BIOTECHNOLOGICAL SULPHATED CHONDROITIN SULPHATE AT POSITION 4 OR 6 ON THE SAME POLYSACCHARIDE CHAIN, AND PROCESS FOR THE PREPARATION THEREOF

摘要

The present invention discloses a process for the production of chondroitin sulphate with an average molecular weight (Mw) of 10-30 kDa by chemical sulphation starting from an unsulphated chondroitin backbone, obtained in turn by acid hydrolysis of capsular polysaccharide K4 made directly from E. coli strain O5:K4:H4, or directly produced from a genetically modified strain of E. coli. Sulphation of the N-acetyl-D-galactosamine residue at position 4 or 6 takes place simultaneously in the same polysaccharide chain, simulating the sulphation pattern observed in natural chondroitin sulphate, unlike the sulphation obtained with the synthesis methods described to date.

主权项

1. Process for the preparation of chondroitin sulphate sodium salt in which all the N-acetyl-D-galactosamine units in the same polysaccharide chain are monosulphated either randomly or at the 4- or 6- position, said process comprising the following steps:
a. transforming chondroitin sodium salt into its free acid or a salt thereof with a quaternary ammonium cation selected from tetramethylammonium, tetraethylammonium or tetrabutyl-ammonium, or into the pyridinium salt or the methyl ester; b. reacting the compound obtained in step a) with an orthoester of formula RC(OR1)3, in which R is selected from hydrogen, methyl, ethyl or phenyl and R1 is selected from methyl or ethyl, in the presence of acid catalysis, to give a compound in which the repeating disaccharide unit present in chondroitin has the formula I in which R and R1 are as defined above; c. protecting the hydroxy groups at the 2′- and 3′-positions of the glucuronic acid units of the compound obtained in the previous step by reaction with an anhydride of formula (R2CO)2O in which R2 is selected from methyl, ethyl or propyl, in the presence of pyridine or an organic tertiary base selected from triethylamine or triisopropylamine and of 4-dimethylaminopyridine (DMAP), to give a compound in which the repeating disaccharide unit present in chondroitin has the formula II in which R, R1 and R2 are as defined above; d. rearranging the orthoester functionality present in the product obtained in step c) with an organic water-soluble acid to give an ester derivative in which the repeating GalNAc units in the polysaccharide consist of triacyl derivatives having formula IIIIa or IIIb in which R and R2 are as defined above; e. mono-sulphating the compound obtained in step d) followed by removal of the O-acyl groups present in compounds IIIa and IIIb obtained in the previous step.