CN103014082A - Biological preparation method of (R)-4-cyano-hydroxybutanoate - Google Patents
Biological preparation method of (R)-4-cyano-hydroxybutanoate Download PDFInfo
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- CN103014082A CN103014082A CN2012105563757A CN201210556375A CN103014082A CN 103014082 A CN103014082 A CN 103014082A CN 2012105563757 A CN2012105563757 A CN 2012105563757A CN 201210556375 A CN201210556375 A CN 201210556375A CN 103014082 A CN103014082 A CN 103014082A
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Abstract
The invention relates to a biological preparation method of (R)-4-cyano-hydroxybutanoate. According to the biological preparation method, (S)-4-chlorine-3-hydroxybutanoate is used as a substrate; under the action of a biocatalyst, the substrate reacts with sodium cyanide to generate a target product (R)-4-cyano-hydroxybutanoate; the biocatalyst is recombination halohydrin dehalogenase; and the reaction is carried out in a water phase with the pH of 6 to 8. According to the invention, by adopting the recombination halohydrin dehalogenase, the concentration of the substrate is improved and the enzyme dosage is reduced, so that production cost is reduced. Moreover, by optimizing the process, both purity and yield of the product are improved.
Description
Technical field
The invention belongs to bio-pharmaceuticals and Green Chemistry field, be specifically related to a kind of biological preparation method of (R)-4-cyano-3-hydroxy ethyl butyrate
Background technology
(atorvastatin, trade(brand)name Lipitor are that first annual sales amount surpasses 10,000,000,000 dollars medicine in the world Lipitor) to atorvastatin.It is by suppressing hydroxymethyl glutaryl CoA reductase enzyme, and the rate-limiting step that hinders cholesterol biosynthesis in the liver reduces the content of Blood Cholesterol.
The committed step of technique is to utilize synthetic (the R)-4-cyano-3-hydroxy ethyl butyrate (A5) (Angew.Chem.Int.Ed.2005,44:362-365) of (S)-4-chloro-3-hydroxyl ethyl butyrate (A4) single stage method now.
In this step reaction, because it is too much to form by product under the alkaline condition that biological process has been avoided existing in the chemical method, therefore the shortcoming of separation and purification difficulty utilizes halohydrin dehalogenase (halohydrin dehalogenase, HHDH) studied from the technique of the synthetic A5 of A4.US Patent No. 7125693 and US 7132267 have announced the research of Codexis company for this project, and its enzyme charging capacity is 1.5% (w/w) of substrate, and thick yield is 93%.Domestic patent CN 102168117A improves this technique afterwards, has realized the recycling of sodium cyanide, but its enzyme charging capacity up to 8.0% (w/w) of substrate, thick yield only is 83.8%.
The defectives such as comprehensive above literature search information in the technique that we find to exist at present, exists enzyme dosage too high, and reaction conditions is inappropriate.
Summary of the invention
Technical problem to be solved by this invention provides a kind of biological preparation method of improved (R)-4-cyano-3-hydroxy ethyl butyrate.
For solving above technical problem, the present invention adopts following technical scheme:
A kind of biological preparation method of (R)-4-cyano-3-hydroxy ethyl butyrate, it is take (S)-4-chloro-3-hydroxyl ethyl butyrate as substrate, this substrate is under the effect of biological catalyst, generate target product (R)-4-cyano-3-hydroxy ethyl butyrate with the sodium cyanide reaction, described biological catalyst is the restructuring halohydrin dehalogenase, the preparation method of described restructuring halohydrin dehalogenase is: with the single colony inoculation of halohydrin dehalogenase in the liquid LB substratum that contains amicillin resistance, in 30-40 ℃ of lower shaking table activation 8-12 hour, the culture that obtains after the activation is transferred in the liquid LB substratum that contains amicillin resistance, reach 0.6-0.8 in 30-40 ℃ of lower enlarged culturing to OD600 value, add inductor, continue to cultivate 8-12 hour in 25-35 ℃, centrifugal collecting precipitate, add phosphate buffered saline buffer and get suspension, suspension was placed the ice bath ultrasonic disruption 8-12 minute, centrifugal again, separation of supernatant, it is-10 ~-25 ℃ with supernatant liquor pre-freeze to temperature, and then freeze-drying 24-48 hour, namely get the restructuring halohydrin dehalogenase, described reaction is that the aqueous phase of 6-8 carries out at pH.
Further, in the reaction system when initial, the concentration of described substrate (S)-4-chloro-3-hydroxyl ethyl butyrate is 150-200mg/ml, and described restructuring halohydrin dehalogenase is the 0.4-1%(w/w of substrate (S)-4-chloro-3-hydroxyl ethyl butyrate).
Further, in the reaction system when initial, the mol ratio of described sodium cyanide and described substrate (S)-4-chloro-3-hydroxyl ethyl butyrate is 1.5-2.5:1.
Further, the transformation efficiency of substrate was 98-100% after reaction finished, and the product optical purity is greater than 99%, and purity is greater than 97%.
Preferably, described reaction is that 7.0 aqueous phase carries out at pH.
Preferably, the restructuring halohydrin dehalogenase described in the described reaction is the 0.5%(w/w of substrate (S)-4-chloro-3-hydroxyl ethyl butyrate).
Preferably, the concentration of substrate (S) in the described reaction-4-chloro-3-hydroxyl ethyl butyrate is 200mg/ml.
Preferably, described inductor is isopropyl-β-D-thiogalactoside(IPTG) or lactose.
Further, described preparation method's implementation process is as follows: add successively entry in reaction vessel, substrate (S)-4-chloro-3-hydroxyl ethyl butyrate, slowly drip sodium cyanide solution after stirring, add again the restructuring halohydrin dehalogenase, at 40-60 ℃ of lower stirring reaction, utilize GC detection reaction process, rate to be transformed is greater than 98% the time, add the sulphur acid for adjusting pH to 2-3, add ethyl acetate extraction, merge organic phase, rotary evaporation namely gets (R)-4-cyano-3-hydroxy ethyl butyrate product.
According to the present invention, except the restructuring halohydrin dehalogenase, all the other raw materials all can be commercially available by general channel.
Beneficial effect of the present invention is:
The present invention is by adopting restructuring halohydrin dehalogenase catalyzed reaction and by the optimization to technique, so that concentration of substrate improves, enzyme dosage reduces, thereby has reduced production cost, and so that the purity of product and yield all increase.
Embodiment
Reaction formula of the present invention is as follows:
The following examples can make the present invention of professional and technical personnel's comprehend, but do not limit the present invention in any way.
Embodiment one
From the glycerine pipe or transform dull and stereotyped single colony inoculation to the liquid LB substratum activation that 4mL contains amicillin resistance spend the night (37 ℃, 200rpm).From overnight culture with 1/100(v/v) inoculum size switching 100mL contains the liquid LB substratum of amicillin resistance, 37 ℃, 200rpm shaking culture to OD600 value reach 0.6-0.8, add IPTG in 30 ℃ of continuation overnight incubation.Centrifugal collecting cell is with 10mL phosphoric acid buffer (2mM, pH7.0) suspension cell.Cell suspending liquid placed the ice bath ultrasonic disruption 10 minutes.Centrifugal, supernatant liquor pre-freeze is spent the night.Freeze-drying 24h-48h.
Embodiment two
In the 50mL there-necked flask, add successively deionized water 23mL, substrate 7g; Rear slowly splashing in the 30% NaCN solution 5mL(dropping process with 20% sulphuric acid soln maintenance system pH 7.2～7.3 stirs), halohydrin dehalogenase powder 35mg, in 50 ° of C, the titration of pH 7.0(30%NaCN solution), under the 800rpm magnetic agitation condition, reaction 24h, GC detects transformation efficiency〉98%, add the sulphur acid for adjusting pH to 2-3, add the equal-volume ethyl acetate, diatomite filtration is collected organic phase, and water adds the equal-volume ethyl acetate extraction twice, merge organic phase, rotary evaporation obtains product 6.3g, purity〉98%, optical purity〉99%.
Embodiment three
In reactor, add successively deionized water 2.3L, substrate 700g; Rear slowly splashing in the 30% NaCN solution 0.5L(dropping process with 20% sulphuric acid soln maintenance system pH 7.2～7.3 stirs), halohydrin dehalogenase powder 3.5g, in 50 ° of C, the titration of pH 7.0(30%NaCN solution), under the mechanical stirring condition, reaction 24h, GC detects transformation efficiency〉98%, add the sulphur acid for adjusting pH to 2-3, add the equal-volume ethyl acetate, diatomite filtration is collected organic phase, water adds the equal-volume ethyl acetate extraction twice, merge organic phase, pass into nitrogen by alkali lye to slough HCN gas, rotary evaporation obtains about product 630g, purity〉98%, optical purity〉99%.
Above-described embodiment only is explanation technical conceive of the present invention and characteristics, and its purpose is to allow the personage who is familiar with technique can understand content of the present invention and according to this enforcement, can not limit protection scope of the present invention with this.All equivalences that spirit is done according to the present invention change or modify, and all should be encompassed within protection scope of the present invention.
Claims (6)
1. the biological preparation method of (R)-4-cyano-3-hydroxy ethyl butyrate, it is take (S)-4-chloro-3-hydroxyl ethyl butyrate as substrate, this substrate is under the effect of biological catalyst, generate target product (R)-4-cyano-3-hydroxy ethyl butyrate with the sodium cyanide reaction, it is characterized in that: described biological catalyst is the restructuring halohydrin dehalogenase, the preparation method of described restructuring halohydrin dehalogenase is: with the single colony inoculation of halohydrin dehalogenase in the liquid LB substratum that contains amicillin resistance, in 30-40 ℃ of lower shaking table activation 8-12 hour, the culture that obtains after the activation is transferred in the liquid LB substratum that contains amicillin resistance, reach 0.6-0.8 in 30-40 ℃ of lower enlarged culturing to OD600 value, add inductor, continue to cultivate 8-12 hour in 25-35 ℃, centrifugal collecting precipitate, add phosphate buffered saline buffer and get suspension, suspension was placed the ice bath ultrasonic disruption 8-12 minute, centrifugal again, separation of supernatant, it is-10 ~-25 ℃ with supernatant liquor pre-freeze to temperature, and then freeze-drying 24-48 hour, namely get the restructuring halohydrin dehalogenase, described reaction is that the aqueous phase of 6-8 carries out at pH.
2. the biological preparation method of (R)-4-cyano-3-hydroxy ethyl butyrate according to claim 1, it is characterized in that: in the reaction system when initial, the concentration of described substrate (S)-4-chloro-3-hydroxyl ethyl butyrate is 150-200 mg/ml, and described restructuring halohydrin dehalogenase is the 0.4-1%(w/w of substrate (S)-4-chloro-3-hydroxyl ethyl butyrate).
3. the biological preparation method of (R)-4-cyano-3-hydroxy ethyl butyrate according to claim 1, it is characterized in that: in the reaction system when initial, the mol ratio of described sodium cyanide and described substrate (S)-4-chloro-3-hydroxyl ethyl butyrate is 1.5-2.5:1.
4. the biological preparation method of (R)-4-cyano-3-hydroxy ethyl butyrate according to claim 1 is characterized in that: described reaction is that 7.0 aqueous phase carries out at pH.
5. the biological preparation method of (R)-4-cyano-3-hydroxy ethyl butyrate according to claim 1, it is characterized in that: described inductor is isopropyl-β-D-thiogalactoside(IPTG) or lactose.
6. the biological preparation method of each described (R)-4-cyano-3-hydroxy ethyl butyrate according to claim 1-3, it is characterized in that: described preparation method's implementation process is as follows: add successively entry in reaction vessel, substrate (S)-4-chloro-3-hydroxyl ethyl butyrate, slowly drip sodium cyanide solution after stirring, add again the restructuring halohydrin dehalogenase, at 40-60 ℃ of lower stirring reaction, utilize GC detection reaction process, rate to be transformed is greater than 98% the time, add the sulphur acid for adjusting pH to 2-3, add ethyl acetate extraction, merge organic phase, rotary evaporation namely gets (R)-4-cyano-3-hydroxy ethyl butyrate product.
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| CN201210556375.7A CN103014082B (en) | 2012-12-20 | 2012-12-20 | Biological preparation method of (R)-4-cyano-hydroxybutanoate |
| PCT/CN2013/077813 WO2014075447A1 (en) | 2012-11-19 | 2013-06-24 | Biological preparation method of ethyl (r)-4-cyano-hydroxybutanoate |
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| CN201210556375.7A CN103014082B (en) | 2012-12-20 | 2012-12-20 | Biological preparation method of (R)-4-cyano-hydroxybutanoate |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014075447A1 (en) * | 2012-11-19 | 2014-05-22 | 苏州汉酶生物技术有限公司 | Biological preparation method of ethyl (r)-4-cyano-hydroxybutanoate |
| CN104372038A (en) * | 2013-08-12 | 2015-02-25 | 南京朗恩生物科技有限公司 | Two-step catalytic preparation method of (R)-4-cyan-3-hydroxyvinyl butyrate |
| CN105567655A (en) * | 2014-10-14 | 2016-05-11 | 南京博优康远生物医药科技有限公司 | Halohydrin dehalogenase and its use in synthesis of statin drug intermediate |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7132267B2 (en) * | 2002-08-09 | 2006-11-07 | Codexis, Inc. | Enzymatic processes for the production of 4-substituted 3-hydroxybutyric acid derivatives and vicinal cyano, hydroxy substituted carboxylic acid esters |
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- 2012-12-20 CN CN201210556375.7A patent/CN103014082B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7132267B2 (en) * | 2002-08-09 | 2006-11-07 | Codexis, Inc. | Enzymatic processes for the production of 4-substituted 3-hydroxybutyric acid derivatives and vicinal cyano, hydroxy substituted carboxylic acid esters |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014075447A1 (en) * | 2012-11-19 | 2014-05-22 | 苏州汉酶生物技术有限公司 | Biological preparation method of ethyl (r)-4-cyano-hydroxybutanoate |
| CN104372038A (en) * | 2013-08-12 | 2015-02-25 | 南京朗恩生物科技有限公司 | Two-step catalytic preparation method of (R)-4-cyan-3-hydroxyvinyl butyrate |
| CN105567655A (en) * | 2014-10-14 | 2016-05-11 | 南京博优康远生物医药科技有限公司 | Halohydrin dehalogenase and its use in synthesis of statin drug intermediate |
| CN105567655B (en) * | 2014-10-14 | 2020-11-20 | 上海弈柯莱生物医药科技有限公司 | Halogen alcohol dehalogenase and application thereof in synthesis of statin drug intermediate |
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