O-sulfated bacterial polysaccharides

Details O-sulfated bacterial polysaccharides O-sulfated bacterial polysaccharides

1999-07-23

2001-09-11

Fonda, Kathleen Kahler (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S056000, C536S018700, C536S021000, C536S123100, C536S124000

Reexamination Certificate

active

06288044

ABSTRACT:

PRIOR ART
The bacterial polysaccharides identified as K4, K5 and K40, consisting of repeating sequences of D-glucuronic acid and amino sugars are known. They are obtained according to known techniques by fermentation of different strains of
E. coli
as it is described in several papers (Manzoni M., Bergomi S., F. and Cavazzoni V. Biotechnology Letters 18 (4) 383-386 (1996); B. Rodriguez M. R. et al., Eur. J. Biochem., 177 (1988) 117-124; and Dengler T. et al., Carb. Res., 150 (1986) 233-240).
Moreover, the O-sulfation processes studied for other polysaccharides as by example for heparin after complete desulfation are known.
Said processes provide for a preliminary treatment suitable to remove from the polysaccharide its natural counter-ion (usually sodium) in order to substitute it with tributyl ammonium hydroxide (Ogamo et al. Carb. Res. 193 (1989) 165-172) or with pyridine (Rej et al., Carb. Res. 210 (1991) 299-310) in order to make the polysaccharide itself soluble in solvents as the dimethylformamide or the dimethylsulfoxide.
Said processes require several steps among which an expensive liophilization step too. Moreover they exhibit several drawbacks as for example the weight doubling due to the counter-ion.
SUMMARY
We found that it is possible to obtain the O-sulfated K4, K5 and K40 polysaccharides by a process wherein said polysaccharides in the form of sodium salts are directly sulfated in a suspension in an organic solvent. The process according to the present invention includes the following steps:
a) suspension of the polysaccharide in the form of sodium salts in an aprotic solvent;
b) O-sulfation with a pyridine-sulphur trioxide or trimethylamine-sulphur trioxide adduct or with chlorosulfonic acid;
c) dilution with water;
d) pH adjustment to a basic value;
e) precipitation by addition of ethanol saturated with sodium acetate;
f) dissolution by a NaCl solution;
g) diafiltration;
h) precipitation of the product by alcohols as ethanol or methanol or isopropanol or by acetone;
i) drying.
Optically K4 is preliminary defructosilated.
By the process according to the present invention polysaccharides having a molecular weight ranging from 4,000 to 35,000 and a sulfates/carboxyls ratio ranging from 0.5 to 4.0 are obtained.
The O-sulfated polysaccharides according to the present invention show an interesting anti-angiogenetic and antiviral as well as anticoagulant activity. Moreover they are effective in the cosmetic art in the prevention of hair falling out.


REFERENCES:
patent: 5550116 (1996-08-01), Lormeau et al.
Biotechnology Letters, vol. 18, No. 4 (Apr. 1996), p. 383-386; “Production and Purification of an Extracellularly Produced K4 Polysaccharide fromE. coli”; M. Manzoni et al.
Eur. J. Biochem., 177 (1988), 117-124; “Structure and Serological Characteristics of the Capsular K4 Antigen ofE. coli05:K4:H4, a Fructose-Containing Polysaccharide with a Chondroitin Backbone”; B. Rodriguez M.R. et al.
Carbohydrate Research, 150 (1986) 233-240; “Structure of the Serine-Containing Capsular Polysaccharide K40 Antigen fromE. coli08:K40:H9”; Thomas Dengler et al.
Carbohydrate Research, 193 (1989) 165-172; “Reactivity Toward Chemical Sulfation of Hydrozyl Groups of Heparin”; Akira Ogamo, et al.
Carbohydrate Research, 210 (1991) 299-310; “Sulfation of Some Chemically-Modified Heparins—Formation of a 3-Sulfate Analog of Heparin”; Rabindra N. Rej et al.

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