CN104830924A - Method for producing thienyl chiral alcohol compound through biological catalysis of carbonyl reductase - Google Patents
Method for producing thienyl chiral alcohol compound through biological catalysis of carbonyl reductase Download PDFInfo
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Abstract
The invention discloses a method for synthesizing corresponding (S) configuration alcohols through asymmetric reduction of five thienyl ketone substrates by taking carbonyl reductase as a biocatalyst. According to the method, the catalyst is easy to prepare, aqueous phase catalysis is realized, the reaction conditions are mild, the reaction system is simple, any side reaction is avoided, most of the ee values are larger than 99 percent, an environment-friendly biological catalysis method is provided for producing thienyl chiral alcohols, and the method has high industrial application potential.
Description
Technical field
The invention belongs to enzyme and biocatalysis field, be specifically related to utilize carbonyl reduction enzymes biocatalysis to produce the method for thienyl chiral alcohol compounds.
Background technology
Duloxetine (Duloxetine) is third generation thymoleptic, one of topmost thymoleptic of market sale, effectively can suppress the re-uptake (Wong of 5-hydroxyl look ammonia and norepinephrine, D.T., et al.Life Sci, 1988,43:2049-2057), and also have good drug effect to the disease such as overall pain, gastrointestinal disturbance.Research shows (S)-duloxetine (chemical name, (S)-(+)-N-methyl-3-(1-naphthyloxy)-3-(2-thiophene)-propylamine) just there is pharmaceutical activity, therefore the acquisition of (S) configuration intermediate becomes the committed step in duloxetine synthesis.
The production of duloxetine chiral intermediate carries out chiral separation after also adopting chemical technology reduction at present, and its main Problems existing has: need a large amount of resolving agents, reactions steps is long, and energy consumption is large; Waste discharge amount is large, and environmental pollution is serious; Productive rate is low, and optical purity of products is not high, heavy-metal residual problem.High based on biocatalysis stereoselectivity, by product is few, reaction conditions is gentle and the unique advantage of the aspect such as environmental friendliness, utilizes the research of biocatalysis production duloxetine. intermediate also to develop rapidly.
Analyze the structure of (S)-duloxetine, according to inverse composition strategy and the easy catalysis of substrate, the principle such as stable, following 5 kinds of listed thienyl ketone substrate S1-S5 can be utilized can to obtain duloxetine chiral intermediate (Tang through asymmetric reduction, C.G., et al.Biotechnol Lett, 2011,33 (7): 1435-1440).
The biocatalysis research of these thienones mainly concentrates on substrate S1, S2 and S5, and S3 and S4 does not also have the report of biocatalysis at present.The main level of the bibliographical information of thienone S1, S2 and S5 biocatalysis is as follows:
(1) substrate S1: concentration of substrate 10g/l, 6h react end, product ee value >98% (Wada, M., et al.BiosciBiotech Bioch, 2004,68 (7): 1481-1488).
(2) substrate S2: the end is with thing Rho dense do degree r1or0ugl/aL, glu is anti-, and ti answers ni6sh, complete thin product born of the same parents thing is that ee urges >ization 98 doses of %, concentration of substrate 30g/l, reaction 48h, transformation efficiency >95%, ee value >99% (Tang, C.G., et al.Biotechnol Lett, 2011,33 (7): 1435-1440); With ChKRED15 crude enzyme liquid for biological catalyst, can transform the substrate of 20g/l in 2h, transformation efficiency is that more than 99%, ee value is greater than 99.9% (CN103740738A).
(3) substrate S5: with Candida tropicalis for biological catalyst, concentration of substrate 1g/l, reaction 60h, product ee value >99% (Soni, P., et al.App Microbiol Biotechnol, 2005,67 (6): 771-777); With alcoholdehydrogenase ADH-LK for biological catalyst, concentration of substrate 100g/l, reaction 24h, product ee value >99% (Codexis, INC.WO2010025238A2).
Although investigators have screened the biocatalysis that multiple-microorganism bacterial strain (or carbonyl reductase) realizes above-mentioned S1, S2 and S5 substrate, all things considered, enzyme source is still less, and most catalytic process take original bacteria as biological catalyst; Meanwhile, substrate feed concentrations is general lower (only have S5 to have patent report to feed intake and can realize 100g/l) also, also has larger distance from application.The more excellent enzyme sources newly of active development, by the biocatalysis synthesis establish a firm foundation for realizing thienyl chiral alcohol compounds.
Summary of the invention
The object of this invention is to provide a kind of with the method for carbonyl reductase for biological catalyst preparing thienyl chiral alcohol compounds.Particularly, the present invention's substrate used is following general structure (I), utilizes different efficient highly selective carbonyl reductases to be biological catalyst, obtains corresponding (S) configuration chiral alcohol through asymmetric reduction.
Biocatalysis process can be expressed as follows:
Wherein, R substituent can for containing ester structure, halogen-substituted alkyl or containing amino structure etc.;
Further, R substituent is COOCH
3, COOCH
2cH
3, CONHCH
3, CH
2cl, CN or CH
2n (CH
3)
2, these substrates obtain S configuration alcohol through carbonyl reduction enzymes biocatalysis, can be used as the chiral intermediate in duloxetine synthesis.
The biological catalyst that the present invention relates to is from the carbonyl reductase work box of this experimental construction.According to the common knowledge of this area, those skilled in the art are by obtaining biological catalyst with under type.First, the information (gene order and aminoacid sequence) of carbonyl reductase is inquired about (http://www.ncbi.nlm.nih.gov/) by the NCBI accession number of this enzyme or knows from the sequence (SEQ ID:No.1-4) specification sheets sequence table, then obtain through the synthesis of gene chemical synthesis company, and build coli expression system with routine techniques means, obtain following enzyme as biological catalyst through overexpression.
The substrate that the present invention is the suitableeest and corresponding carbonyl reductase as follows:
(1)
The carbonyl reductase of catalytic substrate S1 has: (gene order is SEQ ID:No.1 to KRED2157, aminoacid sequence is SEQ ID:No.2), ChKRED07 (NCBI accession number: KC342007), ChKRED10 (NCBI accession number: KC342010), ChKRED11 (NCBI accession number: KC342011), ChKRED12 (NCBI accession number: KC342012) or ChKRED14 (NCBI accession number: KC342014).
The homologue S1-1 of substrate S1, its structural formula is
in the present invention can the above-mentioned carbonyl reductase of catalysis S1 equally can this compound of catalysis, obtain corresponding S configuration alcohol.
(2)
The carbonyl reductase of catalytic substrate S2 has: ChKRED07 (NCBI accession number: KC342007), (gene order is SEQ ID:No.1 to KRED2157, aminoacid sequence is SEQ ID:No.2), ChKRED03 (NCBI accession number: KC342003), ChKRED10 (NCBI accession number: KC342010), ChKRED23 (NCBI accession number: KC342023), ChKRED24 (NCBI accession number: KC342024), ChKRED26 (NCBI accession number: KC342026) or ChKRED14 (NCBI accession number: KC342014).
(3)
The carbonyl reductase of catalytic substrate S3 has: ChKRED07 (NCBI accession number: KC342007) or KRED2935 (gene order is SEQ ID:No.3, and aminoacid sequence is SEQ ID:No.4).
(4)
The carbonyl reductase of catalytic substrate S4 has: ChKRED07 (NCBI accession number: KC342007), ChKRED10 (NCBI accession number: KC342010), ChKRED11 (NCBI accession number: KC342011), ChKRED12 (NCBI accession number: KC342012), ChKRED14 (NCBI accession number: KC342014), (gene order is SEQ ID:No.1 to KRED2157, aminoacid sequence is SEQ ID:No.2), (gene order is shown in SEQ ID:No.3 to ChKRED03 (NCBI accession number: KC342003) or KRED2935, aminoacid sequence is shown in SEQ ID:No.4).
(5)
The carbonyl reductase of catalytic substrate S5 has: ChKRED10 (NCBI accession number: KC342010), ChKRED03 (NCBI accession number: KC342003) or ChKRED05 (NCBI accession number: KC342005).
Biocatalysis system of the present invention and reaction conditions:
(1) when biological catalyst is resting cell, biocatalysis system is: phosphate buffered saline buffer (0.1M, pH6 ~ 8), carbonyl reductase recombinant bacterium resting cell, general structure (I) substrate and glucose.Resting cell concentration is 50 ~ 300g/l (in wet thallus); The final concentration of general structure (I) substrate is 1 ~ 100g/l; The concentration of glucose is 1% ~ 5% (w/v).
Substrate is first dissolved in DMSO and is mixed with 30 ~ 50% (w/v) stock solution.
(2) biological catalyst be the pure enzyme of carbonyl reductase or thick enzyme time, biocatalysis system is:
Phosphate buffered saline buffer (0.1M, pH6 ~ 8), carbonyl reductase, general structure (I) substrate and reduced coenzyme NAD (P) H;
Or biocatalysis system is:
Phosphate buffered (0.1M, pH6 ~ 8), carbonyl reductase, general structure (I) substrate, oxidized coenzyme NAD (P)
+, Hexose phosphate dehydrogenase and glucose.
The enzyme amount of carbonyl reductase can adjust according to concentration of substrate, and preferably concentration is pure enzyme 0.1 ~ 10g/l, thick enzyme 1 ~ 20g/l (with total protein gauge); Final substrate concentrations is 1 ~ 150g/l; NAD (P)
+concentration 0.1 ~ 1g/l; Glucose dehydro enzyme concn 2U/ml ~ 50U/ml; The concentration of glucose is 5% ~ 15% (w/v).
Biocatalysis condition: temperature 20 ~ 35 DEG C, rotating speed 50 ~ 220rpm, transformation time is 1 ~ 24h.
Beneficial effect of the present invention:
The invention discloses and obtain thienyl chiral alcohol compounds with carbonyl reductase Biocatalysis method, the catalyzer of the method is easily prepared, aqueous catalysis, reaction conditions is gentle, reaction system is simple, without side reaction, the ee value overwhelming majority is greater than 99%, produces provide selectable eco-friendly Biocatalysis method for thienyl chirality alcohols.The carbonyl reductase that the present invention relates to is more, for this compounds biocatalysis provides abundant enzyme source.
The chiral intermediate P1-P5 that the present invention obtains all can be used as the intermediate in duloxetine synthesis.To produce P1 product, during ChKRED12 catalytic substrate S1, when concentration of substrate is 100g/l, transformation efficiency >99%, ee value >99%, has very strong industrial application potentiality.
It should be noted that; according to this area common knowledge; due to the degeneracy of the codon of Nucleotide, every above-mentioned carbonyl reductase is through allelic mutation or have one or more aminoacid addition, insertion, disappearance or/and replace and have the enzyme that catalytic structure formula (I) substrate generates corresponding (S) configuration alcohol and all belong to protection scope of the present invention.
Embodiment
The present invention is explained below in conjunction with embodiment.Embodiment for ease of better understanding the present invention, but is not limitation of the present invention.
Embodiment 1: the screening of carbonyl reductase
With method well known in the art, carbonyl reductase gene is connected into pET28a (+) carrier, proceeds to heterogenous expression in e. coli bl21-DE3, obtain resting cell as biological catalyst.During screening, biocatalysis and system are: phosphate buffered saline buffer (0.1M, pH7.0), carbonyl reductase recombinant bacterium resting cell 150g/l (in wet thallus), substrate 1g/l and glucose 2% (w/v).Reaction times is 24h, temperature of reaction 30 DEG C, rotating speed 200rpm.
Meanwhile, pET28 (+) empty carrier not being connected into carbonyl reductase gene is proceeded to heterogenous expression in e. coli bl21-DE3 equally, with this resting cell for blank transforms all kinds of substrate.The selection result of below showing, blank all can not conversion of substrate.
After reaction terminates, with equal-volume extraction into ethyl acetate 3 times, merge organic phase, add anhydrous sodium sulfate drying, then vacuum rotary steam is except desolventizing, adds Virahol (HPLC level) sample dissolution, the transformation efficiency of HPLC working sample and product ee value after high speed centrifugation.
(1) carbonyl reduction substrate for enzymatic activity S1:
Transformation efficiency detects: SIL-100A (4.6 × 250mm); Column temperature: 35 DEG C; Moving phase (v/v): hexane:isopropanol=70:30; Flow velocity: 0.4ml/min; Appearance time: substrate 9.3min, product 10.1min.
Product ee value detects: Daicel CHIRALCEL AD-H (4.6 × 250mm) column temperature: 35 DEG C; Moving phase (v/v): hexane:isopropanol=98:2; Flow velocity: 0.8ml/min; Appearance time: S type 39.0min, R type 40.5min.
The carbonyl reductase of conversion of substrate S1 and transformation efficiency thereof and ee value can see the following form 1:
Table 1
(2) carbonyl reduction substrate for enzymatic activity S1-1:
Transformation efficiency detects: SIL-100A (4.6 × 250mm); Column temperature: 35 DEG C; Moving phase (v/v): hexane:isopropanol=70:30; Flow velocity: 0.4ml/min; Appearance time: substrate 9.9min, product 11.2min.
Product ee value detects: Daicel CHIRALCEL AS-H (4.6 × 250mm) column temperature: 35 DEG C; Moving phase (v/v): hexane:isopropanol=95:5 flow velocity: 0.8ml/min; Appearance time: S type 42.2min, R type 43.3min.
The carbonyl reductase of conversion of substrate S1 and transformation efficiency thereof and ee value can see the following form 2:
Table 2
Due to substrate S1 and S1-1 structure very similar, the carbonyl reductase that therefore can transform both is identical, and just transformation efficiency is variant.
(3) carbonyl reduction substrate for enzymatic activity S2:
Transformation efficiency measures: SIL-100A (4.6 × 250mm); Column temperature: 35 DEG C; Moving phase (v/v): hexane:isopropanol=70:30; Flow velocity: 0.8ml/min; Appearance time: substrate 12.0min, product 8.9min.
Product ee value detects: Daicel CHIRALCEL OJ-H (4.6 × 250mm); Column temperature: 35 DEG C; Moving phase (v/v): hexane:isopropanol=90:10; Flow velocity: 0.5ml/min; Appearance time: S type 22.8min, R type 24.2min.
The carbonyl reductase of conversion of substrate S2 and transformation efficiency thereof and ee value can see the following form 3:
Table 3
(4) carbonyl reduction substrate for enzymatic activity S3:
Transformation efficiency detects: SIL-100A (4.6 × 250mm); Column temperature: 35 DEG C; Moving phase (v/v): hexane:isopropanol=70:30; Flow velocity: 0.4ml/min; Appearance time: substrate 8.9min, product 9.8min.
When product ee value detects: Daicel CHIRALCEL AS-H (4.6 × 250mm); Column temperature: 35 DEG C; Moving phase (v/v): hexane:isopropanol=97:3; Flow velocity: 0.8ml/min; Appearance time: S type 21.6min, R type 23.7min.
The carbonyl reductase of conversion of substrate S3 and transformation efficiency thereof and ee value can see the following form 4:
Table 4
(4) carbonyl reduction substrate for enzymatic activity S4:
Transformation efficiency detects: SIL-100A (4.6 × 250mm); Column temperature: 35 DEG C; Moving phase (v/v): hexane:isopropanol=70:30; Flow velocity: 0.8ml/min; Appearance time: substrate 7.8min, product 5.3min.
Product ee pH-value determination pH: Daicel CHIRALCEL OJ-H (4.6 × 250mm); Column temperature: 35 DEG C; Moving phase (v/v): hexane:isopropanol=80:20; Flow velocity: 0.8ml/min; Appearance time: S type 21.0min, R type 23.7min.
The carbonyl reductase of conversion of substrate S4 and transformation efficiency thereof and ee value can see the following form 5:
Table 5
(5) carbonyl reduction substrate for enzymatic activity S5:
Transformation efficiency detects and product ee value testing conditions: Daicel CHIRALCEL OJ-H (4.6 × 250mm); Column temperature: 35 DEG C; Moving phase (v/v), hexane:isopropanol:triethylamine=95:5:1.5; Flow velocity: 0.8ml/min; Appearance time: substrate 11.2min, S type product 13.5min, R type 12.2min.
The carbonyl reductase of conversion of substrate S5 and transformation efficiency thereof and ee value can see the following form 6:
Table 6
Embodiment 2: the structure of efficient coenzyme circulating system
When making catalyzer with pure enzyme or thick enzyme, Hexose phosphate dehydrogenase and glucose structure coenzyme circulating system can be added.
Coenzyme circulating system can be expressed as (substrate is for S2):
Hexose phosphate dehydrogenase adopts commercial efficient Hexose phosphate dehydrogenase, buys in Sigma (CAS:9028-53-9).For ensureing efficiently carrying out of reaction, the supply of coenzyme must be sufficient.In reaction system, glucose concn is 10% (w/v), and Hexose phosphate dehydrogenase is 20U/ml.Along with the carrying out of reaction, the amount of gluconic acid is progressively accumulated, and reaction system pH declines thereupon.Continue efficiently to make reaction to carry out, the pH of reaction system should control between 6.5-8.0, uses the 10%NaOH aqueous solution to take the mode of interval to regulate pH in practical application.
Embodiment 3: carbonyl reductase ChKRED12 crude enzyme liquid catalysis 10g/l substrate S1
The carbonyl reductase ChKRED12 recombinant bacterium wet thallus getting fresh culture is resuspended in phosphate buffered saline buffer (0.1M, pH8.0), and with clarifixator smudge cells, 13,000rpm, 4 DEG C of centrifugal 20min, gained supernatant is crude enzyme liquid.
Potassium phosphate buffer (0.1M, pH8.0), carbonyl reductase ChKRED12 concentration 10mg/ml (crude enzyme liquid), substrate S1 concentration 10g/l, GDH concentration 20U/ml, glucose 10% (w/v), NADP
+concentration 0.2mM, 30 DEG C, transformation time 2h, in reaction process, every 30min regulates a pH to 8.0.Equal-volume extraction into ethyl acetate termination reaction, sample treatment and detection method identical with embodiment 1 (1).Result shows that in 2h, 10g/l substrate just can transform completely, and the ee value of product is greater than 99.9%.
Embodiment 4: carbonyl reductase ChKRED12 crude enzyme liquid catalysis 20g/l substrate S1
Except S1 concentration of substrate is increased to except 20g/l, remaining reaction system is identical with embodiment 3, transformation time 5h, and in reaction process, every 30min regulates a pH to 8.0.Equal-volume extraction into ethyl acetate termination reaction, sample treatment and detection method identical with embodiment 1 (1).Result shows that in 5h, 10g/l substrate just can transform completely, and the ee value of product is greater than 99.9%.
Embodiment 5: carbonyl reductase ChKRED12 crude enzyme liquid catalysis 50g/l substrate S1
Except S1 concentration of substrate is increased to except 50g/l, remaining reaction system is identical with embodiment 3, transformation time 12h, and in reaction process, every 30min regulates a pH to 8.0.Equal-volume extraction into ethyl acetate termination reaction, sample treatment and detection method identical with embodiment 1 (1).Result shows that in 12h, 50g/L substrate can realize transforming completely, and the ee value of product is greater than 99.9%.
Embodiment 6: carbonyl reductase ChKRED12 crude enzyme liquid catalysis 100g/l substrate S1
Except S1 concentration of substrate is increased to except 100g/l, remaining reaction system is identical with embodiment 3, transformation time 24h, and in reaction process, every 30min regulates a pH to 8.0.Equal-volume extraction into ethyl acetate termination reaction, sample treatment and detection method identical with embodiment 1 (1).Result shows that in 24h, 100g/l substrate S1 can realize transforming completely, and the ee value of product is greater than 99.9%.
Embodiment 7: carbonyl reductase KRED2157 crude enzyme liquid catalysis 10g/l substrate S2
The carbonyl reductase KRED2157 recombinant bacterium wet thallus getting fresh culture is resuspended in phosphate buffered saline buffer (0.1M, pH8.0), and with clarifixator smudge cells, 13,000rpm, 4 DEG C of centrifugal 20min, gained supernatant is crude enzyme liquid.
Potassium phosphate buffer (0.1M, pH8.0), carbonyl reductase KRED2157 concentration 10mg/ml (crude enzyme liquid), substrate S2 concentration 10g/l, GDH concentration 20U/ml, glucose 10% (w/v), NADP
+concentration 0.2mM, 30 DEG C, transformation time 2h, in reaction process, every 2h regulates a pH to 8.0.Equal-volume extraction into ethyl acetate termination reaction, sample treatment and detection method identical with embodiment 1 (3).Result shows that in 24h, 10g/l substrate S2 transforms completely with regard to transforming, and the ee value of product is greater than 99.9%.
Embodiment 8: carbonyl reductase ChKRED07 crude enzyme liquid catalysis 20g/l substrate S3
The carbonyl reductase ChKRED07 recombinant bacterium wet thallus getting fresh culture is resuspended in phosphate buffered saline buffer (0.1M, pH 8.0), and with clarifixator smudge cells, 13,000rpm, 4 DEG C of centrifugal 20min, gained supernatant is crude enzyme liquid.
Potassium phosphate buffer (0.1M, pH8.0), carbonyl reductase ChKRED07 concentration 10mg/ml (crude enzyme liquid), substrate S3 concentration 20g/l, GDH concentration 20U/ml, glucose 10% (w/v), NADP
+concentration 0.2mM, 30 DEG C, transformation time 24h, in reaction process, every 2h regulates a pH to 8.0.Equal-volume extraction into ethyl acetate termination reaction, sample treatment and detection method identical with embodiment 1 (4).Result shows, transformation efficiency is greater than 99%, and the ee value of product is greater than 99.9%.
Embodiment 9: carbonyl reductase ChKRED07 crude enzyme liquid catalysis 20g/l substrate S4
The carbonyl reductase ChKRED07 recombinant bacterium wet thallus getting fresh culture is resuspended in phosphate buffered saline buffer (0.1M, pH 8.0), and with clarifixator smudge cells, 13,000rpm, 4 DEG C of centrifugal 20min, gained supernatant is crude enzyme liquid.
Potassium phosphate buffer (0.1M, pH8.0), carbonyl reductase ChKRED07 concentration 10mg/ml (crude enzyme liquid), substrate S4 concentration 20g/l, GDH concentration 20U/ml, glucose 10% (w/v), NADP
+concentration 0.2mM, 30 DEG C, transformation time 24h, in reaction process, every 2h regulates a pH to 8.0.Equal-volume extraction into ethyl acetate termination reaction, sample treatment and detection method identical with embodiment 1 (5).Result shows, transformation efficiency is greater than 99%, and the ee value of product is greater than 99.9%.
Claims (8)
1. with general structure (I) for substrate, obtain S configuration alcohol through the asymmetric biocatalysis of carbonyl reductase,
Wherein R substituent is COOCH
3, COOCH
2cH
3, CONHCH
3, CH
2cl, CN or CH
2n (CH
3)
2.
2. a carbonyl reductase KRED2157, its gene order is SEQ ID:No.1, and aminoacid sequence is SEQ ID:No.2.
3. a carbonyl reductase KRED2935, its gene order is SEQ ID:No.3, and aminoacid sequence is SEQID:No.4.
4. according to claim 1, it is characterized in that R substituent is COOCH
3or COOCH
2cH
3time, carbonyl reductase used is KRED2157, ChKRED07, ChKRED10, ChKRED11, ChKRED12 or ChKRED14.
5. according to claim 1, it is characterized in that R substituent is CONHCH
3time, carbonyl reductase used is ChKRED07, KRED2157, ChKRED03, ChKRED10, ChKRED23, ChKRED24, ChKRED26 or ChKRED14.
6. according to claim 1, it is characterized in that R substituent is CH
2during Cl, carbonyl reductase used is ChKRED07 or ChKRED2935.
7., according to claim 1, when it is characterized in that R substituent is CN, carbonyl reductase used is ChKRED07, ChKRED10, ChKRED11, ChKRED12, ChKRED14, KRED2157, ChKRED03 or KRED2935.
8. according to claim 1, it is characterized in that R substituent is CH
2n (CH
3)
2time, carbonyl reductase used is ChKRED10, ChKRED03 or ChKRED05.
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| CN108728421A (en) * | 2018-06-19 | 2018-11-02 | 中国科学院成都生物研究所 | A kind of carbonyl reduction enzyme mutant and application thereof |
| CN108728421B (en) * | 2018-06-19 | 2021-12-28 | 中国科学院成都生物研究所 | Carbonyl reductase mutant and application thereof |
| CN112930400A (en) * | 2018-09-26 | 2021-06-08 | 卡拉马祖控股股份有限公司 | Enzymatic production of modified hop products |
| CN112126663A (en) * | 2020-11-04 | 2020-12-25 | 浙江拓普药业股份有限公司 | Production process of duloxetine |
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