JP5946703B2 - Process for producing modified polysaccharides - Google Patents

Description

  The present invention relates to a method for producing a modified polysaccharide.

Among the polysaccharides, hyaluronic acid is abundantly contained in biological tissues such as bovine eyeballs, chicken crowns, animal buffer tissues, placenta, cancer cells and skin. Glucuronic acid and N-acetylglucosamine are β1, This is a high-molecular-weight glucosaminoglycan having a molecular weight of 10 ⁵ to 10 ⁶ Da, which is a linear polysaccharide bonded alternately by 3 bonds and β1,4 bonds. Hyaluronic acid has a high viscosity, a high moisturizing effect, and an excellent lubricating effect against physical friction and an excellent protective effect against invasion of bacteria and the like.
Since hyaluronic acid has these properties, it is widely used as a pharmaceutical agent such as a cosmetic additive, an arthritis therapeutic agent, a wound dressing, an ophthalmic surgical adjuvant, and a post-surgical adhesion inhibitor.

Hyaluronic acid is frequently used as a cosmetic additive because it has particularly excellent moisture retention. However, there is a problem that it flows away from the skin surface due to sweat or the like.
In addition, a method for producing a hyaluronic acid derivative in which hyaluronic acid is chemically modified by reacting a hydroxyl group and / or a carboxyl group in the hyaluronic acid molecule with a carboxylic acid and / or an alcohol for esterification is known. (Patent Document 1). However, when chemical modification such as esterification reaction is performed on hyaluronic acid, the entire molecule is chemically modified, which causes a problem that the hydrophilicity of hyaluronic acid is extremely lowered. There is also a problem that excellent properties such as moisture retention of hyaluronic acid are lost. Therefore, it is desired to develop a method for producing a modified polysaccharide in which a part of a molecule including a hyaluronic acid is chemically modified instead of the whole molecule.

Japanese Patent Laid-Open No. 06-298804

  An object of the present invention is to provide a method for producing a modified polysaccharide partially chemically modified.

The inventor of the present invention has arrived at the present invention as a result of studies to achieve the above object.
That is, the present invention relates to a chemically synthesized polysaccharide (A) which is hyaluronic acid acetylated by a compound having a hydroxyl group and / or a carboxyl group and the following ribonucleoside diphosphate-monosaccharide (B) to a polysaccharide synthase ( This is a method for producing a modified polysaccharide to be reacted in the presence of C).
Ribonucleoside diphosphate-monosaccharide (B): triose (b-1), tetrose (b-2), pentose (b-3), hexose (b-4), heptose (b-5) and the following monosaccharide The hydrogen atom of at least one hydroxyl group contained in at least one monosaccharide (b) selected from the group consisting of (b-6) is substituted with any one functional group of the following chemical formulas (1) to (5) Sugar nucleotides.
Monosaccharide (b-6): a monosaccharide selected from the group consisting of (b-1), (b-2), (b-3), (b-4) and (b-5), A monosaccharide in which at least one selected from the group consisting of a hydrogen atom, a hydroxyl group and a hydroxymethyl group possessed by is substituted with the following substituent (D).
Substituent (D): carboxyl group, amino group, N-acetylamino group, sulfo group, methyl ester group, N-glycolyl group, methyl group, 1,2,3-trihydroxypropyl group, phosphate group and 2- At least one substituent selected from the group consisting of carboxy-2-hydroxyethyl groups;

  The method for producing a modified polysaccharide of the present invention can produce a novel modified polysaccharide that is partially chemically modified.

  In the method for producing a modified polysaccharide of the present invention, a chemically modified polysaccharide (A) and a ribonucleoside diphosphate-monosaccharide (B) are reacted in the presence of a polysaccharide synthase (C).

The chemically modified polysaccharide (A) includes those obtained by chemically binding other compounds to the polysaccharide by a chemical reaction.
The polysaccharide is a series of monosaccharides (b-1) to (b-6) described later, specifically, hyaluronic acid, starch, amylose, amylopectin, glycogen, cellulose, chitin, chitosan, agarose, Carrageenan, heparin, pectin, xyloglucan, xanthan gum and the like are included.
Specific examples of (A) include reaction products obtained by reacting the following compound (E) with polysaccharides.
Compound (E): A compound having a functional group (e) that reacts with a compound having at least one selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, a sulfo group, and a phosphate group.
The compound (E) includes a compound (E1) having at least one selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, a sulfo group, a phosphonic acid group, and a phosphoric acid group as the functional group (e). It is.

(E1) includes the following (E11) to (E15).
(E11): Compound having a hydroxyl group (E12): Compound having a carboxyl group (E13): Compound having an amino group (E14): Compound having a sulfo group (E15): Compound having a phosphonic acid group or a phosphoric acid group (E16): Compound having a hydroxyl group and a carboxyl group

(E11) includes C1-C100 monovalent alcohol (E111) and C2-C100 bivalent or higher alcohol (E112).
(E111) includes monovalent aliphatic alcohol (E111-1), monovalent araliphatic alcohol (E111-2), alkylene oxide adduct (E111-3) of monovalent aliphatic alcohol, and monovalent And an alkylene oxide adduct (E111-4) of a monovalent monoalkyl or a monovalent dialkylamine (E111-5).

  (E111-1) includes aliphatic monools having 1 to 30 carbon atoms, specifically, lower alcohols having 1 to 5 carbon atoms (methanol, ethanol, propanol, butanol, pentanol, etc.), carbon Higher alcohols of several 6 to 30 (hexyl alcohol, octyl alcohol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecyl alcohol, hexadecyl alcohol, heptadecanol, octadecanol, oleyl alcohol, nonadecanol, eicosanol, seryl Alcohol, myricyl alcohol, etc.) and their respective branched chain alcohols. Two or more of these may be combined.

  (E111-2) includes an araliphatic monool having 7 to 30 carbon atoms, and specifically includes benzyl alcohol and the like.

  (E111-3) includes the above-mentioned (E111-1) alkylene oxide (hereinafter abbreviated as AO) adduct (degree of polymerization = 1-100). AO includes those having 2 to 10 carbon atoms. Specifically, ethylene oxide (hereinafter abbreviated as EO), 1,2-propylene oxide (hereinafter abbreviated as PO), butylene oxide and styrene. Examples include oxides. Two or more of these may be used in combination as AO. Specific examples of (E111-3) include oleyl alcohol EOPO adducts and the like.

  (E111-4) includes an AO adduct of an alkylphenol having 7 to 30 carbon atoms (degree of polymerization = 1 to 100). Specific examples of the alkylphenol having 7 to 30 carbon atoms include nonylphenol and octylphenol. AO includes those having 2 to 10 carbon atoms, and examples thereof include EO, PO, butylene oxide, and styrene oxide. Two or more of these may be used in combination as AO. Specific examples of (E111-4) include an EO adduct of nonylphenol and an EO adduct of octylphenol.

  (E111-5) includes an AO adduct of C 1-30 monoalkyl or dialkylamine (degree of polymerization = 1-100). AO includes those having 2 to 10 carbon atoms, and examples thereof include EO, PO, butylene oxide, and styrene oxide. Two or more of these may be used in combination as AO. Specific examples include hexadecylamine EOPO adduct, laurylamine EO adduct, stearylamine EOPO adduct, and the like.

Specifically, as (E112), ethylene glycol, propylene glycol, 1,3 and 1,4-butanediol, 1,6-hexanediol, 1,10-decanediol, diethylene glycol, neopentyl glycol, cyclohexanediol, Dihydric alcohols such as cyclohexanedimethanol, 1,4-bis (hydroxymethyl) cyclohexane and 1,4-bis (hydroxyethyl) benzene; trihydric alcohols such as glycerin and trimethylolpropane; pentaerythritol, sorbitol, mannitol, sorbitan , Diglycerin, dipentaerythritol, etc., 4- to 8-valent alcohols such as sucrose, glucose, mannose, fructose, methylglucoside and derivatives thereof; phenol, phloroglucin, cresol Pyrogallol, catechol, hydroquinone, bisphenol A, bisphenol F, bisphenol S, 1-hydroxynaphthalene, 1,3,6,8-tetrahydroxynaphthalene, anthrol, 1,4,5,8-tetrahydroxyanthracene And phenol such as 1-hydroxypyrene; polybutadiene polyol; castor oil-based polyol and the like.
Of the above (E11), (E111) is preferred from the viewpoint of easy chemical modification.

As a compound (E12) which has a carboxyl group, a C1-C100 monovalent carboxylic acid (E121) and a C1-C100 bivalent or more carboxylic acid (E122) are contained.
(E121) includes monovalent aliphatic carboxylic acid (E121-1), monovalent aromatic carboxylic acid (E121-2), and monovalent araliphatic carboxylic acid (E121-3).

  Examples of (E121-1) include aliphatic monocarboxylic acids having 1 to 100 carbon atoms, specifically, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, Examples include pelargonic acid, capric acid, crotonic acid, isocrotonic acid, sorbic acid, obtusilic acid, and caproleic acid.

  Examples of (E121-2) include aromatic monocarboxylic acids having 7 to 100 carbon atoms, and specific examples include benzoic acid, methylbenzoic acid, propylbenzoic acid, hexylbenzoic acid, and dodecylbenzoic acid. .

  Examples of (E121-3) include araliphatic monocarboxylic acids having 8 to 100 carbon atoms, and specific examples include phenylacetic acid, phenylpropionic acid, and phenyldecanoic acid.

Examples of the divalent or higher carboxylic acid (E122) include alkane dicarboxylic acids having 4 to 100 carbon atoms (such as succinic acid, adipic acid, sebacic acid, azelaic acid, dodecanedicarboxylic acid, octadecanedicarboxylic acid, and decylsuccinic acid); To 100 alicyclic dicarboxylic acids [dimer acid (dimerized linoleic acid) and the like]; alkene dicarboxylic acids having 4 to 100 carbon atoms (alkenyl succinic acids such as dodecenyl succinic acid, pentadecenyl succinic acid and octadecenyl succinic acid) Acid, maleic acid, fumaric acid, citraconic acid, etc.); aromatic polycarboxylic acid having 8 to 100 carbon atoms (phthalic acid, isophthalic acid, terephthalic acid, 2,2'-bibenzyldicarboxylic acid, trimellitic acid, hemirit Acid, trimesic acid, pyromellitic acid and naphthalene-1,4 dicarboxylic acid, naphthalene- , 3,6 tricarboxylic acid, diphenic acid, 2,3-anthracene dicarboxylic acid, 2,3,6-anthracentricarboxylic acid, and C8-C18 aromatic polycarboxylic acid such as pyrene dicarboxylic acid). .
Of the above (E12), (E121) is preferable from the viewpoint of easy chemical modification.

Examples of the compound having an amino group (E13) include an amine having 1 to 100 carbon atoms (E131).
As (E131), a monovalent aliphatic amine (E131-1), a divalent or higher aliphatic amine (E131-2), an alicyclic amine (E131-3), an aromatic amine (E131-4), and Heterocyclic amines (E131-5) are included.

  Specific examples of (E131-1) include monoalkylamines having 1 to 100 carbon atoms (such as methylamine, ethylamine, propylamine and butylamine); dialkylamines having 1 to 100 carbon atoms (dimethylamine, diethylamine, dipropyl) Amines, propylmethylamine, propylethylamine, etc.); C1-C100 trialkylamines (trimethylamine, ethyldimethylamine, diethylmethylamine, triethylamine, tri-n-propylamine, tri-n-butylamine, etc.) and the like.

  (E131-2) includes those having 1 to 100 carbon atoms, specifically 1,2-diaminoethane, 1,2-propanediamine, 1,3-propanediamine, 1,4-butane. Examples include diamine, 1,6-diaminohexane, 1,2-bis (2-aminoethoxy) ethane, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, iminobispropylamine, and bis (hexamethylene) triamine. .

  (E131-3) includes those having 3 to 100 carbon atoms, and specific examples include isophoronediamine and 1,2-cyclohexanediamine.

  (E131-4) includes those having 6 to 100 carbon atoms, specifically, aniline, 1,3-phenylenediamine, 2,4-tolylenediamine, 1,3-xylylenediamine, , 5-naphthalenediamine, 2,3-anthracenediamine, and the like.

(E131-5) includes those having 3 to 100 carbon atoms, and specific examples include piperazine, N-aminoethylpiperazine and 1,4-diaminoethylpiperazine.
Among the above (E13), (E131-1) is preferable from the viewpoint of easy chemical modification.

(E14) includes a C1-C100 compound having one sulfo group (E141) and a C1-C100 compound having two or more sulfo groups (E142).
(E141) is an aliphatic monosulfonic acid having 1 to 100 carbon atoms (methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butanesulfonic acid, hexanesulfonic acid, decanesulfonic acid, undecanesulfonic acid, dodecanesulfonic acid, etc. C6-C30 aromatic monosulfonic acid (benzenesulfonic acid, p-toluenesulfonic acid, o-toluenesulfonic acid, m-toluenesulfonic acid, 4-dodecylbenzenesulfonic acid, 4-octylbenzenesulfonic acid and naphthalene) Sulfonic acid etc.); C1-C20 perfluoroalkanesulfonic acid (trifluoromethanesulfonic acid etc.) etc. are mentioned.

(E142) is an aliphatic polysulfonic acid having 1 to 100 carbon atoms (methionic acid, 1,1-ethanedisulfonic acid, 1,2-ethanedisulfonic acid, 1,1-propanedisulfonic acid, 1,3-propanedisulfonic acid. Acid and polyvinyl sulfonic acid); aromatic polysulfonic acid having 6 to 100 carbon atoms (m-benzenedisulfonic acid, 1,4-naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, 1,6-naphthalenedisulfonic acid, 2 , 6-naphthalenedisulfonic acid, 2,7-naphthalenedisulfonic acid and sulfonated polystyrene, etc.); bis (fluorosulfonyl) imide; bis (perfluoroalkanesulfonyl) imide having 2 to 10 carbon atoms [bis (trifluoromethanesulfonyl) imide Bis (pentafluoroethanesulfonyl) imide, bis (no Perfluorobutane sulfonyl) imide, pentafluoroethanesulfonyl trifluoromethanesulfonyl imide, trifluoromethanesulfonyl heptafluoropropanesulfonyl imide, nonafluorobutanesulfonyl trifluoromethanesulfonyl imide], and the like.
Among the above (E14), (E141) is preferable from the viewpoint of easy chemical modification.

  (E15) includes phosphoric acid, pyrophosphoric acid, triphosphoric acid, polyphosphoric acid, phosphonic acid having 1 to 100 carbon atoms (dimethyl phosphonate, diethyl ethylphosphonate, dipropyl methylphosphonate, tributyl methylphosphonate, dibutyl butylphosphonate, phenyl Diphenyl phosphonate, dicresyl phenylphosphonate, dixylyl phenylphosphonate, phenyl cresyl phenylphosphonate, etc.) and phosphoric acid groups having 1 to 100 carbon atoms (trimethyl phosphate, triethyl phosphate, tripropyl phosphate, triisopropyl phosphate) Tributyl phosphate, triisobutyl phosphate, triphenyl phosphate, tricresyl phosphate, trixylyl phosphate, diphenyl cresyl phosphate, methyl diphenyl phosphate, phenyl diethyl phosphate, and the like.

  (E16) includes hydroxycarboxylic acids having 2 to 100 carbon atoms. Specifically, aliphatic hydroxycarboxylic acids (glycolic acid, lactic acid, 2-hydroxybutyric acid, 6-hydroxyhexanoic acid, 2,8- Dihydroxycaprylic acid, 10-hydroxycapric acid, 12-hydroxylauric acid, 16-hydroxypalmitic acid, 18-hydroxyoleic acid, 22-hydroxybehenic acid, malic acid and citric acid); aromatic hydroxycarboxylic acids ( Salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, 4-hydroxyphthalic acid, 5-hydroxytrimellitic acid, 3-hydroxypyromellitic acid, etc.).

Of the above (E), (E11), (E12) and (E16) are preferred from the viewpoint of easy availability of raw materials and the properties of the resulting modified polysaccharide, more preferably monovalent aliphatic alcohol (E111). -1) and a monovalent carboxylic acid (E121) having 1 to 100 carbon atoms, particularly preferably at least one selected from the group consisting of methanol and acetic acid.
In addition, the chemically modified polysaccharide (A) may be a polysaccharide in which (A) is easily produced and from the viewpoint of the physical properties of the resulting modified polysaccharide, the hydroxyl group and / or carboxyl group of the polysaccharide is esterified. More preferably, it is a polysaccharide in which the hydroxyl group and / or carboxyl group of the polysaccharide is acetylated, and still more preferably hyaluronic acid in which the hydroxyl group and / or carboxyl group of the polysaccharide is acetylated.

In the chemically modified polysaccharide (A), from the viewpoint of obtaining a large amount of modified polysaccharide, (A) at least two terminal monosaccharides constituting the sugar chain of one molecule are chemically modified. It is preferred to include non-polysaccharide molecules. (A) When a molecule | numerator is a polysaccharide molecule by which the terminal monosaccharide was modified, the efficiency which a polysaccharide synthase (C) performs an elongation reaction will worsen. Therefore, it is preferable that (A) includes a polysaccharide molecule in which at least two terminal monosaccharides are not chemically modified.
(A) As a method for preparing a polysaccharide (A) in which the terminal monosaccharide constituting the sugar chain of the molecule is not chemically modified, when synthesizing the chemically modified polysaccharide (A), a substitution rate described later is used. Examples include a method of synthesizing so as to be less than 1. Specifically, among the functional groups {hydroxyl group, carboxyl group, amino group, sulfo group, and phosphate group} of the polysaccharide before chemical modification, the amount of (E) that can react with the hydroxyl group is the hydroxyl group. For example, it may be less than the number of moles.

When the chemically modified polysaccharide (A) is obtained by modifying the carboxyl group of the polysaccharide with the compound (E), the modification with (E11) includes a conventional compound having a carboxyl group and a hydroxyl group. A method of preparing an ester compound from a compound can be used, and as a method of modifying with (E13), a conventional method of preparing an amide compound from a compound having a carboxyl group and a compound having an amino group can be used.
In addition, when (A) is a compound in which the hydroxyl group of the polysaccharide is modified with the compound (E), the modification with (E12) can be carried out by using a conventional compound having a hydroxyl group and a compound having a carboxyl group as an ester compound. As a method of modifying in (E14), a conventional method of creating a sulfonate compound from a compound having a hydroxyl group and a compound having a sulfo group can be used, and the method is modified in (E15). As a method, a conventional method of making a phosphate ester compound from a car having a hydroxyl group and a compound having a phosphonic acid group or a phosphoric acid group can be used.
Further, conventional methods for synthesizing chemically modified polysaccharides (Japanese Patent Laid-Open Nos. 09-110901, 09-124701, 11-012304, etc.) can be used.

  The chemically modified polysaccharide (A) may be selected from at least one selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, a sulfo group, and a phosphate group that the polysaccharide has.

Further, the ratio (substitution rate) in which the chemically modified polysaccharide (A) is modified by (E) is 0.001 to 0.001 from the viewpoint of changing the physical properties of the modified polysaccharide (appropriately imparting hydrophobicity, etc.). 1 is preferable, and 0.1 to 1 is more preferable. Further, from the viewpoint of efficiently obtaining a modified polysaccharide, the substitution rate is preferably less than 1, more preferably 0.9 or less, and still more preferably 0.8 or less.
The substitution rate is obtained by the following formula.
Replacement rate = M2 / M1
M1: Number of moles of functional group {hydroxyl group, carboxyl group, amino group, sulfo group, and phosphate group} that can react with compound (E) in the polysaccharide before chemical modification M2: Compound added to polysaccharide (E The number of moles of (A) can be measured by NMR. For example, when the compound (E) is acetic acid, hyaluronic acid is chemically modified, and (A) is acetylated hyaluronic acid, NMR of the acetylated hyaluronic acid is measured and obtained from the integral value by the following formula.
Substitution rate = (peak integrated value of acetyl group / 3) / {(peak integrated value of hydroxyl group) + (peak integrated value of acetyl group / 3)}

  The weight average molecular weight measured by the liquid chromatography (HPLC) method of the chemically modified polysaccharide (A) is preferably 500 to 10,000,000 from the viewpoint of ease of production of the modified polysaccharide.

In the present invention, ribonucleoside diphosphate-monosaccharide (B) is triose (b-1), tetrose (b-2), pentose (b-3), hexose (b-4), heptose (b-5). ) And at least one monosaccharide (b) selected from the group consisting of the following monosaccharides (b-6) is any one of the following chemical formulas (1) to (5): A sugar nucleotide substituted with a functional group.
Monosaccharide (b-6): a monosaccharide selected from the group consisting of (b-1), (b-2), (b-3), (b-4) and (b-5), A monosaccharide in which at least one selected from the group consisting of a hydrogen atom, a hydroxyl group and a hydroxymethyl group possessed by is substituted with the following substituent (D).
Substituent (D): carboxyl group, amino group, N-acetylamino group, sulfo group, methyl ester group, N-glycolyl group, methyl group, 1,2,3-trihydroxypropyl group, phosphate group and 2- At least one substituent selected from the group consisting of carboxy-2-hydroxyethyl groups;
The monosaccharide (b) includes optical isomers and stereoisomers.

Triose (b-1) is a monosaccharide having 3 carbon atoms, and specific examples include dihydroxyacetone and glyceraldehyde.
Tetrose (b-2) is a monosaccharide having 4 carbon atoms, and specific examples include erythrose, threose and erythrulose.
Pentose (b-3) is a monosaccharide having 5 carbon atoms, and specific examples include arabinose, xylose, ribose, xylulose, ribulose and deoxyribose.
Hexose (b-4) is a monosaccharide having 6 carbon atoms, and specific examples include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fucose, and rhamnose.
Heptose (b-5) is a monosaccharide having 7 carbon atoms, and specific examples thereof include sedheptulose.

The monosaccharide (b-6) substituted with the substituent (D) includes a hydrogen atom (—H) and a hydroxyl group (—OH) in the molecules of the monosaccharides (b-1) to (b-5). And at least one of hydroxymethyl group (—CH ₂ OH) is a carboxyl group, an amino group, an N-acetylamino group, a sulfo group, a methyl ester group, an N-glycolyl group, a methyl group, 1,2,3-tri The following (b-6-1) to (b-6-10) substituted with at least one substituent selected from the group consisting of a hydroxypropyl group, a phosphate group and a 2-carboxy-2-hydroxyethyl group; The following (b-6-11) is included.
(B-6-1): Monosaccharide substituted with a carboxyl group (—COOH) (b-6-2): Monosaccharide substituted with an amino group (—NH ₂ ) (b-6-3): N - monosaccharide substituted with an acetylamino group _{(-NHCOCH 3) (b-6-4} ): monosaccharide substituted with a sulfo group _{(-OSO 3 H) (b-} 6-5): methyl ester group (- monosaccharides substituted by _{COOCH 3) (b-6-6)} : N- monosaccharide substituted with a glycolyl group _{(-NHCOCH 2 OH) (b-} 6-7): monosaccharide substituted with a methyl group ( b-6-8): monosaccharide (b-6-9) substituted with 1,2,3-trihydroxypropyl group (—CHOHCHOHCH ₂ OH): substituted with phosphate group (—OPO ₃ H ₂ ) monosaccharide (b-6-10): substituted with 2-carboxy-2-hydroxyethyl group (-CH ₂ CHOHCOOH) Monosaccharides (b-6-11): (b-1) to (b-5) in which at least two of the hydrogen atom, hydroxyl group and hydroxymethyl group in the molecule are two or more substituents (D) Monosaccharide substituted with

Specific examples of (b-6-1) include uronic acids such as glucuronic acid, iduronic acid, mannuronic acid and galacturonic acid.
Specific examples of (b-6-2) include amino sugars such as glucosamine, galactosamine and mannosamine.
Specific examples of (b-6-3) include N-acetylglucosamine, N-acetylmannosamine and N-acetylgalactosamine.
Specific examples of (b-6-4) include galactose-3-sulfuric acid.
Specific examples of (b-6-5) include glucose methyl ester and methyl ester of carboxylic acid in (b-6-1).
Specifically, as (b-6-11), N-acetylmuramic acid, muramic acid, N-acetylglucosamine-4-sulfuric acid, iduronic acid-2-sulfuric acid, glucuronic acid-2-sulfuric acid, N-acetyl Examples include galactosamine-4-sulfuric acid, sialic acid, neuraminic acid, N-glycolylneuraminic acid, and N-acetylneuraminic acid.

  When (b-6) has at least one selected from the group consisting of a carboxyl group, a phosphate group, a 2-carboxy-2-hydroxyethyl group, and a sulfo group as an anionic group as the substituent (D), The proton of the group may be substituted with an alkali metal (Li, Na and K etc.) cation and / or an alkaline earth metal (Ca etc.) cation.

In the ribonucleoside diphosphate-monosaccharide (B), the hydrogen atom of at least one hydroxyl group of the monosaccharides (b-1) to (b-6) is substituted with the chemical formulas (1) to (5). Sugar nucleotides (B-1) to (B-6).
Specific examples of (B-3) include uridine diphosphate-xylose.
Specific examples of (B-4) include cytidine diphosphate-glucose, guanosine diphosphate-mannose, guanosine diphosphate-fucose, adenosine diphosphate-glucose, uridine diphosphate-glucose, uridine 2-phosphorus. Examples include acid-galactose and uridine diphosphate-mannose.
Specific examples of (B-6) include uridine diphosphate-glucuronic acid, uridine diphosphate-N-acetylglucosamine, uridine diphosphate-uridine diphosphate-N-acetylgalactosamine, and uridine diphosphate- And iduronic acid.

  In the manufacturing method of this invention, (B) may use 1 type and may use 2 or more types. In addition, one type of (B) may be used to produce a modified polysaccharide in which one type of monosaccharide is connected in series. Two types of (B) may be used to determine whether the two types of monosaccharides are random or Alternately modified polysaccharides may be produced, or three or more kinds of (B) may be used to produce modified polysaccharides in which three or more kinds of monosaccharides are randomly or regularly linked. Moreover, you may manufacture multiple types of modified polysaccharide using 2 or more types (A), 2 or more types (B), and 2 or more types (C).

The polysaccharide synthase (C) is an enzyme having an activity of synthesizing a polysaccharide from the above (B). In the production method of the present invention, by reacting (A) and (B) in the presence of (C), (C) catalyzes a reaction in which (B) transfers a sugar to (A), A modified saccharide can be produced by binding a sugar to A). In addition, the modified polysaccharide obtained by the production method of the present invention is such that a plurality of unmodified sugars are bonded to the chemically modified polysaccharide (A) (for example, if the molecule (A) is a straight chain, it has two terminals. A non-chemically modified polysaccharide segment binds to a non-chemically modified polysaccharide segment—a chemically modified polysaccharide segment—an unmodified polysaccharide segment binds to a modified polysaccharide), so that part of the molecule is chemically modified The resulting polysaccharide.
The polysaccharide synthase (C) includes hyaluronic acid synthase (C-1) in which the sugar chain before chemical modification of the chemically modified polysaccharide (A) is hyaluronic acid, and chondroitin synthase (C- 2), xanthan synthase (C-3) which is xanthan, cellulose synthase (C-4) which is cellulose, starch synthase (C-5) which is starch, and heparin synthase (C-6) which is heparin Is included. In addition to (C-1) to (C-6), (C) includes an enzyme having an activity of transferring a sugar from (B) to synthesize a polysaccharide.

Hyaluronic acid synthase (C-1) is ribonucleoside diphosphate-glucuronic acid and ribonucleoside diphosphate-N-acetylglucosamine as a sugar donor, and glucuronic acid is β1,3 linked to N-acetylglucosamine. It is an enzyme that synthesizes a sugar having a repeating structure of β1,4 bonds of linked disaccharide units.
As (C-1), conventional hyaluronic acid synthase can be used, and it is not particularly limited as long as it has hyaluronic acid synthesizing activity. Specific examples include class I and class II hyaluronic acid synthases described in non-patent literature (The Journal of Biological Chemistry, 2007, Vol. 282, No. 51, P36777-36781). Class I and class II are classified according to the homology of the amino acid sequence of the enzyme. Specific examples of class I hyaluronic acid synthase include hyaluronic acid synthase derived from Streptococcus spirogenesis, derived from Streptococcus equisimilis, and algal virus. Specific examples of class II hyaluronic acid synthase include hyaluronic acid synthase derived from Pasteurella multocida.

In the production method of the present invention, when hyaluronic acid is produced using (C-1) as (C), from the viewpoint of the production amount of modified polysaccharide per unit enzyme, (B) used is ribonucleoside 2-phosphorus. Acid-glucuronic acid and ribonucleoside diphosphate-N-acetylglucosamine are preferable, and uridine diphosphate-glucuronic acid and uridine diphosphate-N-acetylglucosamine are more preferable.
Moreover, from the viewpoint of the production amount of the modified polysaccharide per unit enzyme, it is preferable that (A) to be used is a polysaccharide in which hyaluronic acid is chemically modified.

Chondroitin synthase (C-2) is ribonucleoside diphosphate-glucuronic acid and ribonucleoside diphosphate-N-acetylgalactosamine as a sugar donor, and glucuronic acid binds to N-acetylgalactosamine with β1,3 bonds. It is an enzyme that synthesizes a sugar having a repeating structure of β1,4 bonds of the disaccharide unit.
As (C-2), conventional chondroitin synthase can be used, and it is not particularly limited as long as it has chondroitin synthesis activity. Specific examples include chondroitin synthase derived from Streptococcus equisimilis and chondroitin synthase derived from Pasteurella multocida.

In the production method of the present invention, when chondroitin is produced using (C-2) as (C), as (B) to be used, ribonucleoside diphosphate is used from the viewpoint of the production amount of modified polysaccharide per unit enzyme. -Glucuronic acid and ribonucleoside diphosphate-N-acetylgalactosamine are preferred, and uridine diphosphate-glucuronic acid and uridine diphosphate-N-acetylgalactosamine are more preferred.
Moreover, from the viewpoint of the production amount of the modified polysaccharide per unit enzyme, it is preferable that (A) to be used is a polysaccharide in which chondroitin is chemically modified.

Xanthan synthase (C-3) is an enzyme that synthesizes xanthan using ribonucleoside diphosphate-glucose, ribonucleoside diphosphate-mannose and ribonucleoside diphosphate-glucuronic acid as sugar donors.
As (C-3), a conventional xanthan synthase can be used, and it is not particularly limited as long as it has xanthan synthesizing activity. Specific examples include xanthan synthase obtained from xanthan campestris.

In the production method of the present invention, when xanthan is produced using (C-3) as (C), from the viewpoint of the production amount of modified polysaccharide per unit enzyme, (B) used is ribonucleoside diphosphate. -Glucose, ribonucleoside diphosphate-mannose and ribonucleoside diphosphate-glucuronic acid are preferred, and uridine diphosphate-glucose, guanosine diphosphate-mannose and uridine diphosphate-glucuronic acid are more preferred.
Moreover, from the viewpoint of the production amount of modified polysaccharide per unit enzyme, as (A) to be used, xanthan is preferably a chemically modified polysaccharide.

Cellulose synthase (C-4) is an enzyme that forms β1,4-linkages using ribonucleoside diphosphate glucose as a sugar donor.
(C-4) is not particularly limited as long as a conventional cellulose synthase can be used and has cellulose synthesis activity. Specific examples include cellulose synthase derived from acetic acid bacteria.

In the production method of the present invention, when (C) is used to produce cellulose using (C-4), from the viewpoint of the production amount of modified polysaccharide per unit enzyme, (B) used is ribonucleoside diphosphate. -Β-glucose is preferable, and uridine diphosphate-β-glucose is more preferable.
Moreover, from the viewpoint of the production amount of the modified polysaccharide per unit enzyme, it is preferable that (A) to be used is a polysaccharide in which cellulose is chemically modified.

Starch synthase (C-5) is an enzyme that forms α1,6-linkage using ribonucleoside diphosphate-α-glucose as a sugar donor.
As (C-5), a conventional starch synthase can be used, and it is not particularly limited as long as it has starch synthesis activity. Specific examples include corn-derived starch synthase.

In the production method of the present invention, when producing starch using (C-5) as (C), from the viewpoint of the production amount of modified polysaccharide per unit enzyme, (B) used is ribonucleoside 2-phosphorus. Acid-α-glucose is preferable, and uridine diphosphate-α-glucose is more preferable.
Moreover, from the viewpoint of the production amount of modified polysaccharide per unit enzyme, as (A) to be used, it is preferable that starch is a chemically modified polysaccharide.

Heparin synthase (C-6) is ribonucleoside diphosphate-glucuronic acid (β-D-) or ribonucleoside diphosphate iduronic acid (α-L-) and ribonucleoside diphosphate-glucosamine (D-). Glucosamine) is an enzyme that forms a 1,4-bond using a sugar donor.
(C-6) is not particularly limited as long as it can use conventional heparin synthase and has heparin synthesis activity. Specific examples include human-derived heparin synthase.

In the production method of the present invention, when heparin is produced using (C-6) as (C), from the viewpoint of the production amount of modified polysaccharide per unit enzyme, (B) used is ribonucleoside diphosphate. -Preferably glucuronic acid or ribonucleoside diphosphate-iduronic acid and ribonucleoside diphosphate-glucosamine, more preferably uridine diphosphate-glucuronic acid or uridine diphosphate-iduronic acid and uridine diphosphate -Be with glucosamine.
Further, from the viewpoint of the production amount of modified polysaccharide per unit enzyme, as (A) to be used, it is preferable that heparin is a chemically modified polysaccharide.

  The (A), sugar nucleotide (B) and polysaccharide synthase (C) are appropriately selected according to the modified polysaccharide to be produced.

In the production method of the present invention, the concentration of ribonucleoside diphosphate in the reaction solution is less than 100 times the inhibitory concentration IC ₅₀ shown below of polysaccharide synthase (C).
Inhibitory concentration IC _50: ribonucleoside diphosphate - at a concentration of (C) the time of the reaction in the presence of a simple sugar (B) a polysaccharide synthase (C), ribonucleoside diphosphate as substrate - monosaccharide (B ), The concentration of ribonucleoside diphosphate when the enzyme activity of (C) is halved, determined using ribonucleoside diphosphate as an inhibitor.
Inhibitory concentration IC ₅₀ is the same concentration of (C) as when (C) is reacted at any time from the start to the end of the step of reacting (B) in the presence of (C), It can be determined by performing the following measurements under temperature and pH conditions.
<Method of measuring the inhibitory concentration IC _50>
Enzyme reaction solution prepared at a certain temperature and pH containing a certain amount of polysaccharide synthase (C), ribonucleoside diphosphate-monosaccharide (B), ribonucleoside diphosphate, pH adjuster (K) and water ( I) is created.
The temperature of the enzyme reaction solution (I) is adjusted to the same temperature as any temperature from the start to the end of the step of causing (C) to act on (B) during the production process.
The pH of the enzyme reaction solution (I) is adjusted to the same pH as at any time from the start to the end of the step of allowing (C) to act on (B) during the production process.
The molar concentration of (C) in the enzyme reaction solution (I) is adjusted to the same concentration as that at any time from the start to the end of the step of causing (C) to act on (B).
The content (molar concentration) of ribonucleoside diphosphate in the enzyme reaction solution (I) is determined from the enzyme reaction solution (I) in which the ribonucleoside diphosphate is 0 M and the concentration of ribonucleoside diphosphate from 0 M in the polysaccharide synthesis. Four or more kinds of enzyme reaction solutions (I) in which the concentration of ribonucleoside diphosphate is different from the concentration of ribonucleoside diphosphate where the activity of enzyme (C) becomes 0 (polysaccharide production cannot be observed) A total of five or more types of enzyme reaction solutions (I) are prepared. In addition, when the inhibition constant Ki of ribonucleoside diphosphate for similar polysaccharide synthase is known, the concentration range of ribonucleoside diphosphate is 0 M, and the concentration range is less than Ki of similar polysaccharide synthase and greater than 0 M It is sufficient to create two or more types, two or more types within a concentration range of Ki or more and ten times or less of Ki, and a total of five or more types.
The content of the ribonucleoside diphosphate-monosaccharide (B) in the enzyme reaction solution (I) may be selected at one concentration at which the peak area change over time can be observed. When the Michaelis constant Km of the polysaccharide synthase similar to (C) used for the measurement is known, it may be selected between a concentration range of not less than Km and not more than 5 times Km.
The pH adjuster (K) used in the enzyme reaction solution (I) is preferably a Good buffer such as phosphate, borate, HEPES buffer, and MES buffer from the viewpoint of ease of handling and enzyme stability. The content (molar concentration) of (K) in the enzyme reaction solution (I) is 100 to 500 mM.
With respect to the prepared enzyme reaction solution (I), a part of the solution (for example, 100 μL) is taken out immediately after the preparation and every fixed time (for example, 5 minutes), and the extracted solution is heat-treated at 100 ° C. for 1 minute to Stop the reaction. The amount of the modified polysaccharide in the removed reaction solution is quantified using liquid chromatography (hereinafter abbreviated as HPLC). The peak area immediately after preparation of the enzyme reaction solution (I) is P ₀ , the peak area after h hours is P _h, and a calibration curve for the peak area change ΔP (= P _h −P ₀ ) and the peak area of the modified polysaccharide is shown. To calculate the initial enzyme reaction velocity v (M / s).
Furthermore, the enzyme reaction solution (I) having a different ribonucleoside diphosphate concentration is measured in the same manner, and the initial reaction velocity v is calculated.
The inhibitory concentration IC ₅₀ is obtained when the horizontal axis (x axis) is the concentration of each ribonucleoside diphosphate in the enzyme reaction solution (I) and the vertical axis (y axis) is 0 when the concentration of ribonucleoside diphosphate is 0. Relative activity is plotted when the initial reaction rate v is 100 (%). The plots are connected by a straight line, and the concentration of ribonucleoside diphosphate when y = 50 (%) is defined as the inhibitor concentration IC ₅₀ .

In the present invention, the concentration of ribonucleoside diphosphate in the reaction solution is preferably less than 100 times the inhibitory concentration IC ₅₀ and more preferably 10 times or less from the viewpoint of efficiently producing the modified polysaccharide.
When a plurality of types of (C) are used, it is preferable that all (C) inhibition concentrations IC ₅₀ are measured, and at least one (C) inhibition concentration IC ₅₀ falls within the above range.
In the step of reacting (A) and (B) in the presence of (C), the time during which the concentration of ribonucleoside diphosphate is within the above range is obtained by combining (A) and (B) with (C) Is 10 to 100% of the time of the reaction in the presence of, preferably from 30 to 100% from the viewpoint of reaction efficiency, more preferably from 50 to 100%, especially The time is preferably 80 to 100%, and most preferably 90 to 100%.
When (A) and (B) are reacted in the presence of (C), ribonucleoside diphosphate is produced as a by-product. Since the activity of (C) is easily inhibited by ribonucleoside diphosphate, the concentration of ribonucleoside diphosphate is within the above range in the step of reacting (A) and (B) in the presence of (C). Preferably there is.

In the production method of the present invention, examples of the method for setting the concentration of ribonucleoside diphosphate in the above range include the following methods (i) to (iii).
(I) A method of converting ribonucleoside diphosphate to a compound (H) described later using a ribonucleoside diphosphate converting enzyme (G) (ii) A ribonucleoside diphosphate in a reaction solution using a silica gel carrier or the like (Iii) a method of converting ribonucleoside diphosphate into another compound by chemical reaction

In the method (ii), besides the silica gel carrier, any active carbon or zeolite that can adsorb ribonucleoside diphosphate can be used without limitation.
In the method (iii), a generally known chemical reaction can be used without limitation as long as it is a chemical reaction capable of converting ribonucleoside diphosphate into another compound.

  In the present invention, the method of bringing the concentration of ribonucleoside diphosphate into the above range has problems such as high substrate specificity of the reaction and decomposition of the substrate (ribonucleoside diphosphate-monosaccharide (B)). From the viewpoint of being small, the method (i) is preferable.

The method for converting ribonucleoside diphosphate to compound (H) using ribonucleoside diphosphate converting enzyme (G) in (i) specifically includes the following methods.
Ribonucleoside 2 having the activity of converting ribonucleoside diphosphate into the following compound (H) in a method for producing a modified polysaccharide by reacting ribonucleoside diphosphate-monosaccharide (B) with polysaccharide synthase (C) A method for producing a modified polysaccharide in which (C) is allowed to act in the presence of a phosphate converting enzyme (G).
Compound (H): Purine base or pyrimidine base (H-1), ribonucleoside (H-2), ribonucleoside monophosphate (H-3), ribonucleoside triphosphate (H-4), polyribonucleotide ( H-5), at least one compound selected from the group consisting of deoxyribonucleoside diphosphate (H-6) and ribonucleoside diphosphate-monosaccharide (H-7).

(H-1) includes purine bases (such as adenine and guanine) and pyrimidine bases (such as thymine, cytosine and uracil).
(H-2) is a compound in which the base of (H-1) and a monosaccharide are bonded, and specific examples include uridine, adenosine, ribothymidine, cytidine, and guanosine.
(H-3) is a monophosphate ester of (H-2), specifically, uridylic acid (uridine 5′-phosphate), adenosine monophosphate (adenosine 5′-phosphate), ribothymidyl Examples include acids (ribothymidine 5′-phosphate), cytidine monophosphate (cytidine 5′-phosphate), and guanosine monophosphate (guanosine 5′-phosphate).
(H-4) is a triphosphate esterified product of (H-2). Specifically, uridine triphosphate (uridine 5′-triphosphate), adenosine triphosphate (adenosine 5′-triphosphate) Acid), ribothymidine-3-phosphate (ribothymidine 5′-triphosphate), cytidine triphosphate (cytidine 5′-triphosphate), guanosine triphosphate (guanosine 5′-triphosphate) and the like.
(H-5) is a polymer obtained by polymerizing (H-3) with a phosphodiester bond, and specific examples include polyuridylic acid, polyadenylic acid, polythymidylic acid, polycytidylic acid, and polyguanylic acid.
(H-6) is obtained by converting the ribose in the ribonucleoside diphosphate molecule into 2-deoxyribose. Specifically, deoxyuridine diphosphate, deoxyadenosine diphosphate, deoxyguanosine diphosphate , Deoxycytidine diphosphate, and thymidine diphosphate.
(H-7) includes the ribonucleoside diphosphate-monosaccharide (B).

The ribonucleoside diphosphate converting enzyme (G) includes the following (G1) to (G7).
(G1) an enzyme having an activity of converting a ribonucleoside diphosphate into a purine base or a pyrimidine base (G2) an enzyme having an activity of converting a ribonucleoside diphosphate into a ribonucleoside (G3) a ribonucleoside diphosphate into a ribonucleoside Enzyme having activity to convert monophosphate (G4) Enzyme having activity to convert ribonucleoside diphosphate to ribonucleoside triphosphate (G5) Enzyme having activity to convert ribonucleoside diphosphate to polyribonucleotide (G6) an enzyme having an activity of converting ribonucleoside diphosphate to deoxyribonucleoside diphosphate (G7) an enzyme having an activity of converting ribonucleoside diphosphate to ribonucleoside diphosphate-monosaccharide

  (G2) is an enzyme that catalyzes the reaction of hydrolyzing the phosphate ester bond between the sugar and phosphoric acid in ribonucleotides into a nucleoside and phosphoric acid. Specific examples of (G2) include apyrase and apyrase.

  (G3) is an enzyme that catalyzes a reaction in which a phosphate diester such as ribonucleoside diphosphate is hydrolyzed to a phosphate monoester. Specific examples of (G3) include adenosine diphosphate (ADP) -specific phosphofructokinase and nucleotidase.

(G4) is an enzyme that catalyzes a reaction in which a phosphate group is transferred from a compound having a phosphate group to ribonucleoside diphosphate to give ribonucleoside triphosphate.
Specific examples of (G4) include nucleoside diphosphate kinase, polyphosphate kinase, arginine kinase, pyruvate kinase, carbamate kinase, phosphoglycerate kinase, and phosphocreatine kinase.
(G4) includes uridine triphosphate synthase (G4-1). (G4-1) is an enzyme that catalyzes a reaction of synthesizing uridine triphosphate, where the ribonucleoside diphosphate acting is uridine diphosphate.

  Nucleoside diphosphate kinase is an enzyme that transfers a phosphate group from nucleoside triphosphate to nucleoside diphosphate. Specific examples of the nucleoside diphosphate kinase include those derived from organisms {from animals (for example, from human, bovine and rat), from plants (for example, from Arabidopsis and rice). And those derived from microorganisms (for example, those derived from the genus Escherichia, those derived from the genus Saccharomyces, those derived from the genus Bacillus and those derived from the Thermus etc.), Chemically modified (eg, chemically modified by acting at least one selected from the group consisting of carbodiimide compounds, succinic anhydride, iodoacetic acid and imidazole compounds) and those derived from organisms {For example, Smith et al. (Th Journal of Biochemistry, 1998, Vol.253, No.18, such as those obtained by modifying the gene}, and the like using P6551-6560).

  Polyphosphate kinase is an enzyme having an activity of generating ribonucleoside triphosphate and polyphosphate having one degree of polymerization from ribonucleoside diphosphate and polyphosphate. Specific examples of the polyphosphate kinase include those derived from living organisms {from plants (for example, those derived from tobacco) and from microorganisms (from the genus Escherichia, from the genus Corynebacterium, Pseudomonas genus and Thermus genus, etc.], biologically modified ones (for example, selected from the group consisting of carbodiimide compounds, succinic anhydride, iodoacetic acid and imidazole compounds) And those obtained by genetic modification of organisms (for example, the method of Smith et al. (The Journal of Biochemistry, 1998, Vol. 253, No. 18). Those like} and the like obtained by modifying the genes using P6551-6560) and the like.

  Arginine kinase is an enzyme having an activity of generating ribonucleoside triphosphate and L-arginine from ribonucleoside diphosphate and ω-phosphono-L-arginine. Specifically, arginine kinases are derived from organisms (such as those derived from animals (such as those derived from Drosophila, shrimps and fleas), those derived from plants (such as those derived from zelkova), and microorganisms. (For example, those derived from the genus Bacillus, etc.)}, those obtained by chemically modifying organisms (for example, carbodiimide compounds, succinic anhydride, iodoacetic acid, and imidazole compounds) Chemically modified by the action of species, etc.) as well as genetically modified organisms (for example, the method of Smith et al. (The Journal of Biochemistry, 1998, Vol. 253, No. 18, P6551-6560) Etc. obtained by modifying genes using It is.

  Pyruvate kinase is an enzyme having an activity of generating ribonucleoside triphosphate and pyruvate from ribonucleoside diphosphate and phosphoenolpyruvate. Specific examples of pyruvate kinases include organism-derived {animal-derived (eg, human-derived, bovine-derived and rat-derived), plant-derived (eg, Arabidopsis-derived and castor-derived). ) And microorganisms (for example, those derived from the genus Escherichia and Saccharomyces), and those obtained by chemically modifying organisms (for example, carbodiimide compounds, succinic anhydride, iodoacetic acid) And those obtained by genetically modifying organism-derived ones (eg, the method of Smith et al. (The Journal of Biochemistry, 1998, Vol.). .253 No.18, such as those obtained by modifying the gene}, and the like using P6551-6560).

  Carbamate kinase is an enzyme having an activity of generating ribonucleoside triphosphate, carbon dioxide, and ammonia from carbamoyl phosphate and ribonucleoside diphosphate. Specific examples of the carbamate kinase include organism-derived {animal-derived (for example, rat-derived) and microorganism-derived (for example, Pyrococcus genus, and Lactobacillus genus). Etc.), those obtained by chemically modifying organisms (for example, those chemically modified by the action of at least one selected from the group consisting of carbodiimide compounds, succinic anhydride, iodoacetic acid and imidazole compounds) ) And genetically modified organisms (for example, obtained by modifying the gene using the method of Smith et al. (The Journal of Biochemistry, 1998, Vol. 253, No. 18, P6551-6560) Etc.}.

  Phosphoglycerate kinase is an enzyme having an activity of generating ribonucleoside triphosphate and 3-phosphoglycerate from 3-phosphoglycerol phosphate and ribonucleoside diphosphate. Specific examples of the phosphoglycerate kinase include those derived from organisms {from animals (for example, from rats) and from microorganisms (for example, from the genus Saccharomyces)}, A biologically-derived one (for example, a carbodiimide compound, a succinic anhydride, an iodoacetic acid and an imidazole compound that is chemically modified by the action of at least one selected from the group) and a biological-derived one (For example, those obtained by modifying a gene using the method of Smith et al. (The Journal of Biochemistry, 1998, Vol. 253, No. 18, P6551-6560), etc.).

  Phosphocreatine kinase is an enzyme having an activity of generating ribonucleoside triphosphate and creatine from phosphocreatine and ribonucleoside diphosphate. Specific examples of phosphocreatine kinases include those derived from living organisms {animal-derived (eg, rat-derived) and the like}, those obtained by chemically modifying living organisms (eg, carbodiimide compounds, succinic anhydride, And those obtained by genetic modification of organism-derived ones (for example, the method of Smith et al. (The Journal of Biochemistry, 1998). , Vol. 253, No. 18, P6551-6560), etc. obtained by modifying a gene}.

(G4) may use 1 type and may use 2 or more types.
Among (G4), arginine kinase, nucleoside diphosphate kinase, polyphosphate kinase and carbamate kinase are preferred from the viewpoint of high ribonucleoside triphosphate synthesis activity.

When making (G4) act, you may use the phosphate group containing compound (F) which gives a phosphate group to ribonucleoside diphosphate as needed. (F) is a compound having a phosphate group. (F) is preferably a compound capable of donating a phosphate group to ribonucleoside diphosphate from the viewpoint of the substrate specificity of (G4). Examples of (F) include triaminophosphine oxide, phosphorylated amino acid (for example, ω-phosphono-L-arginine), polyphosphoric acid, phosphoenolpyruvic acid and salts thereof (for example, lithium salt, sodium salt and potassium salt) Etc.), carbamoyl phosphate, 3-phosphoglycerol phosphate, phosphocreatine, and nucleoside triphosphate (eg, guanosine triphosphate and adenosine triphosphate).
When the phosphate group-containing compound (F) is used, the combination of (G4) and (F) includes a combination of nucleoside diphosphate kinase and nucleoside triphosphate, a combination of polyphosphate kinase and polyphosphate, arginine Combination of kinase and ω-phosphono-L-arginine, combination of pyruvate kinase and phosphoenolpyruvate and its salt, combination of carbamate kinase and carbamoyl phosphate, phosphoglycerate kinase and 3-phosphoglycerol phosphate , A combination of phosphoglycerate kinase and 3-phosphoglycerol phosphate, and a combination of phosphocreatine kinase and phosphocreatine.

  (G5) is an enzyme that catalyzes a reaction in which a ribonucleoside diphosphate such as ribonucleoside diphosphate is converted into a ribonucleoside monophosphate copolymer (such as polyribonucleotide) and an inorganic phosphate. Specific examples of (G5) include polyribonucleotide nucleotidyl transferase.

  (G6) is an enzyme that catalyzes a reaction in which ribonucleotides such as ribonucleoside diphosphate are reduced to deoxyribonucleotides (deoxyuridine diphosphate, etc.). Specific examples of (G6) include ribonucleoside diphosphoreductase.

  When (G6) is used as the ribonucleoside diphosphate converting enzyme (G), it is necessary to use a reducing agent (g6). As (g6), an electron transfer protein can be used, and examples thereof include reduced thioredoxin.

  (G7) is an enzyme that catalyzes a reaction of synthesizing a sugar nucleic acid (ribonucleoside diphosphate-monosaccharide) from a ribonucleoside diphosphate such as ribonucleoside diphosphate and a sugar or sugar phosphate. Specific examples of (G7) include sucrose synthase and N-acylneulaminate cytidylyltransferase.

When (G7) is used as the ribonucleoside diphosphate converting enzyme (G), it is necessary to use sugar (g7-1) or sugar phosphate (g7-2) as the raw material (g7) of the sugar nucleic acid.
(G7-1) includes monosaccharides, disaccharides and oligosaccharides, and specific examples include sucrose.
(G7-2) is a compound in which one phosphoric acid is bonded to one of the hydroxyl groups of the monosaccharide, such as glucuronic acid monophosphate (1-phospho-α-D-glucuronic acid, etc.), N-acetylglucosamine monophosphate (N-acetyl-D-glucosamine-1-phosphate etc.) etc. are mentioned.
The ribonucleoside diphosphate-monosaccharide synthesized by (G7) may be the same as (B) which is the raw material of the production method of the present invention, or may be different from (B). As the sugar (g7-1) or sugar phosphate (g7-2), the hydroxyl group of the same monosaccharide (b) as the ribonucleoside diphosphate-monosaccharide (B) that is the raw material of the production method of the present invention is 1 In the case of a compound in which two phosphates are bonded, (B) is synthesized.

Among the ribonucleoside diphosphate converting enzymes (G), (G2), (G3), (G4), (G5) and (G7) are from the viewpoint of efficiently producing a modified polysaccharide and from the viewpoint of easy industrialization. More preferably, (G4) and (G7).
(G) may use 1 type and may use 2 or more types together.

The method for producing a modified polysaccharide of the present invention is a method for producing a polysaccharide in which a polysaccharide synthase (C) is allowed to act on a conventional ribonucleoside diphosphate-monosaccharide (B) except that the chemically modified polysaccharide (A) is used. Same as. For example, the method of manufacturing by following process (1)-(3) is included.
(1) A predetermined amount of a chemically modified polysaccharide (A), a ribonucleoside diphosphate-monosaccharide (B), a buffer solution (I) and a polysaccharide synthase (C) are mixed to obtain a reaction solution (L), It is adjusted to a predetermined temperature and a predetermined pH. If necessary, ribonucleoside diphosphate converting enzyme (G) may be added. Moreover, in this process, you may stir as needed.
In the above step, the chemically modified polysaccharide (A), ribonucleoside diphosphate-monosaccharide (B) and buffer solution (I) are mixed, the temperature and pH are adjusted, and then the polysaccharide synthase (C) is added. May be added. Furthermore, (C) may be added as it is, or may be added after diluting with buffer (I).
The reaction solution (L) may contain ribonucleoside diphosphate converting enzyme (G). When (G) to be used is (G-4), (g-4) may be included. When (G) to be used is (G-6), (g-6) is included. When (G) to be used is (G-7), (g-7) is included.
Further, the reaction solution (L) may contain lipid (M), saccharide (N) and oligosaccharide (O).
(2) While adjusting the temperature of the reaction solution (L), chemically modified polysaccharide (A) and ribonucleoside diphosphate-monosaccharide (B) for a predetermined time in the presence of polysaccharide synthase (C) React. In this step, stirring may be performed if necessary.
(3) The produced modified polysaccharide is purified. As a method for purifying a modified polysaccharide, a method of adding a solvent such as an appropriate amount of alcohol (alcohol having 1 to 10 carbon atoms) to precipitate, a method of exchanging a solution using a membrane (specifically, a ceramic membrane, etc.), etc. Is mentioned.

The content (% by weight) of the chemically modified polysaccharide (A) in the reaction solution (L) is 0.000001 from the viewpoint of efficiently producing the modified polysaccharide and allowing the polysaccharide synthase (C) to act efficiently. ˜1% by weight is preferred.
The content (molar concentration) of the ribonucleoside diphosphate-monosaccharide (B) in the reaction solution (L) is from the viewpoint of efficiently producing the modified polysaccharide and from the viewpoint of allowing the polysaccharide synthase (C) to act efficiently. 0.1 mM to 2M is preferred.
The content (unit / L) of the polysaccharide synthase (C) in the reaction solution (L) is 0.1 to 0.1 from the viewpoint of efficiently producing the modified polysaccharide and the action of the polysaccharide synthase (C) efficiently. 10,000,000 units / L are preferred.
One unit is defined as one unit of activity for synthesizing hyaluronic acid from 1 μmol of ribonucleoside diphosphate-monosaccharide (B) per minute.

  The buffer solution (I) includes water containing a buffer having a buffering action. As a buffer having a buffering action, a conventional pH adjusting agent can be used, and borate buffer, phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, sulfuric acid, hydrochloric acid, citric acid, lactic acid, pyruvic acid, formic acid, chloride Sodium, potassium chloride, monoethanolamine, diethanolamine, etc. are mentioned.

  The temperature of the reaction solution (L) is preferably 0 to 100 ° C. from the viewpoints of the stability of (C) and (G) and the reaction rate.

  The pH of the reaction solution (L) is preferably from 3 to 12 from the viewpoint of the optimum reaction conditions.

In the steps (1) and (2), in addition to the chemically modified polysaccharide (A), ribonucleoside diphosphate-monosaccharide (B) and polysaccharide synthase (C), a viewpoint of efficiently producing a modified polysaccharide Therefore, ribonucleoside diphosphate converting enzyme (G) may be used. When (G-4) is used as (G), (g-4) may be used, and when (G-6) is used as (G), (g-6) must be used. When (G-7) is used as (G), it is necessary to use (g-7).
When the ribonucleoside diphosphate converting enzyme (G) is used, the content (unit / L) of (G) in the reaction solution (L) depends on the viewpoint of efficiently producing the modified polysaccharide and the polysaccharide synthase (C). From the viewpoint of causing it to act efficiently, 0.1 to 1,000,000 units / L is preferable.
One unit is defined as one unit of activity that converts 1 μmol of ribonucleoside diphosphate into compound (H) per minute.

  The content (molar concentration) of (g-4), (g-6) or (g-7) in the reaction solution (L) is effective for efficiently producing the modified polysaccharide and the polysaccharide synthase (C). From the viewpoint of making it act well, 0.01 nM to 10 M is preferable.

In addition to the above, lipid (M), saccharide (N) and oligosaccharide (O) may be used from the viewpoint of enzyme stabilization and enzyme activation.
Examples of the lipid (M) include cardiopine and oleic acid.
Examples of the saccharide (N) include glycerin.
Examples of the oligosaccharide (O) include oligohyaluronic acid.

The content (% by weight) of the lipid (M) in the reaction solution (L) is preferably 0 to 1% by weight from the viewpoint of enzyme stabilization and enzyme activation.
The content (% by weight) of the saccharide (N) in the reaction solution (L) is preferably 0 to 30% by weight from the viewpoint of enzyme stabilization and enzyme activation.
The content (% by weight) of the oligosaccharide (O) in the reaction solution (L) is preferably 0 to 1% by weight from the viewpoint of enzyme stabilization and enzyme activation.

  In the step (2), the time during which the polysaccharide synthase (C) is allowed to act is the activity of the polysaccharide synthase (C), the temperature of the reaction solution (L), the polysaccharide synthase (C), and the ribonucleoside diphosphate-monosaccharide. It differs depending on the quantity ratio of (B). The reaction time can be shortened by adjusting the temperature of the reaction solution (L) to a temperature at which the polysaccharide synthase (C) has a high activity and a high reaction rate. In addition, the larger the amount of polysaccharide synthase (C) with respect to ribonucleoside diphosphate-monosaccharide (B) in the reaction solution (L), the faster the reaction and the shorter the reaction time.

  According to the production method of the present invention, if a polysaccharide (A) that has been chemically modified can be used as a substrate by the polysaccharide synthase (C), a modified polysaccharide can be produced. In addition, the availability of (A) as a substrate is such that the substitution rate of the chemically modified polysaccharide (A) is moderate, and the monosaccharide at the end of the chemically modified polysaccharide (A) molecule is not chemically modified. The higher the amount of molecules, the higher the amount of modified polysaccharides that can be obtained.

According to the method for producing a modified polysaccharide of the present invention, it is possible to obtain a novel modified polysaccharide partially modified. In addition, when the polysaccharide is hyaluronic acid, the modified hyaluronic acid obtained by the production method of the present invention maintains the excellent properties of hyaluronic acid while maintaining the substitution rate of chemically modified hyaluronic acid, a compound that chemically modifies ( The physical properties of hyaluronic acid can be adjusted by changing the type of E). Even in polysaccharides other than hyaluronic acid, the physical properties can be adjusted by changing the substitution rate of the chemically modified polysaccharide (A) while maintaining the excellent properties of the polysaccharide.
Therefore, the modified polysaccharide obtained by the production method of the present invention is used as a pharmaceutical, a medical device such as a cosmetic, a quasi-drug, an arthritis therapeutic agent, a wound dressing, an ophthalmic surgical operation adjuvant, and a post-surgical adhesion inhibitor. Can be used widely.

  Hereinafter, although an example and a comparative example explain the present invention further, the present invention is not limited to these. Hereinafter, unless otherwise specified, “%” represents “% by weight” and “parts” represents “parts by weight”.

<Production Example 1>
A plasmid in which a gene encoding an amino acid sequence obtained by fusing a FLAG tag with the hyaluronic acid synthase of SEQ ID NO: 1 derived from Pasteurella multocida was incorporated into pKK223-3 was E. coli E. coli. After transforming into E. coli SURE, culturing and inducing expression, Escherichia coli was recovered using a centrifuge (6000 × g, 15 minutes). The recovered E. coli is resuspended in buffer A {100 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 10 mM magnesium chloride, 10 mM dodecyl maltoside, 5 mM oleic acid} and subjected to ultrasonic disruption (130 W, 10 Min). Then, it refine | purified with the anti- FLAG antibody column, and the aqueous solution of the hyaluronic acid synthase (C-1) which is a polysaccharide synthase (C) was obtained.

<Production Example 2>
A plasmid in which a gene encoding an amino acid sequence in which a FLAG tag is fused to sucrose synthase of SEQ ID NO: 2 derived from broad bean is incorporated into pET22b is transformed into E. coli BL21 (DE3), cultured, induced for expression, and centrifuged. E. coli was recovered using (6000 × g, 15 minutes). The suspension was resuspended in Buffer B {100 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 10 mM magnesium chloride}, and subjected to ultrasonic disruption (130 W, 10 minutes). Thereafter, purification was performed with an anti-FLAG antibody column to obtain an aqueous solution of sucrose synthase, which is a ribonucleoside diphosphate converting enzyme (G).

<Specific activity measurement of hyaluronic acid synthase (C-1) aqueous solution>
Aqueous solution S {1 mM uridine diphosphate-glucuronic acid (uridine diphosphate-glucuronic acid containing a radioisotope labeled with ¹⁴ C, radioactivity 300 mCi / mmol), 1 mM uridine diphosphate-N-acetylglucosamine, 50 mM phosphorus In 1 mL of acid buffer solution (pH 7.0), 100 mM sodium chloride, 10 mM magnesium chloride, 10 mM dodecyl maltoside, 5 mM oleic acid}, 10 μL of 1M sucrose, 10 μL of sucrose synthase aqueous solution obtained in Production Example 2 and Production Example 1 10 μL of the obtained aqueous solution of hyaluronic acid synthase (C-1) was added to obtain a reaction solution (X-1), which was reacted at 30 ° C. for 1 hour. After separation of the substrate and hyaluronic acid unreacted paper chromatography using filter paper, cut out the origin unit, after immersion in a liquid scintillation cocktail to measure the radioisotope using a liquid scintillation counter, ¹⁴ C The amount of hyaluronic acid synthesized was calculated from the amount of glucuronic acid labeled with. As a result, the synthesis amount of hyaluronic acid was 100 μg. Therefore, the specific activity of the aqueous solution of hyaluronic acid synthase (C-1) obtained in Production Example 1 was 0.4 unit / mL.

<Specific activity measurement of sucrose synthase aqueous solution>
To 1 mL of aqueous solution R {100 mM sodium chloride, 10 mM magnesium chloride, 50 mM phosphate buffer (pH 7.0) containing 1 mM uridine diphosphate}, add 10 μL of 1M sucrose aqueous solution and 10 μL of sucrose synthase aqueous solution. Three reaction solutions (1) were prepared and reacted at 30 ° C. for 5 minutes, 10 minutes, and 15 minutes, respectively. The amount of the reaction product (uridine diphosphate-glucose) was developed with TLC (Sidma, PEI-Cellulose plate) (developing solvent: 1M LiCl, aqueous solution containing 1M formic acid), UV light (260 nm) Detected by and determined. When the specific activity of the aqueous sucrose synthase solution was calculated from the relationship between the amount of reaction product and the reaction time, it was 0.3 unit / mL.

<Measurement of inhibitor concentration IC ₅₀ of hyaluronic acid synthase (C-1)>
In Preparation Example 1, add 940 μL of aqueous solution 1 [50 mM phosphate buffer (pH 7.5, 25 ° C.) containing 5 mM magnesium chloride and 0.05 mM sodium uridine diphosphate] in a 1.5 mL capacity tube. 10 μL of the obtained aqueous solution of hyaluronic acid synthase (C-1) was dissolved, and the tube was allowed to stand at 40 ° C. for 20 minutes using a constant temperature water bath. Here, the substrate solution 1 [uridine diphosphate-glucuronic acid sodium salt and uridine diphosphate-N-acetylglucosamine adjusted to 40 ° C. was added to buffer B {50 mM phosphate buffer (pH 7.5, 25 ° C. )} To a concentration of 20 mM each] was added to prepare an enzyme reaction solution (I-1). Immediately after the preparation of (I-1) and every 5 minutes, 100 μL was taken out, and the taken out solution was heated at 100 ° C. for 2 minutes to stop the enzyme reaction. About the solution which stopped the enzyme reaction, it centrifuged using the centrifuge (4 degreeC, 12,000 xg, 10 minutes), and the impurity was precipitated. 80 μL of the supernatant was analyzed by HPLC under the following conditions, and the peak area of hyaluronic acid was recorded.
<Measurement conditions for HPLC>
Apparatus: ACQUITY UPLC system Column: Shodex OHpak SB-806M HQ
Mobile phase: 0.1M NaCO ₃
Flow rate: 1.0 ml / min
Detector: ACQUITY UPLC RID detector Temperature: 40 ° C
In aqueous solution 1, solutions having different concentrations of uridine disodium phosphate {0 mM (aqueous solution 2), 0.15 mM (aqueous solution 3), 0.5 mM (aqueous solution 4), 1 mM (aqueous solution 5), and 3 mM (aqueous solution 6)} Created. Enzyme reaction solutions (I-2) to (I-6) were prepared in the same manner except that the aqueous solutions 2 to 6 were used in place of the aqueous solution 1. For the enzyme reaction solutions (I-2) to (I-6), the peak area of hyaluronic acid was recorded as in the case of the enzyme reaction solution (I-1).
Sodium hyaluronate (Funakoshi, “Hyarose”, molecular mass: 175 kDa) is dissolved in buffer B to concentrations of 0.001 μg / mL, 0.01 μg / mL, 0.1 μg / mL and 5 μg / mL, respectively. Were prepared and used as hyaluronic acid standard solutions (1) to (4). (1) to (4) were analyzed by HPLC, and the peak areas of hyaluronic acid were recorded. Each hyaluronic acid concentration (μg) was plotted on the horizontal axis (x axis), and each peak area P was plotted on the vertical axis (y axis), and the slope “k” of the straight line was calculated.
The peak area of uridine triphosphate immediately after preparation of (I-1) to (I-6) is P ₀ , the peak area after “m” minutes is P _h, and the change in peak area ΔP (ΔP = P _h The initial enzyme reaction velocity v (μg / s) was calculated from the following formula (1) using −P ₀ ) and the slope of the straight line.
v = ΔP / (k × m × 60) (1)
Enzyme reaction measured using enzyme reaction solutions (I-1) and (I-3) to (I-6) with initial reaction rate v measured using enzyme reaction solution (I-2) as 100% The relative value (%) of the initial speed was calculated. Using the calculated relative values, the uridine diphosphate concentration [S] on the horizontal axis (x axis) and the enzyme reaction solutions (I-1) to (I-6) on the vertical axis (y axis) The relative value of the initial reaction rate v was plotted. The concentration of uridine diphosphate at the intersection of the approximate curve of the plot and the straight line y = 50 (%) was the inhibitory concentration IC ₅₀ , and the inhibitory concentration IC ₅₀ was 0.8 mM.

<Example 1>
Aqueous solution X {0.001% acetylated hyaluronic acid (Shiseido; weight average molecular weight of about 100,000, substitution rate with acetic acid 0.15, chemically modified polysaccharide (A-1)), 1 mM uridine diphosphate-glucuronic acid 1 mM uridine diphosphate-N-acetylglucosamine, 50 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 10 mM magnesium chloride} in 1 mL of hyaluronic acid synthase (C-1) obtained in Production Example 1 10 μL of an aqueous solution was added to obtain a reaction solution (L-1), which was reacted at 30 ° C. for 1 hour. Unreacted substrate and modified hyaluronic acid, which is a modified polysaccharide, were purified using an ultrafiltration membrane {Millipore, Amicon Ultra-15 (fractionated molecular weight: 100,000)}. 100 mg of modified hyaluronic acid was obtained.
As a result of analyzing by liquid chromatography about (A-1) and the obtained modified hyaluronic acid, the peak with a molecular weight of 500,000 or more is (A-1) <1%, and the obtained modified hyaluronic acid is 80%. there were.
Moreover, about the obtained modified | denatured hyaluronic acid, the thing of molecular weight 500,000 or more was fractionated using the liquid chromatography, and the modified | denatured hyaluronic acid (V-1) after liquid chromatography fractionation was obtained. As a result of analyzing (V-1) using NMR, the substitution rate was 0.025, which was lower than the substitution rate of 0.15 in (A-1).
Thereafter, the denatured hyaluronic acid (V-1) after separation by liquid chromatography is used by using hyaluronidase (a mixture of β-N-acetylglucosaminase (Seikagaku Corporation) and β-glucuronidase (Sigma)). As a result of fractionation and fractionation with a molecular weight of 1,000 to 100,000 by liquid chromatography and analysis by NMR, the substitution rate of the modified hyaluronic acid after the degradation was 0.155.
Therefore, the modified hyaluronic acid (V-1) after fractionation by liquid chromatography is partially modified by hyaluronic acid segments (glucuronic acid and N-acetylglucosamine) not chemically modified bound to (A-1). It was found to contain modified hyaluronic acid, a modified polysaccharide.

<Example 2>
In Example 1, the acetylated hyaluronic acid in the aqueous solution X was replaced with the acetylated hyaluronic acid (A-2) having a substitution rate of “about 0.15” (Shiseido; weight average molecular weight of about 1). 100,000) was used in the same manner. 3 mg of modified hyaluronic acid was obtained.
As a result of analyzing by (A-2) and the obtained modified hyaluronic acid by liquid chromatography, the peak having a molecular weight of 500,000 or more is (A-2) <1%, and the obtained modified hyaluronic acid is 10%. there were.
With respect to the obtained modified hyaluronic acid, one having a molecular weight of 500,000 or more was fractionated using liquid chromatography to obtain modified hyaluronic acid (V-2) after fractionation by liquid chromatography. As a result of analyzing (V-2) using NMR, the substitution rate was 0.5, which was lower than the substitution rate 1 of (A-2).
Then, the denatured hyaluronic acid (V-2) after fractionation by liquid chromatography is decomposed using hyaluronidase (a mixture of β-N-acetylglucosaminase (Seikagaku) and β-glucuronidase (Sigma)). Then, a fraction having a molecular weight of 1,000 to 100,000 was collected by liquid chromatography and analyzed by NMR. As a result, the substitution rate of denatured hyaluronic acid after decomposition was 0.9.
Therefore, the modified hyaluronic acid (V-2) after fractionation by liquid chromatography is partially modified by hyaluronic acid segments (glucuronic acid and N-acetylglucosamine) not chemically modified bound to (A-2). It was found to contain modified denatured hyaluronic acid.

<Example 3>
In Example 1, the same procedure was performed except that 90 mM uridine diphosphate was contained in the aqueous solution X. 15 mg of modified hyaluronic acid was obtained.
The obtained modified hyaluronic acid was analyzed by liquid chromatography. As a result, the peak having a molecular weight of 500,000 or more was 5%.
About the obtained modified hyaluronic acid, those having a molecular weight of 500,000 or more were fractionated using liquid chromatography to obtain modified hyaluronic acid (V-3) after fractionation by liquid chromatography. As a result of analyzing (V-3) using NMR, the substitution rate was 0.07, which was lower than the substitution rate of 0.15 in (A-1).
Subsequently, the denatured hyaluronic acid (V-3) after fractionation by liquid chromatography is decomposed using hyaluronidase (mixture of β-N-acetylglucosaminase (Seikagaku) and β-glucuronidase (Sigma)). Then, a fraction having a molecular weight of 1,000 to 100,000 was collected by liquid chromatography and analyzed by NMR. As a result, the substitution rate of the modified hyaluronic acid after decomposition was 0.14.
Therefore, the modified hyaluronic acid (V-3) after fractionation by liquid chromatography is partially chemically modified with hyaluronic acid segments (glucuronic acid and N-acetylglucosamine) not chemically modified bound to (A-1). It was found to contain modified denatured hyaluronic acid.

<Example 4>
In Example 1, the acetylated hyaluronic acid in the aqueous solution X was replaced with the acetylated hyaluronic acid (A-3) having a substitution rate of “about 0.15” (A-3) (Shiseido; weight average) This was carried out in the same manner except that a molecular weight of about 100,000) was used. 50 mg of modified hyaluronic acid was obtained.
As a result of analyzing the (A-3) and the obtained modified hyaluronic acid by liquid chromatography, the peak with a molecular weight of 500,000 or more was (A-3) <1%, and the obtained modified hyaluronic acid was 30%. there were.
About the obtained modified hyaluronic acid, those having a molecular weight of 500,000 or more were fractionated using liquid chromatography to obtain modified hyaluronic acid (V-4) after fractionation by liquid chromatography. As a result of analyzing (V-4) using NMR, the substitution rate was 0.05, which was lower than 0.8, which is the substitution rate of the acetylated hyaluronic acid used as a raw material.
Then, the denatured hyaluronic acid (V-4) after liquid chromatography fractionation is decomposed using hyaluronidase (a mixture of β-N-acetylglucosaminase (Seikagaku) and β-glucuronidase (Sigma)). Then, a fraction having a molecular weight of 1,000 to 100,000 was collected by liquid chromatography and analyzed by NMR. As a result, the substitution rate of the modified hyaluronic acid after decomposition was 0.75.
Therefore, the modified hyaluronic acid (V-4) after fractionation by liquid chromatography is partially chemically modified with hyaluronic acid segments (glucuronic acid and N-acetylglucosamine) not chemically modified bound to (A-3). It was found to contain modified denatured hyaluronic acid.

<Example 5>
In Example 1, except that “sucrose (manufactured by Wako Pure Chemical Industries, Ltd.) is further added to the aqueous solution X so that the concentration in the solution becomes 100 mM, and 1 μL of the aqueous solution of sucrose synthase obtained in Production Example 2 is added”. Carried out. 200 mg of modified hyaluronic acid was obtained.
As a result of analyzing the obtained modified hyaluronic acid by liquid chromatography, the peak having a molecular weight of 500,000 or more was 80%.
Moreover, about the obtained modified | denatured hyaluronic acid, the thing of molecular weight 500,000 or more was fractionated using the liquid chromatography, and the modified | denatured hyaluronic acid (V-5) after liquid chromatography fractionation was obtained. As a result of analyzing (V-5) using NMR, the substitution rate was 0.025, which was lower than the substitution rate of 0.15 in (A-1).
Thereafter, the denatured hyaluronic acid (V-5) after separation by liquid chromatography is used by using hyaluronidase (a mixture of β-N-acetylglucosaminase (manufactured by Seikagaku Corporation) and β-glucuronidase (manufactured by Sigma)). As a result of fractionation and fractionation with a molecular weight of 1,000 to 100,000 by liquid chromatography and analysis by NMR, the substitution rate of the modified hyaluronic acid after degradation was 0.15.
Therefore, the hyaluronic acid derivative (V-5) after fractionated by liquid chromatography is partially chemically modified with hyaluronic acid segments (glucuronic acid and N-acetylglucosamine) not chemically modified bound to (A-1). It was found to contain modified hyaluronic acid, a modified polysaccharide.

From the results of Examples 1 to 5, the obtained modified hyaluronic acid had a higher molecular weight than the raw material acetyl hyaluronic acid, and the substitution rate was lower than that of the raw material. Moreover, the modified | denatured hyaluronic acid obtained in Examples 1-5 was decomposed | disassembled by the hyaluronidase, and became the same grade as the substitution rate of a raw material. Therefore, it can be seen that the modified hyaluronic acid obtained in Examples 1 to 5 is a modified hyaluronic acid in which a part of the acetyl hyaluronic acid segment bonded to the hyaluronic acid segment is acetylated.
Therefore, it can be seen that according to the method for producing a modified polysaccharide of the present invention, a partially modified polysaccharide can be produced.
Further, when the substitution rate 1 and Example 2 in which almost all hydroxyl groups were acetylated were compared with Example 1 with a substitution rate of 0.15 and Example 4 with a substitution rate of 0.8, the substitution rate In Examples 1 and 4 in which the value of 0.15 or 0.8 was obtained, the modified polysaccharide could be obtained in a larger amount.
Further, when comparing Example 3 using a solution containing uridine diphosphate, which is ribonucleoside diphosphate 113 times the inhibition concentration IC ₅₀ , with Example 1 in which uridine diphosphate was not added, uridine 2 phosphate In Example 1 where no acid was added, the modified polysaccharide could be obtained in a larger amount. In addition, Example 5 using ribonucleoside diphosphate converting enzyme (G) was able to obtain a larger amount of modified polysaccharide than Example 1 which was not used.

<Ease of flow of hyaluronic acid derivative by sweat>
The modified hyaluronic acids (V-1) to (V-5) obtained by liquid chromatography fractionation in Examples 1 to 5 were converted into ultrafiltration membranes (Amicon Ultra-15 manufactured by Millipore (molecular weight cut off 100,000). ) To obtain 0.1% modified hyaluronic acid aqueous solutions (Y-1) to (Y-5). 500 μL of (Y-1) to (Y-5) was applied to each subject's right hand and allowed to stand for 30 minutes, and then spent 10 minutes in the sauna. After sweating with water easily, the modified hyaluronic acid application site was wiped off with a cloth. Thereafter, the modified hyaluronic acid was extracted from the cloth, and the amount of the modified hyaluronic acid was quantified by liquid chromatography. A similar test was performed on each of the 10 modified hyaluronic acid aqueous solutions (Y-1) to (Y-5), and the amount of the modified hyaluronic acid adhering to the cloth (which remained without sweating) was ( V-1) to (V-5) were all 0.3 ± 0.1 mg.

<Ease of flow of hyaluronic acid by sweat>
Hyaluronic acid was prepared in the same manner as in Example 1 except that hyaluronic acid (weight average molecular weight 100,000) was used instead of acetylated hyaluronic acid.
About the produced hyaluronic acid, the molecular weight vicinity of 1 million was fractionated with the liquid chromatography, and the hyaluronic acid (V'-1) after liquid chromatography fractionation was obtained. Further, the solution was concentrated with an ultrafiltration membrane (Amicon Ultra-15 manufactured by Millipore (fractional molecular weight 100,000)) to obtain a 0.1% hyaluronic acid solution (Y′-1). 500 μL of (Y′-1) was applied to the subject's left hand and left for 30 minutes, and then spent 10 minutes in the sauna. After sweating with water easily, the hyaluronic acid application site was wiped off with a cloth. Thereafter, hyaluronic acid was extracted from the cloth, and the amount of hyaluronic acid was quantified by liquid chromatography. A similar test was performed on 10 people, and the amount of hyaluronic acid adhering to the cloth (which remained without sweating) was <0.1 mg.

From the results of the test of the ease of flow of modified hyaluronic acid and hyaluronic acid by sweat, the modified hyaluronic acid obtained by the production method of the present invention is less likely to be washed away by sweat compared to hyaluronic acid having no chemically modified segment. I understand that.
Therefore, it can be seen that according to the production method of the present invention, a highly functional modified polysaccharide can be obtained.

  According to the method for producing a modified polysaccharide of the present invention, a partially modified polysaccharide can be produced. Moreover, the modified polysaccharide obtained by the production method of the present invention can be used not only for cosmetics but also for quasi drugs, pharmaceuticals and medical devices.

Claims (3)

A chemically modified polysaccharide (A) which is hyaluronic acid acetylated by a compound having a hydroxyl group and / or a carboxyl group and the following ribonucleoside diphosphate-monosaccharide (B) in the presence of polysaccharide synthase (C) A process for producing a modified polysaccharide reacted in
Ribonucleoside diphosphate-monosaccharide (B): triose (b-1), tetrose (b-2), pentose (b-3), hexose (b-4), heptose (b-5) and the following monosaccharide The hydrogen atom of at least one hydroxyl group contained in at least one monosaccharide (b) selected from the group consisting of (b-6) is substituted with any one functional group of the following chemical formulas (1) to (5) Sugar nucleotides.
Monosaccharide (b-6): a monosaccharide selected from the group consisting of (b-1), (b-2), (b-3), (b-4) and (b-5), A monosaccharide in which at least one selected from the group consisting of a hydrogen atom, a hydroxyl group and a hydroxymethyl group possessed by is substituted with the following substituent (D).
Substituent (D): carboxyl group, amino group, N-acetylamino group, sulfo group, methyl ester group, N-glycolyl group, methyl group, 1,2,3-trihydroxypropyl group, phosphate group and 2- At least one substituent selected from the group consisting of carboxy-2-hydroxyethyl groups; The chemically modified polysaccharide (A) includes (A) a polysaccharide molecule in which at least two terminal monosaccharides constituting the sugar chain of one molecule are not chemically modified. The manufacturing method of modified polysaccharide of description. Time for allowing polysaccharide synthase (C) to act on chemically modified polysaccharide (A) and ribonucleoside diphosphate-monosaccharide (B) which are hyaluronic acid acetylated by a compound having a hydroxyl group and / or a carboxyl group The modified polysaccharide according to claim 1 or 2 , wherein the concentration of ribonucleoside diphosphate in the reaction solution is less than 100 times the inhibitory concentration IC ₅₀ of polysaccharide synthase (C) below during a time of 10 to 100% Manufacturing method.
Inhibitory concentration IC _50: ribonucleoside diphosphate - at a concentration of (C) the time of the reaction in the presence of a simple sugar (B) a polysaccharide synthase (C), ribonucleoside diphosphate as substrate - monosaccharide (B ), The concentration of ribonucleoside diphosphate when the enzyme activity of (C) is halved, determined using ribonucleoside diphosphate as an inhibitor.