
  • Glass chromatography column, 24/40 outer joint at top; 2‐mm bore glass stopcock at bottom (VWR Scientific)
  • Glass rods (VWR Scientific)
  • Syringe, 1000 µL, chromatography, removable needle, Ace Glass (VWR Scientific)
  • Bruker 300, 400, or 500 MHz NMR spectrometers (to acquire 1H, 19F, and 13C NMR spectra for compound characterization; 1H and 13C chemical shifts are expressed in ppm with respect to the chemical shift of tetramethylsilane or by chemical shift fixing of HOD to 4.80 ppm)
  • Hitachi EZChrom Elite HPLC system with a L2450 diode array as a detector
  • NUCLEOSIL 100‐5 C 18 (5 µM, 250 × 4.6 mm) column (Macherey‐Nagel)
  • HRMS spectrometer (for compound characterization; our HRMS spectra were obtained by Dr. Cliff Soll of Hunter College Mass Spectrometry Facility)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Belenky, P., Racette, F. G., Bogan, K. L., McClure, J. M., Smith, J. S., & Brenner, C. (2007). Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+. Cell, 129, 473–484. doi: 10.1016/j.cell.2007.03.024.
  Brown, K. D., Maqsood, S., Huang, J. Y., Pan, Y., Harkcom, W., Li, W., … Jaffrey, S. R. (2014). Activation of SIRT3 by the NAD+ precursor nicotinamide riboside protects from noise‐induced hearing loss. Cell Metabolism, 20, 1059–1068. doi: 10.1016/j.cmet.2014.11.003.
  Canto, C., Houtkooper, R. H., Pirinen, E., Youn, D. Y., Oosterveer, M. H., Cen, Y., … Auwerx, J. (2012). The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high‐fat diet‐induced obesity. Cell Metabolism, 15, 838–847. doi: 10.1016/j.cmet.2012.04.022.
  Canto, C., Sauve, A. A., & Bai, P. (2013). Crosstalk between poly(ADP‐ribose) polymerase and sirtuin enzymes. Molecular Aspects of Medicine, 34, 1168–1201. doi: 10.1016/j.mam.2013.01.004.
  Chi, Y., & Sauve, A. A. (2013). Nicotinamide riboside, a trace nutrient in foods, is a vitamin B3 with effects on energy metabolism and neuroprotection. Current Opinion in Clinical Nutrition and Metabolic Care, 16, 657–661. doi: 10.1097/MCO.0b013e32836510c0.
  Chini, E. N. (2009). CD38 as a regulator of cellular NAD: A novel potential pharmacological target for metabolic conditions. Current Pharmaceutical Design, 15, 57–63. doi: 10.2174/138161209787185788.
  Conze, D. B., Crespo‐Barreto, J., & Kruger, C. L. (2016). Safety assessment of nicotinamide riboside, a form of vitamin B3. Human & Experimental Toxicology, 2, 1–12. doi: 10.1177/0960327115626254.
  De Flora, A., Zocchi, E., Guida, L., Franco, L., & Bruzzone, S. (2004). Autocrine and paracrine calcium signaling by the CD38/NAD+/cyclic ADP‐ribose system. Annals of the New York Academy of Sciences, 1028, 176–191. doi: 10.1196/annals.1322.021.
  Felici, R., Lapucci, A., Cavone, L., Pratesi, S., Berlinguer‐Palmini, R., & Chiarugi, A. (2015). Pharmacological NAD‐boosting strategies improve mitochondrial homeostasis in human complex I‐mutant fibroblasts. Molecular Pharmacology, 87, 965–971. doi: 10.1124/mol.114.097204.
  Franchetti, P., Pasqualini, M., Petrelli, R., Ricciutelli, M., Vita, P., & Cappellacci, L. (2004). Stereoselective synthesis of nicotinamide beta‐riboside and nucleoside analogs. Bioorganic & Medicinal Chemistry Letters, 14, 4655–4658. doi: 10.1016/j.bmcl.2004.06.093.
  Gong, B., Pan, Y., Vempati, P., Zhao, W., Knable, L., Ho, L., … Pasinetti, G. M. (2013). Nicotinamide riboside restores cognition through an upregulation of proliferator‐activated receptor‐gamma coactivator 1alpha regulated beta‐secretase 1 degradation and mitochondrial gene expression in Alzheimer's mouse models. Neurobiology of Aging, 34, 1581–1588. doi: 10.1016/j.neurobiolaging.2012.12.005.
  Guan, Y., Wang, S. R., Huang, X. Z., Xie, Q. H., Xu, Y. Y., Shang, D., & Hao, C. M. (2017). Nicotinamide Mononucleotide, an NAD+ Precursor, Rescues Age‐Associated Susceptibility to AKI in a Sirtuin 1‐Dependent Manner. Journal of the American Society of Nephrology, 28, 2337–2352. doi: 10.1681/ASN.2016040385.
  Haigis, M. C., & Sinclair, D. A. (2010). Mammalian sirtuins: Biological insights and disease relevance. Annual Review of Pathology, 5, 253–295. doi: 10.1146/annurev.pathol.4.110807.092250.
  Houtkooper, R. H., Pirinen, E., & Auwerx, J. (2012). Sirtuins as regulators of metabolism and healthspan. Nature Reviews Molecular Cell Biology, 13, 225–238. doi: 10.1038/nrm3293
  Imai, S., Armstrong, C. M., Kaeberlein, M., & Guarente, L. (2000). Transcriptional silencing and longevity protein Sir2 is an NAD‐dependent histone deacetylase. Nature, 403, 795–800. doi: 10.1038/35001622.
  Khan, N. A., Auranen, M., Paetau, I., Pirinen, E., Euro, L., Forsstrom, S., … Suomalainen, A. (2014). Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3. EMBO Molecular Medicine, 6, 721–731. doi: 10.1002/emmm.201403943.
  Klaidman, L., Morales, M., Kem, S., Yang, J., Chang, M. L., & Adams, J. D. Jr (2003). Nicotinamide offers multiple protective mechanisms in stroke as a precursor for NAD+, as a PARP inhibitor and by partial restoration of mitochondrial function. Pharmacology, 69, 150–157. doi: 10.1159/000072668.
  Kulikova, V., Shabalin, K., Nerinovski, K., Dolle, C., Niere, M., Yakimov, A., … Nikiforov, A. (2015). Generation, release, and uptake of the NAD precursor nicotinic acid riboside by human cells. The Journal of Biological Chemistry, 290, 27124–27137. doi: 10.1074/jbc.M115.664458.
  Lu, S. P., Kato, M., & Lin, S. J. (2009). Assimilation of endogenous nicotinamide riboside is essential for calorie restriction‐mediated life span extension in Saccharomyces cerevisiae. The Journal of Biological Chemistry, 284, 17110–17119. doi: 10.1074/jbc.M109.004010.
  Rowen, J. W., & Kornberg, A. (1951). The phosphorolysis of nicotinamide riboside. The Journal of Biological Chemistry, 193, 497–507.
  Ryu, D., Zhang, H., Ropelle, E. R., Sorrentino, V., Mazala, D. A., Mouchiroud, L., … Auwerx, J. (2016). NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation. Science Translational Medicine, 8, 361ra139. doi: 10.1126/scitranslmed.aaf5504.
  Sauve, A. A. (2008). NAD+ and vitamin B3: From metabolism to therapies. The Journal of Pharmacology and Experimental Therapeutics, 324, 883–893. doi: 10.1124/jpet.107.120758.
  Schiedel, M., Robaa, D., Rumpf, T., Sippl, W., & Jung, M. (2017). The current state of NAD+ ‐dependent histone deacetylases (sirtuins) as novel therapeutic targets. Medicinal Research Reviews, 0, 1–54 doi: 10.1002/med.21436.
  Schreiber, V., Dantzer, F., Ame, J. C., & de Murcia, G. (2006). Poly(ADP‐ribose): Novel functions for an old molecule. Nature Reviews Molecular Cell Biology, 7, 517–528. doi: 10.1038/nrm1963.
  Shi, W., Hegeman, M. A., van Dartel, D. A., Tang, J., Suarez, M., Swarts, H., … Keijer, J. (2017). Effects of a wide range of dietary nicotinamide riboside (NR) concentrations on metabolic flexibility and white adipose tissue (WAT) of mice fed a mildly obesogenic diet. Molecular Nutrition & Food Research, 61, 1–11. doi: 10.1002/mnfr.201600878.
  Tanimori, S., Ohta, T., & Kirihata, M. (2002). An efficient chemical synthesis of nicotinamide riboside (NAR) and analogues. Bioorganic & Medicinal Chemistry Letters, 12, 1135–1137. doi: 10.1016/S0960‐894X(02)00125‐7.
  Tempel, W., Rabeh, W. M., Bogan, K. L., Belenky, P., Wojcik, M., Seidle, H. F., … Brenner, C. (2007). Nicotinamide riboside kinase structures reveal new pathways to NAD+. PLoS Biology, 5, e263. doi: 10.1371/journal.pbio.0050263.
  Trammell, S. A., Yu, L., Redpath, P., Migaud, M. E., & Brenner, C. (2016). Nicotinamide riboside is a major NAD+ precursor vitamin in cow milk. Journal of Nutrition, 146, 957–963. doi: 10.3945/jn.116.230078.
  Ummarino, S., Mozzon, M., Zamporlini, F., Amici, A., Mazzola, F., Orsomando, G., … Raffaelli, N. (2017). Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme‐coupled assay. Food Chemistry, 221, 161–168. doi: 10.1016/j.foodchem.2016.10.032.
  Verdin, E. (2015). NAD+ in aging, metabolism, and neurodegeneration. Science, 350(6265), 1208–1213. doi: 10.1126/science.aac4854.
  Weidele, K., Beneke, S., & Burkle, A. (2017). The NAD+ precursor nicotinic acid improves genomic integrity in human peripheral blood mononuclear cells after X‐irradiation. DNA Repair, 52, 12–23. doi: 10.1016/j.dnarep.2017.02.001.
  Yang, T., Chan, N. Y., & Sauve, A. A. (2007). Syntheses of nicotinamide riboside and derivatives: Effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells. Journal of Medicinal Chemistry, 50, 6458–6461. doi: 10.1021/jm701001c.
  Yang, Y., & Sauve, A. A. (2016). NAD+ metabolism: Bioenergetics, signaling and manipulation for therapy. Biochimica et Biophysica Acta, 1864, 1787–1800. doi: 10.1016/j.bbapap.2016.06.014.
  Zhang, H., Ryu, D., Wu, Y., Gariani, K., Wang, X., Luan, P., … Auwerx, J. (2016). NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice. Science, 352, 1436–1443. doi: 10.1126/science.aaf2693.
PDF or HTML at Wiley Online Library