WO2014180085A1

WO2014180085A1 - Biological preparation method for chiral n-protection peridol

Info

Publication number: WO2014180085A1
Application number: PCT/CN2013/083267
Authority: WO
Inventors: 鞠鑫; 郭皓; 李斌; 唐圆圆
Original assignee: 苏州汉酶生物技术有限公司
Priority date: 2013-05-10
Filing date: 2013-09-11
Publication date: 2014-11-13
Also published as: CN103276027A; CN103923957A

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

Biological preparation method of chiral N-protected piperidinol

Technical field

The invention belongs to the technical field of biopharmaceuticals and biochemicals, and particularly relates to a biological preparation method of chiral N-protected piperidinol.

Background technique

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-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-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).

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).

Summary of the invention

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.

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;

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.

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.

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.

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.

According to the present invention, the inducer may be isopropyl-b-D-thiogalactoside (IPTG); the cofactor may be NAD/NADH or NADP/NADPH.

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.

According to a specific and preferred aspect, the structural formula of the chiral N-protected piperidinol is as follows:

as follows:

Its chemical name is 1-tert-butoxycarbonyl-3-piperidinol or N-Boc-3-hydroxypiperidine.

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.

Due to the implementation of the above technical solutions, 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.

DRAWINGS

Figure 1 is a synthetic route of the present invention;

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.

detailed description

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.

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.

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.

Example 1 Recombinant ketone reductase (KRED) enzyme powder shake flask production process

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.

Example 2 One hundred milligrams of preparation process

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%.

Example 3 100 g grade preparation process

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.

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)+ .

Example 4 kg-level preparation process

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.

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)+ .

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

1 . A biological preparation method of chiral N-protected piperidinol, wherein the chiral N-protected piperidinol has the following structural formula:

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, 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 ℃.

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.

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).

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.

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.

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.

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.

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.

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.

10 . The method for preparing a chiral N-protected piperidinol according to claim 1, wherein the chiral N- The structural formula for the protection of piperidinol is as follows:

.