A method for preparing a beta-nicotinamide mononucleotide

Abstract

The present application relates to the technical field of organic synthesis, and particularly relates to a preparation method of beta-nicotinamide mononucleotide. The present application provides a preparation method of beta-nicotinamide mononucleotide, comprising the following steps: mixing a salt corresponding to nicotinamide riboside, phosphorus oxychloride and a polar aprotic organic solvent, performing a phosphorylation reaction, and obtaining beta-nicotinamide mononucleotide. The present application takes nicotinamide riboside as a raw material, takes phosphorus oxychloride as a phosphorylation reagent, and is prepared through a phosphorylation reaction. Compared with a conventional method, the reaction has less by-products and is simple to operate. The subsequent high-purity and high-yield end product can be obtained through simple post-treatment. Meanwhile, the present application is also beneficial to industrial production.

Description

[0001] This application claims priority to Chinese Patent Application No. CN202210913751.7, filed on August 1, 2022, entitled "A Method for Preparing β-Nicotinamide Mononucleotide", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of organic synthesis technology, and in particular to a method for preparing β-nicotinamide mononucleotide. Background Technology

[0003] Nicotinamide adenine dinucleotide (NAD) + NAD+ is an important coenzyme for redox reactions and is central to energy metabolism. + It is also a non-redox NAD + NAD+ dependent enzymes, including important cofactors of deacetylases and poly-ADP-ribonucleases. Besides energy metabolism, NAD+... + It also serves as a cofactor substrate for hundreds of enzymes, thus playing an important role in cellular physiological processes and cellular self-regulation, some of which are still under investigation (Sahar S, Nin V, Barbosa MT, et al. Aging, 2011, 3:794-802). With increasing age, NAD+ levels decrease in various tissues within the human body. + Decreased levels occur in organs including skeletal muscle, liver, adipose tissue, brain, pancreas, spleen, heart, kidneys, and lungs. Many are associated with NAD+. + Enzymes involved in the degradation of biosynthesis, such as poly(ADP-ribose) polymerase, CD38, and Sirtuin, accelerate NAD+ degradation during aging. + Consumption (Camacho Pereira, J. et al. Cell Metab. 23, 1127-1139 (2016)). Additionally, NAD during aging... + The decline in NAD+ is associated with the occurrence and development of age-related diseases, including atherosclerosis, arthritis, hypertension, cognitive decline, diabetes, and cancer (Verdin, E. Science 350, 1208-1213 (2015)). Restoring NAD+ levels in the body is crucial. + Levels of NAD can improve disease conditions and even extend lifespan, therefore NAD is currently being studied. + It has become a star target in the field of anti-aging (Fang, Evandro F, et al. Trends in molecular medicine vol.23,10(2017):899-916).

[0004] NAD in the human body +Besides participating in the tricarboxylic acid cycle and being degraded as a substrate for enzymes, NAD+ is also consumed in other catabolic pathways such as glycolysis (Kulilovava, Gromykodv, Nikiforov AA. Biochemistry (Mosc), 2018, 83(7):800-812). Therefore, organisms need to rely on multiple synthetic pathways to maintain NAD+ levels in the body. + The level of NAD. + There are four precursors: nicotinic acid, nicotinamide, tryptophan, and nicotinamide ribose. The first pathway is the Preiss-Handler pathway, in which the key enzyme is nicotinamide mononucleotide adenylate transferase (NMNAT), which converts nicotinic acid mononucleotide to nicotinic adenine dinucleotide (NAAD) in the presence of ATP. NAAD is then catalyzed to NAD. + (Rajmanl, Chwalek K, Sinclair DA. Cell Metab, 2018, 27(3): 529-547)(Yoshino J, Baur JA, Imai S. Cell Metab, 2018, 27(3): 513-528). The second pathway is the kynurenine pathway, which synthesizes NAD from tryptophan. + Using indoleamine 2,3-dioxygenase or tryptophan 2,3-dioxygenase as key enzymes, tryptophan is converted to N-formylkynurenine, which is then sequentially converted to L-kynurenine, 5-hydroxy-2-aminobenzoic acid, and 2-amino-3-carboxylic acid mucoconic acid-6-semialdehyde (ACMS), finally becoming quinolinic acid. Another key step in this pathway is the conversion of ACMS, NAD... + The synthesized precursor ACMS spontaneously condenses and rearranges to quinolinate. Another key step in this pathway is the conversion of ACMS to NAD+. + The precursor ACMS spontaneously condenses and rearranges to quinolinate. It is then oxidized to acetyl-CoA via the citric acid cycle. If excess tryptophan exceeds the enzymatic capacity of ACMS decarboxylase, quinolinate is converted to NAMN, which then enters the Preiss-Handler pathway (Rajmanl, Chwalek K, Sinclair DA. Cell Metab, 2018, 27(3): 529-547 (Yoshino J, Baur JA, Imai S. Cell Metab, 2018, 27(3): 513-528). A salvage pathway also exists in mammals, which is responsible for the production and maintenance of intracellular NAD. + It is an important pathway for NAMPT levels. Animal studies have shown that NAMPT acts as a pathway for NAD+. + Key enzymes in the salvage pathway, maintaining intracellular NAD +It plays an important role in the horizontal aspect (Yakuk K, Okabe K, Nakagawa T. AgeingRes Rev, 2018, 47: 1-17). NAD + Under the action of consuming enzymes (SIRT, ADPRT, and PARP), nicotinamide, a byproduct of enzyme activity, is produced. Nicotinamide is first converted to nicotinamide mononucleotide (NMN) by NAMPT, and then synthesized into NAD under the catalysis of NMN. + (Li J, Bonkowski MS, Moniot S, et al. Science, 2017, 355(6331):1312-1317). Finally, there is another salvage pathway using NAMPT as the key enzyme to conjugate the adenosine nucleotide portion of ATP to NMN, followed by specific enzyme catalysis to produce NAD. + .

[0005] Currently, the literature reports three methods for synthesizing NMN: fermentation, enzymatic method, and chemical method. The fermentation method mainly uses nicotinamide and lactose as raw materials, and produces NMN by fermentation in Escherichia coli using nicotinamide phosphoribosyltransferase and 5'-phosphoribose pyrophosphate (PRPP) (Marinescu GC, Popescu RG, Stoian G, et al. β-nicotinamide mononucleotide (NMN) production in Escherichia coli. Sci Rep, 2018, (8): 12278). The enzymatic method mainly uses nicotinamide, ATP, and ribose as substrates and raw materials, and prepares the product under the catalysis of nicotinamide phosphoribosyltransferase, ribophosphate pyrophosphate kinase, and ribokinase. There are also literature reports on the preparation of the product using nicotinamide, pyrophosphate, and AMP as raw materials, under the catalysis of nicotinamide phosphoribosyltransferase and adenine phosphoribosyltransferase (ZL201680003975.7, ZL201611245619.4, CN201810940729.5, CN201611245619.4, CN201680003975.7). The chemical method mainly uses nicotinamide or ethyl nicotinate as raw materials, which undergo a condensation reaction with protected ribose to obtain nicotinamide ribose derivatives. After deprotection, nicotinamide ribose is obtained, and finally, it is prepared by phosphorylation reaction with pyrophosphoryl chloride (CN200680051368.4, CN202010144440.X, CN202010531198.1; Jaemoon Lee, Hywyn Churchil, Woo-Baeg Choi, et al. A chemical synthesis of nicotine inamideadenine dinucleotide (NAD) +), Chem. Commun., 1999, 729-730; Shinji Tanimori, Takeshi Ohta, Mistunori Kirihata, An Efficient Chemical Syntosis of Nicotinamide Riboside (NAR) and Analogues, Bioorg. Med. Chem. Lett. 12 (2002): 1135-1137).

[0006] Phosphorylation is the rate-limiting step in the chemical synthesis of NMN. Currently disclosed phosphorylation reagents are typically phosphorylation reagents, with the feed amount controlled at 2-4 equivalents of nicotinamide ribose. Literature reports that phosphorylation with phosphorylation reagents in excess is often necessary; otherwise, nicotinamide ribose phosphorylation will be incomplete. After the reaction, neutralization with alkali at low temperatures is required (theoretically, 1 equivalent of phosphorylation requires 6 equivalents of alkali for neutralization). The quenching process is significantly exothermic, and phosphorylation reagents exhibit slow quenching in cold water but vigorous reaction in hot water. Therefore, large-scale production could potentially lead to temperature runaway and material spillage. Furthermore, phosphorus oxychloride is highly reactive, and the phosphorylation process is often accompanied by many side reactions (such as chlorination and polyphosphorylation), which increase the difficulty of post-processing purification (WO2021092919, WO2021239850, WO2021239850, WO2021214299, WO202119142, CN201810835636.6, CN201680029859.2, CN201910741699.X). The phosphorylation synthesis steps reported above all suffer from low yields, cumbersome post-processing, difficult purification, and difficulties in large-scale production. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing β-nicotinamide mononucleotide, which is simple to operate and has few side reactions.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a method for preparing β-nicotinamide mononucleotide, comprising the following steps:

[0010] The salt corresponding to nicotinamide ribose, pyrophosphoryl chloride, and a polar aprotic organic solvent were mixed and phosphorylated to obtain β-nicotinamide mononucleotide.

[0011] The salt corresponding to the nicotinamide ribose has the structure shown in Formula 1:

[0012]

[0013] Among them, X- It is a monovalent anion.

[0014] Preferably, the X - The ions are chloride ions, bromide ions, iodide ions, p-toluenesulfonate ions, methanesulfonate ions, trifluoromethanesulfonate ions, benzenesulfonate ions, p-bromobenzenesulfonate ions, benzoate ions, formate ions, or acetate ions.

[0015] Preferably, the polar aprotic organic solvent includes one or more of trimethyl phosphate, triethyl phosphate, tetrahydrofuran, and acetonitrile.

[0016] Preferably, the molar ratio of the salt corresponding to the nicotinamide ribose to pyrophosphoryl chloride is 1:(1-5).

[0017] Preferably, the mass ratio of the polar aprotic organic solvent to the salt corresponding to nicotinamide ribose is (1-20):1.

[0018] Preferably, the phosphorylation reaction is carried out at a temperature of -40 to 10°C for a time of 1 to 20 hours.

[0019] Preferably, after the phosphorylation reaction is completed, the process further includes sequential quenching, precipitation of the aqueous phase, vacuum filtration, resolution, extraction, purification, concentration, and alcohol precipitation.

[0020] Preferably, the organic solvent used for precipitation of the aqueous phase includes one or more of methanol, ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, and methyl tert-butyl ether.

[0021] Preferably, the organic solvent used for extraction includes one or more of ethyl acetate, butyl acetate, dichloromethane, and methyl tert-butyl ether;

[0022] The solvent used for the alcohol precipitation includes one or more of methanol, ethanol, and isopropanol.

[0023] Preferably, the quenching agent used in the quenching is water;

[0024] The concentration is nanofiltration concentration; the purification is performed using ion exchange resin.

[0025] This invention provides a method for preparing β-nicotinamide mononucleotide, comprising the following steps: mixing the salt corresponding to nicotinamide ribose, pyrophosphoryl chloride and a polar aprotic organic solvent, and performing a phosphorylation reaction to obtain β-nicotinamide mononucleotide;

[0026] The salt corresponding to the nicotinamide ribose has the structure shown in Formula 1:

[0027]

[0028] Among them, X - It is a monovalent anion.

[0029] This invention uses nicotinamide ribose as a raw material and pyrophosphoryl chloride as a phosphorylation reagent to prepare the product via a phosphorylation reaction. Compared with conventional methods, this reaction produces fewer byproducts and is simpler to operate. It also allows for the acquisition of a high-purity, high-yield final product through simple post-processing. Furthermore, it is beneficial for industrial production. Attached Figure Description

[0030] Figure 1 The H spectrum of the β-nicotinamide mononucleotide described in Example 1;

[0031] Figure 2 The C-chromatogram of the β-nicotinamide mononucleotide described in Example 1 is shown. Detailed Implementation

[0032] This invention provides a method for preparing β-nicotinamide mononucleotide, comprising the following steps:

[0033] The salt corresponding to nicotinamide ribose, pyrophosphoryl chloride, and a polar aprotic organic solvent were mixed and phosphorylated to obtain β-nicotinamide mononucleotide.

[0034] The salt corresponding to the nicotinamide ribose has the structure shown in Formula 1:

[0035]

[0036] Among them, X - It is a monovalent anion.

[0037] In this invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well known to those skilled in the art.

[0038] In this invention, the preparation route of the β-nicotinamide mononucleotide is shown in Formula 2:

[0039]

[0040] In this invention, the X - Preferably, the ions are chloride ions, bromide ions, iodide ions, p-toluenesulfonate ions, methanesulfonate ions, trifluoromethanesulfonate ions, benzenesulfonate ions, p-bromobenzenesulfonate ions, benzoate ions, formate ions, or acetate ions.

[0041] In this invention, the preferred method for preparing the salt corresponding to nicotinamide ribose is the method described in reference Lee J, Churchil H, Choi WB, et al. Chem. Commun., 1999, 8: 729-730; the reaction process is as shown in Formula 3(X) - As shown in the case of bromide ions:

[0042]

[0043] When X - The ions are chloride ions, iodide ions, p-toluenesulfonate ions, methanesulfonate ions, trifluoromethanesulfonate ions, benzenesulfonate ions, p-bromobenzenesulfonate ions, benzoate ions, formate ions, or acetate ions. The only difference from the process shown in Formula 3 is that in step i, when the Ac in the -OAc group is substituted, hydrobromic acid is replaced with hydrochloric acid, hydroiodic acid, p-toluenesulfonate, methanesulfonate, trifluoromethanesulfonate, benzenesulfonate, p-bromobenzenesulfonate, benzoic acid, formic acid, or acetic acid.

[0044] In this invention, the polar aprotic organic solvent preferably includes one or more of trimethyl phosphate, triethyl phosphate, tetrahydrofuran, and acetonitrile, and more preferably trimethyl phosphate; when the polar aprotic organic solvent includes two or more of the above-mentioned specific substances, this invention does not impose any special limitation on the ratio of the specific substances, and they can be mixed in any ratio.

[0045] In this invention, the mixing is preferably carried out under a protective atmosphere; the protective atmosphere is preferably a nitrogen atmosphere. In this invention, the preferred mixing sequence is to first mix the salt corresponding to nicotinamide ribose and a polar aprotic organic solvent, and then add pyrophosphoryl chloride dropwise at the temperature at which the phosphorylation reaction occurs. In this invention, the temperature of the first mixing is preferably 25°C; this invention does not impose any special limitations on the method of the first mixing, and any method well known to those skilled in the art can be used. This invention does not impose any special limitations on the dropwise addition process of the pyrophosphoryl chloride, and any process well known to those skilled in the art can be used. In this invention, the temperature at which the pyrophosphoryl chloride is added dropwise is preferably -40 to 10°C, more preferably -40 to -10°C.

[0046] In this invention, the molar ratio of the salt corresponding to nicotinamide ribose to pyrophosphoryl chloride is preferably 1:(1-5), more preferably 1:(2-4), and most preferably 1:(2.5-3.5).

[0047] In this invention, the mass ratio of the polar aprotic organic solvent to the salt corresponding to nicotinamide ribose is preferably (1-20):1, more preferably (2-8):1, and most preferably (3-6):1.

[0048] In this invention, the phosphorylation reaction is preferably carried out in a protective atmosphere, preferably a nitrogen atmosphere; the temperature of the phosphorylation reaction is preferably -40 to 10°C, more preferably -30 to 5°C, and most preferably -20 to -10°C; the time is preferably 1 to 20 hours, more preferably 5 to 15 hours, and most preferably 8 to 12 hours. In this invention, the phosphorylation reaction time is preferably calculated from the time after the pyrophosphoryl chloride addition is completed.

[0049] In this invention, the preferred criterion for determining whether the phosphorylation reaction is complete is: the mass percentage of the salt corresponding to the nicotinamide ribose is <5% as monitored by central controlled HPLC area normalization method.

[0050] After the phosphorylation reaction is completed, the present invention preferably includes sequential quenching, precipitation of the aqueous phase, vacuum filtration, resolution, extraction, purification, concentration and alcohol precipitation.

[0051] In this invention, the quenching is preferably performed by adding water dropwise at 0°C. After the addition is completed, the invention also preferably includes stirring. The stirring process is not particularly limited and can be performed using a process well known to those skilled in the art.

[0052] In this invention, the organic solvent used for precipitation of the aqueous phase preferably includes one or more of methanol, ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, and methyl tert-butyl ether. When the organic solvent is two or more of the above-mentioned specific selections, this invention does not impose any special limitation on the ratio of the above-mentioned substances, and they can be mixed in any ratio. In this invention, the precipitation process of the aqueous phase is preferably carried out by adding the obtained reaction solution to the organic solvent at 0°C.

[0053] After the aqueous phase has settled, the present invention preferably includes sequential filtration and solid collection; the filtration is preferably vacuum filtration; the present invention does not have any special limitations on the vacuum filtration process, and any process known to those skilled in the art can be used.

[0054] In this invention, the solvent used for the resolution is preferably water.

[0055] After the resolution is completed, the present invention preferably includes adjusting the pH value of the obtained product system to 5; the pH adjuster used is preferably an aqueous solution of sodium bicarbonate, an aqueous solution of potassium carbonate, or an aqueous solution of sodium hydroxide; the present invention does not have any special limitation on the concentration of the aqueous solution of sodium bicarbonate, an aqueous solution of potassium carbonate, or an aqueous solution of sodium hydroxide, and the concentration known to those skilled in the art can be used to ensure that the obtained product system is within the above-mentioned pH value range.

[0056] In this invention, the organic solvent used for extraction preferably includes one or more of ethyl acetate, butyl acetate, dichloromethane, and methyl tert-butyl ether; the number of extractions is preferably three.

[0057] In this invention, the purification is preferably carried out using an ion exchange resin, more preferably a strong base anion exchange resin, and most preferably a strong base anion exchange resin 717.

[0058] In this invention, the concentration is preferably nanofiltration concentration; this invention does not impose any special limitations on the nanofiltration concentration, and any process well known to those skilled in the art can be used.

[0059] In this invention, the solvent used for the alcohol precipitation preferably includes one or more of methanol, ethanol, and isopropanol. The alcohol precipitation is preferably carried out under stirring conditions. This invention does not impose any special limitation on the stirring speed; any speed well-known to those skilled in the art can be used. The alcohol precipitation is preferably carried out overnight at a temperature of -10 to -20°C to precipitate crystals.

[0060] After the alcohol precipitation is completed, the present invention preferably includes filtration; the present invention does not have any special limitations on the filtration process, and any process known to those skilled in the art can be used.

[0061] The preparation method of β-nicotinamide mononucleotide provided by the present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063] In a nitrogen atmosphere at 25°C, 100g of nicotinamide ribose trifluoromethanesulfonate (X) was... - After mixing 500 mL of trimethyl phosphate (trifluoromethanesulfonate ion) and adjusting the temperature of the mixture to -10 °C, 90 mL of pyrophosphoryl chloride (163.8 g) was added dropwise. After the addition was complete, the reaction was carried out at -10 °C for 8 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped, and 100 mL of water was added dropwise at 0 °C with stirring for 1 h. The reaction solution was then added to 4 L of tetrahydrofuran at 0 °C, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 50 mL of water, and the pH was adjusted to 5 by adding sodium bicarbonate aqueous solution. Ethyl acetate was then added for extraction and separation (50 mL × 3). The resulting aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight with 500 mL of cold anhydrous ethanol (0 °C) under stirring at -10 °C. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 85.5% and a purity of 99.1%.

[0064] MS(ESI, m / z): 335.1 [M+H]+ ;

[0065] 1 H-NMR(400MHz,D2O)δ9.37(s,1H),9.20(d,J=6.1Hz,1H),8.89(d,J=7.9Hz,1H),8.22(t,J=7.0Hz,1H),6.13 (d,J=5.3Hz,1H),4.55-4.54(m,1H),4.49-4.46(m,1H),4.35(m,1H),4.23–4.19(m,1H),4.08–4.04(m,1H)(such as Figure 1 (As shown).

[0066] 13 C-NMR (100MHz, D2O) δ 165.75, 145.96, 142.47, 139.85, 133.90, 128.53, 99.96, 87.41, 77.72, 70.99, 64.16 (e.g.) Figure 2 (As shown).

[0067] Example 2

[0068] In a nitrogen atmosphere at 25°C, 50g of nicotinamide ribose chloride (X) was... - After mixing 300 mL of trimethyl phosphate (chloride ions) and the mixture, the temperature of the mixture was adjusted to -20 °C. 180 mL of pyrophosphoryl chloride (327.6 g) was added dropwise. After the addition was complete, the mixture was reacted at -10 °C for 8 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped. 50 mL of water was added dropwise at 0 °C and stirred for 1 h. The reaction solution was added to 2 L of tetrahydrofuran at 0 °C, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 25 mL of water. After adding sodium bicarbonate aqueous solution to pH = 5, ethyl acetate was added for extraction and separation (50 mL × 3). The resulting aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight with 250 mL of cold anhydrous ethanol under stirring at -20 °C. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 90.4% and a purity of 99.0%.

[0069] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0070] Example 3

[0071] In a nitrogen atmosphere at 25°C, 200g of nicotinamide ribose chloride (X) was... -After mixing 800 mL of trimethyl phosphate (chloride ions) and the mixture, the temperature of the mixture was adjusted to -40℃. 90 mL of pyrophosphoryl chloride (163.8 g) was added dropwise. After the addition was complete, the mixture was reacted at -40℃ for 20 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped. 200 mL of water was added dropwise at 0℃ and stirred for 1 h. The reaction solution was added to 5 L of tetrahydrofuran at 0℃, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 200 mL of water. After adding sodium bicarbonate aqueous solution to pH=5, ethyl acetate was added for extraction and separation (100 mL × 3). The resulting aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 1000 mL of cold anhydrous ethanol under stirring at -40℃. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 79.6% and a purity of 98.7%.

[0072] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0073] Example 4

[0074] In a nitrogen atmosphere at 25°C, 200g of nicotinamide ribose chloride (X) was... - After mixing 800 mL of nicotinamide ribose (chloride ions) and 800 mL of trimethyl phosphate, the temperature of the mixture was adjusted to -20°C. 90 mL of pyrophosphoryl chloride (163.8 g) was added dropwise. After the addition was complete, the reaction was carried out at -20°C for 15 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped, and 200 mL of water was added dropwise at 0°C with stirring for 1 h. The reaction solution was then added to 4 L of acetone at 0°C, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 100 mL of water, and the pH was adjusted to 5 by adding potassium carbonate aqueous solution. Methyl tert-butyl ether was then added for extraction and layering (100 mL). X 3) The obtained aqueous phase was purified by strong base anion exchange resin 717, concentrated by nanofiltration, and then 1000 mL of cold anhydrous ethanol was added under stirring at -40℃ overnight to precipitate crystals. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 89.6% and a purity of 99.3%.

[0075] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0076] Example 5

[0077] In a nitrogen atmosphere at 25°C, 100g of nicotinamide ribose chloride (X) was... -After mixing 500 mL of trimethyl phosphate (chloride ions) and the mixture, the temperature of the mixture was adjusted to -20℃. 360 mL of pyrophosphoryl chloride (655.2 g) was added dropwise. After the addition was complete, the mixture was reacted at -30℃ for 18 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped. 100 mL of water was added dropwise at 0℃ and stirred for 1 h. The reaction solution was added to 4 L of acetone at 0℃, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 100 mL of water. After adding potassium carbonate aqueous solution to bring the pH to 5, dichloromethane was added for extraction and separation (100 mL × 3). The resulting aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 500 mL of cold anhydrous ethanol under stirring at -20℃. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 65.8% and a purity of 99.1%.

[0078] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0079] Example 6

[0080] In a nitrogen atmosphere at 25°C, 100g of nicotinamide ribose chloride (X) was... - After mixing 500 mL of trimethyl phosphate (chloride ions) and the mixture, the temperature of the mixture was adjusted to -10℃. 90 mL of pyrophosphoryl chloride (163.8 g) was added dropwise. After the addition was complete, the mixture was reacted at -10℃ for 8 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped. 100 mL of water was added dropwise at 0℃ and stirred for 1 h. The reaction solution was added to 4 L of acetone at 0℃, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 50 mL of water. After adding sodium bicarbonate aqueous solution to pH=5, dichloromethane was added for extraction and separation (50 mL × 3). The resulting aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 500 mL of cold anhydrous ethanol under stirring at -10℃. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 67.35% and a purity of 97.9%.

[0081] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0082] Example 7

[0083] In a nitrogen atmosphere at a temperature of 25°C, 100g of nicotinamide ribose bromide (X) was... -After mixing 500 mL of trimethyl phosphate (for bromide ions), the temperature of the mixture was adjusted to -10℃. 90 mL of pyrophosphoryl chloride (163.8 g) was added dropwise. After the addition was complete, the reaction was carried out at -10℃ for 8 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped. 100 mL of water was added dropwise at 0℃ and stirred for 1 h. The reaction solution was added to 4 L of tetrahydrofuran at 0℃, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 50 mL of water. After adding sodium bicarbonate aqueous solution to pH=5, ethyl acetate was added for extraction and separation (50 mL × 3). The obtained aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 500 mL of cold anhydrous ethanol under stirring at -10℃. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 83.4% and a purity of 99.0%.

[0084] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0085] Example 8

[0086] In a nitrogen atmosphere at 25°C, 100g of nicotinamide ribose chloride (X) was... - After mixing 300 mL of acetonitrile (chloride ions) and the mixture, the temperature of the mixture was adjusted to -40℃. 100 mL of pyrophosphoryl chloride (182.1 g) was added dropwise. After the addition was complete, the mixture was reacted at -40℃ for 12 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped. 300 mL of water was added dropwise at 0℃ and stirred for 0.5 h. The reaction solution was added to 4 L of acetone at 0℃, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 50 mL of water. After adding sodium bicarbonate aqueous solution to pH=5, dichloromethane was added for extraction and separation (50 mL × 3). The resulting aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 500 mL of cold anhydrous ethanol under stirring at -10℃. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 69.9% and a purity of 98.8%.

[0087] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0088] Example 9

[0089] In a nitrogen atmosphere at 25°C, 50g of nicotinamide ribose chloride (X) was... -After mixing 150 mL of acetonitrile (chloride ions), the temperature of the mixture was adjusted to -20℃. 100 mL of pyrophosphoryl chloride (182.1 g) was added dropwise. After the addition was complete, the reaction was carried out at -20℃ for 8 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped. 300 mL of water was added dropwise at 0℃ and stirred for 0.5 h. The reaction solution was added to 2 L of tetrahydrofuran at 0℃, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 25 mL of water. After adding sodium bicarbonate aqueous solution to pH=5, dichloromethane was added for extraction and separation (25 mL × 3). The resulting aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 250 mL of cold anhydrous ethanol under stirring at -10℃. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 96.1% and a purity of 98.0%.

[0090] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0091] Example 10

[0092] In a nitrogen atmosphere at 25°C, 100g of nicotinamide ribose chloride (X) was... - After mixing 200 mL of acetonitrile (chloride ions) and 200 mL of pyrophosphoryl chloride (364.2 g), the temperature of the mixture was adjusted to -10 °C. 200 mL of pyrophosphoryl chloride (364.2 g) was added dropwise. After the addition was complete, the reaction was carried out at -10 °C for 6 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped. 600 mL of water was added dropwise at 0 °C and stirred for 2 h. The reaction solution was added to 8 L of acetone at 0 °C, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 50 mL of water. After adding sodium bicarbonate aqueous solution to pH = 5, dichloromethane was added for extraction and separation (100 mL × 3). The obtained aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 500 mL of cold anhydrous ethanol under stirring at 0 °C. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 61.4% and a purity of 99.1%.

[0093] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0094] Example 11

[0095] In a nitrogen atmosphere at 25°C, 100g of nicotinamide ribose chloride (X) was... -After mixing 300 mL of acetonitrile (chloride ions) and the mixture, the temperature of the mixture was adjusted to -30℃. 150 mL of pyrophosphoryl chloride (273.2 g) was added dropwise. After the addition was complete, the mixture was reacted at -30℃ for 8 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped. 400 mL of water was added dropwise at 0℃ and stirred for 1.5 h. The reaction solution was added to 6 L of tetrahydrofuran at 0℃, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 75 mL of water. After adding sodium bicarbonate aqueous solution to pH=5, dichloromethane was added for extraction and separation (100 mL × 3). The resulting aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 500 mL of cold anhydrous ethanol under stirring at 0℃. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 59.9% and a purity of 98.0%.

[0096] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0097] Example 12

[0098] In a nitrogen atmosphere at 25°C, 200g of nicotinamide ribose chloride (X) was... - After mixing 600 mL of acetonitrile (chloride ions), the temperature of the mixture was adjusted to -10℃. 300 mL of pyrophosphoryl chloride (546.4 g) was added dropwise. After the addition was complete, the reaction was carried out at -10℃ for 18 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped. 550 mL of water was added dropwise at 0℃ and stirred for 2 h. The reaction solution was added to 10 L of tetrahydrofuran at 0℃, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 150 mL of water. After adding sodium bicarbonate aqueous solution to pH=5, dichloromethane was added for extraction and separation (200 mL × 3). The obtained aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 1 L of cold anhydrous ethanol under stirring at 0℃. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 78.5% and a purity of 99.0%.

[0099] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0100] Example 13

[0101] In a nitrogen atmosphere at 25°C, 50g of nicotinamide ribose chloride (X) was... -After mixing 200 mL of acetonitrile (chloride ions), the temperature of the mixture was adjusted to -20℃. 50 mL of pyrophosphoryl chloride (91.6 g) was added dropwise. After the addition was complete, the reaction was carried out at -20℃ for 24 h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped. 125 mL of water was added dropwise at 0℃ and stirred for 0.5 h. The reaction solution was added to 2 L of acetone at 0℃, filtered under reduced pressure, and the solid was collected. The solid was redissolved in 75 mL of water. After adding sodium bicarbonate aqueous solution to pH=5, dichloromethane was added for extraction and separation (50 mL × 3). The resulting aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 250 mL of cold anhydrous ethanol under stirring at 0℃. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 88.4% and a purity of 97.7%.

[0102] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0103] Example 14

[0104] In a nitrogen atmosphere at 25°C, 100g of nicotinamide ribose trifluoromethanesulfonate (X- for trifluoromethanesulfonate ion) and 300mL of acetonitrile were mixed. The temperature of the mixture was adjusted to -40°C, and 200mL of pyrophosphoryl chloride (364.2g) was added dropwise. After the addition was complete, the reaction was carried out at -40°C for 16 hours until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped, and 150mL of water was added dropwise at 0°C with stirring for 0.5 hours. The reaction solution was then cooled at 0°C. The solid was added to 4L of acetone, filtered under reduced pressure, and collected. The solid was redissolved in 100mL of water, and the pH was adjusted to 5 by adding sodium bicarbonate aqueous solution. Then, dichloromethane was added for extraction and separation (75mL×3). The resulting aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 500mL of cold anhydrous ethanol under stirring at 0℃. After filtration, the β-nicotinamide mononucleotide was obtained with a yield of 81.6% and a purity of 99.2%.

[0105] The β-nicotinamide mononucleotide was structurally characterized, and the results were similar to those in Example 1.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing β-nicotinamide mononucleotide, characterized in that, Includes the following steps: In a nitrogen atmosphere at 25°C, 200g of the salt corresponding to nicotinamide ribose and 800mL of trimethyl phosphate were mixed. The temperature of the mixture was adjusted to -20°C, and 163.8g of pyrophosphoryl chloride was added dropwise. After the addition was complete, the reaction was carried out at -20°C for 15h until the nicotinamide ribose content was <5% as monitored by HPLC. The reaction was then stopped, and 200mL of water was added dropwise at 0°C. The mixture was stirred for 1h, and the reaction solution was added to 4L of acetone at 0°C. The mixture was filtered under reduced pressure, and the solid was collected. The solid was redissolved in 100mL of water. After adding potassium carbonate aqueous solution to bring the pH to 5, 100mL of methyl tert-butyl ether was added for extraction and separation three times. The resulting aqueous phase was purified using a strong base anion exchange resin 717, concentrated by nanofiltration, and then precipitated overnight by adding 1000mL of cold anhydrous ethanol under stirring at -40°C. After filtration, the β-nicotinamide mononucleotide was obtained. The salt corresponding to the nicotinamide ribose has the structure shown in Formula 1: Formula 1; Among them, X - It is a chloride ion.

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