JP2003055392A - Method for producing 1-phosphorylated sugar and method for producing nucleocide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly general method for producing a 1-phosphorylated sugar, by which the 1-phosphorylated sugar can be produced in high anomer selectivity without being affected by the skeleton differences of sugars such as furanose and pyranose, by the presence or absence of the substituent of a sugar such as a deoxysugar, or by the kind of a sugar such as a natural sugar or a non-natural sugar. SOLUTION: This method for producing the 1-phosphorylated sugar comprises subjecting an anomer isomer mixture of the 1-phosphorylated sugar to an equilibration reaction in a solvent having a large relative dielectric constant to preferentially increase the existence ratio of one of the anomer isomers. Thereby, the method for producing the 1-phosphorylated sugar in good anomer selectivity is provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing 1-phosphorylated sugar. 1-Phosphorylated saccharides are widely present in the living world, serve as reaction substrates for various enzymes, and serve as raw materials for producing useful substances such as pharmaceuticals and nutritional foods. In addition, non-natural 1-phosphorylated sugar is expected to be used as a raw material for manufacturing antiviral agents and enzyme inhibitors. Furthermore, the present invention relates to a method for producing a nucleoside compound used as a raw material or a drug substance for antiviral drugs, anticancer drugs, antisense drugs and the like.

[0002]

BACKGROUND OF THE INVENTION As a method for producing 1-phosphorylated sugar, 1) a method of condensing 1-brominated sugar and silver phosphate (J.
Biol. Chem. 121, 465 (1937),
J. Am. Chem. Soc. 78,811 (195
6), J. Am. Chem. Soc. 79,5057
(1957)), 2) A method of condensing a 1-halogenated sugar and a triethylamine salt of dibenzyl phosphate (J. Am. Chem. So.
c. 77, 3423 (1955), J. Am. Che
m. Soc. 80, 1994 (1958), J. Am.
Chem. Soc. 106,7851 (1984),
J. Org. Chem. 59,690 (1994)), 3) a method of heat-condensing a 1-acetylated sugar with orthophosphoric acid (J. Org. Chem. 27, 1107 (196).
2), Carbohydrate Res. 3,117
(1966), Carbohydrate Res.
3,463 (1967), Can. J. Bioche
m. 50, 574 (1972)), 4) a method of imidate the 1-position to activate and then condense it with dibenzyl phosphate (Carbohydrate R
es. 61,181 (1978), Tetrahedr
on Lett. 23, 405 (1982)), 5) a method of activating the 1-position as thallium or lithium alcoholate, and then treating with dibenzylphosphoric chloride (Carbohydrate Res. 94, 165).
(1981), Chem. Lett. 23,405 (1
982)), 6) A method for producing a 1-phosphorylated sugar by performing a phosphorolytic reaction of a nucleoside by the action of a nucleoside phosphorylase (J. Biol. Chem. Vol. 18).
4, p437, 1950) are known.

However, there are problems in the following points. A common problem with the chemical production methods 1) -5) is that the α-form /
The β-form anomeric selectivity changes, and it is difficult to establish a general strategy for obtaining the desired isomer with good selectivity. In order to achieve selectivity and high yield, the presence of the 2-position acetoxy group or 2-position acetamino group is essential, and in addition, the 2-position deoxy sugar is unstable. The range is narrow. Therefore, for the synthesis of 1-phosphorylated 2-deoxypyranose, it is difficult to control the anomeric selectivity, and column chromatography purification is required, and only low yields are obtained.
[Chem. Zvesti 28 (1), 115 (19
74), Izv. Akad. Nauk SSSR, Se
r. Khim. 8, 1843 (1975)] Therefore, 2
1-Phosphorylated Deoxypyranose 1-Phosphorus of 2-Deoxyfuranose which is more unstable than the 1-phosphorylated product and whose selectivity is difficult to control
As for the phosphorylated form, no chemical production examples have been reported so far.

With regard to 6), it is difficult to supply nucleosides other than very limited ribonucleosides such as inosine, and only limited 1-phosphorylated sugars such as ribose-1-phosphate can be produced. Can not.
In addition, the cost of the nucleoside itself, which is a raw material, is high, so that the cost is not satisfactory. As described above, the industrial production method of 1-phosphorylated sugar has not been established.

Nucleoside phosphorylase is a general term for enzymes capable of phosphorolytically decomposing N-glycoside bonds of nucleosides in the presence of phosphoric acid, and catalyzes the reaction represented by the following formula.

Nucleoside + phosphoric acid (salt) → base + 1-phosphorylated sugar purine Nucleoside phosphorylase and pyrimidine phosphorylase are broadly distributed in the living world.
It is found in tissues such as mammals, birds and fish, yeast, and bacteria. This enzymatic reaction is reversible, and the synthesis of various nucleosides using the reverse reaction has long been known. For example, from 2'-deoxyribose 1-phosphate and a nucleobase (thymine, adenine or guanine), thymidine (JP-A-01-104190), 2'-deoxyadenosine (JP-A-11-137290) or 2'-deoxy. A method for producing guanosine (JP-A-11-137290) is disclosed.

Further, Agric. Biol. Che
m. , Vol. 50 (1), PP. 121-126,
(1986), inosine in the presence of phosphate, Ent
After decomposing into ribose 1-phosphate and hypoxanthine by a reaction using purine nucleoside phosphorylase derived from E. aerogenes , ribose 1-phosphate and 1,2,4-
Than-triazole-3-carboxamide, also Ent
A technique for producing ribavirin, which is an antiviral agent, has been reported using a purine nucleoside phosphorylase derived from erobacter aerogenes .

However, as described above, 1-
Since an industrial production method of phosphorylated sugar has not been established, a highly versatile industrial production method of nucleoside utilizing the reverse reaction of nucleoside phosphorylase has not been established.

Further, since the reaction for producing a nucleoside from 1-phosphorylated sugar and a base by utilizing the reverse reaction of the enzyme is an equilibrium reaction, there is a technical drawback that the conversion rate is not improved.

[0010]

The first object of the present invention is to provide a sugar skeleton having a different structure such as furanose and pyranose.
It is intended to provide a highly versatile, anomeric selective 1-phosphorylated sugar production method which is not affected by the presence or absence of a substituent such as deoxy sugar or the kind of sugar such as natural type or non-natural type. . A second object of the present invention is to provide a highly versatile method for producing a nucleoside by the action of nucleoside phosphorylase from 1-phosphorylated sugar and a nucleobase, and a method for improving the conversion rate of nucleoside in the same reaction. Is to provide.

That is, an object of the present invention is to provide a method for producing a high-purity nucleoside at low cost by achieving these first and second objects.

[0012]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to achieve the above-mentioned first object and found that 1-phosphorylated sugar exists in an equilibrium state between anomeric isomers under certain conditions. It was found that this equilibrium condition varies depending on the solvent species, and that the larger the relative permittivity of the solvent, the larger the abundance ratio of the anomeric isomers. That is, it was found that the desired anomeric isomer can be obtained with high selectivity and high yield by carrying out the reaction in a solvent having a large relative dielectric constant, and the present invention has been completed based on this finding.

That is, the present invention is as follows. [1] Accompanying isomerization of anomeric phosphate ester, 1-
Anomer selective production method of phosphorylated sugar. [2] The production method according to [1], wherein a solvent having a relative dielectric constant of 10 or more is used as a solvent when isomerizing the anomeric phosphate ester. [3] When isomerizing the anomeric phosphate ester, p
The production method according to [1] or [2], which is carried out under acidic conditions of H6 or lower. [4] 1-phosphorylated sugar is represented by the general formula (1)

[0014]

[Chemical 6]

[In the formula, R1 and R2 each independently represent a hydrogen atom, a methyl group, a protected hydroxymethyl group or a protected carboxyl group, R3 represents an acyl group, and R4 represents a hydrogen atom or a carbon atom. Represents an alkyl group of formulas 1 to 4, R5 represents a hydroxyl-protecting 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,
Represents a sulfur atom or an optionally substituted carbon atom, n
Represents 0 or 1, p and q represent an integer of 0 to 4, and r represents 0 or 1. However, when X is an alkoxy group or an alkylthio group and r is 1 or more, each alkyl group may be bonded to the alkyl group of R4 to form a ring. , Q, r, n
Is p + q + r ≦ when Z is an oxygen atom or a sulfur atom.
2n + 3, p + q + r ≦ 2n + when Z is a carbon atom
5 is satisfied. ] The manufacturing method of Claim 1 to 3 shown by these. [5] After performing the production method described in [1] or [2] above, the elimination reaction of the protecting group is further performed.
-Anomeric selective production of phosphorylated sugars. [6] After carrying out the production method described in the above [4], the protecting group is then eliminated to give a compound of the general formula (2)

[0016]

[Chemical 7]

(In the formula, R1 and R2 each independently represent a hydrogen atom, a methyl group, a hydroxymethyl group or a carboxyl group, R3 represents a hydrogen atom or an acyl group, and R4 represents a hydrogen atom or a carbon number of 1 to 1). 4 represents an alkyl 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 n represents Represents 0 or 1, p and q represent an integer of 0 to 4, r represents 0 or 1, provided that X is an alkoxy group or an alkylthio group;
When r is 1 or more, each alkyl group may combine with the alkyl group of R4 to form a ring, and in this case, p, q, r, and n are each Z is an oxygen atom, When sulfur atom, p + q + r ≦ 2n + 3 is satisfied, and when Z is a carbon atom, p + q + r ≦ 2n + 5 is satisfied. ) The method for producing 1-phosphorylated saccharide. [7] General formula (1a) [Chemical formula 8] which can be obtained by the production method according to any one of [1] to [5] above.

[0018]

[Chemical 8]

(In the formula, R1b and R2b each independently represent a hydrogen atom, a methyl group, a protected hydroxymethyl group, a hydroxymethyl group, a protected carboxyl group or a carboxyl group, and R3a represents a hydrogen atom or an acyl group. R5a represents a hydrogen atom or a hydroxyl-protecting group, R4 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, X represents a halogen atom, an alkoxy group or an alkylthio group, and W represents an oxygen atom or Represents a sulfur atom, Z represents an oxygen atom, a sulfur atom or an optionally substituted carbon atom, n represents 0 or 1, p and q represent an integer of 0 to 4, and r represents 0 or 1. However, when X is an alkoxy group or an alkylthio group and r is 1 or more, each alkyl group is bonded to the alkyl group of R4. May be formed, and in this case, p, q, r, and n satisfy p + q + r ≦ 2n + 3 when Z is an oxygen atom or a sulfur atom, and p + q + r ≦ 2n + 5 when Z is a carbon atom. .) 1-
Phosphorylated sugar derivatives and salts thereof. [8] 1- according to any one of [1] to [6] above
Having a first step of producing a phosphorylated saccharide and a second step of performing an exchange reaction between a phosphate group and a base of 1-phosphorylated saccharide obtained in the first step by the action of nucleoside phosphorylase A method for producing a characteristic nucleoside derivative. [9] The nucleoside derivative has the general formula (3)

[0020]

[Chemical 9]

(In the formula, B independently represents a base selected from the group consisting of pyrimidine, purine, azapurine and deazapurine, which are halogen atom, alkyl group, haloalkyl group, alkenyl group, haloalkenyl group, Alkynyl group, amino group, alkylamino group,
It may be substituted with 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. R1 and R2 each independently represent a hydrogen atom, a methyl group, a protected hydroxymethyl group or a protected carboxyl group, R3 represents an acyl group, R4 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. X is a halogen atom, an alkoxy group or an alkylthio group, W is an oxygen atom or a sulfur atom, Z is an oxygen atom, a sulfur atom or an optionally substituted carbon atom, and n is 0 or 1. , P and q represent an integer of 0 to 4, and r represents 0 or 1. However, when X is an alkoxy group or an alkylthio group,
When r is 1 or more, each alkyl group may combine with the alkyl group of R4 to form a ring, and in this case, p, q, r, and n are each Z is an oxygen atom, When sulfur atom, p + q + r ≦ 2n + 3 is satisfied, and when Z is a carbon atom, p + q + r ≦ 2n + 5 is satisfied. ] The manufacturing method as described in [8] shown by these. [10] By the action of the nucleoside phosphorylase activity, an exchange reaction between the phosphate group and the base of the 1-phosphorylated sugar derivative described in [7] gives the compound represented by the general formula (3)

[0022]

[Chemical 10]

(Wherein B, R1, R2, R3, R4,
X, W, Z, n, p, q and r are as described above. ) A method for producing a nucleoside represented by [11] Nucleoside phosphorylase is a purine nucleoside phosphorylase (EC 2.4.2.1), a guanosine phosphorylase (EC 2.4.2.15), a pyrimidine nucleoside phosphorylase (EC 2.4.2.
2), uridine phosphorylase (EC 2.4.2.
3), thymidine phosphorylase (EC 2.4.2.
4), deoxyuridine phosphorylase (EC2.4.
The production method according to any one of [8] to [10], which uses a nucleoside phosphorylase selected from the group consisting of 2.23). [12] The nucleoside phosphorylase is a purine nucleoside phosphorylase (EC 2.4.2.1), a guanosine phosphorylase (EC 2.4.2.15), a pyrimidine nucleoside phosphorylase (EC 2.4.2.
2), uridine phosphorylase (EC 2.4.2.
3), thymidine phosphorylase (EC 2.4.2.
4), deoxyuridine phosphorylase (EC2.4.
The production method according to any one of [8] to [10], wherein the microorganism expresses at least one nucleoside phosphorylase selected from the group consisting of 2.23). [13] A metal cation capable of forming a sparingly water-soluble salt with a phosphate ion in the reaction solution when the exchange reaction between the phosphate group and the base of the 1-phosphorylated sugar derivative is performed by the action of the nucleoside phosphorylase activity. The manufacturing method according to any one of [8] to [12]. [14] The production method according to [13], wherein the metal cation capable of forming a poorly water-soluble salt with phosphoric acid is at least one metal cation selected from calcium ion, barium ion, magnesium ion, and aluminum ion.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The saccharide used in the present invention is preferably fucose, rhamnose, digitoxose, oleandrose,
6-deoxy sugars such as quinoboses, hexoses such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose, pentoses such as ribose, arabinose, xylose, lyxose, erythrose, threose, etc. D series of amino sugars such as tetrose, glucosamine, daunosamine, uronic acids such as glucuronic acid and galacturonic acid, psicose, fructose, sorbose, tagatose, ketose such as pentulose, and deoxysaccharides such as 2-deoxyribose. Alternatively, a residue derived from a natural monosaccharide consisting of L series, a residue derived from a pyranose-type or a furanose-type non-natural sugar, and a hydroxyl group and / or an amino group thereof have been protected or acylated. Residue derivative, or a hydroxyl group that they have can be mentioned saccharides with a halogenated sugar residue substituted with a halogen atom such as fluorine, but is not limited thereto.

In the present invention, the protecting group in the protected hydroxymethyl group and hydroxyl group protecting group means a protecting group which is removed by a chemical method such as hydrogenolysis, hydrolysis or photolysis. Examples of such a group include a formyl group, an acyl group, a silyl group, an alkyl group, an aralkyl group, and a carbonyl group. Among them, a formyl group, an aliphatic acyl group, an aromatic acyl group, a silyl group, and an alkoxyalkyl group are preferable. , A halogenated alkyl group, an aralkyl group, an alkoxycarbonyl group, and an aralkyloxycarbonyl group.

Examples of the aliphatic acyl group include an alkylcarbonyl group and a halogen-substituted lower alkylcarbonyl group.

Specific examples of the alkylcarbonyl group include acetyl group, propionyl group, butyryl group, isobutyryl group, pentanoyl group, pivaloyl group, valeryl group, isovaleryl group, octanoyl group, nonylcarbonyl group, decylcarbonyl group, 3-methylnonyl group. Carbonyl group, 8
-Methylnonylcarbonyl group, 3-ethyloctylcarbonyl group, 3,7-dimethyloctylcarbonyl group, undecylcarbonyl group, dodecylcarbonyl group, tridecylcarbonyl group, tetradecylcarbonyl group, pentadecylcarbonyl group, hexadecylcarbonyl group , 1-
Examples thereof include a methylpentadecylcarbonyl group, a 14-methylpentadecylcarbonyl group, a 13,13-dimethyltetradecylcarbonyl group, a heptadecylcarbonyl group, a 15-methylhexadecylcarbonyl group and an octadecylcarbonyl group.

Specific examples of the halogen-substituted lower alkylcarbonyl group include a chloroacetyl group, a dichloroacetyl group, a trichloroacetyl group, a trifluoroacetyl group and the like.

As the aromatic acyl group, an arylcarbonyl group, a halogen-substituted arylcarbonyl group, a lower alkylated arylcarbonyl group, a lower alkoxyarylcarbonyl group, a nitrated arylcarbonyl group,
Examples thereof include a lower alkoxycarbonylated arylcarbonyl group and an arylated arylcarbonyl group.

Specific examples of the arylcarbonyl group include benzoyl group, α-naphthoyl group, β-naphthoyl group and the like.

Specific examples of the halogen-substituted arylcarbonyl group include 2-fluorobenzoyl group, 3-fluorobenzoyl group, 4-fluorobenzoyl group, 2-chlorobenzoyl group, 3-chlorobenzoyl group and 4-chlorobenzoyl group. , 2-bromobenzoyl group, 3-bromobenzoyl group, 4-bromobenzoyl group, 2,4-dichlorobenzoyl group, 2,6-dichlorobenzoyl group,
Examples thereof include a 3,4-dichlorobenzoyl group and a 3,5-dichlorobenzoyl group.

Specific examples of the lower alkylated arylcarbonyl group include 2-toluoyl group, 3-toluoyl group and 4
Examples thereof include a toluoyl group and a 2,4,6-trimethylbenzoyl group.

Specific examples of the lower alkoxyarylcarbonyl group include 2-anisoyl group, 3-anisoyl group and 4
-Anisoyl group etc. can be illustrated.

Specific examples of the nitrated arylcarbonyl group include a 2-nitrobenzoyl group, a 3-nitrobenzoyl group, a 4-nitrobenzoyl group and a 3,5-dinitrobenzoyl group.

Specific examples of the lower alkoxycarbonylated arylcarbonyl group include 2- (methoxycarbonyl)
As a specific example of the arylated arylcarbonyl group, a benzoyl group and the like can be exemplified by a 4-phenylbenzoyl group and the like.

Examples of the silyl group include a lower alkylsilyl group and a lower alkylsilyl group substituted with an aryl group.

Specific examples of the lower alkylsilyl group include a trimethylsilyl group, a triethylsilyl group, an isopropyldimethylsilyl group, a tert-butyldimethylsilyl group, a methyldiisopropylsilyl group and a triisopropylsilyl group.

Specific examples of the lower alkylsilyl group substituted with an aryl group include diphenylmethylsilyl group, diphenylisopropylsilyl group and phenyldiisopropylsilyl group.

As the aralkyl group, an aralkyl group substituted with a lower alkyl group, an aralkyl group substituted with a lower alkoxy group, an aralkyl group substituted with a nitro group,
Examples thereof include a halogen-substituted aralkyl group and a cyano group-substituted aralkyl group.

Specific examples of these groups include 2-methylbenzyl group, 3-methylbenzyl group, 4-methylbenzyl group, 2,4,6-trimethylbenzyl group, 2-methoxybenzyl group and 3-methoxy group. Benzyl group, 4-methoxybenzyl group, 2-nitrobenzyl group, 3-nitrobenzyl group, 4-nitrobenzyl group, 2-chlorobenzyl group, 3-chlorobenzyl group, 4-chlorobenzyl group, 2
-Bromobenzyl group, 3-bromobenzyl group, 4-bromobenzyl group, 2-cyanobenzyl group, 3-cyanobenzyl group, 4-cyanobenzyl group and the like can be mentioned.

As the aralkyloxycarbonyl group,
Aralkyloxycarbonyl group substituted with lower alkyl group, aralkyloxycarbonyl group substituted with lower alkoxy group, aralkyloxycarbonyl group substituted with nitro group, aralkyloxycarbonyl group substituted with halogen, cyano group substituted An aralkyloxycarbonyl group may be mentioned.

Specific examples thereof include a 2-methylbenzyloxycarbonyl group, a 3-methylbenzyloxycarbonyl group, a 4-methylbenzyloxycarbonyl group,
2,4,6-trimethylbenzyloxycarbonyl group,
2-methoxybenzyloxycarbonyl group, 3-methoxybenzyloxycarbonyl group, 4-methoxybenzyloxycarbonyl group, 2-nitrobenzyloxycarbonyl group, 3-nitrobenzyloxycarbonyl group, 4-
Nitrobenzyloxycarbonyl group, 2-chlorobenzyloxycarbonyl group, 3-chlorobenzyloxycarbonyl group, 4-chlorobenzyloxycarbonyl group, 2
-Bromobenzyloxycarbonyl group, 3-bromobenzyloxycarbonyl group, 4-bromobenzyloxycarbonyl group, 2-cyanobenzyloxycarbonyl group,
Examples thereof include a 3-cyanobenzyloxycarbonyl group and a 4-cyanobenzyloxycarbonyl group.

Examples of the alkoxycarbonyl group include a lower alkoxycarbonyl group, a halogen-substituted alkoxycarbonyl compound, and an alkoxycarbonyl group substituted with an alkylsilyl group.

Specific examples of the lower alkoxycarbonyl group include methoxycarbonyl group, ethoxycarbonyl group, propoxycarbonyl group, butoxycarbonyl group, sec.
Examples thereof include -butoxycarbonyl group and tert-butoxycarbonyl group.

Specific examples of the halogen-substituted alkoxycarbonyl group include a 2,2,2-trichloroethoxycarbonyl group, and specific examples of the alkoxycarbonyl group substituted with a lower alkylsilyl group include a 2-trimethylsilylethoxycarbonyl group. Can be illustrated.

As the alkyl group, a methoxymethyl group,
An alkoxyalkyl group such as an ethoxymethyl group, a 2-methoxyethyl group, a 2-methoxyethoxymethyl group,
Lower substituted with an aryl group such as a halogenated alkyl group such as a 2,2,2-trichloroethyl group, a benzyl group, an α-naphthylmethyl group, a β-naphthylmethyl group, a diphenylmethyl group and a triphenylmethyl group. An alkyl group is mentioned.

Of these, an aliphatic acyl group, an aromatic acyl group and an aralkyl group are preferable, and a 4-toluoyl group and a 4-chlorobenzoyl group are more preferable.
Or a benzyl group.

The protecting group in the protected carboxyl group as R1 and R2 refers to a protecting group which is removed by a chemical method such as hydrogenolysis, hydrolysis or photolysis. As such a group, preferably, a lower alkyl group such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group or a tert-butyl group, 2- Examples thereof include silylated lower alkyl groups such as (trimethylsilyl) ethyl group and 2- (triethylsilyl) ethyl group, or the above-mentioned aralkyl groups and alkoxyalkyl groups. More preferably, it is a methyl group, a tert-butyl group, or a benzyl group.

The halogen atom represented by X is a fluorine atom,
It represents a chlorine atom, a bromine atom or an iodine atom.

Examples of the alkoxy group and alkylthio group represented by X include the above-mentioned lower alkyl group, aralkyl group, alkoxy group having an alkoxyalkyl group, and alkylthio group. More preferably,
A methoxy group, a methoxyethoxy group, and a methylthio group.

The substitutable carbon atom represented by Z is a carbon atom in which one or two substituents represented by the general formula are substituted, and when not substituted, it is substituted with a hydrogen atom. Refers to a carbon atom.

Examples of the acyl group as R3 include the above-mentioned aliphatic acyl group, aromatic acyl group, alkoxycarbonyl group, aralkyloxycarbonyl group, and lower alkanesulfonyl group such as methanesulfonyl group and trifluoromethanesulfonyl group. And arylsulfonyl groups such as benzenesulfonyl group, p-toluenesulfonyl group. Preferred are an aliphatic acyl group, an aromatic acyl group, and a lower alkanesulfonyl group, and specifically, an acetyl group, a trifluoroacetyl group, a benzoyl group, and a methanesulfonyl group.

The sugar residues having the general formulas (1) to (3) are preferably the above-mentioned natural monosaccharide-derived residues, non-natural sugar-derived residues, sugar residue derivatives, and halogenated sugar residues. Examples thereof include groups, but are not limited thereto.

In the compound (1) of the present invention, the salt means a salt formed by a phosphoric acid group in the molecule of the compound. The salt is preferably an alkali metal salt such as sodium, potassium or lithium, an alkaline earth metal salt such as magnesium, calcium or barium, a metal salt such as aluminum or iron, an ammonium salt, a primary salt. Two
Examples include salts of secondary and tertiary alkylamines.

Examples of primary amines include alkylamines such as methylamine, ethylamine, propylamine, isopropylamine, butylamine, hexylamine, octylamine, cycloalkylamines such as cyclohexylamine, and benzylamine. Can be mentioned.

Examples of the secondary amine include diethylamine, diisopropylamine, dibutylamine, dihexylamine, dialkylamines such as dioctylamine, dicycloalkylamines such as dicyclohexylamine, piperidine, morpholine and N-methylpiperazine. Such cyclic amines can be exemplified.

As the tertiary amine, trimethylamine,
Triethylamine, tripropylamine, N-ethyldiisopropylamine, tributylamine, trihexylamine, trioctylamine, N-ethyldicyclohexylamine, N-methylpiperidine, N-methylmorpholine, N, N, N ', N'- Tertiary alkylamines such as tetramethylethylenediamine, aniline, N,
N-dimethylaniline, N, N-diethylaniline,
Anilines such as N, N-dibutylaniline, N, N-dioctylaniline, pyridine, salts of pyridines such as 2,6-dimethylpyridine, 2,4,6-lutidine and nicotinamide, glycine, alanine, Amino acids such as proline, lysine, arginine, glutamine, cinchonidine, 1- (1-naphthyl) ethylamine, 1-
An optically active amine such as phenylethylamine can be mentioned, and each includes a monovalent or divalent salt.

Further, the compounds (1) to (3) of the present invention.
May absorb water by being left in the atmosphere, and may be adsorbed with water or become a hydrate. Such salts are also included in the present invention.

The anomeric mixture of 1-phosphorylated saccharides before isomerization in the present invention has a reaction formula (I)

[0060]

[Chemical 11]

However, the present invention is not limited to this. In the above formula, R1, R
2, R3, R4, R5, X, W, Z, m, n, p, q and r have the same meaning as in the above formula (1), and Y is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. Represents

Phosphoric acid having a small water content such as orthophosphoric acid or dehydrated phosphoric acid is preferable, but is not particularly limited.

The base is not particularly limited as long as it does not inhibit the reaction and functions as a deoxidizing agent, but as the inorganic base, alkali metal, alkaline earth metal carbonate, hydroxide and the like are preferable. Examples of the organic base include tertiary alkylamines, anilines, pyridines, and optically active amines.

The dehydrating agent can be used when the water mixed from the solvent or the additive adversely affects the reaction. The dehydrating agent is not particularly limited as long as it has a water adsorbing property or a water reacting property, but molecular sieves or phosphorus pentoxide can be preferably used.

Phosphoric acid and base may be added separately, or those prepared as phosphates may be added. When added as a phosphate, the phosphate may be dehydrated in advance by purification or the like.

The reaction is usually carried out in the presence of a solvent.
The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but for example, hexane, aliphatic hydrocarbons such as heptane, benzene, toluene, xylene, anisole. Aromatic hydrocarbons such as, methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, halogenated hydrocarbons such as dichlorobenzene, ethyl formate, ethyl acetate, propyl acetate, n-butyl acetate, diethyl carbonate Ethers such as, diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, ethers such as diglyme,
Nitriles such as acetonitrile, propionitrile, isobutyronitrile, benznitrile, formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone, N, Amides such as N-dimethyl-2-imidazolidinone, acetone, 2-butanone, 2-
Pentanone, 3-pentanone, 4-heptanone, methyl isopropyl ketone, 4-methyl-2-pentanone,
Examples thereof include ketones such as methyl phenyl ketone, cyclopentanone and cyclohexanone, carbonates such as dimethyl carbonate, diethyl carbonate, ethylene carbonate and propylene carbonate, or a mixed solvent of two or three selected from them. it can.

The reaction temperature is not particularly limited and is usually -8.
The temperature is 0 ° C to 60 ° C, preferably -10 ° C to 25 ° C.

The reaction time will differ depending on the starting materials, types of reagents and solvents, reaction temperature, but it is usually 1 minute to 2 minutes.
It is achieved in 4 hours, preferably 10 minutes to 2 hours.

The ratio of the sugar derivative (4) to phosphoric acid in this reaction is not particularly limited, and the reaction can usually be carried out with compound (4): phosphoric acid = 1: 10 to 3: 1.

It is difficult to control the selectivity of the anomeric position only by the reaction represented by the reaction formula (I).
The 1-phosphorylated sugar derivative (1a) or (1b) preferentially containing one of the α-form and the β-form is represented by the reaction formula (II)
[Chemical 12]

[0071]

[Chemical 12]

It can be manufactured by In the above formula, R1, R2, R3, R4, R5, X, W, Z,
m, n, p, q and r have the same meanings as in the above-mentioned formula (1).

The reaction is usually carried out in the presence of a solvent.
The solvent used is preferably one which does not inhibit the reaction and dissolves the starting material to some extent. Further, in order to perform isomerization efficiently, it is preferable that the relative dielectric constant is 10 or more. Examples of such a solvent include halogenated hydrocarbons such as methane chloride, acid anhydrides such as acetic anhydride, nitriles such as acetonitrile, propionitrile, isobutyronitrile, benznitrile, and formamide. , N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N
-Amids such as methylpyrrolidinone, N, N-dimethyl-2-imidazolidinone, acetone, 2-butanone, 2-pentanone, 3-pentanone, 4-heptanone, methyl isopropyl ketone, methyl isobutyl ketone, methyl phenyl ketone Examples thereof include ketones such as cyclopentanone and cyclohexanone, carbonates such as dimethyl carbonate, diethyl carbonate, ethylene carbonate and propylene carbonate, or a mixed solvent composed of two or three kinds selected from them. It is also possible to use a mixed solvent obtained by mixing a solvent having a relative dielectric constant of 10 or more and a solvent having a relative dielectric constant of 10 or less and adjusting the relative dielectric constant to 10 or more.

The reaction temperature is not particularly limited as long as it promotes the equilibrium reaction between the compounds (1a) and (1b), and is usually -80.
C. to 60.degree. C., preferably -10.degree. C. to 25.degree.

The reaction time varies depending on the starting materials, the types of reagents and solvents, and the reaction temperature, but is usually 3 hours to 1 hour.
Achieved in a week, preferably 6 to 24 hours. still,
The ratio of the sugar derivative (1) to phosphoric acid in this reaction is not particularly limited and is usually compound (1): phosphoric acid = 1: 10 to 10.
The reaction can be carried out in the range of 3: 1. At that time, the pH in the reaction system is usually 1 to 6, and preferably 1 to 4 on the acidic side. An acid other than phosphoric acid may be added to adjust the acidity in the reaction system.

Furthermore, 1 having one of α-form and β-form
-The phosphorylated sugar derivative (1a) or (1b) is usually
Since it is unstable in an acidic state, it is preferable to adjust the neutral side to the basic side by adding a base before performing the post-treatment. Further, in the case of purification, it is possible to carry out a salt exchange reaction for purification and take out as a phosphate of a base different from the base used in the reaction system. Examples of the base used here include the aforementioned inorganic bases, primary alkylamines, secondary alkylamines, tertiary alkylamines, anilines, pyridines, amino acids, and optically active amines. The salts formed include monovalent or divalent salts. Furthermore, the elimination reaction of the protecting group is performed according to reaction formula (III)

[0077]

[Chemical 13]

The 1-phosphorylated saccharide (2a) or (2b) can be produced by the following procedure.

In the above formula, R1, R2, R3, R
4, X, W, Z, n, p, q and r have the same meanings as in the above formula (1), and R1 ′ and R2 ′ are each independently a hydrogen atom, a methyl group, a hydroxymethyl group or a carboxyl group. R3 'represents a hydrogen atom or an acyl group. R in compound (1a) or (1b)
As the hydroxymethyl group of 1 and R2 or the hydroxyl protecting group of R4, the above-mentioned aliphatic acyl group, aromatic acyl group, alkoxycarbonyl group, or R1 and R2
When the above-mentioned lower alkyl group is used as the protecting group for the carboxyl group, it can be removed by treating with a base in a water-soluble solvent. The base is preferably an alkali metal carbonate such as sodium carbonate or potassium carbonate, an alkali metal hydroxide such as lithium hydroxide, sodium hydroxide or potassium hydroxide, aqueous ammonia, tetra-n-butylammonium hydroxide. Ammonium hydroxides, such as the aforementioned inorganic bases, primary alkyl amines, secondary alkyl amines,
It can be carried out using a tertiary alkylamine or the like.

The solvent to be used is not particularly limited as long as it can be used in a usual hydrolysis reaction, but preferably water, alcohols such as methanol, ethanol, n-propanol and isopropanol, the above-mentioned. Ethers of can be used. The reaction temperature and the reaction time vary depending on the starting materials, the base used and the like, and are not particularly limited, but it is usually -10 ° C to 100 ° C, and the reaction is completed in 1 hour to 5 days. At this time, the protecting group for R3 can be left as desired or simultaneously removed by adjusting the reaction temperature, the reaction time, or the number of equivalents of the reagent.

R in compound (1a) or (1b)
When the aforementioned aralkyl group is used as the protective group for the hydroxymethyl group of 1 and R2 or the hydroxyl group of R4, the aforementioned aralkyl group, the aralkyloxycarbonyl group or the protective group for the carboxyl group of R1 and R2, for example, It can be removed by catalytic reduction using a metal catalyst.

As the catalyst, preferably, palladium carbon, Raney nickel, platinum oxide, platinum black, rhodium-aluminum oxide, triphenylphosphine-rhodium chloride, palladium-barium sulfate or the like can be used. The pressure is not particularly limited, but the solvent usually used is not particularly limited as long as it is used in a usual hydrogenolysis reaction, but preferably water, methanol,
Alcohols such as ethanol, n-propanol and isopropanol, the above ethers and esters can be used. The reaction temperature and reaction time vary depending on the starting material, the base used, etc. and are not particularly limited,
Usually, it is -10 ° C to 100 ° C and is completed in 1 hour to 5 days. In this case, the protecting group for R3 can usually be left unremoved.

R in compound (1a) or (1b)
When the above-mentioned silyl group is used as the protective group for the hydroxymethyl group of 1 and R2 or the hydroxyl group of R4, or the above-mentioned silylated lower alkyl group is used as the protective group for the carboxyl group of R1 and R2, for example, , Compounds such as tetra-n-butylammonium fluoride, which produce fluorine anions, can be used for removal.

The reaction solvent is not particularly limited as long as it does not inhibit the reaction, but the above ethers can be used. Although the reaction temperature and the reaction time are not particularly limited, the reaction is usually carried out at -10 ° C to 50 ° C for 10 minutes to 10 hours. In this case, the protecting group for R3 can usually be left unremoved.

In removing any protecting group,
The phosphoric acid group in the molecule of the product is obtained as a salt of the base existing in the reaction system, but it can be taken out by changing it to a salt of another base if desired. Examples of the base used at that time include the above-mentioned inorganic bases, primary alkylamines, secondary alkylamines, tertiary alkylamines, anilines, pyridines, amino acids,
An optically active amine can be mentioned, and the salts formed include monovalent or divalent salts.

The 1-phosphorylated saccharide in the present invention is a saccharide or a derivative thereof in which phosphoric acid is ester-bonded to the 1-position.

Specifically, the general formula (1) [Chemical formula 14]

[0088]

[Chemical 14]

(In the formula, R1, R2, R3, R4, R5,
X, W, Z, n, p, q and r are as described above. ).

As the saccharide derived from a natural product which constitutes 1-phosphorylated saccharide, D-arabinose, L-arabinose, D-xylose, L-lyxose, aldopentose such as D-ribose, D-xylose, L-xylose, ketopentose such as D-ribulose, D-galactose, L-galactose, D-glucose, D-
Talose, aldohexose such as D-mannose,
D-tagatose, L-sorbose, D-psicose, D
-Ketohexoses such as fructose, D-2-deoxyribose, D-2,3-dideoxyribose, D-
Fucose, L-fucose, D-rhamnose, L-rhamnose, D-fucopyranose, L-fucopyranose, D-
Rhamnofuranose, L-rhamnofuranose, D-alometylose, D-quinovose, D-antiallose, D-
Deoxy saccharides such as talometylose, L-talometylose, D-dikitarose, D-digitoxose, D-cimalose, tiberose, abecose, paratose, coritose, ascarylose, amino sugars such as glucosamine, daunosamine, glucuronic acid, galacturonic acid. However, the uronic acids are not limited to these.

Further, the base used in the present invention is a natural or unnatural base selected from the group consisting of pyrimidine, purine, azapurine and deazapurine, which are halogen atom, alkyl group, haloalkyl group and alkenyl group. , Haloalkenyl group, alkynyl group, amino group, alkylamino group, hydroxyl group, hydroxyamino group,
It may be substituted with an aminoxy group, an alkoxy group, a mercapto group, an alkylmercapto group, an aryl group, an aryloxy group or a cyano group.

Examples of the halogen atom as the substituent include chlorine, fluorine, iodine and bromine. The alkyl group has 1 to 7 carbon atoms such as methyl, ethyl and propyl.
The lower alkyl group of is exemplified. The haloalkyl group has 1 to 1 carbon atoms such as fluoromethyl, difluoromethyl, trifluoromethyl, bromomethyl and bromoethyl.
Illustrative is a haloalkyl group having 7 alkyls. The alkenyl group has 2 to 2 carbon atoms such as vinyl and allyl.
7 alkenyl groups are exemplified. The haloalkenyl group has 2 to 7 carbon atoms such as bromovinyl and chlorovinyl.
And a haloalkenyl group having alkenyl.
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 alkyl having 1 to 7 carbon atoms such as methylamino and ethylamino. Examples of the alkoxy group include alkoxy groups having 1 to 7 carbon atoms such as methoxy and ethoxy. Examples of the alkylmercapto group include an alkylmercapto group having an alkyl having 1 to 7 carbon atoms such as methylmercapto and ethylmercapto. As the aryl group, 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; dimethylamino 1 to 1 carbon atoms such as phenyl and diethylaminophenyl
Examples thereof include an alkylaminophenyl group having 5 alkylamino; a halogenophenyl group such as chlorophenyl and bromophenyl.

Specific examples of the pyrimidine base include cytosine, uracil, 5-fluorocytosine, 5-fluorouracil, 5-chlorocytosine, 5-chlorouracil, 5-bromocytosine, 5-bromouracil, 5-yo- Docytosine, 5-yo-douracil, 5-methylcytosine, 5-methyluracil (thymine), 5-ethylcytosine, 5-ethyluracil, 5-fluoromethylcytosine, 5-fluorouracil, 5-trifluorocytosine, 5-tri Fluorouracil, 5-vinyluracil,
5-bromovinyluracil, 5-chlorovinyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5
-Propynyluracil, pyrimidin-2-one, 4-hydroxyaminopyrimidin-2-one, 4-aminooxypyrimidin-2-one, 4-methoxypyrimidine-2
-One, 4-acetoxypyrimidin-2-one, 4-fluoropyrimidin-2-one, 5-fluoropyrimidin-2-one and the like can be mentioned.

Specific examples of purine bases 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- Methylpurine, 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 can be mentioned.

Specific examples of the azapurine base and 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 etc. are mentioned.

The nucleoside phosphorylase in the present invention is a general term for enzymes that decompose the N-glycoside bond of nucleoside in the presence of phosphate, and the reverse reaction can be used in the present invention. The enzyme used in the reaction is
It may be of any type and origin as long as it has an activity capable of producing a desired nucleoside from the corresponding 1-phosphorylated sugar and a base. The enzyme is roughly classified into purine type and pyrimidine type. For example, purine type purine nucleoside phosphorylase (EC 2.4.2.1), guanosine phosphorylase (EC 2.4.2.15), pyrimidine type pyrimidine nucleoside phosphorylase ( EC 2.4.2.2), uridine phosphorylase (E
C2.4.2.3), thymidine phosphorylase (EC
2.4.2.4), deoxyuridine phosphorylase (EC 2.4.2.23) and the like.

Microorganisms expressing nucleoside phosphorylase according to the present invention include 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 (EC 2.4.2.23), selected from the group consisting of The microorganism is not particularly limited as long as it is a microorganism expressing the above nucleoside phosphorylase.

Specific examples of such microorganisms include the genus Nocardia , Microbacterium
erium ) genus, Corynebacterium ( Corynebacterium ) genus,
Brevibacterium (Brevibacterium) genus Cellulomonas (Cellulomonas) sp., Flavobacterium (Flabobac
terium) genus, Kuruihera (Kluyvere) genus Mycobacterium (Micobacterium) genus, Haemophilus (Haemophilus)
Genus, Mikopurana (Micopla na) genus, professional data Mino Enterobacter (Pro
taminobacter) genus Candida (Candida) genus Saccharomyces (Saccharomyces) genus Bacillus (Bacillus)
Genus, thermophilic Bacillus, Pseudomonas
s genus, Micrococcus genus, Hafnia ( Haf
nia) genus, Proteus (Proteus) genus Vibrio (Vibrio)
Genus, Staphyrococcus genus, Propionibacterium genus, Zaltina ( Sa
rtina) genus, Plano Lactococcus (Planococcus) genus Escherichia (Escherichia) genus, Kurthia (Kurthia) genus, Rodokokkkasu (Rhodococcus) genus, Acinetobacter (Acinetob
acter) genus, key Santo Enterobacter (Xanthobacter) genus Streptomyces (Streptomyces) genus Rhizobium (Rhizobiu
m) the genus Salmonella (Salmonella) species, Klebsiella (Klebs
iella) genus, Enterobacter (Enterobacter) genus, Erwinia (Erwinia) genus, Earomonasu (Aeromonas) genus, Citrobacter (Citrobacter) sp., Achromobacter (Achrom
bacterium genus, Agrobacterium genus,
Preferable examples include microbial strains belonging to the genus Arthrobacter or the genus Pseudonocardia .

Due to recent advances in molecular biology and genetic engineering, the gene of the protein was obtained from the microbial strain by analyzing the molecular biological properties and amino acid sequence of the nucleoside phosphorylase of the microbial strain described above. , Constructing a recombinant plasmid in which the gene and a control region required for expression are inserted, and introducing this into an arbitrary host, it becomes possible to produce a recombinant bacterium expressing the protein, and It's also relatively easy. In view of such a state of the art, a genetically modified bacterium in which such a nucleoside phosphorylase gene is introduced into an arbitrary host is also included in the microorganism expressing the nucleoside phosphorylase of the present invention.

The term "regulatory region required for expression" as used herein refers to a promoter sequence (including an operator sequence that controls transcription), a ribosome binding sequence (SD sequence), a transcription termination sequence and the like. Specific examples of the promoter sequence include:
E. coli tryptophan operon trp promoter, lactose operon lac promoter, lambda phage-derived PL promoter and PR promoter, and Bacillus subtilis-derived gluconate synthase promoter (g
nt) ・ Alkaline protease promoter (apr)
-Neutral protease promoter (npr) -alpha-amylase promoter (amy) etc. are mentioned. Also,
A uniquely modified / designed sequence such as the tac promoter can also be used. Examples of the ribosome binding sequence include sequences derived from Escherichia coli or Bacillus subtilis, but are not particularly limited as long as the sequences function in a desired host such as Escherichia coli and Bacillus subtilis. For example, 16S ribosomal R
A consensus sequence in which a sequence complementary to 4'or more bases complementary to the 3'terminal region of NA is continuous may be prepared by DNA synthesis and used. The transcription termination sequence is not always necessary, but those independent of ρ factor, such as lipoprotein terminator and trp operon terminator, can be used. Regarding the sequence order of these control regions on the recombinant plasmid, the promoter sequence, the ribosome binding sequence, the gene encoding the nucleoside phosphorylase, and the transcription termination sequence are preferably arranged in this order from the 5′-terminal side upstream.

As a specific example of the plasmid mentioned here,
PBR3 having a region capable of autonomous replication in Escherichia coli
22, pUC18, Bluescript II SK
(+), PKK223-3, pSC101, and pUB110, p having a region capable of autonomous replication in Bacillus subtilis
TZ4, pC194, ρ11, φ1, φ105, etc. can be used as a vector. In addition, as an example of a plasmid capable of autonomous replication in two or more types of hosts, p
HV14, TRp7, YEp7 and pBS7 can be used as vectors.

Escherichia coli is a representative example of the arbitrary host as described below, but it is not particularly limited to E. coli, but Bacillus subtilis and the like. Other microbial strains such as Bacillus, yeast and actinomycetes are also included.

The nucleoside phosphorylase activity in the present invention is not limited to the above-mentioned microbial cells having the enzyme activity, but may also be a treated product of the microbial cells having the enzyme activity or an immobilized product thereof. . The treated cells are, for example, acetone-dried bacterial cells or bacteria disrupted by mechanical disruption, ultrasonic disruption, freeze-thaw treatment, pressure reduction treatment, osmotic treatment, autolysis, cell wall decomposition treatment, surfactant treatment, etc. It may be a crushed body or the like, and if necessary, it may be used after being repeatedly purified by ammonium sulfate precipitation, acetone precipitation, or column chromatography.

In the present invention, the phosphate ion and the metal cation capable of forming a sparingly water-soluble salt form a sparingly water-soluble salt with the phosphate ion by-produced in the reaction and may precipitate in the reaction solution. It is not limited as long as it is one. Such include metal cations such as calcium, magnesium, barium, iron, cobalt, nickel, copper, silver, molybdenum, lead, zinc, lithium. Among them, metal salts that are industrially highly versatile and safe and do not affect the reaction are particularly preferable, and examples of such salts include calcium ion, barium ion, magnesium ion or aluminum ion.

The metal cation capable of forming a sparingly water-soluble salt with a phosphate ion in the present invention is a metal cation capable of forming a sparingly water-soluble salt with phosphoric acid, including a chloride ion, a nitrate ion, a carbonate ion, and a sulfuric acid. It may be added to the reaction solution as a metal salt with one or more kinds of anions selected from ions, hydroxide ions or acetate ions. Specifically, calcium chloride, calcium nitrate, calcium carbonate, calcium sulfate, calcium hydroxide, calcium acetate, barium chloride, barium nitrate, barium carbonate, barium sulfate, barium hydroxide, barium acetate, magnesium chloride, magnesium nitrate, carbonate Magnesium, magnesium sulfate,
Examples include magnesium hydroxide, magnesium acetate, aluminum chloride, aluminum nitrate, aluminum carbonate, aluminum sulfate, aluminum acetate and the like.

Also, the metal cation is pentose-1.
-It may be present in the reaction solution as a salt of phosphoric acid. For example, ribose-1-phosphate calcium salt, 2
-Deoxyribose-1-phosphate / calcium salt, 2,
3-dideoxyribose-1-phosphate / calcium salt,
2,3-Dideoxy-3-fluororibose-1-phosphate-calcium salt, arabinose-1-phosphate-calcium salt, ribose-1-phosphate-barium salt, 2-deoxyribose-1-phosphate-barium Salt, 2,3-dideoxyribose-1-phosphate / barium salt, 2,3-dideoxy-3-fluororibose-1-phosphate / barium salt, arabinose-1-phosphate / barium salt, ribose-1- Phosphoric acid / magnesium salt, 2-deoxyribose-1-phosphoric acid / magnesium salt, 2,3-dideoxyribose-1-phosphoric acid / magnesium salt, 2,3-dideoxy-3-fluororibose-1-phosphoric acid / Magnesium salt, arabinose-1-phosphate / magnesium salt,
Ribose-1-phosphate / aluminum salt, 2-deoxyribose-1-phosphate / aluminum salt, 2,3-dideoxyribose-1-phosphate / aluminum salt, 2,3
-Dideoxy-3-fluororibose-1-phosphate / aluminium salt, arabinose-1-phosphate / aluminum salt, and the like.

In the synthesis reaction of the nucleoside compound in the present invention, the desired nucleoside, 1-phosphorylated sugar and a base which are substrates, nucleoside phosphorylase which is a reaction catalyst or a microorganism having the enzyme activity, and phosphoric acid are used in the reaction system. Depending on the type of the metal salt to be added and its characteristics, the reaction conditions such as pH and temperature and the control range may be selected, but usually pH 5 to 10 and temperature 10
It can be performed in the range of -60 ° C. If the pH is out of the control range, the reaction conversion rate may be lowered due to the stability of the target substance or substrate, the decrease in enzyme activity, the non-formation of a poorly water-soluble salt with phosphoric acid, etc. . During the reaction, pH
If such a change occurs, an acid such as hydrochloric acid or sulfuric acid or an alkali such as sodium hydroxide or potassium hydroxide may be added as needed. The concentration of the 1-phosphorylated sugar and the base used in the reaction is appropriately about 0.1 to 1000 mM, and the molar ratio of the two is 0.1 to 1-phosphorylated sugar or its salt. It can be performed in a 10-fold molar amount. Considering the reaction conversion rate, a molar amount of 0.95 times or less is preferable.

The metal salt capable of forming a sparingly water-soluble salt with phosphoric acid to be added is 0.1 to 10 times the molar amount of 1-phosphorylated sugar used in the reaction, more preferably 0.5. ~ 5
It is better to add a double molar amount. The addition method is not limited, and may be added all at once or sequentially during the reaction. Further, although this reaction basically uses water as a solvent, an appropriate amount of an organic solvent such as alcohol or dimethyl sulfoxide used in a usual enzyme reaction may be added to the reaction system if necessary. In a high-concentration reaction, the base of the substrate or the nucleoside compound of the product may not be completely dissolved and may be present in the reaction solution, but the present invention can also be applied to such a case.

The nucleoside compound thus produced can be isolated by applying a conventional method such as concentration, crystallization, dissolution, electrodialysis treatment, adsorption / desorption treatment with an ion exchange resin or activated carbon.

[0110]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Reference Example 1 Methyl 2,3-dideoxy-
3-fluoro-5-O- (4-phenylbenzoyl)-
Synthesis of D-pentafuranoside (5) Methyl 2,3-dideoxy-3-fluoro-5-O-
Pivaloyl-D-pentafuranoside (JP-A-5-978)
It can be produced by the method described in 85, etc.) 20.42 g
To a solution of (87.2 mmol) in 204 mL of methanol,
4.71 g of sodium methoxide (87.
2 mmol) was added in 3 portions. After stirring at room temperature for 1 hour, 250 mL of ethyl acetate was added, silica gel filtration (50 g, 2.5 cm thickness) was performed, and the mixture was washed 3 times with 100 mL of ethyl acetate. After concentrating the filtrate, adding 100 mL of toluene and concentrating it twice was performed.
L was added and concentrated. 131 mL of pyridine was added to the concentrated residue, and 4-phenylbenzoic acid chloride 1 was added with stirring under ice cooling.
9.5 g (89.8 mmol) was added in 9 portions. After stirring overnight at room temperature, add 2.3 mL of methanol,
The mixture was stirred at room temperature for 1 hour. 240 mL of water was added, and the mixture was extracted twice with 120 mL of isopropyl ether. 10 organic layers
After washing 3 times with 24 mL of 24% sulfuric acid, it was washed with 24 mL of water, and further 3 times with 24 mL of 5% aqueous sodium hydrogen carbonate. The organic layer was concentrated to give 29.2 g of the title compound as a pale yellow oily substance.

Reference Example 2 Synthesis of 1-O-acetyl-2,3-dideoxy-3-fluoro-5-O- (4-phenylbenzoyl) -D-pentafuranoside (6) Obtained in [Reference Example 1]. Compound (5) 17.0 g (51.5
(4 mmol) of acetic anhydride in acetic anhydride (42.5 mL) under stirring with ice-cooling and concentrated sulfuric acid (1.07 mL, 21.0 mmol) of acetic acid (4).
The 2.5 mL solution was added dropwise over 27 minutes. 3 at 4 ° C
After stirring for 2 hours, 28.6 g of sodium acetate trihydrate (21
0 mmol) and 187 mL of water are added, and toluene 1 is added.
Extracted with 70 mL and 100 mL. The organic layer was washed twice with 100 mL of 5% aqueous sodium hydrogen carbonate and then with 100 mL of water,
The organic layer was concentrated to give the title compound (19.0 g) as a pale brown oil.

Reference Example 3 Synthesis of 2,3-dideoxy-3-fluoro-5-O- (4-phenylbenzoyl) -D-pentafuranosyl chloride (7) Compound (6) obtained in [Reference Example 2] 18.4 g (51.3
(mmol) in 37 mL of toluene under ice-cooling with stirring.
4.2% hydrochloric acid-isopropyl ether 29.7 mL (1
15 mmol) was added dropwise. After stirring at 4 ° C for 1 hour, 184 mL of cyclohexane was added, and the mixture was further stirred under ice cooling for 5.
Stir for 5 hours. Under ice-cooled reduced pressure (260 mmHg), 30
After stirring for 1 minute to remove 10.2 g of a low boiling substance containing hydrochloric acid, the precipitate was collected by filtration under a nitrogen atmosphere, and cyclohexane 5
Washed with 0 mL. The obtained crude crystals were dried at room temperature under reduced pressure to give the title compound (11.4 g, yield 66%) as a colorless powder.

Example 1 2,3-Dideoxy-3-fluoro-5-O- (4-phenylbenzoyl) -α-D-pentafuranosyl-1-
Synthesis of phosphoric acid (8) 102 mg of orthophosphoric acid, 69.4 μL of N, N-dicyclohexylamine and 100 m of molecular sieves 4A.
g in various solvents at 0 ° C., and after 50 hours, α-form / β
The body ratio was measured by HPLC. The heats of formation of α-forms were calculated and calculated by the semi-empirical molecular orbital method and the uniform field approximation method. The results are shown in Table-1.

[0115]

[Table 1] [Table-1] Dielectric constant of reaction solvent and anomeric selectivity (α-form: β-form) Reaction solvent Relative permittivity α-form: β-form Heat of formation (kJ / mol) Toluene 2.4 49: 51-1634.9 Acetone 20.7 71: 29-1731.5 2-butanone 18.4 73: 27-1730.1 Acetonitrile 37.5 79: 21-1740.6 propylene Carbonate 65.1 81: 19-1742.9.

As shown in [Table-1], the heat of formation of the α-form has a lower value than the relative permittivity of the solvent, and the α-form exists more stably in the solvent having a large relative permittivity. This result explains the relationship between the anomeric selectivity and the relative dielectric constant of the solvent.

Example 2 2,3-Dideoxy-3-fluoro-5-O- (4-phenylbenzoyl) -α-D-pentafuranosyl-1-
Synthesis of phosphoric acid (8) 102 mg of orthophosphoric acid, 69.4 μL of N, N-dicyclohexylamine and 100 m of molecular sieves 4A.
g in a mixed solvent of each ratio of 5-methyl-2-pentanone and toluene at −5 ° C., and after 24 hours, α-form /
The β body ratio was measured by HPLC. The results are shown in Table-2 (Table 2). However, the relative dielectric constant of 5-methyl-2-pentanone is 1
3.1, relative permittivity of toluene 2.4.

[0118]

[Table 2] [Table-2] Mixing ratio of reaction solvents and anomeric selectivity (α: β) 4-Methyl-2-pentanone: Toluene α-form: β-form                         1: 9 54:46                         2: 8 59:41                         4: 6 70:30                         6: 4 71:29                         8: 2 72:28

Example 3 2,3-Dideoxy-3-fluoro-5-O- (4-phenylbenzoyl) -α-D-pentafuranosyl-1-
Synthesis of phosphoric acid (8) 102 mg of orthophosphoric acid, 69.4 μL of N, N-dicyclohexylamine and 100 m of molecular sieves 4A.
g in a mixed solvent of each ratio of 5-methyl-2-pentanone and chloroform at −5 ° C., and after 24 hours, α
The body / β-body ratio was measured by HPLC. The results are shown in Table-3 (Table 3). However, the relative permittivity of 5-methyl-2-pentanone is 13.1 and the relative permittivity of toluene is 4.8.

[0120]

[Table 3] [Table-3] Mixing ratio of reaction solvents and anomeric selectivity (α-form: β-form) 4-methyl-2-pentanone: chloroform α-form: β-form                         1: 9 30:70                         2: 8 30:70                         4: 6 56:44                         6: 4 71:29                         8: 2 72:28

Example 4 2,3-Dideoxy-3-fluoro-5-O- (4-phenylbenzoyl) -α-D-pentafuranosyl-1-
Synthesis of phosphoric acid (8) Orthophosphoric acid 53.2 g, 5-methyl-2-pentanone
To a mixture of 52 mL and 468 mL of acetonitrile, 43.2 mL of tri-n-butylamine and 52 g of Molecular Sieves 4A were added, and the mixture was cooled to -3 ° C while stirring. 52 g of compound (7) obtained in [Reference Example 3] (wet crystal: methyl 2,3-dideoxy-3-fluoro-5)
-O-pivaloyl-D-pentafuranoside (theoretical amount from 36 g) was added and stirred. After 10 minutes, the α-form / β-form ratio of the title compound (8) in the reaction suspension was 47:53. After further stirring for 8 hours, the α-form / β-form ratio was 78:22. Add 129 mL of tri-n-butylamine,
The molecular sieves were filtered off. 4-methyl- in the filtrate
2-pentanone (520 mL) was added, and the mixture was washed with water (520 mL). The organic layer was ice-cooled and cyclohexylamine 4
4.2 mL was added and the mixture was crystallized with stirring. After 1 hour, the precipitated crystals were collected by filtration and dried under reduced pressure at room temperature to obtain 54.7 g of the dicyclohexylamine salt of the title compound (8) as a colorless powder. Yield 59% (converted into raw material purity: α form: β form = 8
0:20) ¹ H NMR (CD ₃ OD, 400 MHz, 21.8 ° C) δ 8.13 (d, J =
8.3 Hz, 1/5 of 2 H), 8.08 (d, J = 8.3 Hz, 4/5 of 2
H), 7.75 (d, J = 8.3 Hz, 2 H), 7.67 (d, J = 8.3 H
z, 2 H), 7.47 (dd, J = 8.3 & 7.3 Hz, 2 H), 7.39
(m, 1 H), 6.02 (m, 1/5 H), 5.96 (dd, J = 5.6 & 4.6
Hz, 4/5 H), 5.34 (bd, J = 54.6 Hz, 1/5 H), 5.26 (b
d, J = 55.6 Hz, 4/5 H), 4.71 (bd, J = 25.7 Hz, 4/5
H), 4.49 (dd, J = 13.2 & 4.1 Hz, 1 H), 4.42 (dd,
J = 13.2 & 4.1 Hz, 1 H), 3.00 (m, 2 H), 2.54 2.32
(m, 2 H), 2.01 (m, 4 H), 1.78 (m, 4 H), 1.65 (d, J
= 12.4 Hz, 2 H), 1.32 (m, 8 H), 1.20 (m, 2 H): FT-
IR (cm ^-1 ,, KBr) 3424,2938, 2858, 1709, 1612, 1561,
1451, 1390, 1279, 1084, 980, 935, 748, 699: MS (AP
CI) m / z 395 (MH) ^- .

Example 5 Synthesis of 2,3-dideoxy-3-fluoro-α-D-pentafuranosyl-1-phosphate (9) 4 g of the compound (8) obtained in [Example 4] was added to methanol 1
Suspended in a mixed solution of 20 mL and 24 mL of ammonia water,
Stir at room temperature. Further cyclohexylamine 1.92
mL was added and stirred. After 1 month, the precipitated crystals were filtered off, the filtrate was concentrated, and water was added to remove the precipitate. The filtrate was concentrated, ethanol was added, and the mixture was stirred at room temperature for 2 hours.
The precipitated crystals were collected by filtration and dried under reduced pressure at room temperature to give the title compound (9).
2.20 g of the cyclohexylamine salt of was obtained as a colorless powder. Yield 79% (On NMR, α-form: β-form is 5:
1. ) ¹ H NMR (D ₂ O, 400 MHz, 20.8 ° C) δ 5.64 (dd, J = 5.
6 & 5.6 Hz, 1/6 H), 5.61 (dd, J = 5.7 & 5.4 Hz, 5 /
6 H), 5.02 (dd, J = 54.6 & 6.1 Hz, 1 / 6H), 4.96 (d
d, J = 54.9 & 5.1 Hz, 5/6 H), 4.30 (ddd, J = 27.1,
5.1 & 4.4Hz, 5/6 H), 4.06 (ddd, J = 24.2, 5.4 &
6.1 Hz, 1/6 H), 3.5 3.3 (m, 2 H), 2.90 (m, 2 H),
2.2 2.1 (m, 2 H), 1.74 (m, 4 H), 1.56 (m, 4 H),
1.41 (d, J = 11.7 Hz, 2 H), 1.10 (m, 8 H), 0.95
(m, 2 H): FT-IR (cm ^-1 ,, KBr) 2936, 2858, 2560, 220
9, 1625, 1561, 1453, 1392, 1243, 1066, 980, 933, 8
44,732: MS (APCI) m / z 215 (MH) ^- ..

Example 6 Synthesis of 2,3-dideoxy-3-fluoro-α-D-pentafuranosyl-1-phosphate (9) 45.6 g of the compound (8) obtained in [Example 4] was used as 0. .35
Suspended in 460 mL of mM potassium hydroxide aqueous solution, 50 ° C
And stirred for 3 hours. The precipitated crystals were filtered off, and the filtrate was obtained as an aqueous potassium salt solution of the title compound (9).

Example 7 Synthesis of 2 ′, 3′-dideoxy-3′-fluoro-β-D-guanosine (10) Escherichia coli K-12 / XL-10 strain (Str
atagene) inoculated into 50 ml of LB medium and 3
After culturing overnight at 7 ° C, the cells were collected and lysozyme 1 mg / ml
Lysis was performed with a lysate containing After the lysate was treated with phenol, DNA was precipitated by ethanol precipitation by a usual method. The generated DNA precipitate was wound around a glass rod, collected, and then washed to prepare Escherichia coli chromosomal DNA.

[0125] The primer for PCR was prepared by using the known deoD gene nucleotide sequence of Escherichia coli (GenBank ac
cession No. AE000508 (coding region is base number 11531-12
The oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 1 and 2 which were designed based on 250) were used. These primers have restriction enzyme recognition sequences for EcoRI and HindIII near the 5'end and the 3'end, respectively.

Escherichia coli chromosomal DNA 6 ng / μl
And 0.1 ml of PCR reaction solution containing 3 μM each of primers and denaturation: 94 ° C., 1 minute, annealing: 45
PCR was carried out under the condition of 25 cycles of a reaction cycle consisting of 1 ° C for 1 minute and extension reaction: 72 ° C for 1 minute.

The above reaction product and plasmid pUC18
(Takara Shuzo Co., Ltd.) was digested with EcoRI and HindIII, ligated with Ligation High (Toyobo Co., Ltd.), and Escherichia coli DH5α was transformed with the obtained recombinant plasmid. Transformants
Ampicillin (Am) 50 μg / ml and X-Gal
(5-Bromo-4-chloro-3-indolyl-β-D-
It was cultured in LB agar medium containing galactoside) to obtain a transformant strain that was Am-resistant and became a white colony. Extracting a plasmid from the transformant thus obtained,
The plasmid into which the desired DNA fragment was inserted was designated pTA1.
I named it. The transformant thus obtained was named Escherichia coli MT-10905.

Escherichia coli MT-10905
3 strains in 100 ml LB medium containing 50 μg / ml Am
Culture was carried out with shaking at 7 ° C overnight. 1300 of the obtained culture solution
The cells were collected by centrifugation at 0 rpm for 10 minutes and frozen and stored.

An aqueous solution of the potassium salt of compound (9) prepared in [Example 6] was diluted with water, and 7.8 g of guanine and sodium hydroxide were added to a mixture of 6.6 g of the purine nucleoside phosphorylase-producing bacterium prepared above. The resulting aqueous solution was added portionwise and adjusted to pH 11 with calcium chloride hydrate and sodium hydroxide. The total amount of sodium hydroxide used was 7.5 g, and the amount of calcium chloride hydrate was 13.4 g. After completion of the reaction, the mixture was heated and dissolved, the insoluble matter was removed by filtration, and the filtrate was neutralized with hydrochloric acid to pH 7.5. The precipitate was collected by filtration and dried under reduced pressure to give the title compound (10) 8.09.
g was obtained. Yield 45%.

Example 8 3,5-O-bis (4-chlorobenzoyl) -2-deoxy-α-D-pentafuranosyl-1-phosphate (11)
Synthesis of N, N, N ', N'-tetramethylethylenediamine
5.49 g of phosphate and 10 g of 3,5-O-bis (4-chlorobenzoyl) -2-deoxy-α-D-pentafuranosyl chloride were stirred in 100 mL of DMF at room temperature. The α-form / β-form ratio was measured by HPLC. The α-form / β-form ratio immediately after the reaction was 47:53. Furthermore, the α-form / β-form ratio after standing overnight was 70:30.

Example 9 3,5-O-bis (4-chlorobenzoyl) -2-deoxy-α-D-pentafuranosyl-1-phosphate (11)
Synthesis of N, N-dimethylaniline phosphate 5.61 g, imidazole 1.43 g, 3,5-O-bis (4-chlorobenzoyl) -2-deoxy-α-D-pentafuranosyl chloride 10 g DMF 100 mL And stirred at room temperature. The α-form / β-form ratio was measured by HPLC. The α-form / β-form ratio after 30 minutes was 58:42. Furthermore, the α-form / β-form ratio after standing overnight was 68:32.

Example 10 3,5-O-bis (4-chlorobenzoyl) -2-deoxy-α-D-pentafuranosyl-1-phosphate (11)
Synthesis of tri-n-octylamine phosphate 42 mg, tetrabutylammonium iodide 17.2 mg, 3,5-O-
Bis (4-chlorobenzoyl) -2-deoxy-α-D
20 mg of pentafuranosyl chloride in various solvents,
Stir at room temperature. The results of measuring the α-form / β-form ratio by HPLC are shown in [Table-4] (Table 4).

[0133]

[Table 4] [Table-4] Relative permittivity of various solvents and α / β ratio Reaction solvent Dielectric constant α-form: β-form Chloroform 4.8 42:58 Toluene 2.4 51:49 Ethyl acetate 8.6 52:48 2-butanone 18.5 59:41 DMF 36.7 73:27

[0134]

INDUSTRIAL APPLICABILITY The present invention is highly useful as a method for producing anomeric selective 1-phosphorylated sugars and nucleosides, and is expected to be widely used.

Continued front page    (72) Inventor Hironori Kamachi             580-32 Nagaura, Sodegaura-shi, Chiba Mitsui Chemicals, Inc.             Inside the company F-term (reference) 4B024 AA01 BA10 CA03 DA06 GA11                       HA17                 4B064 AF33 CA21 CB30 CD09 DA01                       DA16                 4C057 AA03 AA10 AA18 AA20 BB05                       CC01 DD03 GG06 HH04 LL42                       LL44

Claims (14)

[Claims] 1. A method for the anomeric selective production of 1-phosphorylated saccharide, which involves isomerization of anomeric phosphate ester. 2. The method according to claim 1, wherein a solvent having a relative dielectric constant of 10 or more is used as a solvent when isomerizing the anomeric phosphate ester. 3. The process according to claim 1, wherein the isomerization of the anomeric phosphate ester is carried out under acidic conditions of pH 6 or less. 4. A 1-phosphorylated sugar is represented by the general formula (1): [Chemical Formula 1] [Wherein, R1 and R2 each independently represent a hydrogen atom, a methyl group, a protected hydroxymethyl group or a protected carboxyl group, R3 represents an acyl group, and R4 represents a hydrogen atom or a carbon number of 1 to 1]. 4 represents an alkyl group, R5 represents a hydroxyl-protecting 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 a substituted group. Represents a carbon atom, n represents 0 or 1, p and q represent an integer of 0 to 4, and r
Represents 0 or 1. However, when X is an alkoxy group or an alkylthio group and r is 1 or more, each alkyl group may be bonded to the alkyl group of R4 to form a ring. , Q, r, n are p + q + r ≦ 2n + 3 when Z is an oxygen atom or a sulfur atom.
And when Z is a carbon atom, p + q + r ≦ 2n + 5 is satisfied. ] The manufacturing method of Claim 1 to 3 shown by these. 5. The method according to claim 1 or 2, after which the protecting group elimination reaction is further carried out.
-Anomeric selective production of phosphorylated sugars. 6. After carrying out the production method according to claim 4, a protecting group elimination reaction is then carried out to give a compound of the general formula (2) [Chemical Formula 2] (In the formula, R1a and R2a each independently represent a hydrogen atom, a methyl group, a hydroxymethyl group or a carboxyl group, and R3a represents a hydrogen atom or an acyl group,
R4 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, 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, n represents 0 or 1, p and q represent an integer of 0 to 4, and r represents 0 or 1. However, when X is an alkoxy group or an alkylthio group and r is 1 or more, each alkyl group may be bonded to the alkyl group of R4 to form a ring. ,
q, r and n are p when Z is an oxygen atom or a sulfur atom.
+ Q + r ≦ 2n + 3, and p + q + when Z is a carbon atom
It satisfies r ≦ 2n + 5. ) The method for producing 1-phosphorylated saccharide. 7. A compound represented by the general formula (1a) [Chemical Formula 3] which can be obtained by the production method according to any one of claims 1 to 5. (In the formula, R1b and R2b each independently represent a hydrogen atom, a methyl group, a protected hydroxymethyl group, a hydroxymethyl group, a protected carboxyl group or a carboxyl group, and R3a represents a hydrogen atom or an acyl group. , R5a represents a hydrogen atom or a hydroxyl-protecting group,
R4 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, 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, n represents 0 or 1, p and q represent an integer of 0 to 4, and r represents 0 or 1. However, when X is an alkoxy group or an alkylthio group and r is 1 or more, each alkyl group may be bonded to the alkyl group of R4 to form a ring. ,
q, r and n are p when Z is an oxygen atom or a sulfur atom.
+ Q + r ≦ 2n + 3, and p + q + when Z is a carbon atom
It satisfies r ≦ 2n + 5. ) 1-phosphorylated sugar derivative and its salt shown by these. 8. 1- according to any one of claims 1 to 6.
Having a first step of producing a phosphorylated saccharide and a second step of performing an exchange reaction between a phosphate group and a base of 1-phosphorylated saccharide obtained in the first step by the action of nucleoside phosphorylase A method for producing a characteristic nucleoside derivative. 9. A nucleoside derivative is represented by the general formula (3): [Chemical Formula 4] (In the formula, B independently represents pyrimidine, purine,
Shows a base selected from the group consisting of azapurine and deazapurine, which are halogen atom, alkyl group, haloalkyl group, alkenyl group, haloalkenyl group, alkynyl group, amino group, alkylamino group, hydroxyl group, hydroxyamino group, aminoxy group. , An alkoxy group, a mercapto group, an alkylmercapto group, an aryl group, an aryloxy group or a cyano group. R1 and R2 each independently represent a hydrogen atom, a methyl group, a protected hydroxymethyl group or a protected carboxyl group, R3 represents an acyl group, and R4
Represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, 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, n represents 0 or 1, and p and q represents an integer of 0 to 4, and r represents 0 or 1. However, when X is an alkoxy group or an alkylthio group and r is 1 or more, each alkyl group may be bonded to the alkyl group of R4 to form a ring. , Q,
r and n are p + q when Z is an oxygen atom or a sulfur atom.
+ R ≦ 2n + 3, and when Z is a carbon atom, p + q + r ≦
2n + 5 is satisfied. ] The manufacturing method of Claim 8 shown by these. 10. A nucleoside phosphorylase activity causes an exchange reaction between a phosphate group and a base of the 1-phosphorylated sugar derivative according to claim 7 to give a compound of the general formula (3) [Chemical Formula 5] [Chemical Formula 5] (In the formula, B, R1, R2, R3, R4, X, W, Z,
n, p, q and r are as described above. ) A method for producing a nucleoside represented by 11. A nucleoside phosphorylase is a purine nucleoside phosphorylase (EC2.4.2.1),
Guanosine phosphorylase (EC 2.4.2.15),
Pyrimidine nucleoside phosphorylase (EC2.4.
2.2), uridine phosphorylase (EC 2.4.2.
3), thymidine phosphorylase (EC 2.4.2.
4), deoxyuridine phosphorylase (EC2.4.
The method according to any one of claims 8 to 10, which uses a nucleoside phosphorylase selected from the group consisting of 2.23). 12. A nucleoside phosphorylase is purine nucleoside phosphorylase (EC2.4.2.1),
Guanosine phosphorylase (EC 2.4.2.15),
Pyrimidine nucleoside phosphorylase (EC2.4.
2.2), uridine phosphorylase (EC 2.4.2.
3), thymidine phosphorylase (EC 2.4.2.
4), deoxyuridine phosphorylase (EC2.4.
The method according to any one of claims 8 to 10, wherein the microorganism expresses at least one nucleoside phosphorylase selected from the group consisting of 2.23). 13. A phosphoric acid ion reacts with a metal cation capable of forming a sparingly water-soluble salt during the exchange reaction between the phosphate group and the base of the 1-phosphorylated sugar derivative by the action of nucleoside phosphorylase activity. It exists in a liquid, The manufacturing method of Claim 8 to 12 characterized by the above-mentioned. 14. A metal cation capable of forming a poorly water-soluble salt with phosphoric acid is selected from calcium ion, barium ion, magnesium ion and aluminum ion.
The method according to claim 13, wherein the metal cations are of more than one kind.