, Volume 13, Issue 4, pp 609-619

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

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Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz1H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcAβ1-3GalNAc(4-sulfate) and/or GlcAβ1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)β1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcAβ1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum α-N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.