| 1. |
- Zhang, Tianji, et al.
(författare)
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Characterization of epimerization and composition of heparin and dalteparin using a UHPLC-ESI-MS/MS method
- 2019
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Ingår i: Carbohydrate Polymers. - : ELSEVIER SCI LTD. - 0144-8617 .- 1879-1344. ; 203, s. 87-94
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Tidskriftsartikel (refereegranskat)abstract
- Heparin is a significant anticoagulant that has been used in clinic over decades. Although numerous efforts have been made to characterize the molecular structure of heparin and its derivatives for safety of the medicine, technical barriers still exist because of their structural complexity. In this study, we have established a method capable to evaluate both the epimerization and composition of heparin and dalteparin by a UHPLC-HILIC/WAXMS/MS approach. Ten major disaccharide building blocks retaining the epimerization configuration of parental heparin chains were generated and well separated, 9 of which were unambiguously identified. Isomer identifications were achieved through high-resolution tandem mass spectrometry analysis with reference to elaborately prepared standards. The method was successfully applied for the sameness study of generic dalteparins in combination with an isotopic labelling procedure. We believe this robust method maybe adapted to quality control in pharmaceutical production of heparin and LMWHs.
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| 2. |
- Zhang, Tianji, et al.
(författare)
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Interacting polymer-modification enzymes in heparan sulfate biosynthesis
- 2023
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Ingår i: Carbohydrate Polymers. - : Elsevier. - 0144-8617 .- 1879-1344. ; 299
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Tidskriftsartikel (refereegranskat)abstract
- Glucuronyl 5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) into L-iduronic acid (IdoA) units, through a mechanism involving reversible abstraction of a proton at C5 of hexuronic acid residues. Incubations of a [4GlcA beta 1-4GlcNSO3 alpha 1-]n precursor substrate with recombinant enzymes in a D2O/H2O medium enabled an isotope exchange approach to the assessment of functional interactions of Hsepi with hexuronyl 2-O-sulfotrans-ferase (Hs2st) and glucosaminyl 6-O-sulfotransferase (Hs6st), both involved in the final polymer-modification steps. Enzyme complexes were supported by computational modeling and homogeneous time resolved fluores-cence. GlcA and IdoA D/H ratios related to product composition revealed kinetic isotope effects that were interpreted in terms of efficiency of the coupled epimerase and sulfotransferase reactions. Evidence for a func-tional Hsepi/Hs6st complex was provided by selective incorporation of D atoms into GlcA units adjacent to 6-O -sulfated glucosamine residues. The inability to achieve simultaneous 2-O-and 6-O-sulfation in vitro supported topologically separated reactions in the cell. These findings provide novel insight into the roles of enzyme in-teractions in heparan sulfate biosynthesis.
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| 3. |
- Zhang, Tianji, et al.
(författare)
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Oligosaccharides mapping of nitrous acid degraded heparin through UHPLC-HILIC/WAX-MS
- 2020
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Ingår i: Carbohydrate Polymers. - : Elsevier. - 0144-8617 .- 1879-1344. ; 231
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Tidskriftsartikel (refereegranskat)abstract
- Building blocks characterization is a significant approach for understanding the molecular structure of heparin and its derivatives. Nitrous acid (HONO) depolymerization of heparin generates oligosaccharides that maintain the epimerization conformation on C5 of the uronic acids, reflecting the authentic structure of the parental chain. HONO treatment at pH 1.5 selectively cleaves the bond between N-sulfated glucosamine and hexuronic acid, resulting mainly disaccharides, as well as tetra-, tri-, and mono-saccharides. The tetrasaccharides are derived from the structure of N-acetylated domains while tri-, and mono-saccharides are derived from the reducing or the non-reducing end of the heparin chain. The resulted oligosaccharides were separated and analyzed using a UHPLC-HILIC/WAX-MS method. We succeeded in the identification of 19 tetrasaccharides, 19 trisaccharides and 4 monosaccharides species, majority of which is structurally characterized. By comparing the theoretical possibilities and actual occurrence of the well-characterized tetrasaccharides, we demonstrated that the biosynthesis of heparin is a systematic process.
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