Patent Description:
Nicotinamide adenine dinucleotide (NAD+), and its reduced form <NUM>,<NUM>-dihydronicotinamide adenine dinucleotide (NADH), are key molecules in energy metabolism and mitochondrial function by electron transfer. Moreover, NAD+ is a crucial cofactor in a number of non-redox reactions, by causing adenosine diphosphate ribose (ADP-ribose) to enzymatically catalyze the functions of the two essential protein families, sirtuins (SIRTs) and poly (ADP-ribose) polymerases (PARPs). The sirtuins have several key roles maintaining nuclear, mitochondrial, cytoplasmic or metabolic homeostasis. The most important roles of PARPs are repairing DNA and maintaining chromatin structure and function. Aging, and some disruptive factors such as an acute injury or chronic metabolic or inflammatory conditions, can cause levels of NAD+ to severely decline.

The drop of NAD+ leads to the decrease of energy production which, subsequently, impairs the cellular function, cellular homeostasis, and immune cell function. This phenomenon becomes even more important when the cells are damaged by ordinary environmental factors, because repairing damage requires a large amount of NAD+. If the injury is severe, the damaged cells will not have enough stored energy to provide the NAD+ needed for homeostasis maintenance, and so the damage becomes irreversible. The brain, heart, liver, kidneys and skeletal muscles are the organs with higher numbers of mitochondria, therefore, these vital organs are more susceptible to NAD+ depletion. Therefore, an energy-rich NAD+ precursor is needed to keep the cell with damaged tissue at normal levels of energy. Nicotinamide riboside (NR), nicotinic acid (niacin), and nicotinamide are commercially available, natural compounds used as nutritional supplements to increase the concentrations of NAD+.

NR is a more efficient NAD+ precursor in comparison to niacin and nicotinamide because it is metabolized to NAD+ in mammalian cells in fewer steps (Scheme <NUM>). Studies show that taking NR as a supplement is effective for stimulating NAD+ metabolism and can boost the level of NAD+ by <NUM> percent.

Although NR can increase the NAD+ level of cell and improve cell health, NR must be taken in large quantities to be effective. Recently, Sauve et al. have synthesized <NUM>,<NUM>-dihydronicotinamide riboside (NRH) and demonstrated that this compound is a potent NAD+ concentration enhancer in both in vitro and in vivo conditions. They found that, after administration of NRH to mammalian cells, it increased the NAD+ concentration by <NUM>-<NUM>-fold over control values in just one hour. Their findings demonstrate that the use of NRH is more effective than either NR or NMN. Moreover, NRH considerably enhances the NAD+/NADH ratio in the cultured cells without induction of apoptotic markers or a substantial increases in lactate levels in cells. More recently, Canto et al. have found that, contrary to the NR pathway, NRH uses different steps and enzymes to synthesize NAD+. That explains why NRH is a more effective and a faster NAD+ precursor compared to the NR in mammalian cells. The same researchers have also demonstrated, in experiments with mice, that NRH is orally bioavailable as an NAD+ precursor and prevents cisplatin-induced, acute kidney injury. In addition to increasing NAD+ levels, NRH can also deplete some genotoxins such as hydrogen peroxide and methylmethane sulfonate. As a result, the mouse cells treated with NRH are resistant to cell death.

There are very few methods for the preparation of NRH. This compound can be prepared from dihydronicotinamide mononucleotide (NMNH) by hydrolysis of the <NUM>'-phosphate ester in the presence of alkaline phosphatase. The method from NMNH is time-consuming and is not cost-effective because NMNH as a precursor must be enzymatically hydrolyzed from NADH. Another method for the synthesis of NRH is the reduction of NR in the presence of sodium dithionite (Na<NUM>S<NUM>O<NUM>) as a reducing agent. In this method, nicotinamide riboside triflate is reduced to NRH in an aqueous solution of sodium dithionite and potassium hydrogen phosphate (Scheme <NUM>). Because the aqueous solution of Na<NUM>S<NUM>O<NUM> is very unstable at ambient conditions, this reaction must be carried out at low temperature and under anaerobic, alkaline conditions. Furthermore, the crude product should be immediately purified with HPLC using a C18 resin because NRH is sensitive to both hydrolysis and oxidation at ambient conditions as shown in the results and discussion of Example.

Although this method is suitable to synthesize a small amount of NRH, there are several drawbacks that prevent scaling this to the commercial production of NRH. The precursor, nicotinamide riboside triflate, is very expensive, is a very hygroscopic material, and must be stored at -<NUM> ° C under an inert atmosphere. Moreover, this compound is not food grade because of the presence of the triflate anion in the structure of nicotinamide riboside triflate. Therefore, a complete purification of NRH from the remaining NR (triflate) is required after the reduction.

Another method for the synthesis of NRH is the use of triacetylated nicotinamide riboside triflate instead of nicotinamide riboside triflate (Scheme <NUM>). In the first step of this indirect procedure, triacetylated NR converts to triacetylated NRH with Na<NUM>S<NUM>O<NUM>. In the next step, the NRH is formed by methanolysis of triacetylated NRH while ball-milling. This method might be appropriate for scalable synthesis of NRH because it provides a good yield. However, the use of triacetylated nicotinamide riboside triflate, an expensive and non-food grade substance, may limit this procedure.

It should be noted that most of the nicotinamide ribosides that have been used for the synthesis of NRH are the mixture of two anomeric α- and β- forms. However, only the β-anomer of NR represents bioactivity and medicinal properties. Among NR derivatives, only nicotinamide riboside chloride (NRCl) is commercially available as a dietary supplement.

The present disclosure includes the recognition that using β-NRCl as a precursor to synthesize NRH would represent a breakthrough for the commercialization of the NRH, a desired and valuable product. Indeed, the method of producing NRH disclosed herein has an advantageously high yield from a commercially available NR or its derivatives such as β-NRCl.

Some definitions are provided hereafter. Nevertheless, definitions may be located in the "Embodiments" section below, and the above header "Definitions" does not mean that such disclosures in the "Embodiments" section are not definitions.

All percentages expressed herein are by weight of the total weight of the composition unless expressed otherwise. As used herein, "about," "approximately" and "substantially" are understood to refer to numbers in a range of numerals, for example the range of -<NUM>% to +<NUM>% of the referenced number, preferably -<NUM>% to +<NUM>% of the referenced number, more preferably -<NUM>% to +<NUM>% of the referenced number, most preferably -<NUM>% to +<NUM>% of the referenced number. All numerical ranges herein should be understood to include all integers, whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from <NUM> to <NUM> should be construed as supporting a range of from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, and so forth.

As used in this disclosure and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component" or "the component" includes two or more components.

The words "comprise," "comprises" and "comprising" are to be interpreted inclusively rather than exclusively. Likewise, the terms "include," "including" and "or" should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. Nevertheless, the compositions disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term "comprising" includes a disclosure of embodiments "consisting essentially of" and "consisting of" the components identified.

The term "and/or" used in the context of "X and/or Y" should be interpreted as "X," or "Y," or "X and Y. " Similarly, "at least one of X or Y" should be interpreted as "X," or "Y," or "X and Y. " For example, "at least one dithionite or a functionally similar reducing agent" should be interpreted as "dithionite," or "a functionally similar reducing agent," or "both dithionite and a functionally similar reducing agent.

Where used herein, the terms "example" and "such as," particularly when followed by a listing of terms, are merely exemplary and illustrative and should not be deemed to be exclusive or comprehensive. As used herein, a condition "associated with" or "linked with" another condition means the conditions occur concurrently, preferably means that the conditions are caused by the same underlying condition, and most preferably means that one of the identified conditions is caused by the other identified condition.

The term "a basic solution," as used herein, refers to a solution with a pH greater than <NUM>. For example, a basic solution could have a pH in the range of about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, or preferably about <NUM> to about <NUM>. When reference herein is made to the pH, values correspond to pH measured at <NUM> with standard equipment.

The term "neutral pH," as used herein, refers to a pH of about <NUM>. When reference herein is made to the pH, values correspond to pH measured at <NUM> with standard equipment.

The term "room temperature," as used herein, refers to a temperature commonly used in Physics and Chemistry, namely a temperature of essentially about <NUM> (or about <NUM>).

The term "anaerobic condition," as used herein, refers to a condition in absence of free oxygen.

The term "aerobic condition," as used herein, refers to a condition in presence of free oxygen.

The term "dithionite," as used herein, refers to an anion of S<NUM>O<NUM><NUM>-.

The term "nicotinamide riboside" or "NR," as used herein, refers to a form pyridine-nucleoside of vitamin B3 that functions as a precursor to nicotinamide adenine dinucleotide or NAD+. The Nicotinamide riboside or NR has either an α-form or a β-form. For example, the β-form of the Nicotinamide riboside or NR has a chemical structure Formula I as follows:
<CHM>.

The term "alkali salt" or "basic salt," as used herein, refers to a salt that is the product of the neutralization of a strong base and a weak acid. An alkali salt or basic salt, when hydrolyzes, can form a basic solution. Examples of alkali salts or basic salts may include metal carbonates, metal bicarbonate, metal acetate, metal phosphate derivatives and others.

The term "high yield," as used herein, refers to a total yield of a synthesis at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%. Preferably, a high yield of the present disclosure is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%.

The term "high purity," as used herein, refers to a purity of a compound or substance at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%. Preferably, a high purity of the present disclosure is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%.

The term "effective amount," as used herein, refers to an amount of a substance capable of either increasing (directly or indirectly) the yield of reduction product or increasing selectivity toward <NUM>,<NUM>-dihydronicotinamide riboside (NRH). Optimum amounts of a given substance can vary based on reaction conditions and the identity of other constituents yet can be readily determined in light of the discrete circumstances of a given application.

The term "chromatography," as used herein, refers to any kind of purification technique which separates the compound of interest from other molecules in the mixture, for example, by their differences in partition between a solid substrate and the mobile phase, including but not limited to, solution, buffer or solvent and allows the compound of interested to be isolated.

The term "eluent," as used herein, refers to a solvent or a mixture of two or more solvents used in a chromatography process to move, or elute, one or more substances from a stationary support material. In an embodiment, a polar solvent such as methanol may be used as an eluent to wash out the NRH from the stationary phase during a chromatographic process.

The term "adsorbent" or "stationary phase," as used herein, refers to a material which exists in the fluid stream as a solid form in a chromatographic process. In an embodiment, a mixture of basic alumina and silica may be used as the adsorbent during a chromatographic process.

The term "solvent," as used herein, refers to a substance capable of dissolving another substance (solute).

An aspect of the present disclosure is a method of producing <NUM>,<NUM>-dihydronicotinamide riboside (NRH) from a commercial available chemical such as nicotinamide riboside chloride (NRCl). Nicotinamide riboside chloride (NRCl) is the only commercially available NR derivative (e.g., as a dietary supplement). Thus, the present method represents a new pathway for production and commercialization of <NUM>,<NUM>-dihydronicotinamide riboside (NRH).

Applicant surprisingly found that a reduction of the commercially available nicotinamide riboside chloride (NRCl) with a reducing agent such as a dithionite in a liquid solution can lead to effective formation and production of <NUM>,<NUM>-dihydronicotinamide riboside (NRH). Applicant further surprisingly found that the resulting <NUM>,<NUM>-dihydronicotinamide riboside (NRH) can be isolated and purified with a high yield by using column chromatography.

In an aspect, the present disclosure relates to a method of producing <NUM>,<NUM>-dihydronicotinamide riboside (NRH) from nicotinamide riboside chloride (NRCl).

<FIG> shows an exemplary method (<NUM>) of producing <NUM>,<NUM>-dihydronicotinamide riboside from nicotinamide riboside chloride, according to an embodiment provided by the present disclosure.

As shown in <FIG>, the method <NUM> comprises providing nicotinamide riboside chloride (NRCl) in a liquid solution (<NUM>).

Nicotinamide riboside chloride (NRCl) is commercially available, and could be purchased as, e.g., Niagen™ from ChromaDex.

Nicotinamide riboside chloride (NRCl) includes either α-form NRCl (α-NRCl) or β-form NRCl (β-NRCl). In an embodiment, the nicotinamide riboside chloride (NRCl) of present disclosure comprises β-NRCl. In an embodiment, the nicotinamide riboside chloride (NRCl) of present disclosure is β-NRCl.

In an embodiment, as only the β-anomer of NR represents bioactivity and medicinal properties, the present disclosure uses β-NRCl as the exemplary starting material to produce <NUM>,<NUM>-dihydronicotinamide riboside (NRH). The present method of producing <NUM>,<NUM>-dihydronicotinamide riboside (NRH) can also be applied to using α-NRCl as a starting material.

A chemically effective amount of nicotinamide riboside chloride (β-NRCl) as a form of solid (e.g., a powder) is provided. In an embodiment, the solid form of nicotinamide riboside chloride (β-NRCl) may be used in a solid-state reaction to produce <NUM>,<NUM>-dihydronicotinamide riboside (NRH). An exemplary solid-state reaction was described in the Example.

In another embodiment, the solid form of β-nicotinamide riboside chloride (β-NRCl) is dissolved in a solvent such as water to form a liquid solution for the production of <NUM>,<NUM>-dihydronicotinamide riboside (NRH).

In a preferred embodiment, a chemically effective amount of β-nicotinamide riboside chloride (β-NRCl) is first dissolved in a solution such as water to form a solution for a liquid-based production of <NUM>,<NUM>-dihydronicotinamide riboside (NRH).

As shown in <FIG>, after a chemically effective amount of β-nicotinamide riboside chloride (β-NRCl) is provided in a liquid solution, the method <NUM> may comprise adding a metal dithionite into the liquid solution under a first temperature (<NUM>). As such, a reducing agent such as a metal dithionite may be added into the liquid solution.

In an embodiment, the reducing agent may comprise a metal dithionite such as sodium dithionite (Na<NUM>S<NUM>O<NUM>). In an embodiment, the only reducing agent may be a metal dithionite such as sodium dithionite (Na<NUM>S<NUM>O<NUM>). Scheme <NUM> shows an exemplary reduction reaction of β-nicotinamide riboside chloride (β-NRCl) with sodium dithionite (Na<NUM>S<NUM>O<NUM>) in a liquid solution. <CHM>
<CHM>.

Scheme <NUM> shows a reaction mechanism of NRH synthesis from NR by using Na<NUM>S<NUM>O<NUM> as a reducing agent. As shown in Scheme <NUM>, a reduction reaction of NR by using Na<NUM>S<NUM>O<NUM> initially forms a sulfinate intermediate, which is stable at basic conditions.

After protonation, the sulfinate intermediate forms its sulfinic acid derivative. This sulfinic acid intermediate is unstable at ambient conditions and converts to NRH via SO<NUM> leaving.

As such, applicant notes that a particularly preferred embodiment uses a pH that will not only stabilize Na<NUM>S<NUM>O<NUM> but will also protonate the sulfonate intermediate to produce the NRH. Moreover, because NRH has an N-glycoside bond in its structure, NRH is susceptible to hydrolysis. Consequently, a particularly preferred embodiment adjusts the pH and maintains the pH precisely during the whole course of the reaction so that NRH is not hydrolyzed.

Thus, the present methods include adjusting the pH of the related solutions and maintaining it precisely within a specific range (e.g., <NUM>-<NUM>, preferably <NUM>) for effective production of NRH.

Throughout this disclosure, Na<NUM>S<NUM>O<NUM> was used as an exemplary reducing agent. Other functionally similar reducing agent may also be used. Preferably, the reducing agent is Na<NUM>S<NUM>O<NUM>.

A molar ratio of β-nicotinamide riboside chloride (β-NRCl) to the reducing agent such as Na<NUM>S<NUM>O<NUM> is preferably between about <NUM>:<NUM> and about <NUM>:<NUM>, between about <NUM>:<NUM> and about <NUM>:<NUM>, or between about <NUM>:<NUM> and about <NUM>:<NUM>, preferably between about <NUM>:<NUM> and about <NUM>:<NUM>, more preferably about <NUM>:<NUM>.

As an aqueous solution of Na<NUM>S<NUM>O<NUM> is unstable under aerobic conditions at neutral pH (e.g., pH=<NUM>), preferably the liquid solution for the reduction reaction of β-nicotinamide riboside chloride (β-NRCl) by using Na<NUM>S<NUM>O<NUM> is maintained under anaerobic and basic conditions.

For example, the liquid solution may be purged with an inert gas to remove free oxygen in the solution. An inert gas may include nitrogen (N<NUM>), argon (Ar) or others. In an embodiment, either nitrogen or argon is used as the inert gas. The liquid solution may be purged with an inert gas throughout the reduction reaction.

In an embodiment, the inert gas comprises nitrogen gas. In an embodiment, the only inert gas is nitrogen gas.

In an embodiment, the liquid solution comprises a basic solution. In an embodiment, the liquid solution is a basic solution.

In an embodiment, the basic solution comprises sodium bicarbonate (NaHCO<NUM>). For example, the liquid solution may include at least one alkali salt or basic salt such as sodium bicarbonate (NaHCO<NUM>) to form a basic solution. In an embodiment, the only alkali salt or basic salt is sodium bicarbonate (NaHCO<NUM>). Nevertheless, other alkali salt or basic salt such as sodium carbonate, potassium carbonate, potassium bicarbonate and similar compounds may optionally be used.

In an embodiment, a pH value of the liquid solution may be controlled by the concentration of the at least one alkali salt or basic salt in the solution. For example, by using <NUM>-<NUM>, preferably <NUM>-<NUM>, more preferably <NUM> of NaHCO<NUM>, the pH value of the liquid solution may be controlled in the range of about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, preferably about <NUM>.

In an embodiment, the method <NUM> comprises maintaining a pH value of the NaHCO<NUM> solution between <NUM>-<NUM>. In an embodiment, the method <NUM> comprises maintaining a pH value of the NaHCO<NUM> solution between about <NUM> and about <NUM>. In an embodiment, the method <NUM> comprises maintaining a pH value of the NaHCO<NUM> solution about <NUM>.

In an embodiment, the NaHCO<NUM> solution has a concentration from about <NUM> to about <NUM>. In an embodiment, the NaHCO<NUM> solution has a concentration about <NUM>.

In an embodiment, the reducing agent such as Na<NUM>S<NUM>O<NUM> may be gradually added into the liquid solution of the β-nicotinamide riboside chloride (β-NRCl) under an inert gas atmosphere at a first temperature. In an embodiment, the reducing agent such as Na<NUM>S<NUM>O<NUM> may be pre-dissolved into a solvent such as water and the solution of the reducing agent such as Na<NUM>S<NUM>O<NUM> may be dropped-wise into the liquid solution of the β-nicotinamide riboside chloride (β-NRCl) under an inert gas atmosphere at a first temperature.

In an embodiment, the first temperature is a low temperature such as about <NUM> (e.g., by using ice water to control) so that the reduction reaction can be controlled during the addition of the reducing agent such as Na<NUM>S<NUM>O<NUM>.

Returning to <FIG>, after the addition of the reducing agent such as Na<NUM>S<NUM>O<NUM>, the method <NUM> may comprise reacting the metal dithionite with the nicotinamide riboside chloride (NRCl) in the liquid solution under a second temperature to form a mixture, wherein a portion of the mixture is the <NUM>,<NUM>-dihydronicotinamide riboside (NRH) (<NUM>).

As such, the temperature of the liquid solution is preferably increased from the first temperature to a second temperature. Under the second temperature, the nicotinamide riboside chloride (NRCl) in the liquid solution can completely and effectively react with the reducing agent such as Na<NUM>S<NUM>O<NUM> to form the <NUM>,<NUM>-dihydronicotinamide riboside (NRH). In an embodiment, the second temperature is room temperature.

Under the room temperature, the reducing agent such as Na<NUM>S<NUM>O<NUM> and the β-nicotinamide riboside chloride (β-NRCl) may continuously and completely react to form a mixture, wherein at least portion of the mixture is the <NUM>,<NUM>-dihydronicotinamide riboside (NRH).

In an embodiment, progress of the reduction reaction between the reducing agent such as Na<NUM>S<NUM>O<NUM> and the β-nicotinamide riboside chloride (β-NRCl) may be monitored by using a chromatography technique. For example, as shown in <FIG>, all compounds of β-NRCl, NRH and nicotinamide (NA; a degradation product from NRH; see Scheme <NUM>) were visible at a wavelength of <NUM>. However, at a wavelength of <NUM>, only NRH was visible. By monitoring intensity and position of peaks corresponding to starting material β-NRCl and the resulting product NRH, one can determine progress of the reduction reaction between the reducing agent such as Na<NUM>S<NUM>O<NUM> and the β-nicotinamide riboside chloride (β-NRCl).

After the reduction reaction between the reducing agent such as Na<NUM>S<NUM>O<NUM> and the β-nicotinamide riboside chloride (β-NRCl) is complete, the resulting mixture may be dried to obtain a solid of the mixture. In an embodiment, the resulting mixture may be freeze-dried to obtain a yellow solid of the mixture.

In another aspect of the present disclosure, a purification method for obtaining a high purity of the resulting NRH is disclosed. Applicant surprisingly found that the resulting <NUM>,<NUM>-dihydronicotinamide riboside (NRH) can be isolated and purified with a high yield by using column chromatography. Examples below provide a detailed exemplary purification method by using column chromatography (e.g., with a mixture of basic alumina and silica as the adsorbent).

In an embodiment, a chromatography method may be used to isolate the resulting NRH from the mixture. For example, column chromatography can be used to separate NRH from β-NRCI or other impurity substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions.

In an embodiment, the column chromatograph method uses an alcohol as its eluent. In an embodiment, the eluent comprises methanol. In an embodiment, the only eluent is methanol. Nevertheless, other solvents such as a mixture of methanol and ethanol can optionally be used as eluent additionally or alternatively.

In an embodiment, the adsorbent of the column chromatograph comprises at least basic alumina and silica. In an embodiment, basic alumina and silica are the only adsorbents of the column chromatograph.

The basic alumina and the silica may have a weight ratio of between about <NUM>:<NUM> and about <NUM>:<NUM>, between about <NUM>:<NUM> and about <NUM>: <NUM>, between about <NUM>:<NUM> and about <NUM>:<NUM>, between about <NUM>:<NUM> and about <NUM>:<NUM>, between about <NUM>:<NUM> and about <NUM>:<NUM>, between about <NUM>:<NUM> and about <NUM>:<NUM>, between about <NUM>:<NUM> and about <NUM>:<NUM>, between about <NUM>:<NUM> and about <NUM>:<NUM>, or between about <NUM>:<NUM> and about <NUM>:<NUM>, preferably about <NUM>:<NUM>.

In an embodiment, a column chromatograph purification method may use basic alumina with silica, for example with the weight ratio of about <NUM>:<NUM>, to isolate NRH with a purity at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%, preferably at least <NUM>%.

In an embodiment, the column chromatograph purification method by using basic alumina with silica may remove at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM> of the unreacted NRCl in the mixture. In a preferred embodiment, the column chromatograph purification method by using basic alumina with silica may completely purify the NRH product from the unreacted NRCl in the mixture.

In an embodiment, as demonstrated in Examples, applicant's results show that column chromatography on either silica or basic alumina alone was not effective in separating NR from NRH. Thus, a mixture of basic alumina and silica may be the necessary adsorbent of the column chromatograph.

Basic alumina, as a polar surface with many hydroxyl groups on it, can presumably physically separate NR from NRH to a moderate degree (by difference of their polarities). Moreover, because basic alumina has negative charges on its surface and NR has a positive charge, basic alumina could act as a cationic exchange resin to immobilize NR on the surface of the basic alumina.

One may believe this is the main reason why alumina is a better stationary phase compared to silica for the purification of the NRH. However, Applicant discovered that during the purification of NRH in the column chromatography, basic alumina would clog and result in a very slow flow rate that led to the degradation of NRH.

In an embodiment, the total yield of NRH for the present method may be at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%, preferably at least <NUM>%.

In an embodiment, the present method of producing NRH in a liquid solution produces a significantly higher yield than that of a solid-state method. For example, the present method of producing NRH in a liquid solution produces a yield at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% higher than that of a solid-state method.

Examples below provide a detailed exemplary solid-state method of producing NRH with a yield between about <NUM>% and about <NUM>%.

As shown in the Examples, the exemplary solution-based methods of producing and purifying NRH, according to certain embodiments of the present disclosure, demonstrate a yield in the range of about <NUM>% to about <NUM>%.

Thus, the present method of producing NRH in a liquid solution may produce a yield at least <NUM>% higher than that of the exemplary solid-state method.

In another aspect, the present disclosure relates to stabilities of the NRH as produced from methods as discussed herein.

In an embodiment, a certain percentage of the NRH as produced would hydrolyze to form <NUM>,<NUM>-dihydronicotinamide (DHNA). As shown in the Scheme of <FIG>, <FIG>,<NUM>-dihydronicotinamide (DHNA) was likely formed by hydrolysis of the NRH during the course of the reaction (e.g., pH = <NUM>).

As shown in <FIG>, when pH=<NUM>, there were only trace peaks, corresponding to NA in the <NUM>HNMR of purified NRH. This signifies that only a trace amount of NR hydrolyzes during the course of the reaction to form NA (See Scheme <NUM>).

In an embodiment, a pH value of the liquid solution of the method of producing NRH may be maneuvered to control a reaction rate of the method and hydrolysis of both the starting material of NRCl and the product of NRH.

For example, by increasing the pH from <NUM> to <NUM>, the reaction rate obviously decreases and, subsequently, there is an increased possibility of side reactions, such as hydrolysis of both NR and NRH.

In an embodiment, a pH value about <NUM> may be preferred for increasing the reaction rate of the reduction, while decreasing possibility of side reactions, such as hydrolysis of both NR and NRH.

In an embodiment, the pH of the reaction for producing NRH may be necessarily adjusted to minimize the formation of NA and DHNA to decreasing the reaction time. For example, the retardation factor (Rf) values of these by-products of NA and DHNA are close to the Rf values of NRH. Therefore, NA and DHNA cannot be separated from the NRH product by common column chromatography (see <FIG>). In a preferred embodiment, the pH of the reaction for producing NRH may be adjusted to about <NUM>.

In an embodiment, the NRH is more thermally stable than the NRCl under a nitrogen atmosphere. <FIG> showed that while both NRH and NR show their greatest weight loss at around <NUM> and <NUM>, respectively, NRH shows only around <NUM>% weight loss at this temperature, while NRCl shows around <NUM>% weight loss at this temperature. NRH shows another distinctive weight loss with a peak at around <NUM> which contributes to <NUM>% weight loss.

In an embodiment, NRH may be susceptible to oxidation in the presence of air and NRH may be more stable under a N<NUM> atmosphere than that in the presence of air, as demonstrated in <FIG>. Further, pure NRH (as a powder) may be stable in a refrigerator, in a sealed tube, for a few months without any degradation.

In an embodiment, as demonstrated in <FIG> and <FIG>, the NRH product may be completely degraded in less than one day at pH <NUM>, while the NRH products prepared in the ammonium acetate buffer at pH <NUM> may show a linear decrease of NRH concentration from around <NUM>% in day one to around <NUM>% in day <NUM>.

Further, the NRH products prepared in the carbonate buffer at pH <NUM> may be air stable both in air (e.g., around <NUM>% degradation measured after <NUM> days) and under N<NUM> (e.g., around <NUM>% degradation measured after <NUM> days).

In an embodiment, the NR in an aqueous solution may be very stable with no detectable degradation after <NUM> days, while NRH in an aqueous solution stored in air may show <NUM>% and <NUM>% degradation after <NUM> and <NUM> days, respectively.

In an embodiment, NRH in an aqueous solution stored under a N<NUM> blanket may be fairly stable with no detectable degradation after <NUM> days of storage.

The following non-limiting example presents scientific data developing and supporting the concept of methods and processes for producing <NUM>,<NUM>-dihydronicotinamide riboside from nicotinamide riboside chloride and related purification and stability studies.

In the present work, we describe an efficient method for synthesis and purification of <NUM>,<NUM>-dihydronicotinamide riboside (NRH) from commercially available nicotinamide riboside chloride (NRCl) and in the presence of sodium dithionite as a reducing agent. The method is potentially scalable. NRH is industrially relevant as the most effective, synthetic NAD+ precursor.

We demonstrated that solid phase synthesis cannot be used for the reduction of NRCl to NRH in high yield, whereas a reduction reaction in water at room temperature under anaerobic conditions is shown to be very effective, reaching a <NUM>% isolation yield. For the first time, by using common column chromatography, we were able to highly purify this sensitive bio-compound with good yield. A series of identifications and analyses including HPLC, NMR, LC-MS, FTIR and UV-Vis spectroscopy, were performed on the purified sample, confirming the structure of NRH as well as its purity to be <NUM>%. Thermal analysis of NRH showed higher thermal stability compared to NRCl, and with two major weight losses, one at <NUM> and another at <NUM>. We also investigated the long term stability effects of temperature, pH, light, and oxygen (as air) on the NRH in aqueous solutions. Our results show that NRH can be oxidized in the presence of oxygen, and it hydrolyzed quickly in acidic conditions. We also found that the degradation rate is lower under N<NUM> atmosphere, at lower temperatures, and in basic pHs.

NR chloride (beta form) was a gift from ChromaDex Company. Sodium dithionite was purchased from VWR, sodium hydrogen carbonate was purchased from Aldrich, silica gel (P60, <NUM>-<NUM>, <NUM>Å) was purchased from SiliCycle, and basic alumina (<NUM>-<NUM>, <NUM>Å, pH <NUM>) was purchased from Acros. Methanol (<NUM>%, Certified ACS, Fisher), acetone (<NUM>% Certified ACS, Fisher), sodium hydroxide (certified ACS, Fisher Chemical), Hexanes (≥<NUM>%, GR ACS), ethyl acetate (EtOAc, ><NUM>%, certified ACS) and Silica Gel <NUM> F254 Coated Aluminum-Backed TLC Sheets were purchased from EMD Millipore (Billerica, MA, USA). Deuterated water and dimethyl sulfoxide (DMSO-d6, D, <NUM>%) were purchased from Cambridge Isotope Laboratories, Inc.

Characterization. A <NUM> NMR (Bruker INOVA) spectrometer was used to prepare the <NUM>H and <NUM>C-NMR spectra in deuterated water. Fourier transform infrared spectra (FTIR) were recorded on a Shimadzu IRAffinity-<NUM> spectrophotometer by collecting <NUM> scans with a resolution of <NUM>-<NUM>. UV-vis spectra of the NR and NRH solutions were recorded on a Shimadzu UV-<NUM> spectrophotometer. Thermogravimetric analysis (TGA) thermograms were prepared in the range of <NUM>-<NUM> at a temperature rate of <NUM> min-<NUM> under N<NUM> flow, using a TA Q100 instrument. An Agilent <NUM> LC System equipped with Binary SL Pump & Diode Array Detector and a Shodex RI-<NUM> Refractive Index Detector (single channel) was used to perform the high-performance liquid chromatography (HPLC) measurements. Reversed-phase HPLC was performed on a Discovery C18 Column, <NUM>Å (pore size), <NUM> diameter, <NUM>×<NUM> in dimension. Ammonium acetate (<NUM>) was used as the mobile phase with a flow rate of <NUM> min-<NUM> over <NUM> or <NUM> at <NUM>. All samples were filtrated using a <NUM> Nylon syringe filter with a <NUM> pore size before measurement. For LC-MS analysis, we used LC (Agilent <NUM> series) coupled with a mass spectrometer. Reverse-phase chromatography was used with a Phenomenex Luna Omega (Phenomenex) LC column with the following specifications: <NUM> × <NUM>, <NUM>, polar C18, <NUM>Å pore size with a flow rate of <NUM> min-<NUM>. LC eluents include ammonium acetate <NUM> (solution A) and acetonitrile (solution B) using gradient elution (solution A:B composition change with time: <NUM>: <NUM>:<NUM>, <NUM>: <NUM>:<NUM>, <NUM>: <NUM>:<NUM>, <NUM>: <NUM>:<NUM>, and <NUM> <NUM>:<NUM>). The mass spectrometer (Finnigan LTQ mass spectrometer) was equipped with an electrospray interface (ESI) set in positive electrospray ionization mode for analyzing the NRH. The optimized parameters were a sheath gas flow rate at <NUM> arbitrary unit, spray voltage set at <NUM> kV, capillary temperature at <NUM>, capillary voltage at <NUM> V, and tube lens set at <NUM> V.

<NUM> of NRCI (<NUM> mmol) and <NUM> of NaHCO<NUM> solution (<NUM>) were added to a round bottom flask with a magnetic stir bar. This system was placed in an ice bath, purged of oxygen, and kept under nitrogen gas. Then, <NUM> of sodium dithionite (<NUM> mmol) was gradually added to the reaction mixture. After adding Na<NUM>S<NUM>O<NUM>, the flask was taken out of ice bath, and subsequently the reaction was carried out at room temperature for three extra hours. The reaction mixture was freeze-dried to obtain a yellow solid. Finally, the residue was purified by column chromatography using a mixture of basic alumina and silica with the weight ratio of <NUM>:<NUM> respectively, using methanol as eluent. The extra methanol was removed by rotary evaporator at room temperature to obtain a pale yellow, sticky solid that was next converted to a yellow powder (precipitate) by adding ethyl acetate. Finally, the isolated product was washed with n-hexane and dried under reduced pressure at room temperature to obtain pure NRH in <NUM>% yield (<NUM>). <NUM>H NMR (<NUM>, D<NUM>O), δ ppm: <NUM> (s, <NUM>), <NUM> (dd, J<NUM> = <NUM>, J<NUM>= <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (t, J= <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (dd, J<NUM>= <NUM>, J<NUM>= <NUM>, <NUM>), <NUM> (dd, J<NUM>= <NUM>, J<NUM>= <NUM>, <NUM>), <NUM> (s, <NUM>) (<FIG>). <NUM>C NMR (<NUM>, D<NUM>O), δ ppm: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> (<FIG>). In addition, <NUM>HNMR and <NUM>C NMR spectra of purified NRH in CD<NUM>OD visuals are in the supporting information section (<FIG>) where MS: found m/z = <NUM> (M+<NUM>). Calculated for C<NUM>H<NUM>N<NUM>O<NUM> (M+<NUM>): <NUM> (<FIG> and <FIG>). UV (λmax in H<NUM>O): <NUM> (<FIG>).

In the first step, to decrease the hydrolysis of NRH during the course of the reaction, we set up the reduction reaction in a solvent-free environment. We used SiO<NUM> as a solid support to increase the surface on which the reaction occurs. In this procedure, SiO<NUM> (<NUM>), NRCl (<NUM>, <NUM> mmol), Na<NUM>S<NUM>O<NUM> (<NUM>, <NUM> mmol), and NaHCO<NUM> (<NUM>) were placed in a mortar and ground for <NUM> minutes to obtain a homogeneous powder. Then, <NUM> of DI water was dropwise added to the reaction mixture and the reaction was ground for <NUM> minutes at room temperature (Table <NUM>, entry <NUM>). It should be mentioned that without using the solid support the reaction mixture becomes sticky and is not easily ground. After grinding, the products were extracted by MeOH and the progress of the reaction was followed by TLC (thin layer chromatography). The results showed a trace amount of NRH to be the one product that we could not isolate and purify for further identification. We hypothesized that, in the absence of enough water, the sulfinate intermediate cannot be protonated to produce sulfinic acid intermediate and then NRH as the product (Scheme <NUM>). To test this hypothesis, we repeated this procedure with an alternate final step. At the end, we transferred the reaction mixture to a round bottom flask with a magnetic stir bar, added <NUM> of NaHCO<NUM> solution (<NUM>), and let the reaction continue on for <NUM> at room temperature. Then, the reaction mixture was freeze-dried and the residue was purified by column chromatography in a mixture of basic alumina and silica, using methanol as the eluent. The isolated NRH was obtained with <NUM>% yield (Table <NUM>, entry <NUM>). By changing the solid support (Al<NUM>O<NUM> instead of SiO<NUM>), no remarkable improvement was observed in the yield of the product (Table <NUM>, entries <NUM>, <NUM>).

The ability to work under ambient conditions is an advantage of this procedure, but low yield of the product is a serious drawback. So, in search of a means for producing a high yield, we decided to follow the reaction in aqueous solution under anaerobic conditions (Table <NUM>, entries <NUM>-<NUM>).

In the present study, we introduce a direct procedure for the scalable synthesis of NRH by using commercially available β-NRCl. The reaction was carried out in an aqueous solution of NaHCO<NUM> and Na<NUM>S<NUM>O<NUM> under a nitrogen atmosphere (Scheme <NUM>). We developed a fast, column chromatography method for the purification of the NRH from the reaction mixture that results in <NUM>% purity. Since one of the main applications of NRH is in supplemental beverages, we also investigated the effect of temperature, light, pH, and oxygen of the stability of NRH in aqueous solutions and compared these results to the stability of the NR in similar conditions.

One of the most significant parameters, and one which directly affects the NRH synthesis reaction and the NRH product, is the pH. Adjusting and maintaining a constant pH is important during the course of the reaction. This issue can be understood better by reviewing the mechanism of the reaction (Scheme <NUM>). An aqueous solution of Na<NUM>S<NUM>O<NUM> is unstable under aerobic conditions at pH=<NUM>. Therefore, this reaction must be carried out under anaerobic and alkaline conditions. At first, the reaction between NR and S<NUM>O<NUM><NUM>- leads to the formation of a sulfinate intermediate, which is stable at basic conditions. By protonating this compound, its sulfinic acid derivative is formed. This sulfinic acid intermediate is unstable at ambient conditions and converts to NRH via SO<NUM> leaving. Therefore, it is important to find and establish a pH that will not only stabilize Na<NUM>S<NUM>O<NUM> but will also protonate the sulfonate intermediate to produce the NRH. Moreover, because NRH has an N-glycoside bond in its structure, it is susceptible to hydrolysis. Consequently, it is necessary to adjust the pH and maintain it precisely during the whole course of the reaction so that NRH is not hydrolyzed.

The minimum pH to stabilize the aqueous solution of Na<NUM>S<NUM>O<NUM>, without decreasing its activity as a reducing agent, is between <NUM>-<NUM>. Initially, we set up the reaction at pH <NUM> by employing a solution of NaHCO<NUM> (<NUM>). Next, NRCl was dissolved in this solution and Na<NUM>S<NUM>O<NUM> was gradually added to the reaction mixture, under a nitrogen atmosphere at <NUM>. By adding Na<NUM>S<NUM>O<NUM> to the solution, and throughout its oxidation, the pH decreased. However, the sodium bicarbonate solution with the concentration of <NUM> was enough to keep the pH constant during the course of the reaction. After adding Na<NUM>S<NUM>O<NUM>, the reaction was carried out at room temperature for <NUM> hours. Then, the mixture was freeze-dried to obtain a yellow solid. Finally, the residue was purified by short column chromatography with a mixture of basic alumina and silica (methanol as eluent) to obtain the pure NRH product in <NUM>% yield (Table <NUM>, entry <NUM>). The optimized molar ratio between NRCl and Na<NUM>S<NUM>O<NUM> was <NUM> to <NUM> and an increase in the amount of sodium dithionite did not improve the product yield (Table <NUM>, entry <NUM>). After purification of the crude product, we conducted thin layer chromatography (TLC) with the purified NRH using methanol as a solvent and compared with the pristine NRCl and nicotinamide (NA) (FIG. All compounds were active at a wavelength of <NUM>. However, by switching the wavelength to <NUM>, we found that only NRH fluoresced. It has been reported that NRH is strongly fluorescent around <NUM>. Because the Rf values of NRH and NA were almost the same, the fluorescence property of NRH helped to identify the exact position of this compound during TLC. As shown in FIG. 1a, the purified NRH shows high purity on the TLC plate.

The FT-IR spectrum of the synthesized and purified NRH was studied and compared to the spectrum of the pristine NRCl (<FIG>). In the FT-IR spectrum of NRH, the existence of a specific peak at <NUM>-<NUM> can be attributed to the stretching vibration of C=C band, and confirms the reduction of the NR pyridinium ring. Two peaks at <NUM> and <NUM>-<NUM> refer to asymmetric and symmetric stretching bands of NH<NUM> group in the structure of NRH which implies that the amide group is intact during the course of the reaction. A sharp peak at <NUM>-<NUM> indicates the stretching C=O band of the amide group. Two peaks at <NUM> and <NUM>-<NUM> confirm the stretching vibration of aliphatic C-H in both the ribose and the dihydronicotinamide rings of the NRH structure. The presence of hydroxyl groups in the structure of NRH is confirmed with a broad peak that appears in the range of <NUM> to <NUM>-<NUM>.

In order to further ensure the successful synthesis of NRH and its purity, we also took <NUM>HNMR and <NUM>CNMR spectra of the purified NRH in deuterated water. The existence of eleven protons that are not exchangeable with D<NUM>O completely confirms the structure of synthesized NRH (<FIG>). The chemical shift of each proton and the corresponding coupling constants are in agreement with the ones previously reported in the scientific literature. The <NUM>CNMR and more details are given in <FIG>.

To study the efficiency of the column used for the purification of NRH, we compared the <NUM>HNMR of purified NRH with the <NUM>HNMR of pure NRCl and NA (<FIG>). The major impurity in the reaction mixture was unreacted NR and, after purification, we observed no specific NR peak in the <NUM>HNMR spectrum of purified NRH. By comparing the NRH spectrum with the NA spectrum, we found that there were only trace peaks, thus indicating NA in the <NUM>HNMR of purified NRH. This signifies that only a trace amount of NR hydrolyzes during the course of the reaction to form NA. We also used reverse phase chromatography HPLC to further confirm the purity of the NRH sample (<FIG>). The HPLC findings agreed with <NUM>HNMR data and showed both that the purity of NRH was <NUM>% and the impurity of NA was <NUM>%. Interestingly, no NR was detected with HPLC, although NR was present with <NUM>HNMR. This means that column chromatography on a mixture of basic alumina and silica is very efficient and able to completely purify the NRH product from the remaining NRCl.

Next, we tried to purify crude NRH, using column chromatography on silica and using methanol as the eluent. The results showed that column chromatography on silica alone was not effective in separating NR from NRH. Basic alumina, as a polar surface with many hydroxyl groups on it, can, to a moderate degree, physically separate NR from NRH (by difference of their polarities). Moreover, because basic alumina has negative charges on its surface and NR has a positive charge, it can act as a cationic exchange resin to immobilize NR on its surface. We believe this is the main reason why alumina is a better stationary phase compared to silica for the purification of the NRH. However, we discovered that during the purification of NRH in the column chromatography, basic alumina would clog and result in a very slow flow rate that led to the degradation of NRH. We took steps to easily solved this problem, by mixing basic alumina with silica with the weight ratio of <NUM>:<NUM> respectively. Thus, for the first time, we could quantitatively purify NRH in a preparative mode (without using HPLC) using a common column chromatography method on a mixture of basic alumina and silica, while using methanol as the eluent.

Next, we set up the reduction reaction of NRCl at pH <NUM> by employing a solution of NaHCO<NUM> and Na<NUM>CO<NUM> (Table <NUM>, entry <NUM>). The NRCl was dissolved in this solution, then Na<NUM>S<NUM>O<NUM> was gradually added to the reaction mixture under a nitrogen atmosphere at <NUM>. After the addition of Na<NUM>S<NUM>O<NUM>, the reaction was carried out at room temperature for <NUM> hours. Upon completion of the reaction (followed by TLC), the mixture was freeze-dried to obtain a yellow solid. Finally, the residue was purified by short column chromatography on basic alumina using methanol as the eluent. As a qualitative test, we conducted TLC from this purified NRH and compared with the NR and NA using methanol as a solvent (<FIG>). The results showed that the NRH obtained was not pure after column chromatography. At this point, we observed that the new impurity was positioned only slightly above the NRH on the TLC. Our observation helped to explain why this contamination cannot be separated from the NRH by common column chromatography. Interestingly, the fluorescence property of this new impurity is similar to NRH, causing both of them to fluoresce at <NUM> on TLC. By studying the literature, we found that it was likely <NUM>,<NUM>-dihydronicotinamide (DHNA) formed by hydrolysis of the NRH during the course of the reaction.

For further evidence, we took <NUM>HNMR of this sample in D<NUM>O (<FIG>). The obtained results indicated that there was no residual NRCl in this sample. However, the presence of four peaks with low intensity between <NUM>-<NUM> ppm confirmed the existence of a trace amount of NA in the purified NRH. As shown in <FIG>, four main peaks at <NUM>, <NUM>, <NUM> and <NUM> ppm strongly confirm a <NUM>,<NUM>-dihydronicotinamide ring in the structure of NRH. At the right side of each of the main peaks, there is a peak with lower intensity and a shape is similar to the corresponding main peak. The chemical shifts of these peaks are <NUM>, <NUM> and <NUM> ppm, respectively. The integral of each peak is around <NUM> and the integral of each main peak is <NUM>.

These results demonstrate that around <NUM>% of DHNA exist in the purified NRH. In other words, this means that by increasing the pH, the reaction rate decreases and the NRH is hydrolyzed under the reaction conditions within <NUM> hours. It is clear that by hydrolyzing NRH, D-ribose and DHNA are formed simultaneously. As shown in <FIG>,the corresponding impurity peaks at <NUM> and <NUM> ppm are attributed to α and β anomeric protons of D-ribose, respectively. The other impurity peaks of ribose appear between <NUM> and <NUM> ppm.

By increasing the pH from <NUM> to <NUM>, the reaction rate obviously decreases and, subsequently, there is an increased possibility of side reactions, such as hydrolysis of both NR and NRH.

These observations are in accordance with the reaction mechanism of the reduction of NR to NRH, as seen in Scheme <NUM>. In this mechanism, the reaction involves a sulfinate intermediate which is stable in alkaline conditions and, consequently, it is not easily protonated to produce the NRH. In the process of NRH synthesis, thiosulfate and bisulfite anions are formed as the products of hydrolysis and oxidation of dithionite. These anions, with high nucleophilicity, may attack the carbon <NUM>' in the structure of NRH and substitute instead of DHNA. This would indirectly increase the rate of NRH hydrolysis. By increasing the pH, the possibility of NRH hydrolysis decreases, while simultaneously the existence of some anions, which act as the nucleophile, can increase the NRH hydrolysis. The hydrolysis of NRH by these anions is more severe and increases the reaction time of NRH synthesis. Therefore, as the most important parameter, the pH of the reaction must be adjusted to minimize the formation of NA and DHNA to decreasing the reaction time. This is a very significant factor because the Rf values of these by-products are close to the Rf values of NRH; therefore, they cannot be separated from the NRH product by common column chromatography (<FIG>). In the present work, we found that the optimized pH for the synthesis of NRH from NR was <NUM>; at this pH (<NUM>) we did not observe any DHNA and the amount of NA was negligible.

Thermal stability is very important for drugs and supplements that have the potential for use in food due to the high temperatures that are often needed for industrial processing. In order to study the thermal stability of the NRH, we performed TGA for the pure NRH from <NUM> to <NUM> and the results were compared to the pristine NRCl. <FIG> shows the TGA thermograms in the range of <NUM> to <NUM> for NRH and NRCl. As can be seen in <FIG>, the NRH is clearly more thermally stable than the NRCl. While both NRH and NR show their greatest weight loss at around <NUM> and <NUM>, respectively, NRH shows only around <NUM>% weight loss at this temperature, while NRCl shows around <NUM>% weight loss at this temperature. NRH shows another distinctive weight loss with a peak at around <NUM> which contributes to <NUM>% weight loss. These results confirm that the NRH is more thermally stable compared to the NRCl under a nitrogen atmosphere. It may be due to the existence of chloride ions in the structure of NRCl, that can leave in the form of HCl when the temperature increases.

A stock solution of the freshly synthesized and purified NRH (<NUM>,<NUM> ppm) was prepared in deoxygenated DI water. This stock solution was used for preparation of <NUM> ppm NRH solutions for stability measurements. The effects of light, pH (buffer), temperature, and oxygen (as air) were investigated during a <NUM>-day period of storage. The remaining NRH concentration was measured using HPLC. This procedure was also used for an NR stability measurement and the results were compared to the NRH samples at the same conditions.

NRH is an unstable, sensitive molecule due to its N-glycosidic bond and can undergo degradation, hydrolysis, and oxidation during exposure to high temperatures, nucleophiles, and oxygen. Some of the major products of these degradation reactions for NRH are shown in Scheme <NUM>. Studying the NRH degradation under different conditional variables is important for developing new supplements or potential beverage products. Here, we study the effect of light, temperature, oxygen, and pH on the degradation rate of the freshly synthesized and purified NRH in an aqueous solution during a period of <NUM> days.

The oxidative sensitivity of the NRH, as a dihydronicotinamide derivative, is well known. As the reduced form of the NR, NRH has a high oxidation potential. In order to study the effect of air, as well as ambient light, on the stability of NRH in an aqueous solution, we prepared solutions of freshly purified NRH (by column chromatography) in DI water in air and under a N<NUM> blanket as well as in darkness and ambient light. We monitored the NRH concentration over the course of <NUM> days by HPLC. <FIG> shows the NRH recovery (%) during the <NUM> days of storage based on the HPLC results both for samples in the dark/light and in air/under N<NUM>.

From the result shown in <FIG>, it is obvious that, while the effect of light is negligible on the stability of NRH compared to the samples that have been kept at dark, oxygen (as air) has a dramatic effect on the stability of the NRH. Samples that have been kept at <NUM> in DI water in darkness show faster degradation reaching <NUM>% in <NUM> days under air. By contrast, there is around <NUM>% degradation observed for the sample at the same conditions that have been kept under a N<NUM> blanket. These findings demonstrate that NRH is susceptible to oxidation in the presence of air.

Temperature can have a profound effect on the stability of molecules with an N-glycosidic bond. The NR molecule degradation is temperature dependent following a first order kinetic rule. This is most likely due to the susceptibility of the N-glycosidic bond dissociation because nicotinamide (NA) is a good leaving group. However, the leaving group for NRH is dihydronicotinamide (DHNA) and NRH is not ionic (nitrogen in the ring does not have a positive charge). Therefore, we might anticipate that the NRH should be more stable compared to the NR molecule in terms of spontaneous dissociation of the N-glycosidic bond due to the temperature. However, the oxidative degradation of the NRH also plays an important role for its stability as we showed in previous section.

We study the stability of the NRH at two different temperatures both in air and under a N<NUM> blanket. <FIG> shows the results of the recovery of the NRH in aqueous solutions kept at <NUM> and <NUM> during <NUM> days of storage. <FIG> shows the corresponding HPLC chromatograms of some of the samples in <NUM> days of storage. These results confirm the faster degradation of the NRH at <NUM> compared to <NUM>, and also show the accelerated effect of air on the oxidative degradation of NRH at <NUM>. Samples that have been kept at <NUM> under a N<NUM> blanket do not show any detectable degradation after <NUM> days of storage, while samples at <NUM> in air display around <NUM>% degradation in <NUM> days. For the samples at <NUM>, the degradation is around <NUM>% under a N<NUM> atmosphere and around <NUM>% in air after <NUM> days of storage. Pure NRH (as a powder) was stable in a refrigerator, in a sealed tube, for a few months without any degradation.

Dissociative degradation of the NR molecule is pH independent. The NRH molecule showed relatively good stability in a basic medium (as we also emphasize in the results and discussion) and rapid degradation under acidic conditions during a <NUM> monitoring period. We investigated the effect of pH <NUM>, <NUM>, and <NUM>, made using citrate buffer, ammonium acetate buffer, and carbonate buffer, respectively, on the stability of the NRH in the aqueous solutions kept at <NUM> both in air and under N<NUM>, as well as in darkness, for a period of <NUM> days (<FIG> and <FIG>). Our results from <FIG> confirm the complete degradation of NRH in less than one day at pH <NUM>, while samples prepared in the ammonium acetate buffer at pH <NUM> showed a linear decrease of NRH concentration from around <NUM>% in day one to around <NUM>% in day <NUM>. However, samples prepared in the carbonate buffer at pH <NUM> showed fair stability both in air (around <NUM>% degradation measured after <NUM> days) and under N<NUM> (around <NUM>% degradation measured after <NUM> days), which agrees with the short term data from others.

Comparing <FIG> (<NUM>, pH <NUM> air and N<NUM>) with <FIG> (<NUM>, DI water), it is clear that the rapid degradation of the NRH at pH <NUM> is due to the ammonium acetate salt used to make the buffer. The presence of some anions, such as chloride and acetate, accelerate the oxidation of NRH to NR because the existence of an anion as a counter ion of NR is necessary in this oxidation process. Moreover, the acetate ion may act as a nucleophile and accelerate the NRH hydrolysis. By consuming the acetate anion, the ratio of ammonium to acetate increases and, by hydrolysis of this extra quantity of ammonium, the solution gradually becomes acidic and the NRH hydrolysis rate increases. Therefore, it can be stated that there is a synergistic effect for the degradation of the NRH while at pH <NUM> using a buffer produced with ammonium acetate.

The kinetic graphs for the NRH degradation rate are based on the HPLC data collected during the <NUM> days of storage for samples with rather fast degradation rates. <FIG> shows the kinetic graphs and the first order degradation rates for NRH samples stored at pH <NUM>, at <NUM>, under N<NUM> and air (<FIG>), samples stored at pH <NUM>, at <NUM>, under N<NUM> and air (<FIG>), and for NRH samples that have been kept in DI water, at <NUM>, under N<NUM> and air (<FIG>). The degradation rate for samples that have been kept at <NUM> in DI water under air is <NUM> × <NUM>-<NUM> s-<NUM>, which corresponds to a half-life of <NUM> days. By comparison, for the NRH samples that have been kept at <NUM> in DI water under N<NUM> the degradation rate is <NUM> × <NUM>-<NUM>s-<NUM>, which correspond to a half-life of <NUM> days (<FIG>).

NRH is the reduced form of NR and has a much higher biological activity. However, it is essential to compare the stability of NR to NRH. If the NRH is not as stable as NR, the latter would be useful for more applications. Here, we compare the stability of both NR and NRH in aqueous solutions. Since NR stability in aqueous solutions is pH independent, we studied the NR and NRH stability in DI water at <NUM> and <NUM> for a period of <NUM> days. Figure 6d compares the results of this study for <NUM> and <NUM> days.

It is clear from the results of <FIG> that, at <NUM>, the NR in an aqueous solution is very stable with no detectable degradation after <NUM> days. However, NRH in an aqueous solution stored in air shows <NUM>% and <NUM>% degradation after <NUM> and <NUM> days, respectively, while NRH in an aqueous solution stored under a N<NUM> blanket is fairly stable with no detectable degradation after <NUM> days of storage. On the other hand, the NR solution that has been kept at <NUM> (representing the ambient temperature) shows <NUM>% and <NUM>% degradation after <NUM> and <NUM> days of storage, while this degradation is <NUM>% and <NUM>% for NRH solutions that have been kept under air after <NUM> and <NUM> days and <NUM>% and <NUM>% for and NRH solutions that have been kept under N<NUM> after <NUM> and <NUM> days, respectively. This clearly shows that the thermal degradation is more severe for NR than NRH in aqueous solutions, and storage of NRH away from air improves its stability.

Claim 1:
A method of producing <NUM>,<NUM>-dihydronicotinamide riboside (NRH), comprising:
providing nicotinamide riboside chloride (NRCl) in a liquid solution;
adding a metal dithionite into the liquid solution under a first temperature; and
reacting the metal dithionite with the nicotinamide riboside chloride (NRCl) in the liquid solution under a second temperature to form a mixture, wherein at least a portion of the mixture comprises the <NUM>,<NUM>-dihydronicotinamide riboside (NRH).