JPH0699485B2 - Method for producing synthetic chondroitin polysulfate - Google Patents

Description

TECHNICAL FIELD The present invention relates to a synthetic chondroitin polysulfate and a method for producing the same, and more specifically, the 6-position hydroxyl group of N-acetylgalactosamine residue is selectively highly sulfated. The present invention relates to synthetic chondroitin polysulfate and a method for producing the same.

[Prior Art and its Problems] Chondroitin sulfate E (hereinafter referred to as “CSE”) and chondroitin sulfate H (hereinafter referred to as “CSH”) are chondroitin polysulfates in which the molar ratio of sulfate group to repeating disaccharide is greater than 1, CSE is Seno et al. (N. Seno, et al .; J. Bio
chem. (Tokyo), 60 , 258 (1966)), CSH was found in squid cartilage by Seno et al. (N.Seno, et al.; B).
iochim.Biophys.Acta, 227 , 173 (1971)), and found in the notochord of notel and eel.

CSE and CSH are difficult to collect raw materials and have a low yield.For example, CSE is 100 g of fresh squid head cartilage.
(Approximately 80 individuals) Approximately 60 mg, CSH is 4 from 16 eels
Since it is difficult to obtain a large amount such that only 5 mg can be obtained, the physiological role and biochemical action of CSE and CSH have not been reported in detail. However, Hurle syndrome (Hurle syndrome), which is one of inherited mucopolysaccharide metabolism disorders,
Mucopolysaccharides in the urine of patients with r's syndrome) have an abnormal amount of heparitin sulfate and dermatan sulfate, which is known to be due to a defect in its degrading enzyme. It seems to have some important physiological significance. In addition, when culture medium derived from splenocytes treated with concanavalin A was added to the culture of mouse bone marrow cells, glycosaminoglycan produced in bone marrow-derived mast cells was CSE, Stevens et al. RLStevens, et al.; The Journal of biological C
reported by hemistry, 257 (12), 7229 (1982), and started to investigate the physiological significance of CSE. In addition, Hiromichi Koma et al. (Hiromichi Koma; Japan Dermatological Association Journal, 86, 363 (1976): Hiromichi Koma, Masaru Ohashi, Toshio Kobayashi,
Hisashi Aoyama, Hisashi Iwata; burns, 1, 47 (1976)) is, after the burn,
In the study of the proteoglycan polysaccharide part of the granulation at an important time in the process of formation of scar keloids, we report the sugar of the repeating unit of CSE. As described above, it is becoming very important to examine physiologically and biochemically the existence significance of whole mucopolysaccharides in living organisms and cells.

However, obtaining chondroitin polysulfate having a sulfation rate similar to or higher than these natural chondroitin polysulfates is to selectively sulfate the 6-hydroxyl group of the N-acetylgalactosamine residue. It is not easy because it is difficult.

Therefore, the present inventors have developed a method for selectively sulfating the 6-hydroxyl group of N-acetylgalactosamine residue,
As a result of intensive studies, the 6-hydroxyl group of the N-acetylgalactosamine residue was selected by treating the salt of chondroitin-4-sulfate or dermatan sulfate with a sulfating agent in a uniform solution state in a polar organic solvent. Succeeded in obtaining a highly highly sulfated synthetic chondroitin polysulfate,
The present invention has been completed.

[Structure of the Invention] That is, the synthetic chondroitin polysulfate and its salt obtained by the method of the present invention have the following formula (I): (In the formula, R ₁ O, R ₂ O, R ₃ O and R ₄ O represent a sulfuric acid group or a hydroxyl group, and may be the same or different, but in the molecule, R ₁ O The sulfation rate is 20 to 100%, the sulfation rate of R ₂ O is 20 to 100%, the sulfation rate of each of R ₃ O and R ₄ O is 25% or less, and X is a carboxyl group. And X in the molecule may be the same or different, and Y represents a hydrogen atom when X represents a carboxyl group, and represents a carboxyl group when X represents a hydrogen atom. It is characterized in that the sulfur content in the molecule is 7.3 to 12.5% and the molecular weight is 5,000 to 100,000.

According to the method of the present invention, the above-mentioned synthetic chondroitin polysulfate or a salt thereof can be produced as follows.

That is, a salt of chondroitin-4-sulfate or dermatan sulfate is previously dissolved in a polar organic solvent, and the solution is treated with a sulfating agent at -20 ° C to 20 ° C in a uniform solution state in the polar solvent. be able to.

A salt of chondroitin-4-sulfate usually has a molecular weight of 10
The salt of dermatan sulfate having a molecular weight of 10,000 to 30,000 is usually used. The type of salt varies depending on the type of polar solvent used, and it is necessary to use a salt that can be uniformly dissolved in the solvent used.

As a polar organic solvent, it has polarity and chondroitin-
Any organic solvent may be used as long as it can uniformly dissolve a salt of 4-sulfuric acid or dermatan sulfuric acid, and examples thereof include aromatic heterocyclic bases such as pyridine, quinoline, picoline and lutidine; methylaniline, dimethylaniline, Aromatic bases such as diethylaniline; ethers such as dioxane, tetrahydrofuran, diisopropyl ether;
Carboxylic acid amides such as formamide, acetamide and dimethylformamide; dimethyl sulfoxide, nitromethane, acetonitrile and the like can be mentioned, and pyridine, formamide, dimethylformamide and dimethyl sulfoxide are particularly preferable.

In order to carry out the reaction in a homogeneous system, when formamide, dimethylsulfoxide or the like is used as a polar organic solvent, chondroitin-4-sulfate or dermatan sulfate is a sodium salt, potassium salt, tri-n-butylamine salt, tri-n-butylamine salt or tri-n-butylamine salt. Although it can be used as n-hexylamine salt, tri-n-octylamine salt, etc., as a polar organic solvent,
When using dimethylformamide or the like, chondroitin-4-sulfate or dermatan sulfate is used as a salt with a base such as trialkylamine such as tri-n-butylamine, tri-n-hexylamine and tri-n-octylamine. It is necessary to use.

Examples of the sulfating agent include a complex of sulfur trioxide and a tertiary amine such as pyridine, or dimethylformamide, but it is preferable to use one that can perform the reaction in a homogeneous system depending on the polar organic solvent used. .

The reaction temperature in the sulfation reaction varies depending on the type and dose of the sulfating agent, but is usually -20 ° C to 20 ° C, and particularly 0 to
It is preferably 20 ° C. The reaction time varies depending on the type and dose of the sulfating agent and the reaction temperature, but is usually 1 to 24 hours, and preferably 3 to 8 hours.

After the reaction, the solvent is distilled off from the reaction mixture, or water is added to dilute the mixture, and then dialyzed to remove low-molecular compounds other than the target compound. After that, for example, when the desired product is isolated as a sodium salt, the desired product is converted to a sodium salt by treating with a cation exchange resin (sodium type), and then a water-soluble organic solvent such as The product of the process according to the invention can be obtained by adding ethanol.

[Effects of the Invention] Synthetic chondroitin polysulfate and salts thereof obtained by the method of the present invention have the 6-hydroxyl group of N-acetylgalactosamine residue selectively highly sulfated, which is higher than that of chondroitin-4-sulfate. However, it has an excellent effect on nephritis. In addition, side effects are similar to chondroitin-4-sulfate,
It is useful for treating nephritis.

[Examples of the Invention] Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but these do not limit the scope of the present invention in any way.

In the following examples and the like, salts of chondroitin-4-sulfate and dermatan sulfate derived from whale cartilage and pig skin (manufactured by Seikagaku Corporation) were used.

The physicochemical properties were determined according to the following method.
The contents of galactosamine, glucuronic acid and iduronic acid are respectively S. Gardell (Acta Chem. Scand., 7 , 207).
(1953)), the carbazole method (Z. Dische; J. Biol.
Chem., 167, 189 (1947 )) and orcinol (AHBro
wn; Arch, Biochem.Biophys., 11 , 269 (1946)): JXKhy
m, et al .; J. Am. Chem. Soc., 74 , 3199 (1952)). The sulfation rate is C- in the infrared absorption spectrum.
OS absorption and electrophoresis (cellulose acetate membrane, 0.15M
Formic acid-pyridine (pH 3.0), 0.5 mA / cm, 60 minutes) was used for identification and Dodgson-Price turbidimetric method (KSDodgson, et al .; Bioc
hem.J., 84 , 106 (1962)). Among the synthetic chondroitin polysulfates of the present invention and salts thereof, the position of the sulfate group of the above formula (I) in which X is a carboxyl group is determined by Suzuki et al. (S. Suzuki, et al .; J. Biol. Chem., 243 , 1543
(1968), the position of the sulfate group, although Y is a carboxyl group, was determined by Suzuki et al. (S. Suzuki, et al .; Bi
ochim.Biophys.Acta, 237 , 173 (1971)).

Example 1 1 g of chondroitin-4-sodium sulfate (hereinafter referred to as “CS4SNa”) (molecular weight 20000) was dissolved in 50 ml of purified water and equilibrated at 4 ° C. to prepare a Dowex 50 [H ⁺ ] type column (1.5 ×). 100 cm) and elute with purified water at 4 ° C.
% Tri-n-butylamine ethanol solution to adjust the pH to 5.
Adjusted to 0. The solution was extracted twice with 50 ml of ether to remove excess tri-n-butylamine, and the aqueous layer was concentrated under reduced pressure at 20 ° C. and freeze-dried. Then, it was dried under reduced pressure over phosphorus pentoxide and chondroitin-4-sulfate tri-n-butylamine salt was added.
1.25 g was obtained.

300 mg (0.43 mmol) of the tri-n-butylamine salt was added to 50 ml of dry dimethylformamide (hereinafter referred to as "DMF") at 0 ° C.
Dissolve in, and sulfur trioxide-pyridine complex 445 mg (3.0
10 ml of a dry DMF solution containing 7 mmol) was added, and the mixture was reacted at 0 ° C for 1 hour with stirring.

6 ml of purified water cooled at 0 ° C. was added little by little to the reaction solution, and then the pH was adjusted to 9.0 with 0.1N sodium hydroxide aqueous solution, and dialyzed against purified water at room temperature. Pour this through a Dowex 50 [Na ⁺ ] type column (1.5 x 60 cm),
After eluting with purified water, the mixture was concentrated under reduced pressure at 40 ° C. It was freeze-dried to obtain 219 mg of synthetic chondroitin polysulfate sodium salt (Sample No. Synthetic CS-1).

Molecular weight 24000 Galactosamine content 24.6% Glucuronic acid content 26.5% Sulfur content 12.0% Infrared absorption spectrum 850cm ^-1 (Axial-SO ₄ ) 820cm ^-1 (Equatorial-SO ₄ ) Also, each of the above-mentioned tri-n-butylamine 300 mg salt
(0.43 mmol) was dissolved in 50 ml of DMF, 137 mg (0.86 mmol) of sulfur trioxide-pyridine complex was added, and each was heated at 0 ° C.
Then, after reacting for 1 hour, 3 hours or 5 hours, the same treatment as described above was carried out, and the following synthetic chondroitin polysulfate sodium salt (Sample No. Synthetic CS-1-a, synthetic CS-1-b and synthetic CS-1-b CS-1-c) was obtained.

Example 2 1 g (1.988 mmol) of CS4SNa (molecular weight 35000) was dissolved in 20 ml of formamide, and they were respectively stirred under stirring.
Sulfur trioxide-pyridine complex 0.865 g (5.964 mmol),
Add 1.44 g (9.94 mmol) or 2.88 g (19.88 mmol) and add 6
Reacted at ℃ for 20 hours. The reaction solution was treated according to Example 1 to obtain synthetic chondroitin polysulfate sodium salt, respectively.
0.92g (Sample No. Synthetic CS-2), 1.08g (Sample No. Synthetic CS-)
3) and 1.15 g (sample No. synthetic CS-4) were obtained.

Example 3 1 g (1.988 mmol) of sodium dermatan sulfate (hereinafter referred to as “DSNa”) (molecular weight 20000) was dissolved in 50 ml of formamide, and under stirring, 1.52 g (9.94 mmo) of sulfur trioxide-DMF complex.
l) was added and reacted at 6 ° C. for 20 hours. The reaction solution was used in Example 1.
The synthetic dermatan polysulfate sodium salt (Sample No. Synthetic DS-1) 0.98 g was obtained.

Test Example 1 Identification of Sulfation Rate by Electrophoresis 0.2μg / 0.5μ on cellulose acetate membrane (Separax)
Samples were placed on each and electrophoresed at 0.5 mA / cm for 60 minutes. Buffer: 0.1
5M Formic acid-pyridine (pH 3.0) Staining: Toluidine blue The results are shown in Fig. 1. In FIG. 1, CS4S represents chondroitin-4-sulfate (sulfur content 6.2%), Hep represents heparin (sulfur content 12.8%), and DS represents dermatan sodium sulfate (sulfur content 6.3%).

Test Example 2 Positioning of Sulfate Group of Synthetic Chondroitin Sulfate by Enzymatic Method Synthetic CS1-4 were weighed 1.5 mg each and dissolved in 28 μl of Tris-HCl buffer (pH7.2), and chondroitinase ABC (Proteus vulgaris) was dissolved in each. Origin, Seikagaku Co., Ltd.) 20 units dissolved in 400 μm of the buffer solution were added 80 μm each and reacted at 37 ° C. for 3 hours. This reaction liquid
50μ were separated by paper chromatography (synthetic CS-1,2,3,4-ABC respectively).

In addition, 10 units of chondroitinase ABC digestive fluid contained 1.2 units of chondro-4-sulfatase (derived from proteus vulgaris, manufactured by Seikagaku Corporation) and 1.2 units of chondro-6-sulfatase (derived from proteus vulgaris, manufactured by Seikagaku Corporation)
20 μl of the mixture prepared by dissolving
μ was added to each and the mixture was reacted at 37 ° C. for 3 hours. Add this reaction mixture to 30
Each μ was separated by paper chromatography.

As the paper, Toyo Paper No. 51A.60 × 60 cm was used. The developing solvent is
It was developed for 30 hours at room temperature using n-butanol / acetic acid / 1N aqueous ammonia (2: 3: 1). After unfolding, dry the paper,
Irradiate UV light around 260mμ in a dark room (Toshiba fluorescent inspection lamp FI-3
S-type) and directly observed the UV-absorption spot.

The results are shown in Figure 2. In FIG. 2, St is a standard disaccharide, Δ
Di-0S is ΔDi-4S is ΔDi-6S is A to D are synthetic CS-1,2,3,4 chondroitinase ABC treated solutions, and E to H are synthetic CS-1,2,3,4, respectively.
Represents the one obtained by further treating the chondroitinase ABC treated solution of No. 3 with a mixture of chondro-4-sulfatase and chondro-6-sulfatase.

Test Example 3 Positioning of Sulfate Group of Synthetic DS-1 by Enzymatic Method 1.5 mg of synthetic DS-1 was dissolved in 20 μl of Tris-HCl buffer (pH 7.2), and 20 units of chondroitinase ABC was added to the buffer 400.
80 μ of the one dissolved in μ was added and reacted at 37 ° C. for 3 hours. 50 μl of this reaction solution was separated by paper chromatography (synthetic DS-1-ABC). In addition, the remaining reaction solution 10
A mixture of 1.2 units of chondro-4-sulfatase and 1.2 units of chondro-6-sulfatase was added to 100 μl of the buffer solution.
20 μl of the product dissolved in was added and reacted at 37 ° C. for 3 hours. 30 μl of this reaction solution was separated by paper chromatography (synthetic DS-1-4,6 · Sase). The paper, developing solvent, developing time, and detection method were in accordance with Test Example 2.

The results are shown in Fig. 3. In FIG. 3, A is a synthetic DS-1 chondroitinase ABC treatment solution, and B is a treatment solution of the mixture with chondro-4-sulfatase and chondro-6-sulfatase. St, ΔDi- 0S,
ΔDi-4S and ΔDi-6S have the same meanings as above.

Test Example 4 Positioning of Sulfate Group of Synthetic Chondroitin Polysulfate by Enzymatic Method Synthetic CS-1-a, b, c were treated according to Test Examples 2 and 3,
After unfolding, cut each spot with scissors and add 0.01N hydrochloric acid 1
Finely chop into a test tube containing ~ 2 ml, heat in a warm bath at 50 ° C for 10 minutes, and then centrifuge to remove the supernatant, that is, the extract.
The absorbance at 2 mμ was measured to determine the molar ratio of disaccharide units. The results are shown in the table below.

Test Example 5 Effect of Synthetic Chondroitin Polysulfate on "Kalichromate" Nephritis Kurokawa's Method (Iwao Kurokawa; Journal of the Japanese Society of Internal Medicine, 10 , 363, 507)
(1921)), potassium chromate is dissolved in physiological saline to a concentration of 500 mg / dl, and potassium chromate is 20 mg / kg.
Or 15 mg / kg was subcutaneously injected into a rabbit having an average body weight of 2.5 kg. The third day of the first day of potassium chromate administration
Samples were injected via the ear vein once daily for 14 days from day 16 to day 16. The number of rabbits that survived until the 17th day was counted and the life-prolonging effect was examined. The results are shown in the table below.

A clear life-prolonging effect was observed in the sample-administered group, and the effect was that CS-3SNa was superior to synthetic CS-3. In addition, in the non-administered group, there were many cases of death as a result of progressive weight loss, but in the sample-administered group, it recovered after a temporary mild weight loss. Serum protein levels increased remarkably after a temporary decrease in the non-administered group, but not significantly in the sample-administered group.

[Brief description of drawings]

FIG. 1 shows the results of electrophoresis. 2 and 3 are diagrams showing the results of paper chromatography.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-47-20117 (JP, A) JP-B 31-2170 (JP, B1) JP-B 30-996 (JP, B1) JP-B 52- 35710 (JP, B2) JP-B 50-17049 (JP, B2) JP-B 47-30167 (JP, B1) US Patent 3454560 (US, A) Biochemistry Dictionary First Edition, Tokyo Kagaku Dojin P. 497 ( (1984)

Claims (4)

[Claims] 1. A salt of chondroitin-4-sulfate or dermatan sulfate is previously dissolved in a polar organic solvent, and the solution is a homogeneous solution in the polar solvent at -20 ° C. to 20 ° C. and sulfur trioxide and a tertiary compound. React with complex with amine or dimethylformamide, react with chondroitin-4-sulfate or N- of dermatan sulfate, chondroitin-4-
The following formula is characterized in that the hydroxyl group at the 6-position of the N-acetylgalactosamine residue of sulfuric acid or dermatan sulfate is specifically highly sulfated: (In the formula, R ₁ O, R ₂ O, R ₃ O and R ₄ O represent a sulfuric acid group or a hydroxyl group and may be the same or different, but in the molecule, sulfuric acid of R ₁ O The sulfation rate of R ₂ O is 20 to 100%, the sulfation rate of R ₂ O is 20 to 100%, the sulfation rate of each of R ₃ O and R ₄ O is 25% or less, and X is a carboxyl group. Or X in the molecule may be the same or different, and Y represents a hydrogen atom when X represents a carboxyl group, and represents a carboxyl group when X represents a hydrogen atom. A method for producing a synthetic chondroitin polysulfate or a salt thereof, which comprises a repeating disaccharide unit represented by the formula (1), has a sulfur content in the molecule of 7.3 to 12.5% and a molecular weight of 5,000 to 100,000. 2. The production method according to claim 1, wherein the reaction temperature is 0 ° C. to 20 ° C. 3. The process according to claim 1, wherein the salt of chondroitin-4-sulfate or dermatan sulfate is an alkali metal salt such as sodium salt or potassium salt, and the polar organic solvent is formamide or dimethyl sulfoxide. 4. A salt of chondroitin-4-sulfate or dermatan sulfate is a trialkylamine salt such as tri-n-hexylamine salt, tri-n-butylamine salt or tri-n-octylamine salt, and a polar organic solvent. Is formamide,
Dimethyl sulfoxide, dimethylformamide,
The manufacturing method according to claim 1.