WO2014094462A1

WO2014094462A1 - Biological preparation method of (s)-3-methylamino-1-(2-thienyl)-1-propyl alcohol

Info

Publication number: WO2014094462A1
Application number: PCT/CN2013/083235
Authority: WO
Inventors: 乐庸堂; 鞠鑫
Original assignee: 苏州汉酶生物技术有限公司
Priority date: 2012-12-19
Filing date: 2013-09-10
Publication date: 2014-06-26
Also published as: CN103014088A

Abstract

The present invention provides a biological preparation method of (S)-3-methylamino-1-(2-thienyl)-1-propyl alcohol, wherein 3-methylamino-1-(2-thienyl)-1-acetone is used as a substrate; under a condition that a biocatalyst ketoreductase, a cofactor and a cofactor regeneration system (comprising glucose and glucose dehydrogenase) exist, the substrate undergoes an asymmetric reduction reaction to generate the (S)-3-methylamino-1-(2-thienyl)-1-propyl alcohol; the asymmetric reduction reaction is carried out in a water-phase buffer solution with a pH of 6-8; and in a initial reaction system, concentration of the substrate is 100-150 mg/mL, and a mass ratio of the ketoreductase to the substrate is 1-2%. The present invention improves the concentration of the substrate and reduces enzyme usage.

Description

Biological preparation method of (S)-3-methylamino-1-(2-thienyl)-1-propanol

Technical field:

The invention belongs to the field of biopharmaceuticals and green chemistry, and particularly relates to a biological preparation method of (S)-3-methylamino-1-(2-thienyl)-1-propanol.

Background technique:

Duloxetine is a low side effect drug that can effectively treat mental disorders and metabolic disorders (US patent 5,023,269), the key to the synthesis of duloxetine is to obtain the intermediate (S)-3-methylamino-1-(2-thienyl)-1-propanol (MMAA) containing a chiral center, thus asymmetric reduction 3-methylamino-1-( 2-Thienyl)-1-propanone (MMAK) is one of the most effective and researched methods available. In the chemical reduction method to achieve this route, due to the need for ruthenium metal catalyst catalysis, add 4.5 The economical, safety and environmental friendliness of the MPa hydrogen reaction does not meet the production needs (Tetrahedron Lett. 1990, 31:7101–7104). The chemical resolution method using mandelic acid as a resolving agent cannot be used as the main production method for producing MMAA due to low yield and poor economy (Tetrahedron) Asymm. 2003, 14:1631–1636). Therefore, the use of bioenzymatic reduction of MMAK to obtain MMAA has become the most effective method with its high efficiency and environmental friendliness.

In the biological reduction method, there is the use of Candida The magnoliae recombinant reductase catalyzes an example of the reduction reaction, but the method has a high catalyst addition amount and a low substrate concentration, which is obviously difficult to industrialize (US patent 2008/0220484 A1).

Summary of the invention:

The technical problem to be solved by the present invention is to provide an improved biological preparation method of (S)-3-methylamino-1-(2-thienyl)-1-propanol.

In order to solve the above technical problems, the present invention adopts the following technical scheme: a biological preparation method of (S)-3-methylamino-1-(2-thienyl)-1-propanol, which is 3-methylamino group 1-(2-Thienyl)-1-propanone is a substrate which undergoes asymmetric reduction in the presence of a biocatalyst, cofactor and cofactor regeneration system to form (S)-3-methylamino group- 1-(2-Thienyl)-1-propanol. The biocatalyst is a ketoreductase, the cofactor regeneration system comprises glucose and glucose dehydrogenase, and the asymmetric reduction reaction is carried out in an aqueous phase buffer having a pH of 6-8, in an initial state of reaction. In the system. The concentration of the substrate 3-methylamino-1-(2-thienyl)-1-propanone is 100-150 Mg/mL. The mass ratio of the ketoreductase to the substrate 3-methylamino-1-(2-thienyl)-1-propanone is 1-2%.

Further, in the initial reaction system, the mass ratio of the cofactor: glucose:glucose dehydrogenase: 3-methylamino-1-(2-thienyl)-1-propanone is (0.0005-0.002). ): (1.2-2): (0.01-0.05): 1.

Further, the cofactor is NAD/NADH or NADP/NADPH, and is preferably NADP/NADPH.

Further, the aqueous phase buffer is a triethanolamine buffer solution.

Further, the asymmetric reduction reaction was carried out in an aqueous phase buffer having a pH of 7.0.

Further, the pH of the aqueous phase buffer in which the asymmetric reduction reaction is carried out is adjusted with a sodium hydroxide solution, a sodium carbonate solution or a sodium hydrogencarbonate solution.

Further, the above preparation method can be carried out as follows: in the reaction vessel, a substrate 3-methylamino-1-(2-thienyl)-1-propanone, glucose, an aqueous phase buffer solution is sequentially added, and the mixture is uniformly stirred. The ketone reductase, glucose dehydrogenase and the cofactor are further added, and the reaction is stirred at 25-45 ° C, and the progress of the reaction is detected by HPLC. When the conversion rate reaches 90-99%, the reaction is terminated.

The ketoreductase (Suzhou Hanase Biotechnology Co., Ltd., KRED137) and glucose dehydrogenase (Suzhou Hanzyme Biotechnology Co., Ltd., GDH102) in the present invention are commercially available.

The beneficial effects of the invention are:

The invention optimizes the process conditions, increases the substrate concentration, reduces the enzyme dosage, thereby reducing the production cost, realizing a high-efficiency biotransformation process, and is suitable for industrial production.

detailed description:

The reaction formula of the present invention is as follows:

The following examples are provided to enable those skilled in the art to understand the invention more fully, but are not intended to limit the invention in any way.

Embodiment 1

Add 1.0 g of substrate MMAK and 1.2 g of glucose to a 50 mL reaction three-necked flask, and add 8 to the reaction flask. The pH is 7.0, 0.1 M triethanolamine buffer. Stir at 30 ° C, 900 r / min magnetic stirring water bath, and use 2 M The pH of the NaOH solution was adjusted to 7.0. To stabilize the temperature, add 0.95 mL to the reaction flask and dissolve 20 mg of ketoreductase, 31 mg. GDH triethanolamine buffer. The pH was adjusted to 7.0 with NaOH and flushed with 0.95 mL of triethanolamine buffer and added to the reaction flask. Add 100 μL of 0.5 mg to the reaction flask. The reaction of NADP in triethanolamine buffer begins. The reaction temperature was maintained at 30 °C. The pH of the reaction solution was adjusted by dropwise addition of a 2 M NaOH solution through a pH titrator. The pH is controlled at 7.0 and the initial control point is 6. 5, the final control point is 6.99. Timed sampling for HPLC detection. The reaction was carried out for 23 h with a conversion of >99%.

Embodiment 2

Add 100.0 g of substrate MMAK and 120.0 g of glucose to the reactor and add 800 to the reaction flask. The pH is 7.0, 0.1 M triethanolamine buffer. Stir at 30 ° C in a mechanically stirred water bath and adjust the pH to 7.0 with 2 M NaOH solution. Wait for the temperature to stabilize, add 95 to the reaction flask mL was dissolved in 2 g ketoreductase, 3.1 g GDH in triethanolamine buffer. Adjust the pH to 7.0 with NaOH and use 95 Rinse with triethanolamine buffer and add to the reaction flask. Add 10 mL to the reaction flask and dissolve 50 mg. The reaction of NADP in triethanolamine buffer begins. The reaction temperature was maintained at 30 °C. The pH of the reaction solution was adjusted by dropwise addition of a 2 M NaOH solution through a pH titrator. The pH is controlled at 7.0 and the initial control point is 6. 5, the final control point is 6.99. Timed sampling for HPLC detection. The reaction was carried out for 23 h with a conversion of >98%.

The above embodiments are merely illustrative of the technical concept and the features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the present invention and to implement the present invention, and the scope of the present invention is not limited thereto. Equivalent variations or modifications made in accordance with the spirit of the invention are intended to be included within the scope of the invention.

Claims

A biological preparation method of (S)-3-methylamino-1-(2-thienyl)-1-propanol, which is 3-methylamino-1-(2-thienyl)-1 - Acetone is a substrate which undergoes asymmetric reduction in the presence of a biocatalyst, cofactor and cofactor regeneration system to form (S)-3-methylamino-1-(2-thienyl)-1- Propyl alcohol, characterized in that the biocatalyst is a ketone reductase, the cofactor regeneration system comprises glucose and glucose dehydrogenase, and the asymmetric reduction reaction is buffered in an aqueous phase having a pH of 6-8. In the initial reaction system, the concentration of the substrate 3-methylamino-1-(2-thienyl)-1-propanone is 100-150. The mass ratio of the ketoreductase to the substrate 3-methylamino-1-(2-thienyl)-1-propanone is 1-2%.

The method for biologically preparing (S)-3-methylamino-1-(2-thienyl)-1-propanol according to claim 1, wherein in the initial reaction system, Cofactor: glucose: glucose sugar dehydrogenase: mass ratio of 3-methylamino-1-(2-thienyl)-1-propanone (0.0005-0.002): (1.2-2): (0.01-0.05) ):1.

The biological preparation method of (S)-3-methylamino-1-(2-thienyl)-1-propanol according to claim 1, wherein the cofactor is NAD/NADH. Or NADP/NADPH.

The method for biologically preparing (S)-3-methylamino-1-(2-thienyl)-1-propanol according to claim 1, wherein the aqueous buffer is three Ethanolamine buffer solution.

The method for biologically producing (S)-3-methylamino-1-(2-thienyl)-1-propanol according to claim 1, wherein the asymmetric reduction reaction is at a pH of 7.0. The aqueous phase buffer is carried out.

The method for biologically producing (S)-3-methylamino-1-(2-thienyl)-1-propanol according to any one of claims 1, 4 and 5, which is characterized in that The pH of the aqueous phase buffer in which the symmetric reduction reaction is carried out is adjusted with a sodium hydroxide solution, a sodium carbonate solution or a sodium hydrogencarbonate solution.

The biological preparation method of (S)-3-methylamino-1-(2-thienyl)-1-propanol according to claim 1 or 2, wherein the preparation process is as follows : Add the substrate 3-methylamino-1-(2-thienyl)-1-propanone, glucose, and aqueous buffer to the reaction vessel in sequence, stir evenly, and continue to add ketone reductase, glucose dehydrogenase and The cofactor described above was stirred at 25-45 ° C, and the progress of the reaction was measured by HPLC. When the conversion rate reached 90-99%, the reaction was terminated.