CN101528937A - Process for enzymatic reduction of alkene derivatives - Google Patents
Process for enzymatic reduction of alkene derivatives Download PDFInfo
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- CN101528937A CN101528937A CNA2007800393066A CN200780039306A CN101528937A CN 101528937 A CN101528937 A CN 101528937A CN A2007800393066 A CNA2007800393066 A CN A2007800393066A CN 200780039306 A CN200780039306 A CN 200780039306A CN 101528937 A CN101528937 A CN 101528937A
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Disclosed is a process for the enzymatic preparation of a compound of general formula (2) from an unsaturated alkene derivative of general formula (1) by reduction of the compound of general formula (1) in the presence of a reductase comprising the polypeptide sequence SEQ ID NO: 1. 2 or 3, or a sequence having a sequence identical to SEQ ID NO: 1. 2 or 3, or a functionally equivalent polypeptide sequence having at least 80% identity thereto.
Description
The present invention relates to be used for the method for the ene derivative of enzymatic reduction general formula (1).
Goal of the invention
The present invention seeks to be provided for ethylenic unsaturation derivative, especially prepare the method for the compound of general formula (2) with high chemical yield and fabulous stereoselectivity enzymatic from general formula (1).
Summary of the invention
Solve above-mentioned purpose by the ene derivative that uses reductase enzyme YqjM, OPR1, OPR3 and function equivalent reduction general formula (1) thereof.
Detailed Description Of The Invention
The present invention relates to by in the presence of reductase enzyme, reducing the compound of general formula (1), prepare the method for the compound of general formula (2) from the ethylenic unsaturation derivative enzymatic of general formula (1),
Wherein
A is nitro (NO
2), ketone group (CRO), aldehyde radical (CHO), carboxyl (COOR), R=H or the optional C that replaces
1-C
6-alkyl, R
1, R
2And R
3Be H, C independently of each other
1-C
6-alkyl, C
2-C
6-thiazolinyl, carboxyl, or optional carbocyclic ring or heterocyclic aromatics or non-aromatic group, the perhaps R that replaces
1Be connected to R
3On, to become the part of 4-8 unit ring, perhaps R
1Be connected on the R, to become the part of 4-8 unit ring, condition is R
1, R
2And R
3Inequality, described reductase enzyme (i) comprises at least a among the peptide sequence SEQ ID NO:1,2 or 3 or (ii) has with SEQ IDNO:1,2 or 3 peptide sequence that is equal on the function of at least 80% identity.
In principle, method of the present invention can be used the microorganism of purifying or spissated enzyme itself and natural or recombinant expressed this enzyme, or uses the cell homogenates from it to carry out.
Except as otherwise noted,
-C
1-C
6The concrete nail base of-alkyl, ethyl, propyl group, butyl, amyl group or hexyl, and corresponding single branch or multiple-limb analogue are as sec.-propyl, isobutyl-, sec-butyl, the tertiary butyl, isopentyl or neo-pentyl; Especially preferred C
1-C
4-alkyl;
-C
2-C
6-thiazolinyl specifically refers to have the unsaturated analogue of list of the abovementioned alkyl of 2 to 6 carbon atoms, especially preferred corresponding C
2-C
4-thiazolinyl,
-carboxyl specifically refers to the COOH base,
-carbocyclic ring and heterocyclic aryl or non-aryl rings specifically refer to have 3 to 12 carbon atoms, and have 1 to 4 heteroatoms suitably the time, as N, S and O, and the optional condensed ring of N or O especially.The example that can mention is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, suberyl, its single unsaturated analogue or how unsaturated analogue, and as cyclobutene base, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl, the cycloheptadiene base; Phenyl and naphthyl; And have 1 to 4 heteroatomic 5 yuan to 7 yuan saturated or unsaturated heterocycle base that is selected from O, N and S, wherein said heterocycle can randomly be fused on other heterocycle or the carbocyclic ring.Be to be noted that heterocyclic radical derived from tetramethyleneimine, tetrahydrofuran (THF), piperidines, morpholine, pyrroles, furans, thiophene, pyrazoles, imidazoles, oxazole, thiazole, pyridine, pyrans, pyrimidine, pyridazine, pyrazine, cumarone, indoles and quinoline.Described cyclic group and abovementioned alkyl and thiazolinyl can randomly be substituted once or more times, for example 1,2 or 3 time.The example of the suitable substituent that should mention: halogen, particularly F, Cl, Br;-OH ,-SH ,-NO
2,-NH
3,-SO
3H, C
1-C
4-alkyl and C
2-C
4-thiazolinyl, C
1-C
4-alkoxyl group; And hydroxyl-C
1-C
4-alkyl; Wherein said alkyl and thiazolinyl define as mentioned, and described alkoxyl group is from corresponding alkyl defined above.
R
1And R
3Base also can directly connect each other, forming 4-8 unit ring with to be restored pair of key, and preferred 5-or 6-unit ring, for example cyclopentenes or tetrahydrobenzene structure, it also can be randomly by for example alkyl, and preferable methyl replaces.
R
1Also can directly be connected each other with the R base, forming 4-8 unit ring with to be restored pair of key, preferred 5-or 6-unit ring, for example cyclopentenes or tetrahydrobenzene structure, it also can be randomly by for example alkyl, and preferable methyl replaces.
The first ring of above-mentioned 4-8 can be that carbocyclic ring (promptly only being that carbon atom forms ring) and heterocycle (are heteroatoms, as O; S; N is present in this ring).As needs, these carbocyclic rings or heterocycle also still can be substituted, and promptly hydrogen atom is replaced by heteroatoms.For example, think that N-phenyl succinimide (consulting substrate 3 hereinafter) is the heterocycle of this replacement, it is R
1Form the result who encircles with R.
The embodiment that the present invention is useful especially comprises the corresponding compound that following substrate (compound of general formula 1) enzymatic reduction is become general formula (2):
Substrate 1
Table 1
OPR1 OPR3?wt YqjM
End %
Cofactor E.e.% % E.e.% % E.e.%
Thing
1 NADH ＞99 (R)97 69 (S)82 94 (S)92
1 NADPH ＞99 (R)96 72 (S)87 85 (S)70
1 NAD
+-FDH ＞90 (R)95 40 (S)75 50 (S)85
1 NADP
+-G6PDH 90 (R)98 75 (S)93 47 (S)84
2 NADH ＞99 (S)＞95 90 (S)＞95 70 (S)＞95
2 NADPH ＞99 (S)＞95 90 (S)＞95 73 (S)＞95
2 NAD
+-FDH primary product: geraniol (saturated alcohol)
2 NAD
+-GDH 20 (S)＞95 90 (S)＞95 3 (S)＞95
2 NADP
+-G6PDH 15 (S)＞95 95 (S)＞95 57 (S)＞95
3 NADH 58 (S)61 27 (S)45 50 (S)55
3 NADPH 45 (S)64 19 (S)45 100 (S)66
3 NAD
+-FDH 88 (R)1 65 (R)1 100 (R)2
3 NADP
+-G6PDH 14 (S)61 10 (S)58 72 (S)94
4 NADH n.c. - 1 (S)＞99 0.5 (S)59
4 NADPH n.c. - 2 (S)＞99 n.c. -
4 NAD
+-FDH - - 5 (S)＞99 1 (S)64
4 NADP
+-G6PDH - - 1 (S)＞99 - -
5 NADH n.c. - 3 (S)＞99 n.c. -
5 NADPH n.c. - 2 (S)＞99 n.c. -
5 NAD
+-FDH - - 11 (S)＞99 - -
5 NADP
+-G6PDH - - 1 (S)＞99 - -
6 NADH 99 (R)＞99 99 (R)＞99 99.5 (R)＞99
6 NADPH 99 (R)＞99 99 (R)＞99 98.5 (R)＞99
6 NAD
+-FDH 99 (R)97 99 (R)92 99 (R)92
6 NADP
+-G6PDH 99 (R)96 99 (R)97 99 (R)96
7 NADH ＞99 (R)＞99 n.c. -
7 NADPH ＞99 (R)＞99 n.c. -
7 NAD
+-FDH 2 (R)＞99 n.d. -
7 NADP
+-G6PDH 96 (R)＞99 n.d. -
The inventive method can be especially carried out with the compound of general formula (1), and wherein A is aldehyde radical or ketone group, and R
1Or R
2It is methyl.
The reductase enzyme (it is also referred to as olefin(e) acid (enoate) reductase enzyme sometimes) that is suitable for the inventive method has as the peptide sequence that provides among the SEQ ID NO:1,2 or 3 or has and SEQ ID NO:1,2 or 3 at least 80%, for example at least 90%, or at least 95% and the peptide sequence of at least 97%, 98% or 99% identity especially.
The known polypeptide YqjM (UniprotKB/Swissprot accession number P54550) from subtilis (Bacillussubtilis) by name with SEQ ID NO:1.
Polypeptide with SEQ ID NO:2 is encoded by tomato OPR1 gene (UniprotKB/Swissprot accession number Q9XG54).
Polypeptide with SEQ ID NO:3 is encoded by tomato OYPR3 gene (UniprotKB/Swissprot accession number Q9FEW9).
For purpose described herein, determine sequence identity by " GAP " computer program and the use version 10.3 (canonical parameter that it uses GCG to recommend) of the Genetics Computer Group of Wisconsin university (GCG).
Can begin to obtain this type of reductase enzyme from SEQ ID NO:1,2 or 3 by directed mutagenesis known to the skilled or random mutagenesis methods.Yet, alternative possibility is also can be in microorganism, preferably at those Alishewanella, replace Coccus (Alterococcus), Aquamonas, Aranicola, kill male Pseudomonas (Arsenophonus), Azotivirga, Brenneria, aphid parachorium body (Buchnera) (aphid P-endosymbiont (aphid P-endosymbionts)), Budvicia (Budvicia), Buttiauxella (Buttiauxella), CandidatusPhlomobacter, Cedecea (Cedecea), Citrobacter (Citrobacter), Dickeya, Edwardsiella (Edwardsiella), enterobacter (Enterobacter), erwinia (Erwinia), Escherichia (Escherichia), Ewingella (Ewingella), Grimontella, Hafnia (Hafnia), klebsiella spp (Klebsiella), Kluyvera (Kluyvera), Leclercia (Leclercia), Leminorella (Leminorella), Moellerella (Moellerella), Morganella (Morganella), Obesumbacterium (Obesumbacterium), general Pseudomonas (Pantoea), Pectobacterium (Pectobacterium), Photobacterium (Photorhabdus), Plesiomonas (Plesiomonas), Prague Pseudomonas (Pragia), proteus (Proteus), Providencia (Providencia), Rahnella (Rahnella), Raoultella, salmonella (Salmonella), Samsonia, serratia (Serratia), Shigella (Shigella), Sodalis, Tatumella (Tatumella), Te Labusi Bordetella (Trabulsiella), the fertile Pseudomonas (Wigglesworthia) of lattice Wei, Xenorhabdus belongs to (Xenorhabdus), Yersinia (Yersinia) or Yokenella belong in (Yokenella) the above-mentioned pattern reaction of search catalysis and its aminoacid sequence with SEQ ID NO:1,2 or 3 have the reductase enzyme of required sequence identity or the reductase enzyme that obtains by mutafacient system.
Described reductase enzyme can be with form purifying or partially purified or is used with the form of microorganism itself in addition.Be used for being known by the technician from the method for microorganism acquisition and purifying desaturase.
Utilize the enantioselectivity reduction reaction of reductase enzyme preferably under the situation that has suitable cofactor (being also referred to as cosubstrate), to take place.The cofactor that is generally used for the reduction reaction of ketone is NADH and/or NADPH.Protoenzyme can be used as the cell system that the inherence comprises cofactor in addition, maybe can add alternative redox mediators (A.Schmidt, F.Hollmann and B.B ü hler " Oxidation ofAlcohols ", K.Drauz and H.Waldmann, Enzyme Catalysis in OrganicSynthesis 2002, the III volume, 991-1032, Wiley-VCH, Weinheim).
The cofactor of described reductive agent regenerating oxidation in the reduction reaction process preferably takes place existing under the appropriate reductant in enantioselectivity reduction reaction in addition that utilize reductase enzyme.The example of suitable reductive agent is sugar, particularly hexose, as glucose, seminose, fructose and/or oxidable alcohol, especially ethanol, propyl alcohol or Virahol and formic acid, phosphorous acid or molecular hydrogen.For redox agent and associated regeneration coenzyme, it is possible adding second kind of desaturase, for example when using glucose as reductive agent, adds Hexose phosphate dehydrogenase, maybe when using formic acid as reductive agent, adds hydrogenlyase.It can be used as resolvase or immobilized enzyme uses, or uses with the form of free or immobilized cell.Its preparation can be carried out respectively, or is undertaken by the coexpression in (reorganization) reductase enzyme bacterial strain.
The preferred embodiment of claimed method is by enzyme system regeneration cofactor, uses second kind of desaturase in described enzyme system, preferred especially Hexose phosphate dehydrogenase.
Further advantageously add other additive that promotes reduction reaction, for example metal-salt or sequestrant, for example EDTA.
The reductase enzyme used according to the present invention can use with free or immobilization form.Immobilized enzyme refers to be fixed on the enzyme on the inert support.Suitable carriers material and enzyme fixed thereon are disclosed in EP-A-1149849, EP-A-1 069 183 and DE-A100193773 and the reference wherein quoted.The disclosure of these publications is incorporated herein by reference with its integral body in this regard.The suitable carriers material comprises, for example clay, clay mineral such as kaolin, diatomite, perlite, silicon-dioxide, aluminum oxide, yellow soda ash, lime carbonate, cellulose powder, anion-exchange material, synthetic polymer such as polystyrene, acrylic resin, resol, polyurethane(s) and polyolefine such as polyethylene and polypropylene.Solid support material uses with the particulate form of segmentation usually, to prepare carrier-bound enzyme, preferred porous form.The granular size of described solid support material is no more than 5mm usually, is no more than 2mm (grading curve) especially.Using desaturase to select free on as whole-cell catalyst or the immobilization form is similar possible.The example of solid support material is Protanal TXF 200 and carrageenan.Enzyme and cell also can be directly and glutaraldehyde cross-linking (crosslinked generation CLEAs).Corresponding and other fixing means is described in for example J.Lalonde and A.Margolin " Immobilization of Enzymes ", K.Drauz and H.Waldmann, Enzyme Catalysis in Organic Synthesis 2002, the III volume, 991-1032, Wiley-VCH is among the Weinheim.
Reaction can be carried out in aqueous reaction medium or non-aqueous reaction medium or 2 phase systems or (little) emulsion.Described water reaction medium is preferred buffered soln, and it has 4 to 8 usually, preferred 5 to 8 pH.Aqueous solvent can comprise at least a alcohol beyond dewatering extraly, for example ethanol or Virahol, or dimethyl sulfoxide (DMSO).
Non-aqueous reaction medium refers to, comprises by weight based on the gross weight of liquid reaction medium and is less than 1%, preferably is less than the reaction medium of 0.5% water.Described reaction can be carried out in organic solvent especially.
Appropriate organic solvent is, the aliphatic hydrocrbon that for example preferably has 5 to 8 carbon atoms, as pentane, pentamethylene, hexane, hexanaphthene, heptane, octane or cyclooctane, the halogenated aliphatic hydrocarbon that preferably has 1 or 2 carbon atom, as methylene dichloride, chloroform, tetrachloromethane, ethylene dichloride or tetrachloroethane, aromatic hydrocarbon, as benzene, toluene, dimethylbenzene, chlorobenzene or dichlorobenzene, the aliphatic non-annularity and cyclic ether or the alcohol that preferably have 4 to 8 carbon atoms, as ethanol, Virahol, diethyl ether, methyl tertiary butyl ether, Ethyl Tertisry Butyl Ether, dipropyl ether, Di Iso Propyl Ether, dibutyl ether, tetrahydrofuran (THF), or ester class, as ethyl acetate or n-butyl acetate, or ketone, as methyl iso-butyl ketone (MIBK) Huo diox or its mixture.Preferred especially above-mentioned ether, the especially tetrahydrofuran (THF) of using.
Utilize the reduction reaction of reductase enzyme for example can be, carry out in the water/Virahol of for example any blending ratio (for example 1: 99 to 99: 1 or 10: 90 to 90: 10) or the aqueous reaction medium at water-based organic reaction medium.
In the enzymatic reduction reaction, substrate (1) preferably arrives 500g/l with 0.1g/l, and preferred especially 1g/l uses to the concentration of 50g/l, and can continuous or discontinuous adding.
Enzymatic reduction reaction is usually in the deactivation temperature that is lower than used reductase enzyme and be higher than under-10 ℃ the temperature of reaction and carry out.It is particularly preferably in 0 to 100 ℃ the scope, and is special in 15 to 60 ℃ scope, and especially in 20 to 40 ℃ scope, for example about 30 ℃.
The preferred embodiment of the inventive method is included in and has divalent-metal ion, particularly reacts existing under Ca, Mg, Mn, Zn, Ni, Fe, the Mo ion.Advantageously, the concentration of selection alkaline-earth metal ions is probably identical with the concentration of substrate (ene derivative of general formula I) to be used.About substrate, if particularly described substrate can complexation of metal ions because of its structure, for example under the situation of dicarboxylic acid derivatives, the divalent-metal ion of volumetric molar concentrations such as suggestion adding.
Possible method for example is, for example by stir or shake thorough mixing substrate (1) and reductase enzyme, solvent, coenzyme suitably the time, second kind of desaturase when suitable is with described coenzyme and/or other reductive agent of regenerating.Yet, at reactor, for example in post, fix described reductase enzyme, and make comprise substrate and suitably the time mixture of coenzyme and/or cosubstrate to pass described reactor also be possible.For this reason, make described mixture cycle through described reactor until realizing that the conversion of wanting is possible.
What reduction reaction was carried out the substrate that exists usually in based on mixture is converted at least 70%, and especially preferably at least 85%, and especially at least 95%.The process of reaction, promptly the order restoring of two keys can be ordinary method subsequently, as gas-chromatography or high pressure liquid chromatography.
" function equivalent " of clear and definite disclosed enzyme or analogue are different with described enzyme in the context of the invention but still have the polypeptide of the biological activity of wanting (for example substrate specificity).Therefore, " function equivalent " refers to enzyme, the reaction of its for example catalysis pattern and have one of aminoacid sequence of comprising SEQ ID NO:1, listing for 2 or 3 times enzyme active at least 20%, preferred 50%, preferred especially 75%, preferred very especially 90%.Preferably stable between this external pH 4 to 10 of function equivalent, and advantageously have optimum temperuture in the scope of optimal pH between the pH 5 to 8 and 20 ℃ to 80 ℃.
" function equivalent " also refers in particular to mutant according to the present invention, and it has the amino acid whose amino acid of mentioning especially at least a sequence location that is different from above-mentioned aminoacid sequence, but still has a kind of above-mentioned biological activity.The mutant that " function equivalent " therefore comprises by one or more aminoacid addition, substitutes, disappearance and/or inversion obtain, described modification may take place in any sequence location, has the mass spectral mutant of property of the present invention as long as they cause producing.Particularly when reaction pattern between mutant and the unmodified polypeptide when consistent in nature, when promptly for example same substrate transforms with different rates, also have function equivalent.
The example of suitable amino acid replacement sees following table:
Original residue alternate example
Ala Ser
Arg Lys
Asn Gln；His
Asp Glu
Cys Ser
Gln Asn
Glu Asp
Gly Pro
His Asn；Gln
Ile Leu；Val
Leu Ile；Val
Lys Arg；Gln；Glu
Met Leu；Ile
Phe Met；Leu；Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp；Phe
Val Ile；Leu
" function equivalent " on meaning above also be " precursor " of the polypeptide of describing and " functional deriv ".
" precursor " is natural precursor or the synthetic precursor that has or do not have the bioactive polypeptide of expectation in this context.
Also can be under the help of known technology, the function amino acid side group or they N or C-terminal on " functional deriv " of preparation polypeptide of the present invention.This analog derivative comprises the aliphatic ester class of hydroxy-acid group for example, the amides by the carboxylic acid group that obtained with ammonia or primary amine or secondary amine reaction; By with the N-acyl derivative of the prepared free amine group of acyl group reaction; Or by with the O-acyl derivative of the prepared free hydroxyl group of acyl group reaction.
Under Protein Glycosylation Overview was possible situation, " function equivalent " of the present invention comprised above with de-glycosylation or glycosylation form with by changing the protein of the specified type of modified forms that glycosylation pattern obtains.
" function equivalent " also comprises the polypeptide that obtains the variant of and natural generation biological from other certainly.For example, determine the scope of homologous sequence area, and to determine to be equal to enzyme based on special requirement of the present invention be possible by comparative sequences.
" function equivalent " also comprises fragment, the individual domain or the sequence motifs of preferred polypeptide of the present invention, and it has for example biological function of expectation.
" function equivalent " be fusion rotein still, it comprises the aforementioned polypeptides sequence or from its one of function equivalent, with different with it on the function and be connected at least a other heterologous sequence of (the insignificant mutual function damage that promptly has the fusion rotein part) for functional N or C-terminal.The limiting examples of this type of heterologous sequence is, for example signal peptide or enzyme.
Can identify the proteinic homologue of the present invention by the combinatorial library of screening mutant (for example truncated mutant).For example, can be by combinatorial mutagenesis on nucleic acid level, for example connect the diversified library that the mixture of synthetic oligonucleotide produces protein variant by enzymatic.There is several different methods to can be used for preparing the library of potential homologue from degenerate oligonucleotide sequence.The chemosynthesis of degeneracy gene order can be carried out in automatic dna synthesizer, and institute's synthetic gene can be connected on the suitable expression vector then.The gene of use degeneracy group makes provides the full sequence of the potential protein sequence of coding expectation group to become possibility in a mixture.The method that is used for synthetic degenerate oligonucleotide is (Narang for example, S.A. (1983) Tetrahedron 39:3 known to the technician; Itakura etc. (1984) Annu.Rev.Biochem.53:323; Itakura etc., (1984) Science 198:1056; Ike etc. (1983) Nucleic Acids Res.11:477).
Several technology that are used to screen the gene product of the combinatorial library by point mutation or brachymemma preparation and are used to screen the cDNA library of the gene product with selected character are known in the art.These technology can be suitable for the rapid screening of the gene library that the combinatorial mutagenesis by homologue of the present invention produces.The most common technique that is used for screening big gene library (it carries out high throughput analysis) comprises is cloned into gene library reproducible expression vector, transforms suitable cell with gained vector library and expresses described combination gene under the condition that detects the active carrier of being convenient to separate encoding gene of expectation, and the product of described gene is detected.The overall mutagenesis of technology recurrence (Recursive ensemble mutagenesis) that increases function mutation body frequency in the library (REM) can be used for making up to identify homologue (Arkin and Yourvan (1992) PNAS 89:7811-7815 with shaker test; Delgrave etc. (1993) Protein Engineering 6 (3): 327-331).
The invention still further relates to nucleotide sequence (strand and double-stranded DNA and RNA sequence, for example cDNA and mRNA), its coding has the enzyme of reductase activity of the present invention.Optimized encoding for example is shown in the nucleotide sequence of the aminoacid sequence of SEQID NO:1,2 or 3 characteristic partial sequences.
All nucleotide sequences of herein mentioning Tonghua in a manner known way are synthetic from crossing the preparation of nucleotide structure unit, and for example the fragment condensation by double-helical each overlapping complementary nucleic acid structural unit prepares.The chemosynthesis of oligonucleotide can for example be carried out (Voet, Voet, the 2nd edition, Wiley Press New York, 896-897 page or leaf) by the phosphoramidite method in a known way.The Klenow fragment adding synthetic oligonucleotide of use archaeal dna polymerase and fill up the gap and ligation and general cloning process are described in (1989) such as Sambrook, molecular cloning: laboratory manual, cold spring harbor laboratory publishes.
Be used to carry out other embodiment of enzymatic reduction method of the present invention:
PH advantageously remains on pH 4 to 12 in the inventive method, and preferred pH 4.5 to 9 is between the preferred especially pH 5 to 8.
Minimal Realization 98%ee.
It is possible using the grown cell of the carrier that comprises nucleic acid, nucleic acid construct or coding reductase enzyme for the inventive method.It also is possible using resting cell or ruptured cell.Ruptured cell refers to, for example, by for example solvent treatment permeable cell that become, or handles by enzyme, by mechanical treatment (for example French press or ultrasonic) or the cell by any other method disintegration.The crude extract that obtains in this mode advantageously is suitable for method of the present invention.Enzyme purifying or partially purified also can be used for described method.Immobilized microorganism or enzyme are suitable too, and can be advantageously used in the reaction.
Can be in batches, semi-batch or continue to carry out method of the present invention.
As Biotechnology, the 3rd volume, second edition, Rehm etc. edit (1993), especially describe in the II chapter, can advantageously carry out described method in bio-reactor.
Zhi Bei product can separate from reaction medium by the method that the technician is familiar with in the methods of the invention, and can carry out purifying if desired.These methods comprise distillation method, chromatography, extraction process and crystallization process.As required, can be by making up the purifying that multiple described method significantly improves described product.
Following examples are intended to illustrate the present invention, and not in restriction the present invention.Will be with reference to the accompanying drawings in this context, its demonstration:
Experimental section
The common scheme of asymmetric biological reducing reaction
Use isolating enzyme YqjM, OPR1, OPR3 to carry out the asymmetric biological reducing reaction of substrate according to following common scheme.Because poorly soluble in water, so N-phenyl-2-methyl maleimide adds (final concentration 1%) as 10% solution among the DMF.
To the Tris damping fluid, add zymin (100-200 μ g) and NADH or NADPH cofactor (15mM) in substrate (5mM) solution among the 50mM pH7.5 (0.8ml), and be reflected at 30 ℃ and shake down (140rpm) and carry out.After 48 hours, use the ethyl acetate extraction reaction mixture, and by GC analytical reaction product.
When using the cofactor recirculation system, select following program:
The NADH/FDH system
After adding enzyme (100-200 μ g), with the substrate (5mM) in the 50mM Tris pH of buffer 7.5 (0.8ml), oxidized cofactor NAD
+The mixture of (100 μ M), ammonium formiate (20mM) mixes with FDH (10u), and describedly is reflected at 30 ℃ (140rpm) and carried out 48 hours.
NADH/GDH
After adding enzyme (100-200 μ g), with the substrate (5mM) in the 50mM Tris pH of buffer 7.5 (0.8ml), oxidized cofactor NAD
+The mixture of (100 μ M), glucose (20mM) mixes with (D)-GDH (10u), and describedly is reflected at 30 ℃ (140rpm) and carried out 48 hours.
NADPH/G6PDH
After adding enzyme (100-200 μ g), with the substrate (5mM) in the 50mM Tris pH of buffer 7.5 (0.8ml), oxidized cofactor NADP
+The mixture of (10 μ M), glucose 6-phosphoric acid (20mM) mixes with G6PDH (10u), and describedly is reflected at 30 ℃ (140rpm) and carried out 48 hours.
With H
2Carrying out GC-FID for carrier gas (14.5psi) in Varian 3800 gas chromatographs analyzes.
1-nitro-2-phenyl propylene:
The mensuration that transforms: use 6% cyano group propylbenzene based polysiloxane (cyanopropylphenylpolysiloxane) phase capillary column (Varian CP-1301,30m, 0.25mm, 0.25 μ m film) and analyze the product that is obtained by GC-FID with 30: 1 splitting ratios.Program: 120 ℃/minute to 180 ℃, 20 ℃/minute to 220 ℃, kept 2 minutes.Following: limonene of the residence time (interior mark) 3.81 minutes, 1-nitro-2-phenyl-propane 8.87 minutes, 1-nitro-2-phenyl propylene Z/E 9.55/10.27 minute.
Determine enantiomeric excess and absolute configuration: use cyclodextrin bonded dimethyl polysiloxane capillary column (CP-Chirasil-DEX CB, 25m, 0.32mm, 0.25 μ m film) is mutually determined enantiomeric excess with 25: 1 splitting ratio.Temperature program(me): 105 ℃ kept 5 minutes, and 1 ℃/minute to 120 ℃, kept 6 minutes, 20 ℃/minute to 180 ℃, kept 2 minutes.The residence time is as follows: (S)-and (R)-and 1-nitro-2-phenyl-propane was respectively 12.06 and 12.57 minutes.By inject altogether independent synthetic reference sample determine the absolute configuration of 1-nitro-2-phenyl-propane (J.Org.Chem.1989,54,1802-1804).
Citral
The mensuration that transforms: use polyoxyethylene glycol phase capillary column (Varian CP-Wax 52CB, 30m, 0.25mm, 0.25 μ m film) to analyze the product that is obtained by GC-FID with 20: 1 splitting ratios.Program: 100 ℃ kept 2 minutes, and 15 ℃/minute to 240 ℃, kept 10 minutes.The residence time is as follows: geranial 5.21 minutes, 1-octanol (interior mark) 5.83 minutes, geranic acid 7.53 minutes.
Determine enantiomeric excess and absolute configuration: (hydrodex-β-TBDAc, 25m 0.25mm) determine the enantiomeric excess of geranial to the beta-cyclodextrin capillary column of use modifying.Temperature program(me): 40 ℃ kept 2 minutes, and 4 ℃/minute to 120 ℃, kept 1 minute, 20 ℃/minute to 180 ℃, kept 3 minutes.The residence time is as follows: (S)-and (R)-and geranial was respectively 19.84 and 19.97 minutes.By injecting the absolute configuration that the commercially available reference sample with known absolute configuration is determined geranial altogether.
N-phenyl-2-methyl maleimide:
The mensuration that transforms: the cyano group propylbenzene based polysiloxane phase capillary column (VarianCP-1301,30m, 0.25mm, 0.25 μ m film) of use 6% is analyzed the product that is obtained with 30: 1 splitting ratios by GC-FID.Program: 110 ℃ kept 2 minutes, and 30 ℃/minute to 210 ℃, kept 6 minutes.Following: limonene of the residence time (interior mark) 3.69 minutes, N-phenyl-2-methyl maleimide 8.77 minutes, N-phenyl-2-methyl succinimide 9.89 minutes.
Determine enantiomeric excess and absolute configuration: use Chiralcel OD-H post and normal heptane/alcohol 95: 5 solution is determined enantiomeric excess as elutriant on Shimadzu chirality HPLC.80 μ l carried out isocratic elution 33 minutes at 18 ℃.The residence time is as follows: (S)-and (R)-and N-phenyl-2-methyl succinimide was respectively 27.0 and 29.1 minutes.(J.Mol.Catal.B:Enzym 2005,32,131-134) to determine the absolute configuration of 1-N-phenyl-2-methyl succinimide by CD spectroscopy.
The cyclenes ketone:
The mensuration that transforms: the cyano group propylbenzene based polysiloxane phase capillary column (VarianCP-1301,30m, 0.25mm, 0.25 μ m film) of use 6% is analyzed the product that is obtained with 30: 1 splitting ratios by GC-FID.Program: 80 ℃ kept 10 minutes, and 30 ℃/minute to 200 ℃, kept 3 minutes.The residence time is as follows respectively: 2-methyl-cyclopentanone 4.27 minutes, 3-methyl-cyclopentanone 4.44 minutes, 2-methyl 2-cyclopentenes-1-ketone 5.82 minutes, 3-methylcyclohexanone 7.27 minutes, (R)-limonenes (interior mark) are 8.59 minutes, 3-methyl 2-cyclopentenes-1-ketone 8.77 minutes, 3-methyl 2-tetrahydrobenzene-1-ketone 11.77 minutes.
Determine enantiomeric excess and absolute configuration: (ChiraldexB-TA, 40m 0.25mm) determine enantiomeric excess with 25: 1 splitting ratio to use improved beta-cyclodextrin capillary column.Temperature program(me): 80 ℃ kept 17 minutes, and 30 ℃ minutes to 180 ℃, kept 2 minutes.The residence time is as follows: (S)-and (R)-3-methyl-cyclopentanone 8.08 and 8.29 minutes, and (R)-and (S)-2-methyl-cyclopentanone 6.55 and 6.77 minutes, (R)-and (S)-3-methylcyclohexanone 13.96 and 15.09 minutes.By injecting the absolute configuration that the commercially available reference sample with known absolute configuration is determined 3-methyl-cyclopentanone and 3-methylcyclohexanone altogether.The independent synthetic reference sample that has a known absolute configuration by injection is altogether determined the absolute configuration of 2-methyl-cyclopentanone, and (Tetrahedron:Asymmetry 2001,12,1479-1483).
Sequence table
＜110〉BASF European Co.,Ltd
＜120〉method of enzymatic reduction of alkene derivatives
<130>PF?58579
<160>3
＜170〉PatentIn version 3 .1
<210>1
<211>337
<212>PRT
＜213〉subtilis (Bacillus subtilis)
<400>1
Ala?Arg?Lys?Leu?Phe?Thr?Pro?Ile?Thr?Ile?Lys?Asp?Met?Thr?Leu?Lys
1 5 10 15
Asn?Arg?Ile?Val?Met?Ser?Pro?Met?Cys?Met?Tyr?Ser?Ser?His?Glu?Lys
20 25 30
Asp?Gly?Lys?Leu?Thr?Pro?Phe?His?Met?Ala?His?Tyr?Ile?Ser?Arg?Ala
35 40 45
Ile?Gly?Gln?Val?Gly?Leu?Ile?Ile?Val?Glu?Ala?Ser?Ala?Val?Asn?Pro
50 55 60
Gln?Gly?Arg?Ile?Thr?Asp?Gln?Asp?Leu?Gly?Ile?Trp?Ser?Asp?Glu?His
65 70 75 80
Ile?Glu?Gly?Phe?Ala?Lys?Leu?Thr?Glu?Gln?Val?Lys?Glu?Gln?Gly?Ser
85 90 95
Lys?Ile?Gly?Ile?Gln?Leu?Ala?His?Ala?Gly?Arg?Lys?Ala?Glu?Leu?Glu
100 105 110
Gly?Asp?Ile?Phe?Ala?Pro?Ser?Ala?Ile?Ala?Phe?Asp?Glu?Gln?Ser?Ala
115 120 125
Thr?Pro?Val?Glu?Met?Ser?Ala?Glu?Lys?Val?Lys?Glu?Thr?Val?Gln?Glu
130 135 140
Phe?Lys?Gln?Ala?Ala?Ala?Arg?Ala?Lys?Glu?Ala?Gly?Phe?Asp?Val?Ile
145 150 155 160
Glu?Ile?His?Ala?Ala?His?Gly?Tyr?Leu?Ile?His?Glu?Phe?Leu?Ser?Pro
165 170 175
Leu?Ser?Asn?His?Arg?Thr?Asp?Glu?Tyr?Gly?Gly?Ser?Pro?Glu?Asn?Arg
180 185 190
Tyr?Arg?Phe?Leu?Arg?Glu?Ile?Ile?Asp?Glu?Val?Lys?Gln?Val?Trp?Asp
195 200 205
Gly?Pro?Leu?Phe?Val?Arg?Val?Ser?Ala?Ser?Asp?Tyr?Thr?Asp?Lys?Gly
210 215 220
Leu?Asp?Ile?Ala?Asp?His?Ile?Gly?Phe?Ala?Lys?Trp?Met?Lys?Glu?Gln
225 230 235 240
Gly?Val?Asp?Leu?Ile?Asp?Cys?Ser?Ser?Gly?Ala?Leu?Val?His?Ala?Asp
245 250 255
Ile?Asn?Val?Phe?Pro?Gly?Tyr?Gln?Val?Ser?Phe?Ala?Glu?Lys?Ile?Arg
260 265 270
Glu?Gln?Ala?Asp?Met?Ala?Thr?Gly?Ala?Val?Gly?Met?Ile?Thr?Asp?Gly
275 280 285
Ser?Met?Ala?Glu?Glu?Ile?Leu?Gln?Asn?Gly?Arg?Ala?Asp?Leu?Ile?Phe
290 295 300
Ile?Gly?Arg?Glu?Leu?Leu?Arg?Asp?Pro?Phe?Phe?Ala?Arg?Thr?Al Ala
305 310 315 320
Lys?Gln?Leu?Asn?Thr?Glu?Ile?Pro?Ala?Pro?Val?Gln?Tyr?Glu?Arg?Gly
325 330 335
Trp
<210>2
<211>376
<212>PRT
＜213〉tomato (Lycopersicum esculentum)
<400>2
Met?Glu?Asn?Lys?Val?Val?Glu?Glu?Lys?Gln?Val?Asp?Lys?Ile?Pro?Leu
1 5 10 15
Met?Ser?Pro?Cys?Lys?Met?Gly?Lys?Phe?Glu?Leu?Cys?His?Arg?Val?Val
20 25 30
Leu?Ala?Pro?Leu?Thr?Arg?Gln?Arg?Ser?Tyr?Gly?Tyr?Ile?Pro?Gln?Pro
35 40 45
His?Ala?Ile?Leu?His?Tyr?Ser?Gln?Arg?Ser?Thr?Asn?Gly?Gly?Leu?Leu
50 55 60
Ile?Gly?Glu?Ala?Thr?Val?Ile?Ser?Glu?Thr?Gly?Ile?Gly?Tyr?Lys?Asp
65 70 75 80
Val?Pro?Gly?Ile?Trp?Thr?Lys?Glu?Gln?Val?Glu?Ala?Trp?Lys?Pro?Ile
85 90 95
Val?Asp?Ala?Val?His?Ala?Lys?Gly?Gly?Ile?Phe?Phe?Cys?Gln?Ile?Trp
100 105 110
His?Val?Gly?Arg?Val?Ser?Asn?Lys?Asp?Phe?Gln?Pro?Asn?Gly?Glu?Asp
115 120 125
Pro?Ile?Ser?Cys?Thr?Asp?Arg?Gly?Leu?Thr?Pro?Gln?Ile?Arg?Ser?Asn
130 135 140
Gly?Ile?Asp?Ile?Ala?His?Phe?Thr?Arg?Pro?Arg?Arg?Leu?Thr?Thr?Asp
145 150 155 160
Glu?Ile?Pro?Gln?Ile?Val?Asn?Glu?Phe?Arg?Val?Ala?Ala?Arg?Asn?Ala
165 170 175
Ile?Glu?Ala?Gly?Phe?Asp?Gly?Val?Glu?Ile?His?Gly?Ala?His?Gly?Tyr
180 185 190
Leu?Ile?Asp?Gln?Phe?Met?Lys?Asp?Gln?Val?Asn?Asp?Arg?Ser?Asp?Lys
195 200 205
Tyr?Gly?Gly?Ser?Leu?Glu?Asn?Arg?Cys?Arg?Phe?Ala?Leu?Glu?Ile?Val
210 215 220
Glu?Ala?Val?Ala?Asn?Glu?Ile?Gly?Ser?Asp?Arg?Val?Gly?Ile?Arg?Ile
225 230 235 240
Ser?Pro?Phe?Ala?His?Tyr?Asn?Glu?Ala?Gly?Asp?Thr?Asn?Pro?Thr?Ala
245 250 255
Leu?Gly?Leu?Tyr?Met?Val?Glu?Ser?Leu?Asn?Lys?Tyr?Asp?Leu?Ala?Tyr
260 265 270
Cys?His?Val?Val?Glu?Pro?Arg?Met?Lys?Thr?Ala?Trp?Glu?Lys?Ile?Glu
275 280 285
Cys?Thr?Glu?Ser?Leu?Val?Pro?Met?Arg?Lys?Ala?Tyr?Lys?Gly?Thr?Phe
290 295 300
Ile?Val?Ala?Gly?Gly?Tyr?Asp?Arg?Glu?Asp?Gly?Asn?Arg?Ala?Leu?Ile
305 310 315 320
Glu?Asp?Arg?Ala?Asp?Leu?Val?Ala?Tyr?Gly?Arg?Leu?Phe?Ile?Ser?Asn
325 330 335
Pro?Asp?Leu?Pro?Lys?Arg?Phe?Glu?Leu?Asn?Ala?Pro?Leu?Asn?Lys?Tyr
340 345 350
Asn?Arg?Asp?Thr?Phe?Tyr?Thr?Ser?Asp?Pro?Ile?Val?Gly?Tyr?Thr?Asp
355 360 365
Tyr?Pro?Phe?Leu?Glu?Thr?Met?Thr
370 375
<210>3
<211>396
<212>PRT
＜213〉tomato
<400>3
Met?Ala?Ser?Ser?Ala?Gln?Asp?Gly?Asn?Asn?Pro?Leu?Phe?Ser?Pro?Tyr
1 5 10 15
Lys?Met?Gly?Lys?Phe?Asn?Leu?Ser?His?Arg?Val?Val?Leu?Ala?Pro?Met
20 25 30
Thr?Arg?Cys?Arg?Ala?Leu?Asn?Asn?Ile?Pro?Gln?Ala?Ala?Leu?Gly?Glu
35 40 45
Tyr?Tyr?Glu?Gln?Arg?Ala?Thr?Ala?Gly?Gly?Phe?Leu?Ile?Thr?Glu?Gly
50 55 60
Thr?Met?Ile?Ser?Pro?Thr?Ser?Ala?Gly?Phe?Pro?His?Val?Pro?Gly?Ile
65 70 75 80
Phe?Thr?Lys?Glu?Gln?Val?Arg?Glu?Trp?Lys?Lys?Ile?Val?Asp?Val?Val
85 90 95
His?Ala?Lys?Gly?Ala?Val?Ile?Phe?Cys?Gln?Leu?Trp?His?Val?Gly?Arg
100 105 110
Ala?Ser?His?Glu?Val?Tyr?Gln?Pro?Ala?Gly?Ala?Ala?Pro?Ile?Ser?Ser
115 120 125
Thr?Glu?Lys?Pro?Ile?Ser?Asn?Arg?Trp?Arg?Ile?Leu?Met?Pro?Asp?Gly
130 135 140
Thr?His?Gly?Ile?Tyr?Pro?Lys?Pro?Arg?Ala?Ile?Gly?Thr?Tyr?Glu?Ile
145 150 155 160
Ser?Gln?Val?Val?Glu?Asp?Tyr?Arg?Arg?Ser?Ala?Leu?Asn?Ala?Ile?Glu
165 170 175
Ala?Gly?Phe?Asp?Gly?Ile?Glu?Ile?His?Gly?Ala?His?Gly?Tyr?Leu?Ile
180 185 190
Asp?Gln?Phe?Leu?Lys?Asp?Gly?Ile?Asn?Asp?Arg?Thr?Asp?Glu?Tyr?Gly
195 200 205
Gly?Ser?Leu?Ala?Asn?Arg?Cys?Lys?Phe?Ile?Thr?Gln?Val?Val?Gln?Ala
210 215 220
Val?Val?Ser?Ala?Ile?Gly?Ala?Asp?Arg?Val?Gly?Val?Arg?Val?Ser?Pro
225 230 235 240
Ala?Ile?Asp?His?Leu?Asp?Ala?Met?Asp?Ser?Asn?Pro?Leu?Ser?Leu?Gly
245 250 255
Leu?Ala?Val?Val?Glu?Arg?Leu?Asn?Lys?Ile?Gln?Leu?His?Ser?Gly?Ser
260 265 270
Lys?Leu?Ala?Tyr?Leu?His?Val?Thr?Gln?Pro?Arg?Tyr?Val?Ala?Tyr?Gly
275 280 285
Gln?Thr?Glu?Ala?Gly?Arg?Leu?Gly?Ser?Glu?Glu?Glu?Glu?Ala?Arg?Leu
290 295 300
Met?Arg?Thr?Leu?Arg?Asn?Ala?Tyr?Gln?Gly?Thr?Phe?Ile?Cys?Ser?Gly
305 310 315 320
Gly?Tyr?Thr?Arg?Glu?Leu?Gly?Ile?Glu?Ala?Val?Ala?Gln?Gly?Asp?Ala
325 330 335
Asp?Leu?Val?Ser?Tyr?Gly?Arg?Leu?Phe?Ile?Ser?Asn?Pro?Asp?Leu?Val
340 345 350
Met?Arg?Ile?Lys?Leu?Asn?Ala?Pro?Leu?Asn?Lys?Tyr?Asn?Arg?Lys?Thr
355 360 365
Phe?Tyr?Thr?Gln?Asp?Pro?Val?Val?Gly?Tyr?Thr?Asp?Tyr?Pro?Phe?Leu
370 375 380
Gln?Gly?Asn?Gly?Ser?Asn?Gly?Pro?Leu?Ser?Arg?Leu
385 390 395
Claims (11)
1. the method for preparing the compound of general formula (2) from the ethylenic unsaturation derivative enzymatic of general formula (1), this method is finished by reduce the compound of general formula (1) in the presence of reductase enzyme,
Wherein
A is nitro (NO
2), ketone group (CRO), aldehyde radical (CHO), carboxyl (COOR), R=H or the optional C that replaces
1-C
6-alkyl, R
1, R
2And R
3Be H, C independently of each other
1-C
6-alkyl, C
2-C
6-thiazolinyl, carboxyl, or optional carbocyclic ring or heterocyclic aromatics or non-aromatic group, the perhaps R that replaces
1Be connected to R
3On, to become the part of 4-8 unit ring, perhaps R
1Be connected on the R, to become the part of 4-8 unit ring, condition is R
1, R
2And R
3Inequality, described reductase enzyme
(i) comprise at least a among the peptide sequence SEQ ID NO:1,2 or 3, or
(ii) have with SEQ ID NO:1,2 or 3 peptide sequence that is equal on the function of at least 80% identity is arranged.
2. the process of claim 1 wherein and use NADPH or NADH to carry out described reduction reaction as cofactor.
3. the method for claim 2, wherein the enzymatic described cofactor of regenerating.
4. the method for claim 3 is wherein by Hexose phosphate dehydrogenase or hydrogenlyase or the secondary alcohol described cofactor of regenerating.
5. any one method of aforementioned claim is wherein carried out described reduction reaction in water-based, water-alcohol or alcohol reaction medium.
6. any one method of aforementioned claim, wherein said reductase enzyme is immobilized.
7. any one method of aforementioned claim, wherein said enzyme is selected from the reductase enzyme of subtilis and tomato.
8. any one method of aforementioned claim, the compound of its formula of (1) reacts, wherein R
1Be methyl, and A is a ketone group.
9. any one method of aforementioned claim, wherein said being reflected in 0 to 45 ℃ temperature range and/or 6 to 8 the pH scope carried out.
10. any one method of aforementioned claim, the wherein said divalent-metal ion that is reflected at carries out under existing.
11. by any one general formula (2) compound of method preparation of aforementioned claim as the purposes of chemistry or enzymatic substance synthetic intermediate.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP06124126.1 | 2006-11-15 | ||
| EP06124126 | 2006-11-15 | ||
| EP06126993.2 | 2006-12-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101528937A true CN101528937A (en) | 2009-09-09 |
Family
ID=41095769
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007800393066A Pending CN101528937A (en) | 2006-11-15 | 2007-11-13 | Process for enzymatic reduction of alkene derivatives |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101528937A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103289922A (en) * | 2013-05-17 | 2013-09-11 | 浙江工业大学 | Yokenella sp. and application thereof in preparing alpha, beta-unsaturated enol and aromatic alcohol |
| CN112062767A (en) * | 2020-10-20 | 2020-12-11 | 杭州科巢生物科技有限公司 | Preparation method and intermediate of rumepilone |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005027552A (en) * | 2003-07-11 | 2005-02-03 | Mitsubishi Chemicals Corp | New method for producing optically active 2-hydroxymethyl-3-arylpropionic acid |
-
2007
- 2007-11-13 CN CNA2007800393066A patent/CN101528937A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005027552A (en) * | 2003-07-11 | 2005-02-03 | Mitsubishi Chemicals Corp | New method for producing optically active 2-hydroxymethyl-3-arylpropionic acid |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103289922A (en) * | 2013-05-17 | 2013-09-11 | 浙江工业大学 | Yokenella sp. and application thereof in preparing alpha, beta-unsaturated enol and aromatic alcohol |
| CN103289922B (en) * | 2013-05-17 | 2015-08-26 | 浙江工业大学 | Yokenella and the application in preparation α, β-unsaturated enol and aromatic alcohol thereof |
| CN112062767A (en) * | 2020-10-20 | 2020-12-11 | 杭州科巢生物科技有限公司 | Preparation method and intermediate of rumepilone |
| CN112062767B (en) * | 2020-10-20 | 2022-03-11 | 杭州科巢生物科技有限公司 | Preparation method and intermediate of rumepilone |
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Application publication date: 20090909 |