CA1218986A - Process for the depolymerisation and sulfatation of polysaccharides - Google Patents
Process for the depolymerisation and sulfatation of polysaccharidesInfo
- Publication number
- CA1218986A CA1218986A CA000443861A CA443861A CA1218986A CA 1218986 A CA1218986 A CA 1218986A CA 000443861 A CA000443861 A CA 000443861A CA 443861 A CA443861 A CA 443861A CA 1218986 A CA1218986 A CA 1218986A
- Authority
- CA
- Canada
- Prior art keywords
- depolymerized
- acid
- sulfuric acid
- chlorosulfonic acid
- polysaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
PROCESS FOR THE DEPOLYMERISATION AND
SULFATATION OF POLYSACCHARIDES
ABSTRACT OF THE DISCLOSURE
Process for the depolymerisation and sulfatation of polysaccharides by reaction of said polysaccharides with a sulfuric acid/chlorosulfonic acid mixture.
SULFATATION OF POLYSACCHARIDES
ABSTRACT OF THE DISCLOSURE
Process for the depolymerisation and sulfatation of polysaccharides by reaction of said polysaccharides with a sulfuric acid/chlorosulfonic acid mixture.
Description
The present invention relates to a process for the depolymerization and sulfatation of po.
saccharides.
Sulfated polysaccharides are compounds having a great importance in cosmetic~ textile, alimentary and pharmaceutical industry. More particularly their use is recommended in ~revention of venous throm~osis (I.B. Jacques, Pharmacological Reviews, 1979r 31r 99-166)~
Besides r low molecular weiyht sulfated polvsaccharides have been proposed as antithrombotic non-anticoagulant agents, thus involving a weak hemorraglc risk (D.P. Thomas, Seminars in Hematology, 1978, 15, 1-17).
Low molecular weight sulfated poly-saccharid2s are obtained by sulfatation or low molecularweight polysaccharides. The sulfatation is generally carried Ollt by treatment with chlorosulfonic acid in ~yridine (~.L. Wol rom et al., J. Am. Chem. Soc. lg53, 7S, 1519) or with adducts of sulrur trioxide (sulfuric anhydride) with aprotic solvents (P.L. Whilster, W.W.
Spencer, Methods Carbohydrate Chem., 1964, ~ 297-298;
R.L. Whilstex~ i~id. , 1972, ~, 426-~29).
The low molecular weight polysaccharides are generally obtained by fractionating a whole of species ~5 with various molecular weights or by controlled de~oly-merisation of non-fract.ionated ~olysaccharides ~with nitrous acid~
Ho~ever, the known sulfatation processes ~resent some disadvantages, particularly due to the operating conditions and to the di~ficulty o 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 ~,". ..
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 polysaccharide with a mixture of sulfuric acid and chloro-sulfonic acid both a depolymerisation and a sul:Eatation 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.
In accordance with the present invention, therefore, there is provided a process for the depolymerisation and sulfatation of polysaccharides, which comprises reacting said polysaccharide 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 ra-tio of the two acids is highly variable and may go from traces of chlorosulfonic acid in sulfuric 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 starting product in the sulfuric acid/chlorosulfonic acid mixture may vary according to the nature of the substrate.
F'or example, the poor solubility of cellulose suggests more elevated dilutions, whereas, in the case of chitosan, it is possible to use a higher concen-tration and to carry out the reac-tion at a relatively low temperature.
Generally, the reaction temperature may vary between -20 and +40C; after a period varying from some minutes to
saccharides.
Sulfated polysaccharides are compounds having a great importance in cosmetic~ textile, alimentary and pharmaceutical industry. More particularly their use is recommended in ~revention of venous throm~osis (I.B. Jacques, Pharmacological Reviews, 1979r 31r 99-166)~
Besides r low molecular weiyht sulfated polvsaccharides have been proposed as antithrombotic non-anticoagulant agents, thus involving a weak hemorraglc risk (D.P. Thomas, Seminars in Hematology, 1978, 15, 1-17).
Low molecular weight sulfated poly-saccharid2s are obtained by sulfatation or low molecularweight polysaccharides. The sulfatation is generally carried Ollt by treatment with chlorosulfonic acid in ~yridine (~.L. Wol rom et al., J. Am. Chem. Soc. lg53, 7S, 1519) or with adducts of sulrur trioxide (sulfuric anhydride) with aprotic solvents (P.L. Whilster, W.W.
Spencer, Methods Carbohydrate Chem., 1964, ~ 297-298;
R.L. Whilstex~ i~id. , 1972, ~, 426-~29).
The low molecular weight polysaccharides are generally obtained by fractionating a whole of species ~5 with various molecular weights or by controlled de~oly-merisation of non-fract.ionated ~olysaccharides ~with nitrous acid~
Ho~ever, the known sulfatation processes ~resent some disadvantages, particularly due to the operating conditions and to the di~ficulty o 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 ~,". ..
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 polysaccharide with a mixture of sulfuric acid and chloro-sulfonic acid both a depolymerisation and a sul:Eatation 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.
In accordance with the present invention, therefore, there is provided a process for the depolymerisation and sulfatation of polysaccharides, which comprises reacting said polysaccharide 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 ra-tio of the two acids is highly variable and may go from traces of chlorosulfonic acid in sulfuric 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 starting product in the sulfuric acid/chlorosulfonic acid mixture may vary according to the nature of the substrate.
F'or example, the poor solubility of cellulose suggests more elevated dilutions, whereas, in the case of chitosan, it is possible to use a higher concen-tration and to carry out the reac-tion at a relatively low temperature.
Generally, the reaction temperature may vary between -20 and +40C; after a period varying from some minutes to
2 hours, according to the reaction temperature, the reac-tion is complete and the depolymerized and sulfated poly-sacchar-ide is isolated according to the conventional techniques, for example by neutralization and dialysis, by chroma-tographv or by lyophilisation.
The depolymerized and sulfated polysaccharide mav also be isolated by pouring the reaction mixture in a solvent wherein the end product is insoluble, ror example in a non-polar, aprotic solvent such as diethvl ether, by filtering the precipitate which forms and purifying it according to the techniques known in the sugars chemistrY
The depolymerized and sulrated ?ol~rsaccharides may further be isolated as alkali metal salts tnereof according to the usual methods, for exemple by lyophili-sation or by evaporation under reduced pressure, and characterized according to the ~nown physicochemical methods.
Other salts, such as the calcium salt, may be ob-tained starting from the alkaline salts, preferably from the sodium salt, by exchange reaction with the appropriate salt, for example with a calcium salt, by optionall~
using an ion exchange resin.
In the case of a startin~ polysaccharide having a very high polymerization degree, for exam~le in the case of chitosan, chitin or cellulose, it is advantageous to submit said starting product to a previous depolymerisation according to known methods, for example by treatment with nitrous acid. The product thus ~reviouslv partially depolymerized can be further depolymerized and sulfated according to the ~rocess of the present invention.
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 necessarv to isolate the depolymeri~ed ?roduct; a further amount of the sulfuric acid/chlorosulfonic acid mi~ture can be added to the reaction mixture, for example after the first hour. Surprisingly, this ~rocedure does no involve any degradation or further sulfatation. For example, in the case of cellulose a com~ound de~olymerized and totally sulfated in the 6-position, i.e. on the primary hydroxy group, is obtained accordin~ to this procedure.
The process of the present invention may be carried out on the known polysaccharides. Suitable starting materials are heparin, he~aransulfates, chitosan, chitin, cellulose, starch, guaran, the chondroitinsulfates, the ~olyxylans, inulin, dermatansulfate, ~.eratan, 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.
In the case of heparin, Cor examole, there is obtained a depolymeri~ed and "supersulfated" heparin ha~ing a molecular weight of from 2000 and 9000 and a sulfatat on degree higher than tha~ of the starting heparin. In this depolymerized and "suDersulfated" henarin, all Of the ~rimary hydroxy groups are sulfated.
In the case of chitosan, the reaction with a sul-furic acid/chlorcsulfonic acid mixture according to the present invention provides a chitosan with a depoly-mersation degree which is unknown because the molecular weisht, as that of the starting compound, is too high, but which is suooposecl to be depolymerized. The primary hydro~y groups of this compound is selectively sulfated, without anv variation on the secondary hydroxy grou~
or on the free amino grou~.
In additlon, 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 a~ain, it is possible to obtain a chitosan having a sulfatation degree, selective in the 6-position, higher than ~ero, which can arrive up to 1.
Cellulose, starch and chitin behave as chitosan.
Chondroitinsulfate and dermatansulfate behave as heparin.
In the case of guaran, it is possible to obtain depolymerized guaranes having a sulfate group on the primary hydroxy group of D-mannose.
The depolymerisation degree varies according to the molecular weight of the starting product and the stability.
In the case o-f cellulose and starch, depolymerized and sulfated products having a higher depolymerisation degree are obtained.
Chondroitinsulfate and dermatansulfate are less stable and the depolymerisation may go up to three- and tetrasaccharides.
Generally, the depolymerisation degree may be con-trolled by suitably modifying the sulfuric acid/chlorosul-fonic acid ratio, the reac-tion -time as well as the concen-tratlon of the starting product in the mixture of the two acids.
The following examples illustrate the invention without, however, limiting it. In the following Examples, reference is made to the accompanying drawings, wherein Figures 1 to 10 are graphical representations of electro-phoresis determinations in hydrochloric acid, in which the migration is a function of the sulfatation degree.
. .
To a mixture of 20 ml of 9S% sulfuric acid and 10 ml of chlorosulfonic acid, cooled to a temperature between -4 and 0C, there is added 1 g of heparin from pig intestinal 35 mucosa (PROQUIFIN*, lot 7926-7935, code number : D-212) having a sulfatation degree of 1.95 and a molecular weight 13500, then it is stirred for 1 hour at -the same tempera-ture. After further 60 minutes at room temperature, the * - Trademark mixture is poured into 500 ml of cold diethyl ether (-4 -to 4C), the precipitate is fil-tered and washed with cold diethyl ether. The product thus obtained is dissolved in water, neutralized with 0.5 N sodium hydroxide and dialyzed against distilled water in membranes at 3500 D (THOMAS
DIALYZED TUBING* 3787-H47, 11 mm diameter). Thus a desal~
ting is obtained as well as the ellmination of low molecu-lar fragments. By slow evaporation under reduced pressure, a depolymerized and supersulfated sodium heparin (code number : AH-16) is obtained in 93% yield by weight, as a powder having the following characteristics:
- M.W.: ~ 6000 - Elemental analysis:S:12.93%; C:18.48%; H:3.30%; N:1.76%
- Sulfatation degree (SO3 /COO ):3.0 - IR spectrum:broad band in the region 1300-1200 cm 1, characteristic of the sulfate groups - Electrophoresis in hydrochloric acid: with this technique, the migration is function of -the sulfatation degree. Fig.
1 shows the significant increasing of the electrophoretic migration of the depolymerized and supersulfated heparin compared with the starting heparin.
- Barium acetate electrophoresis: Fig. 2 shows that depolymerized and supersulfated heparin as a "slow-moving"
electrophoretic characteristic, differently from the starting heparin containing both "slow-moving" and "fast moving" components.
- 13C-NMR spectrum: Fig. 3 shows the comparison between the spectrum of the starting heparin and that of depoly-merized and supersulfated heparin. In the spectrum of the new low molecular weight heparin new signals appear, due to the effect of the depolymerisation and of the in-troduc-tion of additional sulfate groups as well as to the disap-pearance of the 6-OH signal. The depolymerized and super-sulfated heparin thus obtained shows a sulfatation degree which is 53% higher than that of starting heparin without any significant decarboxylation.
To a mixture of 10 ml of 98% sulfuric acid and 5 ml * - Trademark ~ J
g~
of chlorosulfonic acid, cooled to a temperature between -4 and 0C, there are added 500 mg of a high molecular weigh-t fraction (M.W. 16500, code number:D-212/B), obtained by precipitation with ethanol and having a sulfatation degree (SO3/COO ) of 2, of heparin PROQUIFIN, lot 7926-7935. The mixture is left to stand 1 hour at room temperature, then it is poured into 250 ml of cold diethyl ether (-10 to 4C) and filtered; the precipitate thus obtained is dis-solved in water, the solution is neutralized with 0.5 N
sodium hydroxide and dialysed against distilled water in membranes at 3500 D (THOMAS DIALYZER TUBING 3787-H47, 11 mm diameter), in order to eliminate the salts and the smallest size reaction products. By evaporation under reduced pressure, a depolymerized and supersulfated sodium heparin (code number:AH-18) is obtained in 60% yield. The product has the following characteristics:
- M.W. : 3000-5000 - Elemental analysis:S:13.56%; C:1~.03%, H:3.00% N:1.70%
- Sulfatation degree (SO3/COO ):2.6 20 - IR spectrum: broad band in the region 1300-1200 cm 1, characteristic of the sulfate groups.
- Barium acetate electrophoresis: Fig. 4 indicates that AH- 18 show "slow-moving" components only, whereas the starting product also shows "fast-moving" components.
.
To a mixture of 10 ml of 98% sulfuric acid and 5 ml of chlorosulfonic acid, cooled to a temperature between -4 and 0C, there are added 500 mg of sodium heparin from pig intestinal mucosa (PROQVIFIN, lot 7926-7935, code number:
D-212) having a sulfatation degree (SO3/COO ) of 1.95.
The mixture is left to stand 1 hour at room tempera-ture, then it is poured into 250 ml of cold diethyl ether (-10 to 4C), and afterwards treated as described in Examples 1 and 2. Thus, a depolymerized and supersulfated sodium heparin (code number:A~-l9) is obtained in 90%
yield. The product has the following characteristics:
- M.W. :~ 6000 - Sulfatation degree (SO3/COO ):3.0
The depolymerized and sulfated polysaccharide mav also be isolated by pouring the reaction mixture in a solvent wherein the end product is insoluble, ror example in a non-polar, aprotic solvent such as diethvl ether, by filtering the precipitate which forms and purifying it according to the techniques known in the sugars chemistrY
The depolymerized and sulrated ?ol~rsaccharides may further be isolated as alkali metal salts tnereof according to the usual methods, for exemple by lyophili-sation or by evaporation under reduced pressure, and characterized according to the ~nown physicochemical methods.
Other salts, such as the calcium salt, may be ob-tained starting from the alkaline salts, preferably from the sodium salt, by exchange reaction with the appropriate salt, for example with a calcium salt, by optionall~
using an ion exchange resin.
In the case of a startin~ polysaccharide having a very high polymerization degree, for exam~le in the case of chitosan, chitin or cellulose, it is advantageous to submit said starting product to a previous depolymerisation according to known methods, for example by treatment with nitrous acid. The product thus ~reviouslv partially depolymerized can be further depolymerized and sulfated according to the ~rocess of the present invention.
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 necessarv to isolate the depolymeri~ed ?roduct; a further amount of the sulfuric acid/chlorosulfonic acid mi~ture can be added to the reaction mixture, for example after the first hour. Surprisingly, this ~rocedure does no involve any degradation or further sulfatation. For example, in the case of cellulose a com~ound de~olymerized and totally sulfated in the 6-position, i.e. on the primary hydroxy group, is obtained accordin~ to this procedure.
The process of the present invention may be carried out on the known polysaccharides. Suitable starting materials are heparin, he~aransulfates, chitosan, chitin, cellulose, starch, guaran, the chondroitinsulfates, the ~olyxylans, inulin, dermatansulfate, ~.eratan, 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.
In the case of heparin, Cor examole, there is obtained a depolymeri~ed and "supersulfated" heparin ha~ing a molecular weight of from 2000 and 9000 and a sulfatat on degree higher than tha~ of the starting heparin. In this depolymerized and "suDersulfated" henarin, all Of the ~rimary hydroxy groups are sulfated.
In the case of chitosan, the reaction with a sul-furic acid/chlorcsulfonic acid mixture according to the present invention provides a chitosan with a depoly-mersation degree which is unknown because the molecular weisht, as that of the starting compound, is too high, but which is suooposecl to be depolymerized. The primary hydro~y groups of this compound is selectively sulfated, without anv variation on the secondary hydroxy grou~
or on the free amino grou~.
In additlon, 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 a~ain, it is possible to obtain a chitosan having a sulfatation degree, selective in the 6-position, higher than ~ero, which can arrive up to 1.
Cellulose, starch and chitin behave as chitosan.
Chondroitinsulfate and dermatansulfate behave as heparin.
In the case of guaran, it is possible to obtain depolymerized guaranes having a sulfate group on the primary hydroxy group of D-mannose.
The depolymerisation degree varies according to the molecular weight of the starting product and the stability.
In the case o-f cellulose and starch, depolymerized and sulfated products having a higher depolymerisation degree are obtained.
Chondroitinsulfate and dermatansulfate are less stable and the depolymerisation may go up to three- and tetrasaccharides.
Generally, the depolymerisation degree may be con-trolled by suitably modifying the sulfuric acid/chlorosul-fonic acid ratio, the reac-tion -time as well as the concen-tratlon of the starting product in the mixture of the two acids.
The following examples illustrate the invention without, however, limiting it. In the following Examples, reference is made to the accompanying drawings, wherein Figures 1 to 10 are graphical representations of electro-phoresis determinations in hydrochloric acid, in which the migration is a function of the sulfatation degree.
. .
To a mixture of 20 ml of 9S% sulfuric acid and 10 ml of chlorosulfonic acid, cooled to a temperature between -4 and 0C, there is added 1 g of heparin from pig intestinal 35 mucosa (PROQUIFIN*, lot 7926-7935, code number : D-212) having a sulfatation degree of 1.95 and a molecular weight 13500, then it is stirred for 1 hour at -the same tempera-ture. After further 60 minutes at room temperature, the * - Trademark mixture is poured into 500 ml of cold diethyl ether (-4 -to 4C), the precipitate is fil-tered and washed with cold diethyl ether. The product thus obtained is dissolved in water, neutralized with 0.5 N sodium hydroxide and dialyzed against distilled water in membranes at 3500 D (THOMAS
DIALYZED TUBING* 3787-H47, 11 mm diameter). Thus a desal~
ting is obtained as well as the ellmination of low molecu-lar fragments. By slow evaporation under reduced pressure, a depolymerized and supersulfated sodium heparin (code number : AH-16) is obtained in 93% yield by weight, as a powder having the following characteristics:
- M.W.: ~ 6000 - Elemental analysis:S:12.93%; C:18.48%; H:3.30%; N:1.76%
- Sulfatation degree (SO3 /COO ):3.0 - IR spectrum:broad band in the region 1300-1200 cm 1, characteristic of the sulfate groups - Electrophoresis in hydrochloric acid: with this technique, the migration is function of -the sulfatation degree. Fig.
1 shows the significant increasing of the electrophoretic migration of the depolymerized and supersulfated heparin compared with the starting heparin.
- Barium acetate electrophoresis: Fig. 2 shows that depolymerized and supersulfated heparin as a "slow-moving"
electrophoretic characteristic, differently from the starting heparin containing both "slow-moving" and "fast moving" components.
- 13C-NMR spectrum: Fig. 3 shows the comparison between the spectrum of the starting heparin and that of depoly-merized and supersulfated heparin. In the spectrum of the new low molecular weight heparin new signals appear, due to the effect of the depolymerisation and of the in-troduc-tion of additional sulfate groups as well as to the disap-pearance of the 6-OH signal. The depolymerized and super-sulfated heparin thus obtained shows a sulfatation degree which is 53% higher than that of starting heparin without any significant decarboxylation.
To a mixture of 10 ml of 98% sulfuric acid and 5 ml * - Trademark ~ J
g~
of chlorosulfonic acid, cooled to a temperature between -4 and 0C, there are added 500 mg of a high molecular weigh-t fraction (M.W. 16500, code number:D-212/B), obtained by precipitation with ethanol and having a sulfatation degree (SO3/COO ) of 2, of heparin PROQUIFIN, lot 7926-7935. The mixture is left to stand 1 hour at room temperature, then it is poured into 250 ml of cold diethyl ether (-10 to 4C) and filtered; the precipitate thus obtained is dis-solved in water, the solution is neutralized with 0.5 N
sodium hydroxide and dialysed against distilled water in membranes at 3500 D (THOMAS DIALYZER TUBING 3787-H47, 11 mm diameter), in order to eliminate the salts and the smallest size reaction products. By evaporation under reduced pressure, a depolymerized and supersulfated sodium heparin (code number:AH-18) is obtained in 60% yield. The product has the following characteristics:
- M.W. : 3000-5000 - Elemental analysis:S:13.56%; C:1~.03%, H:3.00% N:1.70%
- Sulfatation degree (SO3/COO ):2.6 20 - IR spectrum: broad band in the region 1300-1200 cm 1, characteristic of the sulfate groups.
- Barium acetate electrophoresis: Fig. 4 indicates that AH- 18 show "slow-moving" components only, whereas the starting product also shows "fast-moving" components.
.
To a mixture of 10 ml of 98% sulfuric acid and 5 ml of chlorosulfonic acid, cooled to a temperature between -4 and 0C, there are added 500 mg of sodium heparin from pig intestinal mucosa (PROQVIFIN, lot 7926-7935, code number:
D-212) having a sulfatation degree (SO3/COO ) of 1.95.
The mixture is left to stand 1 hour at room tempera-ture, then it is poured into 250 ml of cold diethyl ether (-10 to 4C), and afterwards treated as described in Examples 1 and 2. Thus, a depolymerized and supersulfated sodium heparin (code number:A~-l9) is obtained in 90%
yield. The product has the following characteristics:
- M.W. :~ 6000 - Sulfatation degree (SO3/COO ):3.0
3~
- IR spectrum: broad band in -the region 1300-1200 cm characteristic of the sulfate groups - Barium acetate electrophoresis: Fig. 5 indicates that AH-l9 shows "slow-moving" components only, whereas the starting product also shows ~fast moving" components.
To a mixture of 10 ml of 98% sulfuric acid and 5 ml of 95% chlorosulfonic acid, cooled to a temperature be-tween -4 and 0C, there are added 500 mg of a mean molecu-10 lar weight heparin fraction (M.W.~ 10000, code number:
D-212/A), obtained by fractionation with ethanol of hepar-in PROQUIFIN, lot 7926-7935, said fraction having a sul-fatation degree (SO3/COO ) of 1.5 $
and a barium acetate electroohoretic pattern which shows a very important "fast moving" comronent. The mixture is left to stand 1 hour under stirring at room temperature, then it is poured into 250 ml of cold diethyl ether (-10 to 4C), and afterwards treated as described in Examrles 1 and 2. Thus, a deoolvmerized and suoersulfated sodium her,arin (code number : AH-17) is obtained having the following charasteristics :
- M.W.: 3000-5000 - Elemental analysis: S : 12.70%; C : 17.24~; H : 3.10~;
N :1.67~
- Sul~atation degree (S03/COO ) : 2.S
- XR spectr~ : broad band in the region 1300-1~00 cm characteristic of the sulfate grouos - ~arium acetate electrophoresis : Fig. 6 indicates that AH ~17, compared to the starting herarin fraction, shows a "slow-moving" com?onent onlyn EXAMPLE 5.
To a mixture of 20 ml of 95~ sulfuric acid and 10 ml of 93~ chlorosulfonic acid, cooled to a temnerature between -4 and 0C, there is added 1 g of heparin from pig intestinal mucosa (PROQUIFIN, lot 7926-7935, code number o D-212) having a 5ulratation degree of 1.95, then the reaction mixture is stirred 1 hour at room temperature. The mixture is poured into 500 ml of cold diethyl ether (-4 to 4C), the precipitate is filtered and ~ashed with cold diethvl ether. The product thus obtained is dissolved in 0.1 M calcium chloride a~ueous solution, then O.5 ~ calcium hydroxide is added thereto up to pH 8. The solution is dialysed against 500 ml of 0~1 ~I calcium chloride solution and then against distilled water. By slow evaporation under reduced pressure, a calcium salt of a depolymerized and su~ersulfated herJarin is obtained ~5 as a white powder.
EXAMPLES 6 _o 10.
To a mixture o. 10 ml of 98% sulfuric acid and 5 ml of chlorosulfonic acid, cooled to a temperature between -4 and 0C, there are added 500 mg of heparin from pig intestinal mucosa (PROQUIE'IN, lot 7926-7935, cocle number : D-212) having a sulfatation degree of 1.95 and a molecular weight 13500~ By operating as descri~ed in Example 1, a depolymerized,and supersul'ated heparin (code number:
AH-104) is obtained, in 98~ yield.
The same procedure and conditions are followed in 4 parallel experiments in which the same starting heparin is used~ There are obtained the products desiynated by -their code numbers AH-103, AH-105, AH-106 and AH-107. The characteristics of the products thus obtained as well as those of the 2roduct coded AH 104 are given in Table IV.
TABLE IV
_ . , ,, . . . I
Ex. Product Elemental Analysis Sulfatation Yield bv S~ C% H% N%
deg.ree weight _ _ _ _ 6 AH-104 14.54 15.42 2.84 1.43 2.9 + 0.1 98%
7 AH - 103 14.63 15.53 2.76 1.43 2.8 + 0.1 89 8 AH-105 14.48 15.43 2.61 1.44 3.0 ~ 0.1 67%
9 AH-106 14.54 15.53 2.81 1.46 2.8 + 0.1 96%
AH-107 14.12 15.65 2.80 1.40 3.0 + 0.1 ~ 77 - Molecular weight : ~6000 for the 5 products - IR spectrum : the 5 products show a spectrum identical to that of compound AH-16 described in Exam~le 1.
- Electrophoresis in hydrochloric acid: the electro-phoretic profiles are indentical to those of Eig. 1 for both the starting heparin and the 5 products ,;
- Barium acetate electroohoresis : the electrophoretic .
profiles are identical to those of Fig. 2 for both the starting heparin and the 5 products, apart from the fact that the traces relative to the 5 products do not show the background noise - caused by a temporary defect of the tracing pen or of the paper - observed in the horizontal cart of the graph of Fig. 2 relative to AH-16 - 13C-NMR spectrum: the 5 products and the s-tarting compound present the same spectra as those given in Fig.
3.
The 5 compounds thus obtained are identical each other and identical to the compound described in Example 1 as well.
EXAMPLES 11 to 14 15 In 4 parallel experiments, -to a mixture of 10 ml of 98% sulfuric acid and 5 ml of chlorosulfonic acid, cooled to -4 - 0C, there are added 500 mg of previously lyophil-ized heparin from pig intestinal mucosa (DIOSYNTH* batch CH/N 665, code number:D-479), having a sulfatation degree (SO3/COO ) of 2.1 and a molecular weight of about 11000.
The reaction mixture is left to stand 1 hour at 0C, then it is poured into 250 ml of diethyl ether previously cooled (between -10C and +4C). By operating as des-cribed in Example 1 the products of Table V are ob-tained.
TABLE V
, __ _ _ _ Ex. Product Ele! lental Analysis Sulfatation Yield S% C% H% N% degree by weigh-t __ ___ _ ._ 11 AH-108 14.88 15.29 2.52 1.47 3.1 + 0.1 90%
30 12 AH-109 14.43 15.48 2.72 1.44 3.0 + 0.1 106%
13 AH-110 14.45 15.72 2.76 1.50 2.9 + 0.1 65%
14 AH-lll 14.55 15.08 2.60 1.41 3.0 ~ 0.1 23%
_ . _ __ .......... ___ . _ . .
- Molecular weight: 6000 for the 4 products - IR spectrum:~ broad band between 1300 and 1200 cm 1, characteristlc of the sulfate group - Electrophoresis in hydrochloric acid: Fig. 7 shows the traces of the starting heparin D-479 and of one of the 4 * - Trademark ~ ... .
samples obtained in the different experiments (AH-108).
The traces of the other three compounds are identical.
This figure evidences the significant increase of the electrophoretic migration of the depolymerized and super-sulfated heparin in comparison with the starting heparin - Barium acetate electrophoresis: Fig. 8 shows the traces of the starting heparin D-479 and of AH-108. It results that the depolymerized and supersulfated heparin has a "slow-moving" electrophoretic profile, unlike the starting heparin containing ~slow-moving~ components as well as "fast-moving" components. The traces of the products AH-109, AH-110 and AH-lll are identical to that of AH-108.
To a mixture of 10 ml of 98% sulfuric acid and 5 ml of chlorosulfonic acid, cooled to a temperature between -4 and 0C, there are added 500 mg of heparin from pig intes-tinal mucosa (TEROPMON*, batch 575/018, code number:D-98) having a sulfatation degree of 1.8 and a molecular weight 135.00. By operating as described in Example 1, a depoly-merized and supersulfated heparin is obtained, in 75%yield. The product has the following characteristics:
- M.W.: ~v 6000 - Elemental analysis:S:13.90%; C:15.75%; H:2.96%; N:1.48%
- Sulfatation degree (SO3/COO ):2.8 + 0.1 - IR spectrum: broad band in the region 1300-1200 cm 1, characteristic of the sulfate groups - Electrophoresis in hydrochloric acid: Fig. 9 shows the traces of the starting heparin D-98 and of -the product AH-118. A significant increasing of the electrophoretic migration of AH-L18 compared with the starting heparin D-98, may be observed. Fig. 9 shows also that the com-pound AH-118 possesses a photodensitometric outline analo-gous to those of compounds AH-16 (Example 1, Fig. 1) and AH-17 (Example 4, Fig. 6) whereas the starting heparin D-98 appears very heterogeneous and completely different from the starting heparins utiliæed in Examples 1 and 4.
- Barium acetate electrophoresis: Fig. 10 indica-tes that AH-118 shows a "slow-moving" electrophoretic characteristic * - Trademark ,;'~ :`
, . . .
~, ~2~ r f~
which is different from that of the starting heparin D-98 showing both "slow-moving~ and "fast-moving~ components.
Fig. 10 also confirms that data of Flg. 9 and moreover surprisingly shows that compound AH-118 is not signifi-cantly different from AH-108 of Example 11, though the starting heparins are quite different.
..s~.. .
EXAM~LE 16.
. ~
To a mixture of 20 ml of 95% sul~uric acid lO ml of chlorosulfonic acid, ~reviously cooled to 0-4C, there are added 500 mg of ch1tosan ANIC, lot 116. The mi~ture is stirred at the same tem~erature for about 1 ho~r, then it is ~oured into ~reviouslv cooled diethvl ether. The ?recipitate ~Jhich forms is filtered and neutralized with a ~otassium carhonate solution. After a dial~sis in THO~S DIALYZER T~BI~JG
at 8500 D, a chitosan 6--sulate is obtained, havinc the followins characteristics :
- Substitution degree (conductime'ric ~ethod): l - IR s~ect~-um : ~road band in the reaion 1300-1200 cm 1, characteristic of the sulfate ~rou~s - 13C~N.~R sectru~ : disa~earance of the sianal o~ the ~rimar-~r h~tdxoxy ~rou~ and a~nearance of the sinal relatin~ to the sulfate arou~
EX.~IE~LE 17.
To a mixture of 20 ml of 95~ sulFuric acid and 10 ml of 98~ chlorosulfonis acid, cooled to a temDerature between -4 and 0C, there is added l a o~ chitosan ~NIC, lot 116. The reactlon mixture is left ~o stand 30 minutes at room temoerature, then it ls ?ouxed into 500 ml of ~reviousl-y cooled d~ethyl ether. After filtration, the ~recipitate is ~Jashed in water and neutralized with a solution of 0.5 ~ sodium h~,~dro~ide, then it i.s dialvzed a~ainst distilled water in membranes at 8000D ~TH0~1AS DIALV~E~ TUBI~
and eva~orated under reduced pressure. Thus, a chitosan 5-sul~ate is obtained in 90~ yield. The ~roduct has the follo~7ing characteristics :
- Substitution degree ~conductimetric method~ : 0.5, namely, 50~ only of the hvdrox~ gro1m in 6 ~osition has been sulfated - IR s~ectrum : broad band in the region 1300-1200 cm l, characteristic of the sulfate groups - 13C-NMR spectrum: diminution of the slgnal relating to the primary hydroxy group and apparatus of the signal relating to the sulfate group.
.
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 0.5 M sulfuric acid. The mixture is stirred 12 hours at room temperature, concentrated under reduced pressure and treated with acetone. The precipi-tate which forms is filtered, washed with acetone, dried, dissolved in water and treated with 30 ml of sodium borohydride. After 12 hours at room temperature, the excess of sodium borohydride is destroyed with AMBERLITE*
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 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 be-tween -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. The product is dissolved in water and neu-tralized with a 0.5 M sodium hydroxide solution. After desalting by chromatography on Sephadex* G25, a depolymer-ized chitosan 6-sulfate is obtained in a 90% yield. The product has the following characteristics:
- Substitution degree:l * - Trademarks ~.,,1-- IR s~ectrum : broad band in the resion 1300-1200 cm 1, characteristic of the sulfate grou~s - 13C-NMR spectrum : disa~pearance o~ the signal relating to the primarv hydroxv group and appearance of a ne~l signal due to the sulfate grou~.
EXAMPLE 19.
To a mixture of 20 ml of 95~ sulfuric acid and 10 ml of 98~ chlorosulfonic acid, cooled to 0-4C, there is added 1 g of cellulose microcristalline (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 ~oured into 500 ml of cold diethyl ether, then it is filtered, the preci~itate is washed with diethyl ether and disssolved in water.
By neutralization with a 0.5 M sodium hydroxide solu~ion, dialysis in membranes at 3500 D (TUO~S
DIALYZE?~ TUBING 3787~H 47, 11 m~ diameter) and evapora-tion under ~ressure, a cellulose 6-sulfate is ohtained, with a 36go yield Of dialvsable fraction and 29~ vield of non-dialysable fraction. The ~roduct has the follow-ing characteristics :
- Substitution degree (conductimetric metho~
- IR s~ectrum : broad band in the region 1300-1200 cm 1, ~5 characteristic of the sulfate grou~s - Molecular weight : 3400 E~PLES 20 to 22.
In three parallel ex~eriments, 500 mg of heparin from ~ig intestinal mucosa (DIOSYNTH, batch CH/N 665, code n. D-479) having a sulfatation degree (S03/COo ) 2.1 and a molecular ~eight 11000 are added to 15 ml of a mixture of 98g~ sulfuric acid and 98 chlorosulfonic acid in the following ratios :
Example 20 : 1:4 Example 21 ~
Exam~le 22 : 4:1 By operating as described in Example 1, three depolymer-ized and supersulfated heparins, having the characteris-tics given in Table VI, are obtained.
TABLE VI
Example Sulfatation Molecular Yield (code NO) degree weight % by weight 2.5 4000 89.9 10 (AH-67) 21 2.5 3800 86.8 (AH-65) 22 2.8 4500 77.4 (AH-68) __ To a mlxture of 10 ml of 98% sulfuric acid and 5 ml of 98% chlorosulfonic acid, at the temperature of 0 - 4C, 1 g of guaran (AGOGUM* F-90, lot 433) is added. After 1 hour at *he same temperature, by operating as described in Example 1, a guaran 6-sulfate is isolated as sodium salt (code No . AH-102). The product has the following charac-teristics:
- Substitution degree (conductimetric method):l - IR spectrum: broad band in the region between 1300 and 1200 cm 1, characteristic of the sulfate group.
Yleld: 26% by weight To a mixture of 20 ml of 95% sulfuric acid and 10 ml 30 of 98% chlorosulfonic acid, cooled to 0 - 4C, 1 g of chitin (SIGMA, lot 12 F-7060) is added. The reaction mixture is left to stand 1 hour at the same temperature.
Then, by operating as described in Example 1, after dial-ysis and evaporation under reduced pressure a chitin 6-sulfate is ob-tained and isolated as sodium salt (code No. AH-50). The product has the following characteristics:
- Substitution degree (conductimetric method):l - IR spectrum: broad band in the region between 1300 and * - Trademark 1200 cm 1, characteristic of the sulfate group.
- 13C-NMR spectrum:disappearance of the signal of the primary hydroxy group and appearance of the signal of sulfate groups.
Yield: 70% by weight To a mixture of 15 ml of 95% sulfuric acid: 98%
chlorosulfonic acid 2:1, cooled to 0 - 4C, there are added 500 mg of chondroitinsulfate TAKEDA* (lot BB-185, No. Code:D-267) having a molecular weight 22000 and con-taining 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 dialyzed in tubes at 3500 D (THOMAS DIALYZER TUBING, diameter 15 mm). By evaporating under reduced pressure a depolymerized and supersulfa-ted chondroitinsulfate (code No. AH 69) having the fbllowing characteristics is obtained;
- IR spectrum: broad band in the region 1300-1200 cm 1, characteristic of the sulfate groups - Molecular weight: 2000 To a mixture of 10 ml of 98% sulfuric acid and 5 ml of 98% chlorosulfonic acid, there is added 500 ml of dermatansulfate OPOCRIN* (lot 7-8 HF) having a molecular weight 27000 and a substitution degree (SO3/COO ):1. By operating as described in Example 25, a depolymerized and supersulfated dermatansulfate (code No. AH-79) is obtained.
The product has the following characteristics:
* - Trademarks ~2~
- Substitution de~ree (S03/C00 ), conductimetric method ) : 2.8 - IR s~ectrum broad band in the resion between 1300 and 1200 c~ 1, characteristic of sulfate grou~s - Molecular weight 2000.
- IR spectrum: broad band in -the region 1300-1200 cm characteristic of the sulfate groups - Barium acetate electrophoresis: Fig. 5 indicates that AH-l9 shows "slow-moving" components only, whereas the starting product also shows ~fast moving" components.
To a mixture of 10 ml of 98% sulfuric acid and 5 ml of 95% chlorosulfonic acid, cooled to a temperature be-tween -4 and 0C, there are added 500 mg of a mean molecu-10 lar weight heparin fraction (M.W.~ 10000, code number:
D-212/A), obtained by fractionation with ethanol of hepar-in PROQUIFIN, lot 7926-7935, said fraction having a sul-fatation degree (SO3/COO ) of 1.5 $
and a barium acetate electroohoretic pattern which shows a very important "fast moving" comronent. The mixture is left to stand 1 hour under stirring at room temperature, then it is poured into 250 ml of cold diethyl ether (-10 to 4C), and afterwards treated as described in Examrles 1 and 2. Thus, a deoolvmerized and suoersulfated sodium her,arin (code number : AH-17) is obtained having the following charasteristics :
- M.W.: 3000-5000 - Elemental analysis: S : 12.70%; C : 17.24~; H : 3.10~;
N :1.67~
- Sul~atation degree (S03/COO ) : 2.S
- XR spectr~ : broad band in the region 1300-1~00 cm characteristic of the sulfate grouos - ~arium acetate electrophoresis : Fig. 6 indicates that AH ~17, compared to the starting herarin fraction, shows a "slow-moving" com?onent onlyn EXAMPLE 5.
To a mixture of 20 ml of 95~ sulfuric acid and 10 ml of 93~ chlorosulfonic acid, cooled to a temnerature between -4 and 0C, there is added 1 g of heparin from pig intestinal mucosa (PROQUIFIN, lot 7926-7935, code number o D-212) having a 5ulratation degree of 1.95, then the reaction mixture is stirred 1 hour at room temperature. The mixture is poured into 500 ml of cold diethyl ether (-4 to 4C), the precipitate is filtered and ~ashed with cold diethvl ether. The product thus obtained is dissolved in 0.1 M calcium chloride a~ueous solution, then O.5 ~ calcium hydroxide is added thereto up to pH 8. The solution is dialysed against 500 ml of 0~1 ~I calcium chloride solution and then against distilled water. By slow evaporation under reduced pressure, a calcium salt of a depolymerized and su~ersulfated herJarin is obtained ~5 as a white powder.
EXAMPLES 6 _o 10.
To a mixture o. 10 ml of 98% sulfuric acid and 5 ml of chlorosulfonic acid, cooled to a temperature between -4 and 0C, there are added 500 mg of heparin from pig intestinal mucosa (PROQUIE'IN, lot 7926-7935, cocle number : D-212) having a sulfatation degree of 1.95 and a molecular weight 13500~ By operating as descri~ed in Example 1, a depolymerized,and supersul'ated heparin (code number:
AH-104) is obtained, in 98~ yield.
The same procedure and conditions are followed in 4 parallel experiments in which the same starting heparin is used~ There are obtained the products desiynated by -their code numbers AH-103, AH-105, AH-106 and AH-107. The characteristics of the products thus obtained as well as those of the 2roduct coded AH 104 are given in Table IV.
TABLE IV
_ . , ,, . . . I
Ex. Product Elemental Analysis Sulfatation Yield bv S~ C% H% N%
deg.ree weight _ _ _ _ 6 AH-104 14.54 15.42 2.84 1.43 2.9 + 0.1 98%
7 AH - 103 14.63 15.53 2.76 1.43 2.8 + 0.1 89 8 AH-105 14.48 15.43 2.61 1.44 3.0 ~ 0.1 67%
9 AH-106 14.54 15.53 2.81 1.46 2.8 + 0.1 96%
AH-107 14.12 15.65 2.80 1.40 3.0 + 0.1 ~ 77 - Molecular weight : ~6000 for the 5 products - IR spectrum : the 5 products show a spectrum identical to that of compound AH-16 described in Exam~le 1.
- Electrophoresis in hydrochloric acid: the electro-phoretic profiles are indentical to those of Eig. 1 for both the starting heparin and the 5 products ,;
- Barium acetate electroohoresis : the electrophoretic .
profiles are identical to those of Fig. 2 for both the starting heparin and the 5 products, apart from the fact that the traces relative to the 5 products do not show the background noise - caused by a temporary defect of the tracing pen or of the paper - observed in the horizontal cart of the graph of Fig. 2 relative to AH-16 - 13C-NMR spectrum: the 5 products and the s-tarting compound present the same spectra as those given in Fig.
3.
The 5 compounds thus obtained are identical each other and identical to the compound described in Example 1 as well.
EXAMPLES 11 to 14 15 In 4 parallel experiments, -to a mixture of 10 ml of 98% sulfuric acid and 5 ml of chlorosulfonic acid, cooled to -4 - 0C, there are added 500 mg of previously lyophil-ized heparin from pig intestinal mucosa (DIOSYNTH* batch CH/N 665, code number:D-479), having a sulfatation degree (SO3/COO ) of 2.1 and a molecular weight of about 11000.
The reaction mixture is left to stand 1 hour at 0C, then it is poured into 250 ml of diethyl ether previously cooled (between -10C and +4C). By operating as des-cribed in Example 1 the products of Table V are ob-tained.
TABLE V
, __ _ _ _ Ex. Product Ele! lental Analysis Sulfatation Yield S% C% H% N% degree by weigh-t __ ___ _ ._ 11 AH-108 14.88 15.29 2.52 1.47 3.1 + 0.1 90%
30 12 AH-109 14.43 15.48 2.72 1.44 3.0 + 0.1 106%
13 AH-110 14.45 15.72 2.76 1.50 2.9 + 0.1 65%
14 AH-lll 14.55 15.08 2.60 1.41 3.0 ~ 0.1 23%
_ . _ __ .......... ___ . _ . .
- Molecular weight: 6000 for the 4 products - IR spectrum:~ broad band between 1300 and 1200 cm 1, characteristlc of the sulfate group - Electrophoresis in hydrochloric acid: Fig. 7 shows the traces of the starting heparin D-479 and of one of the 4 * - Trademark ~ ... .
samples obtained in the different experiments (AH-108).
The traces of the other three compounds are identical.
This figure evidences the significant increase of the electrophoretic migration of the depolymerized and super-sulfated heparin in comparison with the starting heparin - Barium acetate electrophoresis: Fig. 8 shows the traces of the starting heparin D-479 and of AH-108. It results that the depolymerized and supersulfated heparin has a "slow-moving" electrophoretic profile, unlike the starting heparin containing ~slow-moving~ components as well as "fast-moving" components. The traces of the products AH-109, AH-110 and AH-lll are identical to that of AH-108.
To a mixture of 10 ml of 98% sulfuric acid and 5 ml of chlorosulfonic acid, cooled to a temperature between -4 and 0C, there are added 500 mg of heparin from pig intes-tinal mucosa (TEROPMON*, batch 575/018, code number:D-98) having a sulfatation degree of 1.8 and a molecular weight 135.00. By operating as described in Example 1, a depoly-merized and supersulfated heparin is obtained, in 75%yield. The product has the following characteristics:
- M.W.: ~v 6000 - Elemental analysis:S:13.90%; C:15.75%; H:2.96%; N:1.48%
- Sulfatation degree (SO3/COO ):2.8 + 0.1 - IR spectrum: broad band in the region 1300-1200 cm 1, characteristic of the sulfate groups - Electrophoresis in hydrochloric acid: Fig. 9 shows the traces of the starting heparin D-98 and of -the product AH-118. A significant increasing of the electrophoretic migration of AH-L18 compared with the starting heparin D-98, may be observed. Fig. 9 shows also that the com-pound AH-118 possesses a photodensitometric outline analo-gous to those of compounds AH-16 (Example 1, Fig. 1) and AH-17 (Example 4, Fig. 6) whereas the starting heparin D-98 appears very heterogeneous and completely different from the starting heparins utiliæed in Examples 1 and 4.
- Barium acetate electrophoresis: Fig. 10 indica-tes that AH-118 shows a "slow-moving" electrophoretic characteristic * - Trademark ,;'~ :`
, . . .
~, ~2~ r f~
which is different from that of the starting heparin D-98 showing both "slow-moving~ and "fast-moving~ components.
Fig. 10 also confirms that data of Flg. 9 and moreover surprisingly shows that compound AH-118 is not signifi-cantly different from AH-108 of Example 11, though the starting heparins are quite different.
..s~.. .
EXAM~LE 16.
. ~
To a mixture of 20 ml of 95% sul~uric acid lO ml of chlorosulfonic acid, ~reviously cooled to 0-4C, there are added 500 mg of ch1tosan ANIC, lot 116. The mi~ture is stirred at the same tem~erature for about 1 ho~r, then it is ~oured into ~reviouslv cooled diethvl ether. The ?recipitate ~Jhich forms is filtered and neutralized with a ~otassium carhonate solution. After a dial~sis in THO~S DIALYZER T~BI~JG
at 8500 D, a chitosan 6--sulate is obtained, havinc the followins characteristics :
- Substitution degree (conductime'ric ~ethod): l - IR s~ect~-um : ~road band in the reaion 1300-1200 cm 1, characteristic of the sulfate ~rou~s - 13C~N.~R sectru~ : disa~earance of the sianal o~ the ~rimar-~r h~tdxoxy ~rou~ and a~nearance of the sinal relatin~ to the sulfate arou~
EX.~IE~LE 17.
To a mixture of 20 ml of 95~ sulFuric acid and 10 ml of 98~ chlorosulfonis acid, cooled to a temDerature between -4 and 0C, there is added l a o~ chitosan ~NIC, lot 116. The reactlon mixture is left ~o stand 30 minutes at room temoerature, then it ls ?ouxed into 500 ml of ~reviousl-y cooled d~ethyl ether. After filtration, the ~recipitate is ~Jashed in water and neutralized with a solution of 0.5 ~ sodium h~,~dro~ide, then it i.s dialvzed a~ainst distilled water in membranes at 8000D ~TH0~1AS DIALV~E~ TUBI~
and eva~orated under reduced pressure. Thus, a chitosan 5-sul~ate is obtained in 90~ yield. The ~roduct has the follo~7ing characteristics :
- Substitution degree ~conductimetric method~ : 0.5, namely, 50~ only of the hvdrox~ gro1m in 6 ~osition has been sulfated - IR s~ectrum : broad band in the region 1300-1200 cm l, characteristic of the sulfate groups - 13C-NMR spectrum: diminution of the slgnal relating to the primary hydroxy group and apparatus of the signal relating to the sulfate group.
.
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 0.5 M sulfuric acid. The mixture is stirred 12 hours at room temperature, concentrated under reduced pressure and treated with acetone. The precipi-tate which forms is filtered, washed with acetone, dried, dissolved in water and treated with 30 ml of sodium borohydride. After 12 hours at room temperature, the excess of sodium borohydride is destroyed with AMBERLITE*
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 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 be-tween -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. The product is dissolved in water and neu-tralized with a 0.5 M sodium hydroxide solution. After desalting by chromatography on Sephadex* G25, a depolymer-ized chitosan 6-sulfate is obtained in a 90% yield. The product has the following characteristics:
- Substitution degree:l * - Trademarks ~.,,1-- IR s~ectrum : broad band in the resion 1300-1200 cm 1, characteristic of the sulfate grou~s - 13C-NMR spectrum : disa~pearance o~ the signal relating to the primarv hydroxv group and appearance of a ne~l signal due to the sulfate grou~.
EXAMPLE 19.
To a mixture of 20 ml of 95~ sulfuric acid and 10 ml of 98~ chlorosulfonic acid, cooled to 0-4C, there is added 1 g of cellulose microcristalline (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 ~oured into 500 ml of cold diethyl ether, then it is filtered, the preci~itate is washed with diethyl ether and disssolved in water.
By neutralization with a 0.5 M sodium hydroxide solu~ion, dialysis in membranes at 3500 D (TUO~S
DIALYZE?~ TUBING 3787~H 47, 11 m~ diameter) and evapora-tion under ~ressure, a cellulose 6-sulfate is ohtained, with a 36go yield Of dialvsable fraction and 29~ vield of non-dialysable fraction. The ~roduct has the follow-ing characteristics :
- Substitution degree (conductimetric metho~
- IR s~ectrum : broad band in the region 1300-1200 cm 1, ~5 characteristic of the sulfate grou~s - Molecular weight : 3400 E~PLES 20 to 22.
In three parallel ex~eriments, 500 mg of heparin from ~ig intestinal mucosa (DIOSYNTH, batch CH/N 665, code n. D-479) having a sulfatation degree (S03/COo ) 2.1 and a molecular ~eight 11000 are added to 15 ml of a mixture of 98g~ sulfuric acid and 98 chlorosulfonic acid in the following ratios :
Example 20 : 1:4 Example 21 ~
Exam~le 22 : 4:1 By operating as described in Example 1, three depolymer-ized and supersulfated heparins, having the characteris-tics given in Table VI, are obtained.
TABLE VI
Example Sulfatation Molecular Yield (code NO) degree weight % by weight 2.5 4000 89.9 10 (AH-67) 21 2.5 3800 86.8 (AH-65) 22 2.8 4500 77.4 (AH-68) __ To a mlxture of 10 ml of 98% sulfuric acid and 5 ml of 98% chlorosulfonic acid, at the temperature of 0 - 4C, 1 g of guaran (AGOGUM* F-90, lot 433) is added. After 1 hour at *he same temperature, by operating as described in Example 1, a guaran 6-sulfate is isolated as sodium salt (code No . AH-102). The product has the following charac-teristics:
- Substitution degree (conductimetric method):l - IR spectrum: broad band in the region between 1300 and 1200 cm 1, characteristic of the sulfate group.
Yleld: 26% by weight To a mixture of 20 ml of 95% sulfuric acid and 10 ml 30 of 98% chlorosulfonic acid, cooled to 0 - 4C, 1 g of chitin (SIGMA, lot 12 F-7060) is added. The reaction mixture is left to stand 1 hour at the same temperature.
Then, by operating as described in Example 1, after dial-ysis and evaporation under reduced pressure a chitin 6-sulfate is ob-tained and isolated as sodium salt (code No. AH-50). The product has the following characteristics:
- Substitution degree (conductimetric method):l - IR spectrum: broad band in the region between 1300 and * - Trademark 1200 cm 1, characteristic of the sulfate group.
- 13C-NMR spectrum:disappearance of the signal of the primary hydroxy group and appearance of the signal of sulfate groups.
Yield: 70% by weight To a mixture of 15 ml of 95% sulfuric acid: 98%
chlorosulfonic acid 2:1, cooled to 0 - 4C, there are added 500 mg of chondroitinsulfate TAKEDA* (lot BB-185, No. Code:D-267) having a molecular weight 22000 and con-taining 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 dialyzed in tubes at 3500 D (THOMAS DIALYZER TUBING, diameter 15 mm). By evaporating under reduced pressure a depolymerized and supersulfa-ted chondroitinsulfate (code No. AH 69) having the fbllowing characteristics is obtained;
- IR spectrum: broad band in the region 1300-1200 cm 1, characteristic of the sulfate groups - Molecular weight: 2000 To a mixture of 10 ml of 98% sulfuric acid and 5 ml of 98% chlorosulfonic acid, there is added 500 ml of dermatansulfate OPOCRIN* (lot 7-8 HF) having a molecular weight 27000 and a substitution degree (SO3/COO ):1. By operating as described in Example 25, a depolymerized and supersulfated dermatansulfate (code No. AH-79) is obtained.
The product has the following characteristics:
* - Trademarks ~2~
- Substitution de~ree (S03/C00 ), conductimetric method ) : 2.8 - IR s~ectrum broad band in the resion between 1300 and 1200 c~ 1, characteristic of sulfate grou~s - Molecular weight 2000.
Claims (26)
1. A process for the depolymerisation and sulfatation of polysaccharides, which comprises reacting said polysaccar-ides with a mixture of sulfuric acid and chlorosulfonic acid.
2. A process as claimed in claim 1 in which the reaction is carried out at temperature of from -20° to +40°C.
3. A process as claimed in claim 1 in which the concen-tration of the two acids is at least 95% by weight.
4. A process as claimed in one of claims 1 to 3 in which the ratio of sulfuric acid:chlorosulfonic acid is from 4:1 to 1:1.
5. A process as claimed in claim 2, in which the concen-tration of the two acids is at least 95% by weight and -the ratio of sulfuric acid:chlorosulfonic acid is from 4:1 to 1 : 1 .
6. A process as claimed in claim 5, in which the ratio of sulphuric acid:chlorosulfonic acid is about 2:1.
7. A process as claimed in one of claims 1 to 3 in which the ratio of sulfuric acid:chlorosulfonic acid is about 2:1.
8. A process as claimed in one of claims 1 to 3 in which the depolymerized and sulfated polysaccharide is isolated as sodium salt.
9. A process as claimed in any one of claims 1 to 3, in which the weight ratio of sulfuric acid:chlorosulfonic acid is from 4:1 to 1:1 and the depolymerized and sulfated polysaccharide is isolated as sodium salt.
10. A process as claimed in any one of claims 1 to 3, in which the weight ratio of sulphuric acid-chlorosulfonic acid is about 2:1 and the depolymerized sulfated polysac-caride is isolated as sodium salt.
11. A process as claimed in claim 5 or 6 in which the depolymerized and sulfated polysaccharide is isolated as sodium salt.
12. A process as claimed in one of claims 1 to 3 in which the depolymerized and sulfated polysaccharide is trans-formed into one of its salts.
13. A process as claimed in any one of claims 1 to 3, in which the weight ratio of sulfuric acid:chlorosulfonic acid is from 4:1 to 1:1 and the depolymerized and sulfated polysaccharide is transformed into one of its salts.
14. A process as claimed in any one of claims 1 to 3, in which the weight ratio of sulfuric acid:chlorosulfonic acid is about 2:1 and the depolymerized and sulfated polysaccharide is transformed into one of its salts.
15. A process as claimed in claim 5 or 6 in which the depolymerized and sulfated polysaccharide is transformed into one of its salts.
16. A process as claimed in any one of claims 1 to 3 in which the depolymerized and sulfated polysaccharide is isolated as sodium salt and is transformed into another salt by exchange with the appropriate salt.
17. A process as claimed in any one of claims 1 to 3, in which the weight ratio of sulfuric acid:chlorosulfonic acid is from 4:1 to 1:1, and the depolymerized and sulfated polysaccharide is isolated as sodium salt and is trans-formed into another salt by exchange with the appropriate salt.
18. A process as claimed in any one of claims 1 to 3, in which the weight ratio of sulfuric acid:chlorosulfonic acid is about 2:1 and the depolymerized and sulfated poly-saccharide is isolated as sodium salt and is transformed into another salt by exchange with the appropriate salt.
19. A process as claimed in claim 5 or 6, in which the depolymerized and sulfated polysaccharide is isolated as sodium salt and is transformed into another salt by ex-change with the appropriate salt.
20. A process as claimed in one of claims 1 to 3 in which the starting polysaccharide is previously depolymerized.
21. A process as claimed in any one of claims 1 to 3, in which the weight ratio of sulfuric acid:chlorosulfonic acid is from 4:1 to 1:1, and the starting polysaccharide is previously depolymerized.
22. A process as claimed in any one of claims 1 to 3, in which the weight ratio of sulfuric acid:chlorosulfonic acid is about 2:1, and the starting polysaccharide is previously depolymerized.
23. A process as claimed in claim 5 or 6, in which the starting polysaccharide is previously depolymerized.
24. The process as claimed in any one of claims 1 to 3, in which the depolymerized and sulfated polysaccharide is isolated as sodium salt, and the starting polysaccharide is previously depolymerized.
25. The process as claimed in any one of claims 1 to 3, in which the depolymerized and sulfated polysaccharide is transformed into one of its salts, and the starting poly-saccharide is previously depolymerized.
26. The process as claimed in any one of claims 1 to 3, in which the depolymerized and sulfated polysaccharide is isolated as sodium salt, and is transformed into another salt by exchange with the appropriate salt, and the star-ting polysaccharide is previously depolymerized.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8221934A FR2538404B1 (en) | 1982-12-28 | 1982-12-28 | |
| FR8221934 | 1982-12-28 | ||
| FR8319506 | 1983-12-05 | ||
| FR8319506A FR2555993B1 (en) | 1983-12-06 | 1983-12-06 | 6-SULPHATE CHITOSANES AND PROCESS FOR THEIR PREPARATION |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1218986A true CA1218986A (en) | 1987-03-10 |
Family
ID=26223212
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000443861A Expired CA1218986A (en) | 1982-12-28 | 1983-12-21 | Process for the depolymerisation and sulfatation of polysaccharides |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0116251B1 (en) |
| KR (1) | KR920003692B1 (en) |
| AU (1) | AU563377B2 (en) |
| CA (1) | CA1218986A (en) |
| DE (1) | DE3374935D1 (en) |
| DK (1) | DK598083A (en) |
| IE (1) | IE56783B1 (en) |
| IL (1) | IL70511A (en) |
| NZ (1) | NZ206698A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4956173A (en) * | 1987-11-25 | 1990-09-11 | Societe Anonyme: Sanofi | Composition and use of ademetionine against ageing of the skin |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2555993B1 (en) * | 1983-12-06 | 1986-11-07 | Anic Spa | 6-SULPHATE CHITOSANES AND PROCESS FOR THEIR PREPARATION |
| FR2538404B1 (en) | 1982-12-28 | 1985-08-23 | Anic Spa | |
| DE3422407A1 (en) * | 1984-06-16 | 1986-03-06 | B. Braun Melsungen Ag, 3508 Melsungen | USE OF HEPARINE DERIVATIVES FOR SELECTIVE EXTRA-CORPORAL PRECIPITATION OF LOW-DENSITY-LIPOPROTEINS FROM FULL SERUM OR PLASMA |
| FR2584728B1 (en) * | 1985-07-12 | 1987-11-20 | Choay Sa | PROCESS FOR THE SULFATION OF GLYCOSAMINOGLYCANS AND THEIR FRAGMENTS |
| JP2511829B2 (en) * | 1987-03-19 | 1996-07-03 | アースロファーム ピーティーワイ．リミティッド | Anti-inflammatory compounds and compositions |
| DE3744119A1 (en) * | 1987-12-24 | 1989-07-06 | Basf Ag | USE OF POLYSULFATED HEPARINES |
| IT1237518B (en) * | 1989-11-24 | 1993-06-08 | Renato Conti | SUPER-SULFATED HEPARINS |
| US6063773A (en) * | 1995-09-29 | 2000-05-16 | Polydex Pharmaceuticals Ltd. | Cellulose sulfate for use as antimicrobial and contraceptive agent |
| US7078392B2 (en) | 2000-06-30 | 2006-07-18 | Polydex Pharmaceuticals Limited | Cellulose sulfate and other sulfated polysaccharides to prevent and treat papilloma virus infection and other infections |
| EP2025687A1 (en) * | 2007-07-23 | 2009-02-18 | Istituto Scientifico di Chimica E Biochimica "G Ronzoni | Process for the preparation of heparanase-inhibiting sulfated hyaluronates and products obtained thereby |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2025073A (en) * | 1934-01-09 | 1935-12-24 | Du Pont | Cellulose derivative and method of making the same |
| US2755275A (en) * | 1952-08-29 | 1956-07-17 | Abbott Lab | Process for sulfating chitin |
| FR1093999A (en) * | 1952-10-15 | 1955-05-11 | Upjohn Co | Process for preparing sulfated chitosan |
| US3454560A (en) * | 1966-03-01 | 1969-07-08 | Seikagaku Kogyo Co Ltd | Process for the production of chondroitin polysulfate |
-
1983
- 1983-12-21 IL IL70511A patent/IL70511A/en unknown
- 1983-12-21 CA CA000443861A patent/CA1218986A/en not_active Expired
- 1983-12-22 NZ NZ206698A patent/NZ206698A/en unknown
- 1983-12-23 DK DK598083A patent/DK598083A/en not_active Application Discontinuation
- 1983-12-23 IE IE3063/83A patent/IE56783B1/en not_active IP Right Cessation
- 1983-12-23 AU AU22856/83A patent/AU563377B2/en not_active Ceased
- 1983-12-26 EP EP83402534A patent/EP0116251B1/en not_active Expired
- 1983-12-26 DE DE8383402534T patent/DE3374935D1/en not_active Expired
- 1983-12-27 KR KR1019830006213A patent/KR920003692B1/en not_active IP Right Cessation
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4956173A (en) * | 1987-11-25 | 1990-09-11 | Societe Anonyme: Sanofi | Composition and use of ademetionine against ageing of the skin |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0116251B1 (en) | 1987-12-16 |
| DK598083A (en) | 1984-06-29 |
| KR920003692B1 (en) | 1992-05-09 |
| AU2285683A (en) | 1984-07-05 |
| EP0116251A1 (en) | 1984-08-22 |
| IE833063L (en) | 1984-06-28 |
| IL70511A0 (en) | 1984-03-30 |
| NZ206698A (en) | 1986-11-12 |
| DK598083D0 (en) | 1983-12-23 |
| DE3374935D1 (en) | 1988-01-28 |
| AU563377B2 (en) | 1987-07-09 |
| IL70511A (en) | 1990-01-18 |
| KR840006813A (en) | 1984-12-03 |
| IE56783B1 (en) | 1991-12-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4948881A (en) | Process for the depolymerization and sulfation of polysaccharides | |
| CA1210760A (en) | Process for preparing depolymerized and supersulfated heparin | |
| US4500519A (en) | Mucopolysaccharides having biological properties, preparation and method of use | |
| US4303651A (en) | Heparin fragments having selective anticoagulation activity | |
| CA1218986A (en) | Process for the depolymerisation and sulfatation of polysaccharides | |
| FI67865C (en) | REFRIGERATOR FOR SUSPENSION AV EN COMPOSITION AV SULFATGRUPPER INNEHAOLLANDE POLYSACKARIDER | |
| FI103050B (en) | Process for the preparation of a composition containing N, O-sulf atherated heparosans | |
| US4814437A (en) | Method for preparation of sulfated polysaccharides by treating the polysaccharide with a reducing agent before sulfation | |
| US4804652A (en) | Mucopolysaccharides having biological properties, preparation and application thereof as drugs | |
| JPH0326701A (en) | Hydroxyethyl sthrch as plasma additives and their manufacture | |
| JP2003523460A5 (en) | ||
| KR910006809B1 (en) | Process for the preparation of neutral heparan sulphate and dermatan sulphate | |
| EP0970130B1 (en) | Glycosaminoglycans having high antithrombotic activity | |
| JP2702377B2 (en) | Semi-synthetic glycosaminoglycans containing α-L-galacturonic acid substituted at the 3-position with a nucleophilic group | |
| DE602004012314T2 (en) | LOW-MOLECULAR POLYSACCHARIDES WITH ANTITHROMBOTIC EFFECT | |
| JPH0643446B2 (en) | Chitosan 6-sulfate and process for producing the same | |
| CA2458651C (en) | Saccharide sulfation methods | |
| WO2003106505A9 (en) | Process for the manufacture of n-acyl-(epi)k5-amine-o-sulfate-derivatives and products thus obtained | |
| EP3724235B1 (en) | Process for the preparation of low molecular weight heparin | |
| US20120253029A1 (en) | Process for the preparation of highly o-sulfated epimerized derivatives of k5 polysaccharide and intermediates therein | |
| KR20050024339A (en) | Process for the manufacture of n-acyl-(epi)k5-amine-o-sulfate-derivatives and products thus obtained | |
| KR20050024349A (en) | Epimerized derivatives of k5 polysaccharide with a very high degree of sulfation | |
| Sutherland | Structural studies in certain animal polysaccharides | |
| ZA200410357B (en) | Process for the manufacture on N-acyl-(EPI)K5-amine-o-sulfate-derivatives and products thus obtained. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |