JPH05230066A - D-biotin intermediate and its production - Google Patents

Abstract

PURPOSE:To obtain a new compound useful as an intermediate for medicines and additives for animals. CONSTITUTION:The objective compound of formula I [R (CO) is acyl], e.g. (+ or -)-(3aalpha,4alpha,6aalpha)-tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thien o[3,4-d]imidazol-2 (3H) -one. This compound of formula I is obtained by acylating a compound of formula II.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel intermediate for the synthesis of D-biotin and a method for producing the same. D-Biotin, also called Vitamin H, is a water-soluble vitamin required by higher animals and many microorganisms. D-
Biotin is a known compound represented by the formula below.

[0002]

[Chemical 20]

[0003]

As a method for synthesizing D-biotin, for example, a method using a sugar as a starting material [Tetrahedron Letters 2765 (1975)] or a method using L-cysteine as a starting material [Journal of American] Chemical Society (J.Am.C
hem.Soc.) 97 , 5936 (1975)] is known.
In these methods, an optically active substance is used as a starting material, and optically active D-biotin is obtained by a stereoselective reaction. Further, as another method for producing D-biotin, for example, the reaction formula:

[0004]

[Chemical 21]

As shown in (5), a method for producing D-biotin using an optically active lactone obtained by selectively reducing only the carboxyl group of a half ester (JP-A-59-59).
No. 84888), reaction formula:

[0006]

[Chemical formula 22]

As shown in (1), there is a method for producing D-biotin using an optically active lactone obtained by esterification, reduction and hydrolysis from an optically active amidecarboxylic acid (Japanese Patent Publication No. 60-3387). Further, JP-A-57-198098 discloses a method for producing an optically active monoester monocarboxylic acid by asymmetrically hydrolyzing a corresponding diester using an ester hydrolase. This optically active monoester monocarboxylic acid is converted into D- by lactonization with lithium borohydride or the like.
Cis-1,3-used as an intermediate for biotin production
It can be converted to dibenzylhexahydro-1H-furo [3,4d] imidazol-2,4-dione. In addition, tetrahedron (Tetrahedron), 46 , 7667 (1
990),

[0008]

[Chemical formula 23]

A method for producing D-biotin by the reaction of and

[0010]

[Chemical formula 24]

Is reduced with triethylsilane and boron trifluoride,

[0012]

[Chemical 25]

A method for producing the above-mentioned halogenated product by subjecting the compound to halogenation is described.

[0014]

All of these methods are not suitable as an industrial production method of D-biotin because of the large number of reaction steps, complicated reaction operation, and low total yield.

[0015]

In view of the above circumstances, the inventors of the present invention have diligently studied a method for producing D-biotin, and have conducted repeated studies on an advantageous method for producing an optically active intermediate. As a result, an enzyme is used as a catalyst. We have found that the kinetic optical resolution method is extremely effective for the preparation of D-biotin intermediate.
As a result of further research based on these findings, a new D-biotin intermediate which is extremely effective for the kinetic resolution method was found, and this was subjected to kinetic resolution in the presence of an enzyme, followed by a series of steps. By carrying out the reaction, it was possible to obtain D-biotin with high optical yield and high optical purity. When using this novel D-biotin intermediate, kinetic optical resolution can be effectively performed for both hydrolysis of this novel intermediate and production of a novel intermediate by acylation by selecting an appropriate enzyme. . From this point of view, recently, an example in which an enzyme such as lipase catalyzes an acylation reaction in an organic solvent has been reported one after another [Journal of American Chemical Society (J. Am. Chem. Soc.),
113, 3166 (1991), Tetrahedron Letters
(Tetrahedron Letters), 33, 3231 (1992)
etc]. However, there are not many enzymes that exhibit activity even in organic solvents, and their application range is limited. Nevertheless, the above acylation reaction can be carried out efficiently. By the way, the biggest drawback of optical resolution is that only half of the raw material can be used. The present inventors have conducted extensive studies to overcome this problem, and the D-biotin intermediate, which is an enantiomer removed by optical resolution, is reused as a raw material for a new D-biotin intermediate by deacyloxy reaction. I found a way to do it. As a method similar to this, the above hydroxyl group is triethylsilane,
A method of reduction with boron trifluoride is known [Tetrahedron, 46, 7667 (199
0)]. However, these reagents are expensive, not easily available, have strong reactivity and are difficult to handle, and since the reaction has to be carried out in an anhydrous state and at a low temperature, it is not suitable as an industrial production method.

The present inventors have also found that certain known intermediates for producing D-biotin can be efficiently converted into other known intermediates useful for producing D-biotin by certain oxidation reactions. .. As the same oxidation method,
Swan oxidation with dimethyl sulfoxide (DMSO) -trifluoroacetic anhydride is known [Tetrahedron
(Tetrahedron), 46, 7667 (1990)]. However, trifluoroacetic anhydride is expensive and
Since it is necessary to carry out the reaction at a temperature as low as 60 ° C, it is not suitable as an industrial production method. On the other hand, the DMSO-activator is known as a cheaper and easier-to-handle oxidizing agent.
This oxidation method known per se as Albright Goldman oxidation has the great advantage that the reaction can be carried out at room temperature, but it is effective only for alcohols with large steric hindrance, and in other cases, methylthio It is known that a methyl ether body etc. is formed as a by-product [Journal of
American Chemical Society (J. Am. Chem.
Soc.), 87, 4214 (1965), Experimental Chemistry Course No. 4
Edition, 23, 318]. When this Albrite-Goldman oxidation was applied to the oxidation of the 4-hydroxyl group of the following compound [II '], acetylation was given priority, and the target compound was produced only very slightly. However, surprisingly, when this oxidation is applied to the above-mentioned intermediate for producing D-biotin under conditions that deviate from the normally used reaction conditions, the production of by-products is suppressed to a minimum and the desired oxidation is achieved. The body was obtained in good yield.

The present invention provides a novel compound useful as an intermediate raw material for producing D-biotin, an industrially advantageous production method thereof, and an industrially advantageous production method of a known intermediate for producing D-biotin. is there. That is, the present invention provides (1) the general formula

[0018]

[Chemical formula 26]

The compound represented by the formula

[0020]

[Chemical 27]

(Including each 4-α optically active isomer of the compound represented by the formula (1)), Acetobacter, Bacillus, Brevibacterium, Pseudomonas, Streptomyces, Amplariella, Candida or Trichosporon having the ability to deacylate more rapidly than the other, or a treated product thereof. [II '] characterized by bringing into contact with
[II '']

[0022]

[Chemical 28]

A method for producing an optically active substance represented by: (3) Formula [II]

[0024]

[Chemical 29]

A compound represented by the formula (wherein the compound includes its racemate and optically active compounds represented by the formulas [II '] and [II "])
A method for producing a compound represented by [I], (4) Formula [III]

[0026]

[Chemical 30]

A method for producing a compound represented by the general formula [I], which comprises reacting the compound represented by the formula with an organic carboxylic acid anhydride; (5) a compound represented by the general formula [I ″] [VI], which is characterized in that

[0028]

[Chemical 31]

(6) A mixture of the compounds represented by the formulas [II '] and [II "], in the presence of an acyl group donor, one of the hydroxyl groups is introduced from the other. A method for producing a compound represented by the general formula [I ′] or [I ″], which comprises contacting a bacterial esterase having the ability to rapidly acylate, (7) in the formula [II ′] Formula characterized by oxidizing the compound represented by dimethylsulfoxide and an activator
[VII]

[0030]

[Chemical 32]

A method for producing the compound represented by the formula (8), and (8) adding one of the hydroxyl groups to the mixture of the compounds represented by the formulas [II '] and [II "] in the presence of an acyl group donor. On the other hand, a compound of the formula [I '] obtained after contacting with a bacterial esterase having the ability to acylate more rapidly and removing the compound represented by the general formula [I'] or [I "]
The present invention provides a method for producing a compound represented by the formula [VII], which comprises subjecting the compound represented by I '] to an oxidation reaction. In the general formulas [I], [I ′] and [I ″],

[0032]

[Chemical 33]

The acyl group represented by is an acyl group derived from an organic carboxylic acid, for example, a formyl group, an alkylcarbonyl group (alkanoyl group), preferably C _1-6.
Alkyl-carbonyl group (eg, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, etc.), arylcarbonyl group (aroyl group), preferably C _6-14 aryl-carbonyl group (eg, benzoyl, 1-) Or 2-naphthoyl etc.), an aralkyl-carbonyl group, preferably a C _7-19 aralkyl-carbonyl group (eg, benzylcarbonyl, 2
-Phenethylcarbonyl, 1- or 2-naphthylmethylcarbonyl, benzhydrylcarbonyl, etc.) are used, and these are nitro, halogen (eg, fluorine, chlorine, bromine, etc.), hydroxyl, oxo, carbamoyl,
C _1-4 alkyl (eg, methyl, ethyl, propyl, isopropyl, butyl, etc.), C _1-4 alkoxy (eg, methoxy,
Ethoxy, propoxy, butoxy, etc.), carboxyl optionally esterified, C _{1-4 alkoxyimino} optionally substituted with carboxyl (eg, methoxyimino, ethoxyimino, carboxymethoxyimino, 1-
(Carboxy-1-methylethoxyimino, etc.) and the like.

[0034]

[Chemical 34]

Is preferably a C _1-6 alkyl-carbonyl group _optionally substituted with a formyl group or a carboxyl group (eg, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, carboxyacetyl, 3 -Carboxypropionyl, etc.). More preferably, it is a C _1-6 alkyl-carbonyl group (eg, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, etc.). The compound [I] has three asymmetric carbon atoms, and there are eight kinds of optical isomers [Ia] to [Ih] shown below (numbers are added to the ring-constituting atoms of [Ia] and [Ib]). ).

[0036]

[Chemical 35]

When the compounds [I], [I '] and [I "] have a carboxyl group in the molecule, they may form a salt with a base. The salt of the compound [I] is an inorganic base. The inorganic bases which can form these salts include, for example, alkali metals (eg, sodium, potassium, etc.), alkaline earth metals (eg, calcium,
Examples of organic bases include trimethylamine, triethylamine, pyridine, picoline, and the like.
N, N-dibenzylethylenediamine, ethanolamine, diethanolamine, trishydroxymethylaminomethane, dicyclohexylamine, morphine, cinchonidine, cinchonine, quinine and the like are used. Compound [I] can be obtained by a method known per se, for example, Journal of Chemical Education (J. Chem. Edu.) 57 ,
220 (1980), Berihite (Berichte) 43, 1401
It can be produced according to the method described in (1910). Further, it can be manufactured by the following manufacturing method 1 or 2. Production Method 1 Compound [I] can be produced by acylating compound [II]. The acylating agent used in this reaction has a general formula

[0038]

[Chemical 36]

A carboxylic acid [IV] represented by the formula [wherein R has the same meaning as described above] or a reactive derivative at the carboxyl group thereof is used. As the reactive derivative at the carboxyl group, for example, an acid halide, an acid anhydride, an active amide, an active ester, an active thioester or the like which can be produced according to a conventional method is used. A concrete description of such a reactive derivative is as follows. 1) Acid halide: For example, acid chloride, acid bromide, etc. are used. 2) Acid anhydrides: eg symmetrical anhydrides, ie (RCO) ₂ O,
Mono C _1-6 alkyl carbonate mixed anhydride or the like is used. 3) Active amides: eg pyrazole, imidazole, 4-
Amides with substituted imidazole, dimethylpyrazole, benzotriazole and the like are used.

4) Active ester: for example, methoxymethyl ester, benzotriazole ester, 4-nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenyl ester, pentachlorophenyl ester and the like, as well as 1-hydroxy-1H-. 2-
Esters with pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide and the like are used. 5) Active thioesters such as 2-pyridylthiol, 2
-Thioesters with heterocyclic thiols such as benzothiazolylthiol, etc. are used. As a method of acylation, for example, a method of acylating the starting compound [II] with a carboxylic acid [IV] in the presence of carbodiimides is used. As the carbodiimides that can be used, any carbodiimide bond may be used as long as it has a carbodiimide bond (-N = C = N-) and can be converted to a urea bond (-NH-CO-NH-) during the acylation reaction, For example, one represented by the following formula [V] is given. R ₁ -N = C = N-R ₂ [V] [R ₁ and R ₂ are each an organic residue capable of converting a carbodiimide bond into a urea bond during the acylation reaction]

Examples of the organic residue represented by R ₁ and R ₂ include di-C _1-6 alkyl, a C _3-7 cycloalkyl group which may have an amino group, and di-C _1-6 alkyl. amino group or a C _1-6 alkyl group which may have a morpholino group or a C _1-6 alkyl group may be selected appropriately from phenyl group which may have,. As the carbodiimides, dicyclohexylcarbodiimide is practically desirable, and other examples include diphenylcarbodiimide and di-o.
-Tolyl carbodiimide, di-p-tolyl carbodiimide, di-tertiary butyl carbodiimide, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide, 1
-Cyclohexyl-3- (4-diethylaminocyclohexyl) carbodiimide, 1-ethyl-3- (2-diethylaminopropyl) carbodiimide and 1-ethyl-
3- (3-Dimethylaminopropyl) carbodiimide or the like is used.

The carboxylic acid [IV] is the starting compound [II] 1
For example, about 1 mol or more may be used, and about 1 mol may be used.
It is preferably about 30 to 30 mol. Carbodiimides [V]
Is, for example, about 1 to 7 per 1 mol of the raw material compound [II].
00 moles may be used, with about 1 to 50 moles often being preferred. Most preferably about 1 to 5 moles.

This reaction is carried out in a solvent which does not inhibit the reaction or in the absence of solvent. Examples of the solvent that does not inhibit the reaction include ketones such as acetone, ethers such as diethyl ether, tetrahydrofuran and dioxane, nitriles such as acetonitrile, hydrocarbons such as benzene, toluene and xylene, dichloromethane, chloroform and 1,2. -Halogenated hydrocarbons such as dichloroethane, esters such as ethyl acetate, amides such as dimethylformamide and dimethylacetamide, such as triethylamine, tributylamine, N-methylmorpholine, N-methylpiperidine, N, N-dimethylaniline and the like The tertiary amines of, for example, pyridines such as pyridine, picoline, lutidine and collidine are used. These may be used alone or as a mixture of two or more kinds at an appropriate ratio.

The present acylation reaction proceeds more advantageously by using a catalyst capable of promoting the acylation of the starting compound [II]. As such a catalyst, for example, a base catalyst or an acid catalyst is used. Examples of the base catalyst include tertiary amines [eg, aliphatic tertiary amines such as triethylamine, pyridine, α-, β- or γ-picoline, 2,6
-Lutidine, 4-dimethylaminopyridine, 4- (1-
Aromatic tertiary amines such as pyrrolidinyl) pyridine, dimethylaniline, diethylaniline], alkali metal halides (eg, potassium fluoride, anhydrous lithium iodide, etc.), organic acid salts (eg, sodium acetate), etc. are used. . Examples of acid catalysts include Lewis acids [eg, anhydrous zinc chloride, anhydrous aluminum chloride (AlCl ₃ ), titanium tetrachloride]
(TiCl ₄ ), tin tetrachloride (SnCl ₄ ), antimony pentachloride, cobalt chloride, cupric chloride, boron trifluoride etherate, etc.], strong inorganic acid (eg sulfuric acid, perchloric acid, hydrogen chloride, hydrogen bromide) Etc.), strong organic acids (eg, benzenesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, trichloroacetic acid, etc.), acidic ion exchange resins (eg, polystyrenesulfonic acid), etc. are used. Among the above catalysts, pyridine, 4-dimethylaminopyridine, sulfuric acid and the like are preferable.

The amount of the catalyst used may be a catalytic amount capable of promoting the acylation of the starting compound [II] with the carboxylic acid [IV], and usually about 0.001 to 10 mol per 1 mol of the compound [IV], It is preferably about 0.001 to 1 mol. The use of such a catalyst often significantly improves the yield of compound [I]. Further, the amount of the carboxylic acid [IV] used can be saved, and for example, it can often be reduced to about 1 to 10 mol per 1 mol of the raw material compound [II]. The reaction temperature is not particularly limited, but is usually about -30 to 100 ° C, preferably 10 to 50 ° C. The reaction time is about several minutes to several tens hours (for example, 5 minutes to 30 hours). The solvent, catalyst and molar ratio in the acylation means using the reactive derivative at the carboxyl group of the carboxylic acid [II] are the same as those in the case of the acylation performed in the presence of the carbodiimides [V]. The reaction temperature is usually about −40 to 100 ° C., more preferably about −20 to 40 ° C., and may be further heated to increase the reaction rate. The reaction time is several minutes to several tens of hours.

Production Method 2 Compound [I] can be produced by reacting compound [III] with an organic carboxylic acid anhydride. The organic carboxylic acid anhydride used in this reaction includes C _1-6
Examples thereof include alkylcarboxylic acid anhydrides such as acetic anhydride, propionic anhydride, butanoic anhydride. This reaction is carried out in a solvent that does not inhibit the reaction or without a solvent. Examples of the solvent that does not inhibit the reaction include ketones such as acetone, diethyl ether, tetrahydrofuran, ethers such as dioxane, nitriles such as acetonitrile, hydrocarbons such as benzene, toluene and xylene, dichloromethane, chloroform, and 1, Halogenated hydrocarbons such as 2-dichloroethane, esters such as ethyl acetate, amides such as dimethylformamide and dimethylacetamide are used. These may be used alone or as a mixture of two or more kinds at an appropriate ratio.

The organic carboxylic acid anhydride is a starting compound [I
II] may be used in an amount of about 1 mol or more, preferably about 1 to 30 mol, per 1 mol. More preferably, it is about 1 to 2 mol. This reaction proceeds more advantageously by using a catalyst that can accelerate the reaction. As such a catalyst, for example, an acid catalyst is used. Examples of the acid catalyst include Lewis acids [eg, anhydrous zinc chloride, anhydrous aluminum chloride (AlCl ₃ ), titanium tetrachloride (TiCl ₄ ), tin tetrachloride (SnCl ₄ ), antimony pentachloride, cobalt chloride,
Cupric chloride, boron trifluoride etherate, etc.], strong inorganic acid (eg, sulfuric acid, phosphoric acid, perchloric acid, hydrogen chloride, hydrogen bromide, etc.), strong organic acid (eg, benzenesulfonic acid, p-toluenesulfonic acid) , Trifluoroacetic acid, trifluoromethanesulfonic acid, trichloroacetic acid, etc.), acidic ion exchange resin
(Eg, polystyrene sulfonic acid, etc.) is used.

The amount of the catalyst used is usually about 0.001 to 10 mol, preferably about 0.01 to 2 mol, per 1 mol of the compound [III]. The reaction temperature is about -20 to 100 ° C, preferably about 10 to 60 ° C. The reaction time is several minutes to several tens hours. Manufacturing method 1 above
Alternatively, the reaction product obtained by the production method 2 can be prepared by known means, for example, solvent extraction, liquid conversion, phase transfer, salting out, crystallization,
It can be isolated and purified by recrystallization, chromatography and the like. Compound [II '] or [II "] is a compound
Acetobacter genus, Bacillus genus, Brevibacterium genus, Pseudomonas genus, Streptomyces sp. Genus,
It is produced by contacting a culture of a microorganism belonging to the genus Candida or the genus Trichosporon or a treated product thereof.

The microorganism used in the present method is a compound
Any microorganism can be used as long as it has the ability to deacylate one of [I '] and compound [I "] more quickly than the other, and bacteria such as bacteria and fungi are used. Genus bacterium, Bacillus
Genus spp., Brevibacterium spp., Pseudomonas spp., Streptomyces
(Streptomyces) genus, Ampullariella
a) Genus bacteria are used. Acetobacter lancens is a genus of Acetobacter.
Bacillus cereus IFO 3003, Bacillus megaterium IFO as Bacillus bacteria such as IFO 3298
13498, Bacillus megaterium IFO 121
Brevibacterium iodinum I as a genus of Brevibacterium such as No. 08.
Pseudomonas aeruginosa is a Pseudomonas species such as FO 3558.
IFO 3445, Pseudomonas aeruginosa IF
O3447, Pseudomonas aeruginosa IFO 3
448 and the like are preferable.

Examples of the genus Streptomyces include Streptomyces roche bar borbilis (Streptomyc
es rochei var.volubilis) IFO 12507 and the like are Ampullariella digitata IFO 1251 as a bacterium belonging to the genus Ampliariella.
2 etc. are used respectively. As the fungus, a genus Candida, a genus Trichosporon and the like are used. Candida guilliermondii
Trichosporon fermentans (Trichosporon fermentans) such as ATCC 14242
fermentans) IFO 1199 and the like are preferable. Strains with the above IFO number are from the Fermentation Research Institute, and strains with the ATCC number are from American Type Culture Collection (American Type Cu).
lture Collection). The above microorganism may be used as it is, but a mutant strain having increased activity or specificity of deacylation ability may be used.

The present method is carried out by bringing the starting material into contact with a culture of the above-mentioned microorganism or a treated product thereof. Microorganisms include glucose, choux cloth, dextrin, soluble starch as a carbon source, meat extract as a nitrogen source, peptone, yeast extract, corn steep liquor, amino acids, ammonium sulfate, ammonium nitrate, ammonium chloride and other organic and inorganic nitrogen-containing substances, It is cultured in a medium containing inorganic substances such as magnesium chloride and sodium chloride. The culture is performed by static culture or aeration-agitation culture. The temperature is about 20 to 45 ° C, more preferably about 28 to 37 ° C.
Is. The pH is about 6-9, more preferably about 6.8.
It is 7.8. The culture time is about 10 to 96 hours, more preferably about 16 to 72 hours. The "culture" used in the present invention is a culture solution obtained by culturing the above-mentioned microorganism. The term "treated product" means bacterial cells or culture supernatant obtained by filtration or centrifugation of the culture, ultrasonic treatment of bacterial cells,
Ammonium sulphate fractionation, ion exchange chromatography, adsorption from cell lysate or cell extract, culture supernatant or cell extract obtained by French press treatment, alumina grinding, lytic enzyme treatment, surfactant, organic solvent treatment, etc. Examples include purified deacylase preparations obtained by chromatography, affinity chromatography, and the like.

In this reaction, the concentration of the starting compound in the reaction solution is about 0.1-100 mg / ml, preferably about 1-10 m.
g / ml. The amount of the culture of the microorganism to be added or the treated product thereof is appropriately 1 to 50 mg per 1 ml of the reaction solution, with the bacterial cells or a corresponding amount of the treated product being the weight of the wet bacterial cells. The reaction temperature is about 15 to 80 ° C, more preferably about 24 to 42 ° C. The pH is about 4-11, more preferably about 6-9. The reaction time is about 10 minutes to 72 hours, more preferably about 1 to 24 hours. If desired, an organic solvent reaction accelerator, an enzyme stabilizer, etc. may be added to the reaction solution. The reaction may be performed under any conditions of standing, shaking, and stirring. If necessary, a deacylation enzyme may be immobilized on a suitable carrier and reacted in a bioreactor.

In the present invention, the compound [I ′] or [I ″] is a mixture of the compounds [II ′] and [II ″] in which either one of the hydroxyl groups is more quickly acylated than the other. It is an esterase derived from a bacterium having the ability to be converted, and can be produced by specifically acylating in the presence of an acyl group donor. Examples of the esterase-producing bacteria include those belonging to the genus Pseudomonas, the genus Streptomyces, the genus Bacillus, and the genus Acetobacter. Specifically, for example, Pseudomonas
PS-21 (FERM-P 7026, JP-B-63-3
594), Bacillus megaterium (Bacillus
megaterium) IFO13498, Pseudomonas aeruginosa IFO3447,
Pseudomonas aerugin
osa) IFO3448, Acetobacter lanceense (Ac
etobacter rancens) IFO 3298, Streptomyces roc
hei var. volubilis) FERM P-6155 and the like. The strains with the above IFO numbers can be obtained from the Fermentation Research Institute.

The esterase can be prepared by a conventional method. To prepare esterase from bacteria, the bacteria are cultivated by an ordinary method, and the obtained culture is centrifuged to obtain a culture supernatant and cells. The cells are disrupted by ultrasonic treatment, French press, alumina crushing, enzyme treatment of cells, etc., and centrifuged to obtain a cell extract. From the above-mentioned culture supernatant and cell extract, esterase precision preparations are obtained by organic solvent precipitation, ammonium sulfate fractionation, ion exchange chromatography, adsorption chromatography, gel filtration, affinity chromatography and the like. Esterase used in this reaction is an unpurified sample (for example, the above-mentioned culture supernatant or cell extract),
Either a partially purified preparation or a single purified preparation may be used.
As the esterase, the culture medium may be used as it is, or cells or culture supernatant prepared from the culture medium may be used.

Examples of the esterase used in the present invention include lipoprotein lipase (LPL) derived from a bacterium of the genus Pseudomonas described in JP-B-63-3594. The above esterase may be used as it is for the acylation reaction, or may be immobilized on a suitable carrier. Examples of the carrier include polysaccharide derivatives such as cellulose, amino acid copolymers, synthetic polymers such as maleic anhydride derivatives and styrene resins, activated carbon, porous glass, diatomaceous earth, alumina, silica gel and other inorganic substances. The acyl group donor in the present invention is
Any compound may be used as long as it has the ability to specifically acylate one of the compounds [II ′] and [II ″]. The acyl group is represented by the formulas [I], [I ′] and [I ″]
In

[0056]

[Chemical 37]

The same as those described above can be mentioned. Examples of the acyl group donor include the same ones as those described above as the acylating agent. When the acyl group is an acetyl group, examples of the acyl group donor include acetic anhydride, ethyl acetate, vinyl acetate, phenyl acetate, acetic acid 2,
2,2-trifluoroethyl or the like is used. When a culture solution or cells are used as the esterase, the acyl group donor may not be used.

Although the acylation reaction in the present invention proceeds in water, it is preferably in an organic solvent. As the organic solvent, a solvent that does not inhibit the acylation reaction is used. Examples of the organic solvent include ketones such as acetone and methyl ethyl ketone, ethers such as diethyl ether and tetrahydrofuran, nitriles such as acetonitrile, hydrocarbons such as benzene and toluene, esters such as ethyl acetate, and amides such as dimethylformamide. And halogenated hydrocarbons such as dichloromethane and chloroform. These may be used alone or in combination of two or more. In this reaction, the compound [I
The concentration of I ′] and [II ″] is about 0.1-100 mg / ml,
It is preferably about 1-10 mg / ml. Reaction temperature is about 15
-80 ° C, preferably about 24-42 ° C. The reaction time is about 10 minutes to 72 hours, preferably about 1 to 24 hours. If desired, an enzyme stabilizer, a dehydrating agent such as molecular sieves, or a water substitute such as DMSO, formamide, or ethylene glycol may be added to the reaction solution. The reaction may be performed under any conditions of standing, shaking, and stirring. When esterase is immobilized on a carrier,
It may be reacted in a bioreactor.

In the above, the compound [I '] is preferably the compound [Ia]. The compound [I "] is preferably a compound
It is [Ib]. The compound [II '] is preferably of the formula [IIa]

[0060]

[Chemical 38]

It is a compound represented by: Compound [II "]
Is preferably of formula [IIb]

[0062]

[Chemical Formula 39]

It is a compound represented by: The reaction product can be isolated and purified by known means, for example, solvent extraction, liquid conversion, phase transfer, salting out, crystallization, recrystallization, chromatography and the like. A method known per se or a method analogous thereto is applied for separating a desired compound (for example, compound [IIa], [IIb] etc.) from the mixture. As such methods, for example, a fractionation method utilizing a difference in solubility or crystallinity, a chromatographic separation method, or the like is used.
For example, a method for obtaining the compound [IIa] using the fractionation method will be shown below. The above-mentioned mixture of optical isomers is heated and dissolved in a specific solvent to prepare a supersaturated solution, which is then cooled or concentrated or a solvent that reduces the solubility of the compound [IIa] is added to obtain only the compound [IIa]. Is separated and crystallized. This fractional crystallization is performed in an inert solvent, but the compound [IIa] can be efficiently crystallized only when a specific solvent is used.

Suitable solvents include ketones such as acetone, alcohols such as isopropyl alcohol, and mixed solvents of esters such as ethyl acetate and hydrocarbons such as hexane. Particularly suitable solvents include ketones such as acetone.
When only compound [IIa] is obtained by fractional crystallization, the compound
Higher content of [IIa] is desirable. When acetone is used as a solvent, the content of the compound [IIa] may be about 14% by weight based on the compound [Ib] in the above mixture. The temperature used in the present invention is about -2.
Compounds [II
Below the crystallization temperature of a] is advantageous. In addition, the compound [II
It is also possible to inoculate a seed crystal of a], but it is not always necessary in this supersaturated solution because spontaneous crystal formation of compound [IIa] occurs. On the other hand, when the unreacted compound [I "] remains in the above mixture, the compound [I"] may be converted into the compound [VI] by deacyloxy reaction. The deacyloxy reaction of the present invention has the following reaction formula

[0065]

[Chemical 40]

As shown in, the compound [I "] is hydrolyzed to the compound [II"], which is then converted into a halogenated compound.
This is reduced to give compound [VI]. The hydrolysis of the compound [I "] shown in the above reaction scheme to the compound [II"] is carried out by usual basic hydrolysis. This hydrolysis can be carried out in any ordinary organic solvent.
Suitable solvents include lower alcohols such as methanol and ethanol, and ethers such as diethyl ether, tetrahydrofuran and dioxane.
As the base, any ordinary base can be arbitrarily used, but suitable bases include, for example, alkali metal carbonates such as sodium carbonate, sodium hydrogen carbonate and potassium carbonate, such as sodium hydroxide, potassium hydroxide and hydroxide. Examples thereof include alkali metal hydroxides such as lithium, and alkaline earth metal hydroxides such as magnesium hydroxide and calcium hydroxide. The amount of base used is about 1 to 1
It is 0 mol, preferably about 1 to 3 mol. In this hydrolysis, the reaction temperature is not particularly limited, and it is about 0 to 10
It is carried out at 0 ° C., preferably about 15-40 ° C. The reaction temperature is about several minutes to several tens of hours.

The compound [II "] can be halogenated with any ordinary halogenating agent. Examples of the halogenating agent include hydrogen halides such as hydrogen chloride, hydrogen bromide and hydrogen iodide, and trichloride. Phosphorus, phosphorus pentachloride, phosphorus tribromide, phosphorus pentabromide, phosphorus triiodide, etc. phosphorus halide, thionyl chloride, thionyl bromide, etc. thionyl halide, sulfuryl chloride, etc. sulfuryl halide, triphenylphosphine Examples thereof include active alkyl halides such as carbon tetrachloride, diphenyltrihalogenophosphorane, triphenylphosphinedihalogenide, triphenyldihalogenide phosphonate, etc. The amount of halogenating agent used is usually about 0. The amount is 0.3 to 10 moles, preferably about 0.3 to 3 moles. Chirueteru, ethers such as tetrahydrofuran and dioxane, nitriles such as acetonitrile, benzene, hydrocarbons such as toluene, esters such as ethyl acetate, dichloromethane, chloroform,
Examples thereof include halogenated hydrocarbons such as 1,2-dichloroethane. These may be used alone or as a mixture of two or more kinds at an appropriate ratio. The reaction temperature is not particularly limited, but is usually about -30 to 100 ° C, preferably-
10 to 30 ° C. The reaction time is several minutes to several tens of hours. Further, although this halogen compound can be isolated, it can be directly subjected to the next step without further purification.

The reduction of the halogen compound can be carried out by catalytic hydrogenation or a hydride reducing agent. Catalytic hydrogenation can be carried out by any conventional method. Examples of the catalyst include palladium on charcoal, palladium on barium sulfate, platinum and Raney nickel.
As the solvent used for the catalytic hydrogenation, usual organic solvents such as lower alcohols such as methanol and ethanol,
Ethers such as diethyl ether, tetrahydrofuran, dioxane, nitriles such as acetonitrile, hydrocarbons such as benzene and toluene, esters such as ethyl acetate, halogenated hydrocarbons such as dichloromethane, chloroform and 1,2-dichloroethane, Organic acids such as acetic acid may be mentioned. These solvents may be used alone or as a mixture of two or more kinds. The reaction temperature and pressure are not particularly limited, but are about 5 ° C. to 100 ° C. and atmospheric pressure to 100 atm, respectively. The reaction time is several minutes to several tens of hours.

For reduction with a hydride reducing agent, sodium borohydride, magnesium borohydride, borohydrides such as diborane, aluminum lithium hydride,
It can be performed with an aluminum hydride such as diisobutylaluminum hydride or diethylaluminum sodium hydride. As the solvent, a solvent which is inactive to the reducing agent under the reaction conditions and in which the reactant is at least partially soluble is preferable, and an ordinary organic solvent such as diethyl ether, tetrahydrofuran, dioxane, and ethers such as diethylene glycol dialkyl ether. Hydrocarbons such as benzene and toluene, and halogenated hydrocarbons such as dichloromethane, chloroform and 1,2-dichloroethane are used. For reducing agents usable in an aqueous solvent such as sodium borohydride, water and alcohols such as methanol, ethanol and diethylene glycol are also effective. These solvents can be used alone or as a mixture of two or more kinds. The reaction temperature is not particularly limited, but is preferably about -10 to 100 ° C.
The reaction time is several minutes to several tens of hours.

The compound [VI] obtained by the above reduction can be prepared by a known method, for example, tetrahedron (Tetrahedron) 4
The compound [II] can be converted by the method described in 6 , 7667 (1990), that is, by chlorination and hydrolysis. In addition, the compound [VI] is, for example, Journal of American Chemical Society (J. Am. Chem. Soc.) 1
00 , 1558 (1978), that is, the compound [III] can be converted by the action of an oxidizing agent such as hydrogen peroxide or sodium periodate. Using these compounds [II] and [III] as starting compounds, compound [IIa] can be obtained according to the above method.

According to the present invention, the compound [II '] is oxidized by using DMSO and an activator,
[VII] can be easily obtained. Examples of the activator include acetic anhydride, a complex of anhydrous sulfuric acid and pyridine, halogen (eg, chlorine, bromine, iodine, etc.), acetyl halide (eg, acetyl chloride, etc.), phosphoric anhydride and the like. One or more of these may be used. When acetic anhydride is used as the activator, in order to selectively obtain the compound [VII], first, a substantial oxidizing agent is generated, that is, DMSO is activated with acetic anhydride, and then the compound [II ′] is used. And oxidize it. That is, this reaction is carried out in two steps: activation of DMSO and oxidation of compound [II ′]. The temperature in the first-step reaction is about 15 ° C to 150 ° C, preferably about 25 ° C to 100 ° C. Further, the reaction time is related to the reaction temperature, and at a relatively low temperature, for example, 25 ° C., it takes 3 to 100 hours, and at a relatively high temperature, for example, 80 ° C., 10 hours or less, usually 3 hours or less are sufficient. The amount of acetic anhydride is also important for the selectivity of the compound [VII], and is about 1 mol to 1 mol of the compound [II '].
It is 20 mol, preferably about 2 mol to 10 mol.

The reaction temperature in the second stage reaction is about 15
C. to 150.degree. C., preferably about 25.degree. C. to 100.degree.
Also, the reaction time is related to the reaction temperature,
For example, at 25 ° C, 10 to 30 hours are required, and at a relatively high temperature, for example, 80 ° C, 15 minutes to 1 hour is sufficient. The compound [VII] obtained by the above-mentioned production method can be isolated and purified by a separation means known per se, ie, solvent extraction, liquid conversion, phase transfer, salting out, crystallization, recrystallization, chromatography and the like. In particular, when the above reaction mixture is added to water, the compound [VII] crystallizes out, and thus the compound [VII] can be isolated by, for example, filtering this. Compound [VII] can be derivatized to D-biotin by the method described in JP-B-53-27279, for example, the following reaction step.

[0073]

[Chemical 41]

D-Biotin is also called Vitamin H and is widely used for the addition of medicines and feeds. In the above production method, the starting compound [II] is prepared by a known method, for example,
Tetrahedron 46 , 7677 (199)
The method described in 0) and the starting compound [III] are known methods, for example, Journal of American Chemical Society (J. Am. Chem. Soc.) 100 , 1558.
It can be produced by the method described in (1978).

[0075]

EXAMPLES The present invention will be described in more detail with reference to examples and reference examples, but the present invention is not limited thereto. The NMR spectrum uses tetramethylsilane as an internal standard. JASCO JNM-GSX27.
Measured with a 0 (270 MHz) type spectrometer,
Values are given in ppm. In the mixed solvent, the value shown in parentheses is the volume mixing ratio of each solvent. Unless otherwise specified,%
Means weight percent. The symbols in the examples have the following meanings. s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, m: multiplet, br .: wide, J: coupling constant, ph: phenyl group, Ac: acetyl group compound [Ia] + Compound [Ib] to Compound [IIa] + Compound
The conversion rate to [IIb] is determined by measuring the content of each component in the reaction solution by high performance liquid chromatography and using the measured value according to the following formula.

[0076]

[Equation 1] The conversion rate from compound [IIa] + compound [IIb] to compound [Ia] + compound [Ib] is determined by the following formula according to the above formula.

[Equation 2]

Specifically, the reaction solution is treated with a suitable organic solvent.
(E.g. ethyl acetate) and extract this extract with a chiral column.
(Eg, Chiralcel OD, manufactured by Daicel Industries, Ltd.) and subjected to high performance liquid chromatography. The stereoselectivity, that is, the enantiomeric excess (ee) is calculated by the following formula in the case of the compound [Ia] and the compound [Ib]. ee = [([Ia] − [Ib]) / ([Ia] + [Ib])] ×
100 Example 1

[0078]

[Chemical 42]

(±)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno
0.5 g of [3,4-d] imidazol-2 (3H) -one was dissolved in 2.0 ml of pyridine, and 2.0 ml of acetic anhydride was added. After stirring at room temperature for 1.5 hours, the solvent was evaporated under reduced pressure. The obtained colorless oil was dissolved in a small amount of ether, and n-hexane was added to the mixture to obtain (±)-(3aα, 4α, 6aα) -tetrahydro-
4-acetoxy-1,3-dibenzyl-1H-thieno
Crystals of [3,4-d] imidazol-2 (3H) -one were obtained. Yield 0.51 g (91% yield). The structure was confirmed by melting point, mass spectrum (MS), infrared absorption spectrum (IR), nuclear magnetic resonance spectrum (NMR), and X-ray structural analysis. Melting point: 72-74 ° C. MS: m / e 382 (M ⁺ ). IR: (KBr) cm ^-1 1745, 1700, 1455, 12
_{. 40,960,700 NMR (CDCl 3):} 1.96 (3H, s, CH 3), 2.96 (1
H, d, J = 10.2, CH ₂ (End) S), 3.03 (1H, dd,
J = 10.2, 3.87, CH ₂ (exo) S), 4.02 (1H,
d, J = 5.8, CHN), 4.2 (1H, m, CHN), 4.19,
4.24, 4.79, 4.84 (1H, d, J = 15.
4, CH ₂ Ph), 6.02 (1H, s, CHOAc), 7.29 (1
0H, m, Ph)

Example 2 (±)-(3aα, 4α, 6aα) -Tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno [3,4-d] imidazol-2 (3H) -one 5. 00 g of acetic anhydride 25 ml
Stir in and add 1 drop of concentrated sulfuric acid. After stirring at room temperature for 1 hour, the solvent was evaporated under reduced pressure. The obtained oil was washed with saturated aqueous sodium hydrogen carbonate, dissolved in a small amount of ether, and n-hexane was added to the mixture to obtain (±)-(3aα, 4α, 6aα) -tetrahydro-4-acetoxy-1,3-dibenzyl. Crystals of -1H-thieno [3,4-d] imidazol-2 (3H) -one 4.
00 g (yield 72%) was obtained. From the mother liquor, 0.93 g (17% yield) of (3aα, 4α, 6aα) -tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3,4-
d] Imidazol-2 (3H) -one was obtained. IR, N
The MR value was the same as that obtained in Example 1.

Example 3 (±)-(3aα, 5α, 6aα) -Tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2
200 mg of (3H) -one-5-oxide was added to 3.0 m of acetic anhydride.
It was dissolved in 1 and stirred at 80 ° C. for 4 hours. After that, this was concentrated under reduced pressure, diethyl ether was added, and the mixture was washed with aqueous sodium hydrogen carbonate. After drying over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crude product. This was chromatographed on silica gel (10 g, hexane /
When subjected to (ethyl acetate = 1: 1), (±)-(3aα, 6a
α) -Tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2 (3H)-
206 mg (92% yield) of colorless oily substance was obtained. By NMR analysis of this product, it was found that the 4α-form and the 4β-form were 55:45.
It was confirmed that it exists in the ratio of.

Example 4 (±)-(3aα, 5α, 6aα) -Tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2
100 mg of (3H) -one-5-oxide was dissolved in 1.0 ml of dichloromethane, 0.060 ml of acetic anhydride and 28 mg of paratoluenesulfonic acid were added, and the mixture was stirred at room temperature for 17 hours. After that, the solvent was evaporated under reduced pressure, diethyl ether was added to the residue, and the mixture was washed with aqueous sodium hydrogen carbonate. After drying over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crude product. This is silica gel chromatography
When subjected to (5 g, hexane / ethyl acetate = 1: 1),
(±)-(3aα, 6aα) -Tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one 94 mg (yield 84%) of colorless oil Got the substance. By NMR analysis of this product,
It was confirmed that the β-form was present in a ratio of 93: 7.

Example 5 (±)-(3aα, 5α, 6aα) -tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2
100 mg of (3H) -one-5-oxide was added to 5.4 m of hydrochloric acid gas.
g was dissolved in 1.0 ml of absorbed dichloromethane, 0.060 ml of acetic anhydride was added, and the mixture was stirred at room temperature for 30 hours. After that, the solvent was evaporated under reduced pressure, diethyl ether was added to the residue, and the mixture was washed with aqueous sodium hydrogen carbonate. After drying over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crude product. This is subjected to silica gel chromatography (5
g, hexane / ethyl acetate = 1: 1),
51 mg (45% yield) of (±)-(3aα, 6aα) -tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one as a colorless oil. Got the substance. By NMR analysis of this product,
It was confirmed that the β-form was present at a ratio of 95: 5.

Example 6 (±)-(3aα, 5α, 6aα) -tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2
100 mg of (3H) -one-5-oxide was dissolved in 1.0 ml of dichloromethane, 0.060 ml of acetic anhydride and 0.0 of phosphoric acid.
86 ml was added, and the mixture was stirred at room temperature for 24 hours. After that, the solvent was evaporated under reduced pressure, diethyl ether was added to the residue, and the mixture was washed with aqueous sodium hydrogen carbonate. After drying over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel chromatography (5 g, hexane / ethyl acetate = 1: 1) to find that (±)-(3a
α, 6aα) -Tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2
100 mg (89% yield) of (3H) -one was obtained as a colorless oily substance. By NMR analysis of this product, it was confirmed that 4α-form and 4β-form were 6
It was confirmed to be present in a ratio of 8:32.

Example 7 (±)-(3aα, 5α, 6aα) -Tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2
100 mg of (3H) -one-5-oxide was dissolved in 1.0 ml of dichloromethane, 0.060 ml of acetic anhydride and 0.07 of sulfuric acid.
8 ml was added, and the mixture was stirred at room temperature for 24 hours. After that, the solvent was evaporated under reduced pressure, diethyl ether was added to the residue, and the mixture was washed with aqueous sodium hydrogen carbonate. After drying over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel chromatography (5 g, hexane / ethyl acetate = 1: 1) to find that (±)-(3a
α, 6aα) -Tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2
81 mg (72% yield) of (3H) -one was obtained as a colorless oily substance. NMR analysis of this product revealed that 9
It was confirmed to be present in a ratio of 3: 7.

Example 8 (±)-(3aα, 5α, 6aα) -Tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2
100 mg of (3H) -one-5-oxide was dissolved in 1.0 ml of dichloromethane, 0.060 ml of acetic anhydride and 0.129 ml of trifluoromethanesulfonic acid were added, and the mixture was stirred at room temperature for 5 hours. After that, the solvent was evaporated under reduced pressure, diethyl ether was added to the residue, and the mixture was washed with aqueous sodium hydrogen carbonate. After drying over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crude product. When this was subjected to silica gel chromatography (5 g, hexane / ethyl acetate = 1: 1), (±)-(3aα, 6aα) -tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3, 4-
d] Imidazole-2 (3H) -one (53 mg, yield 47%) was obtained as a colorless oily substance. 4 by NMR analysis of this product
It was confirmed that the α-form and the 4β-form existed at a ratio of 68:32.

Example 9 (±)-(3aα, 5α, 6aα) -Tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2
100 mg of (3H) -one-5-oxide was dissolved in 1.0 ml of dichloromethane, 0.060 ml of acetic anhydride and 1550 mg of Amberlyst were added, and the mixture was stirred at room temperature for 70 hours. Then, the acidic ion exchange resin (Amberlyst 15, manufactured by Organo) was filtered off, and the solvent was distilled off under reduced pressure. After drying over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crude product. This was chromatographed on silica gel (5 g, hexane / ethyl acetate = 1: 1).
When subjected to (±)-(3aα, 6aα) -tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno
[3,4-d] imidazol-2 (3H) -one (94 mg, yield 84%) was obtained as a colorless oily substance. By NMR analysis of this product, it was confirmed that the 4α-form and the 4β-form were present in a ratio of 93: 7. Example 10

[0088]

[Chemical 43]

Dextrin 1%, glucose 1%, glycerol 1%, peptone 0.5%, yeast extract 0.5%,
Meat extract 0.5%, salt 0.3%, calcium carbonate 0.5
% (Both are W / V%) and pH 7.2, a medium (this medium is called A1 medium) 2 ml was inoculated with various bacteria, and
The cells were cultured at 8 ° C for 2 days with shaking. 2 ml of the obtained culture solution
(±)-(3aα, 4α, 6aα) -tetrahydro-4-acetoxy-1,3-dibenzyl-1H obtained in Example 1
50 µl or 100 µl or 200 µl of a methanol solution of thieno [3,4-d] imidazol-2 (3H) -one (80 mg / ml) was added, and the substrate concentration was 2 mg / ml, respectively.
Alternatively, it was adjusted to 4 mg / ml or 8 mg / ml. This culture solution was shaken at 28 ° C. for 24 hours. To the obtained culture solution, 12 ml of ethyl acetate was added and stirred, and the ethyl acetate layer was diluted with normal hexane-isopropanol (6: 4) and used as a column for CHIRACEL.
OD, manufactured by Daicel Industries, Ltd.), subjected to quantitative analysis by high performance liquid chromatography (HPLC) using normal hexane-isopropanol (6: 4) as a mobile phase (using a detection wavelength of 220 nm), the starting compound and the target product. Quantified
The conversion rate and the ratio of the compounds [IIa] and [IIb] were measured. The results are shown in Table 1.

[0090]

[Table 1]

Also Pseudomonas aeruginosa IF
FIG. 1 shows the HPLC pattern when the reaction was carried out with O 3447 at a substrate concentration of 4 mg / ml. Example 11 2 ml of A1 medium prepared by the same formulation as in Example 10 was inoculated with actinomycetes and cultured at 28 ° C. for 3 days with shaking. 2 ml of the obtained culture solution was added to (±)-(3aα, 4α, 6aα) -tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one. 50 μl or 200 μl of the methanol solution (80 mg / ml) was added to adjust the substrate concentration to 2 mg / ml or 8 mg / ml, respectively. This culture solution was shaken at 28 ° C. for 1 to 24 hours to carry out a reaction. The conversion rate and the ratio between the compounds [IIa] and [IIb] were measured in the same manner as in Example 10. The results are shown in Table 2.

[0092]

[Table 2]

Substrate concentration of 2 mg / ml using Streptomyces rochey bar borbilis IFO 12507
FIG. 2 shows the pattern of HPLC when the reaction was carried out for 1 hour. Example 12 Yeast extract 0.3%, meat extract 0.3%, peptone 0.5
%, Glucose 0.1% (both W / V%) were inoculated with 2 ml of medium (in a test tube), and cultured at 28 ° C. for 3 days with shaking. 2 ml of the obtained culture solution was added to (±)-(3aα,
4α, 6aα) -Tetrahydro-4-acetoxy-1,3-
Dibenzyl-1H-thieno [3,4-d] imidazole-2
50 μl of (3H) -one in methanol (80 mg / ml)
Was added and the culture was shaken at 28 ° C. for 24 hours to carry out the reaction. The conversion rate and the ratio between the compounds [IIa] and [IIb] were measured in the same manner as in Example 10. The results are shown in Table 3.
Shown in.

[0094]

[Table 3] Bacterial species conversion rate (%) [IIa]: [IIb] Candida Gilia Mondee 15 12:88 IFO 14242 Tricos Boron Fermentans 47 12:88 IFO 1199

Example 13 Pseudomonas aerug
inosa) IFO 3447 with dextrin 1%, glucose 1%, glycerol 1%, peptone 0.5%, yeast extract 0.5%, meat extract 0.5%, salt 0.3%, calcium carbonate 0.5% ( Both were inoculated into 40 ml medium (200 ml Erlenmeyer flask) consisting of W / V%) and pH 7.2.
It was cultured under shaking at 8 ° C. for 24 hours. The obtained culture solution 1
ml was transferred to 40 ml of the same medium in a 200 ml Erlenmeyer flask and incubated at 28 ° C. for 48 hours with shaking. 40 ml of the obtained culture solution was added to (±)-(3aα, 4α, 6aα) -tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno.
160 mg of [3,4-d] imidazol-2 (3H) -one (in 2 ml of methanol) was added, and the reaction was carried out at 28 ° C for 18 hours with shaking. This was repeated 13 times, and ethyl acetate was added to the reaction solution obtained and stirred to obtain an ethyl acetate layer.
(A part of this ethyl acetate layer was taken and examined by HPLC. The conversion rate was 40%. Compound [IIa]: Compound [IIb]
The ethyl acetate layer was evaporated to dryness under reduced pressure to obtain 2.32 g of a solid. This is 12 ml of ethyl acetate and n
Crystallization from a mixed solvent of 6 ml of hexane gave (-)-(3a
α, 4α, 6aα) -Tetrahydro-1,3-dibenzyl-
0.613 g (crystal) of 4-hydroxy-1H-thieno [3,4-d] imidazol-2 (3H) -one was obtained.

Melting point: 163-164 ° C., MS: m / e 34
0 (M ⁺ ) IR (KBr) cm ^-1 : 3300,2930,1680 NMR (CDCl ₃ ): 1.66 (1H, br s, OH), 2.86
(1H, d, J = 12.7, CH ₂ (end) S), 3.01 (1H,
dd, J = 12.7,4.7, CH ₂ (exo) S), 4.21 (1
H, dd, C (6a) -H), 4.02 (1H, d, J = 7.9, C (3
a) -H), 4.75,4.67,4.31,4.21 (each 1H, d, J = 15.7, PhCH 2), 5.18 (1H, s, CH O
H), 7.2-7.4 (10H, m, Ph) Specific optical rotation: [α] _D ²⁷ = -68.1 ° (c = 0.78, chloroform) Next, the mother liquor was concentrated under reduced pressure. , An oily product was obtained. This was subjected to silica gel chromatography (100 g, hexane: ethyl acetate = 1: 1) to give (±)-(3aα, 4α, 6aα) -tetrahydro-4-acetoxy-1,3-dibenzyl-1.
H-thieno [3,4-d] imidazol-2 (3H) -one
(Oil) was obtained. Dissolve this in 5 ml of ethanol and add 5%
15 ml of potassium hydroxide in the above was added and the mixture was stirred at 60 ° C. for 1.5 hours. The crystals that were crystallized by ice cooling were collected by filtration, and ethyl acetate 1
Recrystallize with 4 ml and 10 ml of n-hexane to obtain (+)-(3
aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-
There were obtained 0.597 g of crystals of 4-hydroxy-1H-thieno [3,4-d] imidazol-2 (3H) -one. Melting point, MS,
IR and NMR were completely in agreement with the (-) form described in Example 13. Specific rotation: [α] _D ²⁷ = + 67.5 ° (c = 0.78, chloroform)

Example 14 Streptomyces rochey bar borbilis IF
O 12507 (see JP-A-59-183695)
1% dextrin, 1% glucose, 1 glycerol
%, Peptone 0.5%, yeast extract 0.5%, meat extract 0.5%, NaCl 0.3%, calcium carbonate 0.5%, p
40 ml of medium consisting of H7.2 [in a 200 ml Erlenmeyer flask] was inoculated and cultured at 28 ° C. for 3 days under shaking. The obtained culture solution is 10,000 rpm
After centrifugation for 10 minutes, a culture supernatant was obtained. 2 of this culture supernatant
(±)-(3aα, 4α, 6aα) -tetrahydro-4-acetoxy-1,3-dibenzyl-1 obtained in Example 1 in ml.
50 μm of a dimethyl sulfoxide solution (80 mg / ml) containing H-thieno [3,4-d] imidazol-2 (3H) -one
l was added so that the substrate concentration was 2 mg / ml. The reaction solution was incubated at 28 ° C. for 1 hour and 2 hours under shaking. To the reaction solution after the reaction, 2 ml of ethyl acetate was added and stirred, 100 μl of the ethyl acetate layer was taken, and concentrated under reduced pressure. The concentrate was diluted with normal hexane-isopropanol (6: 4) and used as a column for chirasel (CHRA).
CEL OD, manufactured by Daicel Industries, Ltd., high performance liquid chromatography (HPLC) using normal hexane-isopropanol (6: 1) as a mobile phase (detection wavelength: 220 n
The raw material compound and the product were quantified by using m) and the conversion rate and the ratio of the compound [IIa] and the compound [IIb] were measured.
The results are shown in Table 4.

[0098]

[Table 4] Reaction time (hours) Conversion rate (%) [IIa]: [IIb] 1 37 95: 5 2 53 2 92: 8

Example 15 Streptomyces rochey bar borbilis IF
O 12507 with 1% dextrin, 1% glucose,
Glycerol 1%, peptone 0.5%, yeast extract 0.5
%, Meat extract 0.5%, NaCl 0.3%, calcium carbonate 0.5%, pH 7.2 40 ml medium [in a 200 ml Erlenmeyer flask] was inoculated.
The cells were cultured at 8 ° C for 3 days with shaking. 4m of the obtained culture solution
l was transferred to a 1 liter Erlenmeyer flask containing 200 ml of the above indicated medium and the microorganisms were incubated at 28 ° C for 3
Solvented under shaking for one day. The obtained culture solution was centrifuged to obtain a culture supernatant. 3.2 g of (±)-(3aα, 4α, 6a
α) -Tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2 (3H)-
On was dissolved in 40 ml of dimethylsulfoxide, this solution was added to 1.6 liters of the culture supernatant obtained by raising the scale of the above method, and the reaction was carried out at 28 ° C. for 1 hour with shaking. The conversion rate of this reaction and the ratio of [IIa] to [IIb] were determined by the same method as in Example 14, and were 27.7% and 96: 4, respectively.

After the reaction, the reaction solution was extracted with 1.6 liters of ethyl acetate, and the remaining aqueous layer was diluted with 900 ml of ethyl acetate to 2 times.
Extracted twice. Obtained ethyl acetate layer (about 3.5 liters)
Was concentrated under reduced pressure and the concentrate was dissolved in 500 ml of ethyl acetate. The solution was washed with 500 ml of water and then with 500 ml of saturated saline and concentrated under reduced pressure to obtain 3.78 g of a yellow oily substance. The obtained oil (3.78 g) was dissolved by heating in 7.0 ml of acetone, and crystallization started upon cooling at 0 ° C. After about 1.5 hours, crystals were obtained by filtration (344 mg). Also, the mother liquor was left to stand and another 51 mg of crystals were obtained. The melting point, MS, IR and NMR values are shown in Example 13.
It was completely identical to the compound [IIb] shown in. Specific rotation [α] _D ³⁰ = + 62.4 ° (c = 0.78, chloroform)

Example 16 Glucose 3.0%, Proflu (trade name, manufactured by Trader Oil Co.) 1.0%, corn steep liquor 3.5%, magnesium sulfate 0.02%, dibasic potassium phosphate 0.1
%, Soybean oil 0.05%, calcium carbonate 1.5% (percentage display, weight / volume) 500 ml of a medium was adjusted to pH 7.0 using a 20% caustic soda aqueous solution and then distributed to a 2 liter Sakaguchi flask. It was poured, covered with a cotton plug, and then sterilized. Streptomyces rochey bar borbilis (IFO 12507) (ATCC-21250)
(See JP-A-59-183695), after inoculating the slant culture, a reciprocal shaking culture machine (83s
pm). In a 50-liter fermentor, prepare 30 liters of medium having the same composition as the above medium and sterilize
Inoculate 500 ml of the above Sakaguchi flask culture, aeration volume 1V
VM (aeration volume per minute per unit volume), stirring speed 15
A seed culture was obtained by culturing at 0 rpm at 24 ° C. for 24 hours. Two
Glycerin 10%, Proflo in a 00 liter fermentor
(Trade name, manufactured by Trader Oil Co.) 2.0%, corn steep liquor 0.5%, polypeptone 1.0%, sulfuric acid No. 1
100 liters of a medium consisting of iron 0.1%, soybean oil 0.01%, β-cyclodextrin 2.0% (percentage display, weight / volume) was prepared, and a pH of 20% caustic soda solution was used.
After adjusting to 7.0, steam sterilization was performed at 120 ° C. for 20 minutes. The above seed culture (5 liters) was transplanted to this, and the aeration rate was 1 VVM, the stirring speed was 165 rpm, and the temperature was 24 ° C.
Cultured for 6 hours.

60 liters of the thus obtained culture was taken, 20 liters of water and 2 kg of Hyflo Supercell (manufactured by Johns Manville) were added to this, and the mixture was filtered to obtain 70 liters of filtrate. 10 liters of this filtrate was collected in a 60 liter container, 30 liters of ethanol was added thereto, and the mixture was sufficiently stirred with a stirring rod. The mixture is allowed to stand at 5 ° C for 12 hours to cause protein precipitation, and the supernatant liquid is removed by a siphon to obtain a cloudy precipitate. This is 5 ℃, 2,000
It is subjected to cooling centrifugation under the condition of 0 × g, water is removed as much as possible, and ethanol is added for washing. Centrifugation under the same conditions, collecting the precipitate, vacuum drying under conditions of 50 mmHg, 10 ° C, 24 hours, powdered enzyme preparation (200 g)
Got The above enzyme preparation was dissolved in 0.1M phosphate buffer pH 7.0 to various concentrations. (±)-(3aα, 4α, 6aα) -tetrahydro-in the obtained enzyme solution (2 ml)
4-acetoxy-1,3-dibenzyl-1H-thieno
50 μl of dimethylsulfoxide solution (80 mg / ml) containing [3,4-d] imidazol-2 (3H) -one was added,
The substrate concentration was adjusted to 2 mg / ml. 28 this reaction solution
After incubating at ℃ for 1 hour under shaking, the conversion rate and compound [IIa] and compound [IIb] were measured by the method described in Example 14.
Was measured. The results are shown in Table 5.

[0103]

[Table 5] Enzyme concentration (mg / ml) Conversion rate (%) [IIa]: [IIb] 0 − − 0.25 26 96: 4 0.5 37 89:11 1.0 48 84:16

Example 17 Pseudomonas aerug
inosa) IFO 3447 with dextrin 1%, glucose 1%, glycerol 1%, peptone 0.5%, yeast extract 0.5%, meat extract 0.5%, salt 0.3%, calcium carbonate 0.5% ( Both were inoculated into 40 ml of a medium consisting of W / V%) pH 7.2 and cultured at 28 ° C. for 24 hours with shaking. 1 ml of the obtained culture solution was transferred to 40 ml of the same medium as described above in a 200 ml Erlenmeyer flask, and cultured at 28 ° C. for 48 hours with shaking. Culture liquid meter 52 obtained by the above method
0 ml of (±)-(3aα, 4α, 6aα) -tetrahydro-4-
Acetoxy-1,3-dibenzyl-1H-thieno [3,4
1.04 g (in 13 ml of methanol) of -d] imidazol-2 (3H) -one was added, and the reaction was carried out at 28 ° C for 24 hours with shaking. Ethyl acetate (1 liter) was added to the obtained reaction solution and stirred to obtain an ethyl acetate layer. When a part of this reaction solution was taken and analyzed by HPLC, the conversion rate was 53.
%, And the ratio of compound [IIa]: compound [IIb] was 15:85.

The ethyl acetate layer was dried under reduced pressure to give an oily substance. This oil was subjected to silica gel chromatography (8
0 g, hexane: ethyl acetate = 1: 1), and then (±)-(3
aα, 4α, 6aα) -tetrahydro-4-acetoxy-1,
An oil of 3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one was obtained. Dissolve this in a small amount of diethyl ether, add n-hexane and let it stand,
222 mg of crystals were obtained. The melting point, MS, IR and NMR were completely in agreement with the racemate shown in Example 1. Specific rotation [α] _D ²⁹ = + 110 ° (c = 0.76, chloroform) In order to measure the optical purity of this crystal, this crystal was hydrolyzed by the method shown in Example 13 to give a compound [ II]
And analyzed by HPLC. As a result, it was found to be optically pure. Example 18

[0106]

[Chemical 44]

(±)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno
0.50 g of [3,4-d] imidazol-2 (3H) -one was dissolved in 2.0 ml of pyridine and 2.0 ml of propionic anhydride was added. After stirring at room temperature for 1 hour, the solvent was evaporated under reduced pressure. The obtained oily substance was dissolved in a small amount of toluene and washed with aqueous sodium hydrogen carbonate and then with water. The solvent was evaporated under reduced pressure, n-pentane was added, and (±)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-propionyloxy-
Crystals of 1H-thieno [3,4-d] imidazol-2 (3H) -one were obtained. The yield was 0.46 g (78% yield). The structure was confirmed by infrared absorption spectrum (IR) and nuclear magnetic resonance spectrum (NMR). IR (KBr) cm ^-1 : 1740,1690,1475,145
_{5,1245 NMR (CDCl 3): 1.07} (3H, t, J = 7.5, CH 3),
2.22 (2H, q, J = 7.5, CH ₂ CH ₃ ), 2.95 (2H,
_{m, CH 2 S), 3.99} (1H, d, J = 7.8, C (3a) -H),
4.18, 4.24, 4.79, 4.85 (1H, d, J respectively
= 15.4, CH ₂ Ph), 4.2 (1H, m, C (6a) -H), 6.
05 (1H, s, C (4) -H), 7.2-7.3 (10H, m, Ph) Example 19

[0108]

[Chemical 45]

(±)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno
[3,4-d] imidazol-2 (3H) -one (340 mg) was dissolved in dichloromethane (5.0 ml), and triethylamine (0.209 ml) and butyryl chloride (0.154 ml) were dissolved therein.
Was added and the mixture was stirred at room temperature for 1.5 hours. After distilling off the solvent,
Ether was added to remove insoluble materials. The filtrate was concentrated to give an oil. This was purified by silica gel chromatography (hexane: ethyl acetate = 2: 1) to obtain (±)-(3a
α, 4α, 6aα) -Tetrahydro-4-butyryloxy-
147 mg (yield 36%) of crystals of 1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one were obtained. The structure was confirmed by infrared absorption spectrum (IR) and nuclear magnetic resonance spectrum (NMR). IR (KBr) cm ^-1 : 1750,1690,1480,146
0.1250 NMR (CDCl ₃ ): 0.89 (3H, t, J = 7.3, CH ₃ ),
1.58 (2H, m, J = 6.9, CH ₂ ), 2.18 (2H, t, J
= 6.9, COCH ₂ ), 2.99 (2H, m, CH ₂ S), 3.98
(1H, d, J = 7.8, C (3a) -H), 4.18, 4.23, 4.
80,4.87 (1H, d, J = 15.3CH2Ph respectively),
4.2 (1H, m, C (6a) -H), 6.06 (1H, s, C (4)-
H), 7.2-7.3 (10H, m, Ph) Example 20

[0110]

[Chemical 46]

(±)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno
[3,4-d] imidazol-2 (3H) -one (340 mg) was dissolved in diethyl ether (5.0 ml), and triethylamine (0.209 ml) and valeric acid chloride (0.198 ml) were added thereto. , Stirred at room temperature for 1.5 hours. After that, the solvent was distilled off under reduced pressure to obtain an oily substance. This was purified by silica gel chromatography (hexane: ethyl acetate = 2: 1),
(±)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl
-4-valeryloxy-1H-thieno [3,4-d] imidazole-2 (3H)
111 mg (26% yield) of a colorless oil of -one were obtained. Structure confirmation is infrared absorption spectrum (IR), magnetic resonance spectrum
(NMR) and mass spectrum (MS). IR (KBr) cm ^-1 : 1735,1700,1450,124
0 NMR (CDCl ₃ ): 0.87 (3H, t, J = 6.4, CH ₃ ),
1.16-1.62 (4H, m, CH ₂ CH ₂ ), 2.21 (2H,
t, J = 7.4 OCOCH ₂ ), 3.98 (1H, d, J = 7.9,
C (3a) -H), 4.19, 4.22, 4.80, 4.87 (4H,
4d, J = 15.4, CH ₂ Ph), 4.2 (1H, m, C (6a) −
H), 6.06 (1H, s, C (4) -H), 7.21-7.36 (1
OH, m, Ph) MS: m / e 425 (MH ⁺ ) Example 21

[0112]

[Chemical 47]

(±)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno
[3,4-d] imidazol-2 (3H) -one (340 mg) and Meldrum's acid (144 mg) were dissolved in toluene (1.0 ml) and heated to 100 ° C. After 1.5 hours, 5.0 ml each of toluene and aqueous sodium hydrogen carbonate was added. The aqueous layer was taken, made weakly acidic with diluted hydrochloric acid, and extracted with ether. The ether layer was concentrated under reduced pressure to give (±)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-malonyloxy-1H-.
279 mg (yield 65%) of thieno [3,4-d] imidazol-2 (3H) -one oil was obtained. The structure was confirmed by infrared absorption spectrum (IR), nuclear magnetic resonance spectrum (NMR) and mass spectrum (MS). IR (KBr) cm ^-1 : 3420, 2930, 1750, 173
_{0,1700,1660,1455,1245 NMR (CDCl 3): 2.91} (2H, m, CH 2 S), 3.29
(2H, s, CH ₂ COO), 4.05 (1H, d, J = 8.0, C
(3a) -H), 4.18, 4.26, 4.73, 4.76 (4H, 4
d, J = 15.4, CH ₂ Ph), 7.12-7.28 (10H, m,
Ph) MS: m / e 427 (MH ⁺ ).

Example 22 Pseudomonas aeruginosa IFO 3447 was treated with 1% dextrin, 1% glucose, 1% glycerol,
Peptone 0.5%, yeast extract 0.5%, meat extract 0.5
%, Salt 0.3%, calcium carbonate 0.5%, pH 7.2 40 ml medium [200 ml Erlenmeyer (Erl
enmeyer) in a flask] and cultured under shaking at 28 ° C. for 48 hours. 2 ml of the obtained culture solution was used in Examples 1 and 1.
A methanol solution (80 mg / ml) of the compound [I] obtained in 8, 19 and 20 was added. 2 this culture at 28 ℃
It was kept under shaking for 3 hours. After the reaction, the conversion rate of the reaction and the compound [IIa] and the compound [I] were treated in the same manner as in Example 14.
Ib] was calculated. The results are shown in Table 6.

[0115]

[Table 6] Substrate R = substrate concentration (mg / ml) conversion rate (%) [IIa]: [IIb] CH ₃ 2 55 18:82 CH ₃ 4 50 10:90 C ₂ H ₅ 2 57 20:80 C ₃ H ₇ 2 37 43:57 C ₄ H ₉ 2 42 17:83

Example 23 (±)-(3aα, 4α, 6aα) -Tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one (3 .36 g) was hydrolyzed with Pseudomonas aeruginosa IFO 3447, and the reaction solution was extracted with ethyl acetate. The extract was concentrated to dryness under reduced pressure and quantified by HPLC.
-(3aα, 4α, 6aα) -tetrahydro-4-hydroxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one (0.789 g, compound [IIa]
The ratio with [IIb] is 96: 4), (±)-(3aα, 4α, 6a
α) -Tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2 (3H)-
It was found to contain on (2.30 g). The dry solid was then dissolved in acetone (7.0 ml) by heating, cooled and cooled to (+)-(3aα, 4α, 6aα) -tetrahydro-4-hydroxy-1,3-dibenzyl-1H-thieno [3, 4-d]
Crystals of imidazol-2 (3H) -one (0.410 g, compound [IIa] to [IIb] ratio 97: 3) were obtained.

Example 24 (-)-(3aα, 4α, 6aα) -Tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one (5 0.0 g of ethanol (2
5 ml), 75% of 5% potassium hydroxide was added, and the mixture was heated at 60 ° C. for 1.5 hours. The crystals crystallized by ice cooling were filtered and dried to give (−)-(3aα, 4α, 6aα) -tetrahydro-4-hydroxy-1,3-dibenzyl-1H.
-Thieno [3,4-d] imidazol-2 (3H) -one (4.
36 g, yield 98%) was obtained.

Example 25 (-)-(3aα, 4α, 6aα) -Tetrahydro-4-hydroxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one (1 .50 g) was stirred in dichloromethane (50 ml) at 0 ° C. Add phosphorus trichloride (0.
225 ml) was added dropwise. After continuing the reaction for 90 minutes at room temperature,
The solvent was distilled off under reduced pressure. Diethylene glycol dimethyl ether (30 ml) was added to the residue, and the mixture was cooled to 0 ° C. Sodium borohydride (0.85g) was added to this, and the temperature was 80 ° C.
And reacted for 3 hours. Then, this reaction solution was added to ice water, and the precipitated crystals were filtered and dried to give (3aα, 6aα)-
Tetrahydro-1,3-dibenzyl-1H-thieno [3,
4-d] imidazol-2 (3H) -one (1.37 g, 96% yield) was obtained.

Example 26 (-)-(3aα, 4α, 6aα) -Tetrahydro-4-hydroxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one (1 .50 g) was stirred in dichloromethane (50 ml) at 0 ° C. Sulfuryl chloride
(0.485 ml) was added dropwise. After continuing the reaction at room temperature for 90 minutes, the solvent was distilled off under reduced pressure. Diethylene glycol dimethyl ether (50 ml) was added to the residue, and the mixture was cooled to 0 ° C.
Sodium borohydride (1.41 g) was added to this, and 8
The reaction was carried out at 0 ° C for 3 hours. Then, this reaction solution was added to ice water, and the precipitated crystals were filtered and dried to obtain (3aα, 6aα)
-Tetrahydro-1,3-dibenzyl-1H-thieno
[3,4-d] imidazol-2 (3H) -one (1.36 g,
Yield 95%).

Example 27 (-)-(3aα, 4α, 6aα) -Tetrahydro-4-hydroxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one (1 0.5 g) in dichloromethane
Stirred in (50 ml) at 0 ° C. While introducing hydrochloric acid gas, the reaction was carried out at room temperature for 30 minutes. After that, the solvent was distilled off under reduced pressure, chloroform (50 ml) and charcoal-supported palladium (0.25 g) were added to the obtained residue, and the mixture was heated to 50 kg / cm ² at room temperature for 7 minutes.
The time catalytic reduction was performed. After the catalyst was filtered off, the filtrate was concentrated under reduced pressure to give (3aα, 6aα) -tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2.
Crystals of (3H) -one (0.89 g, yield 63%) were obtained. Further, the mother liquor was concentrated to obtain 0.17 g (yield 12%) of crystals. Example 28

[0121]

[Chemical 48]

(±)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno
[3,4-d] imidazol-2 (3H) -one (4.0 mg) was dissolved in benzene (2.0 ml), and immobilized lipoprotein lipase (manufactured by Toyobo) (40 mg) and vinyl acetate (20 μl) were added. In addition, it stirred at 37 degreeC for 20 hours. The obtained reaction solution was diluted with normal hexane / isopropanol (6/4),
The conversion rate determined by high performance liquid chromatography is 55.
%, And the ratio of the compounds [IIa] and [IIb] was 99: 1. The pattern of high performance liquid chromatography is shown in FIG.

Example 29 (±)-(3aα, 4α, 6aα) -Tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno [3,4-d] imidazol-2 (3H) -one (1 Toluene (50 g)
Immobilized lipoprotein lipase (manufactured by Toyobo) (2.5 g), vinyl acetate (1.0 ml), molecular sieves 4A 1/16 (5.0 g) were added to a 1 liter Erlenmeyer flask, The mixture was stirred at 37 ° C for 16 hours. The obtained reaction solution was filtered using a membrane filter (pore size 0.45 μm, manufactured by Advantech) to remove the immobilized lipoprotein lipase and the molecular sieves. The filtrate was analyzed by high performance liquid chromatography to find that the conversion rate was 54% and the ratio of the compounds [IIa] and [IIb] was 9%.
At 9.5: 0.5, (-)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno [3,4-d] imidazole-2 (3H)- No on-peak was observed. The high performance liquid chromatography pattern is shown in FIG. Then, the reaction solution was concentrated under reduced pressure,
The crystallized crystals were filtered to give (+)-(3aα, 4α, 6aα)-
Tetrahydro-1,3-dibenzyl-4-hydroxy-
1H-thieno [3,4-d] imidazol-2 (3H) -one
(344 mg, conversion rate: 33.4%, compounds [IIa] and [II]
The ratio with b] was 99.9: 0.1). The structure was confirmed by melting point, IR, NMR and specific rotation.

Melting point: 166-168 ° C IR (KBr) cm ^-1 : 3300,1680 NMR (CDCl ₃ ): 1.7 (1H, br.s, OH), 2.86
(1H, d, J = 12.7, C (6) -H), 3.01 (1H, dd, J
= 12.7, 4.7, C (6) -H), 4.02 (1H, d, J = 7.
9, C (3a) -H), 4.21 (1H, dd, J = 7.9, 4.6, C
(6a) -H), 4.21, 4.32, 4.66, 4.74 (1H, d, J = 15.5, NCH2), 5.18 (1H, s, C)
(4) -H), 7.2-7.4 (10H, m, Ph) Specific optical rotation: [α] _D ²⁷ = + 71.7 (C = 0.87, chloroform) Furthermore, the mother liquor was concentrated under reduced pressure. To give an oil, which was chromatographed on silica gel (hexane / ethyl acetate = 2).
/) And is purified by (+)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-hydroxy-1.
H-thieno [3,4-d] imidazol-2 (3H) -one
(87 mg, 8.7%, the ratio of the compounds [Ia] and [Ib] was 9
9.0: 1.0) and (-)-(3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-acetoxy-1H
-Thieno [3,4-d] imidazol-2 (3H) -one (6
00 mg, conversion rate: 53.4%).

Example 30 Dimethyl sulfoxide (250 ml) and acetic anhydride (46.3 m)
l) were mixed and heated at 50 ° C. for 1 hour. Then (±) −
(3aα, 4α, 6aα) -Tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno [3,4-d] imidazol-2 (3H) -one (25 g) was added at the same temperature and 1.5. Stir for hours. The reaction solution was poured into ice water (1500 ml), and the precipitated crystals were collected by filtration. High-performance liquid chromatography analysis of this product revealed that (±)-(3aα, 6aα) -tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d].
82.4% imidazol-2 (3H), 4-dione, (3a
α, 4α, 6aα) -Tetrahydro-1,3-dibenzyl-
4-acetoxy-1H-thieno [3,4-d] imidazol-2 (3H) -one was produced in an amount of 9.1%. The crystals were recrystallized from ethyl acetate to give (±)-(3aα, 6aα) -tetrahydro-1,3-dibenzyl-1H-thieno [3,4-
d] 18.18 g (7%) of imidazole-2 (3H), 4-dione
3.2%). Melting point: 125-126 ° C

Example 31 Dimethyl sulfoxide (10 ml) and acetic anhydride (1.85 ml)
Were mixed and stirred at 50 ° C. for 1 hour. (±)-(3a
α, 4α, 6aα) -Tetrahydro-1,3-dibenzyl-
4-Hydroxy-1H-thieno [3,4-d] imidazol-2 (3H) -one (1.0 g) was added, and the mixture was stirred at the same temperature for 3 hours. The reaction solution was poured into ice water (100 ml), and the precipitated crystals were collected by filtration. When this product was analyzed by high performance liquid chromatography, (±)-(3aα, 6aα) -tetrahydro-
81.4% 1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H), 4-dione, (±)-(3a
α, 4α, 6aα) -Tetrahydro-1,3-dibenzyl-
The yield of 4-acetoxy-1H-thieno [3,4-d] imidazol-2 (3H) -one was 3.7%.

Example 32 Dimethyl sulfoxide (10 ml) and acetic anhydride (1.85 ml)
Was mixed and heated at 80 ° C for 15 minutes, and then (±)-
(3aα, 4α, 6aα) -Tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno [3,4-d] imidazol-2 (3H) -one (1.0 g) was added and the mixture was stirred at the same temperature for 15 Stir for minutes. The reaction solution was poured into ice water (100 ml), and the precipitated crystals were collected by filtration. This product was analyzed by high performance liquid chromatography to find that (±)-(3aα, 6aα) -tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H), 4-dione. Is 78.0%, (±)-
7.1% of (3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-acetoxy-1H-thieno [3,4-d] imidazol-2 (3H) -one was produced.

Example 33 Dimethyl sulfoxide (10 ml) and acetic anhydride (0.83 ml)
Were mixed and stirred at 50 ° C. for 1 hour. (±)-(3a
α, 4α, 6aα) -Tetrahydro-1,3-dibenzyl-
4-Hydroxy-1H-thieno [3,4-d] imidazol-2 (3H) -one (1.0 g) was added, and the mixture was stirred at 25 ° C. for 17 hours. The reaction solution was poured into ice water (100 ml) and extracted with dichloromethane. This extract was analyzed by high performance liquid chromatography to find that (±)-(3aα, 6aα) -tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H), 4-dione. Is 78.1%, (±)-
7.8% of (3aα, 4α, 6aα) -tetrahydro-1,3-dibenzyl-4-acetoxy-1H-thieno [3,4-d] imidazol-2 (3H) -one was formed. Example 34 Dimethyl sulfoxide (7.5 ml) was added to (±)-(3aα, 4α,
6aα) -Tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno [3,4-d] imidazole-2 (3
H) -one (1.0 g) was dissolved and triethylamine was added to it.
(3.35 g) was added. The mixture was cooled to 17 to 19 ° C and dissolved in dimethyl sulfoxide (7.5 ml) with stirring. Sulfur trioxide / pyridine complex salt (1.52 g) was added dropwise. After stirring at the same temperature for 45 minutes, pour into ice water (100 ml) and pour with 10% hydrochloric acid.
Adjusted to H4.5. After stirring for 1 hour, the precipitated crystals were filtered, washed with water and dried to give (±)-(3aα, 6aα) -tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazole-2 (3H ), 4-dione (0.82 g, 82.5%)
Was obtained (purity 93.9%).

Reference Example 1 (±)-(3aα, 4α, 6aα) -Tetrahydro-1,3-dibenzyl-4-hydroxy-1H-thieno [3,4-d] imidazol-2 (3H) -one (0 .500g) and (±)-(3a
α, 4α, 6aα) -Tetrahydro-4-acetoxy-1,
3-Dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one (0.500 g) in acetone (5.00 ml)
Was added and melted at 50 ° C. It was cooled to 0 ° C. and crystallized. The yield of the obtained crystals was 0.425 g, and the yield was 85.
% Of (±)-(3aα, 4α, 6aα) -tetrahydro-4-
Hydroxy-1,3-dibenzyl-1H-thieno [3,4
-D] imidazol-2 (3H) -one was in complete agreement in nuclear magnetic resonance spectrum and infrared absorption spectrum. In addition, (±)-(3aα, 4α, 6aα) -tetrahydro-4-acetoxy-1,3-dibenzyl-1H-thieno is included in the crystal.
No [3,4-d] imidazol-2 (3H) -one was observed.

Reference Example 2 (±)-(3aα, 4α, 6aα) -Tetrahydro-4-hydroxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one (0 .200g) and (±)-(3a
α, 4α, 6aα) -Tetrahydro-4-acetoxy-1,
3-Dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one (0.800 g) in acetone (5.00 ml)
Was added and melted at 50 ° C. It was cooled to 0 ° C. and crystallized. The yield of the obtained crystals was 0.13 g, 65%,
(±)-(3aα, 4α, 6aα) -tetrahydro-4-hydroxy-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2 (3H) -one and nuclear magnetic resonance spectrum,
The infrared absorption spectra were in perfect agreement. In addition, (±)-(3aα, 4α, 6aα) -tetrahydro-4-
Acetoxy-1,3-dibenzyl-1H-thieno [3,4
No -d] imidazol-2- (3H) -one was observed.

Reference Example 3 Tetrahydrofuran (5.3 ml) was added to magnesium (0.90).
g) was added and stirred. A few drops of 1,2-dichloroethane were added to this and heated to 42 ° C. to start the reaction. After cooling to 35 ° C, 1,4-dichlorobutane (2.4 g) and tetrahydrofuran (10 ml) were slowly added dropwise. After stirring at the same temperature for 3 hours, the mixture was cooled to -50 ° C. (+)-(3aα,
6aα) -Tetrahydro-1,3-dibenzyl-1H-thieno [3,4-d] imidazol-2- (3H), 4-dione
(2.1 g) and tetrahydrofuran (20 ml) were added dropwise. After stirring at the same temperature for 1 hour, the mixture was cooled to -50 ° C and carbon dioxide was passed through for 30 minutes. 10% Hydrogen chloride water (40 ml) was added dropwise at 10 ° C. Extract with toluene and add concentrated sulfuric acid (0.06g) to it.
Was added and the mixture was stirred at 80 ° C. for 1 hour. The reaction mixture was cooled, washed with saturated brine, dried over sodium sulfate, and concentrated to give an oil. This was dissolved in 5% potassium hydroxide (40 ml) and washed with diethyl ether. After neutralizing with 20% sulfuric acid, it was extracted with ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure to give an oily substance. Add diethyl ether to this,
(+)-(3aα, 6aα) -Tetrahydro-1,3-dibenzyl-2- (ω-carboxybutylidene) -1H-thieno
Crystals of [3,4-d] imidazol-2- (3H) -one (2.
14 g) was obtained. Melting point: 87 ° C. Specific rotation: [α] _D = + 222 (c = 1.0, methanol)

(+)-(3aα, 6aα) -tetrahydro-1,
3-dibenzyl-4- (ω-carboxybutylidene) -1
H-thieno [3,4-d] imidazol-2- (3H) -one
(1.80 g) and isopropanol (100 ml) were placed in a 200 ml autoclave. Hydrogen pressure 50kg / cm ² , 50 ℃
The reduction reaction was carried out for 3 hours. After cooling, the catalyst was removed by filtration, and this was concentrated under reduced pressure to give (-)-(3aα, 6aα)-
Tetrahydro-1,3-dibenzyl-4- (ω-carboxybutyl) -1H-thieno [3,4-d] imidazole-2
-(3H) -one (1.59 g) was obtained. Melting point: 93 ° C. Specific rotation: [α] _D ²² = -26.6 (c = 1.03, methanol)

(-)-(3aα, 4β, 6aα) -tetrahydro-1,3-dibenzyl-4- (ω-carboxybutyl)-
1H-thieno [3,4-d] imidazol-2- (3H) -one (0.95 g), phosphoric acid (9.5 ml), phenol (0.22 m)
l) was stirred at 150 ° C. for 3 hours. After cooling, water (100 ml) was added and the mixture was washed with diethyl ether. Cool the water layer and D
-Biotin crystals (285 mg) were obtained. Melting point: 221-223 ° C. Specific optical rotation: [α] _D ³⁰ = + 87.2 (c = 1.08, N / 10)
Sodium hydroxide)

[0134]

EFFECT OF THE INVENTION Using the compound [I] of the present invention as a starting material, optically active D-biotin can be easily produced with high yield and high purity. An optically active compound [I] is obtained by bringing a culture solution or a treated product of a microorganism into contact with the compounds [I ′] and [I ″].
I '] or [II "] can be easily obtained with high yield.

[Brief description of drawings]

FIG. 1 shows a chromatogram obtained by high performance liquid chromatography of the reaction liquid obtained in Example 10.
The horizontal axis of the figure shows the retention time (unit: minutes), and the corresponding compounds are shown above the peaks.

FIG. 2 shows a chromatogram obtained by high performance liquid chromatography of the reaction liquid obtained in Example 11.
The horizontal axis of the figure shows the retention time (unit: minutes), and the corresponding compounds are shown above the peaks.

FIG. 3 shows a chromatogram obtained by high performance liquid chromatography obtained in Example 28.

FIG. 4 shows a chromatogram obtained by high performance liquid chromatography obtained in Example 29.

[Explanation of symbols]

 A is [Ia ']:

[Chemical 49] B is [Ib ']:

[Chemical 50] C is [IIa]:

[Chemical 51] D is [IIb]:

[Chemical 52]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12R 1: 085) (C12P 17/18 C12R 1:11) (C12P 17/18 C12R 1:13) (C12P 17/18 C12R 1:38) (C12P 17/18 C12R 1:01) (C12P 17/18 C12R 1: 465) (C12P 17/18 C12R 1:72) (C12P 17/18 C12R 1: 645)

Claims (11)

[Claims] 1. A general formula: The compound represented by. 2. A general formula: The compound according to claim 1, which is represented by: 3. A general formula: The compound according to claim 1, which is represented by: 4. A general formula: In a mixture of compounds represented by, Acetobacter, Bacillus, Brevibacterium, Pseudomonas, Streptomyces, Ampliariella, candy having the ability to deacylate any one acyl group more quickly than the other. A general formula characterized by contacting a culture of a microorganism belonging to the genus Da or Trichosporon or a treated product thereof The method for producing an optically active substance represented by. 5. The formula: A compound represented by the general formula: The manufacturing method of the compound represented by. 6. The formula: A compound represented by the following general formula: The manufacturing method of the compound represented by. 7. A general formula: A compound represented by the formula: A method for producing the compound represented by. 8. The formula: A mixture of compounds represented by the formula (1) is contacted with a bacterial esterase having the ability to acylate one hydroxyl group more rapidly than the other in the presence of an acyl group donor. ] The manufacturing method of the compound represented by. 9. The method according to claim 8, wherein the bacterium is Pseudomonas sp., Streptomyces sp., Bacillus sp. Or Acetobacter sp. 10. The formula: Wherein the compound represented by the formula is oxidized by dimethyl sulfoxide and an activator. The manufacturing method of the compound represented by. 11. The formula: In the presence of an acyl group donor, a mixture of compounds represented by is contacted with a bacterial esterase having the ability to acylate one hydroxyl group more quickly than the other,
General formula to generate After removing the compound represented by the formula: A compound represented by the formula: The manufacturing method of the compound represented by.