IE833063L - Process for the depolymerisation and sulfation of¹polysaccharides - Google Patents

Abstract

1. A process for the depolymerisation and sulfatation of polysaccharides other than heparin, characterized in that said polysaccharide is reacted with only one mixture of sulfuric acid and chlorosulfonic acid. [EP0116251A1]

Description

6783 The present invention relates,to a process for the depolymerization and sulfatation of polysaccharides.

Sulfated polysaccharides are compounds having a great importance in cosmetic, textile, alimentary and pharmaceutical industry. More particularly their use is recommended in prevention of venous thrombosis (I.B. Jacques, Pharmacological Reviews, 1979, 3^, 99-166).

Besides, low molecular weight sulfated polysaccharides have been proposed as antithrombotic non-anticoagulant agents, thus involving a weak hemorragic risk (D.P. Thomas, Seminars in Hematology, 1978, 15, 1-17).

Low molecular weight sulfated polysaccharides are obtained by sulfatation of low molecular weight polysaccharides. The sulfatation is generally carried out by treatment with chlorosulfonic acid in pyridine (M.L. Wolfrom et al., J. Am. Chem. Soc. 1953, 75, 1519) or with adducts of sulfur trioxide (sulfuric anhydride) with aprotic solvents (P.L. Whilster, W.W.

Spencer, Methods Carbohydrate Chem., 1964, £, 297-298; R.L. Whilster, ibid. , 1972, 6, 426-429).

The low molecular weight polysaccharides are generally obtained by fractionating a whole of species with various molecular weights or by controlled depoly-merisation of non-fractionated polysaccharides with nitrous acid.

However, the known sulfatation processes present some disadvantages, particularly due to the operating conditions and to the difficulty of controlling the reaction.

The depolymerisation processes, on the other hand, also present the disadvantage of giving a certain percent of inactive products.

In the case of N-sulfated polysaccharides 2 such as heparin, the depolymerisation processes also Involve a hydrolysis of said N-sulfated group, essential to the biological activity of heparin.

It has now surprisingly been found that by reacting a 5 polysaccharide with a mixture of sulfuric acid and chlorosulfonic acid both a depolymerisation and a sulfatation take place concurrently. This finding is particularly surprising, especially because it has also been found that the sulfatation is always total on the possibly present primary hydroxy groups.

Thus, it is an object of the present invention to provide a process for the depolymerisation and sulfatation of polysaccharides other than heparin which comprises reacting said polysaccharide solely with a mixture of sulfuric acid and chlorosulfonic acid.

In the mixture, the two acids are concentrated; preferably their concentration is at least 95% by weight.

The ratio of the two acids is highly variable and may go from traces of chlorosulfonic acid in sulfuric 20 acid up to a ratio sulfuric acid : chlorosulfonic acid 4:1 by volume. Advantageously, the ratio sulfuric acid/ chlorosulfonic acid varies between 4:1 and 1:1, a ratio of about 2:1 being particularly preferred.

The reaction temperature and the concentration of the 25 starting product in the sulfuric acid/chlorosulfonic acid mixture may vary according to the nature of the substrate. For example, the poor solubility of cellulose suggests more elevated dilutions, whereas, in the case of chitosan, it is possible to use a higher concentration 3° and to carry out the reaction at a relatively low temperature.

Generally, the reaction temperature may vary between -20 and +4O'C; after a period varying from some minutes to 2 hours, according to the reaction temperature, the reaction 35 is complete and the depolymerized and sulfated polysacchar- 3 ide is isolated according to the conventional techniques, for example by neutralization and dialysis, by chromatography or by lyophilisation.

The depolymerized and sulfated polysaccharide may 5 also be isolated by pouring the reaction mixture in a solvent wherein the end product is insoluble, for example in a non-polar, aprotic solvent such as diethyl ether, by filtering the precipitate which forms and purifying it according to the techniques known in the sugars 10 chemistry.

The depolymerized and sulfated polysaccharides may further be isolated as alkali metal salts thereof according to the usual methods, for exemple by lyophilisation or by evaporation under reduced pressure, and 15 characterized according to the known physicochemical methods.

Other salts, such as the calcium salt, stay be obtained starting from the alkaline salts, preferably from the sodium salt, by exchange reaction with the appropriate 20 salt, for example with a calcium salt, by optionally using an ion exchange resin.

In the case of a starting polysaccharide having a very high polymerization degree, for example in the case of chitosan, chitin or cellulose, it Is advantageous to 25 submit said starting product to a previous depolymerisation according to known methods,, for example by treatment with nitrous acid. The product thus previously partially depolymerized can be further depolymerized and sulfated according to the process of the present invention. 30 The starting polysaccharide having a very high mole cular weight may also be submitted to the process of the present invention twice. In such a case it is not even necessary to isolate the depolymerized product; a further amount of the sulfuric acid/chlorosulfonic acid mixture 35 can be added to the reaction mixture, for example after 4 the first hour. Surprisingly, this procedure does not involve any degradation or further sulfatation. For example, in the case of cellulose a compound depolymerized and totally sulfated in the 6-position, i.e. on the primary 5 hydroxy group, is obtained according to this procedure.

The process of the present invention may be carried out on the known polysaccharides. Suitable starting materials are heparansulfates, chitosan, chitin, cellulose, starch, guaran, the chondroitinsulfates, 10 the polyxylans, inulin, dermatansulfate, keratan, the mannans, scleroglucan, the galactomannans, the dextrans, the galactans, xanthan.

The process of the present invention is advantageous for its selectivity and conveniences in handling. 15 in the case of chitosan, the reaction with a sul furic acid/chlorosulfonic acid mixture according to the present invention provides a chitosan with a depoly-mersation degree which is unknown because the molecular weight, as that of the starting compound, is too high, 20 but which is suppposed to be depolymerized. The primary hydroxy groups of this compound is selectively sulfated, without any variation on the secondary hydroxy group or on the free amino group.

In addition, according to the process of the present invention it is possible to control the sulfatation degree by suitably varying the reaction temperature and/or time. For example, in the case of chitosan again, it is possible to obtain a chitosan having a sulfatation degree, selective in the 6-position, 5 higher than zero, which can arrive up to 1.

Cellulose, starch and chitin behave as chitosan.

Qiondroitinsulfate and dermatansulfate behave as heparin. 13 In the case of guaran, it is possible to obtain depolymerized guaranes having a sulfate group on the primary hydroxy group of O-mannose.

The depolymerisation degree varies according to the molecular weight of the starting 15 product and the stability .

In the case of cellulose and starch, depolymerized and sulfated products having a higher depolymerisation degree are obtained.

Chondroitinsulfate and dermatan-20 sulfate are less stable and the depolymerisation may go up to three- and tetrasaccharides.

Generally, the depolymerisation degree may be controlled by suitably modifying the sulfuric acid/c'nlorosulfonic acid ratio, the 25 reaction time as well as the concentration of the starting product in the mixture of the two acids.

The following examples illustrate the invention without, however, limiting it.

EXAMPLE 1 • To a mixture of 20 nil of 95% sulfuric acid 10 ml of chlorosulfonic acid, previously cooled to 0-4°C, there are added 500 mg of chitosan ANIC, lot 5 116. The mixture is stirred at the same temperature for about 1 hour, then it is poured into previously cooled diethyl ether. The precipitate which forms is filtered and neutralized with a potassium carbonate solution. After a dialysis in THOMAS DIALYZER TUBING 10 at 8500 D, a chitosan 6-sulfate is obtained, having the following characteristics : - Substitution degree (conductimetric method): 1 - IR spectrum : broad band in the region 1300-1200 cm"*, characteristic of the sulfate groups - 13C-NMR spectrum : disappearance of the signal of the primary hydroxy group and appearance of the signal relating to the sulfate group.

EXAMPLE 2• To a mixture of 20 ml of 95% sulfuric acid 20 and 10 ml of 98% chlorosulfonic acid, cooled to a temperature between -4 and 0SC, there is added 1 g of chitosan ANIC, lot 116. The reaction mixture is left to stand 30 minutes at room temperature, then it is poured into 500 ml of previously cooled diethyl 25 ether. After filtration, the precipitate is washed in water and neutralized with a solution of 0.5 N sodium hydroxide, then it is dialyzed against distilled water in membranes at 8000D (THOMAS DIALY2ER TUBING) and evaporated under reduced pressure. Thus, a 30 chitosan 6-sulfate is obtained in 90% yield. The product has the following characteristics : - Substitution degree (conductimetric method) : 0.5, namely, 50% only of the hydroxy group in 6 position has been sulfated.

- IR spectrum : broad band in the region 1300-1200 cm~^. characteristic of the sulfate groups - 13C-NMR spectrum : diminution of the signal relating to the primary hydroxy group and apparatus of the signal relating to the sulfate group.

EXAMPLE 3. a) To a solution of 1 g of chitosan ANIC, lot 116, in 50 ml of 30% acetic acid, there are added 2.3 ml of 0.5 M nitrous acid, prepared from 10 ml of 0.5 M barium nitrite monohydrate and 10 ml of 10 0.5 M sulfuric acid. The mixture is stirred 12 hours at room temperature, concentrated under reduced pressure and treated with acetone. The precipitate which forms is filtered, washed with acetone, dried, dissolved in water and treated with 30 ml of sodium 15 borohydrlde. After 12 hours at room temperature, the excess of sodium borohydrlde is destroyed with AMBSRLITE IP 120 H+ and the boric acid is eliminated by evaporation under reduced pressure in the presence of methanol.

Thus a depolymerized chitosan is obtained, having a 2o molecular weight much lower than that of the starting chitosan. b) To a mixture of 20 ml of 95% sulfuric acid and 10 ml of 98% chlorosulfonic acid, cooled to a temperature between -4 and O'C, there is added 1 g of depolymerized chitosan, described hereinabove. The reaction mixture is left to stand 1 hour at room temperature, then it is poured into 250 ml of previously cooled diethyl ether; the precipitate which forms is filtered and washed with cold diethyl ether. 30 The product Is dissolved In water and neutralized with a 0.5 M sodium hydroxide solution. After desalting by chromatography on Sephadex G25, a depolymerized chitosan 6-sulfate is obtained in a 90% yield. The product has the following characteristics : 35 - Substitution degree : 1 - IR spectrum : broad band in the region 1300-1200 cm-*, characteristic of the sulfate groups - 13C-NMR spectrum : disappearance of the signal relating to the primary hydroxy group and appearance of a new signal due to the sulfate group.

EXAMPLE 4.

To a mixture of 20 ml of 95% sulfuric acid and 10 ml of 98% chlorosulfonic acid, cooled to 0-4°C, there is added 1 g of cellulose microcristalline 10 (M.W. 20000). The reaction mixture is left 1 hour under stirring, then additional 30 ml of the mixture sulfuric acid:chlorosulfonic acid 2:1 are added thereto. After 30 minutes, the mixture is poured into 500 ml of cold diethyl ether, then it is filtered, the precipitate 15 is washed with diethyl ether and disssolved in water. By neutralization with a 0.5 M sodium hydroxide solution, dialysis in membranes at 3500 D (THOMAS DIALYZER TUBING 3787-H 47, 11 mm diameter) and evaporation under pressure, a cellulose 6-sulfate is obtained, 20 with a 36% yield of dialysable fraction and 23% yield of non-dialvsable fraction. The product has the following characteristics : - Substitution degree (conductimetric method) : 1 - IR spectrum : broad band in the region 1300-1200 cm 25 characteristic of the sulfate groups - Molecular weight : 3400 EXAMPLE 5.

To a mixture of 10 ml of 98% sulfuric acid 15 and 5 ml of 98% chlorosulfonic acid, at the temperature of 0-4#C, 1 g of juaran (AGOGUM F-90, lot 433) is added. After 1 hour at the same temperature, a guaran 6-sulfate is isolated as sodium salt (code No. AH-102). The product 20 has the following characteristics: - Substitution degree (conductimetric method) : 1 - IR spectrum : broad band in the region between 1300 and 1200 cm-3", characteristic of the sulfate group.

Yield s 26% by weight EXAMPLE 6.

To a mixture of 20 ml of 95% sulfuric acid and 10 ml of 98% chlorosulfonic acid, cooled to 0-4°C, 1 g of chitin (SIGMA, lot 12 F-7060) is added. The reaction mixture is left to stand 1 hour at the same 30 temperature. Then, by operating as described in Exarrale 1, after dialysis and evaporation under reduced pressure a chitin 6-sulfate is obtained and isolated as sodium salt (code N° AH-50). The product has the following characteristics : - Substitution degree (conductimetric method) : 1 - IR spectrum : broad band in the region between 1300 5 and 1200 cm"*, characteristic of the sulfate grbup. - 13C-NMR spectrum : disappearance of the signal of the primary hydroxy group and appearance of the signal of sulfate groups.

Yield : 70% by weight 10 EXAMPLE 7.

To a mixture of 15 nl of 95% sulfuric acid: 98% chlorosulfonic acid 2:1, cooled to 0-4°C, there are added 500 mg of choi^oitinsulfate TAKEDA (lot BB-185, No. Code : D-267) having a molecular weight 15 22000 and containing 18% of moisture. After 1 hour at room temperature, the mixture is poured into 500 ml of cold diethyl ether. The precipitate which forms is dissolved in water, the solution is neutralised with 0.5 M sodium hydroxide, then it is dialysed in tubes 20 at 3500 □ (THOMAS DIALYSEK T'JSINC, diarseter 15mm} , By evaporating under reduced pressure- a depolymerized and supersulfated chondroitinsulfate (code No. AH 69) having the following characteristics is obtained : - IR spectrum : broad band in the region 1300-1200 cm 25 characteristic of the sulfate groups - Molecular weight: 2000 EXAMPLE 8.

To a mixture of 10 ml of 98% sulfuric add and 5 ml of 98% chlorosulfonic acid, there is added 30 500 ml of dermatansulfate OPOCRIN (lot 7-8 HF) having a molecular weight 27000 and a substitution degree (SO^/COO-) : 1. By operating as described in Example 25, a depolymerized and supersulfated dermatansulfate (code Na. AH-79) is obtained. The product has the following 35 characteristics : - Substitution degree (SO~/COO~), conductimetric method ) : 2.8 - IR spectrum broad band in the region between 1300 and 1200 cm-*, characteristic of sulfate groups - Molecular weight 2000.

Claims (9)

CLAIMS:

1. A process for the depolymerisation and sulfatation of polysaccharides other than heparin, wherein said polysaccharide is reacted solely with a 5 mixture of sulfuric acid and chlorosulfonic acid.

2. A process according to Claim 1, wherein the reaction is carried out at a temperature of from -20° to +40°C.

3. A process according to either of Claims 1 10 or 2, wherein the concentration of the two acids is at least 95% by weight.

4. A process according to any one of Claims 1 to 3 wherein the ratio sulfuric acid:chlorosulfonic acid is from 4:1 to 1:1. 15 5.

5. A process according to any one of Claims 1 to 4, wherein the ratio sulfuric acid:chlorosulfonic acid is about 2:1. £.

6. A process according to any one of Claims 1 to 5, wherein the depolvmerised and sulfated poly-20 saccharide is isolated in the form of sodium salt.

7. A process according to any of Claims 1 to 6, wherein a polysaccharide previously partially depolvmerised is used as starting product.

8. A process as claimed in Claim 1, for the 25 depolymerisation and sulfation of a polysaccharide, substantially as herein described.

9. A depolymerlsed sulfated polysaccharide, whenever prepared by a process as claimed in any of the preceding claims. MACLACHLAN & DONALDSON Applicants' Agents 47 Merrion Square Dublin 2