CN109207531A - The biological preparation method of Thiamphenicol and Florfenicol key intermediate - Google Patents
The biological preparation method of Thiamphenicol and Florfenicol key intermediate Download PDFInfo
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- CN109207531A CN109207531A CN201710533399.3A CN201710533399A CN109207531A CN 109207531 A CN109207531 A CN 109207531A CN 201710533399 A CN201710533399 A CN 201710533399A CN 109207531 A CN109207531 A CN 109207531A
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Abstract
The present invention provides the biological preparation methods of a kind of Thiamphenicol and Florfenicol key intermediate.Specifically, the method for the present invention is the following steps are included: (a) in liquid reaction system, using compound X as substrate, in the presence of coenzyme, under carbonyl reduction enzymatic, carries out reaction shown in formula A, to form compound Y；(b) compound Y is optionally isolated from the reaction system after the reaction of the previous step.The present invention also provides a kind of reaction systems, comprising: (i) aqueous solvent；(ii) substrate, the substrate are compound X；(iii) coenzyme；(iv) carbonyl reductase；(v) cosubstrate；(vi) is used for the enzyme of regenerating coenzyme.
Description
Technical field
The invention belongs to pharmaceutical technology field more particularly to a kind of alkamine antibacterials Thiamphenicols and Florfenicol
The preparation method of crucial chiral intermediate.
Background technique
Thiamphenicol antibiotics include Thiamphenicol (thiamphenicol, 1) and its fluoro object Florfenicol
(florfenicol, 2).They and antibiotics Chloramphenicol (chloramphenicol, the 3) phase in chemical structure eliminated
Seemingly, belong to beta-alkamine antibiotic[1]。
Alkamine pharmaceutical chemistry structure is as follows:
Thiamphenicol is that nineteen fifty-two is artificial synthesized for the first time, has that antibacterial activity is strong, it is smaller to absorb fast, lasting medicine, toxicity
The characteristics of, clinically it is mainly used for typhoid fever, dysentery, respiratory tract infection, urinary tract infection, liver biliary infection, enteric infection, surgery sense
The diseases such as dye, brucellosis and meningitis have no lethal alpastic anemia and gray syndrome report.It is in livestock and poultry pestilence
Prevention and treatment on significant effect, be mainly used for controlling ox, the respiratory tract of poultry and alimentary infection, and for treating pig, sheep
With the multi-infection disease of fish.
Florfenicol is also known as Florfenicol, and Sch-25298 is that elder generation of U.S. spirit-Schering-Plough is more preferable to seek in the seventies
Chloromycetin derivative and the drug developed, it is main successively in more than 20 country's listings such as Japan, South Korea, Norway, France
The demand in animal health market is supplied, has had mass production in China at present.Florfenicol has passed through U.S. FDA within 1996
Registration becomes and replaces the feeding antibiotic of a new generation of chloramphenicol and Thiamphenicol.Florfenicol is a kind of broad-spectrum antibiotic,
With has a broad antifungal spectrum, good absorbing is distributed the features such as wide, safe and efficient in vivo, is treatment typhoid bacillus, paratyphosum Bacterium, sramana
The choice drug of bacillary Animal diseases caused by Salmonella etc., antibacterial activity are (minimum to inhibit dense better than chloramphenicol and Thiamphenicol
It is 10 times about low to spend MIC), also have to the colon bacteria of resistance to chloramphenicol and Thiamphenicol, staphylococcus aureus, the primary formula bacterium of Cray etc.
Positive effect.
Thiamphenicol and Florfenicol list nearly 30 years, on the one hand due to the structure of good antibacterial effect and amino alcohol
Characteristic attracts always the research interest of various countries synthetic drug scholar；On the other hand, the two chemical structure only hydroxyl and fluorine atom
Difference, at present in existing industrial product route, the two shares a key intermediate 6, and structural formula is as follows:
The prior art reports synthesis Thiamphenicol 20 routes nearly with Florfenicol.
Route one: existing industrial product route prepare compound 6
The structure of Thiamphenicol and Florfenicol contains there are two chiral centre, therefore the key of synthetic route is in chirality
The construction method of the heart.Existing production line is reported in patent DE1938513, US3927054, US5382673, is
It is constructed using the method for classical resolution.First using methyl sulfone benzaldehyde as starting material, under alkaline condition, catalysis of cupric sulphate,
It is condensed to revive the mantoquita of formula compound 3 with glycine, erythro form content is less than 1%.3 and ethyl alcohol at ester after, through L- (+)-tartaric acid
It splits and obtains compound 5, sodium borohydride reduction 5 obtains alkamine key chiral intermediate 6.Reaction step is shown in formula I:
Reagent and reaction condition: (a) glycine, NH3H2O,CuSO4(b)EtOH,SOCl2,H2S(c)L-(+)-TA,MeOH
(d)NaBH4。
The shortcoming of existing production line has at 3 points: (1) copper sulphate is largely used in reaction, copper sulphate color is to water body
Copper sulfide seriously polluted, that copper sulphate and hydrogen sulfide are formed, generates solid waste pollution, therefore the use of mantoquita brings severe ring
Packing pressure；(2) classical resolution theoretical yield 50%；(3) resolving agent needs recovery.
Route two: metal-ligand catalyzed asymmetric syntheses prepare compound 6
Chen Fen academician team of China reports route in Tetrahedron.2016,72:1787-1793 within 2016
Two.The route using methyl sulfone benzoic acid as starting material, with thionyl chloride at acyl chlorides after, through glycine second rouge at amide 10, carbonic acid
Compound 12 is obtained through molecular rearrangement after di tert butyl carbonate protection nitrogen-atoms, the transfer hydrogenation reducing compound in metal ligand presence
12, single step reaction constructs two chiral centres, and researcher is synthesized and screened to ligand, and regrettably reduction is produced
Hydroxyl is configured as unwanted configuration in object 13, thus it is subsequent need to be activated through mesyl chloride, acetic acid anion overturning, hydrolysis obtains
Hydroxyl configuration 17 needed for obtaining, then it is deprotected obtained alkamine key intermediate 6.
The more existing production line of route two has two o'clock advantage: first is that avoiding the introducing of copper sulphate from highway route design；Two
It is the Dynamic Kinetic Resolution method for cleverly designing metal ligand participation in the route, two hands is constructed by single step reaction
Property center, theoretical yield 100%, break through classical resolution 50% limitation.But the middle hydroxyl of sorry obtained reduzate
Be configured as unwanted, need to obtain required configuration by overturning.Reaction step is as shown in formula III:
Reagent and reaction condition: (a) SOCl2,cat.DMF,CH2Cl2, reflux, 2h；(b) glycine ethyl ester hydrochloride,
Na2CO3,EtOAc/H2O,rt,1h；(c)Boc2O,cat.DMAP,CH3CN,rt,3h；(d)t-BuOK,THF,10℃,1h,
71%；(e)RuCl2(arene)2,ligand,HCOONa,Tween 20,25℃；(f)MsCl,Et3N,CH2Cl2,0-10℃,
1h, 94%；(g)LiBH4, THF, rt, 3h, 81%；(h) DBU/AcOH (1:2.2), toluene, 90 DEG C, 15h, 74%；(i)
K2CO3, MeOH, rt, 2h, 90%；(j)TFA,CH2Cl2, rt, 2h, 92%.
Route three: hydroamidase splits prepare compound 6
Bernard et al. is reported in Organic Process Research&Development 1998,2:10-17
A kind of method split using hydroamidase prepares key intermediate 6.The route using methyl sulfone benzaldehyde as starting material, with
Aldol condensation occurs for glycine amide Soviet Union's formula is made to be that de- imines obtains enzyme hydrolysis substrate to principal product 22,22 in acid condition, studies
Person has carried out a large amount of screening operation to hydroamidase, and finishing screen obtains one plant of Ochrobactrum anthropi NCIMB
40321, single configuration 24 (ee > 99%) is obtained with highly-solid selectively, substrate is not hydrolyzed under alkaline condition, equivalent is added
Methyl sulfone benzaldehyde can be realized racemization and obtain 22.24 are made key intermediate 6,6 through sodium borohydride reduction can be by known work
Skill synthesizes Thiamphenicol and Florfenicol.The route shortcoming is to split theoretical yield 50%, secondly uses two equivalent methyl sulfone
Benzaldehyde increases cost of material.Reaction step is as shown in formula IV:
Reagent and reaction condition: (a) .glycinamide hydrochloride, NaOH, H2O-MeOH.；(b).HCl.；
(c).NCIMB 40321amidase.；(d).NaBH4,H2SO4。
There are above-mentioned three point problems for existing production technology, therefore with the increase of environmental protection pressure, are faced with bigger ring
Border challenge.Various countries researcher conducts extensive research it, conjunction especially new with the lasting report of Chen Fen academician team of China
At route.Although the design of route two is rationally ingenious, suitable metal ligand, hydroxyl in transfer hydrogenation reduzate are not screened
Base configuration does not need configuration, needs by cumbersome activation, overturning hydrolyzes three-step reaction.To sum up alignment analysis, there is an urgent need to
Develop a kind of Thiamphenicol with industrial applications prospect and Florfenicol synthetic route.
Summary of the invention
The object of the present invention is to provide the biologies of a kind of Thiamphenicol and Florfenicol key intermediate W (compound 6) to make
Preparation Method.
This method is related to the dynamic reduction kinetics fractionation technology of carbonyl reductase participation, constructs two by single step reaction
Chiral centre.Dynamic reduction kinetics split process committed step is as shown in formula A:
In formula A,
R1And R2Be each independently selected from: H,
R3It is selected from the group:
R4It is selected from the group:
The first aspect of the present invention provides the preparation method of compound Y a kind of, comprising steps of
In liquid reaction system, using compound X as substrate, in the presence of coenzyme, in the enzymatic of (R, S)-carbonyl reduction
Under, reaction shown in formula A is carried out, to form compound Y；
In formula A,
R1And R2Be each independently selected from: H,
R3It is selected from the group:
R4It is selected from the group:
In another preferred example, the R1=H,
In another preferred example, the coenzyme is selected from the group: reducibility coenzyme, oxidisability coenzyme, or combinations thereof.
In another preferred example, the coenzyme is selected from the group: NADH, NADPH, NAD, NADP, or combinations thereof.
In another preferred example, NADH, NADPH, NAD or NADP dosage and substrate dosage ratio be 0.01%~
1.0% (w/w), preferably 0.01%~0.5% (w/w).
In another preferred example, in the reaction system, there is also the enzymes for regenerating coenzyme.
In another preferred example, the enzyme for regenerating coenzyme is selected from the group: alcohol dehydrogenase, hydrogenlyase, Portugal
Grape glucocorticoid dehydrogenase, or combinations thereof.
In another preferred example, in the reaction system, there is also the cosubstrates for regenerating coenzyme.
In another preferred example, the cosubstrate is selected from the group: isopropanol, glucose, ammonium formate, or combinations thereof.
In another preferred example, in the reaction system, the concentration of cosubstrate is 5-30%.
In another preferred example, in step (a), temperature be 10 DEG C -50 DEG C, preferably 20 DEG C -40 DEG C, more preferably 25 DEG C -
35℃。
In another preferred example, in step (a), the time is 0.1-240 hours, preferably 0.5-120 hours, more preferably 1-
72 hours, and more preferably 3-10 hours.
In another preferred example, in step (a), pH 6-10, preferably 7.0-9.0.
In another preferred example, in the reaction system, (R, S)-carbonyl reductase is the enzyme of free form, immobilization
The enzyme of enzyme or thallus form.
In another preferred example, the reaction system is water-based system.
In another preferred example, the reaction system is phosphoric acid buffer salt system.
In another preferred example, the reaction system also contains cosolvent.
In another preferred example, the cosolvent is selected from the group: dimethyl sulfoxide, methanol, ethyl alcohol, isopropanol, acetonitrile,
Toluene, acetone, or combinations thereof.
In another preferred example, the concentration of the cosolvent is 5~30%.
In another preferred example, the method also includes: separate from the reaction system after the reaction of the previous step
Compound Y out.
In another preferred example, the separation includes: addition isopropanol, is centrifuged thallus, partial concentration, the tertiary ether of first or second
Acetoacetic ester extraction, concentration of organic layers.
In another preferred example, in the reaction system after the reaction, the ee value of compound Y (such as compound 13) >=
90%, preferably >=95%, more preferably >=99%；De value >=90%, preferably >=95%, more preferably >=99%.
In another preferred example, in the reaction system after the reaction, compound X (such as compound 12) being converted into
Conversion ratio >=80% of object Y (such as compound 13) is closed, preferably >=85%, more preferably >=95%.
In another preferred example, the carbonyl reductase is selected from the group:
(i) carbonyl reductase EA is derived from, amino acid sequence is as shown in SEQ ID NO.:1；
(ii) amino acid sequence shown in SEQ ID NO.:1 is being kept in enzyme activity range, is carrying out one or more ammonia
Replacement, missing, change, insertion or the increase of base acid, obtained amino acid sequence.
In another preferred example, the coding gene sequence of the carbonyl reductase EA is selected from the group:
(a) sequence shown in SEQ ID NO.:2；
(b) polynucleotides complementary with the sequence that (a) is limited；Or
(c) with (a) limit sequence have at least 70% (preferably at least 75%, 80%, 85%, 90%, more preferably at least
95%, 96%, 97%, 98%, 99%) more than sequence identity any polynucleotides or complementary series.
In another preferred example, the carbonyl reductase is gene constructed on expression vector.
Second aspect of the present invention provides a kind of reaction system, and the reaction system includes:
(i) aqueous solvent；
(ii) substrate, the substrate are compound X；
(iii) coenzyme；
(iv) (R, S)-carbonyl reductase；
(v) cosubstrate；With
(vi) it is used for the enzyme of regenerating coenzyme；
In compound X, R1、R2、R3And R4As defined above.
In another preferred example, in the compound X, R1=H,
In another preferred example, in the compound X, R1=H,
Third aspect present invention provides the preparation method of compound Y a kind of, comprising steps of using such as second party of the present invention
Reaction system described in face carries out reaction shown in formula A under conditions of the enzymatic of (R, S)-carbonyl reductase, to be made
Compound Y:
In formula A:
R1、R2、R3And R4As defined above.
Fourth aspect present invention provides a kind of method for preparing intermediate W, includes the following steps:
(1) method prepare compound Y described in first aspect present invention or third aspect present invention is used, wherein R1, R2Such as
Upper definition,R5For methyl, ethyl, isopropyl or tert-butyl；
(2) intermediate W (compound 6) is prepared by substrate of compound Y.
In another preferred example, in step (2), carbonyl reductase is restored to the R of gained compound Y4In ester bond reduction
For hydroxyl, so that compound Z be made:
In another preferred example, reducing agent used in the step (2) be sodium borohydride and/or potassium borohydride or its with
The composition of magnesium chloride, zinc chloride and/or calcium chloride.
In another preferred example, reaction dissolvent used in the step (2) is common organic solvent.
In another preferred example, the organic solvent be selected from methanol, ethyl alcohol, tetrahydrofuran, methylene chloride, the tertiary ether of first or
A combination thereof.
In another preferred example, R1Compound Z in step (2), is sloughed R by=H2(protecting group), so that intermediate W be made
(compound 6):
In another preferred example, described to slough R2(protecting group) protection is selected from: the hydrogen catalyzed deprotection of palladium charcoal, Bronsted acid are urged
Change and is deprotected under lower deprotection, alkaline condition.
Fifth aspect present invention provides a kind of preparation method of Thiamphenicol, comprising steps of
(1) method prepare compound Y described in first aspect present invention or third aspect present invention is used, wherein R1=H,
R2As defined above,R5For methyl, ethyl, isopropyl or tert-butyl；
(2) intermediate W (compound 6) is prepared by substrate of compound Y；
(3) under the suitable conditions, compound 6 is reacted with methyl dichloroacetate and Thiamphenicol is made, reaction equation is as follows:
In another preferred example, in the preparation method, Thiamphenicol synthetic route is as follows:
Wherein, R1=H, R2As defined above,R5For methyl, ethyl, isopropyl or
Tert-butyl.
Sixth aspect present invention provides a kind of preparation method of Florfenicol, comprising steps of
(1) using method prepare compound Y described in first aspect present invention or third aspect present invention；Wherein R1=H,
R2As defined above,R5For methyl, ethyl, isopropyl or tert-butyl；
(2) intermediate W (compound 6) is prepared by substrate of compound Y；
(3) under the suitable conditions, by compound 6 through protecting, fluorine replaces, and methyl dichloroacetate is at amide, and reaction equation is such as
Under:
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 shows the liquid phase figure of four isomers of raceme compound Y in embodiment 3.
Fig. 2 shows the liquid phase figure of the chiral purity of enzymatic conversion reaction prepare compound Y in embodiment 3.
Specific embodiment
The present inventor after extensive and in-depth study, is surprised to find that a kind of compound Y (such as compound 13) for the first time
Biological preparation method.Specifically, biological preparation method of the invention with compound X (such as compound 12) be raw material, with carbonyl
Base reductase is biocatalyst, in the presence of coenzyme, efficiently prepares compound Y (such as the chemical combination with three-dimensional conformation
Object 13) (reduction yield > 98%, chiral ee value > 99%, chiral de value > 97%), single step reaction constructs two chiral centres, from
And production efficiency is greatlyd improve, reduce production cost.In addition, the method for the present invention has significantly simplified subsequent processing, and with it is existing
There is production line to compare, get rid of the use of copper sulphate, substantially reduce or eliminate the use of all kinds of pollution chemicals, thus aobvious
Work reduces risk of environmental pollution.
The present invention only needs extracting operation, easy to operate, low in cost, environmentally protective, and being more suitable for industrialized production has height
Chemical purity and high-optical-purity key intermediate W, that is, compound 6, with the system for the drugs such as Thiamphenicol and Florfenicol
It is standby.
Term
Enantiomeric excess (ee, enantiomeric excess): commonly used to an enantiomerism in characterization chiral molecules
Excessive value of the body relative to another enantiomter.
Diastereomeric excess (de, diastereomeric excess): commonly used to characterization two or more chiral centre
Molecule in excessive value of the diastereomer relative to another diastereomer.
(R, S)-carbonyl reductase
In the present invention, " stereoselectivity carbonyl reductase " refers to the latent chirality of stereoselectivity catalytic asymmetric reduction
Ketone is the enzyme of chiral alcohol.
Typically, in the present invention, the stereoselectivity carbonyl reductase is preferably (R, S)-carbonyl reductase, is stood
Body is selectively defined as enantiomeric excess (ee) >=80%, diastereomeric excess (de) >=80%.
With should (R, R)-carbonyl reductase when, stereoselectivity is defined as enantiomeric excess (ee) >=80%, diastereomeric
Body is excessive (de) >=80%, and so on.
In the present invention, the definition of configuration is using compound Y as reference, and wherein hydroxyl is configured as R, and amino is configured as S,
The amino of S- configuration in the identification compound X of all energy stereoselectivities, and be R configuration hydroxyl by the carbonyl reduction in compound X
The carbonyl reductase of base is defined as (R, S)-carbonyl reductase in the technical solution of the present invention.
In the present invention, the carbonyl reductase can be wild type or saltant type.Furthermore, it is possible to separation,
It is also possible to recombination.
Carbonyl reductase for use in the present invention can come from different plant species.A kind of amino acid of typical carbonyl reductase
Sequence is as shown in SEQ ID No.:1, and encoding gene is as shown in SEQ ID No.:2.
SEQ ID No.:1
MKYTVITGASSGIGYETAKLLAGKGKSLVLVARRTSELEKLRDEVKQISPDSDVILKSVDLADNQNVHD
LYEGLKELDIETLINNAGFGDFDLVQDIELGKIEKMLRLNIEALTILSSLFARDHHDIEGTTLVNISSLGGYRIVPN
AVTYCATKFYVSAYTEGLAQELQKGGAKLRAKVLAPAATETEFVDRARGEAGFDYSKNVHKYHTAAEMAGFLHQLIE
SDAIVGIVDGETYEFELRGPLFNYAG
SEQ ID No.:2
aagtacacggtcattacaggagcaagttcaggaattggatatgagacagcaaaactactcgcaggaaaaggaaaatc
actcgtcctcgtcgcacggcggacgtctgagctcgaaaaacttcgggatgaagtcaaacaaatctcaccagatagtg
atgtcatcctcaagtcggtcgatctcgcagataaccaaaatgtccatgatttatatgagggactaaaggaactcgac
atcgagacgctcatcaacaatgctggattcggcgattttgatctcgtccaggacattgagctcgggaaaatcgagaa
aatgctccgcttgaacatcgaggcgctgacgattctatcgagtctgttcgcacgcgatcatcatgacatcgaaggaa
cgacactcgtcaatatctcgtcactcggtggctaccggatcgttccgaacgcggtcacgtattgcgcgacgaagttc
tatgtcagtgcctatacggaagggctagcgcaagaactgcaaaaaggcggggcaaaactccgggcgaaagtactggc
accagctgcgactgagacagagtttgtcgatcgtgcacgcggcgaagcagggttcgactacagcaagaacgtccata
agtaccatacggcggctgagatggcaggcttcttgcatcagttgatcgaaagtgacgcgatcgtcggcatcgtcgac
ggtgagacgtatgagttcgaattgcgtggtccgttgttcaactacgcaggataa
Due to the degeneracy of codon, the base sequence of amino acid sequence shown in SEQ ID NO.1 not only office is encoded
It is limited to SEQ ID NO.2.Those skilled in the art can be obtained by being suitably introduced into replacement, missing, change, insertion or increase
The homologue of the base sequence, the present invention cover these homologues, as long as the recombinase of its expression keeps the catalysis to compound X
Reduction activation.In the present invention homologue of polynucleotide can by one to base sequence SEQ ID NO.2 or
Multiple bases are replaced, lack or increase in holding enzyme activity range to be made.
Carbonyl reductase of the invention further includes keeping enzymatic activity model to amino acid sequence shown in SEQ ID NO.:1
In enclosing, replacement, missing, change, insertion or the increase of one or more amino acid, obtained amino acid sequence are carried out.
In the present invention, (R, the S)-carbonyl reductase can use in a variety of manners.This hair is expressed for example, can be used
The various differences such as crude enzyme liquid, pure enzyme or thick enzyme powder also can be used in the resting cell or wet thallus of the bright carbonyl reductase
Form, or use immobilised enzymes.
Preferably, to obtain higher transformation efficiency and reduce cost, it is preferable to use crude enzyme liquid.
Carbonyl reduction enzyme dosage and substrate dosage ratio are preferably 0.1-20%, and preferably 1%~6% (w/w) is (by enzyme
The quality meter of quality and substrate) or resting cell quality and substrate quality ratio be 1-200%, preferably 10-100%.
Coenzyme
In the present invention, " coenzyme " refers to the coenzyme that can be realized electron transmission in redox reaction.
Typically, coenzyme of the invention is reducibility coenzyme NADH, NADPH or oxidisability coenzyme NAD+、NADP+.Due to also
The Costco Wholesale of originality coenzyme is expensive, preferably oxidisability coenzyme NAD+、NADP+。
When selective oxidizing coenzyme, the method for needing to select to realize regenerating coenzyme, main includes that three kinds of (1) glucose are de-
Hydrogen enzyme substrate glucose together；(2) alcohol dehydrogenase and cosubstrate isopropanol；(3) hydrogenlyase and cosubstrate ammonium formate.
In a preferred embodiment, coenzyme NADP+, Cofactor Regeneration Systems are glucose dehydrogenase, oxidisability coenzyme
NADP+Dosage and substrate dosage ratio are 0.01%~0.5% (w/w), and buffer system is 0.1mol/L phosphate-buffered salt.Buffering
The pH of liquid is 6.0-10.
Cosolvent
In the present invention, cosolvent can be added or not added in the reaction system.
As used herein, term " cosolvent " refers to slightly solubility substance and the third substance being added formed in a solvent can
Complex compound, associated matter or double salt between soluble molecule etc., to increase the solubility of slightly solubility substance in a solvent.It is this third
Substance is known as cosolvent.
In the present invention, substrate compounds 12 are insoluble in water, when concentration of substrate increases, seriously affect reaction conversion ratio.Cause
This, need to improve substrate dissolubility by the way that cosolvent is added, react conversion situation to improve.Optional cosolvent is that dimethyl is sub-
Sulfone, methanol, ethyl alcohol, isopropanol, acetonitrile, toluene, acetone, concentration are preferably 5-30% (v/v), preferably dimethyl sulfoxide, first
Alcohol, ethyl alcohol, isopropanol.
Dynamic reduction kinetics resolution reaction principle
It is another while by prochiral ketone (such as R- configuration) of the reduction single configuration of carbonyl reductase stereoselectivity
The prochiral ketone (such as S- configuration) of configuration is realized the racemization of alpha-position chiral configuration, is restored and disappeared by the tautomerism of carbonyl
Rotationization carries out under same reaction condition, realizes that efficiently building contains the purpose of two chiral centre secondary alcohol.
Typically, the carbonyl reductase for only identifying X-R (R configuration of compound X) is obtained in the present invention by screening, to carbonyl
The reduction for carrying out stereoselectivity obtains chiral hydroxyl group, is converted without identified X-S (S configuration of compound X) by racemization
For X-R, racemization is connected propulsion with reduction process, is converted with this, theoretically can chiral product needed for 100% acquisition.Pass through
Carbonyl reductase restores latent chiral carbonyl substrate, while through tautomerism racemization, the ingenious combination of the two, single step reaction height
Effect two chiral centres of economic building, are demonstrated by good application and development prospect.
Intermediate W (compound 6) and its application in synthesis downstream product
After synthetic intermediate 6, Thiamphenicol and Florfenicol can be synthesized by already known processes.
Typically, Thiamphenicol is made at amide in intermediate 6 and methyl dichloroacetate.
Typically, the existing production line of Florfenicol and Thiamphenicol share key intermediate 6, compound 6 and two chloroethenes
Nitrile reacts to form oxazole ring 7-1 and 7-2, and compound 7-2 can be converted into 7-1 under alkaline condition, and 7-1 is introduced through fluoro reagent
Fluorine atom, rear open loop directly obtain Florfenicol.Reaction step is as shown in Formula II:
Reagent and reaction condition: (a) Cl2CHCN,H2SO4,i-PrOH,70℃,1.5h；(b) it is saturated NH3/i-PrOH,60-
80 DEG C, 20h, 95%；(c) Ishiawa reagent, CH2Cl2,100℃,2h；(d) AcOK, i-PrOH, H2O, pH=3.5-4.0,
Rt, 10h, 85.7%.
The synthesis of Thiamphenicol and Florfenicol is referring also to DE1938513, US3927054, US5382673.
Main advantages of the present invention are:
(1) the key intermediate W i.e. biological preparation method of compound 6 is provided, this method relates to carbonyl reductase Jie
The dynamic reduction kinetics led is split, and restores yield > 98%, chiral ee value > 99%, chiral de value > 97%.
(2) after making a breakthrough on synthesis key intermediate, the new synthesis route of Thiamphenicol and Florfenicol is provided.
(3) biological preparation method has green, environmental protection, and economic feature is achieved compared with the prior art and significantly improved, is
A large amount of mantoquitas and water pollution problem existing for existing production technology are solved, the promising resolving ideas of tool is provided.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and
Number is calculated by weight.Experimental material involved in the present invention can obtain unless otherwise specified from commercially available channel.
Material
Total gene synthesis is completed by hundred power lattice of Shanghai.
It synthesizes to obtain encoding gene by commercialized full genome, encoding gene is then built into expression vector, import
Host strain, inducing expression obtain carbonyl reductase.
The preparation of enzyme reduction substrate compounds X can be found in Tetrahedron.2016,72:1787-1793 the method.
Method
1. the preparation method of enzyme
By this field routine techniques, the glucose dehydrogenase of above-mentioned realization regenerating coenzyme and target gene are constructed same
On one plasmid pET28a (+) carrier, expressive host Escherichia coli are then introduced into, by inducing expression, are obtained containing purposeful double enzymes
Thallus.Centrifugation can be used directly and obtain thallus, its broken wall can also be obtained to crude enzyme liquid, thick enzyme powder is used for subsequent bioconversion
Reaction.
2. the method for biocatalytic reduction compound X prepare compound Y
The present invention provides the methods of carbonyl reduction enzymatic reducing compound X prepare compound Y a kind of.Reaction equation is such as
Under:
Wherein the biocatalysis system includes carbonyl reductase, coenzyme.Heretofore described carbonyl reductase
Coding gene sequence is SEQ ID NO.:2, and the amino acid sequence of carbonyl reductase is SEQ ID NO.:1.According to this field one
As common sense, above-mentioned carbonyl reduction enzyme gene can synthesize to obtain by commercialized full genome.
According to above-mentioned preferred system, the implementation process of the preparation method is as follows: substrate being substantially dissolved in cosolvent, such as
Dimethyl sulfoxide or isopropanol, are then added in phosphate buffer, after mixing evenly, be added thallus, crude enzyme liquid, thick enzyme powder or
Coenzyme NAD P is added in pure enzyme+, cosubstrate glucose maintains 20 DEG C~40 DEG C, TLC or HPLC monitoring, until starting material left <
2%, terminate reaction.It is added isopropanol into reaction solution, centrifugation or crosses ceramic membrane, remove thallus, take supernatant, supernatant is with having
Solvent extraction, organic solvent are chosen as methyl tertiary butyl ether(MTBE), toluene, ethyl acetate, isopropyl acetate, methylene chloride, 2- first
Base tetrahydrofuran, n-butanol.Aqueous layer extracted 2-3 times merges organic phase；With saturated common salt water washing 2~3 times, obtained after concentration
Light yellow oil.
The final concentration of 10-200g/L of substrate compounds (VI) in system, reaction temperature are 20-40 DEG C, and revolving speed is
200rpm/min, reaction time about 3-10h are varied according to concentration of substrate or monitor raw material conversion situation by HPLC,
When starting material left < 2%, reaction is terminated.
3. the chiral positive monitoring method of compound Y:
HPLC condition: Daicel IC-3 (250 × 4.6mm, 3 μm)；Flow velocity 0.8ml/min；Mobile phase: n-hexane: isopropyl
Alcohol=80:20；Ultraviolet detection wavelength 260nm；25 DEG C of column temperature；Sample is dissolved in methanol, concentration 10mg/ml；2 μ l of sampling volume.
4. the reverse phase monitoring method of compound Y:
HPLC condition: phenomenex Gemini 5u C18 110A, 250 × 4.6mm, 5 μm；Flow velocity: 1ml/min；Stream
Dynamic phase gradient such as following table；Ultraviolet detection wavelength: 260nm；Column temperature: 30 DEG C；Sample concentration: 10mg/ml；10 μ l of sampling volume.
Eluent gradient:
| Time (min) | H2O-0.1%TFA (%) | ACN-0.1%TFA (%) |
| 0 | 80 | 20 |
| 15 | 20 | 80 |
| 35 | 20 | 80 |
| 35.1 | 80 | 20 |
| 40 | 20 | 80 |
The building of embodiment 1, carbonyl reduction enzyme engineering bacteria
By EA carbonyl reductase target gene, glucose dehydrogenase target gene entrusts commercialization company to carry out full genome conjunction
At being cloned into pET28a (+) carrier, be transferred to bacillus coli DH 5 alpha competent cell, plate culture, picking positive transformant single bacterium
After falling and extracting plasmid order-checking determination, recombinant plasmid is extracted, is imported in BL21 (DE3) bacterial strain, LB culture, acquisition can induce table
Up to the genetic engineering bacterium of recombination carbonyl reductase and alcohol dehydrogenase.
Embodiment 2, recombination carbonyl reductase, the preparation of glucose dehydrogenase
Previous step is stored in the genetic engineering bacterium in glycerol, is inoculated into the LB liquid medium containing kanamycins, 37
DEG C, 220rpm, cultivate 13h, obtain seed culture medium, by seed culture fluid in 1.5% ratio be inoculated into card containing 50ug/ml that
On the fluid nutrient medium of chloramphenicol resistance, then 37 DEG C, 220rmp is cultivated to OD600Final concentration of 1.0% lactose is added in value > 2.0,
25 DEG C are cooled to, continues to cultivate 3h, final concentration of 0.5% lactose is added, cultivate 20h, put tank, be centrifuged to obtain thallus, for biology
Conversion is prepared.
Fermentating formula is as follows:
| Raw material | Mass content (%) |
| Yeast extract | 2.4 |
| Soy peptone | 1.2 |
| Sodium chloride | 0.3 |
| Glycerol | 0.5 |
| Dipotassium hydrogen phosphate | 0.2 |
| Epsom salt | 0.05 |
Embodiment 3, biocatalysis prepare compound Y (being configured as (R, S))
Wherein R1For H；R2ForR3ForR4For
The phosphate buffer (100ml) of 0.1M is taken, NADP is added+(0.1g) is added glucose 25g, above-mentioned hair is added
Thallus EA (5g) obtained by ferment, 1M sodium hydrate aqueous solution adjust pH to 7.3-8.0, are vigorously stirred and add compound X (10g) in batches
DMSO (30ml) solution, 25 DEG C, 220rpm, shaking table reaction, interval 0.5h adjusts pH, when HPLC monitors reaction conversion ratio > 98%,
Terminate reaction.
Centrifugation removes thallus, takes supernatant, and the tertiary ether of isopropanol/first (v/v=1/3,100ml) extraction, isopropanol/first is added
Tertiary ether aqueous layer extracted merges organic layer, saturated common salt washing, and anhydrous sodium sulfate dries, filters, and give light yellow oil is concentrated,
Product configuration is (R, S) (compound Y).
In order to obtain highly-solid selectively, the carbonyl reductase of high reaction conversion ratio, inventor carries out carbonyl reductase
Screening.
When due to QNR and KETONE carbonyl reduction substrate for enzymatic activity, less than 30%, chiral purity is difficult to measure conversion ratio,
When CK03 carbonyl reduction substrate for enzymatic activity, conversion ratio 95%, ee:99.2% > 80%, but de:20.3%, it is unsatisfactory for institute of the present invention
(R, S)-carbonyl reductase of definition, therefore it is not belonging to (R, S)-carbonyl reductase.
Embodiment 4, biocatalysis prepare compound Y ' (being configured as (R, R))
Wherein R1For H；R2ForR3ForR4For
The phosphate buffer (25ml) of 0.1M is taken, NAD is added+(0.1g) is added isopropanol 7.5ml, above-mentioned hair is added
Thallus LK (1g) obtained by ferment, 1M sodium hydrate aqueous solution adjust pH to 7.3-8.0, are vigorously stirred and add compound X (2g) in batches
DMSO (3ml) solution, 25 DEG C, 220rpm, shaking table reaction when HPLC monitors reaction conversion ratio > 98%, terminates reaction.
Centrifugation removes thallus, takes supernatant, and the tertiary ether of isopropanol/first (v/v=1/3,100ml) extraction, isopropanol/first is added
Tertiary ether aqueous layer extracted merges organic layer, saturated common salt washing, and anhydrous sodium sulfate dries, filters, and concentration give light yellow oil produces
Object is configured as (R, R) (compound Y '), ee value 99.7%, de value 98.2%.
Embodiment 5, biocatalysis prepare compound Y (being configured as (R, S))
Wherein R1For H；R2ForR3ForR4For
The phosphate buffer (25ml) of 0.1M is taken, NADP is added+(0.01g) is added glucose 5g, above-mentioned fermentation is added
Gained thallus EA (1g), 1M sodium hydrate aqueous solution adjust pH to 7.3-8.0, are vigorously stirred and add compound X (2g) in batches
DMSO (30ml) solution, 25 DEG C, 220rpm, shaking table reaction, every 0.5h adjusts pH, when HPLC monitors reaction conversion ratio > 98%, eventually
Only react.
Centrifugation removes thallus, takes supernatant, and the tertiary ether of isopropanol/first (v/v=1/3,100ml) extraction, isopropanol/first is added
Tertiary ether aqueous layer extracted merges organic layer, saturated common salt washing, and anhydrous sodium sulfate dries, filters, and give light yellow oil is concentrated,
Product configuration is (R, S) (compound Y), ee value 99.4%, de value 98.5%.
The synthesis of 6 compound 17 of embodiment
Under ice bath, compound 13 (5.0) is dissolved in methanol (40ml), sodium borohydride is added portionwise to 5 DEG C in temperature control
50 DEG C are risen to after (0.5g), LCMS monitoring reaction to raw material conversion completely, adds water quenching to go out, partial concentration methanol, extracting n-butyl alcohol 3
Secondary, washing, anhydrous magnesium sulfate, which dries, filters, is concentrated to give light yellow solid 4.18g (compound 17).
The synthesis of 7 compound 6 of embodiment
Above-mentioned light yellow solid (compound 17) is dissolved in methanol, the methanolic hydrogen chloride solution (10ml) of saturation is added,
40 DEG C are heated to, LCMS monitoring is reacted to raw material conversion completely, and reaction is concentrated to doing to obtain light yellow solid (compound 6).
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, those skilled in the art can be right after having read above content of the invention
The present invention makes various changes or modifications, and these equivalent forms also fall within the scope of the appended claims of the present application.
Sequence table
<110>Shanghai Institute of Pharmaceutical Industry
China State Institute of Pharmaceutical Industry
<120>biological preparation method of Thiamphenicol and Florfenicol key intermediate
<130> P2017-0095
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 249
<212> PRT
<213>artificial sequence
<400> 1
Met Lys Tyr Thr Val Ile Thr Gly Ala Ser Ser Gly Ile Gly Tyr Glu
1 5 10 15
Thr Ala Lys Leu Leu Ala Gly Lys Gly Lys Ser Leu Val Leu Val Ala
20 25 30
Arg Arg Thr Ser Glu Leu Glu Lys Leu Arg Asp Glu Val Lys Gln Ile
35 40 45
Ser Pro Asp Ser Asp Val Ile Leu Lys Ser Val Asp Leu Ala Asp Asn
50 55 60
Gln Asn Val His Asp Leu Tyr Glu Gly Leu Lys Glu Leu Asp Ile Glu
65 70 75 80
Thr Leu Ile Asn Asn Ala Gly Phe Gly Asp Phe Asp Leu Val Gln Asp
85 90 95
Ile Glu Leu Gly Lys Ile Glu Lys Met Leu Arg Leu Asn Ile Glu Ala
100 105 110
Leu Thr Ile Leu Ser Ser Leu Phe Ala Arg Asp His His Asp Ile Glu
115 120 125
Gly Thr Thr Leu Val Asn Ile Ser Ser Leu Gly Gly Tyr Arg Ile Val
130 135 140
Pro Asn Ala Val Thr Tyr Cys Ala Thr Lys Phe Tyr Val Ser Ala Tyr
145 150 155 160
Thr Glu Gly Leu Ala Gln Glu Leu Gln Lys Gly Gly Ala Lys Leu Arg
165 170 175
Ala Lys Val Leu Ala Pro Ala Ala Thr Glu Thr Glu Phe Val Asp Arg
180 185 190
Ala Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val His Lys Tyr
195 200 205
His Thr Ala Ala Glu Met Ala Gly Phe Leu His Gln Leu Ile Glu Ser
210 215 220
Asp Ala Ile Val Gly Ile Val Asp Gly Glu Thr Tyr Glu Phe Glu Leu
225 230 235 240
Arg Gly Pro Leu Phe Asn Tyr Ala Gly
245
<210> 2
<211> 747
<212> DNA
<213>artificial sequence
<400> 2
aagtacacgg tcattacagg agcaagttca ggaattggat atgagacagc aaaactactc 60
gcaggaaaag gaaaatcact cgtcctcgtc gcacggcgga cgtctgagct cgaaaaactt 120
cgggatgaag tcaaacaaat ctcaccagat agtgatgtca tcctcaagtc ggtcgatctc 180
gcagataacc aaaatgtcca tgatttatat gagggactaa aggaactcga catcgagacg 240
ctcatcaaca atgctggatt cggcgatttt gatctcgtcc aggacattga gctcgggaaa 300
atcgagaaaa tgctccgctt gaacatcgag gcgctgacga ttctatcgag tctgttcgca 360
cgcgatcatc atgacatcga aggaacgaca ctcgtcaata tctcgtcact cggtggctac 420
cggatcgttc cgaacgcggt cacgtattgc gcgacgaagt tctatgtcag tgcctatacg 480
gaagggctag cgcaagaact gcaaaaaggc ggggcaaaac tccgggcgaa agtactggca 540
ccagctgcga ctgagacaga gtttgtcgat cgtgcacgcg gcgaagcagg gttcgactac 600
agcaagaacg tccataagta ccatacggcg gctgagatgg caggcttctt gcatcagttg 660
atcgaaagtg acgcgatcgt cggcatcgtc gacggtgaga cgtatgagtt cgaattgcgt 720
ggtccgttgt tcaactacgc aggataa 747
Claims (13)
1. a kind of preparation method of compound Y, it is characterised in that:
In liquid reaction system, using compound X as substrate, in the presence of coenzyme, under the enzymatic of (R, S)-carbonyl reduction, into
Row reaction shown in formula A, to form compound Y；
In formula A,
R1And R2Be each independently selected from: H,
R3It is selected from the group:
R4It is selected from the group:
2. preparation method according to claim 1, which is characterized in that R1=H,
3. according to claim 1 or preparation method described in 2, which is characterized in that the coenzyme is selected from the group: reproducibility is auxiliary
Enzyme, oxidisability coenzyme, or combinations thereof.
4. according to claim 1 or preparation method described in 2, which is characterized in that the coenzyme is selected from the group: NADH,
NADPH, NAD, NADP, or combinations thereof.
5. any preparation method according to claim 1 or in 2, which is characterized in that in the reaction system, there is also
Enzyme for regenerating coenzyme.
6. preparation method according to claim 5, which is characterized in that in the reaction system, there is also be used for coenzyme
Regenerated cosubstrate.
7. preparation method according to claim 1 or 2, which is characterized in that the carbonyl reductase is selected from the group:
(i) carbonyl reductase EA is derived from, amino acid sequence is as shown in SEQ ID NO.:1；
(ii) amino acid sequence shown in SEQ ID NO.:1 is being kept in enzyme activity range, is carrying out one or more amino acid
Replacement, missing, change, insertion or increase, obtained amino acid sequence.
8. preparation method according to claim 7, which is characterized in that the coding gene sequence of the carbonyl reductase EA
It is selected from the group:
(a) sequence shown in SEQ ID NO.:2；
(b) polynucleotides complementary with the sequence that (a) is limited；Or
(c) with (a) limit sequence have at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least
95%, any polynucleotides or complementary series of 96%, 97%, 98%, 99% or more sequence identity.
9. a kind of reaction system, which is characterized in that the reaction system includes:
(i) aqueous solvent；
(ii) substrate, the substrate are compound X；
(iii) coenzyme；
(iv) (R, S)-carbonyl reductase；
(v) cosubstrate；With
(vi) it is used for the enzyme of regenerating coenzyme；
In compound X, R1、R2、R3And R4As defined above.
10. a kind of preparation method of compound Y, which is characterized in that comprising steps of using reactant as claimed in claim 9
System, under conditions of the enzymatic of (R, S)-carbonyl reductase, carries out reaction shown in formula A, so that compound Y be made:
In formula A:
R1、R2、R3And R4As defined above.
11. a kind of method for preparing intermediate W, which comprises the steps of:
(1) method prepare compound Y described in claim 1 or 10 is used, wherein R1, R2As defined above,R5For methyl, ethyl, isopropyl or tert-butyl；
(2) intermediate W i.e. compound 6 is prepared by substrate of compound Y.
12. a kind of preparation method of Thiamphenicol, which is characterized in that comprising steps of
(1) method prepare compound Y described in claim 1 or 10 is used, wherein R1=H, R2As defined above,R5For methyl, ethyl, isopropyl or tert-butyl；
(2) intermediate W i.e. compound 6 is prepared by substrate of compound Y；
(3) under the suitable conditions, compound 6 is reacted with methyl dichloroacetate and Thiamphenicol is made, reaction equation is as follows:
13. a kind of preparation method of Florfenicol, which is characterized in that comprising steps of
(1) using method prepare compound Y described in claim 1 or 10；Wherein R1=H, R2As defined above,R5For methyl, ethyl, isopropyl or tert-butyl；
(2) intermediate W i.e. compound 6 is prepared by substrate of compound Y；
(3) under the suitable conditions, by compound 6 through protecting, fluorine replaces, and for methyl dichloroacetate at amide, reaction equation is as follows:
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| CN113249417A (en) * | 2021-04-20 | 2021-08-13 | 复旦大学 | Preparation method of chiral cis-beta-aryl-beta-hydroxy-alpha-amino ester compound |
| WO2023011627A1 (en) * | 2021-08-05 | 2023-02-09 | 上海医药工业研究院有限公司 | Carbonyl reductase mutant and application thereof |
| CN116024187A (en) * | 2022-10-26 | 2023-04-28 | 合肥学院 | Enzymatic preparation method of Violet Luo Zhongjian body |
| CN116024187B (en) * | 2022-10-26 | 2024-04-26 | 合肥大学 | Enzymatic preparation method of vilantrum Luo Zhongjian |
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