JPS645043B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides acidic mucopolysaccharides
mucopolysaccharides (hereinafter abbreviated as AMPS)
In particular, the present invention relates to a method for depolymerizing AMPS that selectively cleaves β-D-hexosaminide bonds. Such AMPS include, for example, hyaluronic acid,
chondroitin-4-sulfate, chondroitin-6
- Sulfuric acid, dermatan sulfate, etc. are present. These AMPS are polysaccharides in which uronide bonds and β-D-hexosaminide bonds are alternately repeated, but according to the depolymerization method of the present invention, the β-D-hexosaminide bonds are selectively cleaved and the uronide bonds are Since the bond is difficult to cleave, disaccharide constituent units having hexosamine at the reducing end and oligosaccharides composed thereof are produced. At this time, AMPS containing sulfate groups
In this case, desulfation proceeds preferentially. N-
Elimination of the acetyl group does not occur. Among these AMPS, Table 1 shows the chemical structures and approximate molecular weight ranges of hyaluronic acid, chondroitin sulfate, and dermatan sulfate, as well as oligosaccharides obtained by the method of the present invention. In addition, dermatan sulfate was used as a uronic acid component until a few years ago.
-It was thought to contain only iduronic acid, but now it contains L-iduronic acid and D-iduronic acid as uronic acid components.
It has been revealed that dermatan sulfate contains both glucuronic acid and the ratio of both in dermatan sulfate varies depending on the raw material. Therefore, there are two types of constituent disaccharides derived from dermatan sulfate, and a variety of oligosaccharides exist with different content ratios and arrangement orders of both uronic acids. [Table] Although these disaccharides and oligosaccharides, the constituent units of AMPS, are extremely important as raw materials for the production of a group of substances expected to have various physiological activities,
Since a manufacturing method for supplying these has not been developed, there has been almost no application of oligosaccharides derived from these acidic mucopolysaccharides to the pharmaceutical field. Physiological activities expected by appropriate chemical modification of these oligosaccharides include, for example, bifidus factor-like activity in the case of hyaluronic acid, and polysulfation of the oligosaccharides of these systems in the case of chondroitin sulfate and dermatan sulfate. These include anti-coagulation effects such as enhancement of antithrombin activity in the blood coagulation system or direct inhibition of thrombin activity, and effective lipid serum clarifying activity against hyperlipidemia, which is a major cause of arteriosclerosis. Furthermore, by phosphoric acid esterification of these disaccharides and oligosaccharides, it would be possible to synthesize a group of substances that induce interferon production ability. In addition, if multiple chemical reactions are applied to these oligosaccharides, it will be possible to produce useful substances with even more possibilities. In this way, the disaccharide that constitutes AMPS and the oligosaccharide composed of it are useful substances that are expected to be used in the pharmaceutical field.
Methanolysis of AMPS using hydrochloric acid, methanol, etc., or hydrolysis in an acidic aqueous solution has been carried out on a laboratory scale. However, all of these methods involve removal of the N-acetyl group,
Furthermore, in the case of hydrochloric acid/methanol, there was a problem in that it involved methyl esterification of uronic acid. Above all, the biggest drawback of these methods is the presence of strong acids (approximately PH
1) Since the reaction was carried out in a system, the decomposition of the uronic acid moiety was significant. In the end, the constituent unit disaccharides of AMPS, including the oligosaccharides listed in the right column of Table 1,
In particular, oligosaccharides made of these oligosaccharides, with the exception of some of them, have not been obtained by conventional methods, and could be obtained for the first time by the depolymerization method of the present invention. The purpose of the present invention is to selectively cleave the β-D-hexosaminide bond of such AMPS, and to produce a disaccharide as a constituent unit and an oligosaccharide composed thereof, which are expected to be used in the pharmaceutical field as described above.
The object of the present invention is to provide a method for depolymerizing AMPS. The depolymerization method of the present invention is particularly characterized by the treatment conditions for hydrolysis.
It is characterized by treatment in dimethyl sulfoxide (hereinafter abbreviated as DMSO) containing water under the following acidic conditions. What is important in the present invention is that the solvent contains water.
Using DMSO and controlling the reaction process to a pH of 2 or higher
It is to maintain acidic conditions below 5.5. According to these conditions, desulfation of AMPS and subsequent cleavage of the β-D-hexosaminide bond proceed.
A depolymerization reaction proceeds to produce constituent disaccharides and oligosaccharides composed of them. This depolymerization reaction is
First, the desulfation reaction proceeds rapidly, followed by β-
It is believed that the D-hexosaminide bond is selectively cleaved. If the reaction is carried out under strongly acidic conditions with a pH of less than 2, the N-acetyl group will be removed, the uronide bond will be broken, and the uronic acid moiety will also be decomposed.
It is extremely difficult to obtain the desired disaccharide, which is the constituent unit of AMPS, and oligosaccharides composed of it. In addition, when the reaction is carried out using only water without using aqueous DMSO as a solvent, desulfation does not proceed and there is not much difference in the reactivity of cleavage of β-D-hexosaminide bonds and cleavage of uronide bonds. As a result, a mixture of sulfuric acid-bonded monosaccharides and various oligosaccharides is produced. Therefore, in the present invention, the liquid property is adjusted to pH2 or higher.
Maintaining acidic conditions below 5.5 and using aqueous DMSO as the solvent are essential requirements.
In addition, even under acidic conditions, the higher the PH value, the slower the depolymerization rate tends to be, so for practical purposes, PH3
Acidic conditions of ~5.5 are generally preferred. The reason why β-D-hexosaminide bonds are selectively cleaved by the depolymerization method of the present invention is as follows.
This is thought to be because DMSO acts catalytically on the decomposition reaction of this bond. The amount of water contained in DMSO is 20% by weight, which is more than the theoretical amount required for hydrolysis of β-D-hexosaminide bonds and sulfate bonds.
The following are preferred. Excessive presence of water causes disadvantages in that desulfation does not proceed, the selectivity of the cleavage of sugar chains decreases, and uronide bonds are also more likely to be cleaved. AMPS is usually available in the form of sodium salt, but the AMPS treated by the method of the present invention is
It may be in the form of such a salt or in the form of a free acid. PH condition during reaction process is PH2 or higher 5.5
The condition of the starting materials does not matter, provided they are kept under acidic conditions and uniformly dissolved in the solvent. Appropriate bases and acids can be used to adjust the PH of the reaction solution, but using a weak base can be expected to have a buffering effect in the acidic region, thereby suppressing fluctuations in the PH value during the reaction. There are advantages. Such weak bases include pyridine, quinoline, collidine,
Organic weak bases such as picoline are preferred. Note that dermatan sulfate with a high content of L-iduronic acid (for example, derived from pig skin)
Compared to AMPS, L-iduronic acid (monosaccharide) is more easily produced as a by-product, so the PH value of the reaction solution is slightly increased, and the PH
It is better to adjust it to 4.0 or higher. If depolymerization is carried out under such conditions, the by-product of L-iduronic acid can be suppressed. Although the depolymerization of AMPS by the method of the present invention proceeds even at room temperature, it is highly temperature dependent. For example, chondroitin-6-pyridinium sulfate salt in 10% aqueous DMSO (PH3.8-4.0) at 95°C or
Figure 1 shows the gel permeation chromatogram of the reaction product when heated at 105°C for 18 hours (solid line: 95°C, broken line: 105°C). When the reaction temperature was 95℃, the unit disaccharide and tetrasaccharide (Table 1-
2, n=0 and 1) as main products (22.3 and
21.5%), and oligosaccharides with n≧2 (56.2%).
%) is generated. On the other hand, when the reaction temperature was 105°C, the main product was unit disaccharide (54.8%), and n
≧1 oligosaccharide (42.5%) was produced. As seen in this example, by choosing the reaction temperature
The degree of depolymerization of AMPS can be adjusted. Dermatan sulfate (Tables 1-3) is similar in chemical structure to chondroitin-4- and 6-sulfate, but is more resistant to depolymerization. For example, dermatan sulfate pyridinium salt made from chicken crest is 10%
Figure 3 shows the gel permeation chromatogram of the reaction product when heated at 105°C for 18 or 30 hours in DMSO containing 10% water (PH3.8-4.0) (solid line: 105°C x
18hr, dashed line: 105℃ x 30hr). When the reaction time is 18 hours, the units disaccharide and tetrasaccharide (Table 1-3, n=0,
1) is the main product (20.4 and 23.1%), and oligosaccharides with n≧2 (34.7%) are also produced. On the other hand, when the reaction was carried out for 30 hours, unit disaccharides were the main products (63.4%), and oligosaccharides containing tetrasaccharides and hexasaccharides as main components (30.8%) were produced (Example 3). In this way, the degree of depolymerization can also be adjusted by extending or contracting the reaction time. Therefore, the PH, reaction temperature and time of the reaction process are the starting materials
Although the reaction temperature is selected depending on the type of AMPS and the intended product, a suitable reaction temperature is room temperature to 120°C, and practically 80 to 120°C is preferable. Since the depolymerization method of the present invention cleaves only the β-N-acetylhexosaminide bond with almost no decomposition of uronic acid, the main product can be obtained by allowing a sufficient reaction time in the above temperature range. is a unit disaccharide (Table 1-1~
3, n = 0), the yield is good, and the recovery rate of uronic acid from the total oligosaccharides produced is almost
It is 100%. Hyaluronic acid has these sulfate groups
Although it has a much higher molecular weight than AMPS, under similar conditions (water content, pH, temperature, time), oligosaccharide mixtures with unit disaccharide as the main product (Table 1-1 , n=0 and n≧1) (see FIG. 4 and Example 4). The present invention will be explained in more detail by showing examples below. Example 1 Chondroitin-6-pyridinium sulfate salt (500 mg) was dissolved in 10% aqueous DMSO (200 ml),
Heated at 105° C. for 18 hours with stirring. During this time, the pH of the reaction solution was 3.8 to 4.0. After the reaction was completed, equal amounts of water and 0.5M NaOH solution were added to adjust the pH to 6.8, and the mixture was concentrated under reduced pressure (approximately 10 ml). This was transferred to the upper end of a Cephadex G-25 (trade name, manufactured by Pharmacia AB) column (2 x 92 cm) and eluted with 0.1M NaCl. Figure 1 shows the analytical scale (sample amount 10 mg,
A chromatogram performed using a column size of 1.6 x 88 cm) is shown. (Dashed line ---). Fractions corresponding to disaccharides (Di), tetrasaccharides (Tetra), and hexasaccharides (Hexa) were collected, lyophilized, and then individually dissolved in 10% ethanol. ) Column (2.5 x 90 cm)/Desalted with 10% ethanol. By lyophilizing each fraction, sodium salts of disaccharide, tetrasaccharide, and hexasaccharide were obtained in yields of 193, 97, and 53 mg, respectively. In addition, an analytical scale chromatogram corresponding to the above (Sephadex G-25 Column, elution degree: 0.1M NaCl) is shown in Figure 1 by a solid line. Example 2 Chondroitin-6-pyridinium sulfate salt (500 mg) was dissolved in 10% aqueous DMSO (125 ml),
Heated at 90° C. for 14 hours with stirring. During this time, the pH of the reaction solution was in the range of 3.6 to 3.9. After the reaction is complete,
Add equal amounts of water and 0.5 M NaOH solution to adjust the pH to 6.8, concentrate the solution under reduced pressure (approximately 10 ml), and apply the anion exchange resin AG1 x 4 (trade name, BIORAD, manufactured by BioRad) to a Cl ^- type column (2.5 x 47 cm). The concentration of the eluent LiCl solution was increased linearly from 0M to 0.4M as shown by the broken line (---) in Figure 2-1. The disaccharide (n=0) to octasaccharide (n=3) fractions were separated and concentrated to 10
% ethanol, Sephadex G-15
Column (2.5 x 90 cm): Desalted with 10% ethanol and then lyophilized to give 33.2, 41.4, 42.6, and 36.1 mg of each lithium salt of disaccharide, tetrasaccharide, hexasaccharide, and octasaccharide as pale yellow-white powder, respectively. I got it. The fraction containing more than 10 saccharides (n = 4) in Figure 2-1 was desalted using a Sephadex G-15 column, and then concentrated.
153 mg of an oligosaccharide mixture containing more than 10 saccharides was obtained. Dissolve this in a small amount of water and use the anion exchange resin AG1 again.
Separation was performed using a ×2 (Cl ^- type) column (1.5 × 88 cm) using a LiCl solution as an eluent while linearly changing the concentration from 0.2 M to 0.4 M.
As a result, various oligosaccharide fractions from decasaccharides to decasaccharides were obtained as shown in FIG. 2-2. Concentrate each fraction, dissolve in 10% ethanol and use Sephadex G.
-15 column/10% ethanol and then lyophilized to obtain the respective lithium salts as white powders in yields of 35.3, 33.4, 26.8, 16.0, 8.7, and 7.3 mg. Desulfated structural unit disaccharide (N-acetylchondrosin) of chondroitin-6-sulfate obtained in this example and oligosaccharide (disaccharide to 20 saccharide, n=0 to 2 in Table 1-2) 9) was subjected to the following chemical analysis and physical property measurements. (1) [α] ²⁹ _D Solvent: Water, Concentration: 0.13-0.15 (g/100
ml) (2) RGluA (relative mobility to glucuronic acid by paper electrophoresis) Buffer: Pyridine-acetic acid-1-butanol-water (5:1:5:250 volume ratio), PH5.8, 23V/
cm, time 60 minutes, temperature 4℃ (3) Thin layer chromatography (Rf value) Ascent method development, cellulose plate used Solvent:
n-Butanoic acid-0.5N ammonia water (5:3)
Mixed liquid (4) Content of uronic acid and hexosamine ●Uronic acid: Yoshizawa et al.'s improved carbazole method (M.
Kosakai, Z.Yosizawa, Anal.Biochem.
93 (1979) 295-298) ●Hexosamine: Antonopoulos method (CA
Antonopoulos, Arkiv.Kemi., 25 (1966)
243) (5) Molar ratio of hexosamine to uronic acid The method of A. Linker et al. (A. Linker, K. Meyer,
P.Hoffman, J.Biol.Chem., 181 (1949) 149−
151) to reduce oligosaccharides. By reduction with NaBH ₄ , N-acetylgalactosamine at the reducing end position of N-acetylchondrosin and oligosaccharides consisting of it is converted to N-acetylgalactosamine.
It becomes acetylgalactosaminitol, and as a result, it is no longer necessary to quantify amino sugars by the method (4) above. Therefore, the degree of polymerization of each oligosaccharide can be determined by measuring the uronic acid/hexosamine molar ratio before and after reduction. (6) Molar ratio of N-acetylhexosamine to uronic acid ●Method of Reissig et al. (JLReissig, JL
Strominger, LFLeloir, J.Biol.Chem.
217 (1955) 959-966), the content of N-acetylhexosamine was measured, and the molar ratio was determined from the amount of uronic acid determined by the method (4) above. Table 2 summarizes the measurement results. Free N-
Measured values for a standard product of acetylchondrosin (obtained by N-acetylating a commercially available standard product of chondrosin (manufactured by Seikagaku Corporation) by the method of Danishefsky et al.) are also listed. [Table] [Table] *1, *2 Examples of calculated values are in parentheses 3 Dermatan sulfate pyridinium salt derived from chicken crest (200 mg) was dissolved in 10% aqueous DMSO (100 ml) and heated at 105°C for 30 hours with stirring. Heated. During this time, the pH of the reaction solution was in the range of 3.8 to 4.0.
After the reaction was completed, equal amounts of water and 0.5M NaOH solution were added to adjust the pH to 6.8, and the mixture was concentrated under reduced pressure (approximately 10 ml). According to the analytical gel perchromatogram (sample amount 10 mg, column size 1.6 x 88 cm) using Sephadex G-25/0.1M NaCl (dashed line in Figure 3-1), the reaction product mainly consists of disaccharides. It was a mixture of tetrasaccharides, hexasaccharides, and a small amount of monosaccharides. The concentrated solution obtained above was treated with anion exchange resin AG1×4 (Cl ^-
The product was treated in the same manner as in Example 2 using a column (type), and separated into disaccharide, tetrasaccharide, and hexasaccharide fractions (Figure 3-2). Each fraction was lyophilized and each fraction was
After desalting with 15 columns/10% ethanol,
Lyophilization was performed again to obtain lithium salts of disaccharides, tetrasaccharides, and hexasaccharides in yields of 81, 32.4, and 11.2 mg, respectively. The chromatogram indicated by the solid line in FIG. 3-1 is an analytical gel permeation chromatogram obtained when depolymerization was carried out under the same conditions as in the above example except that the reaction time was 18 hours. Example 4 Hyaluronic acid (free form) (500mg) was added to DMSO
(225 ml), add 0.05 M pyridine sulfate aqueous solution (25 ml), and heat at 105°C for 30 minutes with stirring.
heated for an hour. During this time, the pH of the reaction solution is 3.6-3.9
It was hot. After the reaction, equal volumes of water and 0.5M NaOH
The solution was added to adjust the pH to 6.8, and concentrated under reduced pressure (approximately 10
ml). Analytical gel perchromatogram using Sephadex G-25/0.1M NaCl (sample volume approx.
4.0 mg, column size 1.6 x 84 cm), it is a mixture of disaccharides, tetrasaccharides, and hexasaccharides (Fig. 4, solid line --). The concentrate obtained above is treated with anion exchange resin.
It was treated in the same manner as in Example 3 using an AG1×4 (Cl ⁻ type) column and separated into disaccharide, tetrasaccharide, and hexasaccharide fractions.
Each fraction was lyophilized and each fraction was
After desalting with -15/10% ethanol, the mixture was freeze-dried again to obtain lithium salts of disaccharide, tetrasaccharide, and hexasaccharide in yields of 239, 108, and 36 mg, respectively. The chromatogram indicated by the broken line in Figure 4 shows depolymerization under the same conditions as in the above example except that pyridine hydrochloride was used instead of pyridine sulfate (PH during this period was in the range of 3.5 to 3.8). This is an analytical gel permeation chromatogram obtained in the same manner. Example 5 Dermatan sulfate pyridinium salt derived from pig skin (100 mg) was added to 0.3% pyridine aqueous solution/DMSO
(1:9) dissolved in 50 ml and heated to 105℃ for 30 minutes with stirring.
heated for an hour. During this time, the pH of the reaction solution was in the range of 4.4 to 4.6. After the reaction was completed, it was treated in the same manner as in Example 3 and subjected to analytical gel perchromatography using Cephadex G-25/0.1M NaCl (Figure 5).
It is a mixture of hexasaccharides, small amounts of octasaccharides, and monosaccharides. The entire amount of the product obtained in the above reaction was transferred to an anion exchange resin AG1 x 4 (HCOO ^- type) column (1.6 x 90 cm).
As shown in Figure 6, disaccharides (separated into N-acetyldermosine (a) and N-acetylchondrosine (b)), tetrasaccharides, and hexasaccharides were eluted with 0.3M HCOOH and 1M HCOOH. Each of the sugars and monosaccharides (L-iduronic acid (c) and D-glucuronic acid (d)) was fractionated. Each fraction was lyophilized and free oligosaccharides and monosaccharides were obtained in the following yields. N-acetyldermosine (20.5mg), N-acetylchondrosin (6.7mg), L-iduronic acid (6.1mg), tetrasaccharide (2 peaks) 18.1mg, hexasaccharide (11.2
mg).

[Brief explanation of the drawing]

Figure 1 shows chondroitin-6-pyridinium sulfate salt in 10% aqueous DMSO at 95°C for 18 hours and at 105°C.
Figure 2 is a Sephadex G-25 gel permeation chromatogram of each reaction product at
-1 and 2-2 are anion exchange chromatograms of the reaction products at 90°C for 14 hours. Figure 3
-1 is a gel permeation chromatogram of the reaction product of dermatan sulfate pyridinium salt derived from chicken crest in 10% aqueous DMSO at 105°C for 18 hours and at 105°C for 30 hours. Figure 3-2 is an anion exchange chromatogram of the reaction product obtained at 105°C for 30 hours. Figure 4 shows a pyridine sulfate aqueous solution of hyaluronic acid (free form): DMSO (1:9),
105℃, 30hr and pyridine hydrochloride aqueous solution/DMSO
(1:9), 105° C., and gel permeation chromatogram of each reaction product for 30 hours. Figure 5 shows dermatan sulfate pyridinium salt derived from pig skin in 0.3% pyridine aqueous solution/DMSO (1:9).
This is a gel permeation chromatogram of the reaction product at 105°C for 30 hours. Figure 6 is an anion exchange chromatogram of the reaction product obtained at 105°C for 30 hours.

Claims (1)

[Scope of Claims] 1. A method for depolymerizing acidic mucopolysaccharides, which comprises treating acidic mucopolysaccharides in dimethyl sulfoxide containing water under acidic conditions with a pH of 2 or more and 5.5 or less. 2. The depolymerization method according to claim 1, wherein the acidic mucopolysaccharide is composed of a constituent disaccharide consisting basically of uronic acid and hixosamine. 3 Acidic mucopolysaccharides include hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate,
or dermatan sulfate, the depolymerization method according to claim 2. 4 The water content in dimethyl sulfoxide is β-D
- The depolymerization method according to claim 1, wherein the amount is more than the theoretical amount necessary for hydrolysis of the hexosaminide bond and the sulfuric ester bond and less than 20% by weight. 5. The depolymerization method according to claim 1, wherein the acidic conditions are PH3 to 5.5.