Reduced Nicotinamide Riboside (NRH): A Review of Generation, Synthesis, and Biological Functions
Abstract
Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme central to cellular metabolism and homeostasis. Its precursor, nicotinamide riboside (NR), has been extensively studied for boosting NAD+ levels. Recently, its reduced form, dihydronicotinamide riboside, commonly termed reduced nicotinamide riboside (NRH), has emerged as a potent, distinct NAD+ precursor. This review outlines the chemical identity of NRH, summarizes strategies for its biological and chemical synthesis, and explores its superior efficacy in enhancing NAD+ pools, along with its downstream biological functions in energy metabolism, DNA repair, signaling, and potential therapeutic applications.
- Introduction: From NR to NRH
NAD+ is a fundamental cofactor involved in redox reactions (glycolysis, TCA cycle), DNA repair (via PARPs), epigenetic regulation (via sirtuins), and calcium signaling. Age-related decline in NAD+ is linked to various pathologies. Supplementing NAD+ precursors is a promising intervention strategy.
NR, a natural dietary component, is phosphorylated by nicotinamide riboside kinases (NRK1/2) to nicotinamide mononucleotide (NMN), which is then converted to NAD+. NRH (chemical name: 1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,4-dihydropyridine-3-carboxamide) is the reduced form of NR, where the pyridinium ring of the nicotinamide moiety is converted to a dihydropyridine. This structural alteration enables a unique and more efficient bypass pathway for NAD+ biosynthesis, independent of the rate-limiting NRK step.
- Generation and Synthesis of NRH
2.1 Enzymatic and Biocatalytic Preparation
As NRH is not abundant in nature, in vitro enzymatic synthesis is a key method for research-grade production.
Key Enzymes: Engineered or homologous nicotinamide riboside kinases (NRKs) can utilize NRH as a substrate, phosphorylating it to reduced NMN (NMNH). This process can be optimized for NRH synthesis or detection.
Strategies: One approach involves chemical reduction of NR (e.g., using sodium dithionite) to yield crude NRH, followed by enzymatic purification. Alternatively, a fully enzymatic cascade can be designed using cheaper starting materials like dihydronicotinamide and ribose derivatives, catalyzed by kinases and phosphorylases. While offering excellent stereoselectivity under mild conditions, this method is currently cost-intensive and suited for laboratory-scale preparation.
2.2 Chemical Synthesis
Chemical synthesis is the primary route for scalable NRH production.
Core Challenge: The key step is the stereoselective formation of the glycosidic bond between a dihydronicotinamide base and a protected ribose moiety.
Glycosylation Strategy: A common method involves coupling a protected dihydronicotinamide derivative with an activated ribosyl donor (e.g., 1-acetoxy ribose) under carefully controlled conditions.
Reduction Strategy: Another practical path involves the selective reduction of pre-synthesized NR. Catalytic hydrogenation or reduction with mild agents like sodium borohydride targets the pyridinium ring. This requires precise protection/deprotection of the ribose hydroxyl groups to prevent side reactions.
Purification and Characterization: The product is purified via chromatography (HPLC or column). Structure is confirmed by nuclear magnetic resonance (NMR) and mass spectrometry (MS). High-purity NRH (often as a chloride or iodide salt) requires storage under dark, dry, and cold conditions due to its sensitivity to oxidation.
- Biological Functions of NRH
NRH’s functions are primarily mediated through its exceptional ability to elevate cellular NAD+ and NADH pools.
3.1 A Potent and Distinct NAD+ Biosynthetic Pathway
Unlike NR, NRH enters an alternative pathway. It is rapidly phosphorylated, potentially by adenosine kinase (ADK) or other kinases, to form NMNH. NMNH is then directly converted to NADH by nicotinamide mononucleotide adenylyltransferases (NMNATs). This NADH can be oxidized to NAD+ via the mitochondrial electron transport chain, simultaneously boosting both NAD(H) pools (Fig. 2). Studies show NRH increases intracellular NAD+ levels 10- to 100-fold more efficiently than NR in various cell lines and mouse tissues.
3.2 Cellular Energy Metabolism and Redox Homeostasis
By rapidly expanding the NAD(H) pool, NRH enhances mitochondrial respiration and ATP production, crucial for high-energy tissues (brain, heart, muscle). It also helps maintain the critical NAD+/NADH ratio, influencing metabolic flux and cellular redox balance.
3.3 Activation of NAD+-Consuming Enzymes
Elevated NAD+ directly activates major NAD+-dependent enzymes:
Sirtuin Activation: Boosts activity of SIRT1 (regulating transcription, metabolism, stress resistance) and the mitochondrial SIRT3 (optimizing oxidative metabolism, reducing ROS).
PARP Activation: Supports DNA repair by PARP1. Under genotoxic stress, NRH efficiently replenishes NAD+ pools depleted by PARP overactivation, preserving genomic integrity.
3.4 Potential Therapeutic Applications in Disease Models
Preclinical studies highlight NRH’s therapeutic potential:
Acute Tissue Injury: In cisplatin-induced acute kidney injury, NRH protects renal function by supporting PARP-mediated repair.
Metabolic Disorders: Improves glucose tolerance and hepatic steatosis in diet-induced obese mice.
Neuroprotection: Exerts protective effects in models of Alzheimer’s and Parkinson’s disease by enhancing mitochondrial function via SIRT3.
Muscle Function: Restores NAD+ levels and improves exercise capacity in aged mice.
Cancer Therapy Sensitization: Emerging evidence suggests NRH may alter tumor metabolism, potentially sensitizing cancer cells to chemotherapy or radiotherapy.
- Challenges and Future Perspectives
Despite its promise, NRH faces several hurdles:
Stability: NRH is more chemically labile than NR, prone to oxidation in solution, demanding advanced formulation strategies.
Pharmacokinetics and Safety: Comprehensive ADME (Absorption, Distribution, Metabolism, Excretion) and long-term toxicology profiles are required.
Tissue-Specific Delivery: Effective delivery to specific organs (e.g., crossing the blood-brain barrier) remains a challenge for clinical translation.
Dual Effect: The concurrent sharp increase in both NAD+ and NADH may have complex, context-dependent effects on cellular redox state.
Future research will focus on developing more stable NRH prodrugs or analogs, conducting rigorous preclinical safety studies, and advancing toward clinical trials to evaluate its efficacy against age-related diseases, metabolic disorders, and mitochondrial pathologies.
Figure Legends
Fig. 1: Chemical structures of NR and NRH. The inset highlights the reduction of the nicotinamide ring from a pyridinium (oxidized) in NR to a dihydropyridine (reduced) in NRH.
Fig. 2: Comparative intracellular metabolic pathways of NR and NRH. The classic NR Pathway: NR → (NRK) → NMN → (NMNAT) → NAD+. The efficient NRH Pathway: NRH → (ADK/other kinase) → NMNH → (NMNAT) → NADH → (Electron Transport Chain) → NAD+.
References
Giroud-Gerbetant, J., et al. (2019). A reduced form of nicotinamide riboside defines a new path for NAD+ biosynthesis and acts as an orally bioavailable NAD+ precursor. Molecular Metabolism, 30, 192-202. (Seminal paper first characterizing NRH)
Yang, Y., et al. (2019). NAD+ repletion restores mitophagy and limits accelerated aging in Werner syndrome. Nature Communications, 10(1), 5284.
Ratajczak, J., et al. (2016). NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells. Nature Communications, 7, 13103.
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For detailed chemical synthesis protocols, refer to specialized journals in organic and medicinal chemistry.