JP2002191392A - Method for producing nucleoside derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for the effective mass-synthesis of an L-type nucleoside derivative free from the defects of conventional production process and enabling the supply of the derivative without limitation. SOLUTION: The separation and purification of the objective compound are easily performed by the selective decomposition of specific isomers using a nuclease.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for selectively producing optical isomers and stereoisomers of nucleoside derivatives. In particular, the present invention relates to a method for selectively producing a nucleoside derivative having an L-shaped configuration. Antiviral activity of L-type nucleoside derivatives has been reported, and development of a large number of derivatives is expected.

[0002]

2. Description of the Related Art Hitherto, as a method for producing an optical isomer of an L-type nucleoside derivative, two methods are roughly classified. (1) A method of chemically synthesizing a sugar having an L-type configuration, using the sugar as a raw material, and condensing it with a nucleoside base to synthesize an L-type nucleoside derivative. (2) A method of synthesizing a nucleoside derivative as a mixture of optical isomers and optically resolving the mixture to synthesize an L-type nucleoside derivative.

When the method (1) is used, there is a problem in the step of condensing a nucleoside base. In this step, the same reaction result is obtained for both the L-form and the D-form. Therefore, a method for synthesizing a D-type nucleoside derivative, which has been known for a long time, has been used. Methods are known. (1) -1.6-Chloropurine sodium salt is L-2
-A method of condensing with a deoxyribosyl-1-chloride derivative and then ammonolytically or hydrolyzing chlorine on the purine to obtain an L-type purine nucleoside. Further, after condensation with silylated uracil, the carbonyl of uracil is converted to a sulfur adduct, and further subjected to ammonia decomposition to obtain L-type 2-deoxycytidine [Nucleos
ides & Nucleotides, 11 (2-
4), 341-349 (1992)]. (1) The silyl form of -2.6-chloropurine was converted to L-2,
A method in which a 3-deoxy-3-fluoro-1-O-methylribose derivative is condensed with a Lewis acid in the presence, and then chlorine on the purine is subjected to ammonia decomposition or hydrolysis to obtain an L-purine nucleoside. Further, a method of obtaining an L-type cytidine derivative by condensing with a silylated cytosine derivative and then hydrolyzing a protecting group on the cytosine [Carbohyd]
rateResearch, 328, 49-59 (20
00)]. (1) -3. A method of condensing an optically active oxathiolane derivative having an L-type configuration with a silylated cytosine, and separating and purifying the resulting cis-trans stereoisomer mixture by column chromatography [J. Org. Che
m. , 60, 2621-2623 (1995); Tet.
rahedron: Asymmetry, 6 (2), 3
93-396 (1995); Org. Chem. ,
56, 6503-6505 (1991)].

However, in any of the reported examples, since the stereoselectivity in the step of condensing the nucleoside base is low, an extra step such as conversion to sulfur is required, and purification by column chromatography cannot be avoided. However, even if it can be purified by recrystallization, the production method is industrially unsuitable, such as a large purification loss.

As the method (2), the following method is known. (2) -1. An optical isomer mixture of an oxathiolan-5-ylcytosine derivative was converted to an HPL using an optical resolution column.
Separation method by C [registered patent 2927546]. (2) -2. The optical isomer mixture of the oxathiolan-5-ylcytosine derivative is converted into the uracil derivative by the immobilized cytidine deaminase, and the remaining L-form oxathiolan-5-ylcytosine derivative is separated by an ion exchange resin. Purification method [registered patent 2927546]. (2) -3. Once the mixture of optical isomers of the oxathiolan-5-ylcytosine derivative is once converted to a carboxylic acid ester, the ester of the D-type optical isomer is hydrolyzed by pig liver esterase, and the remaining L-type optical isomer is left. Are separated by an extraction operation. Furthermore, a method of obtaining an L-type oxathiolan-5-ylcytosine derivative by hydrolyzing the ester [Registered Patent 2901160].

[0006] Among the above methods, the method (2) -1 has a limit in mass supply. (2) In the method of -2,
In order to convert the D-type isomer into a compound having relatively similar properties, an industrially difficult purification and separation method such as an ion exchange resin is required. Further, since the isomeric nucleoside is separated and removed as a uracil adduct, a relatively expensive nucleoside base cannot be recovered and reused. In the method (2) -3, two additional steps of a step of converting the ester to an ester form and a step of hydrolyzing the ester again are required. Further, in order to reuse the D-type isomer, more steps are required.

[0007]

As described above, conventionally, it is difficult to separate and purify stereoisomers or optical isomers.
There has been no method for producing an L-type nucleoside derivative that can be supplied efficiently and in large quantities. The present invention provides a production method for efficiently producing an L-type nucleoside derivative without restriction on the supply amount.

[0008]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, by treating a stereoisomeric or optical isomer mixture of a nucleoside derivative with a nuclease, The present inventors have found that an L-type nucleoside derivative can be efficiently synthesized, and have completed the present invention.
That is, the present invention provides [1] a nucleolytic enzyme, a microorganism having the enzyme activity, or a processed product thereof on a mixture of isomers of nucleoside derivatives to decompose a specific isomer of the mixture of isomers. And a process for producing an isomer-selective nucleoside derivative, characterized in that only one type of isomer remains, and [2] a general formula [1]
1]

[0009]

[Formula 11]

Wherein B is a base selected from the group consisting of pyrimidine, purine, azapurine and deazapurine, which are halogen, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, amino It may be substituted by a group, an alkylamino group, a hydroxyl group, a hydroxyamino group, an aminoxy group, an alkoxy group, a mercapto group, an alkylmercapto group, an aryl group, an aryloxy group or a cyano group. X
Represents a halogen atom, an alkoxy group or an alkylthio group, W represents an oxygen atom or a sulfur atom, Z represents an oxygen atom, a sulfur atom or an optionally substituted carbon atom, p represents an integer of 0 to 2, q represents an integer of 0 to 4. (However, when Z is an oxygen atom or a sulfur atom, p + q ≦ 2 is satisfied, and when Z is a carbon atom, p + q ≦ 4 is satisfied.) A mixture of isomers of a nucleoside derivative represented by the following formula: Or a microorganism having the enzyme activity or a treated product thereof, wherein a specific isomer in the mixture of isomers is decomposed and only one type of isomer remains, and the general formula [2] [Formula 12]

[0011]

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(Wherein B, X, W, Z, p, and q are as defined above), which is a method for producing a nucleoside derivative represented by the following general formula [3]:

[0013]

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[In the formula, B has the same meaning as [1] described in [2], and W and Z each independently represent an oxygen atom or a sulfur atom. From the isomer mixture of the nucleoside derivative represented by the general formula [4]

[0015]

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(Wherein B, W, and Z have the same meanings as described above), which is a production method according to [2], wherein [4] a general formula [5] [formula 15]

[0017]

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[Wherein B and X have the same meanings as [1] described in [2], p represents an integer of 0 to 2, and q represents 0 to 4
Represents an integer. (However, p + q ≦ 4 is satisfied.) From the isomer mixture of the nucleoside derivative represented by the general formula [6]

[0019]

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(Wherein B, X, p, and q have the same meanings as described above), which is a production method according to [2], wherein [5] a general formula [7] 17]

[0021]

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[Wherein, B, W and Z have the same meanings as [3] of the third aspect. By treating the optical isomer mixture of the nucleoside derivative represented by the above with a nuclease, the D-type isomer of the optical isomer mixture is decomposed,
A compound represented by the following general formula [8], wherein only the L-type isomer remains:

[0023]

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(Wherein B, W, and Z have the same meanings as described above), which is a method for producing an L-type nucleoside derivative represented by the following general formula:

[9] [Chemical Formula 19]

[0025]

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[Wherein, B has the same meaning as [1] described in [2]. By treating the stereoisomeric mixture of the nucleoside derivative represented by the above with a nuclease, the trans isomer of the isomer mixture is decomposed, leaving only the cis isomer remaining, Formula [10] [Formula 2
0]

[0027]

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(Wherein B has the same meaning as defined above), which is a method for producing a cis-type nucleoside derivative represented by the formula:
[7] The production method according to [1] to [6], wherein the nuclease is a nucleoside phosphorylase,
[8] Nucleoside phosphorylases include purine nucleoside phosphorylase (EC 2.4.2.1), guanosine phosphorylase (EC 2.4.2.15), and pyrimidine nucleoside phosphorylase (EC 2.4.2.
2), uridine phosphorylase (EC 2.4.2.
3), thymidine phosphorylase (EC 2.4.2.
4), deoxyuridine phosphorylase (EC 2.4.
The production method according to any one of [1] to [6], which is one or more nucleoside phosphorylases selected from the group consisting of 2.23).

[0029]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. As the base in the nucleoside derivative represented by the general formula [1], a natural or unnatural base selected from the group consisting of pyrimidine, purine, azapurine and deazapurine is shown, which includes a halogen atom, an alkyl group, a haloalkyl Group, alkenyl group, haloalkenyl group, alkynyl group, amino group, alkylamino group, hydroxyl group, hydroxyamino group, aminoxy group, alkoxy group, mercapto group, alkylmercapto group, aryl group, aryloxy group or cyano group May be.

Examples of the halogen atom as a substituent include chlorine, fluorine, iodine and bromine. Examples of the alkyl group include those having 1 to 7 carbon atoms such as methyl, ethyl and propyl.
Are exemplified. Examples of the haloalkyl group include those having 1 to 1 carbon atoms such as fluoromethyl, difluoromethyl, trifluoromethyl, bromomethyl, and bromoethyl.
An example is a haloalkyl group having 7 alkyls. Examples of the alkenyl group include those having 2 to 2 carbon atoms such as vinyl and allyl.
7 alkenyl groups are exemplified. Haloalkenyl groups include bromovinyl, chlorovinyl and the like having 2 to 7 carbon atoms.
Examples are haloalkenyl groups having an alkenyl of
Examples of the alkynyl group include alkynyl groups having 2 to 7 carbon atoms, such as ethynyl and propynyl. Examples of the alkylamino group include alkylamino groups having 1 to 7 carbon atoms such as methylamino and ethylamino. Examples of the alkoxy group include an alkoxy group having 1 to 7 carbon atoms such as methoxy and ethoxy. Examples of the alkyl mercapto group include an alkyl mercapto group having 1 to 7 carbon atoms such as methyl mercapto and ethyl mercapto. Examples of the aryl group include a phenyl group; an alkylphenyl group having 1 to 5 carbon atoms such as methylphenyl and ethylphenyl, an alkoxyphenyl group having 1 to 5 carbon atoms such as methoxyphenyl and ethoxyphenyl, and dimethylamino. 1 to 1 carbon atoms such as phenyl and diethylaminophenyl
Examples thereof include an alkylaminophenyl group having an alkylamino of 5, and a halogenophenyl group such as chlorophenyl and bromophenyl.

Specific examples of pyrimidine bases include cytosine, uracil, 5-fluorocytosine, 5-fluorouracil, 5-chlorocytosine, 5-chlorouracil, 5-bromocytosine, 5-bromouracil, and 5-yo- Dotocytosine, 5-iodouracil, 5-methylcytosine, 5-methyluracil (thymine), 5-ethylcytosine, 5-ethyluracil, 5-fluoromethylcytosine, 5-fluorouracil, 5-trifluorocytosine, 5-trito Fluorouracil, 5-vinyluracil,
5-bromovinyluracil, 5-chlorovinyluracil, 5-ethynylcytosine, 5-ethynyluracil,
-Propynyluracil, pyrimidin-2-one, 4-hydroxyaminopyrimidin-2-one, 4-aminooxypyrimidin-2-one, 4-methoxypyrimidin-2
-One, 4-acetoxypyrimidin-2-one, 4-fluoropyrimidin-2-one, 5-fluoropyrimidin-2-one and the like.

Specific examples of the purine base include purine, 6-aminopurine (adenine), 6-hydroxypurine, 6-fluoropurine, 6-chloropurine, 6-methylaminopurine, 6-dimethylaminopurine, 6-trifluoromethylaminopurine, 6-benzoylaminopurine, 6-acetylaminopurine, 6-hydroxyaminopurine, 6-aminooxypurine, 6-methoxypurine, 6-acetoxypurine, 6-benzoyloxypurine, 6- Methyl purine, 6-ethylpurine, 6-trifluoromethylpurine, 6-phenylpurine, 6-mercaptopurine, 6-methylmercaptopurine, 6-aminopurine-1-oxide, 6-hydroxypurine-1-
Oxide, 2-amino-6-hydroxypurine (guanine), 2,6-diaminopurine, 2-amino-6-chloropurine, 2-amino-6-iodopurine, 2-aminopurine, 2-amino-6 -Mercaptopurine, 2-amino-6-methylmercaptopurine, 2-amino-6-hydroxyaminopurine, 2-amino-6-methoxypurine, 2-amino-6-benzoyloxypurine, 2-amino-6-acetoxy Purine, 2-amino-6-methylpurine, 2-amino-6-cyclopropylaminomethylpurine, 2-amino-6-phenylpurine, 2-amino-8-bromopurine, 6-cyanopurine, 6-amino- 2-chloropurine (2-chloroadenine), 6-amino-2-fluoropurine (2-fluoroadenine) and the like.

Specific examples of the azapurine base and the deazapurine base include 6-amino-3-deazapurine,
6-amino-8-azapurine, 2-amino-6-hydroxy-8-azapurine, 6-amino-7-deazapurine, 6-amino-1-deazapurine, 6-amino-2-
Azapurin and the like.

Nucleoside derivatives consisting of these bases include 2'-deoxythymidine, 2'-deoxycytidine, 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxy-2'-fluorothymidine,
2'-deoxy-2'-fluorocytidine, 2'-deoxy-2'-fluoroadenosine, 2'-deoxy-
2'-fluoroguanosine, 3'-thia-2 ', 3'-
Dideoxythymidine, 3'-thia-2 ', 3'-dideoxycytidine, 3'-thia-2', 3'-dideoxyadenosine, 3'-thia-2 ', 3'-dideoxyguanosine, 3'-thia- 2 ′, 3′-dideoxy-5-fluorocytidine, 3′-oxa-2 ′, 3′-dideoxythymidine, 3′-oxa-2 ′, 3′-dideoxycytidine, 3′-oxa-2 ′, 3 '-Dideoxyadenosine, 3'-oxa-2', 3'-dideoxyguanosine and 3'-oxa-2 ', 3'-dideoxy-5-fluorocytidine can be exemplified, but not limited thereto.

As the isomer mixture, both the cis-trans stereoisomer between the 1-position and the 4-position corresponding to the furan ring and the mixture of the D-form and the L-form optical isomer corresponding to the natural nucleoside are provided. Or a mixture containing both.

[0036] Nucleolytic enzyme is a generic term for enzymes that degrade N-glycoside bonds of nucleosides in the presence of phosphoric acid. The enzyme used in the reaction may be of any type and origin as long as it has an activity capable of decomposing a specific isomer of the corresponding nucleoside derivative. The enzyme is roughly classified into a purine type and a pyrimidine type. For example, purine nucleoside phosphorylase (EC 2.4.
2.1), guanosine phosphorylase (EC 2.4.
2.15), pyrimidine nucleoside phosphorylase (EC 2.4.2.2), uridine phosphorylase (EC 2.4.2.3), thymidine phosphorylase (EC 2.4.2.4), deoxyuridine phosphorylase (EC2) as pyrimidine type 4.2.2.23). The microorganism expressing the nucleoside phosphorylase in the present invention includes purine nucleoside phosphorylase (EC 2.4.2.1), guanosine phosphorylase (EC 2.4.2.15), and pyrimidine nucleoside phosphorylase (EC 2.4.2.2). ), Uridine phosphorylase (EC 2.4.2.3), thymidine phosphorylase (EC 2.4.2.4), deoxyuridine phosphorylase (EC 2.4.2.23), and at least one nucleoside selected from the group consisting of The microorganism is not particularly limited as long as it is a microorganism expressing phosphorylase.

Specific examples of such microorganisms include the genus Nocardia and Microbacteria.
erium) genus Corynebacterium (Corynebacterium) genus,
Brevibacterium (Brevibacteriu m) genus Cellulomonas (Cellulomonas) genus Flavobacterium (Flabob
acterium) genus, Kuruihera (Kluyvere) genus Mycobacterium (Micobacterium) genus, Haemophilus (Haemophilus)
Genus, Mikopurana (Micoplana) genus, professional data Mino Enterobacter (Pr
otaminobacter) genus Candida (Candida) genus Saccharomyces (Saccharomyces) genus Bacillus (Bacillus)
Genus, thermophilic Bacillus, Pseudomonas
s) belonging to the genus, Micrococcus (Micrococcus) genus, hafnia (Haf
nia) genus, Proteus (Proteus) genus Vibrio (Vibrio)
Sp., Staphylococcus (Staphyrococcus) genus Propionibacterium (Propionibacterium) genus, Zaruchina (Sa
rtina) genus, Plano Lactococcus (Planococcus) genus Escherichia (Escherichia) genus, Kurthia (Kurthia) genus, Rodokokkkasu (Rhodococcus) genus, Acinetobacter (Acinetob
acter) genus, key Santo Enterobacter (Xantho bacter) genus Streptomyces (Streptomyces) genus Rhizobium (Rhizobi
um) genus Salmonella (Salmonella) species, Klebsiella (Kleb
siella ), Enterobacter ( Enterobacter ), Erwinia ( Erwinia ), Aeromonas ( Aeromonas ), Citrobacter ( Citrobacter ), Achromobacter ( Achrobacter)
omobacter ), Agrobacterium
Microorganism strains included in the genera, the genus Arthrobacter or the genus Pseudonocardia can be mentioned as preferred examples.

According to recent advances in molecular biology and genetic engineering, the gene of the protein is obtained from the microorganism strain by analyzing the molecular biological properties, amino acid sequence, and the like of the nucleoside phosphorylase of the microorganism strain described above. Constructing a recombinant plasmid into which the gene and the control region necessary for expression have been inserted, introducing this into any host, and making it possible to produce a genetically modified bacterium expressing the protein, and It's also relatively easy. In view of the state of the art, genetically modified bacteria having such a nucleoside phosphorylase gene introduced into an arbitrary host are also included in the microorganism expressing the nucleoside phosphorylase of the present invention. The control region necessary for expression herein includes a promoter sequence (including an operator sequence for controlling transcription), a ribosome binding sequence (SD sequence), a transcription termination sequence, and the like. Specific examples of the promoter sequence include trp promoter of tryptophan operon derived from Escherichia coli, lac promoter of lactose operon, PL promoter and PR promoter derived from lambda phage, gluconate synthase promoter (gnt) derived from Bacillus subtilis, and alkaline protease promoter. (A
pr) · neutral protease promoter (npr) · α
-Amylase promoter (amy) and the like.
In addition, sequences that have been independently modified and designed, such as the tac promoter, can also be used. As the ribosome binding sequence,
Examples include sequences derived from Escherichia coli or Bacillus subtilis, but are not particularly limited as long as they function in a desired host such as Escherichia coli or Bacillus subtilis. For example, a consensus sequence in which a sequence complementary to the 3 ′ terminal region of 16S ribosomal RNA is continuous for 4 bases or more may be prepared by DNA synthesis and used. Although a transcription termination sequence is not necessarily required, a p-factor independent one such as a lipoprotein terminator or a trp operon terminator can be used. The sequence of these control regions on the recombinant plasmid is preferably arranged in the order of the promoter sequence, the ribosome binding sequence, the gene encoding nucleoside phosphorylase, and the transcription termination sequence from the 5 'terminal side upstream.

Specific examples of the plasmid referred to herein include:
PBR3 having an autonomously replicable region in Escherichia coli
22, pUC18, Bluescript II SK
(+), PKK223-3, pSC101, and pUB110, pSC110 which has a region capable of autonomous replication in Bacillus subtilis.
TZ4, pC194, ρ11, φ1, φ105 and the like can be used as vectors. Examples of plasmids capable of autonomous replication in two or more hosts include p
HV14, TRp7, YEp7 and pBS7 can be used as vectors. The arbitrary host referred to herein, E. coli (Escherichia coli), but may be mentioned as a typical example, in particular Bacillus subtilis (Bacillus subtilis s) such as not of being limited to E. coli, as described in Examples below Other microbial strains such as fungi, yeasts and actinomycetes are also included.
The nucleoside phosphorylase activity in the present invention includes not only the above-mentioned microbial cells having the enzymatic activity, but also the processed cells of the microbial cells having the enzymatic activity or their immobilized products. What is treated with cells
For example, acetone-dried cells and mechanical disruption, ultrasonic crushing, freeze-thaw treatment, pressure-reduced pressure treatment, osmotic pressure treatment, autolysis, cell wall decomposition treatment, cell crushed substances prepared by surfactant treatment, etc. Further, if necessary, those obtained by repeating purification by ammonium sulfate precipitation, acetone precipitation or column chromatography may be used.

As described above, according to the present invention, the nucleoside derivative [2] can be produced more efficiently without mixing of isomers.

[0041]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Reference Example 1 Benzyl 5-acetoxy-1,3-oxathiolane
2-Carboxylate 2-DMAP28.9 was added to a solution of 28.9 g of 5-acetoxy-1,3-oxathiolane-2-carboxylic acid and 17.1 mL of benzyl alcohol in 434 mL of dichloromethane.
In the presence of mg, 37.2 g of DCC was added at room temperature. After 3 hours, the precipitate was removed by filtration, washed with dichloromethane, and the filtrate was concentrated. The concentrated residue was purified by column chromatography, and from ethyl acetate / n-hexane (1: 4) stream,
Benzyl 5-acetoxy-1,3-oxathiolane-2
-13.3 g of carboxylate were obtained as an oil. The yield was 31%. ¹ H NMR (CDCl ₃ , 270 MHz) d 7.36 (s, 5 H), 6.79 (d,
J = 4.0 Hz, 1 H), 5.69 (s, 1 H), 5.20 (m, 2 H), 3.4
3 (dd, J = 4.0 & 12.0 Hz, 1 H), 3.16 (d, J = 12.0 H
z, 1 H), 2.09 (s, 3 H).

Reference Example 2 Benzyl 5-cytosine-1'-yl-1,3-oxathiolane-2-carboxylate 4.0 g of 5-fluorocytosine and 80 m of ammonia sulfate
80 g of HMDS was added to g, and the mixture was heated and stirred at 120 ° C for 3 hours. After completion of the reaction, the mixture was concentrated to dryness. The obtained white solid and 7.0 g of benzyl 5-acetoxy-1,3-oxathiolane-2-carboxylate were dissolved in 210 mL of dichloromethane, and trimethylsilyl triflate was added under ice-cooling. 38 mL was added dropwise. After standing on a water bath overnight, saturated aqueous sodium hydrogen carbonate was added, and the mixture was extracted with 5% methanol / dichloromethane. The organic layer was dried over magnesium sulfate and concentrated. 25 mL of methanol and 5 mL of ethyl acetate
0 mL and 100 mL of n-hexane were added, and the mixture was stirred at room temperature overnight. The precipitate was collected by filtration, washed with ethyl acetate / n-hexane (1: 4) and dried, and 3.03 g of benzyl 5-cytosin-1′-yl-1,3-oxathiolane-2-carboxylate was obtained as a colorless powder. As obtained. Yield 35
%Met. ¹ H NMR (DMSO-d ₆ , 270 M Hz) d 8.15 (d, J = 7.1 Hz,
1 H), 7.93 (s, 1 H), 7.68 (s, 1 H), 7.40 (m, 5 H),
6.30 (ddd, J = 2.0, 5.1 & 6.6 Hz, 1 H), 5.79 (s,
1 H), 5.29 (d, J = 12.2 Hz, 1 H), 5.23 (d, J = 12.
2 Hz, 1 H), 3.55 (dd, J = 5.1 & 12.2 Hz, 1 H), 3.20 (dd, J = 6.6 &
12.2 Hz, 1 H): FT-IR (cm ^-1 , KBr) 3382, 3072, 1742,
1694, 1631, 1508, 1182, 1079.

Reference Example 3 Mixture of optical isomers of 3'-thia-2 ', 3'-dideoxycytidine benzyl 5-cytosin-1'-yl-1,3-oxathiolane-2-carboxylate 2.83 g of methanol / To a mixed solution of 113 mL of dichloromethane (1: 1),
649 mg of lithium borohydride was added under ice cooling. After stirring at room temperature for 2 hours, 22 g of silica gel was added, and the mixture was further stirred overnight. The silica gel was removed by filtration, and the filtrate was concentrated. The concentrated residue was crystallized by adding ethyl acetate,
It was suspended in 15 mL of methanol and 70 mL of ethyl acetate. The precipitate was collected by filtration and dried under reduced pressure to give 3′-thia-2 ′,
1.18 g of 3'-dideoxycytidine was obtained as a mixture of optical isomers. The yield was 46%. Optical purity is
Optical resolution HPLC (column: Cyclobond I2000
Ac). ¹ H NMR (DMSO-d ₆ , 270 MHz) d 8.19 (d, J = 7.3 Hz, 1
H), 7.79 (s, 1 H), 7.53 (s, 1 H), 6.15 (ddd, J =
2. 0, 5.0 & 6.0 Hz, 1 H), 5.41 (dd, J = 5.6 & 6.0 H
z, 1 H), 5.19 (dd, J = 3.6 & 4.0 Hz, 1 H), 3.82 (d
dd, J = 3.6, 6.0 & 12.4 Hz, 1 H), 3.75 (ddd, J =
4.0, 5.6 & 12.4 Hz, 1 H), 3.43 (dd, J = 6.0 & 12.0
Hz, 1 H), 3.11 (dd, J = 5.0 & 12.0 Hz, 1 H): FT-IR
(cm ^-1 , KBr) 3447, 3246, 3061, 1690, 1621, 1542, 15
09, 1154, 1062.

Example 1 Synthesis of (-)-3'-thia-2 ', 3'-dideoxycytidine Escherichia coli K-12 / XL-10 strain (Stra
(Tagene) in 100 ml of LB medium.
The culture solution cultured overnight at 7 ° C. was incubated at 12,000 rpm for 1 hour.
The cells were collected by centrifugation for 5 minutes. The cells were suspended in 10 mL of 10 mM Tris-HCl buffer (pH 7.5) and disrupted by ultrasonication. 500 μm of a 20 mM aqueous solution of the optical isomer mixture of 3′-thia-2 ′, 3′-dideoxycytidine obtained above.
was mixed with a 0.5 M potassium phosphate buffer (pH 7.5) in an amount of 400 μl, and 100 μl of the above-mentioned disrupted cell suspension was added thereto.
Was added and the reaction was carried out at 50 ° C. for 20 hours. When the reaction mixture was analyzed under the following HPLC conditions (1), a decrease in the peak of the optical isomer mixture of 3′-thia-2 ′, 3′-dideoxycytidine and a new peak of 5-fluorouracil were observed. Was. HPLC analysis conditions (1) Column: Develosil ODS-MG-525
0 × 4.6 mmI. D. Column temperature: 40 ° C. Pump flow rate: 1.0 ml / min Detection: UV 254 nm, Eluent: 25 mM monopotassium dihydrogen phosphate: methanol = 875: 125 (V / V) Further, the following HPLC conditions using an optical resolution column In (2), the enantiomeric excess of (−)-3′-thia-2 ′, 3′-dideoxycytidine and the recovery of 5-fluorouracil were determined to be 84% and 50%, respectively.
Met. HPLC analysis conditions (2) Column; Cyclobond I 2000 Ac 250 ×
4.6 mmI. D. Column temperature; 18 ° C Pump flow rate; 0.3 ml / min detection; UV 262 nm, eluent; 0.2% ammonia acetate aqueous solution

Reference Example 4 L-3 ', 5'-bis (4-chlorobenzoyl) -2'
-Β / α mixture of deoxyadenosine 80 mL of methanol and 389 mg of sodium hydroxide were added to 1.38 g of adenine, and suspended under ultrasonic waves. After dissolution, the solvent was distilled off under reduced pressure. Toluene was added to the concentrated residue, and the solvent was distilled off under reduced pressure. This operation was repeated twice. The obtained white powder was treated with 4-methyl-2-pentanone 4
1. Suspension in 2 mL and at room temperature 3,5-di (4-chlorobenzoyl) -1-chloro-2-deoxy-L-ribose.
09 g was added. After stirring at room temperature for 5 hours, the insolubles were filtered through silica gel and washed with 4-methyl-2-pentanone. The organic layer was washed three times with water and once with saturated saline. The organic layer was dried over magnesium sulfate, concentrated to dryness,
1.41 g of 3 ′, 5′-bis (4-chlorobenzoyl) -2′-deoxy-β-adenosine was obtained. The yield is 55
%, And the β- / α-form ratio was 97/3.

Reference Example 5 β / α anomeric isomer mixture of L-2′-deoxyadenosine 3 ′, 5′-O-bis (4-chlorobenzoyl) -2 ′
-Deoxy-L-adenosine 607 mg (1.15 m
(mol) in methanol (12 mL) was added with 28% aqueous ammonia (2.4 mL), and the mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was concentrated, water was added, and the mixture was washed twice with ethyl acetate and twice with chloroform. The aqueous layer was concentrated to give the title compound 25
6 mg was obtained as a pale brown oil. Yield 89%. β / α ratio was 91/9.

Example 2 L-2'-deoxy-β-adenosine The β / α anomeric isomer mixture of L-2'-deoxyadenosine obtained above was treated with a cell homogenate and analyzed by HPLC. Decomposition of the α-form was confirmed, and L-2′-deoxy-β-adenosine was obtained. HPLC analysis conditions Column; YMC-Pack ODS-AM AM312
150 × 6.0 mmI. D. Column temperature; 25 ° C. Pump flow rate; 1.0 ml / min detection; UV 254 nm, eluent; 10 mM monosodium dihydrogen phosphate: methanol = 9: 1 (V / V)

[0049]

According to the present invention, it has become possible to produce an L-type nucleoside derivative more efficiently than a conventional method by a method capable of mass production.

Continued on front page (72) Inventor Takahiro Yamauchi 1144 Togo, Mobara-shi, Chiba F-term (reference) 4B064 AF34 CA21 CD27 in Mitsui Chemicals, Inc.

Claims (8)

[Claims] 1. A mixture of isomers of a nucleoside derivative,
A nuclease, or a microorganism having the enzyme activity, or a processed product thereof is acted on, and a specific isomer is decomposed in a mixture of isomers, leaving only one type of isomer. A method for producing a body-selective nucleoside derivative. 2. A compound represented by the general formula [1]: [In the formula, B represents a base selected from the group consisting of pyrimidine, purine, azapurine and deazapurine, and these are halogen atoms, alkyl groups, haloalkyl groups, alkenyl groups, haloalkenyl groups, alkynyl groups, amino groups, alkyl groups. Amino group, hydroxyl group, hydroxyamino group,
It may be substituted by an aminoxy group, an alkoxy group, a mercapto group, an alkylmercapto group, an aryl group, an aryloxy group or a cyano group. X represents a halogen atom, an alkoxy group or an alkylthio group, W represents an oxygen atom or a sulfur atom, Z represents an oxygen atom, a sulfur atom or an optionally substituted carbon atom, and p represents an integer of 0 to 2. , Q represents an integer of 0 to 4. (However, when Z is an oxygen atom or a sulfur atom, p + q ≦ 2
Satisfies p + q ≦ 4 when Z is a carbon atom. )] Is reacted with a nuclease, a microorganism having the enzyme activity, or a processed product thereof to decompose a specific isomer of the mixture of isomers. A compound represented by the general formula [2], wherein only the isomer remains: (Wherein, B, X, W, Z, p, and q have the same meanings as described above). 3. A compound of the general formula [3] Wherein B is as defined in [1] of claim 2,
And Z each independently represent an oxygen atom or a sulfur atom. From the isomer mixture of the nucleoside derivative represented by the general formula [4] (Wherein B, W, and Z have the same meanings as described above). 4. A compound of the general formula [5] [Wherein, B and X have the same meanings as [1] in claim 2, p represents an integer of 0 to 2, and q represents an integer of 0 to 4. (However, p + q ≦ 4 is satisfied.)] From the isomer mixture of the nucleoside derivative represented by the general formula [6]
[Formula 6] (Wherein B, X, p, and q have the same meanings as described above). 5. A compound of the general formula [7] [Wherein B, W and Z have the same meanings as [3] in claim 3. By treating a mixture of optical isomers of the nucleoside derivative represented by the above with a nuclease, thereby decomposing the D-type isomer of the optical isomer mixture and leaving only the L-type isomer. The general formula [8] [Formula 8] (Wherein B, W, and Z have the same meanings as described above). 6. A compound of the general formula [9] [Wherein, B has the same meaning as [1] in claim 2. By treating the stereoisomeric mixture of the nucleoside derivative represented by the above with a nuclease, the trans isomer of the isomer mixture is decomposed, leaving only the cis isomer remaining, Formula [10] [Formula 10] (Wherein B has the same meaning as described above.) A method for producing a cis-type nucleoside derivative represented by the formula: 7. The production method according to claim 1, wherein the nucleolytic enzyme is a nucleoside phosphorylase. 8. The method according to claim 8, wherein the nucleoside phosphorylase is purine nucleoside phosphorylase (EC 2.4.2.1),
Guanosine phosphorylase (EC 2.4.2.15),
Pyrimidine nucleoside phosphorylase (EC 2.4.
2.2), uridine phosphorylase (EC 2.4.4.2.
3), thymidine phosphorylase (EC 2.4.2.
4), deoxyuridine phosphorylase (EC 2.4.
7. One or more nucleoside phosphorylases selected from the group consisting of 2.23).
The manufacturing method as described.