WO2014180085A1 - Biological preparation method for chiral n-protection peridol - Google Patents
Biological preparation method for chiral n-protection peridol Download PDFInfo
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- WO2014180085A1 WO2014180085A1 PCT/CN2013/083267 CN2013083267W WO2014180085A1 WO 2014180085 A1 WO2014180085 A1 WO 2014180085A1 CN 2013083267 W CN2013083267 W CN 2013083267W WO 2014180085 A1 WO2014180085 A1 WO 2014180085A1
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- C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
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- the invention belongs to the technical field of biopharmaceuticals and biochemicals, and particularly relates to a biological preparation method of chiral N-protected piperidinol.
- (S)-N-Boc-3-hydroxypiperidinol can be obtained from N-Boc-3- Hydroxypiperidone is prepared while introducing a chiral center.
- Traditional ketone synthesis methods have chemical reduction and biological reduction. The use of chemical catalysis to achieve this process has been reported, but due to low efficiency, harsh reaction conditions, resulting in three wastes, not able to achieve industrial applications (US patent 2011092698 A1, WO 2011036280 A1).
- the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide an improved biological preparation method of chiral N-protected piperidinol.
- the present invention provides a biological preparation method of chiral N-protected piperidinol, and the structural formula of chiral N-protected piperidinol is as follows: Wherein R represents a protecting group;
- the preparation method comprises the N-protected piperidone as a substrate, and the substrate is asymmetrically reduced in the presence of a biocatalyst, a cofactor and a hydrogen donor to form a chiral N-protected piperidinol, which is characterized in that:
- the biocatalyst is a recombinant ketoreductase prepared by inoculating a single colony of recombinant Escherichia coli containing a ketoreductase gene into a liquid LB medium containing ampicillin resistance at 35-40 ° C.
- the culture obtained after activation was inoculated into ampicillin-resistant liquid LB medium, cultured at 35-40 ° C, and cultured until the OD 600 value reached 0.6-0.8.
- the agent is further cultured at 25 ⁇ 33 °C for 8 ⁇ 12 hours, centrifuged, and the precipitate is collected.
- the suspension is added with phosphate buffer solution.
- the suspension is ultrasonically disrupted in an ice water bath for 8 ⁇ 12 minutes, and then centrifuged.
- the supernatant is pre-frozen until the temperature drops to -10 °C ⁇ -25 °C, and then freeze-dried for 24 to 48 hours to obtain a freeze-dried powdery recombinant ketoreductase; the hydrogen donor is isopropanol, Asymmetric reduction in aqueous buffer at pH 7.0 ⁇ 9.0 The temperature is 25 ° C ⁇ 45 ° C.
- the concentration of the substrate in the reaction system at the start of the reaction is 3% to 20% (w/v).
- the amount of the recombinant ketone reductase is 2% to 8% (w/w) of the mass of the substrate, and the amount of the isopropanol is 1.5 to 6 times the amount of the substrate, and the amount of the cofactor is the substrate. 0.02% ⁇ 1% of quality (w/w).
- the concentration of the substrate in the reaction system at the start of the reaction is 4% to 10% (w/v), and the amount of the recombinant ketoreductase is 4% to 6% (w/w) of the mass of the substrate.
- the amount of the substance of the isopropanol is 4 to 6 times the amount of the substance of the substrate, and the amount of the cofactor is 0.4% to 0.6% (w/w) of the mass of the substrate.
- the preparation process is as follows: first, N- Protect the piperidone in a part of isopropyl alcohol, then add the aqueous buffer, stir evenly, add the recombinant ketoreductase and cofactor, and maintain at 25 °C ⁇ 45 °C, start At the same time, the air pump was turned on to inflate the air into the reaction system, and the remaining isopropanol was added at a constant rate, and the reaction was monitored by HPLC until the reaction conversion rate reached 99.0% or more, and the reaction was terminated.
- the inducer may be isopropyl-b-D-thiogalactoside (IPTG); the cofactor may be NAD/NADH or NADP/NADPH.
- IPTG isopropyl-b-D-thiogalactoside
- the cofactor may be NAD/NADH or NADP/NADPH.
- the aqueous phase buffer may be Tri-HCl buffer, TEA-HCl buffer or phosphate buffer.
- the pH of the aqueous buffer is preferably 7 to 7.5.
- the temperature of the asymmetric reduction reaction is preferably from 28 ° C to 32 ° C.
- the present invention has the following advantages compared with the prior art:
- the method of the invention successfully prepares a chiral with high optical purity by using a specific recombinant ketoreductase instead of the source unstable catalyst used in the existing biological method.
- N-protected piperidinol The method has mild reaction conditions, high reaction efficiency and simple operation. In particular, the concentration of the reaction substrate can be increased to 20%, and the preparation of chiral N-protected piperidinol is greatly improved. The efficiency and cost of the reaction are reduced, and have important practical industrial application value.
- Figure 1 is a synthetic route of the present invention
- Example 2 is an HPLC chromatogram of a sample sampled in Example 2 for 1 h reaction
- Figure 3 is a HPLC chromatogram of the sample obtained by sampling in 20 hours of the reaction in Example 2.
- the present invention aims to provide a method for preparing a chiral N-protected piperidinol by asymmetric reduction of N-protected piperidone with a recombinant ketone reductase and isopropanol.
- the structural formula of the chiral N-protected piperidinol is as follows : Wherein R represents a protecting group and may be tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, benzyl or the like.
- Asymmetric reduction can be carried out in an aqueous buffer at pH 7.0 ⁇ 9.0 at a temperature of 25 °C It is carried out at ⁇ 45 ° C, the reaction conditions are mild, and the operation is simple.
- a single colony of recombinant E. coli containing the ketoreductase gene was inoculated from 4 g of ampicillin-resistant liquid LB medium from a glycerol tube or a transformation plate overnight (37 ° C, 200 rpm). 100 mL of ampicillin-resistant liquid LB medium was transferred from the overnight culture at 1/100 inoculation, and cultured at 37 ° C, 200 rpm to an OD 600 value of 0.6-0.8, and IPTG was added to continue incubation at 30 ° C overnight. The cells were collected by centrifugation and suspended in 10 mL of phosphate buffer (2 mM, pH 7.0).
- the cell suspension was ultrasonically disrupted in an ice bath for 10 minutes, centrifuged, and the supernatant was pre-frozen overnight, and lyophilized for 24 h to 48 h to obtain a freeze-dried powdery ketone reductase KRED.
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Abstract
Provided is a biological preparation method for chiral N- protection peridol, in which N-protection piperidone is used as a substrate, and the substrate is subjected to an asymmetric reduction reaction in the presence of a reconstructed ketone reduction enzyme, a cofactor, and isopropyl alcohol to generate chiral N- protection peridol. The asymmetric reduction reaction takes place in an aqueous buffer having a pH value of 7.0 to 9.0 at the temperature of 25°C to 45°C, so as to successfully prepare chiral N- protection peridol having a high optical purity. The method has temperate reaction conditions, high reaction efficiency, and simple operations; especially, the concentration of the reaction substrate can be increased to 20%, so that the method has important and practical industrial application value.
Description
技术领域 Technical field
本发明属于生物制药和生物化工技术领域,具体涉及一种手性 N- 保护哌啶醇的生物制备方法。 The invention belongs to the technical field of biopharmaceuticals and biochemicals, and particularly relates to a biological preparation method of chiral N-protected piperidinol.
背景技术 Background technique
大量具备重要生物活性的天然和非天然物质的结构中都包含有一个哌啶环。因此,在 3-
位上的碳原子衍生化之后形成的手性中心对于这些生物活性物质的性质有着非常重要的影响。在如何引入此手性中心的问题上,大量工作研究了还原 3- 位上的 酮酯
( J. Chem. Soc.,
Perkin Trans. 1998, 22, 3673-3684, Acta Chem. Scand. 1998, 52, 461-468.)
。然而,对于并不含有酯结构的手性哌啶醇,特别是手性 N- 保护哌啶醇这类具有重要药物合成价值的化合物,对其如何合成却缺乏必要的研究。A large number of natural and non-natural substances with important biological activities contain a piperidine ring. Therefore, the chiral centers formed after the carbon atom derivatization at the 3-position have a very important influence on the properties of these bioactive substances. On the issue of how to introduce this chiral center, a lot of work has been done to reduce the ketoester at the 3-position. (J. Chem. Soc., Perkin Trans. 1998, 22, 3673-3684, Acta Chem. Scand. 1998, 52, 461-468.). However, for chiral piperidinol which does not contain an ester structure, especially a compound having important drug synthesis value such as chiral N-protected piperidinol, there is no necessary study on how to synthesize it.
(S)-N-Boc-3- 羟基哌啶醇 ( 结构式:
)
可以用于合成一种非天然药物抗充血性心力衰竭药卡莫瑞林 (Bio. Med. Chem. 2003, 11, 581-590) 、 BTK 抑制剂
ibrutinib 、天然物质异白刺啉淋胺 (isonitramine) 和小果白刺碱 (sibirine ) 等药物前体 (Tetrahedron
Lett. 1989, 30, 2301-2304, J. Org. Chem. 1984, 49, 1688-1691 等 )
,因此该类化合物具有广泛的应用前景。(S)-N-Boc-3-hydroxypiperidinol (Structure: Can be used to synthesize a non-natural drug anti-congestive heart failure drug carmerilin (Bio. Med. Chem. 2003, 11, 581-590), BTK inhibitor ibrutinib, natural substance isoalporin ( Isonitramine) and prodrugs such as sibirine (Tetrahedron Lett. 1989, 30, 2301-2304, J. Org. Chem. 1984, 49, 1688-1691, etc.), therefore such compounds have a wide range of Application prospects.
(S)-N-Boc-3- 羟基哌啶醇可以由 N-Boc-3-
羟基哌啶酮制得,同时引入手性中心。传统的酮合成法有化学还原和生物还原。利用化学催化方法实现这一过程已有报道,但是由于效率较低,反应条件苛刻,产生的三废较多,没有能够实现工业化应用
(US patent 2011092698 A1, WO 2011036280 A1) 。 (S)-N-Boc-3-hydroxypiperidinol can be obtained from N-Boc-3-
Hydroxypiperidone is prepared while introducing a chiral center. Traditional ketone synthesis methods have chemical reduction and biological reduction. The use of chemical catalysis to achieve this process has been reported, but due to low efficiency, harsh reaction conditions, resulting in three wastes, not able to achieve industrial applications
(US patent 2011092698 A1, WO 2011036280 A1).
虽然利用生物转化实现还原反应生产手性仲醇已经成为成熟可靠的技术,但是通过文献检索,只有一个利用胡萝卜整细胞催化该还原反应的实例,所用催化剂较多 (
占底物质量的 23.4%) ,且来源不稳定。植物细胞反应涉及到品种、产地和季节,含有复杂的成分,对下游分离提取不利。更重要的是,该反应底物浓度较低 (3.3
mM) ,产物光学纯度只有 98% ,不具备实际工业应用价值 (Org. Lett. 2009, 11, 1245-1248) 。
Although the use of biotransformation to reduce the production of chiral secondary alcohols has become a mature and reliable technology, only one example of the use of carrot whole cells to catalyze this reduction reaction has been used in the literature search.
It accounts for 23.4% of the substrate quality and the source is unstable. Plant cell reactions involve varieties, origins, and seasons, and contain complex components that are detrimental to downstream separation and extraction. More importantly, the reaction substrate concentration is low (3.3
mM), the product optical purity is only 98%, does not have practical industrial application value (Org. Lett. 2009, 11, 1245-1248).
发明内容 Summary of the invention
本发明所要解决的技术问题是克服现有技术的不足,提供一种改进的手性 N- 保护哌啶醇的生物制备方法。 The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide an improved biological preparation method of chiral N-protected piperidinol.
为解决以上技术问题,本发明 提供一种手性 N- 保护哌啶醇的生物制备方法 ,手性 N- 保护哌啶醇的结构式
如下:
, 其中 R 代表保护基;In order to solve the above technical problems, the present invention provides a biological preparation method of chiral N-protected piperidinol, and the structural formula of chiral N-protected piperidinol is as follows: Wherein R represents a protecting group;
该制备方法以 N- 保护哌啶酮为底物,使该底物 在生物催化剂、辅因子及氢供体的存在下发生不对称还原反应生成
手性 N- 保护哌啶醇,其特征在于:所述生物催化剂为重组酮还原酶,该酮还原酶的制备方法为:将含有酮还原酶基因的重组大肠杆菌单菌落接种到含氨苄青霉素抗性的液体
LB 培养基中,于 35~40℃下活化8~12 小时,将活化后得到的培养物接种到含氨苄青霉素抗性的液体 LB 培养基中,于
35~40℃下振荡培养,培养至OD600 值达到 0.6~0.8 时,加入诱导剂,于 25~33℃下继续培养8~12
小时,离心,收集沉淀物,加入磷酸盐缓冲液得悬浮液,将悬浮液置于冰水浴中超声破碎 8~12 分钟,再离心,将上清液预冻至温度降至 -10 ℃
~-25℃,然后再冻干24~48 小时,即得冻干粉状的重组酮还原酶;所述的氢供体为异丙醇,所述不对称还原反应在 pH 值为 7.0~9.0
的水相缓冲液中、温度 25 ℃ ~45℃下进行。The preparation method comprises the N-protected piperidone as a substrate, and the substrate is asymmetrically reduced in the presence of a biocatalyst, a cofactor and a hydrogen donor to form a chiral N-protected piperidinol, which is characterized in that: The biocatalyst is a recombinant ketoreductase prepared by inoculating a single colony of recombinant Escherichia coli containing a ketoreductase gene into a liquid LB medium containing ampicillin resistance at 35-40 ° C. After activation for 8 to 12 hours, the culture obtained after activation was inoculated into ampicillin-resistant liquid LB medium, cultured at 35-40 ° C, and cultured until the OD 600 value reached 0.6-0.8. The agent is further cultured at 25~33 °C for 8~12 hours, centrifuged, and the precipitate is collected. The suspension is added with phosphate buffer solution. The suspension is ultrasonically disrupted in an ice water bath for 8~12 minutes, and then centrifuged. The supernatant is pre-frozen until the temperature drops to -10 °C ~ -25 °C, and then freeze-dried for 24 to 48 hours to obtain a freeze-dried powdery recombinant ketoreductase; the hydrogen donor is isopropanol, Asymmetric reduction in aqueous buffer at pH 7.0~9.0 The temperature is 25 ° C ~ 45 ° C.
根据本发明的进一步实施方案, 反应起始时的反应体系中,底物的浓度为 3%~20% (w/v)
,重组酮还原酶的用量为底物质量的 2%~8% (w/w) ,异丙醇的物质的量为底物的物质的量的 1.5~6 倍,辅因子的用量为底物质量的 0.02%~1%
(w/w) 。 进一步优选地, 反应起始时的反应体系中,底物的浓度为 4%~10% (w/v) ,重组酮还原酶的用量为底物质量的 4%~6% (w/w)
,异丙醇的物质的量为底物的物质的量的 4~6 倍,辅因子的用量为底物质量的 0.4%~0.6% (w/w) 。 According to a further embodiment of the present invention, the concentration of the substrate in the reaction system at the start of the reaction is 3% to 20% (w/v).
The amount of the recombinant ketone reductase is 2% to 8% (w/w) of the mass of the substrate, and the amount of the isopropanol is 1.5 to 6 times the amount of the substrate, and the amount of the cofactor is the substrate. 0.02%~1% of quality
(w/w). Further preferably, the concentration of the substrate in the reaction system at the start of the reaction is 4% to 10% (w/v), and the amount of the recombinant ketoreductase is 4% to 6% (w/w) of the mass of the substrate.
The amount of the substance of the isopropanol is 4 to 6 times the amount of the substance of the substrate, and the amount of the cofactor is 0.4% to 0.6% (w/w) of the mass of the substrate.
根据本发明的一个具体实例,制备方法的实施过程如下:先将 N-
保护哌啶酮溶解在一部分异丙醇中,然后加入水相缓冲液,搅拌均匀后,加入重组酮还原酶与辅因子,维持在 25 ℃ ~45℃,开始
反应,同时开启充气泵向反应体系中鼓入空气,恒速流加剩余的异丙醇, HPLC 监控,至反应转化率达到 99.0 % 以上,终止反应。 According to a specific example of the present invention, the preparation process is as follows: first, N-
Protect the piperidone in a part of isopropyl alcohol, then add the aqueous buffer, stir evenly, add the recombinant ketoreductase and cofactor, and maintain at 25 °C ~ 45 °C, start
At the same time, the air pump was turned on to inflate the air into the reaction system, and the remaining isopropanol was added at a constant rate, and the reaction was monitored by HPLC until the reaction conversion rate reached 99.0% or more, and the reaction was terminated.
进一步地,反应终止后,
向反应液中加入硅藻土搅拌,过滤,滤液用乙酸乙酯萃取,滤饼返回反应釜,加入乙酸乙酯搅拌过滤,合并有机相;有机相用饱和食盐水洗涤 2~3
次,浓缩后得到黄色油状物,加石油醚于黄色油状物中,于 45~50 ℃下搅拌至均相,于-10℃~-20℃下静置,过滤后得到 手性 N- 保护哌啶醇。 Further, after the reaction is terminated,
To the reaction mixture, celite was added and stirred, and the filtrate was extracted with ethyl acetate. The filter cake was returned to the reaction vessel, and ethyl acetate was added thereto, and the organic phase was combined; the organic phase was washed with saturated brine.
Then, after concentration, a yellow oil is obtained. Add the petroleum ether to a yellow oil, stir at 45 to 50 ° C until homogeneous, and let stand at -10 ° C to -20 ° C to obtain a chiral N-protected pipe. Pyridinol.
根据本发明, 诱导剂可以为异丙基 - b -D- 硫代半乳糖苷( IPTG ) ; 辅因子可以为
NAD/NADH 或 NADP/NADPH 。 According to the present invention, the inducer may be isopropyl-b-D-thiogalactoside (IPTG); the cofactor may be
NAD/NADH or NADP/NADPH.
在根据本发明的优选实例中,水相缓冲液可以为 Tri-HCl 缓冲液、 TEA-HCl 缓冲液或磷酸缓冲液。
水相缓冲液的 pH 值优选为 7~7.5 。 不对称还原反应的温度优选为 28 ℃ ~32℃。 In a preferred embodiment according to the invention, the aqueous phase buffer may be Tri-HCl buffer, TEA-HCl buffer or phosphate buffer.
The pH of the aqueous buffer is preferably 7 to 7.5. The temperature of the asymmetric reduction reaction is preferably from 28 ° C to 32 ° C.
根据一个具体和优选方面, 手性 N- 保护哌啶醇的结构式如下: According to a specific and preferred aspect, the structural formula of the chiral N-protected piperidinol is as follows:
, 其化学名称为 1- 叔丁氧羰基 -3- 哌啶醇或N-Boc-3- 羟基哌啶。 Its chemical name is 1-tert-butoxycarbonyl-3-piperidinol or N-Boc-3-hydroxypiperidine.
本说明书中公开的所有特征,或公开的所有方法或过程中的步骤,除了互相排斥的特征和 /
或步骤以外,均可以以任何方式组合。 All features disclosed in this specification, or steps in all methods or processes disclosed, except for mutually exclusive features and /
In addition to or in addition to the steps, they may be combined in any manner.
由于以上技术方案的实施,本发明与已有技术相比具有如下优势: Due to the implementation of the above technical solutions, the present invention has the following advantages compared with the prior art:
本发明方法通过采用特定的重组酮还原酶来代替现有生物法所用的来源不稳定的催化剂,成功制备出光学纯度高的手性
N- 保护哌啶醇。该方法反应条件温和,反应效率高,操作简便,特别是,反应底物的浓度可以提高至 20% ,大大提高了制备手性 N- 保护哌啶醇
的效率和降低了反应的成本,具有重要的实际工业应用价值。 The method of the invention successfully prepares a chiral with high optical purity by using a specific recombinant ketoreductase instead of the source unstable catalyst used in the existing biological method.
N-protected piperidinol. The method has mild reaction conditions, high reaction efficiency and simple operation. In particular, the concentration of the reaction substrate can be increased to 20%, and the preparation of chiral N-protected piperidinol is greatly improved.
The efficiency and cost of the reaction are reduced, and have important practical industrial application value.
附图说明 DRAWINGS
图 1 为本发明的合成路线; Figure 1 is a synthetic route of the present invention;
图 2 为实施例 2 中,反应 1h 取样所得样品的 HPLC 图谱; 2 is an HPLC chromatogram of a sample sampled in Example 2 for 1 h reaction;
图 3 为实施例 2 中,反应 20h 取样 所得样品的 HPLC 图谱。 Figure 3 is a HPLC chromatogram of the sample obtained by sampling in 20 hours of the reaction in Example 2.
具体实施方式 detailed description
本发明旨在提供一种利用重组酮还原酶和异丙醇不对称还原 N- 保护哌啶酮制备 手性 N- 保护哌啶醇的方法
,所述手性 N- 保护哌啶醇的结构式如下:
, 其中 R
代表保护基,可以是叔丁氧羰基,苄氧羰基,乙酰基,苄基等。The present invention aims to provide a method for preparing a chiral N-protected piperidinol by asymmetric reduction of N-protected piperidone with a recombinant ketone reductase and isopropanol. The structural formula of the chiral N-protected piperidinol is as follows : Wherein R represents a protecting group and may be tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, benzyl or the like.
不对称还原反应可以在 pH 为 7.0~9.0 的水相缓冲液中、温度 25 ℃
~45℃下进行,反应条件温和,操作简便。 Asymmetric reduction can be carried out in an aqueous buffer at pH 7.0~9.0 at a temperature of 25 °C
It is carried out at ~45 ° C, the reaction conditions are mild, and the operation is simple.
以 R 代表叔丁氧羰基的 手性 N- 保护哌啶醇为例,其合成路线如图 1 所示。 Taking the chiral N-protected piperidinyl group represented by R as a tert-butoxycarbonyl group as an example, the synthetic route is shown in Fig. 1.
下面结合具体实施例对本发明做进一步详细的说明,但本发明并不限于以下实施例。 The present invention will be further described in detail below with reference to specific embodiments, but the invention is not limited to the following examples.
实施例 1 重组酮还原酶( KRED )酶粉摇瓶生产工艺 Example 1 Recombinant ketone reductase (KRED) enzyme powder shake flask production process
从甘油管或转化平板将含有酮还原酶基因的重组大肠杆菌单菌落接种到 4 mL 含氨苄青霉素抗性的液体 LB
培养基活化过夜( 37 ℃ , 200 rpm )。从过夜培养物以 1/100 接种量转接 100 mL 含氨苄青霉素抗性的液体 LB 培养基, 37 ℃ 、
200 rpm 振荡培养至 OD600 值达到 0.6-0.8 ,加入 IPTG 于 30 ℃ 继续培养过夜。离心收集细胞,用 10
mL 磷酸缓冲液( 2 mM , pH 7.0 )悬浮细胞。细胞悬浮液置于冰浴中超声波破碎 10 分钟,离心,上清液预冻过夜,冻干 24h-48h
,即得冻干粉状的酮还原酶 KRED 。A single colony of recombinant E. coli containing the ketoreductase gene was inoculated from 4 g of ampicillin-resistant liquid LB medium from a glycerol tube or a transformation plate overnight (37 ° C, 200 rpm). 100 mL of ampicillin-resistant liquid LB medium was transferred from the overnight culture at 1/100 inoculation, and cultured at 37 ° C, 200 rpm to an OD 600 value of 0.6-0.8, and IPTG was added to continue incubation at 30 ° C overnight. The cells were collected by centrifugation and suspended in 10 mL of phosphate buffer (2 mM, pH 7.0). The cell suspension was ultrasonically disrupted in an ice bath for 10 minutes, centrifuged, and the supernatant was pre-frozen overnight, and lyophilized for 24 h to 48 h to obtain a freeze-dried powdery ketone reductase KRED.
实施例 2 百毫克级制备工艺 Example 2 One hundred milligrams of preparation process
在 5ml 反应瓶中加入 150 mg N-Boc- 哌啶酮及 0.1 ml
异丙醇,搅拌至底物完全溶解,随后加入 1.2 ml TEA-HCl 缓冲液 (0.1M , pH 7.0) 、 7.5 mg 酮还原酶粉 (0.5 U/mg
,溶于 0.1 ml 缓冲液 ) 、 NAD+0.75 mg ( 溶于 0.1 ml 缓冲液 ) ,在 30 ℃ 下磁力搅拌反应 1h
和 20 h 后取样进行 HPLC 分析,图谱分别见图 2 (反应 1 小时样品,保留时间 5.86 分钟为产物, 10.75 分钟为底物)和图 3 (反应
20 小时样品, 5.86 分钟为产物),最终转化率 >99.5% 。Add 150 mg of N-Boc-piperidone and 0.1 ml of isopropanol to a 5 ml reaction flask, stir until the substrate is completely dissolved, then add 1.2 ml of TEA-HCl buffer (0.1 M, pH 7.0) and 7.5 mg of ketone. Enzyme powder (0.5 U/mg, dissolved in 0.1 ml buffer), NAD + 0.75 mg (dissolved in 0.1 ml buffer), magnetically stirred at 30 °C for 1 h and 20 h, and then sampled for HPLC analysis. 2 (reaction 1 hour sample, retention time 5.86 minutes for product, 10.75 minutes for substrate) and Figure 3 (reaction 20 hours sample, 5.86 minutes for product), final conversion > 99.5%.
实施例 3 百克级制备工艺 Example 3 100 g grade preparation process
将 450 g (2.26 mol) N-Boc-3- 哌啶酮加至 20 L 反应釜中,加入 0.3 L
异丙醇,搅拌至完全溶解;加入 9.0 L Tris-HCl 缓冲液 (0.1 M, pH 7.0) ,于 30 ℃ 下搅拌 (450 rpm)
均匀;然后依次加入 22.5 g 酮还原酶粉 (0.5 u/mg) 与 2.25 g NAD+
,开始反应;同时开启充气泵向反应体系中鼓入空气,恒速流加异丙醇 0.6 L , 20 h 滴完。 HPLC 监控,反应 24 h 后,转化率 99.3 %
,终止反应。Add 450 g (2.26 mol) of N-Boc-3-piperidone to the 20 L reactor, add 0.3 L of isopropanol, stir until completely dissolved; add 9.0 L of Tris-HCl buffer (0.1 M, pH 7.0) Stir at 30 °C (450 rpm); then add 22.5 g of ketone reductase powder (0.5 u/mg) and 2.25 g of NAD + to start the reaction; simultaneously turn on the air pump to blow air into the reaction system. Speed flow plus isopropanol 0.6 L, 20 h drop. The reaction was monitored by HPLC, and after 24 h of reaction, the conversion rate was 99.3 %, and the reaction was terminated.
向反应液中加入硅藻土( 50g )搅拌 0.5 h ,过滤,滤液用乙酸乙酯萃取 (3.0 L × 2)
,滤饼返回反应釜,加入乙酸乙酯搅拌过滤,合并有机相;有机相用饱和食盐水洗涤两次,浓缩后得到 0.49 kg 黄色油状物。加 0.5 L
石油醚于黄色油状物中,于 50 ℃ 下搅拌至均相,于 -20 ℃ 下静置 8 h ,过滤后得到 N-Boc-3- 羟基哌啶 0.37 kg ,收率 81.4
% , GC 纯度 98.7 % , ee 值 99.5 % ; 1 H-NMR (400 MHz ,
CDCl3): δ = 1.443 - 1.458 [m, 11 H, -(CH3)3,
-CH2], 1.727 - 1.883 [d, 2 H, -CH2], 3.039 - 3.125 [m, 2
H, -CH2], 3.539 [s, 1 H, -CHOH], 3.731 - 3.765 [m, 2 H,
-CH2] ; MS(ESI): 202 (M+H)+ 。Add diatomaceous earth (50g) to the reaction mixture, stir for 0.5 h, filter, extract the filtrate with ethyl acetate (3.0 L × 2), return the filter cake to the reaction kettle, add ethyl acetate and filter, and combine the organic phase; It was washed twice with saturated brine and concentrated to give a white oil. Add 0.5 L of petroleum ether in a yellow oil, stir to a homogeneous phase at 50 ° C, and let stand at -20 ° C for 8 h. After filtration, N-Boc-3-hydroxypiperidine 0.37 kg is obtained, the yield is 81.4 %. GC purity 98.7 %, ee value 99.5 %; 1 H-NMR (400 MHz , CDCl 3 ): δ = 1.443 - 1.458 [m, 11 H, -(CH3) 3 , -CH 2 ], 1.727 - 1.883 [d, 2 H, -CH 2 ], 3.039 - 3.125 [m, 2 H, -CH 2 ], 3.539 [s, 1 H, -CHOH], 3.731 - 3.765 [m, 2 H, -CH 2 ] ; MS (ESI ): 202 (M+H)+ .
实施例 4 公斤 级制备工艺 Example 4 kg-level preparation process
将 1.35 kg (6.78 mol) N-Boc-3- 哌啶酮加至 50 L 反应釜中,加入 0.9
L 异丙醇,搅拌至完全溶解;加入 27.0 L Tris-HCl 缓冲液 (0.1 M, pH 7.0) ,于 30 ℃ 下搅拌 (450 rpm)
均匀;然后依次加入 67.5 g 酮还原酶粉 (0.5 u/mg) 与 6.75 g NAD+
,开始反应;同时开启充气泵向反应体系中鼓入空气,恒速流加异丙醇 1.8 L , 20 h 滴完。 HPLC 监控,反应 24 h 后,转化率 99.2 %
,终止反应。Add 1.35 kg (6.78 mol) of N-Boc-3-piperidone to the 50 L reactor, add 0.9 L of isopropanol, stir until completely dissolved; add 27.0 L of Tris-HCl buffer (0.1 M, pH 7.0) Stir at 30 °C (450 rpm); then add 67.5 g of ketoreductase powder (0.5 u/mg) and 6.75 g of NAD + in sequence to start the reaction; simultaneously turn on the air pump to blow air into the reaction system. Speed flow plus isopropanol 1.8 L, 20 h drop. After HPLC monitoring, the reaction rate was 99.2%, and the reaction was terminated.
向反应液中加入硅藻土 (150g) 搅拌 0.5 h ,过滤,滤液用乙酸乙酯萃取 (9.0 L × 2)
,滤饼返回反应釜,加入乙酸乙酯搅拌过滤,合并有机相;有机相用饱和食盐水洗涤两次,浓缩后得到 1.45 kg 黄色油状物。加 1.45 L
石油醚于黄色油状物中,于 50 ℃ 下搅拌至均相,于 -20 ℃ 下静置 8 h ,过滤后得到 N-Boc-3- 羟基哌啶 1.09 kg ,收率 80.1
% , GC 纯度 99.2 % , ee 值 99.5 % 。 1 H-NMR (400 MHz ,
CDCl3): δ = 1.443 - 1.458 [m, 11 H, -(CH3)3,
-CH2], 1.727 - 1.883 [d, 2 H, -CH2], 3.039 - 3.125 [m, 2
H, -CH2], 3.539 [s, 1 H, -CHOH], 3.731 - 3.765 [m, 2 H,
-CH2] ; MS(ESI): 202 (M+H)+ 。Add diatomaceous earth (150g) to the reaction mixture, stir for 0.5 h, filter, extract the filtrate with ethyl acetate (9.0 L × 2), and return the filter cake to the reaction kettle, add ethyl acetate and filter, and combine the organic phase; It was washed twice with saturated brine and concentrated to give 1.45 g of yellow oil. Add 1.45 L of petroleum ether in a yellow oil, stir at 50 °C until homogeneous, and let stand at -20 °C for 8 h. After filtration, N-Boc-3-hydroxypiperidine 1.09 kg is obtained, the yield is 80.1%. The GC purity was 99.2% and the ee value was 99.5 %. 1 H-NMR (400 MHz , CDCl 3 ): δ = 1.443 - 1.458 [m, 11 H, -(CH 3 ) 3 , -CH 2 ], 1.727 - 1.883 [d, 2 H, -CH 2 ], 3.039 - 3.125 [m, 2 H, -CH 2 ], 3.539 [s, 1 H, -CHOH], 3.731 - 3.765 [m, 2 H, -CH 2 ] ; MS (ESI): 202 (M+H)+ .
上述实施例只为说明本发明的技术构思及特点,其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内。
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 (10)
1 、一种手性 N- 保护哌啶醇的生物制备方法 ,所述手性 N- 保护哌啶醇的结构式如下: 1 . A biological preparation method of chiral N-protected piperidinol, wherein the chiral N-protected piperidinol has the following structural formula:
所述制备方法以 N- 保护哌啶酮为底物,使该底物 在生物催化剂、辅因子及氢供体的存在下发生不对称还原反应生成 手性 N-
保护哌啶醇,其特征在于:所述生物催化剂为重组酮还原酶,该重组酮还原酶的制备方法为:将含有酮还原酶基因的重组大肠杆菌单菌落接种到含氨苄青霉素抗性的液体 LB
培养基中,于 35~40℃下活化8~12 小时,将活化后得到的培养物接种到含氨苄青霉素抗性的液体 LB 培养基中,于
35~40℃下振荡培养,培养至OD600 值达到 0.6~0.8 时,加入诱导剂,于 25~33℃下继续培养8~12
小时,离心,收集沉淀物,加入磷酸盐缓冲液得悬浮液,将悬浮液置于冰水浴中超声破碎 8~12 分钟,再离心,将上清液预冻至温度降至 -10 ℃
~-25℃,然后再冻干24~48 小时,即得冻干粉状的重组酮还原酶;所述的氢供体为异丙醇,所述不对称还原反应在 pH 值为 7.0~9.0
的水相缓冲液中、温度 25 ℃ ~45℃下进行。The preparation method comprises the N-protected piperidone as a substrate, and the substrate is asymmetrically reduced in the presence of a biocatalyst, a cofactor and a hydrogen donor to form a chiral N-protected piperidinol, which is characterized in that The biocatalyst is a recombinant ketoreductase, and the recombinant ketoreductase is prepared by inoculating a single colony of recombinant Escherichia coli containing a ketoreductase gene into a liquid LB medium containing ampicillin resistance at 35~ After activation at 40 ° C for 8 to 12 hours, the culture obtained after activation is inoculated into ampicillin-resistant liquid LB medium, cultured at 35-40 ° C, and cultured until the OD 600 value reaches 0.6-0.8. Add the inducer, continue to culture at 25~33 °C for 8~12 hours, centrifuge, collect the precipitate, add the phosphate buffer to obtain the suspension, sonicate in the ice water bath for 8~12 minutes, then centrifuge. The supernatant is pre-frozen until the temperature drops to -10 °C ~ -25 °C, and then freeze-dried for 24 to 48 hours to obtain a freeze-dried powdery recombinant ketoreductase; the hydrogen donor is isopropanol. The asymmetric reduction reaction is slow in the water with a pH of 7.0 to 9.0. Solution, carried out at a temperature of 25 ℃ ~ 45 ℃.
2 、根据权利要求 1 所述的手性 N- 保护哌啶醇的生物制备方法 ,其特征在于: 反应起始时的反应体系中,底物的浓度为
3%~20% (w/v) ,重组酮还原酶的用量为底物质量的 2%~8% (w/w) ,异丙醇的物质的量为底物的物质的量的 1.5~6
倍,辅因子的用量为底物质量的 0.02%~1% (w/w) 。The method for biologically preparing a chiral N-protected piperidinol according to claim 1, wherein the concentration of the substrate in the reaction system at the start of the reaction is
3%~20% (w/v), the amount of recombinant ketoreductase is 2%~8% (w/w) of the substrate mass, and the amount of isopropanol is 1.5~ of the amount of the substrate. 6
The cofactor is used in an amount of 0.02% to 1% (w/w) of the substrate mass.
3 、根据权利要求 2 所述的手性 N- 保护哌啶醇的生物制备方法 ,其特征在于: 反应起始时的反应体系中,底物的浓度为
4%~10% (w/v) ,重组酮还原酶的用量为底物质量的 4%~6% (w/w) ,异丙醇的物质的量为底物的物质的量的 4~6 倍,辅因子的用量为底物质量的
0.4%~0.6% (w/w) 。The method for biologically preparing a chiral N-protected piperidinol according to claim 2, wherein the concentration of the substrate in the reaction system at the start of the reaction is
4%~10% (w/v), the amount of recombinant ketoreductase is 4%~6% (w/w) of the substrate mass, and the amount of isopropanol is the amount of the substrate. 6 times, the amount of cofactor is the mass of the substrate
0.4%~0.6% (w/w).
4 、 根据权利要求 2 或 3 所述的手性 N- 保护哌啶醇的生物制备方法 ,其中, 制备方法的实施过程如下:先将 N-
保护哌啶酮溶解在一部分异丙醇中,然后加入水相缓冲液,搅拌均匀后,加入重组酮还原酶与辅因子,维持在 25 ℃ ~45℃,开始
反应,同时开启充气泵向反应体系中鼓入空气,恒速流加剩余的异丙醇, HPLC 监控,至反应转化率达到 99.0 % 以上,终止反应。The method for preparing a chiral N-protected piperidinol according to claim 2 or 3, wherein the preparation method is as follows: firstly, N-
Protect the piperidone in a part of isopropyl alcohol, then add the aqueous buffer, stir evenly, add the recombinant ketoreductase and cofactor, and maintain at 25 °C ~ 45 °C, start
At the same time, the air pump was turned on to inflate the air into the reaction system, and the remaining isopropanol was added at a constant rate, and the reaction was monitored by HPLC until the reaction conversion rate reached 99.0% or more, and the reaction was terminated.
5 、 根据权利要求 4 所述的手性 N- 保护哌啶醇的生物制备方法 ,其特征在于:反应终止后,
向反应液中加入硅藻土搅拌,过滤,滤液用乙酸乙酯萃取,滤饼返回反应釜,加入乙酸乙酯搅拌过滤,合并有机相;有机相用饱和食盐水洗涤 2~3
次,浓缩后得到黄色油状物,加石油醚于黄色油状物中,于 45~50 ℃下搅拌至均相,于-10℃~-20℃下静置,过滤后得到 手性 N-
保护哌啶醇。The method for preparing a chiral N-protected piperidinol according to claim 4, wherein after the reaction is terminated,
To the reaction mixture, celite was added and stirred, and the filtrate was extracted with ethyl acetate. The filter cake was returned to the reaction vessel, and ethyl acetate was added thereto, and the organic phase was combined; the organic phase was washed with saturated brine.
After concentration, a yellow oil is obtained, and petroleum ether is added to a yellow oil. The mixture is stirred at 45 to 50 ° C until homogeneous, and allowed to stand at -10 ° C to -20 ° C to obtain chiral N-
Protect piperidinol.
6 、 根据权利要求 1 所述的手性 N- 保护哌啶醇的生物制备方法 ,其特征在于: 诱导剂为异丙基 - b -D-
硫代半乳糖苷 ; 辅因子为 NAD/NADH 或 NADP/NADPH 。The method for biologically preparing a chiral N-protected piperidinol according to claim 1, wherein the inducer is isopropyl-b-D-
Thiogalactoside; cofactor is NAD/NADH or NADP/NADPH.
7 、 根据权利要求 1 所述的手性 N- 保护哌啶醇的生物制备方法 ,其特征在于: 水相缓冲液为 Tri-HCl
缓冲液、 TEA-HCl 缓冲液或磷酸缓冲液。The method for preparing a chiral N-protected piperidinol according to claim 1, wherein the aqueous phase buffer is Tri-HCl.
Buffer, TEA-HCl buffer or phosphate buffer.
8 、 根据权利要求 1 或 7 所述的手性 N- 保护哌啶醇的生物制备方法 ,其特征在于:水相缓冲液的 pH 值为
7~7.5 。The method for biologically preparing chiral N-protected piperidinol according to claim 1 or 7, wherein the pH of the aqueous phase buffer is
7~7.5.
9 、 根据权利要求 1 至 3 中任一项所述的手性 N- 保护哌啶醇的生物制备方法 ,其特征在于:
不对称还原反应的温度为 28 ℃ ~32℃。The method for biologically preparing chiral N-protected piperidinol according to any one of claims 1 to 3, wherein:
The temperature of the asymmetric reduction reaction is from 28 °C to 32 °C.
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---|---|---|---|---|
CN111378704A (en) * | 2020-03-23 | 2020-07-07 | 焦作健康元生物制品有限公司 | Method for producing 4-AA intermediate by ketoreductase |
CN111549011A (en) * | 2020-06-03 | 2020-08-18 | 重庆迪维斯生物科技有限公司 | Transaminase mutant derived from aspergillus terreus and application thereof |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103276027A (en) * | 2013-05-10 | 2013-09-04 | 苏州汉酶生物技术有限公司 | Method for biologically preparing chiral N-protective pipradrol |
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012007965A1 (en) * | 2010-07-14 | 2012-01-19 | Cadila Healthcare Limited | Enzyme for the production of optically pure 3 - quinuclidinol |
CN102341501A (en) * | 2009-03-05 | 2012-02-01 | Iep有限责任公司 | Method for stereoselectively and enzymatically reducing keto compounds |
CN102925501A (en) * | 2012-11-19 | 2013-02-13 | 苏州汉酶生物技术有限公司 | Biological preparation method of (S)-4-chloro-3-hydroxybutyrate ethyl |
CN102978249A (en) * | 2012-11-19 | 2013-03-20 | 苏州汉酶生物技术有限公司 | Biological preparation method of 6-cyan-(3R, 5R)- dihydroxy-hexanoic acid tert-butyl ester |
CN103276027A (en) * | 2013-05-10 | 2013-09-04 | 苏州汉酶生物技术有限公司 | Method for biologically preparing chiral N-protective pipradrol |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102351781A (en) * | 2011-08-08 | 2012-02-15 | 宿迁联盛化学有限公司 | Synthesis technology of tetramethylpiperidine |
CN102965403A (en) * | 2012-11-19 | 2013-03-13 | 苏州汉酶生物技术有限公司 | Biological preparation method of tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate |
-
2013
- 2013-05-10 CN CN2013101730882A patent/CN103276027A/en active Pending
- 2013-09-11 WO PCT/CN2013/083267 patent/WO2014180085A1/en active Application Filing
-
2014
- 2014-05-08 CN CN201410192536.8A patent/CN103923957A/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102341501A (en) * | 2009-03-05 | 2012-02-01 | Iep有限责任公司 | Method for stereoselectively and enzymatically reducing keto compounds |
WO2012007965A1 (en) * | 2010-07-14 | 2012-01-19 | Cadila Healthcare Limited | Enzyme for the production of optically pure 3 - quinuclidinol |
CN102925501A (en) * | 2012-11-19 | 2013-02-13 | 苏州汉酶生物技术有限公司 | Biological preparation method of (S)-4-chloro-3-hydroxybutyrate ethyl |
CN102978249A (en) * | 2012-11-19 | 2013-03-20 | 苏州汉酶生物技术有限公司 | Biological preparation method of 6-cyan-(3R, 5R)- dihydroxy-hexanoic acid tert-butyl ester |
CN103276027A (en) * | 2013-05-10 | 2013-09-04 | 苏州汉酶生物技术有限公司 | Method for biologically preparing chiral N-protective pipradrol |
Non-Patent Citations (3)
Title |
---|
KIZAKI NORIYUKI ET AL.: "Characterization of novel alcohol dehydrogenase of Devosia riboflavina involved in stereoselective reduction of 3-pyrrolidinone derivatives", JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC, vol. 51, 9 November 2007 (2007-11-09), pages 73 - 80 * |
LACHERETZ ROMAIN ET AL.: "Daucus carota Mediated-Reduction of Cyclic 3-Oxo-amines", ORGANIC LETTERS, vol. 11, no. 6, 12 February 2009 (2009-02-12), pages 1245 - 1248 * |
TEWARI Y. B. ET AL.: "A thermodynamic study of ketoreductase- catalyzed reactions 5. Reduction of substituted ketones in n-hexane", J. CHEM.THERMODYNAMICS, vol. 40, 7 November 2007 (2007-11-07), pages 661 - 670 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111378704A (en) * | 2020-03-23 | 2020-07-07 | 焦作健康元生物制品有限公司 | Method for producing 4-AA intermediate by ketoreductase |
CN111549011A (en) * | 2020-06-03 | 2020-08-18 | 重庆迪维斯生物科技有限公司 | Transaminase mutant derived from aspergillus terreus and application thereof |
CN111549011B (en) * | 2020-06-03 | 2023-05-23 | 卡柔恩赛生物技术湖北有限公司 | Transaminase mutant from aspergillus terreus and application thereof |
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