阅读说明：本技术 一种填充床连续流不对称合成(S)-1-Boc-3-氨基哌啶的方法 (Method for asymmetric synthesis of (S) -1-Boc-3-aminopiperidine by continuous flow of packed bed ) 是由 魏东芝 王华磊 汪湘湘 于 2020-07-09 设计创作，主要内容包括：本发明提供一种填充床连续流不对称合成(S)-1-Boc-3-氨基哌啶的方法,所述方法包括：将一定量的固定化转氨酶填充于填充床中构成反应器,所述反应器水浴保温,自所述反应器的一端将反应液以一定流速泵入所述反应器中,在所述反应器的另一端收集流出物,实现(S)-1-Boc-3-氨基哌啶的合成；其中,所述固定化转氨酶通过将ω-转氨酶固定在氨基-环氧树脂上制得,所述反应液包含：N-Boc-3-哌啶酮、异丙胺以及磷酸吡哆醛。根据本发明,采用的连续流催化模式能够原位去除产物,有效降低产物抑制的影响,提高催化效率,具有高时空产率、可持续等特点,反应条件温和,操作简便,延长固定化酶使用寿命,具有良好的应用前景。(The invention provides a method for asymmetric synthesis of (S) -1-Boc-3-aminopiperidine by continuous flow of a packed bed, which comprises the following steps: filling a certain amount of immobilized transaminase into a packed bed to form a reactor, keeping the temperature of the reactor in a water bath, pumping reaction liquid into the reactor from one end of the reactor at a certain flow rate, and collecting the effluent from the other end of the reactor to realize the synthesis of (S) -1-Boc-3-aminopiperidine; wherein the immobilized transaminase is produced by immobilizing ω -transaminase on an amino-epoxy resin, and the reaction solution contains: N-Boc-3-piperidone, isopropylamine and pyridoxal phosphate. According to the invention, the adopted continuous flow catalysis mode can remove the product in situ, effectively reduce the influence of product inhibition, improve the catalysis efficiency, has the characteristics of high space-time yield, sustainability and the like, is mild in reaction conditions, simple and convenient to operate, prolongs the service life of the immobilized enzyme, and has good application prospect.)

1. A method for continuous flow asymmetric synthesis of (S) -1-Boc-3-aminopiperidine by packed bed, comprising: filling a certain amount of immobilized transaminase into a packed bed to form a reactor, keeping the temperature of the reactor in a water bath, pumping reaction liquid into the reactor from one end of the reactor at a certain flow rate, and collecting the effluent from the other end of the reactor to realize the synthesis of (S) -1-Boc-3-aminopiperidine; wherein the immobilized transaminase is produced by immobilizing ω -transaminase on an amino-epoxy resin, and the reaction solution contains: N-Boc-3-piperidone, isopropylamine and pyridoxal phosphate.

2. The continuous flow asymmetric synthesis method of (S) -1-Boc-3-aminopiperidine according to claim 1, wherein the packed bed is a glass column having a length of 10-30 cm and an inner diameter of 10-20 mm, and the immobilized transaminase is used in an amount of 1-10 g.

3. The continuous flow-through packed bed asymmetric (S) -1-Boc-3-aminopiperidine synthesis according to claim 1, wherein the concentration of N-Boc-3-piperidone in the reaction solution is 20 to 200mM, isopropylamine is 1 to 5-fold equivalent of N-Boc-3-piperidone, and pyridoxal phosphate is 0.1 to 1 mM.

4. The continuous flow-through packed bed asymmetric synthesis method of (S) -1-Boc-3-aminopiperidine according to claim 1, wherein the flow direction of the reaction solution in the reactor is from top to bottom, and the average residence time of the reaction solution in the reactor is 5-15 min.

5. The continuous flow asymmetric packed bed process for the synthesis of (S) -1-Boc-3-aminopiperidine according to claim 1, wherein the reactor is maintained at 20-60 ℃ in a water bath.

6. The packed bed continuous flow asymmetric synthesis of (S) -1-Boc-3-aminopiperidine according to claim 1, wherein the immobilized transaminase is prepared by the following method:

s1: fermenting and culturing Escherichia coli containing recombinant plasmid ATA-W12-pET-28a, collecting wet thallus, suspending in 100mM phosphate buffer solution, ultrasonically crushing, and centrifuging to obtain crude enzyme solution of omega-transaminase;

s2: weighing a certain amount of amino-epoxy resin, adding a proper amount of omega-transaminase crude enzyme liquid and a buffer solution, and treating in a shaking table for a certain time to realize immobilization of omega-transaminase on the amino-epoxy resin;

s3: filtering, draining, washing with phosphate buffer solution to remove protein which can not be fixed, adding 50mM phosphate buffer solution containing 3M glycine, and treating in a shaking table for a certain time to block unreacted epoxy sites; and

s4: and (3) carrying out suction filtration and washing to obtain the immobilized transaminase, adding 100mM phosphate buffer solution into the immobilized transaminase, and storing the immobilized transaminase in a refrigerator at 4 ℃ to obtain the immobilized transaminase.

7. The continuous flow-through packed bed asymmetric synthesis method of (S) -1-Boc-3-aminopiperidine according to claim 6, wherein in step S2 of the immobilized transaminase preparation method, the protein concentration of the crude ω -transaminase is 0.4 to 3mg/mL, the amount of the crude ω -transaminase added is 1 to 6mL/1g of amino-epoxy resin, and the buffers are a citrate-sodium citrate buffer containing pyridoxal phosphate at an ionic concentration of 50mM to 1M and pH of 5.0 to 10.0, a sodium phosphate buffer, a Tris-HCl buffer and a glycine-NaOH buffer, wherein the pyridoxal phosphate is 0.1 to 1 mM.

8. The continuous flow-through packed bed asymmetric synthesis method of (S) -1-Boc-3-aminopiperidine according to claim 6, wherein the immobilization time in step S2 is 1-18 h, and the amino-modification density of the amino-epoxy resin used is 1-70 μmol/g.

9. The packed bed continuous flow asymmetric synthesis of (S) -1-Boc-3-aminopiperidine according to claim 1 or 6, characterized in that the amino-epoxy resin is prepared by the following method:

a1: weighing a certain amount of epoxy resin, adding deionized water for washing at least three times, and washing off impurities;

a2: mixing the washed epoxy resin with an ethylenediamine modification solution according to the proportion of 1 (8-12) (w/v), and treating for a period of time under the condition of a shaking table; and

a3: after the reaction is finished, performing suction filtration, repeatedly washing for more than 5 times by using deionized water, removing unreacted ethylenediamine, draining, and storing in a refrigerator at 4 ℃.

10. The continuous flow asymmetric packed bed-based method for synthesizing (S) -1-Boc-3-aminopiperidine according to claim 9, wherein the pH of the ethylenediamine modification solution in the step A2 is 8.0-10.0, the concentration is 0.1-1M, and the reaction time is 1-6 h.

Technical Field

The invention relates to the technical field of biocatalysis, in particular to a method for asymmetrically synthesizing (S) -1-Boc-3-aminopiperidine by using continuous flow of a packed bed.

Background

Enantiomerically pure chiral amines, including primary, secondary and tertiary amines, are important components in the synthesis of a variety of biologically active compounds, such as agrochemicals and pharmaceuticals, and have been widely used in the pharmaceutical industry, in agrochemicals, in materials and other fields (mathews., et al, 2012, ACS Catalysis; Koszelewski d., et al, 2010, Trends in biotechnology).

3-aminopiperidine (3APi) with optical activity and derivatives thereof are very important medical intermediates, and can be used for synthesizing medicines for treating type II diabetes, such as alogliptin, linagliptin and the like. At present, 3-aminopiperidine is mainly synthesized by a chemical method (Decosta B R, et al, 1992, Journal of medicinal Chemistry), however, the chemical synthesis method not only has complex operation and lower yield and optical purity of products, but also wastes time and labor in the processes of later treatment, including the recycling of a resolving agent, the recovery of enantiomers and the like, so that the preparation of 3-aminopiperidine by a biocatalytic conversion method has good research value.

As biocatalysts are increasingly used for the industrial scale synthesis of drug substances, a number of innovative technologies have been seen as complementary tools for the development of intensive and industrially relevant biocatalytic processes. In particular, there is an increasing interest in carrying out biocatalytic conversions in continuous flow reactors, with the following advantages: (1) reducing the inhibition of the enzyme by continuously removing the product; (2) when the immobilized biocatalyst is used, downstream treatment (separation is facilitated) is easy to carry out; (3) the total number of transitions (TTNs) is increased. Luccaraftift h.r. et al synthesized 2-aminophenyloxazin-3-one (APO) from nitrobenzene in a continuous flow mode in three steps in a microfluidic device, however substrate concentration (1mM), overall yield (19%) and productivity needed to be further improved (luccararfift H R, et al, 2007, Biotechnology and Biotechnology). To some extent, the basis for achieving the application of biocatalysis in continuous flow is the continuing development of enzyme immobilization technology.

Immobilized enzymes (immobilized enzymes) are enzymes bound to water-insoluble carriers by physical or chemical means or confined in a space, but retain their catalytic activity, are capable of continuous reaction, and are capable of recovering reusable biocatalysts.

Immobilized enzymes can be prepared by whole living cells, dead cells, crude enzyme, or purified enzyme, depending on the type and application of the enzyme. The traditional immobilization method comprises an adsorption method, a cross-linking method, an embedding method and a covalent bonding method, wherein the covalent bonding method is used for obtaining an immobilized enzyme molecule which is firmly connected with a carrier, the situation of protein shedding is not easy to occur in the reaction process, and the immobilized enzyme molecule has excellent stability and reusability, but the covalent action can influence the protein structure of the enzyme and even destroy the active center of a biocatalyst, so that the enzyme activity is obviously reduced, and the general enzyme activity recovery rate is only about 30-50%.

Among the carriers used in the covalent bonding method, epoxy-activated carriers are almost ideal immobilization materials, and immobilization of proteins and enzymes can be easily performed on a laboratory and industrial scale. The epoxy-based carrier has good stability, can be stored for a long time under the condition of low-temperature drying, and can be kept stable in a neutral humid environment, which is an important prerequisite for ensuring that an immobilized enzyme with stable property is obtained. Furthermore, under very mild experimental conditions (e.g., pH 7.0), epoxy-activated carriers are capable of forming very stable covalent bonds directly with different protein groups (e.g., amino, thiol, and phenolic, etc.). For example, in 2016, De Souza et al immobilized ω -TAVf from Vibrio fluvialis in epoxy-activated cellulose for asymmetric synthesis of (S) -phenylethylamine, active in the temperature range of 30 to 60 ℃, but only able to reuse 4 batches (De Souza, et al, 2016, RSC Advances).

However, immobilization on conventional epoxy resin carriers requires an environment of high ionic strength, and hydrophobic interaction between the enzyme and the carrier may affect the activity and stability of the enzyme. Furthermore, even under weakly alkaline conditions, the reaction between epoxide groups and soluble proteins is extremely slow. Therefore, modification of the epoxy carrier is an effective means for obtaining a novel immobilized carrier with higher performance, and more carrier choices can be provided for enzyme immobilization.

Disclosure of Invention

The invention aims to provide a method for asymmetrically synthesizing (S) -1-Boc-3-aminopiperidine by continuous flow of a packed bed, thereby solving the problems that the chemical synthesis method of (S) -1-Boc-3-aminopiperidine in the prior art is complicated to operate, and the enzyme activity and stability of the biocatalytic synthesis method are easily influenced by various factors, so that the yield is low.

In order to solve the problems, the invention adopts the following technical scheme:

the invention provides a method for asymmetric synthesis of (S) -1-Boc-3-aminopiperidine by continuous flow of a packed bed, which comprises the following steps: filling a certain amount of immobilized transaminase into a packed bed to form a reactor, keeping the temperature of the reactor in a water bath, pumping reaction liquid into the reactor from one end of the reactor at a certain flow rate, and collecting the effluent from the other end of the reactor to realize the synthesis of (S) -1-Boc-3-aminopiperidine; wherein the immobilized transaminase is produced by immobilizing ω -transaminase on an amino-epoxy resin, and the reaction solution contains: N-Boc-3-piperidone, isopropylamine and pyridoxal phosphate.

According to the method provided by the invention, the reaction process of (S) -1-Boc-3-aminopiperidine asymmetric synthesis by transaminase is shown in figure 1, the immobilized transaminase [email protected] EES packed bed continuous flow reaction system for realizing the reaction is shown in figure 2, and the schematic diagram of the mode transaminase reaction is shown in figure 3.

According to a preferable scheme of the invention, the packed bed is a glass chromatographic column with the length of 10-30 cm and the inner diameter of 10-20 mm, and the dosage of the immobilized transaminase is 1-10 g.

Preferably, the concentration of N-Boc-3-piperidone in the reaction solution is 20-200 mM, isopropylamine is 1-5 times equivalent of N-Boc-3-piperidone, and the content of pyridoxal phosphate is 0.1-1 mM. Most preferably, isopropylamine is 2 times that of N-Boc-3-piperidone.

Preferably, the reaction solution further comprises a cosolvent dimethyl sulfoxide, and the dosage of the cosolvent dimethyl sulfoxide is 5-20% (V/V).

Preferably, the flowing direction of the reaction liquid in the reactor is from top to bottom, and the average residence time of the reaction liquid in the reactor is 5-15 min, preferably 8-12 min.

According to a preferred embodiment of the present invention, when the amount of the immobilized transaminase in the glass column is 2.5g, the flow direction of the reaction solution is from top to bottom, and the flow rate of the reaction solution is 0.2 to 1.5 mL/min.

Preferably, the reactor is subjected to water bath heat preservation at the temperature of 20-60 ℃.

Preferably, the continuous flow reaction system uses sodium phosphate buffer solution, Tris-HCl buffer solution and glycine-NaOH buffer solution with the ion concentration of 50 mM-1M and the pH value of 7.0-9.0.

According to another preferred embodiment of the present invention, the packed bed is a glass chromatography column having a length of 10cm and an inner diameter of 10mm, the amount of the immobilized transaminase in the glass chromatography column is 2.5g, the flow direction of the reaction solution is from top to bottom, and the flow rate of the reaction solution is 0.4 mL/min.

According to the process provided by the invention, the immobilized transaminase is prepared by the following method: s1: fermenting and culturing Escherichia coli containing recombinant plasmid ATA-W12-pET-28a, collecting wet thallus, suspending in 100mM phosphate buffer solution, ultrasonically crushing, and centrifuging to obtain crude enzyme solution of omega-transaminase; s2: weighing a certain amount of amino-epoxy resin, adding a proper amount of omega-transaminase crude enzyme liquid and a buffer solution, and treating in a shaking table for a certain time to realize immobilization of omega-transaminase on the amino-epoxy resin; s3: filtering, draining, washing with phosphate buffer solution to remove protein which can not be fixed, adding 50mM phosphate buffer solution containing 3M glycine, and treating in a shaking table for a certain time to block unreacted epoxy sites; and S4: and (4) carrying out suction filtration and washing to obtain the immobilized transaminase, adding 100mM phosphate buffer solution into the immobilized transaminase, and storing the immobilized transaminase in a refrigerator at 4 ℃.

Preferably, in step S2, the protein concentration of the crude enzyme solution of ω -transaminase is 0.4-3 mg/mL, and the addition amount of the crude enzyme solution of ω -transaminase is 1-6 mL/1g of amino-epoxy resin.

Preferably, in step S2, the buffer solution is a citric acid-sodium citrate buffer solution, a sodium phosphate buffer solution, a Tris-HCl buffer solution or a glycine-NaOH buffer solution with pH of 5.0-10.0, and the ion concentration of each buffer solution is 50 mM-1M, and each buffer solution contains 0.1-1 mM pyridoxal phosphate.

Preferably, the immobilization time in step S2 is 1-18 h, and the amino-modified density of the amino-epoxy resin is 1-70 μmol/g.

Alternatively, the 100mM sodium phosphate buffer solution pH8.0 containing 0.1mM pyridoxal phosphate in step S1, the 50mM phosphate buffer solution pH8.5 containing 3M glycine in step S3, and the 100mM sodium phosphate buffer solution pH8.0 containing 0.1mM pyridoxal phosphate in step S4.

According to a preferred embodiment of the present invention, in step S2, 1g of amino-epoxy resin was weighed, 4mL of the crude enzyme solution with a protein concentration of 1.8mg/mL was added, 100mM of pH8.0 and 0.1mM of pyridoxal phosphate in sodium phosphate buffer was used, the immobilization time was 6 hours, and the amino-epoxy resin used had an amino group modification density of 20. mu. mol/g.

The catalysis temperature of the immobilized transaminase is 20-60 ℃, and preferably 37 ℃.

The immobilized transaminase catalytic reduction pH is 7.0-10.0, preferably 8.0.

According to the method provided by the invention, the amino-epoxy resin is prepared by the following method: a1: weighing a certain amount of epoxy resin, adding deionized water for washing at least three times, and washing off impurities; a2: mixing the washed epoxy resin with an ethylenediamine modification solution according to the proportion of 1 (8-12) (w/v), and treating for a period of time under the condition of a shaking table; a3: after the reaction is finished, performing suction filtration, repeatedly washing for more than 5 times by using deionized water, removing unreacted ethylenediamine, draining, and storing in a refrigerator at 4 ℃.

Preferably, the pH of the ethylenediamine modification solution in the step A2 is 8.0-10.0, the concentration is 0.1-1M, and the reaction time is 1-6 h.

According to a preferred embodiment of the present invention, the pH of the ethylenediamine modified solution in step A2 is 8.5, the concentration of ethylenediamine is 0.3M, and the washed epoxy resin and the ethylenediamine modified solution are mixed at a ratio of 1:10(w/v), and treated at 20 ℃ and 200rpm for 1 hour.

The creativity of the invention mainly lies in that 1) the epoxy resin is modified, so that the amino-epoxy resin with higher performance is obtained, and further the omega-transaminase fixed on the amino-epoxy resin has obviously improved enzyme activity and stability; 2) by providing a process method for asymmetrically synthesizing (S) -1-Boc-3-aminopiperidine in a continuous flow mode by using immobilized transaminase, the method realizes the high-efficiency and low-cost biocatalytic synthesis of (S) -1-Boc-3-aminopiperidine for the first time and has good application prospect.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a process method for asymmetrically synthesizing (S) -1-Boc-3-aminopiperidine in a continuous flow mode by using immobilized transaminase, wherein the transaminase is immobilized on amino-epoxy resin modified by ethylenediamine, the recovery rate of the enzyme activity is 75%, the transaminase has good stability, no obvious loss of the enzyme activity after being stored for 30 days in a refrigerator at 4 ℃, 85% of the activity is kept after the transaminase is continuously and repeatedly used for 20 times, and the transaminase shows stronger tolerance than free enzyme in a pH range of 5-10. The catalytic efficiency of the continuous flow reaction system is 6 times of that of the intermittent reaction, no obvious enzyme activity loss is seen after the system continuously works for 24 hours, and the space-time yield is 930.73 g.L^-1·d^-1The method proves that the asymmetric production of (S) -1-Boc-3-aminopiperidine in a continuous flow mode has very high feasibility, and theoretically, the system can be applied to the synthesis of other more important pharmaceutical intermediates, and has very high research value and good application prospect.

In conclusion, according to the method for continuous flow asymmetric synthesis of (S) -1-Boc-3-aminopiperidine by using a packed bed, which is provided by the invention, the invention relates to an amination modification of epoxy resin and a preparation method of immobilized transaminase, the invention adopts immobilized enzyme [email protected] EES as a catalyst, so that the enzyme activity loss is less, the stability is strong, the product can be removed in situ in a continuous flow catalysis mode, the influence of product inhibition is effectively reduced, the catalysis efficiency is improved, the method has the characteristics of high space-time yield, sustainability and the like, the reaction condition is mild, the operation is simple and convenient, the service life of the immobilized enzyme is prolonged, the economic cost is reduced, and the method has a good application prospect.

Drawings

FIG. 1 is a diagram showing the process of asymmetric synthesis of (S) -1-Boc-3-aminopiperidine by transaminase ATA-W12;

FIG. 2 is a schematic representation of a packed bed continuous flow reaction system for immobilized transaminase [email protected] EES;

FIG. 3 is a schematic diagram of a transaminase mode reaction;

FIG. 4 is a graph of epoxy carrier amino modification density as a function of EDA reaction time;

FIG. 5 is a protein gel electrophoresis chart of ATA-W12 crude enzyme solution;

FIG. 6 shows the effect of the density of carrier amino group modifications on the enzymatic activity of immobilized transaminase;

FIG. 7 is the results of optimization of carrier protein loading;

FIG. 8 shows the optimization results of immobilization time;

FIG. 9 is a graph showing the effect of reaction pH on the enzyme activity of free and immobilized enzymes;

FIG. 10 is a graph showing the effect of reaction temperature on enzymatic activity of immobilized enzyme;

FIG. 11 shows the results of comparison of storage stability of free enzyme and immobilized enzyme;

FIG. 12 shows the results of comparison of pH stability of free enzyme and immobilized enzyme;

FIG. 13 shows the results of the operational stability of the immobilized enzyme;

FIG. 14 shows the optimization of the flow direction of the reaction solution in the continuous flow system;

FIG. 15 shows the optimization results of the amount of immobilized enzyme packing in a continuous flow system;

FIG. 16 shows the optimization results of the flow rate of the reaction solution in the continuous flow system.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.