METHOD FOR PREPARING NICOTINAMIDE MONONUCLEOTIDE BY USING NICOTINAMIDE AS RAW MATERIAL

Abstract

The invention relates to a method for preparing nicotinamide mononucleotide by using nicotinamide as a raw material, which comprises: in acetonitrile, dichloromethane, 1,2-dichloroethane or liquid sulfur dioxide as a solvent, allowing nicotinamide and tetraacetyl ribose to react as catalyzed by trimethylsilyl trifluoromethanesulfonate or tin tetrachloride, adjusting a pH value thereof to 3-5, adding a sodium methoxide solution thereto to react at −15° C. to 5° C., adjusting a pH value thereof to 3-5, and subjecting the reaction mixture to microfiltration and nanofiltration using a membrane concentrator, thereby obtain a nicotinamide ribose solution; allowing the nicotinamide ribose solution to react as catalyzed by nicotinamide ribokinase in the presence of Mg ions, ATP and a buffer, thereby obtaining nicotinamide mononucleotide. The method of the invention omits the step of refining nicotinamide ribose, and thus has simpler process, lower cost and less time consumption, and has the advantages of faster reaction speed and lower enzyme consumption compared with the enzyme catalytic process directly using refined nicotinamide ribose solid.

Claims

1. A method for preparing nicotinamide mononucleotide by using nicotinamide as a raw material, wherein the method comprises steps of: 1) allowing nicotinamide and tetraacetyl ribose to react in acetonitrile, dichloromethane, 1,2-dichloroethane or liquid sulfur dioxide as a solvent as catalyzed by trimethylsilyl trifluoromethanesulfonate or tin tetrachloride at a temperature of 20-40° C. to obtain a first reaction mixture; 2) adding sodium bicarbonate, sodium carbonate or sodium hydroxide into the first reaction mixture to adjust a pH value thereof to 3-5 to obtain a second reaction mixture; 3) adding a sodium methoxide solution into the second reaction mixture to react at a temperature of −15° C. to 5° C. to obtain a third reaction mixture; 4) adding hydrochloric acid into the third reaction mixture to adjust a pH value thereof to 3-5 to obtain a fourth reaction mixture; 5) performing microfiltration and nanofiltration on the fourth reaction mixture in sequence by using a membrane concentrator to obtain a nicotinamide ribose solution; and 6) allowing the nicotinamide ribose solution obtained in the step 5) to react as catalyzed by nicotinamide ribokinase at a temperature of 35-39° C. in the presence of Mg ions, ATP and a buffer, wherein a pH value of the reaction mixture is controlled at 7.5-8.0 during the reaction, and nicotinamide mononucleotide is obtained after the reaction is complete.

2. The method for preparing nicotinamide mononucleotide by using nicotinamide as a raw material according to claim 1, wherein the reaction in the step 1) is carried out at a temperature of 25-35° C.

3. The method for preparing nicotinamide mononucleotide by using nicotinamide as a raw material according to claim 1, wherein in the step 1), a molar ratio of the trimethylsilyl trifluoromethanesulfonate to the nicotinamide to the tetraacetyl ribose is 1.2-5:1.2-2:1.

4. The method for preparing nicotinamide mononucleotide by using nicotinamide as a raw material according to claim 1, wherein the reaction in the step 3) is carried out at a temperature of −10° C. to −5° C.

5. The method for preparing nicotinamide mononucleotide by using nicotinamide as a raw material according to claim 1, wherein in the step 3), a molar ratio of the sodium methoxide to the tetraacetyl ribose is 1-5:1.

6. The method for preparing nicotinamide mononucleotide by using nicotinamide as a raw material according to claim 1, wherein during the step 4), the temperature of the reaction mixture is maintained at −10° C. to −5° C.

7. The method for preparing nicotinamide mononucleotide by using nicotinamide as a raw material according to claim 1, wherein in the step 5), a microfiltration membrane with a pore size of 0.2-1 μm is used in the microfiltration process, and a hollow fiber membrane with a molecular weight cutoff of 150-250 is used in the nanofiltration process.

8. The method for preparing nicotinamide mononucleotide by using nicotinamide as a raw material according to claim 1, wherein in the step 6), the Mg ion, the ATP, the buffer, the nicotinamide ribose and the nicotinamide ribokinase are added at an amount of 10-50 mM, 10-30 mM, 20-100 mM, 9-27 mM and 0.2-1 g/L respectively.

9. The method for preparing nicotinamide mononucleotide by using nicotinamide as a raw material according to claim 1, wherein in the step 6), the Mg ion is MgCl.sub.2.

10. The method for preparing nicotinamide mononucleotide by using nicotinamide as a raw material according to claim 1, wherein in the step 6), the buffer is K.sub.2HPO.sub.4 buffer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The invention is further described in detail below with reference to specific examples, which are illustrative of the invention, and the invention is not limited to the following examples.

[0028] The raw materials and reagents used in the following examples, unless otherwise specified, were purchased from the market.

EXAMPLE 1

Preparation of Intermediate Product Nicotinamide Ribose Solution

[0029] Under nitrogen protection, 100 g of tetraacetyl ribose (314.5 mmol, 1.0 eq), 115.1 g of nicotinamide (943.4 mmol, 3 eq) and 700 ml of acetonitrile were sequentially added into a 2 L three-necked flask, and stirred at 20° C. to obtain a suspension. 170.3 ml (943.4 mmol, 3 eq) of trimethylsilyl trifluoromethanesulfonate was slowly added dropwise, during which the suspension became clear and then turned cloudy. The stirring was continued for 45 min at 20° C., and at that time, sample dispensing on the plate showed that the raw materials were completely converted. Sodium bicarbonate was added into the reaction mixture to adjust the pH value to 3-5, and the mixture was concentrated by distillation to remove the solvent, thereby obtaining a pale yellow oily liquid (yield 100%).

[0030] The obtained pale yellow oily liquid was dissolved in 500 ml of methanol, cooled to −10° C. with stirring, and 314.5 ml (1572.5 mmol, 5 eq) of a sodium methoxide solution was slowly added dropwise thereto while keeping the internal temperature of the reaction mixture below −5° C. Thereafter, the reaction was carried out at −5° C. to −10° C. for 40 min, and at that time, high performance liquid chromatography (HPLC) showed that the raw materials were completely converted. To the reaction mixture, 250 mL (1500 mmol) of 6 M hydrochloric acid was slowly added dropwise to adjust the pH value to 3-4, and the reaction mixture was concentrated to obtain a pale yellow liquid (yield 100%).

[0031] The obtained pale yellow liquid was dissolved in water, and then fed into a membrane concentrator to receive microfiltration and nanofiltration in sequence, wherein a microfiltration membrane with a pore size of 0.2 μm was used in the microfiltration process, and a hollow fiber membrane with a molecular weight cutoff of 150 was used in the nanofiltration process, thereby obtaining a nicotinamide ribose solution (with residual organic solvent of less than 0.5%) for later use.

EXAMPLE 2

Preparation of Intermediate Product Nicotinamide Ribose Solution

[0032] Under nitrogen protection, 100 g of tetraacetyl ribose (314.5 mmol, 1.0 eq), 76.7 g of nicotinamide (628.7 mmol, 2 eq) and 700 ml of dichloromethane were sequentially added into a 2 L three-necked flask, and stirred at 25° C. to obtain a suspension. 113.5 ml (628.7 mmol, 2 eq) of trimethylsilyl trifluoromethanesulfonate was slowly added dropwise thereto, followed by stirring at 25° C. for 50 min, whereupon the raw materials were completely converted. Sodium bicarbonate was added into the reaction mixture to adjust the pH value to 3-5, and the mixture was concentrated by distillation to remove the solvent, thereby obtaining a pale yellow oily liquid (yield 100%).

[0033] The obtained pale yellow oily liquid was dissolved in 500 ml of methanol, cooled to −10° C. with stirring, and 188.7 ml (943.5 mmol, 3 eq) of a sodium methoxide solution was slowly added dropwise thereto while keeping the internal temperature of the reaction mixture below −5° C. Thereafter, the reaction was carried out at −5° C. to −10° C. for 60 min, whereupon the raw materials were completely converted. To the reaction mixture, 150 mL (900 mmol) of 6 M hydrochloric acid was slowly added dropwise to adjust the pH value to 3-4, and the reaction mixture was concentrated to obtain a pale yellow liquid (100% yield).

[0034] The obtained pale yellow liquid was dissolved in water, and then fed into a membrane concentrator to receive microfiltration and nanofiltration in sequence, wherein a microfiltration membrane with a pore size of 0.5 μm was used in the microfiltration process, and a hollow fiber membrane with a molecular weight cutoff of 200 was used in the nanofiltration process, thereby obtaining a nicotinamide ribose solution (with residual organic solvent of less than 0.5%) for later use.

EXAMPLE 3

Preparation of Intermediate Product Nicotinamide Ribose Solution

[0035] Under nitrogen protection, 100 g of tetraacetyl ribose (314.5 mmol, 1.0 eq), 57.5 g of nicotinamide (471.3 mmol, 1.5 eq) and 700 ml of 1,2-dichloroethane were added into a 2 L three-necked flask, and stirred at 35° C. to obtain a suspension. 85.1 ml (471.3 mmol, 1.5 eq) of tin tetrachloride was slowly added dropwise thereto, followed by stirring at 35° C. for 45 min, whereupon the raw materials were completely converted. Sodium carbonate was added into the reaction mixture to adjust the pH value to 3-5, and the mixture was concentrated by distillation to remove the solvent, thereby obtaining a pale yellow oily liquid (yield: 100%).

[0036] The obtained pale yellow oily liquid was dissolved in 500 ml of methanol, cooled to −10° C. with stirring, and 125.8 ml (629 mmol, 2 eq) of a sodium methoxide solution was slowly dropwise added thereto while keeping the internal temperature of the reaction mixture below −5° C. Thereafter, the reaction was carried out at −5° C. to −10° C. for 90 min, whereupon the raw materials were completely converted. To the reaction mixture, 100 mL (600 mmol) of 6 M hydrochloric acid was slowly added dropwise to adjust the pH value to 3-5, and the reaction mixture was concentrated to obtain a pale yellow liquid (100% yield).

[0037] The obtained pale yellow liquid was dissolved in water, and then fed into a membrane concentrator to receive microfiltration and nanofiltration in sequence, wherein a microfiltration membrane with a pore size of 0.7 μm was used in the microfiltration process, and a hollow fiber membrane with a molecular weight cutoff of 200 was used in the nanofiltration process, thereby obtaining a nicotinamide ribose solution (with residual organic solvent of less than 0.5%) for later use.

EXAMPLE 4

Preparation of Intermediate Product Nicotinamide Ribose Solution

[0038] Under nitrogen protection, 100 g of tetraacetyl ribose (314.5 mmol, 1.0 eq), 46 g of nicotinamide (377 mmol, 1.2 eq) and 700 ml of liquid sulfur dioxide were added into a 2 L three-necked flask, and stirred at 40° C. to obtain a suspension. 68.0 ml (377 mmol, 1.2 eq) of trimethylsilyl trifluoromethanesulfonate was slowly added dropwise thereto, followed by stirring at 40° C. for 45 min, whereupon the raw materials were completely converted. Sodium hydroxide was added into the reaction mixture to adjust the pH value to 3-5, and the mixture was concentrated by distillation to remove the solvent, thereby obtaining a pale yellow oily liquid (yield: 100%).

[0039] The obtained pale yellow oily liquid was dissolved in 500 ml of methanol, cooled to −10° C. with stirring, and 94.3 ml (471.7 mmol, 1.5 eq) of a sodium methoxide solution was slowly dropwise added thereto while keeping the internal temperature of the reaction mixture below −5° C. Thereafter, the reaction was carried out at −5° C. to −10° C. for 120 min, whereupon the raw materials were completely converted. To the reaction mixture, 75 mL (450 mmol) of 6 M hydrochloric acid was slowly added dropwise to adjust the pH value to 3-5, and the reaction mixture was concentrated to obtain a pale yellow liquid (100% yield).

[0040] The obtained pale yellow liquid was dissolved in water, and then fed into a membrane concentrator to receive microfiltration and nanofiltration in sequence, wherein a microfiltration membrane with a pore size of 1 μm was used in the microfiltration process, and a hollow fiber membrane with a molecular weight cutoff of 250 was used in the nanofiltration process, thereby obtaining a nicotinamide ribose solution (with residual organic solvent of less than 0.5%) for later use.

EXAMPLE 5

[0041] The results of various parameters in the preparation of the intermediate product nicotinamide ribose solution are shown in Table 1 to Table 5.

TABLE-US-00001 TABLE 1 Effects of nicotinamide/trimethylsilyl trifluoromethanesulfonate dosage on reaction conversion Trimethylsilyl trifluoromethane- Nicotinamide sulfonate/tin Reaction Experiment dosage tetrachloride dosage conversion 1 1.0 eq 1.0 eq Excessive tetraacetyl ribose 2 1.2 eq 1.2 eq Complete reaction 3 1.5 eq 2.0 eq Complete reaction 4 2.0 eq 5.0 eq Complete reaction

TABLE-US-00002 TABLE 2 Effects of pH adjustment with sodium bicarbonate on the long-term stability of nicotinamide ribose solution Experiment pH Temperature Storage time Stability 1 3 2-4° C. 45 days Substantially no degradation 2 4 2-4° C. 45 days 2.4% degradation 3 5 2-4° C. 45 days 3.7% degradation

TABLE-US-00003 TABLE 3 Effects of temperature on reaction in step 3) Sodium Reaction Temperature methoxide dosage time Reaction result 0-5° C. 2.0 eq 0.5 h Complete reaction and 9.2% product degradation −5° C. to 2.0 eq 1.0 h Complete reaction and 0° C. 19.6% product degradation −10° C. to 2.0 eq 1.5 h Complete reaction and −5° C. substantially no product degradation −15° C. to 2.0 eq 5.0 h Complete reaction and −10° C. substantially no product degradation

TABLE-US-00004 TABLE 4 Effects of sodium methoxide dosage on reaction in step 3) Sodium methoxide Reaction Reaction dosage temperature time Reaction result 1.2 eq −10° C. to −5° C. 24.0 h Complete reaction and substantially no product degradation 1.5 eq −10° C. to −5° C. 2.0 h Complete reaction and substantially no product degradation 2.0 eq −10° C. to −5° C. 1.5 h Complete reaction and substantially no product degradation 3.0 eq −10° C. to −5° C. 1.0 h Complete reaction and substantially no product degradation 5.0 eq −10° C. to −5° C. 40 min Complete reaction and substantially no product degradation

TABLE-US-00005 TABLE 5 Effects of pH adjustment with hydrochloric acid on the long-term stability of nicotinamide ribose solution Storage Experiment temperature pH Storage time Stability 1 0-4° C. 3 45 days Substantially no degradation 2 0-4° C. 4 45 days 1.3% degradation 3 0-4° C. 5 45 days 2.1% degradation

EXAMPLE 6

Preparation of Nicotinamide Mononucleotide

[0042]

TABLE-US-00006 TABLE 6 Amount per Material Concentration 100 mL K2HPO4 50 mM 2.28 g MgCl2 10 mM 0.41 g ATP 20 mM 1.10 g Commercially available NR 18 mM 0.65 g/6 ml solid (content 80%)/NR solution prepared in example of the invention (content 300 mM) Nicotinamide ribokinase 0.33-0.5 g/L

[0043] 1 g of nicotinamide ribokinase was weighed and prepared into an enzyme working solution with 10 mL of pure water, and stored at 4° C.

[0044] The first three materials were each weighed according to Table 6 and dissolved with about 70 mL of pure water respectively, and then the commercial NR solid (content 80%) or the NR solution prepared according to the example of the invention (content 300 mM) was added thereto respectively. The mixture was adjusted to a pH value of 7.5-8.0 with 3 M NaOH, and made up to a volume of 100 mL. After preheating the mixture at 37° C., 330-500 μL of the enzyme working solution was added and the mixture was subjected to a constant-temperature oscillation reaction at 220 rpm. After the reaction was completed, nicotinamide mononucleotide was obtained.

[0045] Samples were taken every half hour during the reaction, and the product formation was analyzed by HPLC, with pH monitored and regulated, and the results are shown in Table 7:

TABLE-US-00007 TABLE 7 Enzyme NR.fwdarw.NMN conversion Substrate type amount 0.5 h 1 h 1.5 h Commercially available 0.5 g/L 66.4% 91.6% 98.5% NR solid NR solution prepared by 0.33 g/L  61.4% 92.7% 99.3% example of the invention NR solution prepared by 0.5 g/L 79.4% 96.5% 99.2% example of the invention

[0046] As can be seen from table 7, that NR solution prepared in the example of the invention has a faster initial reaction speed and requires a lower amount of enzyme than the commercially available NR solid.

EXAMPLE 7

Preparation of Nicotinamide Mononucleotide

[0047]

TABLE-US-00008 TABLE 8 Amount per Material Concentration 100 mL K2HPO4 100 mM 4.56 g MgCl2 20 mM 0.82 g ATP 20 mM 1.10 g Commercially available NR 18 mM 1.05 g/3.15 ml solid (content 50%)/NR solution prepared in example of the invention (content 570 mM) Nicotinamide ribokinase 0.65 g/L

[0048] 1 g of nicotinamide ribokinase was weighed and prepared into an enzyme working solution with 10 mL of pure water, and stored at 4° C.

[0049] The first three materials were each weighed according to Table 8 and dissolved with about 70 mL of pure water respectively, and then the commercial NR solid (content 50%) or the NR solution prepared according to the example of the invention (content 570 mM) was added thereto respectively. The mixture was adjusted to a pH value of 7.5-8.0 with 3 M NaOH, and made up to a volume of 100 mL. After preheating the mixture at 37° C., 650 μL of the enzyme working solution was added and the mixture was subjected to a constant-temperature oscillation reaction at 220 rpm. After the reaction was completed, nicotinamide mononucleotide was obtained.

[0050] Samples were taken every half hour during the reaction, and the product formation was analyzed by HPLC, with pH monitored and regulated, and the results are shown in Table 9:

TABLE-US-00009 TABLE 9 Enzyme NR.fwdarw.NMN conversion Substrate type amount 0.5 h 1 h 1.5 h Commercially available 0.65 g/L 76.3% 85.0% 94.1% NR solid - lot 1 Commercially available 0.65 g/L 29.7% 34.9% 36.7% NR solid - lot 2 NR solution prepared in 0.65 g/L 87.2% 97.0% 99.6% example of the invention - lot 1 NR solution prepared in 0.65 g/L 89.6% 98.1% 99.8% example of the invention - lot 2

[0051] As can be seen from table 9, as compared with the commercial NR solid, the NR solution prepared in the example of the invention has a faster initial reaction speed, and can avoid the phenomenon of reaction inhibition due to the presence of certain impurities that occurred in the commercial NR solid—lot 2.

EXAMPLE 8

Preparation of Nicotinamide Mononucleotide

[0052]

TABLE-US-00010 TABLE 10 Material Concentration Amount per 100 mL K.sub.2HPO.sub.4 20 mM 0.91 g MgCl.sub.2 50 mM 2.05 g ATP 30 mM 1.65 g Commercially available NR 27 mM 0.82 g/2.65 ml solid (content 98%)/NR solution prepared in example of the invention (content 1020 mM) Nicotinamide ribokinase 1 g/L

[0053] 1 g of nicotinamide ribokinase was weighed and prepared into an enzyme working solution with 10 mL of pure water, and stored at 4° C.

[0054] The first three materials were each weighed according to Table 10 and dissolved with about 70 mL of pure water respectively, and then the commercial NR solid (content 98%) or the NR solution prepared according to the example of the invention (content 1020 mM) was added thereto respectively. The mixture was adjusted to a pH value of 7.5-8.0 with 3 M NaOH, and made up to a volume of 100 mL. After preheating the mixture at 37° C., 1 mL of the enzyme working solution was added and the mixture was subjected to a constant-temperature oscillation reaction at 220 rpm. After the reaction was completed, nicotinamide mononucleotide was obtained.

[0055] Samples were taken every half hour during the reaction, and the product formation was analyzed by HPLC, with pH monitored and regulated, and the results are shown in Table 11:

TABLE-US-00011 TABLE 11 Enzyme NR.fwdarw.NMN conversion Substrate type amount 0.5 h 1 h 1.5 h Commercially 1 g/L 77.1% 83.8% 86.2% available NR solid NR solution 1 g/L 82.3% 83.7% 86.5% prepared in example of the invention

[0056] As can be seen from table 11, as compared with the commercial NR solid, the NR solution prepared in the example of the invention has a slightly faster initial reaction speed, and the conversion will reduce when the substrate concentration is increased.