JPS6259711B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to alkyl glycosides of acidic mucopolysaccharide (hereinafter abbreviated as AMPS) constituent unit disaccharides and oligosaccharides composed of them, and methods for producing them, and in particular, the present invention relates to a method for producing them, in particular a method for producing them in an animal body. Hyaluronic acid, chondroitin sulfate, dermatan sulfate, etc. present in connective tissues (skin, tendons, blood vessel walls, cartilage, etc.)
This invention relates to a method for producing constitutional unit disaccharides and alkyl glycosides of oligosaccharides composed of them, using AMPS as a raw material and depolymerizing it by alcoholysis using the solvent effect of dimethyl sulfoxide (hereinafter abbreviated as DMSO). The alkyl glycoside of the present invention has the general formula (): (In the formula, R is a lower alkyl group (preferably an alkyl group having 1 to 5 carbon atoms), Ac is an acetyl group, and n is an integer of 0 to 9). Specifically, the alkyl glycosides shown in the right column of Table 1 are included, and these are the constituent unit disaccharides derived from hyaluronic acid, chondroitin sulfate, and dermatan sulfate, and oligosaccharides composed of these, respectively, shown in the left column of the same table. It is an alkyl glycoside of sugar. In addition,
Until a few years ago, dermatan sulfate was thought to contain only L-iduronic acid as a uronic acid component, but
Currently, L-iduronic acid and D are used as uronic acid components.
-glucuronic acid. Therefore, there are two types of disaccharide constituent units derived from dermatan sulfate, and a variety of oligosaccharides exist with different content ratios and arrangement orders of both uronic acids.

【table】

[Table] Although the constituent disaccharides and oligosaccharides of AMPS according to the present invention are extremely important as raw materials for producing 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 by enhancing the activity of antithrombin in the blood coagulation system, and effective lipid serum clarifying activity against hyperlipidemia, which is the main 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 constituent disaccharides of AMPS and the oligosaccharides composed of them are useful substances that are expected to be used in the pharmaceutical field.
Attempts have been made and used to depolymerize AMPS by methanolysis with hydrochloric acid-methanol or by acidic hydrolysis. However, all of these methods involve removal of the N-acetyl group, and furthermore, in the case of hydrochloric acid-methanol, they involve methyl esterification of uronic acid. Above all, the biggest drawback of these methods is the strong acidity (approximately PH1
Since the reaction was carried out in a system (before and after), the decomposition of the uronic acid moiety was significant. In conclusion, the alkyl glycoside of the present invention represented by formula () has not been obtained by conventional attempts, and is a novel substance that can be produced for the first time by the production method disclosed herein. As mentioned above, the object of the present invention is to provide alkyl glycosides of constituent disaccharides and oligosaccharides, which are new substances that could not be produced in practice in the past, and furthermore, to provide a method for producing them. It is about providing. Chondroitin-4- or 6-sulfate or dermatan sulfate (hereinafter referred to as "sulfated")
The method for producing these structural unit disaccharides and oligosaccharides using sulfated AMPS as a raw material is characterized by reacting the weak base salts of sulfated AMPS in DMSO containing a lower alcohol.
Here, the weak base is preferably, for example, pyridine, quinoline, collidine, picoline, etc., and the lower alcohol is, for example, a monohydric alcohol having 1 to 5 carbon atoms (methanol, ethanol, propanol, butanol, pentanol).
The content of these lower alcohols is generally 0.5 to 20
% by weight is preferred. The method for producing hyaluronic acid using hyaluronic acid as a raw material is characterized by reacting free hyaluronic acid or a weak base salt of hyaluronic acid in DMSO containing a lower alcohol in the presence of a weak base sulfate. Here, examples of the weak base and lower alcohol are the same as in the case where sulfated AMPS is used as a raw material, and the content of the lower alcohol is also the same. In any of the above methods, AMPS depolymerizes without any decomposition side reactions and produces alkyl glycosides of their constituent disaccharides represented by formula () and oligosaccharides composed of them. generate. When sulfated AMPS is used as a raw material, the reaction is generally carried out by heating, but the formula () using chondroitin-6-sulfate pyridinium salt as an example: [ Kinzo Nagasawa, Yuko Inoue, Toshio Kamata, Carbohydr.Res., 58 (1977) 47-55]. At this time, the pH of the reaction solution is approximately 3.5. In addition, when using hyaluronic acid as a raw material, the pH of the reaction solution is 3.5 ~
It is 3.6. When such a reaction solution is heated at various temperatures, alcoholysis occurs at the β-N-acetylhexosaminide bonding portion depending on the reaction time, with DMSO acting as a catalyst, and depolymerization of the AMPS sugar chain progresses. Under these reaction conditions, the N-acetyl group is stable and the uronic acid carboxyl group remains free without being alkyl esterified. Furthermore, the greatest advantage of the method of the present invention is that there is almost no decomposition of uronic acid, which is a constituent sugar of AMPS. These effects are due to the fact that the pH of the reaction solution is not on the strongly acidic side as in conventional methods, and that DMSO exhibits an excellent solvent effect. Therefore, the uronic acid recovery rates for the constituent unit disaccharides and oligosaccharides produced by the method of the present invention are as follows in Examples 1 and 2.
(in the case of chondroitin-6-sulfate), it is 100%. Although the depolymerization of AMPS according to the method of the present invention proceeds even at room temperature, it is highly temperature dependent. for example,
Chondroitin-6-pyridinium sulfate salt at 90℃
The anion exchange chromatograms of the reaction products (products of Examples 1 and 2) when heated at 95° C. for 18 hours are shown in FIGS. 1 and 2. When the reaction temperature is 90° C., the main product is the unit disaccharide (Table 1-2, n=0) as shown in FIG. 1, and other oligosaccharides with n=1 to 4 are produced. On the other hand, when the reaction temperature was 95°C, unit disaccharide was the main product as shown in Figure 2, and only n=1 oligosaccharide was produced as a by-product. As seen in this example, the degree of depolymerization of AMPS can be largely controlled by selecting the reaction temperature, and the degree of depolymerization can be finely controlled by extending or contracting the reaction time. Therefore, the reaction temperature and time are
The reaction temperature is appropriately selected depending on the type of the reaction mixture and the desired product, but the reaction temperature is suitably between room temperature and 110°C, and practically preferably between 80 and 110°C. Since the production method of the present invention cleaves only the β-N-acetylhexosaminide bond with almost no decomposition of uronic acid,
If sufficient reaction time is allowed in the above temperature range, the main product will be the unit disaccharide alkyl glycoside (Table 1
-1 to 3, n=0), the yield is almost stoichiometric, and the recovery rate of uronic acid is almost 100%. Dermatan sulfate (Tables 1-3) is similar to chondroitin-4- and 6-sulfate but is somewhat resistant to depolymerization (see Example 3). Hyaluronic acid is
It has a much higher molecular weight than these sulfated AMPS, and under similar conditions (methanol concentration, temperature, time),
There is a tendency to produce a mixture of more oligosaccharide alkyl glycosides (Table 1-1, n=0-4) in addition to unit disaccharides (see Example 4). The unit disaccharide and oligosaccharide alkyl glycosides of AMPS obtained by the production method of the present invention are a mixture of α-type and β-type, and as shown in Figures 1 and 2, the main peaks of Di, Tetra, Hexa, etc. are both β
(see Example 2, analytical data: specific rotation [α]D), and the small peaks immediately before the main peak are presumed to be the respective α types. The starting material AMPS is usually supplied as a sodium salt, but any type of salt must first be converted into a pyridinium salt, quinolinium salt, collidinium salt, picolinium salt, etc. (in the case of hyaluronic acid) may be a free acid or a pyridinium salt).
This is because this reaction does not proceed when AMPS forms a strong salt, such as a sodium salt or calcium salt, with a metal cation or a strong organic base. To make AMPS into a pyridinium salt, etc., either convert it into a free acid using a cation exchange resin (free type), and then freeze-dry it by adding pyridine or the like, or use a cation exchange resin (pyridinium type, quinolinium type, etc.). It is convenient to directly obtain a pyridinium salt solution, quinolinium salt solution, etc. by passing through the solution, and freeze-dry this solution. In the case of hyaluronic acid, the weak base sulfates used for addition, such as pyridine sulfate, quinoline sulfate, collidine sulfate, and picoline sulfate, are added by adding 1 mole of sulfuric acid (as dilute sulfuric acid) to 1 mole of pyridine, etc. It can be prepared by vacuum concentration drying or freeze drying. 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% methanol/DMSO (200 ml) and heated at 90°C for 18 hours with stirring. 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 applied to an anion exchange resin column (AG1−X4 (product name,
BIO・RAD), Cl ^- type, 2 x 92
cm), and as shown in Figure 1 by the dotted line (...), increase the concentration of the eluent LiCl solution linearly, first from OM to 0.21M, then from 0.21M to 0.4M. The sample was injected and eluted to obtain the chromatogram shown in FIG. The disaccharide (Di), tetrasaccharide (Tetra), hexasaccharide (Hexa), octasaccharide (Octa), and decasaccharide (Deca) methyl glycoside fractions shown in Figure 1 were collected, freeze-dried, and individually 10% It was dissolved in ethanol and desalted using a Cephadex G-15 (trade name, manufactured by Pharmacia AB) column (2.5 x 90 cm)/10% ethanol. By freeze-drying each fraction, disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, and decasaccharide methyl glycosides (all α-type and β-type mixtures) were obtained as lithium salts at 169, 74, 51, and 28, respectively. , with a yield of 17 mg. Example 2 Chondroitin-6-pyridinium sulfate salt (500 mg) was dissolved in 10% methanol/DMSO (200 ml) and heated at 95°C for 18 hours with stirring. After the reaction, equal amounts of water and 0.5M NaOH solution were added to adjust the pH to 6.8, and the solution (approximately 10 ml) concentrated under reduced pressure was transferred to an anion exchange resin column (same as in Example 1), and the eluent was LiCl solution. The dotted line in Figure 2 indicates the concentration of
), the elution was carried out linearly increasing from OM to 0.2M, and the chromatogram shown in Figure 2 was obtained. Fraction 3 of the disaccharide (Di) fraction (Fig. 2,
Di-Fr.3) and tetrasaccharide (Tetra) fraction 4 (Fig. 2, Tetra-Fr.4) were each lyophilized and dissolved in 10% ethanol. Sephadex G-15 column (2.5 x 90 cm)/10% ethanol was used to desalt the disaccharide methyl glycoside (β
type) lithium salt, 170 mg (43.0%) and tetrasaccharide methyl glycoside (β type) lithium salt, 41 mg (10.4%)
I got it. In order to make the obtained products even analytically pure, they were subjected to silicic acid column chromatography (column size: 3 x 30 cm; eluent: acetone/methanol (4:1) → (3:1)). ) → (2:
1) stepwise elution) and recrystallization of the resulting powder from methanol/acetone ether (1:1) and methanol/acetone, respectively, to obtain the disaccharide methyl glycoside as an analytically pure product. body (β type), 126 mg (31.9%), [α]
^29D = _-120.7 ° (CH ₃ OH) and tetrasaccharide methyl glycoside (β type), 35 mg (8.9%), [α] ²⁹ _D = -47.9° (CH ₃ OH) were obtained. Example 3 Dermatan sulfate (500 mg) in 10% methanol,
Dissolve in DMSO (200 ml) and heat at 95 °C with stirring.
Heated for 18 hours. After the reaction is complete, add equal amounts of water and
The pH was adjusted to 6.8 by adding 0.5M NaOH solution, and the mixture was concentrated under reduced pressure (approximately 10 ml). Sefadex G-25 (product name,
Pharmacia AB)/Analytical gel permeation chromatogram using 0.1M NaCl (sample amount approx. 1.0mg,
Column size: 1.6 x 84 cm) (Figure 3), it is a mixture of tetrasaccharides, hexasaccharides, and octasaccharides, with disaccharides as the main component. The concentrated solution obtained above was treated with an anion exchange resin column in the same manner as in Example 1 to obtain disaccharides, tetrasaccharides, hexasaccharides,
It was separated into octasaccharide fractions. Each fraction was freeze-dried, desalted by Cephadex G-15/10% ethanol treatment, and then freeze-dried again. β
type mixture) as lithium salt 151, 111, 69, 20
Obtained with a yield of mg. Example 4 Hyaluronic acid (free form) (500mg) was added to DMSO
(225 ml), 0.05M pyridine sulfate/methanol solution (25 ml) was added thereto, and the mixture was heated at 95°C for 18 hours with stirring. After the reaction, equal amounts of water and
The pH was adjusted to 6.8 by adding 0.5M NaOH solution, and the mixture was concentrated under reduced pressure (approximately 10 ml). Sefadex G-25/0.1M
According to the analytical gel perchromatogram using NaCl (sample amount approximately 1.0 mg, column size 1.6 x 84 cm), it is a mixture of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides (Figure 4). The concentrate obtained above was treated with an anion exchange resin column in the same manner as in Example 1, and separated into disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, and decasaccharide fractions. Each fraction was lyophilized and each fraction was added to Sephadex G-15/10
After desalting by % ethanol treatment, freeze-drying again, disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, and decasaccharide methyl glycosides (all mixtures of α and β types) were converted into lithium salts83, 105, The yields were 112, 83, and 67 mg. Example 5 Chondroitin-4-pyridinium sulfate salt (500 mg) was dissolved in 10% methanol/DMSO (200 ml) and heated at 95°C for 18 hours with stirring. Thereafter, the reaction solution was treated in the same manner as in Example 2, and then separated into a disaccharide fraction and a tetrasaccharide fraction using an anion exchange resin column. Sephadex G-
Desalt with 15/10% ethanol to obtain 238 mg (66%) and 63 mg of lithium salts of disaccharide and tetrasaccharide methyl glycosides (both α-type and β-type mixture), respectively.
(16%) yield. Example 6 Chondroitin-6-pyridinium sulfate salt (500 mg) was dissolved in 5% methanol/DMSO (200 ml) and heated at 90°C for 18 hours with stirring. Thereafter, the reaction solution was treated in the same manner as in Example 1, fractionated using an anion exchange resin column, and Sephadex G-
After desalting with 15 columns, each methyl glycoside (all α-type,
β-type mixture) lithium salt 152, 93, respectively
The yields were 64, 41, and 32 mg. Example 7 Chondroitin-6-pyridinium sulfate salt (500 mg) was dissolved in 10% ethanol/DMSO (200 ml) and heated at 90°C for 18 hours with stirring. Thereafter, the reaction solution was treated in the same manner as in Example 1, and then each ethyl glycoside fraction from disaccharide to decasaccharide was separated using an anion exchange resin column. After freeze-drying each fraction, it was desalted using a Sephadex G-15 column, and then the ethyl glycosides of disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, and decasaccharide (all mixtures of α-type and β-type) Lithium salts were obtained in yields of 150, 79, 64, 35, and 28 mg, respectively.

[Brief explanation of the drawing]

Figure 1 is an anion exchange chromatogram of the reaction product of chondroitin-6-pyridinium sulfate in 10% methanol/DMSO at 90°C for 18 hours, and Figure 2 is an anion exchange chromatogram of the reaction product of chondroitin-6-pyridinium sulfate at 90°C for 18 hours. This is a chromatogram. Figure 3 shows dermatan sulfate pyridinium salt in 10% methanol/DMSO at 95°C.
Figure 2 is a gel permeation chromatogram of the 18 hour reaction product. Figure 4 is a gel permeation chromatogram of the reaction product of hyaluronic acid in 10% methanol/DMSO at 95°C for 18 hours in the presence of pyridine sulfate.

Claims (1)

[Claims] 1. General formula: (In the formula, R is a lower alkyl group, Ac is an acetyl group, and n is an integer from 0 to 9). Alkyl glycosides of sugars. 2 General formula characterized by reacting hyaluronic acid or a weak base salt of hyaluronic acid in dimethyl sulfoxide containing a lower alcohol in the presence of a weak base sulfate: (In the formula, R is a lower alkyl group, Ac is an acetyl group, and n is an integer from 0 to 9). Body alignment method. 3 General formula characterized by reacting a weak base salt of chondroitin-4-sulfate, chondroitin-6-sulfate or dermatan sulfate in dimethyl sulfoxide containing a lower alcohol: (In the formula, R is a lower alkyl group, Ac is an acetyl group, and n is an integer from 0 to 9). How the body is manufactured.