JP3563440B2 - Method for producing acylated hyaluronic acid - Google Patents

Description

[0001]
[Industrial applications]
The present invention relates to a method for acylating a hydroxyl group of a polymer hyaluronic acid. Hyaluronic acid is naturally used in animal tissues, has bioresorbability, and has no toxicological or immunological effects, and is itself used as a drug or cosmetic. Further applications can be expected by making modifications. For example, a drug or a bioactive peptide bound to hyaluronic acid is an excellent drug delivery system (hereinafter, referred to as DDS) drug. In addition, when ultraviolet light is applied to a substance obtained by binding cinnamic acid or a uracil derivative to hyaluronic acid, an insoluble crosslinked hyaluronic acid having a three-dimensional network structure is obtained by an intermolecular crosslinking reaction, and is biodegradable and biocompatible. It is an excellent medical material.
[0002]
[Prior art]
Conventionally, as a method for acylating a water-soluble polysaccharide, a method in which hyaluronic acid is suspended in N, N-dimethylformamide (hereinafter referred to as DMF) and reacted with an acid chloride using pyridine as a catalyst (Eur. J. Biochem. , 1, 46-50 (1967), Chem. Express, 6 (9), 647-650 (1991)), replacing the sodium salt of glycosaminoglycan with a quaternary or tertiary ammonium salt to make it soluble in DMF. After that, a method of reacting with acid anhydride using dimethylaminopyridine as a catalyst (Carbohydr. Res., 236, 107-119 (1992)), and a method of heating pullulan with benzoyl chloride using pyridine as a catalyst in DMF (Japanese Unexamined Patent Application Publication No. 52-78286) and the like.
[0003]
However, hyaluronic acid is usually obtained in the form of a sodium salt, which is insoluble in an organic solvent.If sodium hyaluronate is used as a raw material, the reaction becomes heterogeneous, and the acylation reaction is very slow. It is difficult to control the number of introductions, and the introduction positions are likely to be uneven. In addition, the carboxyl group of hyaluronic acid reacts with the acid chloride to form an acid anhydride, which is accompanied by undesirable side reactions such as regio-specifically forming an intramolecular or intermolecular ester bond with the hydroxyl group.
[0004]
A method in which the sodium salt of hyaluronic acid is replaced with a quaternary or tertiary ammonium salt to make it soluble in DMF, and then reacted with an acid anhydride using dimethylaminopyridine as a catalyst (Carbohydr. Res., 236, 107-119). (1992)), however, this salt exchange operation is very complicated and generates a large amount of DMF waste liquid, which is environmentally and industrially problematic. Furthermore, most of these methods require heating in order to increase the solubility and the reaction rate. In the case of high-molecular-weight hyaluronic acid, the molecular weight is rapidly reduced.
[0005]
In addition, in the chemical modification of high-molecular-weight hyaluronic acid with high viscosity, it is necessary to repeat the reaction and concentration under high-dilution conditions. Not suitable for providing medical materials.
[0006]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to firstly provide a simple solubilizing method in an environmentally friendly aqueous system which is not only a best solubilizing solvent but also suitable for environmental protection without reducing the molecular weight of the hydroxyl group of hyaluronic acid. The second is to provide a method for uniformly introducing an arbitrary number of acyl groups by the method described above. Secondly, a DDS material having applicability and versatility based on them, and photocrosslinkable hyaluronic acid in an endotoxin-free state. , As medical materials.
[0007]
[Means for Solving the Problems]
As a result of intensive studies, the inventors of the present application have succeeded in achieving the above object by the following configuration. That is, the present invention is as follows.
1) The hydroxyl group of hyaluronic acid or a salt thereof is converted into an aqueous solution containing water alone or a water-miscible organic solvent in the presence of an acylation catalyst.Acylating agentAnd acylating the product.
2)The method for producing an acylated hyaluronic acid according to the above 1), wherein the acylating agent is an acid anhydride or an acid halide of a carboxylic acid.
[0008]
3) Reaction temperatureIs 0The method for producing an acylated hyaluronic acid according to the above 1), wherein the temperature is from 50 to 50 ° C.
4) The method for producing an acylated hyaluronic acid according to the above 1), wherein the acylation catalyst is an N, N-dialkylaminopyridine-based catalyst.
5) The method for producing an acylated hyaluronic acid according to 1) above, wherein the acylation catalyst is 4-dimethylaminopyridine or 4-pyrrolidinopyridine.
[0009]
6) Average molecular weight of hyaluronic acid or its saltIs 1The method for producing an acylated hyaluronic acid according to the above 1), wherein the amount is from 10,000 to 5,000,000.
7) The carboxylic acid is an amino acid, a peptide or a derivative thereof, cinnamic acid or a derivative having a substituent on the phenyl group, orotic acid, uracil-5-carboxylic acid, 1- (carboxyalkyl) thymine, salicylic acid or acetylsalicylic acid. Characterized in that there is2The method for producing an acylated hyaluronic acid according to the above).
[0010]
8The above-mentioned, wherein the derivative of the amino acid or the peptide has an amino-protecting group.7The method for producing an acylated hyaluronic acid according to the above).
9After the acylation reaction,Characterized by the elimination of a mino protecting groupThe above 8)A method for producing an acylated hyaluronic acid.
10The method for producing an acylated hyaluronic acid according to the above 1), wherein the acylation reaction is carried out in the presence of an acid neutralizing agent, and a tertiary amine or an inorganic base is used as the acid neutralizing agent.
[0011]
11) The aqueous solution is a water-miscible organic solvent0The method for producing an acylated hyaluronic acid according to the above 1), wherein the amount is contained in a range of from 50% to 50%.
12) reacting the hydroxyl group of hyaluronic acid or a salt thereof with an acylating agent in an aqueous solution containing water alone or a water-miscible organic solvent in the presence of an acylation catalyst, and then obtaining the acyl group of the resulting acylated hyaluronic acid Is obtained by partially saponifying the compound to obtain an acylated hyaluronic acid into which an arbitrary number of acyl groups have been introduced.
[0012]
13) The number of acyl groups introduced into the acylated hyaluronic acid obtained by partial saponification is equivalent to the number of disaccharide units constituting hyaluronic acid.R0. 12. The method for producing an acylated hyaluronic acid according to the above item 12), which is 005 to 0.5.
14) Molecule in water miscible organic solvent aqueous solutionQuantity 1A water-miscible organic solution containing citric anhydride or cinnamic chloride, 4-dimethylaminopyridine or 4-pyrrolidinopyridine, and an acid neutralizing agent in a solution in which 100,000 to 5,000,000 sodium hyaluronate is dissolved. Add a solvent solution to react, molecular weightIs 1A method for producing cinnamoylated hyaluronic acid, wherein the hyaluronic acid has a hydroxyl group of from 100,000 to 5,000,000 and is hydroxyl-esterified with cinnamic acid.
[0013]
15) The water-miscible organic solvent is dioxane, dimethylformamide, N-methylpyrrolidone, acetamide or pyridine, and the acid neutralizing agent is triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine or sodium hydrogen carbonate. Said 14)) The method for producing cinnamoylated hyaluronic acid according to the above.
[0014]
Hereinafter, the present invention will be described in detail.
[0015]
The hyaluronic acid used in the present invention is not limited in origin and molecular weight, but is generally derived from natural products, preferably vertebrates or microorganisms, and has an average molecular weight ofIs 1The carboxyl group may be in the form of a salt, or may be used in the form of either an alkali metal salt or an alkaline earth metal salt. Preferably, as such salts, generally available sodium or potassium salts are convenient.
[0016]
The acylation reaction in the present invention is carried out by adding an acid anhydride or an acid halide of a carboxylic acid as an acylating agent to a hyaluronic acid solution dissolved in water alone or an aqueous solution containing a water-miscible organic solvent. . Specifically, water or a water-miscible organic solventOf 0This is carried out by adding an acylating agent dissolved in an anhydrous organic solvent to a solution of hyaluronic acid or a salt thereof dissolved in an aqueous solution containing 〜50% while stirring. Here, the mixing ratio (%) of the organic solvent in water = 100 · organic solvent volume / mixed aqueous solution volume.
[0017]
The reaction system in the acylation reaction of the present invention is not particularly limited and any method can be used as long as the above conditions are satisfied.In particular, hyaluronic acid or a salt thereof is first dissolved in water and then dissolved in an organic solvent. A solution of an acylating agent, an acid neutralizing agent (base), and an acylating catalyst in an organic solvent without or with the addition of0It is preferable to carry out the acylation reaction by dropping at a temperature in the range of -50 ° C, preferably 0 ° C or higher and room temperature or lower, more preferably under ice cooling.
[0018]
According to this method, it is a great advantage that the acylation of hyaluronic acid can be achieved without any complicated operation such as salt exchange or solvent exchange, which is required for the conventional reaction in an organic solvent.
[0019]
When the acylating agent is added in the present invention, it is preferable to use an N, N-dialkylaminopyridine-based catalyst, particularly 4-dimethylaminopyridine or 4-pyrrolidinopyridine, as an acylation catalyst. For the purpose of neutralization, it is preferable to use a tertiary amine such as triethylamine, diisopropylethylamine, pyridine or N-methylmorpholine or an inorganic base such as sodium hydrogencarbonate as a neutralizing agent for the acid. These are preferably added together with the acylating agent, but may be added in advance to the sodium hyaluronate solution.
[0020]
The acylation reaction temperatureIs 0The temperature is from 0 to 50 ° C, preferably from 0 ° C to room temperature (below 5 to 35 ° C as room temperature in JIS K0050), more preferably under ice-cooling, and the reaction time is 3 minutes to 2 hours. Thereafter, the acylation reaction is performed by stirring for a necessary time, for example, 30 minutes to 2 hours. Thereafter, low-molecular impurities are removed by a known purification method such as ethanol precipitation or dialysis. By keeping the reaction temperature low in this way, even high molecular weight hyaluronic acid, which is liable to be degraded, can be acylated while maintaining the original molecular weight.
[0021]
The molecular weight of the acylated hyaluronic acid can be determined by gel filtration (GPC) or the like after removing all the acyl groups by saponification.
[0022]
The reaction solvent is water alone or an aqueous solution containing a water-miscible organic solvent, and preferably has a composition in which all the components are dissolved during the dropwise addition of the acylating agent. Absent.
Here, the water-miscible organic solvent means an organic solvent that is uniformly mixed at a free ratio with water and does not inhibit the acylation reaction.
[0023]
Specific examples of the water-miscible organic solvent used in the present invention include dioxane, tetrahydrofuran (THF), N, N-dimethylacetamide, acetamide, DMF, dimethylsulfoxide (DMSO), hexamethylphosphoric triamide (HMPA), N-methylpyrrolidone, pyridine and the like can be mentioned. Further, as the reaction solvent, a total of at least one kind selected from any of the water-miscible organic solvents exemplified above.At 0It is preferable to use an aqueous solution containing 50% to 50%, in which all components are dissolved during the dropwise addition of the acylating agent. In the reaction in the water-containing system of the present invention, the acylated hyaluronic acid is insolubilized when the rate of introduction of the hydrophobic acyl group increases with the progress of the reaction, so that the number of acyl groups introduced is not excessively increased. . Further, the target substance can be easily purified by separating it with a usual solid-liquid separation means and washing it with water and / or an organic solvent.
[0024]
The amount of the acylating agent and the acylating catalyst with respect to the hyaluronic acid can be appropriately selected depending on the type of the desired acylated hyaluronic acid, that is, the molecular weight of the hyaluronic acid or the type of the acylating agent and the desired number of acyl groups to be introduced. Generally, an excess amount should be used in consideration of the loss due to decomposition of the reagent during the reaction. The number of acyl groups introduced into the acylated hyaluronic acid (hereinafter, referred to as DS) can be defined as the number introduced per hyaluronic acid-constituting disaccharide unit. DS can be determined by measuring the integrated intensity of proton NMR or the absorbance at 280 nm (see Example 1).
[0025]
In the present invention, DS can be controlled to a desired value., 0. 005-0.5, preferably about 0.05-0.5.
[0026]
If the acylation product precipitates during the acylation reaction, the precipitate is separated by solid-liquid separation means such as filtration using a glass filter or the like, centrifugation, decanting, etc. The target substance can be purified and isolated by a simpler purification operation such as washing with successive solvents. In general, the DS is insolubilized when the DS is high, but the acylated hyaluronic acid thus obtained is partially saponified with a dilute alkali such as an aqueous sodium hydroxide solution or an aqueous potassium hydroxide solution under ice-cooling to obtain an arbitrary DS value. Can also be obtained.
[0027]
As a specific method of the saponification, for example, an insoluble acylated hyaluronic acid is suspended in water, a necessary amount of an aqueous sodium hydroxide solution is dropped at 0 ° C. in order to obtain a desired DS, and dissolved at room temperature. Stir until done. In this case, sodium hydroxide is used in an equimolar amount to the acyl group to be saponified.
When the product does not precipitate even after completion of the reaction, the usual post-treatment of this type of reaction, namely, organic solvent precipitation using ethanol or the like, dialysis, salting-out, and, if desired, various processes such as GPC, affinity, ion exchange, etc. The acylated hyaluronic acid can be purified by chromatography or the like.
[0028]
The acylating agent used in the acylation reaction of the present invention is a carboxylic acid anhydride (symmetric acid anhydride, mixed acid anhydride) or an acid halide. Further, it may be one which changes into an acid anhydride or an acid halide at least during the acylation reaction. The type of the carboxylic acid is not limited, but preferred examples include a peptide or a derivative thereof, cinnamic acid or a derivative having a substituent on the phenyl group (eg, p-aminocinnamic acid), orotic acid, uracil-5. -Carboxylic acid, 1- (carboxyalkyl) thymine (for example, 1- (carboxyethyl) thymine), salicylic acid, acetylsalicylic acid, and the like.
[0029]
When the acyl group to be bonded to hyaluronic acid is a residue of an amino acid or a peptide, the amino group contained therein is commonly used in peptide chemistry such as a benzyloxycarbonyl group (Z) or a t-butoxycarbonyl group (Boc). The amino group must be protected with a protecting group (Biochemical Dictionary, 2nd edition, pp. 1471-1472, Nov. 22, 1990, issued by Tokyo Chemical Doujin Co., Ltd.). The obtained acylated hyaluronic acid having a protecting group may be used depending on the purpose.handIt can be deprotected to a free amino group. By linking the amino group of the acylated hyaluronic acid having the free amino group with a desired drug, amino acid, peptide or the like, a useful DDS drug or the like can be produced.
[0030]
The carboxylic acid is subjected to the reaction in the form of an acylating agent such as an acid anhydride or an acid halide of the carboxylic acid, and a method for activating the carboxyl group to obtain the acid anhydride of the carboxylic acid is a carbodiimide. (For example, dicyclohexylcarbodiimide, 1-ethyl-3- (dimethylaminopropyl) -carbodiimide, etc.) or a mixed acid anhydride method using dimethylphosphinothioyl chloride or the like.
[0031]
For example, carboxylic acids whose symmetric acid anhydrides are suitable include cinnamic acid anhydride, and those whose mixed acid anhydrides are suitable include t-butoxycarbonylated glycine and benzyloxycarbonylated glycine.
Then, the benzyloxy group of the obtained benzyloxycarbonylglycinylated hyaluronic acid is deprotected by catalytic hydrogen transfer reduction or catalytic reduction to produce a glycylated hyaluronic acid having a free amino group.
[0032]
When the acyl group is a cinnamoyl group, cinnamic acid chloride and cinnamic anhydride are effective. Further, when the acyl group is derived from 1-carboxyethylthymine, activation by an acid anhydride is preferable, and when the acyl group is derived from orotic acid, activation by an acid chloride is more preferable.
The acylating agent used in the present invention can be selected from other carboxylic acids. The carboxylic acid may be any organic compound having at least one carboxyl group (including aliphatic, aromatic, and heterocyclic compounds), and may have a plurality of carboxyl groups.
[0033]
In particular, if a useful agent such as salicylic acid, acetylsalicylic acid, or melphalan (4- [bis (2-chloroethyl) amino] -L-phenylalanine) is used as the carboxylic acid, the acylated hyaluronic acid can be used as a DDS preparation. It is.
[0034]
This method, which is carried out in a water-containing solvent, is much simpler than the conventional glycosaminoglycan acylation method.By using endotoxin-free water, the target substance substantially free of endotoxin can be obtained. Can be easily obtained. It also has the advantage of greatly reducing the amount of organic waste liquid that is industrially problematic.
[0035]
【Example】
Hereinafter, the present invention will be described in detail with reference to examples.
[0036]
Example 1
Production of cinnamoylated hyaluronic acid in aqueous dioxane solution
After dissolving 500 mg of sodium hyaluronate having an average molecular weight of 1,000,000 (1.25 mmol as a disaccharide unit) in 200 ml of water, 100 ml of dioxane was added. To this solution, a solution of 1.7 g (6.25 mmol) of cinnamic anhydride, 865 μl (6.25 mmol) of triethylamine and 763 mg (6.25 mmol) of 4-dimethylaminopyridine in 10 ml of dioxane was added under ice cooling for 30 minutes. After dropping over a period of one minute, stirring was continued at room temperature for 1.5 hours. The resulting solution was poured into 1.7 L (liter) of saturated sodium acetate ethanol and the resulting precipitate was centrifuged. The obtained precipitate was further purified three times by the ethanol precipitation method, and dried under reduced pressure to obtain 417 mg of the above-mentioned partially cinnamoylated hyaluronic acid as a white powder. As a result of the proton NMR measurement at 500 MHz, the intensity ratio of the signal derived from the acetyl group of hyaluronic acid (δ = 2 ppm) to the signal derived from the benzene ring of cinnamate and the double bond (δ = 6.5-7.9 ppm) ( 3: 2.2), the DS of the cinnamoyl group per disaccharide unit of hyaluronic acid was 0.37 (see FIG. 1).
[0037]
Example 2
Production of cinnamoylated hyaluronic acid by partial saponification method
After dissolving 80 mg of sodium hyaluronate having an average molecular weight of 1,000,000 (0.2 mmol as a disaccharide unit) in 20 ml of water, 10 ml of dioxane was added. To this solution, a solution of cinnamate chloride 144 μl (1.0 mmol), triethylamine 139 μl (1.0 mmol) and 4-dimethylaminopyridine 122 mg (1.0 mmol) in 2 ml of dioxane was added dropwise over 25 minutes under ice-cooling. After that, stirring was continued at room temperature for 30 minutes. The resulting precipitate was collected on a glass filter, thoroughly washed with ethanol, and dried. The obtained solid was made into a fine powder, suspended in 20 ml of water, and 2 ml of a 0.05 N sodium hydroxide solution was added dropwise under ice cooling. Further, after stirring for 1 hour and 45 minutes under ice cooling, the obtained homogeneous solution was purified by the same ethanol precipitation method as in Example 1 to obtain 55 mg of the title compound. The DS of the cinnamoyl group determined by NMR was 0.46.
[0038]
FIG. 2 shows the results of the GPC analysis of the change in molecular weight of hyaluronic acid as a raw material of cinnamoylated hyaluronic acid subjected to partial saponification and cinnamoylated hyaluronic acid obtained by partial saponification. FIG. 2 (a) shows the hyaluronic acid as a raw material, and FIG. 2 (b) shows the hyaluronic acid after partial saponification. FIG. 2 shows that hyaluronic acid was not hydrolyzed by the partial saponification. This indicates that the molecular weight was not reduced.
[0039]
The GPC measurement conditions are as follows.
Column temperature: 35 ° C
Column: Tosoh TSKgel-G6000PWXL
Flow rate: 0.5 mL / min
Detection wavelength: 210 nm
Sample injection: 30 μL
Solvent: 0.2 M aqueous sodium chloride solution
[0040]
Example 3
Production of cinnamoylated hyaluronic acid in DMF aqueous solution
After dissolving 80 mg (0.2 mmol as a disaccharide unit) of sodium hyaluronate having an average molecular weight of 1,000,000 in 20 ml of water, 10 ml of DMF was added. A solution of 278 mg (1.0 mmol) of cinnamic anhydride, 139 μl (1.0 mmol) of triethylamine and 122 mg (1.0 mmol) of 4-dimethylaminopyridine in 2 ml of DMF was added dropwise to the solution over 15 minutes under ice-cooling. After that, stirring was continued for 1.5 hours at room temperature. 250 ml of sodium acetate saturated ethanol were poured into the resulting solution and the resulting precipitate was centrifuged. The obtained precipitate was further purified three times by the ethanol precipitation method, and dried under reduced pressure to obtain 42 mg of the title cinnamoylated hyaluronic acid as a white powder. The DS of the cinnamoyl group determined by NMR was 0.46.
[0041]
Example 4
Preparation of cinnamoylated hyaluronic acid in aqueous pyridine solution
After dissolving 80 mg of sodium hyaluronate having an average molecular weight of 1,000,000 (0.2 mmol as a disaccharide unit) in 20 ml of water, 10 ml of pyridine was added. To this solution, a pyridine solution of 278 mg (1.0 mmol) of cinnamic anhydride and 122 mg (1.0 mmol) of 4-dimethylaminopyridine was added dropwise over 15 minutes under ice-cooling, followed by stirring at room temperature for 1.5 hours. Continued. 250 ml of sodium acetate saturated ethanol were poured into the resulting solution and the resulting precipitate was centrifuged. The obtained precipitate was further purified by ethanol precipitation three times and dried under reduced pressure to obtain 67 mg of the above-mentioned partially cinnamoylated hyaluronic acid as a white powder. The DS of the cinnamoyl group determined by NMR was 0.09.
[0042]
Example 5
Preparation of cinnamoylated hyaluronic acid using diisopropylethylamine as base
Cinnamoylated hyaluronic acid was synthesized according to the method of Example 3, except that 80 mg of sodium hyaluronate having an average molecular weight of 1,000,000 (0.2 mmol as a disaccharide unit) and diisopropylethylamine were used instead of triethylamine. After purification, the residue was dried under reduced pressure to obtain 74 mg of the above-mentioned partially cinnamoylated hyaluronic acid as a white powder. The DS of the cinnamoyl group determined by NMR was 0.54.
[0043]
Example 6
Production of cinnamoylated hyaluronic acid with cinnamic acid chloride
After dissolving 80 mg (0.2 mmol as a disaccharide unit) of sodium hyaluronate having an average molecular weight of 1,000,000 in 20 ml of water, 10 ml of dioxane was added. To this solution, a solution of 144 μl (1.0 mmol) of cinnamic acid chloride, 139 μl (1.0 mmol) of triethylamine and 122 mg (1.0 mmol) of 4-dimethylaminopyridine in 2 ml of dioxane was added dropwise over 15 minutes under ice-cooling. After that, stirring was continued for 1.5 hours at room temperature. 250 ml of sodium acetate saturated ethanol were poured into the resulting solution and the resulting precipitate was centrifuged. The obtained precipitate was further purified by ethanol precipitation three times and then dried under reduced pressure to obtain 98 mg of the above-mentioned partially cinnamoylated hyaluronic acid as a white powder. The DS of the cinnamoyl group determined by NMR was 0.25.
[0044]
Example 7
Production of cinnamoylated hyaluronic acid using 4-pyrrolidinopyridine as acylation catalyst
80 mg (0.2 mmol as a disaccharide unit) of sodium hyaluronate having an average molecular weight of 1,000,000 was dissolved in 20 ml of water. A solution of 278 mg (1.0 mmol) of cinnamic anhydride, 139 μl (1.0 mmol) of triethylamine and 148 mg (1.0 mmol) of 4-pyrrolidinopyridine in 2 ml of dioxane was added to this solution under ice-cooling for 15 minutes. After the dropwise addition, stirring was continued for 1.5 hours at room temperature. 250 ml of sodium acetate saturated ethanol were poured into the resulting solution and the resulting precipitate was centrifuged.
[0045]
The obtained precipitate was further purified three times by the ethanol precipitation method, and dried under reduced pressure to obtain 55 mg of the above-mentioned partially cinnamoylated hyaluronic acid as a white powder. The DS of the cinnamoyl group determined by NMR was 0.95.
[0046]
Example 8
Production of cinnamoylated hyaluronic acid (molecular weight 150,000)
After dissolving 800 mg (2.0 mmol as a disaccharide unit) of sodium hyaluronate having an average molecular weight of 150,000 in 200 ml of water, 100 ml of dioxane was added. A solution of 2.78 g (10 mmol) of cinnamic anhydride, 1.38 ml (10 mmol) of triethylamine and 1.22 g (10 mmol) of 4-dimethylaminopyridine in 25 ml of dioxane was added to this solution under ice-cooling for 1 hour. After dropping, stirring was continued at room temperature for 1 hour. To the resulting solution was poured 750 ml of saturated sodium acetate ethanol and the resulting precipitate was centrifuged. The resulting precipitate was further purified by ethanol precipitation three times, and then converted to an aqueous solution to obtain 300 ml of a 2.7 g / l aqueous solution of the above-mentioned partially cinnamoylated hyaluronic acid. The DS of the cinnamoyl group determined by NMR was 0.60.
[0047]
Example 9
Production of cinnamoylated hyaluronic acid in aqueous solution substantially free of organic solvents
80 mg (0.2 mmol as a disaccharide unit) of sodium hyaluronate having an average molecular weight of 1,000,000 was dissolved in 20 ml of water. 139 μl (1.0 mmol) of triethylamine and 122 mg (1.0 mmol) of 4-dimethylaminopyridine were added to this solution under ice-cooling, and the mixture was stirred uniformly. 144 μl of cinnamic acid chloride was added, and the mixture was stirred at room temperature for 1.5 hours. Continued. 250 ml of sodium acetate saturated ethanol were poured into the resulting solution and the resulting precipitate was centrifuged. The obtained precipitate was further purified by ethanol precipitation three times and then dried under reduced pressure to obtain 58 mg of the above-mentioned partially cinnamoylated hyaluronic acid as a white powder. The DS of the cinnamoyl group determined by NMR was 0.22.
[0048]
Example 10
Production of t-butoxycarbonylglycylated hyaluronic acid
After dissolving 80 mg of sodium hyaluronate having an average molecular weight of 1,000,000 (0.2 mmol as a disaccharide unit) in 20 ml of water, 10 ml of dioxane was added. In this solution, 175 mg (1.0 mmol) of t-butoxycarbonylglycine, in which the carboxyl group was activated as a mixed acid anhydride by a dimethylphosphinothioyl group, 139 μl (1.0 mmol) of triethylamine and 122 mg of 4-dimethylaminopyridine ( (1.0 mmol) in 3 ml of a dimethylformamide solution was added under ice-cooling, and stirring was continued at room temperature for 1.5 hours. 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 entitled t-butoxycarbonylglycylated hyaluronic acid as a white powder. The DS of t-butoxycarbonylglycine determined from the intensity ratio of the signal derived from the hyaluronic acid acetyl group (δ = 2 ppm) and the signal derived from the tertiary butyl group (δ = 1.5 ppm) in NMR was 0.11. .
[0049]
Example 11
Production of benzyloxycarbonylglycylated hyaluronic acid
The same method as in Example 10 was followed, except that 209 mg (1.0 mmol) of benzyloxycarbonylglycine (also referred to as Z glycine) was used instead of t-butoxycarbonylglycine.
[0050]
After purification, the residue was dried under reduced pressure to obtain 74 mg of the title partial benzyloxycarbonylglycylated hyaluronic acid as a white powder. The DS of benzyloxycarbonylglycine determined from the intensity ratio of the signal derived from the hyaluronic acid acetyl group in NMR (δ = 2 ppm) and the signal derived from the benzyl group in benzyloxycarbonylglycine (δ = 7.5 ppm) was 0.11. there were.
[0051]
Example 12
Production of glycylated hyaluronic acid (de-Zation of benzyloxycarbonylglycylated hyaluronic acid)
60 mg (0.15 mmol / unit) of Z-glycylated hyaluronic acid synthesized in Example 11 was dissolved in 50 ml of water, and 15 mg of 10% palladium (Pd) activated carbon and 19 mg (0.3 mmol) of ammonium formate were added under an argon atmosphere. After stirring for 6 hours, the same amounts of Pd-activated carbon and ammonium formate were added again and stirred. Six hours later, the same operation was performed again, the activated carbon was removed with a 0.22 μm filter, the solution was dialyzed for 2 days, and lyophilized to obtain 51 mg of a white substance. NMR confirmed the disappearance of the peak derived from the benzyl group (δ = 7.5 ppm).
[0052]
Example 13
Production of t-butoxycarbonylglycylated hyaluronic acid (molecular weight 150,000) After dissolving 80 mg (0.2 mmol as a disaccharide unit) of sodium hyaluronate having an average molecular weight of 150,000 in 20 ml of water, 10 ml of dioxane was added. In this solution, 175 mg (1.0 mmol) of t-butoxycarbonylglycine in which the carboxyl group of t-butoxycarbonylglycine was activated as a mixed acid anhydride with a dimethylphosphinothioyl group, 139 μl (1.0 mmol) of triethylamine and 4 A solution of 122 mg (1.0 mmol) of dimethylaminopyridine in dimethylformamide was added under ice-cooling, and stirring was continued at room temperature for 1.5 hours. 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 an ethanol precipitation method, and dried under reduced pressure to obtain 55 mg of the title t-butoxycarbonylglycylated hyaluronic acid as a white powder. The DS of t-butoxycarbonylglycine determined by NMR was 0.05.
[0053]
Example 14 Production of Hyaluronic Acid with Cinnamoylation of Partially Endotoxin-Free Hydroxyl Groups
5 g of sodium hyaluronate having an average molecular weight of 1,000,000 (12.5 mmol as a disaccharide unit) was placed in a dry-sterilized 3 L Erlenmeyer flask, dissolved in 2 L of water for injection (endotoxin-free), and then 500 ml of dioxane was added. To this solution, a solution of 17.4 g (62.5 mmol) of cinnamic anhydride, 8.65 ml (62.5 mmol) of triethylamine, and 7.63 g (62.5 mmol) of 4-dimethylaminopyridine in 250 ml of dioxane was ice-cooled. After the dropwise addition for 30 minutes, stirring was continued at room temperature for 1 hour. The resulting solution was poured into 11 L of saturated sodium acetate ethanol and the resulting precipitate was centrifuged. The obtained precipitate was further purified three times by an ethanol precipitation method, and dried under reduced pressure to obtain 4.63 g of the above-mentioned partially cinnamoylated hyaluronic acid as a white powder. As a result of 500 MHz proton NMR measurement, the intensity ratio of the signal derived from the acetyl group of hyaluronic acid (δ = 2 ppm) and the signal derived from the benzene ring and double bond of cinnamic acid (δ = 6.5-7.9 ppm) was obtained. The DS of the cinnamoyl group was 0.35. The endotoxin content determined by Limulus test (Toxicolor, measured by Seikagaku Corporation) was 26.0 pg per mg of cinnamoylated hyaluronic acid.
[0054]
【The invention's effect】
As described above, in the present invention, acylated hyaluronic acid having biocompatibility, biodegradability and useful as a material for DDS and photocrosslinkable medical materials is not accompanied by a decrease in molecular weight and does not adversely affect the environment. It can be provided in a simple operation using water as a solvent and in an endotoxin-free state.
[Brief description of the drawings]
FIG. 1 shows a 500 MHz proton NMR spectrum of cinnamoylated hyaluronic acid (DS = 0.37) in Example 1.
FIG. 2 is a chromatogram showing a comparison between cinnamoylated hyaluronic acid and raw material hyaluronic acid by GPC (gel filtration method) in Example 2;

Claims (15)

A method for producing an acylated hyaluronic acid, which comprises reacting a hydroxyl group of hyaluronic acid or a salt thereof with an acylating agent in an aqueous solution containing water alone or a water-miscible organic solvent in the presence of an acylation catalyst to effect acylation. Law. The method for producing an acylated hyaluronic acid according to claim 1, wherein the acylating agent is an acid anhydride or an acid halide of a carboxylic acid. The method for producing an acylated hyaluronic acid according to claim 1, wherein the reaction temperature is 0 to 50 ° C. The method for producing an acylated hyaluronic acid according to claim 1, wherein the acylation catalyst is an N, N-dialkylaminopyridine catalyst. The method for producing an acylated hyaluronic acid according to claim 1, wherein the acylation catalyst is 4-dimethylaminopyridine or 4-pyrrolidinopyridine. The method for producing an acylated hyaluronic acid according to claim 1, wherein the average molecular weight of the hyaluronic acid or a salt thereof is 100,000 to 5,000,000. The carboxylic acid is an amino acid, a peptide or a derivative thereof, a cinnamic acid or a derivative having a substituent on the phenyl group, orotic acid, uracil-5-carboxylic acid, 1- (carboxyalkyl) thymine, salicylic acid, or acetylsalicylic acid The method for producing an acylated hyaluronic acid according to claim 2, wherein The method for producing an acylated hyaluronic acid according to claim 7 , wherein the amino acid or peptide derivative has an amino protecting group. After acylation reaction, preparation of acylated hyaluronic acid according to claim 8, characterized in that desorbs amino protecting group. The method for producing an acylated hyaluronic acid according to claim 1, wherein the acylation reaction is carried out in the presence of an acid neutralizing agent, and a tertiary amine or an inorganic base is used as the acid neutralizing agent. The method for producing an acylated hyaluronic acid according to claim 1, wherein the aqueous solution contains a water-miscible organic solvent in a range of 0 to 50%. After reacting the hydroxyl group of hyaluronic acid or a salt thereof with an acylating agent in the presence of an acylation catalyst in an aqueous solution containing water alone or a water-miscible organic solvent, the acyl group of the resulting acylated hyaluronic acid is partially reacted. A process for producing an acylated hyaluronic acid, which comprises obtaining an acylated hyaluronic acid into which an arbitrary number of acyl groups have been introduced by saponification. 0 Number introduction of an acyl group of the acylated hyaluronic acid obtained by partially saponifying Ri per hyaluronic acid disaccharide units. 13. The process for producing an acylated hyaluronic acid according to claim 12, wherein the ratio is from 005 to 0.5. A solution prepared by dissolving sodium hyaluronate having a molecular weight of 100,000 to 5,000,000 in a water-miscible organic solvent aqueous solution is mixed with cinnamic anhydride or cinnamic chloride and 4-dimethylaminopyridine or 4-pyrrolidinopyridine and acid. A water-miscible organic solvent solution containing a neutralizing agent is added and reacted to obtain hyaluronic acid having a molecular weight of 100,000 to 5,000,000 and a hydroxyl group esterified with cinnamic acid. For the production of activated hyaluronic acid. The water-miscible organic solvent is dioxane, dimethylformamide, N-methylpyrrolidone, acetamide or pyridine, and the acid neutralizing agent is triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine or sodium hydrogen carbonate. claims 1 to 4, the preparation of cinnamoyl hyaluronic acid according to.

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