JPH09188705A - Glycosaminoglycan derivative and production thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for chemically modifying glycosaminoglycan(GAG) under mild conditions in a short time, and obtain a GAG derivative by the above method. SOLUTION: This GAG derivative is obtained (1) by reaction between a hydroxyl group- or amino group-bearing GAG and a mixed acid anhydride prepared by reaction of a carboxylic acid with a halogenated di-lower alkyl phosphinothioyl (Rpt-X) to effect formation ester linkage or amido linkage between the carbonyl group of the mixed acid anhydride and the hydroxyl or amino group of the above GAG. (2) The mixed acid anhydride is obtained by reaction between a carboxyl-bearing GAG and the Rpt-X. (1) Another version of the method for obtaining the GAG derivative is as follows: the mixed acid anhydride mentioned in (2) is reacted with a primary or secondary amine to form amido linkage.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a glycosaminoglycan derivative and a method for producing the same.

[0002]

2. Description of the Related Art Glycosaminoglycans (hereinafter referred to as "GA
"G"), such as anti-thrombotic agent heparin and arthritis therapeutic agent hyaluronic acid, whose usefulness is already clear, and chondroitin sulfate, etc. have been sufficiently developed for use. There are some that are not. On the other hand, peptides and proteins are a group of compounds that have the potential to be useful drugs because they show various excellent pharmacological actions, but in many cases it is difficult to put them to practical use due to their instability in vivo. . Therefore, it is known that these compounds are modified with a polymer compound to enhance the stability in vivo, and GAG and amino acids, peptides, proteins, lipids and other low molecular weight organic compounds having excellent biocompatibility are known. It is considered that the complex (polysaccharide hybrid) with etc. is suitable for such a purpose, and it is possible to increase the usefulness of both and to provide a new function.

For example, hyaluronic acid is naturally used in animal tissues, has bioresorbability, and has no toxicological or immunological actions. Therefore, hyaluronic acid is itself used as a drug or a cosmetic product. Those to which bioactive peptides are bound are excellent drugs for drug delivery systems. Moreover, by making hyaluronic acid insoluble by chemical modification, it becomes a medical material having excellent biocompatibility.

GA for obtaining the above-mentioned polysaccharide hybrid
As a method for chemically modifying G, since GAG is insoluble in an organic solvent, GAG is suspended in dimethylformamide (DMF) and reacted with acid chloride using pyridine as a catalyst (Eur.J.Biochem., 1, 46-50 (1967), Chem.Express, 6 (9),
647-650 (1991)) and GAG have a carboxyl group and are water-soluble, and thus activated in the presence of N-hydroxysuccinimide (HONSu) or the like as a water-soluble carbodiimide (WSC). A method of binding GAG and amines by treating with a condensing agent is known.

Further, there is known a method in which the sodium salt of heparin is replaced with an ammonium salt or a tertiary amine salt, which is solubilized in DMF, and then reacted with symmetric acid anhydride of carboxylic acid using dimethylaminopyridine as a catalyst. There is (Ca
rbohydr. Res. , 236 , 107-119
(1992)). On the other hand, as a method for chemically synthesizing peptides,
A reaction of condensing amino acids by a mixed acid anhydride method using dimethylphosphinothioyl chloride (Mpt-Cl) is known (Chem. Lett., 1 , p45-48 (1982)). This method is a method in which the hydroxyl group of an amino acid can be reacted without protection in peptide synthesis, and can also be reacted in an alcohol solvent. Also known is a method for producing an N-acylamino sugar in which a mixed acid anhydride derived from Mpt-Cl and a carboxylic acid such as acetic acid, benzoic acid, glycolic acid, or glucuronic acid is reacted with an amino sugar such as glucosamine or galactosamine. (Japanese Patent Laid-Open No. 61-197589).

However, the mixed acid anhydride method using a halogenated di (lower alkyl) phosphinothioyl oil has not been used for the chemical modification of a polysaccharide containing glycosaminoglycan.

[0007]

The conventional chemical modification method of GAG requires severe reaction conditions such as heating and long-time reaction, and often involves reduction of molecular weight and side reactions.
Further, the method using an activated condensing agent such as WSC is
It is difficult to control the amidation rate because the activated condensing agent and the activated carboxyl group are gradually decomposed by reacting with water and the reaction is slow and an excess reagent is used. In addition, it is often a problem because it is difficult to remove the activated condensing agent used in excess, its decomposition product, and particularly acylurea by-produced by acyl transfer.

Further, a method of reacting an ammonium salt or a tertiary amine salt of heparin with a symmetrical acid anhydride of a carboxylic acid is a method in which the symmetrical acid anhydride is unstable, so that the carboxylic acid is decomposed during the reaction, so that the desired amount of the carboxylic acid is heparinized. There was a problem that it was difficult to introduce. The object of the present invention is to
It is intended to provide a method of chemically modifying G in a short time under mild conditions and a GAG derivative which can be produced by the method.

[0009]

As a result of earnest research, the present inventors have succeeded in solving the above problems by the following constitution. That is, the present invention provides: 1) a mixed acid anhydride obtained by reacting a carboxylic acid with a halogenated di (lower alkyl) phosphinothioyl oil, and a glycosaminoglycan having a hydroxyl group or an amino group, thereby reacting the mixed acid. Glycosamim in which a carboxylic acid and the glycosaminoglycan are ester-bonded or amide-bonded, wherein a carbonyl group of an anhydride and a hydroxyl group or an amino group of the glycosaminoglycan are ester-bonded or amide-bonded. The method for producing a noglycan derivative, 2) the carboxylic acid has a functional group other than a carboxyl group protected with a protecting group, and the protecting group is removed after the ester bond reaction or the amide bond reaction. Method for producing glycosaminoglycan derivative of 3), 3) Carboxyl group-containing glycosaminoglycan and halogenation A glycosaminoglycan characterized by producing a glycosaminoglycan mixed acid anhydride comprising the glycosaminoglycan and a halogenated di-lower alkylphosphinothioyl oil by reacting with a lower alkylphosphinothioyl oil. Method for producing derivative, 4) Glycosaminoglycan mixed acid anhydride obtained by reacting glycosaminoglycan having carboxyl group with halogenated di-lower alkylphosphinothioyl oil, and 1
1) The glycosaminoglycan, which is characterized by reacting with a primary amine or a secondary amine to form an amide bond between the carbonyl group of the mixed acid anhydride and the amino group of the amine.
A method for producing a glycosaminoglycan derivative having an amide bond with a primary amine or a secondary amine, 5) reacting in an anhydrous organic solvent in the presence of a neutralizing agent, 1) to 4) above 6) The method according to 1) or 4) above, wherein 6) the reaction is carried out in an aqueous solution containing a water-miscible organic solvent, 7-1) the glycosaminoglycan A chondroitin sulfate, hyaluronic acid, or a spacer compound-bound glycosaminoglycan in which a spacer compound having a hydroxyl group or an amino group is bound to these, and the carboxylic acid is 1
Alternatively, a glycosamino obtained by forming an ester bond or an amide bond between a carboxylic acid produced by the method described in 1) above and a glycosaminoglycan having a hydroxyl group or an amino group, which is a physiologically active substance having two or more carboxyl groups. Glycan derivative, 7-2) The glycosaminoglycan is a spacer compound-bonded glycosaminoglycan having an amino group in which a diamine is covalently bonded as a spacer compound to chondroitin sulfate, hyaluronic acid or their carboxyl groups, and the carboxylic acid is Indomethacin, deoxycholic acid, acetylsalicylic acid, salazosulfapyridine, methotrexate, leucine enkephalin, leucine,
Glycosaminoglycan derivative obtained by forming an ester bond or an amide bond between a carboxylic acid produced by the method 1) which is serine, glycine or glutamine, and a glycosaminoglycan having a hydroxyl group or an amino group, 8-1) the above glyco Saminoglycan is chondroitin sulfate, hyaluronic acid or a spacer compound-bonded glycosaminoglycan in which a spacer compound having a carboxyl group is bonded to these, and the primary amine or secondary amine is a primary amino group or secondary amino group. A glycosaminoglycan derivative which is a physiologically active substance having a carboxyl group-containing glycosaminoglycan produced by the method described in 4) above and an amide bond between a primary amine or a secondary amine, 8-2) above Glycosaminoglycan is chondroitin sulfate or hyal Acid and the primary amine or secondary amine is bestatin, tranexamic acid, adriamycin, methotrexate, phenylalanine or glutamine, and a glycosaminoglycan having a carboxyl group produced by the method described in 4) above and a primary amine or It is a glycosaminoglycan derivative formed by amide bond with a secondary amine.

The present invention will be described in detail below. Carboxylic acid and halogenated di (lower alkyl) phosphinothioyl oil used in the present invention (hereinafter referred to as Rpt-X. X is a halogen and Rpt is a di (lower alkyl) phosphinothioyl group: general formula R ₂ P = S ( In the formula, R represents an alkyl group having 1 to 4 carbon atoms, preferably a methyl group), and a mixed acid anhydride (hereinafter, also referred to as “mixed acid anhydride A”) obtained by reacting It can be obtained by reacting a carboxylic acid and Rpt-X at -25 ° C to 40 ° C, preferably 0 ° C to 25 ° C for 1 minute to 1 hour, preferably 1 minute to 10 minutes. Further, a glycosaminoglycan mixed acid anhydride (hereinafter, also referred to as “mixed acid anhydride B”) obtained by reacting a glycosaminoglycan having a carboxyl group with Rpt-X is the GAG having a carboxyl group. And Rpt-X are reacted under the same conditions as those for preparing the mixed acid anhydride A described above.

The reaction for producing the mixed acid anhydride A is represented by the following formula (1). R'-COOH + Rpt-X → R'-COORpt (1) (R 'is a residue excluding one carboxyl group of the carboxylic acid.) The reaction for producing the mixed acid anhydride B is as follows. ). gag-COOH + Rpt-X → gag-COORpt (2) (gag is a residue excluding one carboxyl group of GAG.) In the present invention, GAG means GAG itself and GAG.
It is a concept including a derivative in which a spacer compound or the like is introduced into itself.

The mixed acid anhydride B of the above formula (2), that is, g
ag-COORpt is a GA produced by the method of the present invention.
It is a kind of G derivative and is also a raw material of a novel GAG derivative obtained by reacting it with a primary amine or a secondary amine.

The carboxylic acid used in the reaction of the above formula (1) includes all compounds having a carboxyl group capable of reacting with Rpt-X, and functional groups other than the carboxyl group of the compound (for example, amino group, A compound in which a hydroxyl group or the like) is protected by a protective group may be used. Specific examples of the carboxylic acid include amino acids with protected amino groups (Fmoc-Gln-OH, F
moc-Leu-OH, Z-Leu-OH, Boc-Leu-OH, Fmoc-Pro-OH, Bo
c-Gly-OH, Z-Gly-OH, Fmoc-Ser (O ^t Bu) -OH, etc., where F
moc- is a 9-fluorenylmethyloxycarbonyl group, Z
-Indicates a benzyloxycarbonyl group, Boc-indicates a tertiary butoxycarbonyl group, and ^t Bu indicates a tertiary butyl group. ), A peptide in which an amino group is protected in the same manner as in the case of the above amino acid, a physiologically active substance having a carboxyl group in the molecule and having a useful pharmacological action (eg, indomethacin,
Deoxycholic acid, acetylsalicylic acid, salazosulfapyridine, nicotinic acid, thioctic acid, indole-3
Acetic acid, retinoic acid, methotrexate, etc.), a dicarboxylic acid as a spacer compound is bound to a compound having an amino group or a hydroxyl group by a known method to have a carboxyl group, an aliphatic carboxylic acid (palmitic acid, linoleic acid, linolene) Acid, etc.), cinnamic acid, salicylic acid, trans-epoxysuccinic acid and the like.

As the GAG having a carboxyl group used in the reaction of the above formula (2), hyaluronic acid,
Examples thereof include chondroitin sulfate, chondroitin, dermatan sulfate, heparin and heparan sulfate. Especially hyaluronic acid and chondroitin sulphate are biocompatible, G
It is preferable because the AG derivative is excellent in terms of activity persistence and the like.
The above GAG is not limited in origin and molecular weight, but generally has a molecular weight of about 10,000 to 5,000,000, preferably about 20,000 to.
2 million. In addition, free or salt GAG
Any of the above can be used, but alkali salts such as alkali metal salts, alkaline earth metal salts, amine (for example, tertiary amines such as triethylamine, N-methylmorpholine, diisopropylethylamine, etc.) salts are preferred, and particularly generally The readily available sodium salt or potassium salt is preferred.

The GAG having a carboxyl group can also be synthesized by binding a known compound such as a spacer compound having a carboxyl group so as to have at least a carboxyl group by a known method. As the spacer compound used in the synthesis,
Examples thereof include dicarboxylic acids, amino acids, and derivatives in which a protecting group is introduced into these carboxyl groups.

Examples of the spacer compound include R ^{1
_{- (CH 2) n -R 2}} (R 1 = R 2 = carboxyl group,
Alternatively, R ^{1 is a} protected carboxyl group and R ^{2 is} an amino group, and n is 2 to 18), but is not limited thereto. Here, the protective group may be any group as long as it can deprotect the protective group for the carboxyl group after the GAG having the spacer compound bound thereto is prepared.

In the present invention, for convenience, "GAG having a carboxyl group" is defined as not included in "carboxylic acid". As Rpt-X, known ones can be used, but chloride, bromide and the like are preferably used, and as the lower alkyl group, an alkyl group having 1 to 4 carbon atoms, preferably a methyl group is used. Specifically, dimethyl phosphinothioyl chloride (Mpt-Cl) is exemplified.

In the reaction for producing the mixed acid anhydride A or B,
When the carboxyl group is a free acid, a base for neutralizing hydrochloric acid generated by the formation of the mixed acid anhydride A or B (activation of the carboxyl group), preferably triethylamine, N-methylmorpholine, diisopropyl It is preferable to add a tertiary amine such as ethylamine in an equimolar amount to Rpt-X. When the carboxyl group is a salt of the base, it is not necessary to add a base.

The above-mentioned formation reaction is carried out by dimethylformamide (DMF), N, N-dimethylacetamide (DM
A), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), chloroform, dichloromethane or the like in a single or mixed organic solvent, preferably an anhydrous organic solvent. For example, the carboxylic acid is Rpt-
When the mixed acid anhydride A is produced by reacting (activating) with X, the reaction is carried out in an aprotic organic solvent such as chloroform or DMF. GA having a carboxyl group
Similarly, by activating the carboxyl group of G, mixed acid anhydride B
In the case of producing, by substituting an ammonium salt or a tertiary amine salt for an alkali metal salt (sodium salt, potassium salt, etc.) or an alkaline earth metal salt of a carboxyl group or a sulfate group of GAG by a known method. A solubilized product in an aprotic organic solvent such as DMF can be used for the reaction. In this case, if GAG is dissolved in an organic solvent and then residual water is removed using a molecular sieve or the like, decomposition of the mixed acid anhydride is suppressed. The introduction rate of the primary amine is improved.

Although it is possible to use Rpt-X in excess with respect to carboxylic acid or GAG having a carboxyl group, it is usually 1.0 to 1.2 equivalents, preferably equimolar (1.0 equivalents). If used, the desired mixed acid anhydride can be obtained. As the acid anhydride method, a symmetrical acid anhydride method and a mixed acid anhydride method are known. When the symmetrical acid anhydride method is used to obtain a desired acid anhydride, 2 equivalents or more of carboxylic acid is required. On the other hand, in the mixed acid anhydride method of the present invention, a carboxylic acid and a GAG having a carboxyl group in about equimolar amounts may be used.

The mixed acid anhydride thus obtained is
It is possible to isolate it, but as it is, mixed acid anhydride A
The GAG having a hydroxyl group or an amino group and the mixed acid anhydride B can be reacted with a primary amine or a secondary amine to produce a glycosaminoglycan derivative. The reaction between the mixed acid anhydride A and GAG containing a hydroxyl group is represented by, for example, the following formula (3).

R′-COORpt + HO-gag → R′-COO-gag (3) The reaction between the mixed acid anhydride A and GAG having an amino group is
For example, it is represented by the following equation (4). R′-COORpt + H ₂ N-gag → R′-CONH-gag (4) The reaction of the mixed acid anhydride B with the primary amine or the secondary amine is, for example, the reaction with a primary amine as follows. It is represented by the equation (5).

Gag-COORpt + H ₂ N-A → gag-CONH-A (5) (A represents a residue obtained by removing one amino group from an amine.) By the reaction of the above formula (3), a carvone is obtained. When an acid is introduced into GAG having a hydroxyl group, it can be introduced by reacting the mixed acid anhydride A with the hydroxyl group of GAG to form an ester bond. At this time, the solvent used in the reaction includes an aqueous solution containing a water-miscible organic solvent and an anhydrous organic solvent, but an anhydrous organic solvent is preferable in view of good reaction efficiency.

Specific examples of the water-miscible organic solvent include dioxane, tetrahydrofuran (THF), N, N-dimethylacetamide (DMA), acetamide, DM.
F, dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide (HMPA), N-methylpyrrolidone, pyridine and the like can be mentioned. Examples of the aqueous solution include aqueous solutions containing one or more kinds selected from any of the water-miscible organic solvents exemplified above in a range of about 0 to 50% in total.

Specific examples of the anhydrous organic solvent include the water-miscible organic solvents exemplified above and a single or mixed solvent such as chloroform and dichloromethane.
Examples of GAG having a hydroxyl group include GAGs such as hyaluronic acid, chondroitin sulfate, chondroitin, dermatan sulfate, heparin, heparan sulfate, and keratan sulfate, and their derivatives. Further, it may be a compound in which a spacer compound having a hydroxyl group is bound to these GAGs.

As a specific method, the mixed acid anhydride A is
GAG (free form), GAG sodium salt or potassium salt, or GAG obtained by substituting GAG sodium salt or potassium salt with ammonium salt or tertiary amine salt, and -25 ° C to 60 ° C, preferably 0 ° C to 2
The reaction is carried out at 5 ° C for 5 minutes to 24 hours. At this time, N, N
-It is preferable to add a dialkylaminopyridine-based catalyst, particularly a catalyst for esterification reaction such as 4-dimethylaminopyridine (DMAP) or 4-pyrrolidinopyridine. Addition of this catalyst enables reaction under milder conditions. Become.

For the purpose of neutralizing the acid produced by the reaction, a tertiary amine such as triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine or an inorganic base such as sodium hydrogen carbonate is added as an acid neutralizing agent. Preferably. The mixed acid anhydride A and GAG having an amino group are prepared by the reaction of the above formula (4),
Alternatively, the mixed acid anhydrides B and 1 can be obtained by the reaction of the above formula (5).
In the case of reacting with a primary amine or a secondary amine (hereinafter, “GAG having an amino group, primary amine or secondary amine” may be collectively referred to as an amino component),
-25 ° C to 60 ° C, preferably 0 ° C to 25 ° C, 5
The reaction can be carried out for from minutes to 24 hours, preferably from 30 minutes to 2 hours, in the above anhydrous organic solvent or in an aqueous solution containing a water-miscible organic solvent. At this time, an acid (di-lower alkylthiophosphinic acid) produced as the reaction progresses
For the purpose of neutralizing the above, it is preferable to add the above neutralizing agent as an acid neutralizing agent. The water content of the aqueous solution is 0 to
80% by volume can be mentioned.

GA having an amino group used in the present invention
As G, a compound in which a substituted amino group (acetylamino group, sulfoamino group, etc.) of the amino sugar of GAG exemplified as the above-mentioned GAG having a hydroxyl group is converted into an amino group,
Examples include deacetylated derivatives. Also, GA
A spacer compound having an amino group at a hydroxyl group or a carboxyl group of G, for example, a diamine, an amino acid or the like is covalently bonded to form a spacer compound-bonded GAG having an amino group, or a spacer compound having an amino group at the reducing end of GAG. G having an amino group can also be obtained by introducing G to form a spacer compound-bound GAG.
It can be AG. When GAG having a spacer compound having an amino group attached to the reducing end of GAG is used, it becomes possible to attach a carboxylic acid to the reducing end of GAG, that is, at a specific one position.

The spacer compound can be introduced, for example, by esterifying the carboxyl group of the amino group protected amino group as a spacer compound with the hydroxyl group of GAG, followed by deprotection, or one amino group of diamine. A method of amide-bonding with the carboxyl group of GAG, and reducing GAG and diamine are mixed by mixing the reducing end of GAG and diamine in a suitable solvent to form a Schiff base, and preferably further treated with a reducing agent. Examples include a method of binding by amination.

As the amino acid with the amino group protected as the spacer compound, any naturally occurring amino acid such as glycine and leucine can be used. Further, ω-amino acid having an arbitrary carbon number such as 6-aminocaproic acid can be used, and in this case, the degree of freedom as a spacer can be increased. Examples of the amino-protecting group include a benzyloxycarbonyl (Z) group which can be removed by catalytic reduction, a 9-fluorenylmethyloxycarbonyl (Fmoc) group which can be removed by a base, and a trifluoroacetic acid aqueous solution. A 3-butoxycarbonyl (Boc) group and the like are preferable.

When a diamine is used as the spacer compound, a diamine represented by H ₂ N- (CH ₂ ) _n -NH ₂ (n is preferably 2-18) (eg 1,6-
(Hexanediamine etc.) and lysine which is an amino acid and its ester form can be used. Alternatively, a spacer compound-bonded GAG having an amino group may be obtained by using an amino alcohol protected with an amino group as a spacer compound to form an ester bond with the carboxyl group of GAG and then deprotecting.

As a method of introducing the spacer compound, a known method of using carbodiimides to form an amide bond or an ester bond with the carboxyl group of GAG is excellent in the ease of operation and cost. Further, the carboxyl group of GAG is activated by Rpt-X to prepare a mixed acid anhydride B.
And may be reacted with a diamine by the reaction of the above formula (5).

Mixed acid anhydride B with the carboxyl group of GAG
Examples of primary amines or secondary amines that are activated as and react with primary amines or secondary amines include amino acids (glutamine, phenylalanine, etc.), amino acid esters (phenylalanine benzyl ester, etc.), peptides, peptide esters. , Peptide amide, protein, physiologically active substance having amino group in the molecule and having useful pharmacological action (tranexamic acid, cycloserine, bestatin, aminocephalosporinic acid, pyridoxamine, adriamycin, methotrexate, etc.), spacer with amino acid or diamine Examples of the compound include a compound having an amino group bonded thereto, an amino sugar (glucosamine, galactosamine, etc.), a lipid having an amino group (phosphatidylethanolamine, etc.) and the like.

Reaction of the mixed acid anhydride A with GAG having a hydroxyl group or an amino group, or mixed acid anhydrides B and 1
After the reaction with a primary amine or a secondary amine, it can be purified by a known method such as ethanol precipitation or dialysis. When the product precipitates after the reaction, the target product can be obtained by a simpler purification operation such as collecting by filtration with a glass filter or the like and sequentially washing with an appropriate solvent such as sodium bicarbonate water, water, and ethanol. .

In the reactions of the above formulas (3) to (5), the amount of the mixed acid anhydride and the catalyst with respect to the GAG having a hydroxyl group or the amino component is determined by the kind of the desired GAG derivative, that is, the kind, molecular weight or mixture of GAG. It can be appropriately selected depending on the type of acid anhydride and the desired introduction number of carboxylic acid or primary amine or secondary amine with respect to GAG. Generally, when forming an amide bond with an amino component, the desired introduction number of 1. The mixed acid anhydride may be used in an amount of 0 to 3.0 times, and in the case of forming an ester bond with the hydroxyl group of GAG, the mixed acid anhydride may be used in an amount of 2.0 to 10 times the desired introduction number. The introduction rate of the carboxylic acid or primary amine or secondary amine into the GAG derivative can be defined as the percentage of the number of moles introduced per GAG-constituting disaccharide unit. The number of moles introduced can be determined by measuring the integrated intensity of proton NMR.

Further, according to the production method of the present invention, an amino acid having an amino group protected is selected as the carboxylic acid, and the mixed acid anhydride A is reacted by the method of the above formula (4) to have an amino group. After the carboxyl group of the amino acid whose amino group is protected by the method of the present invention is bound to the amino group of GAG, the amino protecting group is removed and the operation of binding the N-protected amino acid again by the method of the present invention is repeated. It is also possible to synthesize a GAG-peptide conjugate by performing peptide synthesis in which amino acids are sequentially elongated on GAG. In this case, as the amino-protecting group, for example,
Can be quickly removed with 10% aqueous solution of diethylamine 9
A -fluorenylmethyloxycarbonyl (Fmoc) group and the like are preferable.

[0037]

The present invention will be described in detail with reference to the following examples. Preparation Example 1 Introduction of 1,6-Hexanediamine as a Spacer into Chondroitin Sulfate Sodium chondroitin sulfate (10
g) is dissolved in water (100 ml) and DMF (100 ml)
Was added. N-hydroxybenzotriazole (HOB
t) (40 mmol) in DMF (30 ml) and 1,
After adding 6-hexanediamine dihydrochloride (20 mmol), dicyclohexylcarbodiimide (DC
A solution of C) (20 mmol) in DMF (40 ml) was added. After stirring overnight at room temperature, the precipitated dicyclohexylurea was washed off with ethanol on a glass filter. After washing with ethanol, the precipitate obtained on the glass filter was further purified three times by an ethanol precipitation method, and then dried under reduced pressure to give chondroitin sulfate into which 1,6-hexanediamine was introduced as white powder 7.67.
g was obtained. As a result of measurement of 270 MHz proton NMR,
The signal derived from the acetyl group of chondroitin sulfate and (δ
= 2 ppm) and the signal (δ = 1.3-1.7 ppm) derived from the four methylene groups of the spacer (3:
From 2.60), the introduction rate of the diamine per 32 sugar units constituting chondroitin sulfate was 32%.

Here, the introduction rate is expressed by the following equation. Introduction rate (%) = 2 moles of diamine introduced per sugar unit × 100 Preparation Example 2 Introduction of lysine methyl ester as a spacer into chondroitin sulfate Sodium chondroitin sulfate having an average molecular weight of 30,000 (3
g) was dissolved in water (30 ml) and DMF (30 ml) was added. Lysine (Z) methyl ester hydrochloride (3 mmol) and HOBt (6 mmol) were added, and then a DMF solution (10 ml) of DCC (6 mmol) was added under ice cooling. After stirring overnight at room temperature, the mixture was poured into saturated sodium acetate ethanol and the resulting precipitate was centrifuged. The obtained precipitate was further purified three times by the ethanol precipitation method, and then water (10 m
It was dissolved in 1) and dialyzed against deionized water for 3 days. 0.22
After filtration through a micron filter, freeze-drying gave 3.08 g of the title compound as a white powder. 270 MH
As a result of proton NMR measurement of z, a signal derived from the acetyl group of chondroitin sulfate (δ = 2 ppm) and a signal derived from the benzene ring of the Z group (δ = 7.3-7.5 pp)
From the intensity ratio (3: 1.23) of m), the introduction rate of lysine (Z) methyl ester per disaccharide unit of chondroitin sulfate was 25%. This product was subjected to catalytic reduction with ammonium formate and 10% palladium carbon in an aqueous solution to remove the Z group, and lysine methyl ester-bonded chondroitin sulfate was obtained.

Example 1 Synthesis of deoxycholic acid-modified chondroitin sulfate Deoxycholic acid (0.1 mmol) in DMF solution (1
ml), a solution of triethylamine (0.2 mmol) and dimethylphosphinothioyl chloride (Mpt-Cl) (0.1 mmol) in DMF (0.2 ml) was added at room temperature. After 5 minutes, this product had a modification rate of 25 synthesized in Preparation Example 2.
% Lysine methyl ester linked chondroitin sulfate (5
0 mg) in water-DMF (1: 1, v / v) solution (2 m
It was added to 1) under ice cooling. After stirring overnight at room temperature, the mixture was poured into saturated sodium acetate ethanol and the resulting precipitate was centrifuged. The obtained precipitate was further purified three times by the ethanol precipitation method, dissolved in water (10 ml) and washed with deionized water.
Dialysis for one day. After filtration through a 0.22 micron filter, lyophilization gave the title compound as a white powder, 5
8 mg were obtained. As a result of measurement of proton NMR at 270 MHz, the intensity ratio of the signal derived from the acetyl group of chondroitin sulfate (δ = 2 ppm) and the signal derived from one methyl group of deoxycholic acid (δ = 1.3-0.7 ppm). From (3: 0.81), the introduction rate of deoxycholic acid per constituent disaccharide unit of chondroitin sulfate was 27%.

Here, the introduction rate is expressed by the following equation. Introduction rate (%) = Number of moles of deoxycholic acid introduced per disaccharide unit × 100 Example 2 Synthesis of acetylsalicylic acid-modified chondroitin sulfate Lysine methyl ester-bonded chondroitin sulfate (50 mg) synthesized in Preparation Example 2 and having a modification rate of 25% Was used in place of deoxycholic acid of Example 1, and acetylsalicylic acid (aspirin) was used to perform the same operation. However, when preparing the mixed acid anhydride, acetylsalicylic acid, triethylamine, Mpt-Cl and their solvents were used in 5 times the amount of Example 1. Yield 56 mg. The introduction rate of aspirin (acetylsalicylic acid) was 28%.

Example 3 Synthesis of chondroitin sulfate having Z-amino acid bound thereto Z-leucine (0.5 mmol) in DMF solution (2 m)
1) triethylamine (0.5 mmol) and Mpt-
A solution of Cl (0.5 mmol) in DMF (0.5 ml) was added at room temperature. After 5 minutes of this, triethylamine salt of chondroitin sulfate (100 mg) and 4-dimethylaminopyridine (DMAP) (0.5 mmol) in D
To the MF solution (2 ml) was added under ice cooling, and further triethylamine (0.5 mmol) was added. After stirring overnight at room temperature, 5% sodium hydrogen carbonate solution (5 ml) was added, and the mixture was poured into saturated sodium acetate ethanol and the resulting precipitate was centrifuged. The obtained precipitate was further purified three times by the ethanol precipitation method, dissolved in water (10 ml) and dialyzed against deionized water for 3 days. After filtration through a 0.22 micron filter and freeze-drying, 107 mg of the title compound was obtained as a white powder. As a result of proton NMR measurement at 270 MHz, the intensity ratio (3 = 1.9) of the signal derived from the acetyl group of chondroitin sulfate (δ = 2 ppm) and the signal derived from the benzene ring of Z-leucine (3: 1.9) indicates the constituent disaccharide of chondroitin sulfate. The introduction rate of Z-leucine per unit was 38%.

Example 4 Binding of Protected Amino Acid to the Reducing End of Hyaluronic Acid A) A solution of Fmoc-Ser (O ^t Bu) -OH (0.1 mmol) in DMF (1 ml) with triethylamine (0.1 mmol). Mpt-Cl (0.1 mmol)
Of DMF solution (0.1 ml) was added under ice cooling. After 5 minutes of this, 1,6-hexanediamine was bound to the reducing terminal by a reductive amination method using sodium cyanoborohydride by a conventional method, and water of hyaluronic acid (100 mg) having an average molecular weight of 40,000 was added. DMF (1: 1) solution (8m
l) under ice-cooling, and further triethylamine (0.
1 mmol). After stirring overnight at room temperature, 5% sodium hydrogen carbonate solution (5 ml) was added, and the mixture was poured into saturated sodium acetate ethanol and the resulting precipitate was centrifuged.
The obtained precipitate was further purified three times by the ethanol precipitation method, dissolved in water (10 ml) and dialyzed against deionized water for 7 days. After filtering through a 0.22 micron filter, it was freeze-dried to obtain 106 mg as a white powder. As a result of proton NMR measurement at 270 MHz, a signal derived from the acetyl group of hyaluronic acid (δ = 2 ppm) and tertiary butyl ( ^t
From the intensity ratio of the signals derived from the (Bu) group (3: 0.162), the hyaluronic acid per molecule of serine is 56 disaccharide units.
It was united.

B) Binding of Protected Amino Acid to the Reducing End of Chondroitin Sulfate A similar reaction was carried out using chondroitin sulfate having an average molecular weight of 30,000 instead of hyaluronic acid of Example 4A), and 1,6-hexanediamine was obtained. 98 mg of the desired product was obtained from chondroitin sulfate (100 mg) bound with γ.
According to NMR, chondroitin sulfate per molecule of serine was bound by 60 units of disaccharide units.

Example 5 Synthesis of salazosulfapyridine-modified hyaluronic acid A sodium salt of hyaluronic acid having an average molecular weight of 40,000 was converted into a triethylamine salt by an ordinary method using an ion exchange resin. This hyaluronic acid triethylamine salt (200m
To DMF solution (15 ml) of g) to which DMAP (0.2 mmol) was added, mixed acid anhydride of salazosulfapyridine (0.2 mmol) prepared by the same method as in Example 1 was added, The mixture was stirred overnight at room temperature. After the same post-treatment as in Example 3, 117 mg of the title compound was obtained as a yellow powder. The introduction rate of salazosulfapyridine determined by NMR was 5%.

Example 6 Synthesis of indomethacin-modified chondroitin sulfate (introduced by amide bond) A solution of indomethacin (1 mmol) in DMF (2 ml) was cooled with ice and triethylamine (1 mmol) and Mp were added.
DMF (2 ml) in t-Cl (1 mmol) was added.
After 5 minutes of this, 1,6-hexanediamine was bound as a spacer and chondroitin sulfate having an average molecular weight of 30,000 (200 mg, prepared by the same method as in Preparation Example 1, introduction rate 32%) in water-DMF (1: 1) To the solution (8 ml) was added followed by triethylamine (1 mmol).
After stirring at room temperature overnight, the same treatment as in Example 3 was carried out to obtain 242 mg of the title compound as a white powder. The introduction rate determined by NMR was 24%.

Example 7 Synthesis of indomethacin-modified hyaluronic acid (introduced by ester bond) Indomethacin (1 mmol) in DMF solution (1 ml)
Triethylamine (1 mmol) and Mpt-Cl (1
A DMF solution (1 ml) of (mmol) was added under ice cooling. After 5 minutes of this, a triethylamine salt of hyaluronic acid having an average molecular weight of 800,000 (100 mg) / DMF solution 5
0 ml and 4-dimethylaminopyridine (DMAP)
To the mixed solution of (0.5 mmol) / DMF solution (1 ml) was added under ice cooling, and further triethylamine (1 mmol) was added. After stirring overnight at room temperature, 5% sodium hydrogen carbonate solution (2 ml) was added, and the mixture was poured into saturated sodium acetate ethanol and the resulting precipitate was centrifuged. The resulting precipitate is washed 5 times with 80% ethanol, purified,
After drying under reduced pressure, 140 mg of the title compound was obtained as a white powder.

The indomethacin introduction rate calculated by comparing the indomethacin content obtained from the calibration curve previously calculated by the absorptiometric method with the hyaluronic acid content obtained by the carbazole sulfate method was 29.8%. Example 8 Relationship between the amount of indomethacin charged and the rate of introduction According to the method described in Example 7, the relationship between the amount of indomethacin charged (reaction equivalent) and the rate of introduction was examined. Also, hyaluronic acid having an average molecular weight of 50,000, 300,000 and 800,000 was used.

The results are shown in FIG. It can be seen from FIG. 1 that the relationship between the charging amount and the introduction rate is almost linear. Further, when viewed in terms of the average molecular weight of hyaluronic acid, 50,000 and 300,000 showed almost the same behavior, but it can be seen that the reactivity of 800,000 was lowered as a whole.
It is considered that this may be due to the increase in viscosity due to the high molecular weight.

Example 9 Preparation of t-butoxycarbonylglycine modified hyaluronic acid 80 mg of sodium hyaluronate having an average molecular weight of 1,000,000
(0.2 mmol as disaccharide unit) was dissolved in 20 ml of water and then 10 ml of dioxane was added. To this solution, 175 mg of t-butoxycarbonylglycine whose carboxyl group was activated as a mixed acid anhydride with Mpt-Cl.
(1.0 mmol) and 139 μl of triethylamine
(1.0 mmol) and 4-dimethylaminopyridine 12
After adding 3 ml of a 2 mg (1.0 mmol) dimethylformamide solution under ice cooling, stirring was continued for 1 hour and a half at room temperature. 150 ml of sodium acetate saturated ethanol was poured into the obtained solution, and the resulting precipitate was centrifuged. The obtained precipitate was further purified three times by the ethanol precipitation method and then dried under reduced pressure to obtain 75 mg of the title t-butoxycarbonylglycine-modified hyaluronic acid as a white powder.
The signal derived from the acetyl group of hyaluronate in NMR and (δ
= 2 ppm) and a signal derived from a tert-butyl group (δ = 1.
The rate of introduction of t-butoxycarbonylglycine per disaccharide unit constituting hyaluronic acid was 11%, which was determined from the intensity ratio of 5 ppm).

Example 10 Production of benzyloxycarbonylglycine-modified hyaluronic acid and glycine-modified hyaluronic acid Benzyloxycarbonylglycine (also referred to as Z-glycine) 209 m instead of t-butoxycarbonylglycine
A procedure similar to that of Example 7 was followed using g (1.0 mmol).

After purification, it was dried under reduced pressure to obtain 74 mg of the title benzyloxycarbonylglycine-modified hyaluronic acid as a white powder. Signal from acetyl hyaluronate acetyl group (δ = 2 ppm) and signal from benzyl group of benzyloxycarbonylglycine (δ
= 7.5 ppm), the introduction ratio of benzyloxycarbonylglycine per disaccharide unit constituting hyaluronic acid was 11%.

60 mg (0.15 mmol / unit) of benzyloxycarbonylglycine-modified hyaluronic acid was dissolved in 50 ml of water, and under an argon atmosphere, 15 mg of 10% palladium (Pd) activated carbon and 19 m of ammonium formate were dissolved.
After adding g (0.3 mmol) and stirring at room temperature for 6 hours,
Again, the same amounts of Pd activated carbon and ammonium formate were added and stirred. After 6 hours, the same operation was performed again, and then 0.22 μm
The activated carbon was removed with the filter of No. 1, the solution was dialyzed with deionized water for 2 days, and then freeze-dried to obtain 51 mg of a white product. Peak derived from benzyl group by NMR (δ = 7.
5 ppm) disappearance was confirmed.

Example 11 Preparation of t-butoxycarbonylglycine-modified hyaluronic acid (molecular weight 150,000) 80 mg of sodium hyaluronate having an average molecular weight of 150,000
(0.2 mmol as disaccharide unit) was dissolved in 20 ml of water and then 10 ml of dioxane was added. T in this solution
-Mp the butoxycarbonylglycine carboxyl group
A solution of 175 mg (1.0 mmol) of t-butoxycarbonylglycine, 139 μl (1.0 mmol) of triethylamine, and 122 mg (1.0 mmol) of 4-dimethylaminopyridine in dimethylformamide activated as a mixed acid anhydride with t-Cl. Was added under ice cooling, and stirring was continued at room temperature for 1 hour and a half. 150 ml of sodium acetate saturated ethanol was poured into the obtained solution, and the resulting precipitate was centrifuged. The obtained precipitate was further purified by an ethanol precipitation method three times and then dried under reduced pressure to obtain 55 mg of the title t-butoxycarbonylglycine-modified hyaluronic acid as a white powder. The introduction rate of t-butoxycarbonylglycine per disaccharide unit constituting hyaluronic acid determined by NMR was 5%.

Example 12 A) Synthesis of chondroitin sulfate mixed acid anhydride and bestatin-modified chondroitin sulfate A sodium salt of chondroitin sulfate having an average molecular weight of 30,000 was converted into a triethylamine salt by a conventional method using an ion exchange resin. To a solution of this chondroitin sulfate triethylamine salt (50 mg) in DMF (2 ml) was added M under ice cooling.
DMF solution of pt-Cl (0.1 mmol) (0.2 m
1) was added to prepare chondroitin sulfate mixed acid anhydride.

After 5 minutes, bestatin (0.05 mmol)
And triethylamine (0.05 mmol) and water (0.5 ml) were added, and the mixture was stirred at room temperature overnight. After adding a 5% sodium hydrogen carbonate solution (5 ml), the mixture was poured into ethanol saturated with sodium acetate and the resulting precipitate was centrifuged. The obtained precipitate was further purified three times by the ethanol precipitation method, dissolved in water (10 ml), and dialyzed against deionized water for 4 days. After filtration through a 0.22 micron filter, freeze-drying gave 57 mg of the title compound as a white powder.
Obtained. As a result of proton NMR measurement at 270 MHz, a signal derived from chondroitin sulfate acetyl group and (δ = 2
ppm) and the intensity ratio of the signal derived from the benzene ring of bestatin (3: 0.41), the introduction rate of bestatin per disaccharide unit of chondroitin sulfate was 8%.

B) Synthesis of Tranexamic Acid-Modified Chondroitin Sulfate The same operation was performed using tranexamic acid instead of bestatin of Example 10A), and the title compound was obtained with an introduction rate of 15%. Example 13 Synthesis of Phenylalanine Benzyl Ester-Modified Chondroitin Sulfate A sodium salt of chondroitin sulfate having an average molecular weight of 30,000 was dissolved in DMF as a triethylamine salt by an ordinary method using an ion exchange resin, 4A of molecular sieve was added, and the solution was dried. did. This chondroitin sulfate triethylamine salt (100 mg) in DMF solution (2 ml)
And DM of Mpt-Cl (0.2 mmol) under ice cooling.
F solution (0.4 ml) was added. After 5 minutes, tosyl salt of phenylalanine benzyl ester (0.2 mmol) and triethylamine (0.4 mmol) were added and the mixture was stirred at room temperature for 1 minute.
Stirred overnight. After adding a 5% sodium hydrogen carbonate solution (5 ml), the mixture was poured into ethanol saturated with sodium acetate and the resulting precipitate was centrifuged. The obtained precipitate was further purified three times by an ethanol precipitation method, and then dissolved in water (10 ml),
It was dialyzed against deionized water for 3 days. After filtration through a 0.22-micron filter and freeze-drying, 103 mg of the title compound was obtained as a white powder. As a result of measurement of proton NMR at 270 MHz, the intensity ratio (3: 2.5) of the signal derived from the acetyl group of chondroitin sulfate and the signal derived from the benzene ring of phenylalanine benzyl ester to (δ = 2 ppm) indicates that the disaccharide of chondroitin sulfate is The introduction rate of phenylalanine benzyl ester per unit is 2
5%.

In the same experiment as above, when the DMF solution was not dried by the molecular sieve, the introduction rate was 13%. Example 14 Synthesis of adriamycin-modified chondroitin sulfate A sodium salt of chondroitin sulfate having an average molecular weight of 30,000 was used as a tetrabutylammonium salt according to a conventional method using an ion exchange resin. A solution of this chondroitin sulfate tetrabutylammonium salt (50 mg) in DMF (10 ml) was added with DM of Mpt-Cl (0.05 mmol) under ice cooling.
F (0.1 ml) and triethylamine (0.1 mmol) were added. After 10 minutes adriamycin (10 mg)
(1 ml) was added, and the mixture was stirred overnight at room temperature. Treatment according to a conventional method gave the title compound as a red powder, 5
0 mg was obtained. The introduction rate determined by NMR was 9%.

Adriamycin could not be introduced into chondroitin sulfate by the known carbodiimide method. Example 15 Synthesis of methotrexate modified hyaluronic acid (introduced by ester bond) A solution of methotrexate (0.25 mmol) in DMF (1 ml) was added with a solution of triethylamine (0.25 mmol) and Mpt-Cl (0.25 mmol) in DMF ( 1m
1) was added under ice cooling. After 5 minutes, triethylamine salt of hyaluronic acid having an average molecular weight of 800,000 (100
(50 mg) / DMF solution and a mixed solution of 4-dimethylaminopyridine (DMAP) (0.125 mmol) / DMF solution (1 ml) were added under ice cooling, and triethylamine (0.25 mmol) was further added. After stirring overnight at room temperature, 5% sodium hydrogen carbonate solution (2 ml) was added, and the mixture was poured into saturated sodium acetate ethanol and the resulting precipitate was centrifuged. The resulting precipitate was washed three times with 80% ethanol. The obtained precipitate was dissolved in water, dialyzed overnight with deionized water, and freeze-dried to obtain 77 mg of the title compound as a white powder.

The introduction rate of methotrexate calculated by comparing the methotrexate content obtained from the calibration curve previously calculated by the absorptiometry with the hyaluronic acid content obtained by the carbazole sulfate method was 2.7%. Example 16 Synthesis of mixed acid anhydride of hyaluronic acid and methotrexate-modified hyaluronic acid (introduced by amide bond) A sodium salt of hyaluronic acid having an average molecular weight of 800,000 was converted into a triethylamine salt by an ordinary method using an ion exchange resin. This hyaluronic acid triethylamine salt (100m
In 50 ml of DMF solution of g), Mpt-Cl under ice cooling.
A DMF solution (0.25 ml) of (0.25 mmol) was added to prepare a hyaluronic acid mixed acid anhydride.

After 5 minutes, 1 ml of a solution of methotrexate (0.25 mmol) in DMF and triethylamine (0.2
(5 mmol) was added and the mixture was stirred at room temperature overnight. After adding a 5% sodium hydrogen carbonate solution (5 ml), the mixture was poured into ethanol saturated with sodium acetate and the resulting precipitate was centrifuged.
The obtained precipitate was washed three times with 80% ethanol, dissolved in water (10 ml), and dialyzed against deionized water for 3 days. Lyophilization gave 83 mg of the title compound as a white powder.

The introduction rate of methotrexate calculated by comparing the methotrexate content obtained from the calibration curve previously calculated by the absorptiometric method with the hyaluronic acid content obtained by the carbazole-sulfuric acid method was 3.0%. Example 17 Synthesis of glutamine-modified hyaluronic acid (introduced by ester bond) A solution of Fmoc-Gln-OH (1 mmol) in DMF (1 ml) containing triethylamine (1 mmol) and Mpt-Cl (1 mmol) in DMF (1 ml). Was added under ice cooling.
After 5 minutes of this, a triethylamine salt of hyaluronic acid having an average molecular weight of 800,000 (100 mg) / DMF solution 50 m
l and 4-dimethylaminopyridine (DMAP) (0.5
To a mixed solution of (mmol) / DMF solution (1 ml) under ice cooling, triethylamine (1 mmol) was further added. After stirring at room temperature for 5 hours, 5% sodium hydrogen carbonate solution (2 ml) was added, and the mixture was poured into saturated sodium acetate ethanol, and the generated precipitate was centrifuged. The precipitate obtained was washed three times with 80% ethanol and then washed with water (10 m
It was dissolved in 1) and dialyzed against deionized water for 1 day. Freeze-dried Fmoc-Gln-hyaluronic acid as white powder 12
7 mg were obtained.

The glutamine introduction rate calculated by comparing the Fmoc-Gln-OH content obtained from the calibration curve previously calculated by the absorptiometry with the hyaluronic acid content obtained by the carbazole sulfate method was 31.3%. Moreover, this Fmoc-Gln-
A 57% aqueous solution of diethylamine (20 ml) was added to 57 mg of hyaluronic acid under ice cooling, and the mixture was stirred at room temperature for 1 hour.
A 5% sodium hydrogen carbonate solution (2 ml) was added, the mixture was poured into ethanol saturated with sodium acetate, and the resulting precipitate was centrifuged. After washing the obtained precipitate with 80% ethanol,
It was dissolved in water (10 ml) and dialyzed against deionized water for 1 day.
Lyophilized to give the title compound as a white powder, 39 m
g was obtained.

Example 18 Elongation of Peptide on Chondroitin Sulfate Fmoc-leucine (0.5 mmol) was reacted with Mpt-Cl in the same manner as in Example 3 to prepare a mixed acid anhydride. A water-DMF (1: 1) solution (4 ml) of hexanediamine-bonded chondroitin sulfate (average molecular weight 30,000, spacer introduction rate 32%, 100 mg) synthesized in Preparation Example 1 was added dropwise to this, and further triethylamine (0. 5
Mmol). Post-treatment was carried out in the same manner as in Example 3 to obtain 99 mg of Fmoc-Leu-hexanediamine-chondroitin sulfate as a white powder. The leucine introduction rate determined by proton NMR at 270 MHz was 18% with respect to the carboxyl groups of chondroitin sulfate (100% with respect to the spacer). To Fmoc-Leu-hexanediamine-chondroitin sulfate (95 mg) was added 10% aqueous diethylamine solution (5 ml) under ice cooling,
It was left for 1 hour. 0.22 after dialysis with deionized water for 2 days
After filtering with a micron filter and freeze-drying, L
85 mg of eu-hexanediamine-chondroitin sulfate was obtained as a white powder. 270 MHz proton NMR
As a result of the measurement, the signal derived from leucine remained and Fmo
The signal derived from the c group had disappeared.

Similarly, Fmoc-phenylalanine, Fmoc-glycine, Fmoc-glycine, Fmo
The binding of c-tyrosine and the removal of the Fmoc group were repeated to finally obtain leucine enkephalin-modified chondroitin sulfate.

[0065]

INDUSTRIAL APPLICABILITY As described above, the present invention is useful as a new method for producing a GAG derivative, which is a promising raw material for DDS and medical materials. Furthermore, the production method of the present invention is
Since it is a reaction under mild conditions, there are few side reactions such as lowering the molecular weight of GAG, the reaction time is short, and the mixed acid anhydride is relatively stable, so it is relatively easy to control the modification rate. It is an excellent method in some respects. Furthermore, the reaction (acylation) can be carried out in a water-containing organic solvent.

Further, the GAG derivative to which a physiologically active substance such as indomethacin, adriamycin, salazosulfapyridine, etc., which is produced by the method of the present invention is combined with the physiologically active substance alone in view of water solubility, activity sustainability and the like. An excellent effect is expected compared to.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the amount of indomethacin charged and the introduction rate in Example 8. In the figure (mol / mo
l) is the number of moles of indomethacin / hyaluronic acid (2
Sugar) Indicates the number of moles.

Claims (8)

[Claims] 1. A mixed acid anhydride obtained by reacting a carboxylic acid with a halogenated di-lower alkylphosphinothioyl oil is reacted with a glycosaminoglycan having a hydroxyl group or an amino group, and the mixed acid anhydride is reacted. Characterized by forming an ester bond or an amide bond between the carbonyl group and the hydroxyl group or amino group of the glycosaminoglycan.
A method for producing a glycosaminoglycan derivative in which a carboxylic acid and the glycosaminoglycan are ester-bonded or amide-bonded. 2. The glycosamino according to claim 1, wherein the carboxylic acid has a functional group other than a carboxyl group protected with a protecting group, and the protecting group is removed after the ester bond reaction or the amide bond reaction. Method for producing glycan derivative. 3. A glycosamino comprising a glycosaminoglycan having a carboxyl group and a halogenated di (lower alkyl) phosphinothioyl oil to react with the glycosaminoglycan and the halogenated di (lower alkyl) phosphinothioyl. A method for producing a glycosaminoglycan derivative, which comprises producing a glycan mixed acid anhydride. 4. A glycosaminoglycan mixed acid anhydride obtained by reacting a glycosaminoglycan having a carboxyl group with a halogenated di (lower alkyl) phosphinothioyl oil is reacted with a primary amine or a secondary amine. And a carbonyl group of the mixed acid anhydride and an amino group of the amine are amide-bonded to each other, wherein the glycosaminoglycan is amide-bonded to a primary amine or a secondary amine. Method for producing glycan derivative. 5. The production method according to claim 1, wherein the reaction is carried out in an anhydrous organic solvent in the presence of a neutralizing agent. 6. The method according to claim 1, wherein the reaction is carried out in an aqueous solution containing a water-miscible organic solvent. 7. The glycosaminoglycan is a chondroitin sulfate, hyaluronic acid, or a spacer compound-bound glycosaminoglycan having an amino group in which a diamine is covalently bound to these carboxyl groups as a spacer compound, and the carboxylic acid is indomethacin, Deoxycholic acid, acetylsalicylic acid, salazosulfapyridine, methotrexate, leucine enkephalin, leucine, serine, glycine or glutamine. The carboxylic acid produced by the method according to claim 1 and a glycosaminoglycan having a hydroxyl group or an amino group. A glycosaminoglycan derivative in which is an ester bond or an amide bond. 8. The method according to claim 4, wherein the glycosaminoglycan is chondroitin sulfate or hyaluronic acid, and the primary or secondary amine is bestatin, tranexamic acid, adriamycin, methotrexate, phenylalanine or glutamine. A glycosaminoglycan derivative in which a glycosaminoglycan having a carboxyl group and a primary amine or a secondary amine are amide-bonded.