The method for preparing orlistat intermediate

Abstract

The invention belongs to the technical field of biopharmaceuticals and biochemical industries, and in particular relates to a method for synthesizing (R)-β-hydroxytetradecanoic acid ester compounds. The method is to convert β-carbonyl myristate in ketoreductase, glucose, glucose dehydrogenase, NADP + React with buffer to obtain (R)-β-hydroxytetradecanoic acid esters. The method is an enzyme-catalyzed biosynthesis method, is simple to operate, uses conventional equipment, is environmentally friendly, and produces a product with high purity, high yield and high ee value.

Description

technical field

[0001] The present invention belongs to the field of biopharmaceutical and biochemical technology, and specifically relates to a biosynthesis method of (R)-β-hydroxymyristate compounds. Background technique

[0002] Drugs for the treatment of obesity are mainly divided into two categories, namely central nervous action weight loss drugs and non-central nervous action weight loss drugs. The weight-reducing effect of central nervous action weight-reducing medicine is obvious, but side effect is relatively large, and clinical application is greatly restricted. Among them, fenfluramine was withdrawn from the market in 1997 because it could cause pulmonary hypertension and hypertrophic heart valve disease. Sibutramine was announced by the State Food and Drug Administration in 2010 to stop its production, sales and use due to its possible increase in serious cardiovascular risks. Locaserin (trade name Belviq) was approved for marketing by the FDA in June 2012, bu...

Examples

Embodiment 1

[0071] Weigh 1g of methyl β-carbonyltetradecanoate and 1.5g of glucose into a 100mL three-neck flask, then add 50mL of PBS buffer solution with a concentration of 50mM and a pH value of 7.0; put the three-necked flask into a constant temperature water bath , the stirring speed was adjusted to 800rpm, the temperature was 35°C, and then 10mg NADP was added + , 25mg of glucose dehydrogenase GDH enzyme powder (shown in SEQ ID NO:3), and 100mg of ketoreductase JR3789 enzyme powder (shown in SEQ ID NO:1), to obtain a mixed reaction solution, adjust the pH with 2M NaOH solution The temperature was maintained at 7.0-7.5, and the temperature was maintained at 35° C., and the reaction progress was monitored by HPLC. After 9 hours, the reaction was completed, and the conversion rate was measured to be >99%.

[0072] After the reaction, first raise the temperature to 60°C and keep it warm for 15min, then cool down to 20-25°C, add 80mL of ethyl acetate to extract and stir for 20min, filter...

Embodiment 2

[0074] Weigh 1g of ethyl β-carbonyltetradecanoate and 1.5g of glucose into a 100mL three-necked flask, and then add 50mL of PBS buffer solution with a concentration of 50mM and a pH value of 7.0; put the three-necked flask into a constant temperature water bath , the stirring speed was adjusted to 900rpm, the temperature was 35°C, and then 10mg NADP+, 35mg glucose dehydrogenase GDH enzyme powder (shown in SEQ ID NO:3), and 150mg ketoreductase JR3789 enzyme powder (shown in SEQ ID NO:1) were added respectively Shown), to obtain a mixed reaction solution, adjust the pH with 2M NaOH solution to maintain between 7.0-7.5, the temperature is maintained at 35 ° C, using HPLC to monitor the reaction process, 10h after the end of the reaction, and measured conversion> 99%.

[0075] After the reaction, first raise the temperature to 60°C and keep it warm for 15min, then cool down to 20-25°C, add 80mL of ethyl acetate to extract and stir for 20min, filter, and separate the filtrate to tak...

Embodiment 3

[0077] Weigh 5g of methyl β-carbonyltetradecanoate and 7.5g of glucose into a 100mL three-necked flask, then add 50mL of PBS buffer solution with a concentration of 50mM and a pH value of 7.0; put the three-necked flask into a constant temperature water bath , the stirring speed was adjusted to 800rpm, the temperature was 35°C, and then 50mg NADP+, 125mg glucose dehydrogenase GDH enzyme powder (shown in SEQ ID NO:3), and 500mg ketoreductase JR3789 enzyme powder (shown in SEQ ID NO:1) were added respectively Shown), the mixed reaction solution was obtained, the pH was adjusted with 2M NaOH solution to maintain between 7.0-7.5, the temperature was maintained at 35 ° C, the reaction progress was monitored by HPLC, the reaction was completed after 13 hours, and the conversion rate > 99% was measured.

[0078] After the reaction, first raise the temperature to 60°C and keep it warm for 15min, then cool down to 20-25°C, add 80mL of ethyl acetate to extract and stir for 20min, filter,...

technical field

[0001] The present invention belongs to the field of biopharmaceutical and biochemical technology, and specifically relates to a biosynthesis method of (R)-β-hydroxymyristate compounds. Background technique

[0002] Drugs for the treatment of obesity are mainly divided into two categories, namely central nervous action weight loss drugs and non-central nervous action weight loss drugs. The weight-reducing effect of central nervous action weight-reducing medicine is obvious, but side effect is relatively large, and clinical application is greatly restricted. Among them, fenfluramine was withdrawn from the market in 1997 because it could cause pulmonary hypertension and hypertrophic heart valve disease. Sibutramine was announced by the State Food and Drug Administration in 2010 to stop its production, sales and use due to its possible increase in serious cardiovascular risks. Locaserin (trade name Belviq) was approved for marketing by the FDA in June 2012, bu...

Examples

Embodiment 1

[0071] Weigh 1g of methyl β-carbonyltetradecanoate and 1.5g of glucose into a 100mL three-neck flask, then add 50mL of PBS buffer solution with a concentration of 50mM and a pH value of 7.0; put the three-necked flask into a constant temperature water bath , the stirring speed was adjusted to 800rpm, the temperature was 35°C, and then 10mg NADP was added + , 25mg of glucose dehydrogenase GDH enzyme powder (shown in SEQ ID NO:3), and 100mg of ketoreductase JR3789 enzyme powder (shown in SEQ ID NO:1), to obtain a mixed reaction solution, adjust the pH with 2M NaOH solution The temperature was maintained at 7.0-7.5, and the temperature was maintained at 35° C., and the reaction progress was monitored by HPLC. After 9 hours, the reaction was completed, and the conversion rate was measured to be >99%.

[0072] After the reaction, first raise the temperature to 60°C and keep it warm for 15min, then cool down to 20-25°C, add 80mL of ethyl acetate to extract and stir for 20min, filter...

Embodiment 2

[0074] Weigh 1g of ethyl β-carbonyltetradecanoate and 1.5g of glucose into a 100mL three-necked flask, and then add 50mL of PBS buffer solution with a concentration of 50mM and a pH value of 7.0; put the three-necked flask into a constant temperature water bath , the stirring speed was adjusted to 900rpm, the temperature was 35°C, and then 10mg NADP+, 35mg glucose dehydrogenase GDH enzyme powder (shown in SEQ ID NO:3), and 150mg ketoreductase JR3789 enzyme powder (shown in SEQ ID NO:1) were added respectively Shown), to obtain a mixed reaction solution, adjust the pH with 2M NaOH solution to maintain between 7.0-7.5, the temperature is maintained at 35 ° C, using HPLC to monitor the reaction process, 10h after the end of the reaction, and measured conversion> 99%.

[0075] After the reaction, first raise the temperature to 60°C and keep it warm for 15min, then cool down to 20-25°C, add 80mL of ethyl acetate to extract and stir for 20min, filter, and separate the filtrate to tak...

Embodiment 3

[0077] Weigh 5g of methyl β-carbonyltetradecanoate and 7.5g of glucose into a 100mL three-necked flask, then add 50mL of PBS buffer solution with a concentration of 50mM and a pH value of 7.0; put the three-necked flask into a constant temperature water bath , the stirring speed was adjusted to 800rpm, the temperature was 35°C, and then 50mg NADP+, 125mg glucose dehydrogenase GDH enzyme powder (shown in SEQ ID NO:3), and 500mg ketoreductase JR3789 enzyme powder (shown in SEQ ID NO:1) were added respectively Shown), the mixed reaction solution was obtained, the pH was adjusted with 2M NaOH solution to maintain between 7.0-7.5, the temperature was maintained at 35 ° C, the reaction progress was monitored by HPLC, the reaction was completed after 13 hours, and the conversion rate > 99% was measured.

[0078] After the reaction, first raise the temperature to 60°C and keep it warm for 15min, then cool down to 20-25°C, add 80mL of ethyl acetate to extract and stir for 20min, filter,...

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