CN112779300B

CN112779300B - Method for preparing (S) -3-butyn-2-amine by biocatalysis

Info

Publication number: CN112779300B
Application number: CN202011468892.XA
Authority: CN
Inventors: 李�杰; 孙丰来; 唐大林; 蔡小飞; 郑晨抗; 谢磊; 周丹; 朱景仰; 傅小勇; 陈民章
Original assignee: Shanghai STA Pharmaceutical R&D Ltd
Current assignee: Shanghai STA Pharmaceutical R&D Ltd
Priority date: 2020-12-15
Filing date: 2020-12-15
Publication date: 2022-10-21
Anticipated expiration: 2040-12-15
Also published as: CN112779300A

Abstract

The invention discloses a method for preparing (S) -3-butyn-2-amine through biocatalysis, which is characterized in that 3-alkynyl-2-butanone is used as a substrate in a liquid phase reaction system, and under the condition of coenzyme existence, carbonyl of the 3-alkynyl-2-butanone is reduced into chiral amino through catalysis of imine reductase to prepare the (S) -3-butyn-2-amine. The method has the advantages of simple operation, mild and easily-controlled reaction conditions, short reaction time, high conversion rate of the substrate and high chiral purity of the obtained product.

Description

Method for preparing (S) -3-butyn-2-amine by biocatalysis

Technical Field

The invention relates to the field of biocatalytic chemical reactions, in particular to a method for preparing enantiomerically pure (S) -3-butyn-2-amine through biocatalysis.

Background

(S) -3-butyne-2-amine (shown as a formula II) is a very important chiral building block and is applied to synthesis of a plurality of medical intermediates. For example, (S) -3-butyn-2-amine is an important drug intermediate in the preparation route of drugs for inhibiting neurodegenerative diseases.

At present, the method for preparing (S) -3-butyn-2-amine is shown in the following figure, and the (R) - (+) -3-butyn-2-ol is used as a raw material in the literature (Chin.J. chem.2013,31, 173-181), and the virulent and unstable raw materials such as triphenyl phosphorus and hydrazine are used for carrying out a one-step inversion reaction and a one-step deprotection reaction to obtain the product (S) -3-butyn-2-amine. However, (R) - (+) -3-butyn-2-ol as a raw material is expensive and is not suitable for industrial mass production from an economic viewpoint.

The bio-enzyme catalysis method has the advantages of high reaction efficiency, good stereoselectivity, mild reaction conditions, low energy consumption, environmental friendliness and the like, and is more and more widely concerned and deeply researched.

At present, no relevant patent report exists on the biological preparation method of (S) -3-butyn-2-amine.

Disclosure of Invention

The invention aims to provide a preparation method of (S) -3-butyn-2-amine with high yield, good selectivity and simplified route, and mainly solves the technical problems of complicated step of catalyzing enamine or imine hydrogenation by transition metal, low conversion rate or low selectivity in the steps of the existing route.

In order to solve the technical problems, the invention adopts the technical scheme that

A method for preparing (S) -3-butyn-2-amine by biocatalysis, in a liquid phase reaction system, 3-alkynyl-2-butanone is taken as a substrate, under the condition of coenzyme existence, the pH value of the reaction system is 8.0-9.0, the reaction temperature is 20-40 ℃, and the (S) -3-butyn-2-amine is prepared by using imine reductase to catalyze and reduce the carbonyl of the 3-alkynyl-2-butanone into chiral amino.

The reaction formula is as follows:

in a preferred embodiment, the imine reductase enzyme

The amino acid sequence of (A) is shown in SEQ ID NO. 1.

In a preferred embodiment, the gene sequence encoding the imine reductase is selected from the group consisting of:

(a) A nucleotide sequence shown as SEQ ID NO. 2;

(b) A polynucleotide complementary to the sequence defined in (a); or

(c) Any polynucleotide or complementary sequence having at least 70% (preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%) or more sequence identity to the sequence defined in (a).

In a preferred embodiment, the gene workpiece encoding the imine reductase is on an expression vector.

In the present invention, the sources of the imine reductase include, but are not limited to, vibrio, arthrobacter and Chromobacterium. Among them, preferred transaminases are derived from a novel imine reductase (AoIRED) found in Amycolatopsis orientalis (Uniprot R4SNK 4) or from the genetic recombination of said species in Escherichia coli.

In a preferred embodiment, the reaction system has a reaction starting concentration of the 3-alkynyl-2-butanone of 1g/L to 5g/L.

In a preferred embodiment, the imine reductase is used in an amount of 1 to 3 times the mass of the substrate.

In a preferred embodiment, the coenzyme is an oxidative coenzyme, and an enzyme for coenzyme regeneration is also present in the reaction system.

In a preferred embodiment, the oxidative coenzyme is selected from NAD, NADP, or a combination thereof. Preferably, the coenzyme is selected from the group consisting of the oxidative coenzyme NADP.

In a preferred embodiment, the ratio of the amount of coenzyme to the amount of substrate is 0.01% to 1.0% (w/w). Preferably, the ratio of the amount of coenzyme to the amount of substrate is 0.01% to 0.5% (w/w).

In a preferred embodiment, a co-substrate is also present in the reaction system.

In a preferred embodiment, the co-substrate is selected from: one or more of isopropanol, glucose, and ammonium formate. Preferably, the co-substrate is selected from glucose.

In a preferred embodiment, the concentration of the cosubstrate in the reaction system is 5 to 30%.

In a preferred embodiment, the enzyme for coenzyme regeneration is selected from the group consisting of: one or more of alcohol dehydrogenase, formate dehydrogenase and glucose dehydrogenase. Preferably, the enzyme for coenzyme regeneration is selected from Glucose Dehydrogenase (GDH).

In a preferred embodiment, the enzyme for coenzyme regeneration is used in an amount of 1% to 10% by mass of the coenzyme in the reaction system.

In a preferred embodiment, the reaction system is a buffered saline solution system. The pH of the reaction system was controlled by buffering the aqueous salt solution. Commonly used buffered saline solutions include, but are not limited to, ammonium chloride (NH) ₄ Cl), ammonium formate (HCOONH) ₄ ) Or ammonium acetate (CH) ₃ COONH ₄ ) And (4) a buffer solution.

In a preferred embodiment, the reaction system further comprises a cosolvent. The cosolvent is an organic solvent.

In a preferred embodiment, the co-solvent is selected from the group consisting of: dimethyl sulfoxide, methanol, ethanol, isopropanol, acetone, or combinations thereof. Preferably, the co-solvent is selected from dimethyl sulfoxide. The co-solvent used should be miscible with water to further increase the solubility of the substrate.

In a preferred embodiment, the reaction temperature is 25 ℃ to 35 ℃; more preferably 30 to 35 ℃.

In a preferred embodiment, the reaction time is from 1 to 24 hours.

In a preferred embodiment, preferably, the reaction system has a pH of 8.5. + -. 0.3; more preferably, the pH is 8.5 + -0.2; more preferably, the pH is 8.5 + -0.1; more preferably, the pH is 8.5.

In a preferred embodiment, the preparation process according to the invention further comprises the step of isolating the hydroxy product (S) -3-butyn-2-amine.

Under preferred conditions, (S) -3-butyn-2-amine (II) prepared by the process according to the invention has an enantiomeric excess of not less than 99% and a conversion of not less than 81%. More preferably, the conversion is not less than 99%.

The invention realizes the purpose of preparing enantiomer pure (S) -3-butyne-2-amine by a novel enzyme catalysis technology. The method starts from 3-alkynyl-2-butanone, uses imine reductase with a screened specific amino acid sequence to catalyze the reaction of (S) -3-butyn-2-amine, can also add reduced auxiliary NADPH as electron transfer in the reaction, and can also cooperate with glucose dehydrogenase to carry out coenzyme circulation. The technical route has simple steps, high conversion rate, green and environment-friendly technology and potential of industrial production.

The method has the advantages that the technical route for catalyzing and reacting S-3-butyn-2-amine by using the novel imine reductase is simple and easy in operation steps, high in yield, good in selectivity, high in substrate conversion rate (more preferably 99% under the condition) of more than 81%, the chiral purity of the obtained product reaches 99%, the synthetic route is simple and efficient, the reaction condition is mild and easy to control, the reaction time is short, the method is green and environment-friendly, new ideas and opportunities are provided for industrial production of (S) -3-butyn-2-amine, and the method has potential for industrial popularization.

Drawings

FIG. 1 is a high performance liquid chromatography profile of a racemate of 3-butyn-2-amine. The left peak is in the R configuration and the right peak is in the S configuration.

FIG. 2A is a chiral high performance liquid chromatography profile of S-3-butyn-2-amine after conversion according to the method of example 1 of the present invention. The only peak in the figure is the target compound S-3-butyn-2-amine.

FIG. 2B is a chart of gas chromatography of the conversion of (S) -3-butyn-2-amine after conversion according to the method of example 1 of the invention (ammonium chloride pH 8.5). The only peak in the figure is the target compound S-3-butyn-2-amine.

FIG. 3 is a chart of gas chromatography for the conversion of (S) -3-butyn-2-amine after conversion according to the method of example 2 of the invention (ammonium chloride pH 8.5).

FIG. 4 is a chart of gas chromatography for the conversion of (S) -3-butyn-2-amine after conversion according to the method of example 3 of the present invention (ammonium chloride pH 10.0).

FIG. 5 is a chart of gas chromatography for the conversion of (S) -3-butyn-2-amine after conversion according to the method of example 4 of the present invention (ammonium chloride pH 6.0).

FIG. 6 is a gas chromatography chromatogram of the conversion of (S) -3-butyn-2-amine after conversion by the method of example 5 according to the invention (ammonium chloride pH 7.0).

FIG. 7 is a gas chromatography chromatogram of the conversion of (S) -3-butyn-2-amine after conversion by the method of example 6 according to the invention (ammonium chloride pH 9.5).

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

In an 8mL glass reaction flask, 4mL of ammonium chloride buffer was added. Adding a small amount of ammonia water for multiple times, continuously stirring by using a glass rod, immersing the electrode into the reaction liquid after the reaction liquid is uniform, measuring the pH value of the reaction liquid again, adjusting the pH value to 8.5, then slightly shaking the glass reaction bottle, after the electrode is immersed into the reaction liquid, waiting for the digital stability, reading, parallelly measuring for three times, recording, and finally taking the average value of the measured values of the three times, wherein the pH value of the reaction liquid is 8.5. And (5) cleaning the electrode by using distilled water after the measurement is finished, sucking the distilled water by using filter paper, and sleeving the composite electrode sleeve on the electrode, wherein the measurement step is finished. Then 50mg of imine reductase freeze-dried powder and 1mg of Nicotinamide Adenine Dinucleotide Phosphate (NADP), 5mg of Glucose Dehydrogenase (GDH) and 100mg of glucose are added into the reaction solution, stirred fully, added with 20mg of substrate, put into a full-temperature constant-temperature culture shaker for adjustment, the reaction temperature is controlled to be 35 ℃, and stirred continuously for 24 hours, so that the mixture reacts fully.

And after the reaction is finished, adding 1ml of acetonitrile into 1ml of reaction solution, dripping 1 drop of benzoyl chloride, placing the mixture into a shaking table, oscillating for 15 minutes, placing the mixture into a centrifugal machine for centrifugal treatment, and taking a layer required by the upper layer after layering to obtain a solution of a crude product. And (3) introducing nitrogen into the crude product solution, drying by blowing, removing impurities, adding 1ml of ethanol, entering a high performance liquid chromatograph for analysis, and detecting the reaction selectivity condition. Analysis conditions were as follows: shimadzu LC-20A liquid chromatograph and ultraviolet detector, CHIRALPAK OD-3R,150mm × 4.6mm,3 μm chiral chromatographic column, mobile phase: acetonitrile containing 0.05% trifluoroacetic acid-water containing 0.05% trifluoroacetic acid. Equilibrating at 30 ℃ under 1mL/min, and detecting the wavelength at 220nm.

Because the product has no ultraviolet absorption, the chiral purity of the product is detected by adopting a derivatization method and combining liquid chromatography. The chiral spectrum of the product by liquid chromatography is shown in FIG. 2A. Fig. 2A shows that the only peak at t =9.5 is the target compound (S) -3-butyn-2-amine. After the reaction is finished, the chiral purity of the obtained product is more than 99 percent by detecting the solution of the crude product obtained by treatment.

And in addition, 1ml of reaction solution is taken and added with 1ml of dichloromethane for extraction, after the extraction is finished, the extraction mixture is put into a centrifuge for centrifugal treatment, and after layering, the layer required by the lower layer is taken, namely the solution of a crude product. And (4) entering a high-efficiency gas chromatograph for analysis, and detecting the reaction purity condition. The analysis conditions are as follows: shimadzu GC-2010Plus gas chromatograph with FID detector, DB-FFAP (30m 0.32mm id 1.0 μm) or equivalent column, H2:40ml/min, air:400ml/min, injection port temperature: 180 ℃, sample injection split ratio: 50, column flow rate: 30.0cm/sec, constant linear velocity mode.

Fig. 2B is a gas chromatography spectrum for confirming the conversion rate of the reaction. Fig. 2B shows that the only peak at t =5.4 is the target compound (S) -3-butyn-2-amine, with a conversion of 99% or more.

Example 2

First, 21.40g of ammonium chloride was added to 1L of purified water, and the mixture was sufficiently stirred until the solid became clear, thereby preparing 400mmol of ammonium chloride buffer solution. The buffer concentration was measured by a pH meter, and the initial pH was 7.5. In an 8ml glass reaction flask, 4ml of ammonium chloride buffer was added. Ammonia was added to adjust the pH to 8.5 in an 8ml glass reaction flask. Then, 50mg of imine reductase (AoIRED) lyophilized powder and 1mg of nicotinamide adenine dinucleotide phosphate (oxidized state) (NADP), 5mg of Glucose Dehydrogenase (GDH), and 100mg of glucose were added to the reaction solution, and after stirring well, 20mg of substrate alkynone was added. Putting the mixture into a full-temperature constant-temperature culture shaking table, adjusting and controlling the reaction temperature to be 30 ℃, and continuously stirring for 24 hours at constant temperature to ensure that the mixture fully reacts.

Adding 1ml of dichloromethane into 1ml of reaction liquid for extraction, after extraction is finished, putting the extraction mixture into a centrifuge for centrifugal treatment, and taking a layer required by the lower layer after layering to obtain a solution of a crude product. And (4) entering a high-efficiency gas chromatograph for analysis, and detecting the reaction purity condition. Analysis conditions were as follows: shimadzu GC-2010Plus gas chromatograph with FID detector, DB-FFAP (30m 0.32mm id 1.0 μm) or equivalent column, H2:40ml/min, air:400ml/min, injection port temperature: 180 ℃, sample injection split ratio: 50, column flow rate: 30.0cm/sec, constant linear velocity mode.

Vapor phase chromatography was used to confirm the conversion of the reaction. The product has a chiral pattern of gas chromatography shown in FIG. 3. Fig. 3 shows that the peak at t =5.1 is the starting material, the peak at t =5.5 is the target compound (S) -3-butyn-2-amine, and the conversion rate is more than 81%.

Example 3

First, 21.40g of ammonium chloride was added to 1L of purified water, and the mixture was sufficiently stirred until the solid became clear, thereby preparing 400mmol of ammonium chloride buffer solution. The buffer concentration was measured by a pH meter, and the initial pH was 7.5. In an 8ml glass reaction flask, 4ml of ammonium chloride buffer was added. Ammonia was added to adjust the pH to 10 in an 8ml glass reaction flask. Then, 50mg of imine reductase (AoIRED) lyophilized powder and 1mg of nicotinamide adenine dinucleotide phosphate (oxidized state) (NADP), 5mg of Glucose Dehydrogenase (GDH), and 100mg of glucose were added to the reaction solution, and after stirring sufficiently, 20mg of substrate alkynone was added. Putting the mixture into a full-temperature constant-temperature culture shaking table, adjusting and controlling the reaction temperature to be 30 ℃, and continuously stirring for 24 hours at constant temperature to ensure that the mixture fully reacts.

The product has a chiral pattern of gas chromatography shown in FIG. 4. Fig. 4 shows that t =5.1 shows a peak as starting material and t =5.571 shows a peak as target compound (S) -3-butyn-2-amine with a conversion of only 4%. It is found that when the pH of the reaction mixture is 10, the reaction is not favorably carried out.

Comparative example:

according to examples 2-3, the effect of different phs on the reaction conversion rate was examined under otherwise identical reaction conditions, and the experimental results are shown below:

serial number	Ph	Conversion rate
			Example 4	6.0	0
Example 5	7.0	0
			Example 6	9.5	38

As shown in FIGS. 5 to 7, at pH6.0 and 7.0, the reaction was not facilitated; at pH9.5, the conversion rate of the reaction was also not high.

In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> Shanghai Hequan drug development Co Ltd

<120> method for preparing (S) -3-butyn-2-amine by biocatalysis

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Gly Ser Ala Leu Ala Ala Ala Leu Leu Asp Arg Gly His Asp Val Thr

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Val Trp Asn Arg Ser Pro Gly Lys Ala Ala Pro Leu Val Ala Lys Gly

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Ala Arg Gln Ala Asp Asp Ile Val Asp Ala Val Ser Ala Ser Arg Leu

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Leu Val Val Cys Leu Ala Asp Tyr Asp Ala Leu Tyr Ser Ala Leu Gly

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Pro Ala Arg Glu Ala Leu Arg Gly Arg Val Val Val Asn Leu Asn Ser

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Gly Thr Pro Lys Glu Ala Asn Glu Ala Leu Arg Trp Ala Glu Arg His

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Gly Thr Gly Tyr Leu Asp Gly Ala Ile Met Val Pro Pro Ala Met Val

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Gly His Pro Gly Ser Val Phe Leu Tyr Ser Gly Ser Ala Glu Val Phe

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Glu Glu Tyr Lys Glu Thr Leu Ala Gly Leu Gly Asp Pro Val His Leu

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Gly Thr Glu Ala Gly Leu Ala Val Leu Tyr Asn Thr Ala Leu Leu Ser

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Met Met Tyr Ser Ser Met Asn Gly Phe Leu His Ala Ala Ala Leu Val

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Gly Ser Ala Gly Val Pro Ala Ala Glu Phe Thr Lys Leu Ala Val Asp

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Val Ala Ala Gly Phe Gly Glu Ser Ser Tyr Tyr Ser Val Ile Glu Leu

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gccgccgccc tgctcgaccg cggtcacgac gtgaccgtgt ggaaccgctc ccccggcaag 120

gccgccccct tggtggcgaa aggcgcgcgg caggcggacg acatcgtgga cgcggtgtcc 180

gcgagccgtc tgctcgtggt ctgcctcgcc gactacgacg cgctttactc cgcgctcggc 240

cccgcgcggg aagccttgcg cggccgcgta gtggtgaacc tgaattccgg cacgccgaag 300

gaggcgaacg aagctctccg atgggccgag cgacacggaa cgggctatct cgacggcgcc 360

atcatggttc cccccgcgat ggtcggccac cccggctcgg tcttcctcta cagcggttcc 420

gccgaggttt tcgaggaata caaggagaca ttggccggtc tgggtgatcc ggtccatctc 480

ggcacggaag ccggcctcgc cgtgctgtac aacaccgcgt tgctgagcat gatgtactcg 540

tcgatgaacg gtttcctcca cgccgccgcg ctggtcggca gtgccggggt cccggcggcc 600

gaattcacga agctcgccgt cgactggttc ctgcccgcgg tgatcggaca gatcatcaag 660

gcggaggcgc ccaccatcga cgaaggcgtg taccccggtg acgccggttc gctggaaatg 720

aacgtcacga cactgaagca catcatcgga accagccagg agcagggcgt cgacaccgag 780

atcccggtcc gcaacaagga acttctggac cgggccgtcg ccgccgggtt cggcgagagc 840

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Claims

1. A method for preparing (S) -3-butyn-2-amine through biocatalysis is characterized in that in a liquid phase reaction system, 3-alkynyl-2-butanone is used as a substrate, the pH value of the reaction system is 8.0 to 9.0 under the condition that coenzyme exists, and carbonyl of the 3-alkynyl-2-butanone is reduced to chiral amino through catalysis of imine reductase to prepare (S) -3-butyn-2-amine;

the amino acid sequence of the imine reductase is shown as SEQ ID NO. 1.

2. The method of claim 1, wherein the imine reductase encoding gene sequence is selected from the group consisting of:

(a) A nucleotide sequence shown as SEQ ID NO. 2;

(b) A polynucleotide complementary to the sequence defined in (a); or

(c) Any polynucleotide or complementary sequence having at least 70% sequence identity to a sequence defined in (a).

3. The method according to claim 1, wherein the reaction system has a reaction initiation concentration of the 3-alkynyl-2-butanone of 1g/L to 5g/L.

4. The method of claim 1, wherein the imine reductase is used in an amount of 1 to 3 times the mass of the substrate.

5. The method of claim 1, wherein the coenzyme is an oxidative coenzyme and an enzyme for coenzyme regeneration is further present in the reaction system.

6. The method of claim 5, wherein the oxidative coenzyme is selected from NAD, NADP, or a combination thereof.

7. The method of claim 5, wherein the coenzyme is selected from the group consisting of the oxidative coenzymes NADP.

8. The method of claim 1, wherein in the reaction system, a co-substrate is also present; the co-substrate is selected from: one or more of isopropanol, glucose and ammonium formate.

9. The method of claim 8, wherein the co-substrate is selected from glucose.

10. The method of claim 5, wherein the enzyme for coenzyme regeneration is selected from the group consisting of: one or more of alcohol dehydrogenase, formate dehydrogenase and glucose dehydrogenase.

11. The method of claim 10, wherein the enzyme for coenzyme regeneration is selected from glucose dehydrogenases.

12. The method of claim 1, wherein the reaction system is a buffered saline solution system; the buffered saline solution is selected from ammonium chloride, ammonium formate or ammonium acetate buffer.

13. The method of claim 1, wherein the reaction system further comprises a co-solvent; the cosolvent is an organic solvent.

14. The method of claim 13, wherein the co-solvent is selected from the group consisting of: dimethyl sulfoxide, methanol, ethanol, isopropanol, acetone, or combinations thereof.

15. The method of claim 14, wherein the co-solvent is selected from dimethyl sulfoxide.

16. The process of claim 1, wherein the reaction temperature is from 25 ℃ to 35 ℃.

17. The process of claim 1, wherein the reaction temperature is from 30 ℃ to 35 ℃.

18. The process of claim 1, wherein the reaction time is from 1 to 24 hours.

19. The method of claim 1, wherein the pH is 8.5 ± 0.3.

20. The method of claim 1, wherein the pH is 8.5 ± 0.2.

21. The method of claim 1, wherein the pH is 8.5 ± 0.1.

22. The process of claim 1, further comprising the step of isolating the product (S) -3-butyn-2-amine.