CN111378695B - Method for synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol, phenylephrine and eye drops - Google Patents
Method for synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol, phenylephrine and eye drops Download PDFInfo
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Abstract
The invention relates to the field of bioengineering, and particularly discloses a method for synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol, phenylephrine and an eye drop, wherein the method for synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol comprises the following steps: 2-chloro-1- (3-hydroxyphenyl) ethanone is taken as a substrate, ketoreductase is taken as a catalyst, and a reaction system consisting of a hydrogen donor, a buffer solution and coenzyme is added for catalytic reaction, so that phenylephrine intermediate is obtained. The ketoreductase provided by the embodiment of the invention has high activity and stereoselectivity, and the synthesis method provided by the embodiment of the invention adopts the ketoreductase to perform enzyme catalysis synthesis of phenylephrine intermediate, only one-step reaction is needed, the used reagents are few, the synthesis steps are greatly simplified, the problem of complex steps in the existing R-3- (2-chloro-1-hydroxyethyl) phenol synthesis method is solved, the reaction condition is mild, the optical purity is high, and the method is green and environment-friendly.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a method for synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol, phenylephrine and an eye drop.
Background
Phenylephrine is a powerful vasoconstrictor and widely applied to hypotension emergency treatment or ophthalmic examination as a mydriasis drug, and because the agonism of R-type isomer is far higher than that of S-type isomer, chiral selectivity problem needs to be considered in preparing phenylephrine, wherein R-3- (2-chloro-1-hydroxyethyl) phenol is a common phenylephrine intermediate and belongs to R-type isomer.
At present, the synthesis method of R-3- (2-chloro-1-hydroxyethyl) phenol is usually synthesized by a resolution method, namely, a phenylephrine intermediate is synthesized by two steps of resolution and turnover, and the method cannot directly solve the chiral problem in the phenylephrine synthesis process, and is more in reagents, complex in operation and more in three wastes.
Therefore, the technical scheme has the following defects in actual operation: most of the existing synthesis methods of phenylephrine intermediates are complex in steps, so that chiral problems in the phenylephrine synthesis process cannot be directly solved.
Disclosure of Invention
The embodiment of the invention aims to provide a method for synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol, phenylephrine and eye drops, which are used for solving the problem that the existing R-3- (2-chloro-1-hydroxyethyl) phenol synthesis method provided in the background art has complex steps.
In order to achieve the above object, the embodiment of the present invention provides the following technical solutions:
a method for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol, comprising the following steps:
2-chloro-1- (3-hydroxyphenyl) ethanone is taken as a substrate, ketoreductase is taken as a catalyst, a reaction system is formed by adding a hydrogen donor, a buffer solution and coenzyme to perform catalytic reaction, and after the reaction is completed, the reaction solution is extracted to obtain the R-3- (2-chloro-1-hydroxyethyl) phenol.
Specifically, the synthetic route of the method for catalyzing and synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol is as follows:
as a further scheme of the invention: the ketoreductase is reductase coded by a ketoreductase gene, wherein the nucleotide sequence of the ketoreductase gene is shown as SEQ ID No.1, and the amino acid sequence of the ketoreductase is shown as SEQ ID No. 2.
Specifically, the nucleotide sequence shown in SEQ ID No.1 is as follows:
atggcagaaa tcccggaaaa acagacggcg ttcgtgttca aaaatggctc gttcgcactg
gaaaaaaaag aaatcgaagt cccgaaaccg gatgccggca aagtcctgct gaaagtggcg
gccgcaggtg tttgtcatag cgatctgcat gtcctgcacg gcggtctgcc gtatccggac
ggcctgattc tgggtcatga aattgcaggc cacatcgtcg cttatggcga tggtgtggac
aaagctgcgt ttccgtctga tgcactgtac gctgtggttg gcccgaaccc gtgcggcatg
tgtaaagcgt gccgtaccgg tgccgataat gtgtgcgaag acccgagtcg tacgcacatg
ggcctgggtt ccccgggcgg ttatgaacag tacacccaag tgagcgcgcg taacattacg
aaagttccgg aaggtatccc ggccgcagtc gctgcggcct ctaccgatgc cgttctgacg
ccgtatcacg cgctgaaacg tgccggcatt aacggtatga cccgtctgct gatcgtgggc
ctgggcggtc tgggtattaa tgccgttcag atcgcgaaag ccttcggcag ctacgttatc
gcagtcgatc cgaaagaaag ctctcgtgac ctggcaaaac agtatggtgc taatgaagtg
tacgcgaaac tgccggaaga atcactggat gtcgacgtgg cagctgattt ttatggctcg
cagggtacct tcgacctgtg tcagaaacat gtgaaagcac aaggcattct gctgccggtt
ggtctgcaag atccgaaaat cacctttgac ctgaaccacc tggcgttccg cgaatacacg
attatcggca atttttgggg taccagtcag gatcaaacgg aagttttcga actggtcaaa
aaaggtctgg tgaccccgca ggttgaaacc acgtcctggc tgaacgtgaa taaagttctg
aaagacctgg acgaaggcaa aatcaaatct cgcatggtgc tggtgcataa cgaagacaac
tagtaa。
further, the amino acid sequence shown in SEQ ID No.2 is as follows:
Met Ala Glu Ile Pro Glu Lys Gln Thr Ala Phe Val Phe Lys Asn Gly
Ser Phe Ala Leu Glu Lys Lys Glu Ile Glu Val Pro Lys Pro Asp Ala
Gly Lys Val Leu Leu Lys Val Ala Ala Ala Gly Val Cys His Ser Asp
Leu His Val Leu His Gly Gly Leu Pro Tyr Pro Asp Gly Leu Ile Leu
Gly His Glu Ile Ala Gly His Ile Val Ala Tyr Gly Asp Gly Val Asp
Lys Ala Ala Phe Pro Ser Asp Ala Leu Tyr Ala Val Val Gly Pro Asn
Pro Cys Gly Met Cys Lys Ala Cys Arg Thr Gly Ala Asp Asn Val Cys
Glu Asp Pro Ser Arg Thr His Met Gly Leu Gly Ser Pro Gly Gly Tyr
Glu Gln Tyr Thr Gln Val Ser Ala Arg Asn Ile Thr Lys Val Pro Glu
Gly Ile Pro Ala Ala Val Ala Ala Ala Ser Thr Asp Ala Val Leu Thr
Pro Tyr His Ala Leu Lys Arg Ala Gly Ile Asn Gly Met Thr Arg Leu
Leu Ile Val Gly Leu Gly Gly Leu Gly Ile Asn Ala Val Gln Ile Ala
Lys Ala Phe Gly Ser Tyr Val Ile Ala Val Asp Pro Lys Glu Ser Ser
Arg Asp Leu Ala Lys Gln Tyr Gly Ala Asn Glu Val Tyr Ala Lys Leu
Pro Glu Glu Ser Leu Asp Val Asp Val Ala Ala Asp Phe Tyr Gly Ser
Gln Gly Thr Phe Asp Leu Cys Gln Lys His Val Lys Ala Gln Gly Ile
Leu Leu Pro Val Gly Leu Gln Asp Pro Lys Ile Thr Phe Asp Leu Asn
His Leu Ala Phe Arg Glu Tyr Thr Ile Ile Gly Asn Phe Trp Gly Thr
Ser Gln Asp Gln Thr Glu Val Phe Glu Leu Val Lys Lys Gly Leu Val
Thr Pro Gln Val Glu Thr Thr Ser Trp Leu Asn Val Asn Lys Val Leu
Lys Asp Leu Asp Glu Gly Lys Ile Lys Ser Arg Met Val Leu Val His
Asn Glu Asp Asn。
as still further aspects of the invention: the ketoreductase is an E.coli expression product and the host cell is E.Coli, BL21 (DE 3), which is purchased from Beijing full gold Biotechnology Co., ltd.
As still further aspects of the invention: the reaction temperature of the catalytic reaction is 20-50 ℃, preferably 30 ℃.
As still further aspects of the invention: the concentration of the coenzyme in the reaction system is 0.003-1g/L.
Preferably, the concentration of the coenzyme in the reaction system is 0.1g/L.
As still further aspects of the invention: the coenzyme is NAD (nicotinamide adenine dinucleotide ) or NADP (nicotinamide adenine dinucleotide phosphate, nicotinamide adenine dinucleotide phosphate).
As still further aspects of the invention: the hydrogen donor in the catalytic reaction is isopropyl alcohol (IPA) or 1, 3-butanediol, ethanol, 2-butanol, 2, 3-butanediol, etc.
As still further aspects of the invention: the buffer is 0.1mol/L PBS buffer (namely phosphate buffer salt solution), and the pH of the PBS buffer ranges from 7 to 9, and more preferably pH=7.
As still further aspects of the invention: the ketoreductase used in the reaction system is coliphage, cell disruption supernatant or enzyme powder which expresses the ketoreductase.
As still further aspects of the invention: the method for synthesizing the R-3- (2-chloro-1-hydroxyethyl) phenol by catalysis adopts two reaction coupling modes, takes cheaper coenzyme NAD as a hydrogen electron carrier, takes isopropanol as a hydrogen donor, and realizes the recycling of NAD.
Another object of the embodiment of the invention is to provide a phenylephrine intermediate prepared by the method for synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol by catalysis.
Preferably, the phenylephrine intermediate is R-3- (2-chloro-1-hydroxyethyl) phenol.
It is another object of an embodiment of the present invention to provide phenylephrine prepared from the above-described R-3- (2-chloro-1-hydroxyethyl) phenol.
It is another object of an embodiment of the present invention to provide an eye drop comprising, in part, phenylephrine as described above.
Compared with the prior art, the invention has the beneficial effects that:
the method for synthesizing the R-3- (2-chloro-1-hydroxyethyl) phenol adopts ketoreductase to perform enzymatic synthesis on the phenylephrine intermediate R-3- (2-chloro-1-hydroxyethyl) phenol, can efficiently synthesize the phenylephrine intermediate by only one-step reaction, directly solves the chiral problem in the phenylephrine synthesis process, simultaneously uses fewer reagents, greatly simplifies the synthesis steps, has extremely high activity and stereoselectivity, has the conversion rate of more than 99 percent and the chiral selectivity of more than 99.7 percent, is suitable for a plurality of production scenes, solves the problem of complex steps in the existing R-3- (2-chloro-1-hydroxyethyl) phenol synthesis method, and has mild reaction conditions, high optical purity, simple and convenient operation and environmental protection.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
Example 1
Coli expressing ketoreductase, wherein the ketoreductase is reductase coded by a ketoreductase gene, the amino acid sequence of the ketoreductase is shown as SEQ ID No.2, and the nucleotide sequence of the ketoreductase gene is shown as SEQ ID No. 1.
Specifically, the construction and culture method of the escherichia coli expressing the ketoreductase are as follows:
1) Cutting the artificially synthesized ketoreductase gene DNA fragment with restriction enzymes NdeI and EcoRI for 8 hours at 37 ℃, purifying by agarose gel electrophoresis, and recovering the target fragment by using an agarose gel DNA recovery kit;
2) Under the action of T4DNA ligase, the target fragment is connected with plasmid pET24a which is also subjected to restriction enzyme digestion by restriction enzyme NdeI and restriction enzyme EcoRI at 25 ℃ overnight, and the connection product is converted into recombinant escherichia coli;
3) Inoculating recombinant escherichia coli into a culture dish containing 30mg/L of chloramphenicol-resistant LB solid medium, culturing at 37 ℃ for 20 hours, selecting single colony, inoculating the single colony into 50mL of chloramphenicol-resistant LB liquid medium, culturing for 20 hours under shaking, transferring bacterial liquid into 250mL of TB liquid medium after culturing is finished, culturing for 2.5 hours, diluting the bacterial liquid, detecting OD value to be 0.7, adding 0.1mmol/L of IPTG induction protein for expression, culturing at 30 ℃ under shaking for 18 hours, and centrifugally collecting bacterial bodies at 5000 rpm.
Example 2
The cells prepared in example 1 were reconstituted with 0.1mol/L PBS buffer (ph=7.0), homogenized and broken, and the enzyme supernatant was collected by centrifugation and lyophilized to obtain ketoreductase (enzyme powder), wherein the PBS buffer was added in the following amounts by weight: PBS buffer = 1: 5.
Example 3
The ketoreductase prepared in example 2 is used as a catalyst for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol, and specifically, the method for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol comprises the following steps: the method comprises the steps of taking 2-chloro-1- (3-hydroxyphenyl) ethanone as a substrate, taking ketoreductase prepared in the embodiment 2 as a catalyst, adding a hydrogen donor, a buffer solution and coenzyme NAD to form a reaction system, and carrying out catalytic reaction in a reaction bottle, specifically, placing the reaction bottle after adding a magneton in the reaction bottle on a reactor preheated to 30 ℃ in advance, regulating the rotating speed of the reactor to 400rpm for stirring reaction, and adding 5mL of acetonitrile after 24 hours for terminating reaction to obtain a reaction solution.
In an embodiment of the invention, the phenylephrine intermediate is R-3- (2-chloro-1-hydroxyethyl) phenol.
In the example of the present invention, the total volume of the catalytic system in which the coenzyme NAD was 1g/L of coenzyme NAD (i.e., the final concentration of coenzyme NAD in the final catalytic system was 0.1 g/L), the hydrogen donor was 2.5mL of isopropanol, the addition amount of 2-chloro-1- (3-hydroxyphenyl) ethanone was 2g (concentration was 400 g/L), and finally the system was complemented with 0.1mol/L of PBS buffer (pH=7).
In the embodiment of the invention, the reaction conditions of the catalytic reaction are as follows: the reaction was carried out at 30℃with magnetic stirring at 400rpm for 24 hours.
Example 4
The synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol was carried out in the same manner as in example 3, wherein the ketoreductase prepared in example 2 was used as a catalyst in an amount of 0.05g, 0.09g and 0.125g, respectively, which was divided into three groups of 0.05g, 0.09g and 0.125g, was reacted, and the reaction solutions obtained in the reaction groups of 0.05g, 0.09g and 0.125g were diluted with 50% acetonitrile (v/v), respectively, and then subjected to HPLC detection analysis, and the specific HPLC detection analysis results are shown in Table 1. Wherein the conditions for the HPLC detection and analysis are: the instrument model was Agilent 1100, the column was ZORBAX SB-C18 (4.6X105 mm gauge, particle size 5 μm), column temperature was 45 ℃, detection wavelength was 215nm, mobile phase A was 0.4% perchloric acid, mobile phase B was Acetonitrile (ACN), and mobile phase A: mobile phase B=80% (mass): 20% (mass), flow rate was 2mL/min, retention time of reaction product was 2.6min, retention time of substrate was 5.3min.
Table 1 HPLC assay results table
| Ketoreductase mass | Conversion rate | Chiral/%ee |
| 0.05g | 40.1％ | 99.74 |
| 0.09g | 75.2％ | 99.72 |
| 0.125g | 99.4％ | 99.77 |
According to the data in Table 1, it can be seen that the conversion rate can reach more than 99% and the chiral selectivity can reach more than 99.7% by using the ketoreductase prepared in the embodiment of the invention as a catalyst for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol, and the whole reaction only needs one step of synthesis, so that the reagent is less, the complicated step of synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol by using a chemical process is greatly simplified, and the method is a more economic and environment-friendly synthesis method.
Example 5
The ketoreductase prepared in example 2 is used as a catalyst for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol, and specifically, the method for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol comprises the following steps: taking 2-chloro-1- (3-hydroxyphenyl) ethanone as a substrate, taking ketoreductase prepared in the embodiment 2 as a catalyst, adding a hydrogen donor, a buffer solution and coenzyme NAD to form a reaction system, and carrying out catalytic reaction in a reaction bottle, specifically, placing the reaction bottle after adding a magneton in the reaction bottle on a reactor preheated to 30 ℃ in advance, regulating the rotating speed of the reactor to 400rpm for stirring reaction, adding 5mL of acetonitrile after 24 hours for stopping reaction to obtain a reaction solution, and extracting the reaction solution to obtain the R-3- (2-chloro-1-hydroxyethyl) phenol; wherein the total volume of the catalytic system in which the hydrogen donor was 2.5mL of isopropanol, the addition of 2-chloro-1- (3-hydroxyphenyl) ethanone was 2g (concentration 400 g/L), the addition of ketoreductase was 0.05g, and finally the system was complemented with 0.1mol/L of PBS buffer (ph=7).
In the embodiment of the invention, the final concentration of the coenzyme NAD in the reaction system is respectively 0.003g/L, 0.1g/L, 0.5g/L and 1g/L, after the reaction is completed, the reaction solutions obtained by the reaction sets of 0.003g/L, 0.1g/L, 0.5g/L and 1g/L are respectively diluted by 50% acetonitrile (v/v), and then HPLC detection analysis is carried out, and the specific HPLC detection analysis results are shown in Table 2. Wherein the conditions for the HPLC detection and analysis are: the instrument model was Agilent 1100, the column was ZORBAX SB-C18 (4.6X105 mm gauge, particle size 5 μm), column temperature was 45 ℃, detection wavelength was 215nm, mobile phase A was 0.4% perchloric acid, mobile phase B was Acetonitrile (ACN), and mobile phase A: mobile phase B=80% (mass): 20% (mass), flow rate was 2mL/min, retention time of reaction product was 2.6min, retention time of substrate was 5.3min.
TABLE 2 optimization results of coenzyme concentration
| Coenzyme concentration | 24 hour conversion/% |
| 0.003g/L | 38.9 |
| 0.1g/L | 41.5 |
| 0.5g/L | 40.7 |
| 1g/L | 42.1 |
As is clear from the data in Table 2, increasing the coenzyme concentration on the basis of 0.1g/L coenzyme concentration does not significantly contribute to the improvement of the reaction rate, and the amount of 0.1g/L coenzyme is the optimum condition from the viewpoints of the enzyme catalytic rate and the cost.
Example 6
The ketoreductase prepared in example 2 is used as a catalyst for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol, and specifically, the method for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol comprises the following steps: taking 2-chloro-1- (3-hydroxyphenyl) ethanone as a substrate, taking ketoreductase prepared in the example 2 as a catalyst, adding a hydrogen donor, a buffer solution and coenzyme NAD to form a reaction system, and carrying out catalytic reaction in a reaction bottle, specifically placing the reaction bottle after adding a magneton in the reaction bottle on a reactor preheated to 30 ℃ in advance, regulating the rotating speed of the reactor to 400rpm for stirring reaction, and adding 5mL of acetonitrile after 24 hours for terminating reaction to obtain a reaction solution; wherein the total volume of the catalytic system is 5mL, in the catalytic system, the hydrogen donor is 2.5mL of isopropanol, the addition amount of the 2-chloro-1- (3-hydroxyphenyl) ethanone is 2g (the concentration is 400 g/L), the addition amount of the ketoreductase is 0.05g, the final concentration of the coenzyme NAD in the reaction system is 0.1g/L, and finally the system is complemented by 0.1mol/L of PBS buffer.
In the embodiment of the present invention, the PBS buffers are respectively five groups of ph=6, ph=7, ph=8, ph=9 and ph=10, and after the reaction is completed, the reaction solutions obtained by the reaction groups of ph=6, ph=7, ph=8, ph=9 and ph=10 are respectively diluted with 50% acetonitrile (v/v), and then are subjected to HPLC detection analysis, and the specific HPLC detection analysis results are shown in table 3. Wherein the conditions for the HPLC detection and analysis are: the instrument model was Agilent 1100, the column was ZORBAX SB-C18 (4.6X105 mm gauge, particle size 5 μm), column temperature was 45 ℃, detection wavelength was 215nm, mobile phase A was 0.4% perchloric acid, mobile phase B was Acetonitrile (ACN), and mobile phase A: mobile phase B=80% (mass): 20% (mass), flow rate was 2mL/min, retention time of reaction product was 2.6min, retention time of substrate was 5.3min.
TABLE 3 buffer pH optimization results Table
| Buffer pH | 24 hour conversion/% |
| 6 | 32.5 |
| 7 | 42.7 |
| 8 | 39.4 |
| 9 | 34.3 |
| 10 | 30.3 |
From the data in Table 3, the enzyme catalytic rate was the fastest at buffer pH 7.
Example 7
The ketoreductase prepared in example 2 is used as a catalyst for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol, and specifically, the method for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol comprises the following steps: taking 2-chloro-1- (3-hydroxyphenyl) ethanone as a substrate, taking the ketoreductase prepared in the example 2 as a catalyst, adding a hydrogen donor, a buffer solution and coenzyme NADP to form a reaction system, and carrying out catalytic reaction in a reaction bottle, specifically placing the reaction bottle after adding a magneton in the reaction bottle on a reactor preheated to 50 ℃ in advance, regulating the rotating speed of the reactor to 400rpm for stirring reaction, and adding 5mL of acetonitrile after 24 hours for terminating reaction to obtain a reaction solution; wherein the total volume of the catalytic system is 5mL, in the catalytic system, the addition amount of 2-chloro-1- (3-hydroxyphenyl) ethanone is 0.05g (concentration is 10 g/L), the addition amount of ketoreductase is 0.1g, the final concentration of coenzyme NADP in the reaction system is 0.003g/L, the hydrogen donor is 1, 3-butanediol, the addition amount of 1, 3-butanediol is 2.5mL, and after the reaction is completed, the system is finally complemented with 0.1mol/L PBS buffer (pH=7).
In the examples of the present invention, the obtained reaction solution was diluted with 50% acetonitrile (v/v) and then subjected to HPLC detection analysis, and the result showed that the conversion rate was 99.2% in 24 hours of the reaction. Wherein the conditions for the HPLC detection and analysis are: the instrument model was Agilent 1100, the column was ZORBAX SB-C18 (4.6X105 mm gauge, particle size 5 μm), column temperature was 45 ℃, detection wavelength was 215nm, mobile phase A was 0.4% perchloric acid, mobile phase B was Acetonitrile (ACN), and mobile phase A: mobile phase B=80% (mass): 20% (mass), flow rate was 2mL/min, retention time of reaction product was 2.6min, retention time of substrate was 5.3min.
Example 8
The ketoreductase prepared in the example 2 is used as a catalyst for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol, and the specific method comprises the following steps:
the preparation method comprises the steps of taking 2-chloro-1- (3-hydroxyphenyl) ethanone as a substrate, taking ketoreductase prepared in the embodiment 2 as a catalyst, adding a hydrogen donor, a buffer solution and coenzyme NAD to form a reaction system, and carrying out catalytic reaction in reaction bottles, specifically preparing a plurality of reaction bottles, respectively adding magnetons into the reaction bottles, placing the reaction bottles on a preheated reactor with the temperature of 20 ℃,30 ℃,40 ℃ and 50 ℃, regulating the rotation speed of the reactor to 400rpm for stirring reaction, and adding 5mL of acetonitrile after 24 hours for terminating the reaction to obtain a reaction solution.
In the example of the present invention, the total volume of the catalytic system in which the coenzyme NAD was 1g/L of coenzyme NAD (i.e., the final concentration of coenzyme NAD in the final catalytic system was 0.1 g/L), the hydrogen donor was 2.5mL of isopropanol, the addition amount of 2-chloro-1- (3-hydroxyphenyl) ethanone was 0.05g (concentration was 10 g/L), and finally the system was complemented with 0.1mol/L of PBS buffer (pH=7).
In the embodiment of the invention, the reaction conditions of the catalytic reaction are as follows: the temperature was 20℃and 30℃and 40℃and 50℃respectively, and the reaction was carried out by magnetic stirring at 400rpm for 24 hours.
In the examples of the present invention, the obtained reaction solution was diluted with 50% acetonitrile (v/v) and then subjected to HPLC detection analysis, and the specific HPLC detection analysis results are shown in Table 4. Wherein the conditions for the HPLC detection and analysis are: the instrument model was Agilent 1100, the column was ZORBAX SB-C18 (4.6X105 mm gauge, particle size 5 μm), column temperature was 45 ℃, detection wavelength was 215nm, mobile phase A was 0.4% perchloric acid, mobile phase B was Acetonitrile (ACN), and mobile phase A: mobile phase B=80% (mass): 20% (mass), flow rate was 2mL/min, retention time of reaction product was 2.44min, retention time of substrate was 3.70min.
TABLE 4 Table of results of reactions at different temperatures
| Reaction temperature | Conversion at 24 hours |
| 20℃ | 98.2％ |
| 30℃ | 99.4％ |
| 40℃ | 99.1％ |
| 50℃ | 98.2％ |
Example 9
The ketoreductase prepared in the example 2 is used as a catalyst for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol, and the specific method comprises the following steps:
the method comprises the steps of taking 2-chloro-1- (3-hydroxyphenyl) ethanone as a substrate, taking ketoreductase prepared in the embodiment 2 as a catalyst, respectively adding hydrogen donors (the hydrogen donors can be one of ethanol, isopropanol, 2-butanol, 1, 3-butanediol and 2, 3-butanediol), buffer solution and coenzyme NAD to form a reaction system, carrying out catalytic reaction in a reaction bottle, specifically adding a magneton into the reaction bottle, placing the reaction bottle on a reactor preheated to 30 ℃ in advance, regulating the rotation speed of the reactor to 400rpm for stirring reaction, and adding 5mL of acetonitrile after 24 hours for terminating reaction to obtain a reaction solution.
In the example of the present invention, the total volume of the catalytic system in which the coenzyme NAD was 1g/L of coenzyme NAD (i.e., the final concentration of coenzyme NAD in the final catalytic system was 0.1 g/L) was 5mL, the addition amount of 2-chloro-1- (3-hydroxyphenyl) ethanone was 0.05g (concentration was 10 g/L), and finally the system was complemented with 0.1mol/L of PBS buffer (pH=7).
In the embodiment of the invention, the reaction conditions of the catalytic reaction are as follows: the reaction was carried out at 30℃with magnetic stirring at 400rpm for 24 hours.
In the examples of the present invention, the obtained reaction solution was diluted with 50% acetonitrile (v/v) and then subjected to HPLC detection analysis, and the specific HPLC detection analysis results are shown in Table 5. Wherein the conditions for the HPLC detection and analysis are: the instrument model was Agilent 1100, the column was ZORBAX SB-C18 (4.6X105 mm gauge, particle size 5 μm), column temperature was 45 ℃, detection wavelength was 215nm, mobile phase A was 0.4% perchloric acid, mobile phase B was Acetonitrile (ACN), and mobile phase A: mobile phase B=80% (mass): 20% (mass), flow rate was 2mL/min, retention time of reaction product was 2.44min, retention time of substrate was 3.70min.
TABLE 5 reaction results of different hydrogen donors
| Hydrogen donor | Conversion at 24 hours |
| Ethanol | 72.3％ |
| Isopropyl alcohol | 99.8％ |
| 2-butanol | 99.2％ |
| 1, 3-butanediol | 99.2％ |
| 2, 3-butanediol | 99.6％ |
Example 10
The ketoreductase prepared in example 2 is used as a catalyst for catalyzing and synthesizing phenylephrine intermediate, and specifically, the method for catalyzing and synthesizing phenylephrine intermediate comprises the following steps:
the method comprises the steps of taking 2-chloro-1- (3-hydroxyphenyl) ethanone as a substrate, taking ketoreductase prepared in the embodiment 2 as a catalyst, adding hydrogen donor (IPA (circulating with self-ketoreductase), glucose (circulating with GDH), ammonium formate (circulating with FDH), buffer solution and coenzyme NAD and circulating enzymes respectively taking self-ketoreductase, GDH and FDH as circulating enzymes into a reaction bottle to perform catalytic reaction, specifically, placing the reaction bottle after adding a magneton on a reactor preheated to 30 ℃, regulating the rotating speed of the reactor to 400rpm to perform stirring reaction, adding 5mL of acetonitrile after 24 hours to terminate the reaction to obtain a reaction solution, wherein the total volume of the catalytic system is 5mL, the adding amount of 2-chloro-1- (3-hydroxyphenyl) ethanone in the catalytic system is 0.05g (the concentration is 10 g/L), the adding amount of the ketoreductase is 0.1g, the final concentration of the coenzyme in the reaction system is 0.003g/L, the rotating speed of the hydrogen donor is 2.5mL of isopropanol, the adding amount of the hydrogen donor is 2.5mL of the buffer solution is 0.0.7 times the final concentration of the circulating enzyme (the concentration of the buffer solution is 0.0.0.0 time of the final concentration of the buffer solution is 0.0 time of the circulating enzyme).
In the examples of the present invention, the obtained reaction solution was diluted with 50% acetonitrile (v/v) and then subjected to HPLC detection analysis, and the specific HPLC detection analysis results are shown in Table 6. Wherein the conditions for the HPLC detection and analysis are: the instrument model was Agilent 1100, the column was ZORBAX SB-C18 (4.6X105 mm gauge, particle size 5 μm), column temperature was 45 ℃, detection wavelength was 215nm, mobile phase A was 0.4% perchloric acid, mobile phase B was Acetonitrile (ACN), and mobile phase A: mobile phase B=80% (mass): 20% (mass), flow rate was 2mL/min, retention time of reaction product was 2.44min, retention time of substrate was 3.70min.
TABLE 6 results of different circulation systems
| Coenzyme circulation system | Conversion at 24 hours |
| Self ketoreductase circulation system | 99.2％ |
| GDH circulation system | 99.4％ |
| FDH circulation system | 99.4％ |
As can be seen from the results, the ketoreductase prepared by the embodiment of the invention has extremely high activity and stereoselectivity, is a ketoreductase with industrialization potential, and is used for synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol by enzyme catalysis, and the method has the advantages of mild reaction conditions, high optical purity, simple and convenient operation, environmental protection and extremely competitive future; moreover, the single reaction substrate feeding amount can be more than 400g/L, the conversion rate can be more than 99%, the chiral selectivity can be more than 99.7%, and the ketoreductase used in the reaction has excellent performances of high temperature resistance, high organic resistance and the like, is suitable for a plurality of production scenes, and the whole reaction only needs one-step synthesis, uses fewer reagents, and greatly simplifies the complicated steps of synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol by adopting a chemical process.
While the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Sequence listing
<110> Ningbo enzyme Sitting bioengineering Co., ltd
<120> method for synthesizing R-3- (2-chloro-1-hydroxyethyl) phenol, phenylephrine, and eye drops
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1026
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atggcagaaa tcccggaaaa acagacggcg ttcgtgttca aaaatggctc gttcgcactg 60
gaaaaaaaag aaatcgaagt cccgaaaccg gatgccggca aagtcctgct gaaagtggcg 120
gccgcaggtg tttgtcatag cgatctgcat gtcctgcacg gcggtctgcc gtatccggac 180
ggcctgattc tgggtcatga aattgcaggc cacatcgtcg cttatggcga tggtgtggac 240
aaagctgcgt ttccgtctga tgcactgtac gctgtggttg gcccgaaccc gtgcggcatg 300
tgtaaagcgt gccgtaccgg tgccgataat gtgtgcgaag acccgagtcg tacgcacatg 360
ggcctgggtt ccccgggcgg ttatgaacag tacacccaag tgagcgcgcg taacattacg 420
aaagttccgg aaggtatccc ggccgcagtc gctgcggcct ctaccgatgc cgttctgacg 480
ccgtatcacg cgctgaaacg tgccggcatt aacggtatga cccgtctgct gatcgtgggc 540
ctgggcggtc tgggtattaa tgccgttcag atcgcgaaag ccttcggcag ctacgttatc 600
gcagtcgatc cgaaagaaag ctctcgtgac ctggcaaaac agtatggtgc taatgaagtg 660
tacgcgaaac tgccggaaga atcactggat gtcgacgtgg cagctgattt ttatggctcg 720
cagggtacct tcgacctgtg tcagaaacat gtgaaagcac aaggcattct gctgccggtt 780
ggtctgcaag atccgaaaat cacctttgac ctgaaccacc tggcgttccg cgaatacacg 840
attatcggca atttttgggg taccagtcag gatcaaacgg aagttttcga actggtcaaa 900
aaaggtctgg tgaccccgca ggttgaaacc acgtcctggc tgaacgtgaa taaagttctg 960
aaagacctgg acgaaggcaa aatcaaatct cgcatggtgc tggtgcataa cgaagacaac 1020
tagtaa 1026
<210> 2
<211> 340
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Ala Glu Ile Pro Glu Lys Gln Thr Ala Phe Val Phe Lys Asn Gly
1 5 10 15
Ser Phe Ala Leu Glu Lys Lys Glu Ile Glu Val Pro Lys Pro Asp Ala
20 25 30
Gly Lys Val Leu Leu Lys Val Ala Ala Ala Gly Val Cys His Ser Asp
35 40 45
Leu His Val Leu His Gly Gly Leu Pro Tyr Pro Asp Gly Leu Ile Leu
50 55 60
Gly His Glu Ile Ala Gly His Ile Val Ala Tyr Gly Asp Gly Val Asp
65 70 75 80
Lys Ala Ala Phe Pro Ser Asp Ala Leu Tyr Ala Val Val Gly Pro Asn
85 90 95
Pro Cys Gly Met Cys Lys Ala Cys Arg Thr Gly Ala Asp Asn Val Cys
100 105 110
Glu Asp Pro Ser Arg Thr His Met Gly Leu Gly Ser Pro Gly Gly Tyr
115 120 125
Glu Gln Tyr Thr Gln Val Ser Ala Arg Asn Ile Thr Lys Val Pro Glu
130 135 140
Gly Ile Pro Ala Ala Val Ala Ala Ala Ser Thr Asp Ala Val Leu Thr
145 150 155 160
Pro Tyr His Ala Leu Lys Arg Ala Gly Ile Asn Gly Met Thr Arg Leu
165 170 175
Leu Ile Val Gly Leu Gly Gly Leu Gly Ile Asn Ala Val Gln Ile Ala
180 185 190
Lys Ala Phe Gly Ser Tyr Val Ile Ala Val Asp Pro Lys Glu Ser Ser
195 200 205
Arg Asp Leu Ala Lys Gln Tyr Gly Ala Asn Glu Val Tyr Ala Lys Leu
210 215 220
Pro Glu Glu Ser Leu Asp Val Asp Val Ala Ala Asp Phe Tyr Gly Ser
225 230 235 240
Gln Gly Thr Phe Asp Leu Cys Gln Lys His Val Lys Ala Gln Gly Ile
245 250 255
Leu Leu Pro Val Gly Leu Gln Asp Pro Lys Ile Thr Phe Asp Leu Asn
260 265 270
His Leu Ala Phe Arg Glu Tyr Thr Ile Ile Gly Asn Phe Trp Gly Thr
275 280 285
Ser Gln Asp Gln Thr Glu Val Phe Glu Leu Val Lys Lys Gly Leu Val
290 295 300
Thr Pro Gln Val Glu Thr Thr Ser Trp Leu Asn Val Asn Lys Val Leu
305 310 315 320
Lys Asp Leu Asp Glu Gly Lys Ile Lys Ser Arg Met Val Leu Val His
325 330 335
Asn Glu Asp Asn
340
Claims (4)
1. A method for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol, comprising the steps of: 2-chloro-1- (3-hydroxyphenyl) ethanone is taken as a substrate, ketoreductase is taken as a catalyst, a hydrogen donor, a buffer solution and coenzyme are added to form a reaction system for catalytic reaction, and after the reaction is completed, the reaction solution is extracted to obtain the R-3- (2-chloro-1-hydroxyethyl) phenol;
the nucleotide sequence of the ketoreductase gene is shown as SEQ ID No.1, and the amino acid sequence of the ketoreductase is shown as SEQ ID No. 2; the concentration of the 2-chloro-1- (3-hydroxyphenyl) ethanone is 400g/L; the coenzyme is nicotinamide adenine dinucleotide, and the concentration of the coenzyme in the reaction system is 0.003. 0.003 g/L; the coenzyme circulatory system, enzyme is ketoreductase itself, or other coenzyme circulatory system, GDH, FDH and corresponding hydrogen donor.
2. The method for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol according to claim 1, wherein the reaction temperature of the catalytic reaction is 20-50 ℃.
3. The method for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol according to claim 1, wherein the hydrogen donor is any one of isopropanol, 1, 3-butanediol, ethanol, 2-butanol or 2, 3-butanediol.
4. The method for catalytic synthesis of R-3- (2-chloro-1-hydroxyethyl) phenol according to claim 1, wherein the pH of the buffer is in the range of 7-9.
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