Method for preparing nicotinamide mononucleotide by using nicotinamide as raw material

Preparation of Intermediate Product Nicotinamide Ribose Solution

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%).

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%).

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.

Preparation of Intermediate Product Nicotinamide Ribose Solution

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%).

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).

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.

Preparation of Intermediate Product Nicotinamide Ribose Solution

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%).

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).

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.

Preparation of Intermediate Product Nicotinamide Ribose Solution

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%).

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).

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.

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

Preparation of Nicotinamide Mononucleotide

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.

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.

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:

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.

Preparation of Nicotinamide Mononucleotide

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.

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.

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:

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.

Preparation of Nicotinamide Mononucleotide

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.

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.

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:

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.