Process for the preparation of polysaccharides

Abstract

The present invention relates to a process for the preparation of a polysaccharide composed of D-xylose units of formula (III) linked together via beta 1,4 glycosidic bonds wherein R.sub.1 is hydrogen or acetyl, R.sub.2 is hydrogen, acetyl or a 4-O-methyl glucuronic acid unit, wherein, when R.sub.2 is a 4-O-methyl glucuronic acid unit, the R.sub.1 group on the same saccharide unit is defined as G, wherein G is hydrogen or acetyl, and wherein the sugar unit at the reducing end of sun such polysaccharide is xylose, lyxose or xylulose, said process comprising the following steps: selective deacetylation of xylan extracted from beech wood; and isomerization of the selectively deacetylated xylan achieved in step or the following steps: isomerization of xylan extracted from beech wood; and selective deacetylation of isomerized xylan achieved in step. The process is useful for the preparation of pentosan polysulfate or pharmaceutically acceptable salts thereof for pharmaceutical use.

Claims

1. Process for the preparation of a polysaccharide composed of D-xylose units of formula (III) linked together via beta 1,4 glycosidic bonds ##STR00006## wherein R.sub.1 is hydrogen or acetyl, R.sub.2 is hydrogen, acetyl or a 4-O-methyl glucuronic acid unit, wherein, when R.sub.2 is a 4-O-methyl glucuronic acid unit, the R.sub.1 group on the same saccharide unit is defined as G, wherein G is hydrogen or acetyl, and wherein the sugar unit at the reducing end of the polysaccharide is, lyxose or xylulose, said process comprising the following steps: a) selectively deacetylating xylan extracted from beech wood; and b) isomerizing the selectively deacetylated xylan achieved in step a) or the following steps: c) isomerizing xylan extracted from beech wood; and d) selectively deacetylating isomerized xylan achieved in step c).

2. The process according to claim 1 wherein step a) or step d) is performed in the presence of a basic reagent.

3. The process according to claim 2 wherein step a) or step d) is performed in aqueous environment at a pH comprised in the range between pH=8 and pH=12.

4. The process according to claim 1 wherein-step b) is performed by heating deacetylated xylan achieved in step a) in presence of pyridine.

5. The process according to claim 1 wherein step c) is performed by heating xylan extracted from beech wood in presence of pyridine.

6. The process according to claim 4 wherein that step b) or step c) is performed in the presence of at least one additional substance selected from alumina, aluminum acetate, calcium chloride and calcium acetate.

7. The process according to claim 1 wherein step a) and step b) are performed in one-pot.

8. The process according to claim 7 wherein the one-pot reaction is performed in the presence of a basic reagent and of at least one substance containing polyvalent metals in aqueous environment.

9. The process according to claim 8 wherein said at least one substance containing polyvalent metals is selected from the group consisting of-calcium chloride, calcium acetate and aluminum acetate.

10. The process according to claim 1 further comprising the step of converting the polysaccharide into pentosan polysulfate by sulfation reaction.

11. The process according to claim 10 further comprising the step of converting pentosan polysulfate into a pharmaceutically acceptable salt.

12. Process according to claim 1, wherein the polysaccharide has the formula (II) ##STR00007## wherein R is hydrogen or acetyl, G is hydrogen or acetyl, A is a, lyxose or xylulose unit.

13. A polysaccharide composed of D-xylose units of formula (III) linked together via beta 1,4 glycosidic bonds ##STR00008## wherein R.sub.1 is hydrogen or acetyl, R.sub.2 is hydrogen, acetyl or a 4-O-methyl glucuronic acid unit, wherein, when R.sub.2 is a 4-O-methyl glucuronic acid unit, the R.sub.1 group on the same saccharide unit is defined as G, wherein G is hydrogen or acetyl, and wherein the sugar unit at the reducing end of such polysaccharide is, lyxose or xylulose.

14. A polysaccharide of formula (II) ##STR00009## wherein R is hydrogen or acetyl, G is hydrogen or acetyl, A is a, lyxose or xylulose unit.

Description

EXAMPLES

Example 1

(1) Method of Analysis for Acetyl Groups

(2) Xylan sample (about 25 mg) is solubilized in 0.7 mL D2O. For the acquisition of the .sup.1H-NMR spectra an Agilent Mercury 200 MHz equipment was used.

(3) Signal attributions for the hydrogen atoms of the relevant acetyl groups are the following ones: 2.20 ppm: Acetyl group bonded to position 3 of a xylose unit linked in position 2 with a 4-O-methyl glucuronic acid unit 2.14 ppm: Acetyl group bonded to position 3 of a xylose unit not linked to 4-O-methyl glucuronic acid units 2.09 ppm: Acetyl group bonded to position 2 of a xylose unit

Example 2

(4) Method of Analysis for Monosaccharides

(5) Xylan sample (about 25 mg) is hydrolyzed in 4 mL sulfuric acid 1M at 110 C. for 1.5 hours. After hydrolysis the mixture is neutralized with sodium hydroxide. For the derivatization 0.1 mL of the neutralized solution are mixed with 0.4 mL of 0.23 M phosphate buffer (pH=8) and 0.2 mL of a 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP) 87 mg/mL solution in methanol.

(6) The mixture is heated at 70 C. for 1 hour and extracted at room temperature three times with 0.7 mL chloroform. The derivatized solution is prepared diluting 0.1 mL of the aqueous phase with 1 mL of Phase B (see Eluents). For the HPLC analysis Aeris Peptide XB-C18 4.6250 mm (Phenomenex) is used. The chromatographic conditions are:

(7) TABLE-US-00001 Flow: 0.8 mL/min Column T: 35 C. Gradient: t = 0 min Phase A 30% Phase B 70% t = 1 min Phase A 30% Phase B 70% t = 41 min Phase A 0% Phase B 100% t = 42 min Phase A 30% Phase B 70% t = 55 min Phase A 30% Phase B 70% Eluents: Phase A = 100% Na2HPO4 buffer 0.1M pH 7 Phase B = 80% Na2HPO4 buffer 0.1M pH 7 20% acetonitrile Detector: UV 250 nm Injection: 5 L of the derivatized soluzion Retention times: Lyxose about 25 min Xylose about 37 min

Example 3

(8) Extraction of Beech Xylan

(9) Sodium hydroxide 15% solution is added until pH=4 to 270 mL of a 10% solution of peracetic acid. Beech sawdust (14 g) is added under stirring and the mixture is heated to 85 C. Stirring is maintained at 85 C. for 30 minutes. Decolorization is observed. After cooling at room temperature the mixture is filtered and the recovered solids are washed with fresh water. After drying under vacuum at 40 C., 7.5 g are obtained. To the dried product 450 mL of DMSO are added. The suspension is heated under nitrogen at 60 C. and stirred for 24 hours, cooled at room temperature and filtered. DMSO extraction is repeated on the solid. Each of the DMSO solutions are added with 2 L ethanol, cooled at 4 C. and maintained for 3 days. The solids are filtered, mixed together and dried yielding in total 0.33 g of beech xylan.

Example 4

(10) Extraction of Beech Xylan

(11) Xylan extraction is carried out using an accelerated solvent extractor equipment (Dianox ASE 150 model from Thermo Scientific). The 100 mL stainless steel cell of the instrument is charged with 30 g of beech sawdust. Deionized water is used as extraction solvent. The following instrumental parameters are applied to execute the extractions:

(12) TABLE-US-00002 Temperature 150 C. Static time 90 min Rinse volume 220 sec Static cycle 1 Reduce relief off Preheat time 0 min Preheat purge off Bypass heatup off

(13) This extraction procedure is repeated 10 times and the collected brown solutions are mixed together. DMSO (60 mL) is added. Solution is concentrated under vacuum with external bath temperature set at 65 C., once most of water is evaporated, xylan is precipitated adding ethanol (1500 mL) under stirring. Stirring is continued for 1 hour and the product is filtered and washed with ethanol (two times, each one with 200 mL). After drying 29 g beech xylan are obtained.

Example 5

(14) Selective Deacetylation of Xylan

(15) Water (160 mL) and beech xylan (10 g) are mixed under stirring. The temperature is brought to 10 C. with a thermostatic bath. Sodium hydroxide 30% water solution is added till pH=11.0. Stirring is continued and temperature is maintained while sodium hydroxide 5% water solution is automatically added to maintain pH in the range between pH=11.0 and pH=11.1. The addition of sodium hydroxide solution is controlled the by means of a pH-stat system.

(16) After 8 hours in total 10.6 g of the sodium hydroxide 5% solution were added. Acetic acid is added for neutralization. To the reaction mixture DMSO (20 mL) is added and the mixture is concentrated under vacuum till residual weight is about 35 g. The concentrated solution is added to ethanol (600 mL). A precipitate is formed, the slurry is stirred at room temperature for 2 hours and filtered. After drying 7.8 g of slightly colored powder are obtained.

(17) Starting material (beech xylan) and the obtained selectively deacetylated xylan product are both analyzed for acetyl distribution by .sup.1H-NMR. Acetyl group linked to a xylose unit bearing also a 4-O-methyl-glucuronic acid unit resulted to be 10% of total acetyl groups in the starting material and 83% of total acetyl groups in the selectively deacetylated product.

Example 6

(18) Isomerization of Beech Xylan

(19) Beech xylan of example 5 (5 g) is mixed with 50 mL water and 50 mL pyridine. The mixture is heated to reflux and temperature maintained under stirring for 8 hours. Solvent is removed under vacuum until residual weight is 10 g. Ethanol is added and again solvent is removed under vacuum until residual weight is about 10 g. Ethanol (150 mL) is added and the mixture is stirred at room temperature for 2 hours. The solid is filtered and dried under vacuum at 45 C. obtaining 4.3 g. Both starting material (beech xylan) and product (isomerized beech xylan) were analyzed for monosaccharide composition: the result for lyxose in the starting material was 0 (lyxose not detectable) and 0.9% in the obtained product.