WO 2015014031
Biological preparation method of
(S)-3-(dimethylamino)-1-(thiophene-2-radical)-1-propyl alcohol by EnzymeWorks Inc
Enzymeworks,Inc., 苏州汉酶生物技术有限公司 |
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The present invention relates to a biological preparation method of (S)-3-(dimethylamino)-1-(thiophene-2-radical)-1-propyl alcohol. According to the method, (S)-3-(dimethylamino)-1-(thiophene-2-radical)-1-acetone or salt thereof is used as a substrate, and the substrate is subjected to asymmetric reduction reaction in the presence of biocatalyst, cofactor and hydrogen donor to produce (S)-3-(dimethylamino)-1-(thiophene-2-radical)-1-propyl alcohol. Particularly, the biocatalyst is a combination of ketoreductase (KRED) and glucose dehydrogenase, the hydrogen donor is glucose, and the asymmetric reduction reaction is conducted at pH 6.8-7.0 and 25ºC-35ºC. Compared with the biological method in the prior art, the present invention has higher processing stability, and is simpler, more efficient and safer. During the reaction, the generation of highly toxic organic solvents such as acetone can be avoided, the application principle of green chemistry is met, and industrialized application is facilitated.
Enzyme catalyzed synthesis of (S)-3-(dimethylamino)-1-(thiophene-2-radical)-1-propanol, by asymmetric reduction of its corresponding ketone, using ketoreductase and glucose dehydrogenase. Useful as an intermediate in synthesis of duloxetine (first claimed in EP273658), which was developed and launched by Eli Lilly, in collaboration with Shionogi and ex-licensee Boehringer Ingelheim.
The advantage of the present process is its high efficiency and green chemistry approach in synthesis of the drug. Follows on from WO2014094462, claiming a similar method for preparing duloxetine intermediates.
Duloxetine (Duloxetine) is a low side effect, can effectively treat mental disorders and metabolic disorders drugs (US 5,023,269).The key is to obtain the synthesis of duloxetine intermediates contain a chiral center (S) -3- (dimethylamino) -1- (thiophen-2-yl) -1-propanol (DMAA). Thus the asymmetric reduction of 3- (dimethylamino) -1- (thiophen-2-yl) -1-propanone (DMAK) to give the intermediate is one of the most effective, most studied methods. In achieving this pathway chemical reduction method, due to the need catalytic ruthenium metal catalyst through reaction with hydrogen, its economy, safety and environmental friendliness does not meet the needs of production. Use of chiral resolving agents as chemical resolving agent split method, due to low yield and poor economic reasons, it can not serve as the main production methods. Therefore, the use of enzymatic reduction target product, with its high efficiency and environmental friendliness has become the most effective method.
[5]
In the biological reduction method, U.S. Patent US 2010/0151534 A1 discloses a ketoreductase (KRED) produced (S) -3- (dimethylamino) -1- (thiophen-2-yl) -1- propanol method, the process requires strict control of temperature and reaction, and adding isopropanol, while the reaction of the need to introduce the negative reaction of acetone to remove byproducts, complex operation, it is difficult to repeat, and acetone are dangerous goods, easy volatile, flammable.
[6]
SUMMARY
[7]
Technical problem to be solved by the present invention is to overcome the deficiencies of the prior art, to provide an improved (S) -3- (dimethylamino) -1- (thiophen-2-yl) -1-propanol biological Methods.
[8]
To solve the above technical problem, the present invention takes the following technical solutions: one (S) -3- (dimethylamino) -1- (thiophen-2-yl) -1-propanol biological preparation method, the method 3- (dimethylamino) -1- (thiophen-2-yl) -1-propanone (DMAK) or a salt thereof as a substrate, so that the substrate in the presence of a biocatalyst, cofactor, and a hydrogen donor asymmetric reduction reaction of (S) -3- (dimethylamino) -1- (thiophen-2-yl) -1-propanol, wherein the biological catalyst is ketoreductase (KRED) and glucose dehydrogenase combination, the hydrogen donor is glucose, the asymmetric reduction reaction at a pH of 6.8 to 7.0 and a temperature of 25 ℃ ~ 35 ℃ in performed.
[9]
According to a particular embodiment of the invention embodiment, the biocatalyst, a hydrogen donor, cofactors, substrates feed weight ratio can be 0.02 to 0.1: 1.5 to 6: 0.01 to 0.1: 1, preferably 0.04 to 0.08: 1.5 to 3 : 0.01 to 0.05: 1.
[10]
Preferably, the biocatalyst ketoreductase and glucose dehydrogenase weight ratio of 1: 1 ~ 3. More preferably, the ketoreductase and glucose dehydrogenase weight ratio of 1: 2.
[11]
According to a preferred embodiment of the present invention, may be a cofactor NAD / NADH or NADP / NADPH.
[12]
Asymmetric reduction reaction temperature is preferably from 28 ℃ ~ 32 ℃. Asymmetric reduction reaction is preferably carried out in an aqueous buffer phase.
WO2014094462
http://www.google.com/patents/WO2014094462A1?cl=en
Example one
Three to 50 mL reaction flask add 1.0 g substrate MMAK and 1.2g glucose, 8 mL reaction flask again with a good pH of 7.0,0.1 M triethanolamine buffer. Placed in 30 ℃, 900 r / min magnetic stirring water bath with stirring and treated with 2 M NaOH solution to adjust pH to 7.0. When the temperature was stabilized, the reaction flask was added 0.95 mL solution of 20 mg ketoreductase, 31 mg GDH triethanolamine buffer. PH adjusted to 7.0 with NaOH, and washed with 0.95 mL triethanolamine buffer was added to the reaction flask. To the reaction flask was added 100 μL of a solution of 0.5 mg NADP triethanolamine buffer, the reaction started. The reaction temperature was maintained at 30 ℃. The pH of the reaction solution was added dropwise 2 M NaOH solution was adjusted by a pH titrator. Control pH 7.0, initial control point 6.5, final control point is 6.99. HPLC assay for controlling timing of sampling. Reaction 23 h, the conversion rate of> 99%.
Example two
Add 100.0 g and 120.0 g of substrate MMAK glucose into the reactor, was added to the reaction flask with 800 mL of a good pH 7.0,0.1 M triethanolamine buffer. Placed in 30 ℃, mechanically stirred water bath with stirring and treated with 2 M NaOH solution to adjust pH to 7.0. When the temperature was stabilized, the reaction flask was added 95 mL a solution of 2 g ketoreductase, 3.1 g GDH triethanolamine buffer. PH adjusted to 7.0 with NaOH, and washed with 95 mL of triethanolamine buffer was added to the reaction flask. To the reaction flask with 10 mL of a solution of 50 mg NADP triethanolamine buffer, the reaction started. The reaction temperature was maintained at 30 ℃. The pH of the reaction solution was added dropwise 2 M NaOH solution was adjusted by a pH titrator. Control pH 7.0, initial control point 6.5, final control point is 6.99. HPLC assay for controlling timing of sampling. Reaction 23 h, the conversion rate of> 98%.
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| CN101993828A * | Nov 8, 2010 | Mar 30, 2011 | 江南大学 | Method for improving transformation efficiency of (S)-phenyl glycol by coupling glucose-6-phosphate dehydrogenase and (S)-carbonyl reductase |
| CN102206686A * | Apr 19, 2011 | Oct 5, 2011 | 华东理工大学 | Preparation method of methyl (R)-o-chloromandelate utilizing biocatalytic asymmetric reduction |
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substituted alkanones such as 3-methylamino-1-(2-thienyl)-propan-1-one;
biosynthesis; expression cassettes; host cells; for production of
duloxetine |
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