JPH05230090A - Method for selectively desulfating and oxidizing sulfated saccharide - Google Patents

Abstract

PURPOSE:To obtain a new sulfated saccharide useful as a raw material for an improver for lipid metabolism by reacting an organic base salt of sulfated saccharide with N,O-bis(trimethylsilyl)acetamide and selectively desulfating a sulfate group bonded to primary hydroxyl group. CONSTITUTION:An aqueous solution of a sulfated saccharide (e.g. methyl-alpha- galactopyranoside-6 ammonium sulfate) is treated with an anion exchange resin and substituted into a free sulfuric ester type and the anion exchange resin is removed. The solution is mixed with an organic base such as pyridine to prepare an organic base salt of sulfated saccharide (e.g. methyl-alpha- galactosylpyranoside-6 pyridinium sulfate salt). An N,O-bis(trimethylsilyl) acetamide is added in the presence of the organic base salt, reacted at 80 deg.C for about one hour and sulfate group bonded to primary hydroxyl group of the sulfated saccharide is selectively desulfated to give a sulfated saccharide containing a sulfuric ester at a specific position which has not been known.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for selectively desulfating a sulfated sugar, and more particularly to a primary hydroxyl group (having 5 or 6 monosaccharides as a constituent sugar unit) among sulfated esters of a sulfated sugar. In the case of consisting of 5 and 6 respectively represent a hydroxyl group) and a method for selectively desulfating a sulfate group bonded to each.

[0002]

2. Description of the Related Art Various methods for desulfating sulfated sugars have been studied in order to provide sulfated sugars having biological activity.
As a method for desulfating sulfated sugar, a method of desulfating with an acid catalyst in hydrogen chloride methanol is known (Kantor
TG and Schubert M., J. Amer. Chem. Soc. 79 , 152
(1957)). However, this method cannot desulfurize only a specific sulfate group, and also the sugar chain is cleaved by the methanolysis of glycosidic bond, resulting in a low molecular weight and a low yield of the reaction product having the original chain length. To do.

As a method for desulfurization with a high yield, an aprotic solvent such as dimethyl sulfoxide, N, N-dimethylformamide or pyridine (Usov AI, Adamyant
s KS, Miroshnikova LI, Shaposhnikova AA and
Kochetkov NK, Carbohydr. Res. 18 , 336 (1971)) or in dimethyl sulfoxide containing a small amount of water or methanol (Nagasawa K., Inoue Y. and Kamata T., Carbohyd.
r. Res., 58 , 47 (1977), Nagasawa K., Inoue Y. and
Tokuyasu T, J. Biochem., 86 , 1323 (1979)). It is known that the reaction mechanism of solvolysis is the reverse reaction of the sulfation reaction performed using a complex of sulfur trioxide and an amine in an aprotic solvent.
This reaction is controlled by controlling the reaction conditions.
It can be used as a method for selectively desulfating a sulfate group. However, in the case of O-sulfate group, the sulfate group bonded to the primary or secondary hydroxyl group could not be selectively eliminated. Further, to date, no desulfation enzyme having position specificity is known.

[0004]

The development of a specific elimination method for a sulfate group bonded to a primary hydroxyl group provides a sulfated sugar for the purpose of creating a drug having a biological activity preferable to humans. Is very important to do. For example, sulfated polysaccharides such as dextran sulfate, xylan sulfate, chondroitin sulfate, and heparins are used as lipid metabolism improving agents and antithrombotic agents, but those artificially introduced sulfate groups have introduced sulfate groups. It is also known that the position cannot be specified, and that the introduction of a large amount of sulfate groups causes a side effect of increasing the tendency of bleeding from the tissue. In addition, naturally occurring sulfated sugars have different positions and amounts of sulfate groups depending on their origins, and the physiological activities of the sulfated sugars are also slightly different. The present inventor has desulfated a sulfate ester at a specific position of a sulfated sugar to remove a regioselective sulfate ester for reducing hemorrhagic action and for expressing a different biological activity with respect to the original sulfated sugar. The present invention has been achieved through intensive studies aimed at sulfuric acid.

[0005]

The method of the present invention is a method of sulfating an organic base salt of a sulfated sugar by reacting it with N, O-bis (trimethylsilyl) acetamide in the presence of the organic base. A method for producing a desulfated saccharide, which comprises selectively desulfating a sulfate group bonded to a primary hydroxyl group of a sugar.

According to the method of the present invention, it is bonded to the primary hydroxyl group of the sulfated sugar (in the case where the constitutional sugar unit is composed of 5 monosaccharides or 6 monosaccharides, the hydroxyl group is at the 5th or 6th position, respectively). It is possible to selectively perform desulfation or partial desulfation of all the sulfate groups.

The sulfated sugar to which the method of the present invention is applied is a sulfate ester of the sugar regardless of whether it is a monosaccharide, an oligosaccharide, a polysaccharide or a complex polysaccharide. Examples of organic base salts of sulfated sugars used in this method include aromatic amines such as pyridine; aliphatic tertiary amines such as trimethylamine and triethylamine; N
Examples thereof include salts with organic bases such as N-alkylheterocyclic amines such as -methylpyrimidine, N-ethylpyrimidine, N-methylmorpholine and N-ethylmorpholine.

The sulfated sugar is dissolved using an organic base as a solvent,
Form a salt with the organic base. Then, in the presence of an anhydrous organic base, N, O-bis (trimethylsilyl) acetamide (hereinafter abbreviated as BTSA) which is a trimethylsilylating reagent, N, O-bis (trimethylsilyl) trifluoroacetamide (BTSFA), N, O- The selective desulfation of the present invention can be achieved by adding bis (trimethylsilyl) difluoroacetamide and N, O-bis (trimethylsilyl) monofluoroacetamide and reacting at a reaction temperature of room temperature to 100 ° C.

When desulfating all of the sulfate groups bonded to the primary hydroxyl group, it is 16 with respect to the total amount of hydroxyl groups including free hydroxyl groups and sulfate esterified hydroxyl groups in the sulfated sugar.
In the case of partially desulfating the sulfate group bonded to the primary hydroxyl group in an amount equal to or more than twice the molar amount and leaving a part of the amount of the sulfuric acid group remaining, a 3 to 16-fold molar amount of trimethylelsilylation A reagent may be used.

Specifically, for example, in the case of desulfating all the sulfate groups bonded to the primary hydroxyl group, it is preferably 40 ° C. to 80 ° C. in the presence of an amount of trimethylsilylating agent satisfying the above conditions. By reacting within 3 hours,
All of the objectives can be desulfated. Further, in the case of partially desulfating the sulfate group bonded to the primary hydroxyl group, the reaction is carried out in the presence of a predetermined amount of trimethylsilylating agent, preferably at 30 ° C to 80 ° C for 2 hours or more, by combining appropriate conditions. By doing so, the purpose can be achieved.

Subsequently, water is added to the reaction solution to stop the reaction by making the excess trimethylsilylating agent hydro-non-reactive, and then the solvent and the non-reactive trimethylsilylating agent are removed by dialysis as much as possible. .. Next, the dialysis inner solution is boiled at 100 ° C. for 30 minutes to 1 hour to hydrolyze and remove the O-trimethylsilyl group of the O-trimethylsilylated sugar derivative remaining in the dialysis inner solution.

When the sulfated sugar used contains a sulfate ester with a secondary hydroxyl group in addition to a sulfate ester with a primary hydroxyl group, or when all sulfate groups bonded to the primary hydroxyl group are removed. When reaction conditions that do not desulfate are selected, in order to convert these remaining sulfate groups into Na salts, sodium hydroxide is added to adjust pH to around 9, dialyzate, and then lyophilize to form sodium sulfated sugar. A salt can be obtained.

[0013]

EXAMPLES The present invention will be described in more detail with reference to Examples and Test Examples. Identification of sulfated sugars was carried out based on the following Test Examples. The examples do not limit the scope of the invention in any way.

Test Example 1) Quantification of Sulfate Ion The quantification of sulfate groups in sulfated and desulfated sugars was carried out according to the method described in Shinsei Chemistry Experimental Course 3, Carbohydrate II, p37, p81 (published by Tokyo Kagaku Dojin). The sulfate group was converted to a free sulfate ion by acid hydrolysis, and the ion was adsorbed on an ion exchange resin, and then eluted with a solvent, and the free sulfate ion was detected and quantified using an electric conductivity meter.

That is, 3 N- was added to 300 μg of sulfated sugar.
Hydrochloric acid (1 ml) was added and the mixture was hydrolyzed at 100 ° C. for 18 hours. The solution was dried and then dissolved in 1 ml of water. After filtering with a Millipore ultrafiltration membrane (0.45 μm), the sulfate ion content was analyzed by ion exchange chromatography by HPLC (Waters ILC-1). IC Pak Anion (W
aters, 4.6 mm x 5 cm) and 0.8 ml / min using a mobile phase of composition 1.3 mM sodium tetraborate, 1.5 mM sodium gluconate, 6 mM boric acid, 0.25% glycerin and 12% acetonitrile. Was developed at a flow rate of. An electric conductivity meter (Waters 430) was used to detect sulfate ions.

2) ¹³ C-NMR Nuclear magnetic resonance spectrum of skeletal carbon was measured for the purpose of determining the properties of the hydroxyl group of the sulfated sugar. The spectrum of 10% sugar in heavy aqueous solution was 80 ° C, 75.4 MHz (General Electoric
Company, QE-300).

3) Structural Analysis by Enzyme Digestion Shinsei Chemistry Laboratory Lecture 3, Carbohydrate II p.
It carried out as follows with reference to "Structural analysis combining glycosaminoglycan degrading enzyme and HPLC".

A) Digestion with chondroitinase ABC An aqueous solution of dermatan sulfate and chondroitin sulfate (10 mg / m 2) according to the method of Yoshida et al. (Biochemistry, 57 , 1189 (1985)).
l) 20 μl, 0.4 M Tris-hydrochloric acid (pH 8.0) 20 μl
, 0.4 M sodium acetate (20 μl), 0.1% bovine serum albumin (20 μl) and water (120 μl), chondroitinase ABC (5 U / ml) (20 μl) were added, and the mixture was reacted at 37 ° C. for 2 hours.

B) Digestion with heparin-degrading enzyme M, W, Mclean method (MWMclean, “Proc. 9th Int. Sy
mp. Glycoconjugates ”, p.B41 (1987)), dissolve 0.1 mg of heparin in 220 μl of 20 mM sodium acetate (pH 7.0) containing 2 mM calcium acetate to give 20 mU
Of heparinase, and 20 mU of heparinase I and II were added and reacted at 37 ° C for 2 hours.

C) Analysis by HPLC 50 μl of the solution after digestion with chondroitinase ABC or heparin-degrading enzyme was analyzed by HPLC (medical science, model).
852) was used for analysis. Ion exchange column (Shimadzu P
The absorbance at 232 nm was measured using an NH ₂ column, 4.0 mm × 250 mm). At a flow rate of 1 ml / min, 16 mM sodium hydrogen phosphate was eluted by increasing it from 0% to 50% in 60 minutes.

4) Gel filtration 10 μl of a 3% solution of sulfated sugar was analyzed by gel filtration by HPLC. Column is TSKgel-G4000PW _X1 (Tosoh,
7.8 mm x 30 cm) and 0.2 M potassium sulfate was used as the eluent, and the flow rate was 1.0 ml / min. A differential refractometer (Shimadzu, AID-2A) was used to detect the sulfated sugar.

Example 1 Amberlite IR-12 was added to an aqueous solution of about 2 mg / 3 ml of methyl-α-galactopyranoside-6 sulfate ammonium salt.
5 ml of OH type resin was added to convert to free sulfate ester type. After removing the resin, 2 ml of pyridine was added to form a pyridinium salt. Residual pyridine was distilled off under reduced pressure, and the residue was freeze-dried to obtain dry methyl-α.
-Galactopyranoside-6 pyridinium sulfate salt was prepared. This sample was divided into 0.2 mg aliquots, and about 0.05 ml of dry pyridine was added, and the molar amount of all hydroxyl groups of methyl-α-galactopyranoside-6-pyridinium sulfate ([-OH]
^{_{+ [- O-SO 3 -}} ]) molar amount of BTSA for were prepared different reaction system of up to 0-20 times. After adding BTSA to each system, the mixture was heated and reacted at 80 ° C. for about 1 hour. The desulfation amount was calculated by measuring the trimethylsilylated methylgalactopyranoside in the reaction mixture of each system by direct gas chromatography, and the results are shown in FIG. 1.

Example 2 The desulfurization rate was examined with time by setting the molar amount of BTSA of Example 1 to 16 times and 8 times and changing the reaction temperature to 40 ° C., 60 ° C. and 80 ° C., respectively. The results are shown in Fig. 2.

As is clear from Examples 1 and 2, BT
By reacting SA at a reaction amount of 40 ° C. to 80 ° C. within 3 hours at a reaction amount of 16 times or more with respect to the molar amount of all hydroxyl groups including free hydroxyl groups and sulfated hydroxyl groups of sulfated sugar,
It is possible to desulfate all the sulfate groups bonded to the primary hydroxyl group. When the sulfate group bonded to the primary hydroxyl group is partially desulfated, BTSA is used in a molar amount of 3 to 16 times, the reaction temperature is 30 ° C. to 80 ° C., and the reaction time is appropriately selected. By doing so, the purpose can be achieved.

Examples 3 to 9 α-D-galactopyranoside-6-sulfate (Example 3), α
-D-galactopyranoside-2 sulfate (Example 4), funoran (Example 5), porphyran (Example 6), chondroitin sulfate (Example 7), dermatan sulfate (Example 8), heparin (Example) Desulfation of the seven types of sulfated sugars of 9) was performed with BSTA as follows.

200 mg of each sodium salt of sulfated sugar was applied to an IR-120 (H ⁺ type) column (1 x 10 cm), and excess pyridine was added to the eluate to neutralize the pH to 8, and freeze-dried. A pyridine salt of sulfated sugar was prepared. The pyridine salt of each sulfated sugar was dissolved in 20 ml of dehydrated pyridine to prepare a reaction sample. A 20-fold molar amount (about 4 ml) of BTSA was added to the calculated equivalent of all hydroxyl groups in the sugar skeleton of each sample, and the mixture was stirred at 60 ° C. for 2 hours. 20 ml after the reaction
The reaction was stopped by adding water to make unreacted BTSA unhydrolyzable and dialyzed using a Millipore ultrafiltration membrane.

Next, in order to hydrolyze and remove the O-trimethylsilyl group, the dialyzed solution was heated and boiled at 100 ° C. for 30 minutes to 1 hour (until the solution became transparent). After adjusting the pH to 9 to 9.5 by adding sodium hydroxide, it was dialyzed.
The dialyzed solution was freeze-dried to obtain 140 mg of each desulfated product of each sulfated sugar. Table 1 shows the quantitative values of sulfate ions before and after the treatment of each sugar.

[0027]

[Table 1]

Example 10, Comparative Examples 1 to 3 According to Examples 3 and 4, B was used as a trimethylsilylating agent.
TSFA, trimethylsilyl imidazole (TSI),
Table 2 shows the results of the reaction using trimethylsilane iodide (ITS) and N, O-bis (trimethylsilyl) carbamate (BSC).

[0029]

[Table 2]

As can be seen from Examples 3 and 4, the method of the present invention is a method for selectively desulfating the sulfate group bonded to the primary hydroxyl group, and the desulfation of the secondary hydroxyl group does not occur. I know there isn't one.

The funoran of Example 5 is -3 D Gal (6-SO ₄ ).
It has a structure in which the disaccharide β1-4 (3,6-anhydro) L-Gal α1- is repeated, and the sulfate group was almost eliminated by the BTSA treatment. Porphyran of Example 6 was -3 D Gal β1-4 LGal (6
-SO ₄ ) α1-disaccharide is a repeating structure
Almost all sulfate groups were eliminated by the treatment. Example 5
The NMR analysis in and 6 confirmed a high magnetic field shift of the ¹³ C signal at the C-6 position and a low magnetic field shift at the C-5 position, and there was no change in other sites.
Report by B. (The Polysaccharides Vol.1, p133-193 (
Academic Press)).

The chondroitin sulfate of Example 8 was derived from a shark, and the composition disaccharide analysis using chondroitinase revealed that the amount ratio of 6-sulfate: 4-sulfate was 90:10. The chondroitin sulfate and heparin of Examples 8 and 9 contain not only 6-sulfate but also 4-sulfate, disulfate, etc.
The sulfate ion content after TSA treatment is 6.6% and 2 respectively.
A value of 4.9% was shown. Almost all of the dermatan sulfate of Example 7 was 4-sulfate, and therefore the change in the sulfate ion content before and after the BTSA treatment was small.

Dermatan sulfate, chondroitin sulfate and heparin were subjected to enzymatic digestion into disaccharide units and analyzed by HPLC in order to investigate further detailed structural changes.
New experiments were carried out using the corresponding sulfated sugars of Examples 7, 8 and 9 and sugars after desulfation treatment, chondroitin sulfate ABC for chondroitin sulfate and dermatan sulfate, and heparin degrading enzyme for heparin. Biochemistry Course 3,
It processed according to the method of sugar II p54-59 (Tokyo Kagaku Dojin). The HPLC fraction chromatogram of the resulting unsaturated disaccharide isomer is shown in FIG.

Most of the dermatan sulfuric acid used in Example 7 was 4-sulfuric acid and a small amount of 6-sulfuric acid was mixed, but desulfurization occurred only in 6-sulfuric acid by the BTSA treatment.
No change was observed except for the disaccharide residue having no sulfate group bonded. The chondoitin sulfate used in Example 8 was a mixture of 6-sulfate, 4-sulfate, disulfate and the like, but when BTSA treatment was performed, only the change caused by the elimination of 6-sulfate was observed. Next, regarding the heparin used in Example 9, when the digested and released disaccharide residues were compared, only 6-sulfate was eliminated and the sulfate groups at other positions were not changed at all.

Further, dermatan sulfate, chondroitin sulfate and heparin were examined for changes in molecular weight before and after BTSA treatment by gel filtration. As shown in FIG. 4, almost no change in the molecular weight distribution was observed.

From the above, it is clear that the desulfation reaction by BTSA acts specifically on the sulfate group bonded to the primary hydroxyl group and does not affect the sulfate group and glycoside bond at other positions. Is.

As can be seen from Examples 1 and 10 and Comparative Examples 1, 2, and 3, silylating agents applicable in the method of the present invention include organic carboxylic acids such as N, O-bis (trimethylsilyl) acetamides. It can be specified as an N, O-bis (trimethylsilyl) compound of acid amide.

[0038]

INDUSTRIAL APPLICABILITY According to the present invention, a sulfate group having different properties bonded to a hydroxyl group of a sugar skeleton is converted to a primary hydroxyl group and a secondary hydroxyl group.
By recognizing the difference in the primary hydroxyl groups and desulfating, a sulfated sugar having a hitherto unknown position-specific sulfate ester can be produced.

[Brief description of drawings]

FIG. 1 is a graph showing the desulfation rate of methyl-α-galactopyranoside-6 sulfate as the amount of BTSA changes.

FIG. 2 is a graph showing a time-dependent desulfation rate of methyl-α-galactopyranoside-6 sulfuric acid at each temperature.
The solid line shows the results with 16 times molar amount of BTSA, and the broken line shows the results with 8 times molar amount of BTSA.

FIG. 3 is a fractional chromatogram showing changes in sugar composition due to BTSA treatment.

FIG. 4 is a chromatogram of HPLC (gel filtration) showing changes in molecular weight distribution due to BTSA treatment. The solid line shows before BTSA processing, and the broken line shows after BTSA processing.

Claims (1)

[Claims] 1. An organic base salt of a sulfated sugar is reacted with N, O-bis (trimethylsilyl) acetamide in the presence of the organic base to bind to the primary hydroxyl group of the sulfated sugar. A method for producing a desulfated sugar, which comprises selectively desulfating sulfate groups present in the product.