Technical field
[0001]
　The present invention relates to a manufacturing method of rosuvastatin calcium and intermediates thereof.
Background technique
[0002]
　Rosuvastatin is an enzyme 3-hydroxy-3-methylglutaryl - an inhibitor of coenzyme A reductase (HMG-CoA reductase), for example useful in the treatment of hypercholesterolemia and mixed dyslipidemia. Rosuvastatin, (E) -7- [4- (4- fluorophenyl) -6-isopropyl-2- [methyl (methylsulfonyl) amino] pyrimidin-5-yl] - (3R, 5S) -3,5- it is a generic name of hydroxy-6-heptenoic acid. Rosuvastatin, in the treatment, it is administered as the calcium salt. Rosuvastatin calcium is a trade name CRESTOR (TM), sold as HMG-CoA reductase inhibitor. Rosuvastatin calcium has the following chemical formula.
[0003]
[Formula 1]

[0004]
　Patent Document 1, rosuvastatin, its sodium salt and calcium salt, and methods for their preparation are disclosed. According to Patent Document 1, rosuvastatin and salts thereof, (3R) -3 - [(tert- butyldimethylsilyl) oxy] -5-oxo-6-triphenylphosphoranylidene hexanoic acid methyl 4- (4- fluorophenyl) -6-isopropyl-2-(N-methyl -N- methanesulfonylamino) -5-pyrimidine carboxylate aldehyde condensation was introduced a side chain having one asymmetric center, then the 3-hydroxy group deprotection, obtained by performing 5-asymmetric reduction of an oxo group, and hydrolysis. In this method, since the cryogenic conditions at the time of the asymmetric reduction (preferably -85 ℃ ~ -70 ℃) is needed, not necessarily industrially preferred method.
[0005]
　In a similar way, it is also known a method of introducing a side chain having two asymmetric centers (such as Patent Documents 2 and 3). In these methods, since the extremely low temperature condition (e.g. about -75 ° C.) is needed in carrying out the Wittig reaction, not necessarily industrially preferred method.
[0006]
　Also known is a method of introducing an asymmetric center with an optically active titanium catalyst (Patent Document 4). In these methods, it uses an expensive optically active catalyst, also because the extremely low temperature condition at the time of the asymmetric reduction (about -80 ℃ ~ -50 ℃) is required, necessarily industrially preferred method Can not say.
[0007]
　Non-Patent Documents 1 and 2, a method for producing a dihydroxy ester derivative by reducing a diketo ester derivatives. However, the in Non-Patent Documents 1 and 2 are specifically shown is only reduced by the organic synthesis reaction, also diketo ester derivative or dihydroxy ester derivative is only a compound is a tert- butyl ester.
[0008]
　Patent Documents 5 and 6, as a manufacturing method of pitavastatin, manufacturing method using carbonyl reductase have been described. However, Patent Documents 5 and 6, no description about rosuvastatin, also, while the rosuvastatin having a pyrimidine ring substituted with a sulfonylamino group, pitavastatin has a quinoline ring, different their chemical structure greatly ing.
CITATION
Patent literature
[0009]
Patent Document 1: Japanese Patent No. 2648897
Patent Document 2: WO 2010/047296 Patent
Patent Document 3: WO 2005/042522 Patent
Patent Document 4: WO 2008/065410 Patent
Patent Document 5: International Publication No. 2002 / 063,028 Patent
Patent Document 6: WO 2003/078634
Non-Patent Document
[0010]
非特許文献1 : ＩＰ．ｃｏｍ　ｎｕｍｂｅｒ：ＩＰＣＯＭ０００１４４０２６Ｄ、Ｄｅｃｅｍｂｅｒ　１４，２００６
非特許文献2 : ＩＰ．ｃｏｍ　ｎｕｍｂｅｒ：ＩＰＣＯＭ０００１４５６２３Ｄ、Ｊａｎｕａｒｙ　１９，２００７
Summary of the invention
Problems that the Invention is to Solve
[0011]
　Conventional method of manufacturing a rosuvastatin, to use cryogenic reaction or expensive asymmetric catalyst, the development of a more economical production method is desired. The present invention, rosuvastatin calcium, and intermediates thereof, can be efficiently produced at low cost and with high purity, an object of the present invention to provide a novel method.
Means for Solving the Problems
[0012]
　The present inventors have found a result of intensive studies to solve the above problems, the manufacturing method and / or intermediates following, economical reaction conditions efficiently rosuvastatin calcium, found to be able and prepared in high purity, the present invention a has been completed.
　That is, the present invention is as follows.
[1] (i) the following general formula (1):
[0013]
[Of 2]

[0014]
(Wherein, R.-X-represents a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms 1 and -X 2 are independently each represents -OH or = O , -X 1 and / or -X 2 is = O.)
to the compound represented by the microorganisms or cells capable of producing the enzyme, an enzyme having an activity capable of reducing the carbonyl group stereoselectively , a step of reduction by the action of culture medium containing the enzyme obtained by culturing processed product of the microorganism or cell, and / or the microorganism or cell;
characterized by having a following general formula (2 ):
[0015]
[Formula 3]

[0016]
(Wherein, R represents R a is as defined. Of the general formula (1))
the production method of the compound represented by.
[2] the enzyme following (A), an enzyme comprising any of the polypeptides shown in (B) or (C), the method of producing the above-mentioned [1].
(A) Ogataea minuta var. nonfermentans NBRC1473-derived carbonyl reductase (OCR1) (SEQ ID NO: 2) a polypeptide
having, consisting of an amino acid sequence having the amino acid sequence 80% or more homology as described in (B) SEQ ID NO: 2, the general formula (1 a compound represented by) a polypeptide having an activity of converting the compound represented by the general formula (2),
in the amino acid sequence set forth in (C) SEQ ID NO: 2, one or several amino acids are substituted, comprising a deletion or addition of amino acid sequences, and a polypeptide having an activity of converting the compound represented by the general formula (1), the compound represented by the general formula (2).
[3] the gene encoding the enzyme, the following (D), a DNA comprising the nucleotide sequence shown in (E) or (F), the production method according to [1].
(D) the nucleotide sequence set forth in SEQ ID NO: 1,
is represented by a DNA which hybridizes with the DNA under stringent conditions consisting of the complementary sequence of the nucleotide sequence described in (E) SEQ ID NO: 1 and the general formula (1) that acts on the compound, nucleotide sequence encoding a polypeptide having an activity of converting the compound represented by the general formula (2),
(F) 1 or several nucleotides in the nucleotide sequence set forth in SEQ ID NO: 1 polypeptide but having an activity of converting substitution comprises a deletion or addition of nucleotide sequence, and acts on the compound represented by the general formula (1), the compound represented by the general formula (2) nucleotide sequence encoding.
[4] the step of (i), in the presence of a polyhydric alcohol, The process according to any one of [1] to [3].
[5] (ii) the following formula (3):
[0017]
[Of 4]

[0018]
A compound represented by the following general formula (4):
[0019]
[Of 5]

[0020]
(Wherein, R 1 represents a straight-chain or branched alkyl group having 1 to 8 carbon atoms.)
The compound represented by the presence of a base, the process of condensing;
characterized by having a following the general formula (1):
[0021]
[Of 6]

[0022]
(Wherein, R.-X-represents a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms 1 and -X 2 are independently each represents -OH or = O , -X 1 and / or -X 2 is = O.)
production method of the compound represented by.
[6] (iia) the following formula (3):
[0023]
[Chemical Formula 7]

[0024]
A compound represented by the following general formula (4a):
[0025]
[Of 8]

[0026]
(Wherein, R 2 represents a branched alkyl group having 3 to 8 carbon atoms, the above-mentioned R are different groups.) The compound represented by the presence of a base, step condensation; and
(iib) the following general formula obtained in the step (iia) (5):
[0027]
[Formula 9]

[0028]
(Wherein, R 2 is R in the general formula (4a) 2 is synonymous with.)
And a compound represented by, R-OH (wherein, R 1 alkyl group or a C 1 to 8 carbon atoms . the number of 3 to represent a secondary alkyl group of 6) reacting the alcohol represented by;
characterized by having a production method according to [5].
[7] (iia) the following formula (3):
[0029]
[Of 10]

[0030]
A compound represented by the following general formula (4a):
[0031]
[Of 11]

[0032]
(Wherein, R 2 represents a branched alkyl group having 3 to 8 carbon atoms, wherein the R are different groups.) The compound represented by the presence of a base, step condensation;
(iib) the the following general formula obtained in step (iia) (5):
[0033]
[Of 12]

[0034]
(Wherein, R 2 is R in the general formula (4a) 2 is synonymous with.)
And a compound represented by, R-OH (wherein, R 1 alkyl group or a C 1 to 8 carbon atoms . representing a secondary alkyl group having 3 to 6) a step to react an alcohol represented by; and
(ia) the step (iib) the following general formula obtained in (1a) compound represented by:
[0035]
[Of 13]

[0036]
(Wherein, R represents. A primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms)
, the enzyme having an ability capable of reducing the carbonyl group stereoselectively, the enzyme microorganisms or cells having the ability to produce, processed product of the microorganism or cell, and / or the microorganism or the cells by the action of culture medium containing the enzyme obtained by culturing reduced to the following general formula (1b ) and / or (1c):
[0037]
[Of 14]

[0038]
(Wherein, R represents the same as R in the general formula (1a).)
[0039]
[Of 15]

[0040]
(Wherein, R represents the same as R in the general formula (1a).)
To obtain a compound represented by;
characterized by having a production method according to [5].
[8] wherein said enzyme following (A), an enzyme comprising any of the polypeptides shown in (B) or (C), the method of producing the above-mentioned [7].
(A) Ogataea minuta var. nonfermentans NBRC1473-derived carbonyl reductase (OCR1) (SEQ ID NO: 2) a polypeptide
having, consisting of an amino acid sequence having the amino acid sequence 80% or more homology as described in (B) SEQ ID NO: 2, the general formula (1 a compound represented by) a polypeptide having an activity of converting the compound represented by the general formula (2),
in the amino acid sequence set forth in (C) SEQ ID NO: 2, one or several amino acids are substituted, comprising a deletion or addition of amino acid sequences, and a polypeptide having an activity of converting the compound represented by the general formula (1), the compound represented by the general formula (2).
[9] the gene encoding the enzyme, the following (D), a DNA comprising the nucleotide sequence shown in (E) or (F), the production method according to [7].
(D) the nucleotide sequence set forth in SEQ ID NO: 1,
is represented by a DNA which hybridizes with the DNA under stringent conditions consisting of the complementary sequence of the nucleotide sequence described in (E) SEQ ID NO: 1 and the general formula (1) that acts on the compound, nucleotide sequence encoding a polypeptide having an activity of converting the compound represented by the general formula (2),
(F) 1 or several nucleotides in the nucleotide sequence set forth in SEQ ID NO: 1 polypeptide but having an activity of converting substitution comprises a deletion or addition of nucleotide sequence, and acts on the compound represented by the general formula (1), the compound represented by the general formula (2) nucleotide sequence encoding.
[10] the step of (ia), carried out in the presence of a polyhydric alcohol, The process according to any one of [7] to [9].
[11] a compound represented by the following general formula (1a).
[0041]
[Of 16]

[0042]
(Wherein, R represents a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms having 1 to 8 carbon
atoms.) [12] a compound represented by the following general formula (1b) or (1c).
[0043]
[Of 17]

[0044]
(Wherein, R represents a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms.)
[0045]
[Of 18]

[0046]

(. Wherein, R representing a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms having 1 to 8 carbon atoms) [13] following formula:
[0047]
[Of 19]

[0048]
A in the compounds represented by the crystal, 2θ = 8.7 °, 16.3 ° , 19.7 °, 21.2 °, characteristic peaks in the 21.3 ° (± 0.2 °) crystal having a powder X-ray diffraction pattern shown.
[14] (iiia) the general formula obtained by the production method according to [1] (2):
[0049]
[Of 20]

[0050]
(Wherein, R 1 alkyl group, or represents. A secondary alkyl group having 3 to 6 carbon atoms having 1 to 8 carbon atoms)
reacting a After hydrolysis with a base a compound represented by the calcium compound ;
characterized by having the following formula (6):
[0051]
[Of 21]

[0052]
Ross server method of manufacturing a statin calcium shown in.
In [15] the step (iiia), the hydrolysis, and a polar solvent, an ether solvent, a hydrocarbon solvent, and be carried out in the presence of a mixed solvent of at least one solvent selected from the group consisting of halogen solvent and wherein, the manufacturing method of rosuvastatin calcium according to the above [14].
[16] In the step (iiia), pH and characterized by a pH 5 ~ 10, the manufacturing method of rosuvastatin calcium according to the above [14] or [15] for starting the reaction of the calcium compound.
[. 17] (iiib) wherein the general formula obtained by the production method according to [1] (2):
[0053]
[Of 22]

[0054]
(Wherein, R represents. A secondary alkyl group of primary alkyl group or a C 3-6 1-8 carbon atoms)
After hydrolysis with a base a compound represented by, treated with an acid, to give was the following formula (8):
[0055]
[Of 23]

[0056]
In the compound represented by is reacted with an amine compound, obtained by the following general formula (9):
[0057]
[Of 24]

[0058]
(Wherein, R 3 and R 4 are each independently, represents an alkyl group having 1 to 8 carbon atoms.)
After the compound represented by the salt exchange with a base, reacting with the calcium compound;
having the following formula which is characterized in that (6):
[0059]
[Of 25]

[0060]
Ross server method of manufacturing a statin calcium shown in.
[18] (iiic) wherein the general formula obtained by the production method according to [1] (2):
[0061]
[Of 26]

[0062]
(Wherein, R represents. A primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms)
After hydrolysis with a base a compound represented by the presence of an acid catalyst or engaged intramolecular dehydration condensation in the absence, resulting the following formula (10):
[0063]
[Of 27]

[0064]
Step of the compound represented by is reacted with a calcium compound;
formula characterized by having a (6):
[0065]
[28 *]

[0066]
Ross server method of manufacturing a statin calcium shown in.
[19] 2θ = 19.8 °, characterized by having a powder X-ray diffraction pattern showing characteristic peaks at 22.9 ° (± 0.2 °), (E) -7- [4- (4-fluorophenyl) -6-isopropyl-2- [methyl (methylsulfonyl) amino] pyrimidin-5-yl] - (3R, 5S) of 3,5-hydroxy-6-heptenoic acid n- propylamine salt crystal.
[20] 2θ = 6.6 °, characterized by having a powder X-ray diffraction pattern showing characteristic peaks at 17.0 ° (± 0.2 °), (E) -7- [4- (4-fluorophenyl) -6-isopropyl-2- [methyl (methylsulfonyl) amino] pyrimidin-5-yl] - (3R, 5S) -3,5- hydroxy-6-heptenoic acid crystals dimethylamine salt.
[21] the following formula (11):
[0067]
[Of 29]

[0068]
Characterized in that it contains in compound represented by 1ppm or 1500ppm or less, rosuvastatin calcium.
[22] the following general formula (2):
[0069]
[Of 30]

[0070]
(Wherein, R 1 grade represents. A secondary alkyl group of the alkyl group or a carbon 3-6 1-8 carbon atoms)
in a mixed solvent of an organic solvent a compound represented by, or an organic solvent and water It was dissolved, by cooling below a cooling rate of 15 ° C. / hour, characterized in that to precipitate crystals of the compound represented by the formula (2) is represented by the general formula (2) purification methods of that compound.
[23] (B) the following general formula (12):
[0071]
[Of 31]

[0072]
(. Wherein, M is an alkali metal element, an alkaline earth metal element, or a hydrogen)
a compound represented by the following formula (13):
[0073]
[Of 32]

[0074]
In the step of converting the compound represented
and having a method for producing rosuvastatin calcium.
[24] The prior to step (Bs), (Aa) following general formula (14):
[0075]
[Of 33]

[0076]
(. Wherein, R representing a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms)
and a compound represented by the following general formula (15):
[0077]
[Of 34]

[0078]
(. Wherein, R is as defined above)
a mixture comprising a compound represented by the presence of a base, and hydrolyzing the following general formula (16):
[0079]
[Of 35]

[0080]
(Wherein, M is an alkali metal element, an alkaline earth metal element, or a hydrogen.)
With a compound represented by the step of converting the mixture containing the compound represented by the general formula (12)
and characterized in that it has, the manufacturing method of rosuvastatin calcium according to [23].
[25] after the step (B), (C) and having a step of removing the compound represented by the formula (13), of rosuvastatin calcium according to [23] or [24] Production method.
[26] after the step (C), (D) and the step compound obtained by (C), characterized by having a step of reacting a calcium compound, rosuvastatin calcium according to the above [25] method of manufacturing.
[27] the following general formula (12):
[0081]
[Of 36]

[0082]
(Wherein, M is an alkali metal element, an alkaline earth metal element, or hydrogen.)
In a method of purifying rosuvastatin calcium containing compound represented,
is represented by (B) formula (12) that compound, the following formula (13):
[0083]
[Of 37]

[0084]
In the step of converting the compound represented
and having a purification method of rosuvastatin calcium.
[28] Prior to the step (Bs), according to which (Ab) and having a step of dissolving the rosuvastatin calcium comprising said compound represented by Formula (12) in a solvent, the above-mentioned [27] purification process of rosuvastatin calcium.
[29] after the step (B), (C) and having a step of removing the compound represented by the formula (13), of rosuvastatin calcium according to [27] or [28] purification methods.
[30] after the step (C), (D) and the compound obtained in Step (C), characterized by having a step of reacting a calcium compound, rosuvastatin calcium according to the above [29] method of purification.
[31] the following formula (13):
[0085]
[Of 38]

[0086]
Which is characterized by containing a compound represented by 1ppm or 1000ppm or less, rosuvastatin calcium.
Effect of the invention
[0087]
　According to the production method of the present invention, without using a cryogenic reaction or expensive asymmetric catalyst, an economical high purity rosuvastatin calcium in conditions and its intermediates, efficient production to an industrial scale it is possible.
Brief description of the drawings
[0088]
[1] The compound obtained in Example 2 (DOXP ((E) -7- [4- (4- fluorophenyl) -6-isopropyl-2- [methyl (methylsulfonyl) amino] pyrimidin-5-yl ] is a graph illustrating a powder X-ray diffraction pattern of 3,5-dioxo-6-heptenoic acid n- propyl ester)). The vertical axis is the intensity, and the horizontal axis represents the 2θ (°).
It is a diagram illustrating a powder X-ray diffraction pattern [Figure 2] the compound obtained in Example 2 '(DOXP). The vertical axis is the intensity, and the horizontal axis represents the 2θ (°).
[3] The compound obtained in Example 5 (DOLP (((3R) , (5S), (6E)) - 7- [4- (4- fluorophenyl) -6-isopropyl-2- [methyl ( it is a diagram showing a methylsulfonyl) amino] pyrimidin-5-yl] -3,5-dihydroxy-6-heptenoic acid n- propyl ester)) of the powder X-ray diffraction pattern. The vertical axis is the intensity, and the horizontal axis represents the 2θ (°).
It is a diagram illustrating a powder X-ray diffraction pattern [Figure 4] the compound obtained in Example 11 (DOLP). The vertical axis is the intensity, and the horizontal axis represents the 2θ (°).
5 is a diagram illustrating a powder X-ray diffraction pattern of the propylamine salt obtained in Example 7. The vertical axis is the intensity, and the horizontal axis represents the 2θ (°).
6 is a diagram illustrating a powder X-ray diffraction pattern of dimethylamine salt obtained in Example 9. The vertical axis is the intensity, and the horizontal axis represents the 2θ (°).
DESCRIPTION OF THE INVENTION
[0089]
　Below, it will be described in detail the terms used in the present specification.
　As used herein, the term "primary alkyl group having 1 to 8 carbon atoms", a methyl group, an ethyl group, n- propyl group, n- butyl group, n- pentyl group, n- hexyl, n- octyl group It means.
　As used herein, the term "primary alkyl group having 1 to 4 carbon atoms" refers to a methyl group, an ethyl group, n- propyl group, an n- butyl group.
　As used herein, the term "secondary alkyl group having 3 to 6 carbon atoms", an isopropyl group, a cyclopropyl group, sec- butyl group, 1-methylbutyl group, 1-methylheptyl group, 1-ethylpropyl group, 1 - means ethylbutyl group.
　As used herein, the term "secondary alkyl group having 3 to 4 carbon atoms", an isopropyl group, a cyclopropyl group, refers to a sec- butyl group.
　As used herein, the term "linear or branched alkyl group having 1 to 8 carbon atoms", a methyl group, an ethyl group, n- propyl group, n- butyl group, n- pentyl group, n- hexyl group, n- octyl group, an isopropyl group, a cyclopropyl group, sec- butyl group, 1-methylbutyl group, 1-methylheptyl group, tert-butyl group, means a tert-amyl group.
　As used herein, "branched alkyl group having 3 to 8 carbon atoms", an isopropyl group, a cyclopropyl group, sec- butyl group, 1-methylbutyl group, 1-methylheptyl group, tert-butyl group, tert It means amyl group.
　As used herein, "calcium compound" refers to calcium chloride, such as calcium acetate, a compound of the carboxylic acid can be converted to calcium salts of carboxylic acids. Preferably, the calcium compound is calcium chloride.
　As used herein, "amine compound" is meant n- propylamine, isopropylamine, such as dimethyl amine, the compound of the carboxylic acid can be converted to an amine salt of a carboxylic acid. Preferably, the amine compound is an n- propylamine or dimethylamine.
　Incidentally, the compounds according to the present invention, the salts of the compounds, anhydrides, hydrates, solvates and the like are also included.
[0090]
　In the present specification, "an enzyme having an activity capable of stereoselectively reducing the carbonyl group", an enzyme having an activity of converting the optically active alcohols by asymmetric reduction of the carbonyl group of the carbonyl group-containing compound It means.
　Whether they have a "activity capable of stereoselectively reducing the carbonyl group", an activity of converting the optically active alcohols by asymmetric reduction of the carbonyl group of the carbonyl group-containing compound, by conventional assays which can be determined by measuring. For example, the amount of the compound represented by the general formula (1), reacted with the enzyme of interest of the measurement, the compound represented by the general formula (1) converted from the compound represented by the general formula (2) the by directly measuring, it is possible to confirm the enzymatic activity.
　Further, the "enzyme" herein, (including partially purified enzyme.) Purified enzyme or those immobilized using conventional immobilization techniques, such as polyacrylamide, carriers such as carrageenan gel thing was immobilized, etc. are also included.
　As used herein, "microbial or cell capable of producing an enzyme having activity which can reduce the carbonyl group stereoselectively" (hereinafter, may be referred to as "microbial or cell of the present invention".) The particularly limited as long as it has the "activity capable of reducing the carbonyl group stereoselectively" does not, may be a microorganism or cells having an endogenously the activity was granted the activity by breeding microorganisms or it may be a cell. As means for imparting the activity by breeding, including genetic engineering processes (transformation) and mutagenesis, it is possible to employ a known method. As a method for transformation, it is used methods such as introducing a gene of interest, enhance the expression of the enzyme gene in the biosynthetic pathway of organic compounds, which reduces the expression of an enzyme gene in-product biosynthetic pathway it can.
　As the kind of "microbial or cell" include those described in a host organism or host cell to be described later. "Microbial or cell" may also be used in a state of being frozen. Included Further, in the present specification, the "microbial or cell capable of producing an enzyme having the activity" is not limited to living microorganisms or cells, it is dead as living body having an enzymatic activity It is.
　Further, microorganisms or cells of the present invention can be prepared by the method described in WO 2003/078634.
　In the present specification, the type of organism "host organism" is not particularly limited, Escherichia coli, Bacillus subtilis, coryneform bacteria, bacteria belonging to the genus Pseudomonas, Bacillus bacteria, Rhizobium bacteria, lactobacilli, succinonitrile Bacillus bacteria, Ana erotic bios pyridinium Lamb bacteria belonging to the genus, Actinobacillus bacteria belonging to the genus Bacillus such as prokaryotic, yeast, fungi such as filamentous fungi, plants, and the like eukaryotic organisms such as animals. Among them, preferably, E. coli, yeast, a coryneform bacterium, particularly preferably E. coli.
　In the present specification, the type of cells to be "host cell" is not particularly limited, and may be an animal cell, a plant cell, an insect cell and the like.
　As used herein, "expression vector", by introducing into the host organism incorporate polynucleotide encoding a protein having the desired function, in order to replicate and express a protein having the desired function in the host organism is a genetic factor to be used. For example, a plasmid, virus, phage, but cosmids, and the like without limitation. Preferably, the expression vector is a plasmid.
　As used herein, "transformant", the expression vector is introduced, it means a microorganism or cell has become possible to represent a desired trait associated with a protein having the desired function.
　As used herein, the term "processed product of the microorganism or cells", culturing microorganisms or cells, the microorganism or cell, 1) those treated by an organic solvent or the like, 2) a lyophilized, 3) a carrier, such as which was immobilized, 4) are those physically or enzymatically disrupted, and means such as those containing a protein having the desired function.
　In the present specification, "culture medium containing the enzyme obtained by culturing microorganisms or cells", 1) the culture broth of a microorganism or cell, 2) was treated with an organic solvent such as a culture broth of a microorganism or cell culture, 3) means a culture medium are physically or enzymatically disrupt the cell membrane of the microorganism or cell.
[0091]
[Production method of the present invention]
　will now be described in detail the production method of the present invention. In the following, w / v means weight / volume.
　The production method of the present invention, as shown below, in the step of converting the general formula (1) compounds represented by the compound represented by the general formula (2) (i), and general formula (2) step of converting the compound represented by the rosuvastatin calcium of the formula (6) (iiia), ( iiib) ((iiib-1) ~ (iiib-3)) or (iiic) ((iiic-1 ) ~ ( iiic-2)) are included.
[0092]
[Of 39]

[0093]
　In the formula, R represents a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms, preferably 2 grade primary alkyl group or a C 3-4 1-4 carbon atoms It represents an alkyl group. As the R, a methyl group, an ethyl group, n- propyl group, an isopropyl group or n- butyl are preferred. Among them, from the viewpoint can be carried out efficiently process a (i), examples of R, more preferably n- propyl group or an isopropyl group, n- propyl group is particularly preferred.
　-X 1 and -X 2 are each independently a -OH or = O, -X 1 and / or -X 2 is = O.
　R 3 and R 4 are each independently, a hydrogen atom or an alkyl group having 1 to 8 carbon atoms, preferably a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.
[0094]
　The production method of the present invention, as shown below, as a manufacturing method of the general formula (1) compounds represented by used in step (i), the compound represented by formula (3) and general formula (4 a step of converting the compound represented by the compounds represented by the general formula (1)) (ii) contains.
　Further, a preferred embodiment of step (ii), a step of converting the compound represented by the compound represented by formula (3) and general formula (4a) to the compound represented by the general formula (5 of 5) (iia), and it contains a compound represented by the general formula (5) is a step of converting the compound represented by the general formula (1) (iib).
[0095]
　Further, as another embodiment of step (i), the step of converting the compound represented by the general formula a compound represented by the compound represented by general formula (1a) (1b) and / or the general formula (1c) (ia), and general formula (1b) step of converting a compound represented by and / or the general formula (1c) compounds represented by the compound represented by the general formula (2) (ib) also according to the invention It is included in the production method.
[0096]
[Of 40]

[0097]
　Wherein, R, -X 1 and -X 2 are as defined above definition.
　R 1 represents a linear or branched alkyl group having 1 to 8 carbon atoms, preferably an primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms, more preferably carbon It represents a primary alkyl group or a secondary alkyl group having a carbon number of 3 or 4 of the number of 1 to 4. R 1 as a methyl group, an ethyl group, n- propyl group, an isopropyl group or n- butyl are preferred. Among them, from the viewpoint can be carried out efficiently process a (i), R 1 as is more preferably n- propyl group or an isopropyl group, n- propyl group is particularly preferred.
　R 2 represents a branched alkyl group having 3 to 8 carbon atoms, are different groups from the above R. R 2 is preferably an isopropyl group, s- butyl group, tert-butyl group, a tert-amyl group, particularly preferably a tert-butyl group.
[0098]
　Described in detail in the following each step of the production method of the present invention.
[0099]
Step (i):
　step (i), the compound represented by the general formula (1), microorganisms or cells capable of producing the enzyme, an enzyme having an activity capable of reducing the carbonyl group stereoselectively ( microorganisms or cells of the present invention), processed product of the microorganism or cell, and / or the microorganism or cell culture medium containing the enzyme obtained by culturing (hereinafter "enzyme or the like of the present invention" them collectively may be referred to as.) was reduced by the action of a step for obtaining a compound represented by the general formula (2).
[0100]
[Of 41]

[0101]
　Wherein, R, -X 1 and -X 2 are as defined above definition.
[0102]
　As the enzyme used in step (i), those having the amino acid sequence set forth in SEQ ID NO: 2 (hereinafter sometimes referred to as "OCR1") or can be used homolog of the amino acid sequence. Specifically, the following (the A), it includes (B) or enzyme comprising any of the polypeptides (C), the or their homologs.
(A) described in Japanese Patent No. 4270918 Ogataea minuta var. nonfermentans NBRC1473-derived carbonyl reductase (OCR1) (SEQ ID NO: 2) polypeptide having the
consist (B) an amino acid sequence having the amino acid sequence 80% or more homology of SEQ ID NO: 2, the general formula (1) a compound represented by a polypeptide having an activity of converting the compound represented by the general formula (2)
in the amino acid sequence set forth in (C) SEQ ID NO: 2, one or several amino acids are substituted, deleted or added polypeptides having been comprises an amino acid sequence, and a compound represented by the general formula (1), is converted to the compound represented by the general formula (2) activity
[0103]
　Homologues of above (B) is SEQ ID NO: 2 amino acid sequence full length as shown in at least 80% or more, preferably 85% or more, more preferably 90% or more, more preferably a protein having homology of 95% or more .
　Furthermore, homologues of the above (C) is within a range which does not impair the activity capable of reducing the carbonyl group stereoselectively, one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 are deleted, added or substituted and those having an amino acid sequence. Here, "one or several amino acids", in particular more than 20, preferably no more than 10, more preferably 5 or less amino acids.
　Gene encoding the enzyme, the following (D), a DNA containing a base sequence, or their homologs shown in (E) or (F).
(D) the nucleotide sequence set forth in SEQ ID NO: 1
hybridizes with (E) consisting of the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 1 DNA under stringent conditions, and a compound represented by the general formula (1) It acts on the general formula (2) a nucleotide sequence encoding a polypeptide having an activity of converting the compound represented by
one or several bases are substituted in the nucleotide sequence described in (F) SEQ ID NO: 1, deleted includes loss or addition of nucleotide sequence, and acts on the compound represented by the general formula (1), the general formula (2) nucleotide sequences encoding polypeptides having the activity of converting the compound represented by
　wherein in, the term "hybridizes the base sequence under stringent conditions" above (E), DNA used as a probe, under stringent conditions, colony hybridization, plaque hybridization, or Southern blot high It means a nucleotide sequence of the obtained DNA by the use of a hybridization method, or the like. As the stringent conditions, for example, in a colony hybridization and plaque hybridization, using a filter immobilized with DNA or fragment of the DNA derived from a colony or plaque, 0.7mol / L ~ 1.0mol / the presence of sodium chloride aqueous solution of L, after the hybridization at 65 ℃, the composition of the 0.1 ~ 2 × SSC solution (1 × SSC, 150mmol / L aqueous solution of sodium chloride, 15mmol / L aqueous solution of sodium citrate ) was used, mention may be made of the conditions for washing the filter under a condition of 65 ° C..
　Each hybridization, Molecular Cloning: A laboratory Mannual, 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. , 1989. It can be carried out according to the method described in like.
　Furthermore, homologues of the above (F) is within a range which does not impair the activity capable of reducing the carbonyl group stereoselectively, 1 or several nucleotides into the nucleotide sequence set forth in SEQ ID NO: 1 by deletion, addition or substitution and it has a base sequence. The term "one or several bases", in particular 60 or fewer, preferably 30 or less, more preferably 15 or fewer bases.
　In step (i), an enzyme or the like of the present invention is excellent in handling property, also, since it is added to the reaction system is easy, it can also be used in a state of frozen. When using enzymes like frozen present invention, the shape is not particularly limited, for example, can be prismatic, cylindrical, massive, spherical, and the like.
[0104]
　In step (i), the compound represented by the general formula (1) as a reaction substrate is generally 0.01% substrate concentration w / v ~ 20% w / v, preferably 0.1% w / v used in the range of ~ 10% w / v. Reaction substrate may be added at once at the start of the reaction. Further, when there is substrate inhibition of the enzyme reduces its impact, also from the viewpoint of improving the storage density of the product can be continuously or intermittently added that.
　In the step (i), coenzyme NAD (P) + is preferably carried out in the presence of or NAD (P) H, in this case, the coenzyme, usually, 0.001mmol / L ~ 100mmol / L , preferably preferably added to a concentration of 0.01mmol / L ~ 10mmol / L.
　In the case of adding the coenzyme, in the reaction system, NAD (P) NAD generated from H (P) + to be regenerated to NAD (P) H preferable because production efficiency improved. As playback
method, 1) microbes or cells themselves NAD (P) of the present invention + ability to generate from the NAD (P) H, i.e., NAD (P) + a method of utilizing the reducing
ability, 2) NAD (P ) + microorganisms and treated products thereof having the ability to produce NAD (P) H from, or glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate enzymes available for regeneration of NAD (P) H such dehydrogenase, etc.) (hereinafter, a method of adding the "regenerating enzyme" hereinafter) and the one or more reaction,
3) producing a microorganism or cell of the present invention Upon which, the gene of the reproduction enzyme more than one type, method, and the like to be introduced into the host organism or host cell together.
　In the method of the above 1), glucose to the reaction system, ethanol is preferably added and 2-propanol or formic acid.
　Further, in the above method 2), those microorganisms having the ability to produce the regenerating enzyme, the microorganism was acetone treatment, those freeze-dried, physically or enzymatically treated product of a microorganism such as those crushed , which was taken out enzyme Motoga fraction as a crude product or a purified product, and further, these polyacrylamide gels, may be used such as those immobilized on a carrier such as carrageenan gel, also be a commercially available enzyme good.
　In this case, the used amount of the regenerating enzyme, the carbonyl group of the present invention as compared to the enzyme of the carbonyl reduction activity which has the ability capable of stereoselectively reducing, usually 0.01 to 100 times the enzyme activity, preferably added so as to be 0.5 times to about 20 times.
　Further, the above becomes a substrate for the regenerating enzyme compounds, for example, the addition of such as ethanol or isopropanol in the case of using glucose in the case of using glucose dehydrogenase, formic acid in the case of utilizing formate dehydrogenase, alcohol dehydrogenase is also required. as the addition amount of the compound represented by the general formula (1) which is a reaction raw material, usually 0.1 equivalents to 20 equivalents, preferably added 1 equivalent to 10 equivalents.
　Further, in the method of the above 3), a method of incorporating together with DNA encoding the enzymes used in step (i) the DNA of the regenerating enzyme into the chromosome, introducing both DNA into a single expression vector, a host organism or methods of transforming cells, or both DNA and after introduction into separate expression vectors, respectively, the host organism or host cell it is possible to use a method of transforming. For a method for transforming a host organism or host cells after introducing both DNA into separate expression vectors, respectively, it is necessary to select an expression vector in consideration of the incompatibility between the both expression vectors.
　When introducing a plurality of genes into a single expression vector, it is expressed as an operon containing multiple cistrons, such as the methods and the lactose operon which connects the region involved in such expression control promoter and terminator for each gene it is also possible.
[0105]
　Step (i), culturing the compound and the enzyme represented by the general formula (1), microorganisms or cells capable of producing the enzyme, processed product of the microorganism or cell, and / or the microorganism or cell culture containing the enzyme obtained on, as well as various coenzymes necessary (the reproduction system, i.e., it is more preferable. be enabled to play a coenzyme) containing, in an aqueous medium or performed in a mixture of aqueous medium and the organic solvent. The compound represented by the general formula (1) can be produced by a method described later.
　As the aqueous medium, water or potassium phosphate buffer, sodium citrate buffer, a buffer such as Tris-HCl buffer, and the like.
　The organic solvent is represented by ethyl acetate, isopropyl acetate, butyl acetate, toluene, chloroform, n- hexane, n- heptane, dimethyl sulfoxide, methanol, ethanol, n- propanol, 2-propanol or the like, the general formula (1) what the high solubility of the compounds can be used that. Among them, as the organic solvent, because of its high solubility of the compound represented by the general formula (1), dimethyl sulfoxide, methanol, ethanol is preferred. Dimethylsulfoxide because of high further conversion rate is more preferable.
　Also, step (i) can be glycerol, ethylene glycol, propylene glycol, erythritol, inositol, sorbitol, be carried out in the presence of polyhydric alcohols xylitol. Polyhydric alcohols mentioned above may be a polymer or a derivative thereof, also possible to use a type can also be used as a mixture of two or more. When the process (i) is carried out in the presence of a polyvalent alcohol, there is a tendency that the conversion rate is improved. Among them, glycerin is considered to be able to maintain the enzymatic activity by maintaining the conformation of the enzyme, further, preferred for their easy availability. The amount of glycerin, preferably at least 40 g / L, more preferably at least 170 g / L, also less preferred 600 g / L, more preferably at most 400 g / L.
　Incidentally, later steps (ia) and / or step (ib) may also be carried out in the presence of polyhydric alcohols described above.
　Step (i) is usually 4 ℃ ~ 70 ℃, preferably at a reaction temperature of 20 ℃ ~ 60 ℃, usually pH3 ~ 11, is preferably carried out in pH4 ~ 8. The reaction time is 0.5 hour to 48 hr, preferably 0.5 to 24 hours. It is also achieved utilizing such film reactor.
　The compound represented by the general formula obtained in step (i) (2), after separation of the cells and the polypeptide such as by centrifugal separation or filtration, and adjusted to an appropriate pH, hexane, ethyl acetate, extraction with an organic solvent such as toluene, purification by column chromatography can be purified by appropriately combining such crystallization.
[0106]
　If a compound represented by the general formula (2) is purified by crystallization, the organic solvent which can be used, cyclohexane, n- hexane, n- heptane, hydrocarbon solvents such as toluene, chlorobenzene, dichlorobenzene, etc. halogen solvents, tert-butyl methyl ether, tetrahydrofuran (THF), ether solvents such as cyclopentyl methyl ether (CPME), methanol, ethanol, n- propanol, an alcohol solvent such as isopropanol, N- methyl-2-pyrrolidone, N, N - can be used dimethylformamide, N, N-dimethylacetamide, polar solvents such as dimethyl sulfoxide, the solvent is highly soluble compound represented by the general formula (2). These organic solvents may be used alone may also be used a mixed solvent of these organic solvents and water.
　When purifying a compound represented by the general formula (2) by crystallization, after which the above formula (2) compound was dissolved in a mixed solvent of an organic solvent, or an organic solvent and water, 15 ° C. / hr by cooling below the cooling rate, that of the general formula (2) crystals it is preferable to precipitate the compound represented by (this cooling the step of precipitating crystals, hereinafter referred to as "cooling step "hereinafter).
　In the cooling step, the temperature to start cooling is preferably from 15 ℃ ~ 60 ℃, more preferably from 20 ℃ ~ 55 ℃.
　The cooling rate in the cooling step is preferably 15 ° C. / hour or less, more preferably less. 9 ° C. / hour, more preferably less. 6 ° C. / hour, particularly preferably not more than 5 of 5 ° C. / hour. This is to increase the purity of the compound represented by the obtained formula (2).
　Note that in the cooling step, it is also possible to change the cooling rate in the middle. In particular, preferably 45 ℃ or less, more in preferably 40 ℃ or less of temperature range, it is preferable to cool slowly. Specifically, it is more preferable to set the cooling rate and. 9 ° C. / hour, more preferably to less. 6 ° C. / hour, and particularly preferably not more than 5 of 5 ° C. / hour.
[0107]
　Further, a compound represented by the general formula (2) even after dissolved in a solvent described above, prior to said cooling step, a step of aging (hereinafter, referred to as "ripening") it is preferable to provide a . Ripening step preferably has a low-temperature aging step of performing a high-temperature aging step, an aging at a temperature lower than said high temperature aging step. In the aging step, the order of the high-temperature aging step and low temperature aging step, is not particularly limited, preferably be provided with a high-temperature aging step after the low temperature aging step. Further, the low-temperature aging step and the high-temperature aging step, if necessary, several times, also be carried out repeatedly.
[0108]
　The low-temperature aging step in the ripening process, the general formula wherein the organic solvent a compound represented by (2), or after dissolving in a mixed solvent of an organic solvent and water, dissolved in said organic solvent such as lower than the temperature at, and is a step of performing ripening at a temperature lower than the aging temperature of the hot aging step described below.
　Aging temperature in the low temperature aging step, it is preferably at least 1 ℃ lower than the temperature at which dissolved in the organic solvent or the like, it is more preferably 5 ℃ or less, and particularly preferably 10 ° C. or higher low. Specific examples of the aging temperature, preferably from 0 ℃ ~ 59 ℃, more preferably from 5 ℃ ~ 50 ℃.
　Further, in the low-temperature aging step, it may be varied temperature on the way. If you want to change the temperature, for example, first to a relatively high temperature (for example, 35 ℃ ~ 45 ℃) aged for 5 minutes to 12 hours, then, a relatively low temperature (for example, 30 ℃ ~ 40 ℃) ~ 10 minutes it can be 5 hours aged.
　Further, the low-temperature aging step, preferably 10 minutes to 24 hours, more preferably from 20 minutes to 10 hours, carried out.
　In the low-temperature aging step, not only to maintain the temperature, if necessary, stirring or may be or seeded.
[0109]
　The high-temperature aging step in the ripening step is a step of performing aging at a temperature higher than the aging temperature of the above-mentioned low-temperature aging step.
　Aging temperature in the high-temperature aging process, high it is preferably not less than 1 ℃ than the aging temperature of the low-temperature aging process, it is more preferably 3 ℃ or more high, it is particularly preferred high 5 ℃ or more. Specific examples of the aging temperature, preferably from 20 ℃ ~ 60 ℃, more preferably from 25 ℃ ~ 55 ℃. Usually, (if you do more than once a high-temperature aging process, the temperature of the last of the high-temperature aging process) aging temperature of the high-temperature aging process consists of aging temperature of to start the cooling process. Further, even at a high temperature aging step may change the temperature on the way.
　Further, the high-temperature aging step, preferably 10 minutes to 24 hours, more preferably from 20 minutes to 10 hours, carried out.
　In the high-temperature aging step, not only to maintain the temperature, if necessary, may be stirred.
　By providing such a ripening step, the effect is obtained that the purity of the target compound filterability is improved can be improved.
　If a compound represented by the general formula (2) is purified by crystallization, the compound represented by the general formula (2) was dissolved in a mixed solvent of an organic solvent, or an organic solvent and water, wherein the aging step (the high temperature aging step, and the low-temperature aging step), by carrying out the cooling step, it is preferably purified.
　By crystallizing this way, it is possible to further improve the purity of the crystals obtained.
[0110]
　Also, step (i), as follows, may be performed in two stages of step (ia) and step (ib).
[0111]
Step (ia):
　step (ia) are the compounds of formula (1), -X 1 and -X 2 in the compound represented by the = O in which formula (1a), stereoselectively reducing the carbonyl group to act enzymes, microorganisms or cells capable of producing the enzyme, processed product of the microorganism or cell, and / or the culture medium containing the enzyme obtained by cultivating the microorganism or cell having a can activity reduction on the general formula (1), -X 1 is -X with -OH 2 compound represented by the general formula (1b) is = O, and / or -X 1 -X in the = O 2 There is a step of obtaining a compound represented by the general formula (1c) is -OH.
　Reduction of the compound represented by the general formula (1a) can be employed in the same manner as step (i).
[0112]
[Of 42]

[0113]
　In the formula, R has the same meaning as defined above.

The scope of the claims
[Claim 1]
　(I) the following general formula (1):
[Chemical formula 1]

(wherein, R is.-X-represents a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms 1 and -X 2 each independently represent -OH or = O, -X 1 and / or -X 2 is = O.)
to a compound represented by the activity capable of reducing the carbonyl group stereoselectively a step of reductase, a microorganism or cells capable of producing the enzyme, processed product of the microorganism or cell, and / or the microorganism or in the cell by the action of culture medium containing the enzyme obtained by culturing with ;
characterized by having a following general formula (2):
[formula 2]

(wherein, R is R a is as defined in the general formula (1).)
the production method of the compound represented by.
[Claim 2]
　Wherein said enzyme following (A), an enzyme comprising any of the polypeptides shown in (B) or (C), the method of producing according to claim 1.
(A) Ogataea minuta var. nonfermentans NBRC1473-derived carbonyl reductase (OCR1) (SEQ ID NO: 2) a polypeptide
having, consisting of an amino acid sequence having the amino acid sequence 80% or more homology as described in (B) SEQ ID NO: 2, the general formula (1 a compound represented by) a polypeptide having an activity of converting the compound represented by the general formula (2),
in the amino acid sequence set forth in (C) SEQ ID NO: 2, one or several amino acids are substituted, comprising a deletion or addition of amino acid sequences, and a polypeptide having an activity of converting the compound represented by the general formula (1), the compound represented by the general formula (2).
[Claim 3]
　Gene encoding the enzyme, the following (D), a DNA comprising the nucleotide sequence shown in (E) or (F), The method according to claim 1.
(D) the nucleotide sequence set forth in SEQ ID NO: 1,
is represented by a DNA which hybridizes with the DNA under stringent conditions consisting of the complementary sequence of the nucleotide sequence described in (E) SEQ ID NO: 1 and the general formula (1) that acts on the compound, nucleotide sequence encoding a polypeptide having an activity of converting the compound represented by the general formula (2),
(F) 1 or several nucleotides in the nucleotide sequence set forth in SEQ ID NO: 1 polypeptide but having an activity of converting substitution comprises a deletion or addition of nucleotide sequence, and acts on the compound represented by the general formula (1), the compound represented by the general formula (2) nucleotide sequence encoding.
[Claim 4]
　The step of (i), in the presence of a polyhydric alcohol, The process according to any one of claims 1-3.
[Claim 5]
　(Ii) the following formula (3):
[Formula 3]

a compound represented by the following general formula (4):
[Formula 4]

(wherein, R 1 represents a linear or branched alkyl having 1 to 8 carbon atoms . represents a group)
a compound represented by the presence of a base, the process of condensing;
characterized by having a following general formula (1):
[chemical formula 5]

(wherein, R represents a C1- represents an 8 primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms.-X- 1 and -X 2 are each independently a -OH or = O, -X 1 and / or -X 2 is = O.)
the production method of the compound represented by.
[6.]
　(Iia) the following formula (3):
[Chemical Formula 6]

a compound represented by the following general formula (4a):
[Chemical Formula 7]

(wherein, R 2 represents a branched alkyl group having 3 to 8 carbon atoms, the above-mentioned R are different groups a compound represented by), the presence of a base, step condensation; and.
(iib) the step (the following general formula obtained in iia) (5 of 5):
[formula 8]

(wherein, R 2 is R in the general formula (4a) 2 is synonymous with.)
and a compound represented by, R-OH (wherein, R 1 alkyl group or a C 1 to 8 carbon atoms reacting the alcohol represented by the representative) a secondary alkyl group having 3 to 6;.
and having a method according to claim 5.
[7.]
　(Iia) the following formula (3):
[Formula 9]

a compound represented by the following general formula (4a):
[Formula 10]

(In the formula, R 2 represents a branched alkyl group having 3 to 8 carbon atoms, the and R are different groups a compound represented by), the presence of a base, step condensation;.
(iib) the step (the following general formula obtained by iia) (5 of 5):
[formula 11]

( in the formula, R 2 is R in the general formula (4a) 2 is synonymous with.)
and a compound represented by, R-OH (wherein, R 1 alkyl group or a carbon number of 1 to 8 carbon atoms . to 3-6 secondary alkyl group represents a step reacting the alcohol represented by); and
(ia) the step (the following general formula obtained in iib) (1a) compound represented by:
[formula 12 ]

(wherein, R represents. a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms having 1 to 8 carbon atoms)
, the enzyme having an ability capable of reducing the carbonyl group stereoselectively, the microbial or cell, processed product of the microorganism or cell capable of producing the enzyme, and / or the microorganism or the cells by the action of culture medium containing the enzyme obtained by culturing reduced by the following general formula ( 1b) and / or (1c):
[formula 13]

. (wherein, R formula (1a) is the same as R in)
[formula 14]

(wherein, R formula (1a) of . R and is synonymous)
to obtain a compound represented by;
characterized by having a process according to claim 5.
[8.]
　Wherein said enzyme following (A), an enzyme comprising any of the polypeptides shown in (B) or (C), the method of producing according to claim 7.
(A) Ogataea minuta var. nonfermentans NBRC1473-derived carbonyl reductase (OCR1) (SEQ ID NO: 2) a polypeptide
having, consisting of an amino acid sequence having the amino acid sequence 80% or more homology as described in (B) SEQ ID NO: 2, the general formula (1 a compound represented by) a polypeptide having an activity of converting the compound represented by the general formula (2),
in the amino acid sequence set forth in (C) SEQ ID NO: 2, one or several amino acids are substituted, comprising a deletion or addition of amino acid sequences, and a polypeptide having an activity of converting the compound represented by the general formula (1), the compound represented by the general formula (2).
[9.]
　Gene encoding the enzyme, the following (D), a DNA comprising the nucleotide sequence shown in (E) or (F), The method according to claim 7.
(D) the nucleotide sequence set forth in SEQ ID NO: 1,
is represented by a DNA which hybridizes with the DNA under stringent conditions consisting of the complementary sequence of the nucleotide sequence described in (E) SEQ ID NO: 1 and the general formula (1) that acts on the compound, nucleotide sequence encoding a polypeptide having an activity of converting the compound represented by the general formula (2),
(F) 1 or several nucleotides in the nucleotide sequence set forth in SEQ ID NO: 1 polypeptide but having an activity of converting substitution comprises a deletion or addition of nucleotide sequence, and acts on the compound represented by the general formula (1), the compound represented by the general formula (2) nucleotide sequence encoding.
[10.]
　The step of (ia), carried out in the presence of a polyhydric alcohol, The process according to any one of claims 7-9.
[11.]
　Compound represented by the following general formula (1a).
[Formula 15]

(In the formula, R represents a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms.)
[12.]
　Compound represented by the following general formula (1b) or (1c).
[Formula 16]

(In the formula, R represents a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms.)
[Formula 17]

(wherein, R 1 to 8 carbon atoms It represents a primary alkyl group or a secondary alkyl group having 3 to 6 carbon atoms.)
[13.]
　Following formula:
[Formula 18]

A crystal of a compound represented by, 2θ = 8.7 °, 16.3 ° , 19.7 °, 21.2 °, 21.3 ° (± 0.2 ° ) to the crystal having a powder X-ray diffraction pattern showing characteristic peaks.
[14.]
　(Iiia) the general formula obtained by the production method according to claim 1 (2):
[Formula 19]

(wherein, R represents 2 of primary alkyl group or a C 3-6 1 to 8 carbon atoms . representing the class alkyl group)
after hydrolysis with a base a compound represented by the step of reacting a calcium compound;
formula characterized by having a (6):
[formula 20]

of rosuvastatin calcium represented by Production method.
[15.]
　In the step (iiia), the hydrolysis and a polar solvent, an ether solvent, a hydrocarbon solvent, and characterized by performing in the presence of a mixed solvent of at least one solvent selected from the group consisting of halogen solvent the manufacturing method of rosuvastatin calcium according to claim 14.
[16.]
　In step (iiia), pH and characterized by a pH 5 ~ 10, the manufacturing method of rosuvastatin calcium according to claim 14 or 15 when starting the reaction of the calcium compound.
[17.]
　(Iiib) the general formula obtained by the production method according to claim 1 (2):
[Formula 21]

(wherein, R represents 2 of primary alkyl group or a C 3-6 1 to 8 carbon atoms .) representing the class alkyl group
after the compound represented by hydrolyzing with a base, is treated with an acid, the resulting formula (8):
[formula 22]

a compound represented by is reacted with an amine compound, the resulting following general formula (9):
[formula 23]

(wherein, R 3 and R 4 each independently represents an alkyl group having 1 to 8 carbon atoms.)
with a base a compound represented by after salt exchange, reacting with the calcium compound;
formula characterized by having a (6):
[formula 24]

the manufacturing method of rosuvastatin calcium represented by.
[18.]
　(Iiic) wherein the general formula obtained by the production method according to claim 1 (2):
[Formula 25]

(wherein, R represents 2 of primary alkyl group or a C 3-6 1 to 8 carbon atoms . representing the class alkyl group)
after hydrolysis with a base a compound represented by the intramolecular dehydration condensation in the presence or absence of an acid catalyst, the resulting formula (10):
[formula 26]

with reacting a compound of calcium compound represented;
following formula and having a (6):
[formula 27]

the manufacturing method of rosuvastatin calcium represented by.
[19.]
　2θ = 19.8 °, characterized by having a powder X-ray diffraction pattern showing characteristic peaks at 22.9 ° (± 0.2 °), (E) -7- [4- (4- fluorophenyl) -6-isopropyl-2- [methyl (methylsulfonyl) amino] pyrimidin-5-yl] - (3R, 5S) -3,5- hydroxy-6 crystals heptenoic acid n- propylamine salt.
[20.]
　2θ = 6.6 °, characterized by having a powder X-ray diffraction pattern showing characteristic peaks at 17.0 ° (± 0.2 °), (E) -7- [4- (4- fluorophenyl) -6-isopropyl-2- [methyl (methylsulfonyl) amino] pyrimidin-5-yl] - (3R, 5S) -3,5- hydroxy-6-heptenoic acid crystals dimethylamine salt.
[21.]
　Following formula (11):
[Formula 28]

characterized by containing 1ppm or more 1500ppm or less of a compound represented by, rosuvastatin calcium.
[22.]
　Following general formula (2):
[Formula 29]

(wherein, R represents a secondary alkyl group of primary alkyl group or a C 3-6 1 to 8 carbon atoms.)
The organic solvent a compound represented by or it was dissolved in a mixed solvent of an organic solvent and water, by cooling below a cooling rate of 15 ° C. / hour, and characterized in that to precipitate crystals of the compound represented by the formula (2) the purification method of the compound represented by the general formula (2).
[23.]
　(B) under note general formula (12):
[for 30]

(wherein, M ha, manufactured Hikaru ka ri metal elements, manufactured Hikaru ka ri earth metal element, and wa Hydrogenでthou ru.)
でtable Connecticut made me cry ru compound wo, under-recording (13):
[of 31]

でcompound table Connecticut made me cry ru ni su ru CONVERSION engineering
wo su ru koとwo have special Zhiとsu ru, ro ba su su ta chi nn grades HikaruのシウRousseau manufacturing method.
[24.]
　Prior to the step (Bs), (Aa) following general formula (14):
[Formula 32]

a (wherein, R 1 alkyl group or a secondary alkyl group having 3 to 6 carbon atoms 1 to 8 carbon atoms . representing)
a compound represented by the following general formula (15):
[formula 33]

. (wherein, R is as defined above)
a mixture comprising a compound represented by the presence of a base under is hydrolyzed, the following general formula (16):
[formula 34]

(. wherein, M is an alkali metal element, an alkaline earth metal element, or hydrogen)
and a compound represented by the general step of converting the mixture containing the compound represented by the formula (12)
and having a method for producing rosuvastatin calcium according to claim 23.
[25.]
　After the step (B), (C) and having a step of removing the compound represented by the formula (13), the manufacturing method of rosuvastatin calcium according to claim 23 or claim 24.
[26.]
　After the step (C), (D) and the step compound obtained by (C), characterized by having a step of reacting a calcium compound, process for producing rosuvastatin calcium according to claim 25.
[27.]
　Under remember general formula (12):
[of 35]

(wherein, M ha, manufactured Hikaru ka ri metal elements, manufactured Hikaru ka ri earth metal element, and wa Hydrogenでthou ru.)
でtable Connecticut made me cry ru compound wo containingむro su ba su ta chi nn grades HikaruシウRousseauのpurification methodでthou ~ te,
(B) of the formula (12)でtable Connecticut made me cry ru compound wo, under-recording (13):
[of 36]

でtable Connecticut made me cry ru compound ni CONVERSION su ru engineering
wo have su ru koとwo special Zhiとsu ru, ro su ba su ta chi nn grades HikaruシウRousseauのpurification method.
[28.]
　Prior to the step (Bs), (Ab), characterized in that the rosuvastatin calcium containing compound represented by the general formula (12) has a step of dissolving in the solvent, purification of rosuvastatin calcium according to claim 27 Method.
[29.]
　After the step (B), (C) and having a step of removing the compound represented by the formula (13), the purification method of rosuvastatin calcium according to claim 27 or claim 28.
[30.]
　After the step (C), (D) and the compound obtained in Step (C), characterized by having a step of reacting a calcium compound, the purification method of rosuvastatin calcium according to claim 29.
[31.]
　Following formula (13):
[Formula 37]

characterized by containing 1ppm or more 1000ppm or less the compound represented by, rosuvastatin calcium