title: Determination of substitution positions in hyaluronic acid hydrogels using NMR and MS based methods.
abstract: In hydrogels of cross-linked polysaccharides, the total amount of cross-linker and the degree of cross-linking influence the properties of the hydrogel. The substitution position of the cross-linker on the polysaccharide is another parameter that can influence hydrogel properties; hence methods for detailed structural analysis of the substitution pattern are required. NMR and LC-MS methods were developed to determine the positions and amounts of substitution of 1,4-butanediol diglycidyl ether (BDDE) on hyaluronic acid (HA), and for the first time it is shown that BDDE can react with any of the four available hydroxyl groups of the HA disaccharide repeating unit. This was achieved by studying di-, tetra-, and hexasaccharides obtained from degradation of BDDE cross-linked HA hydrogel by chondroitinase. Furthermore, amount of linker substitution at each position was shown to be dependent on the size of the oligosaccharides. For the disaccharide, substitutions were predominantly at ΔGlcA-OH2 and GlcNAc-OH6 while in the tetra- and hexasaccharides, it was mainly at the reducing end GlcNAc-OH4. In the disaccharide there was no substitution at this position. Since chondroitinase is able to completely hydrolyse non-substituted HA into unsaturated disaccharides, these results indicate that the enzyme is prevented to cleave on the non-reducing side of an oligosaccharide substituted at the reducing end GlcNAc-OH4. The procedure can be adopted for the determination of substitution positions in other types of polymers.
authors.affiliation: Department of Chemistry and Biotechnology, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden. Electronic address: Frida.Wende@slu.se.
authors.forename: Frida J
authors.initials: FJ
authors.lastname: Wende
authors.affiliation: Department of Chemistry and Biotechnology, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden. Electronic address: Suresh.Gohil@slu.se.
authors.forename: Suresh
authors.initials: S
authors.lastname: Gohil
authors.affiliation: Research A&C Galderma, Seminariegatan 21, SE-752 28 Uppsala, Sweden. Electronic address: Hotan.Mojarradi@galderma.com.
authors.forename: Hotan
authors.initials: H
authors.lastname: Mojarradi
authors.affiliation: Galderma R&D, Les Templiers, 2400 route des Colles BP 87, 06410 Biot Cedex, France. Electronic address: Thibaud.Gerfaud@galderma.com.
authors.forename: Thibaud
authors.initials: T
authors.lastname: Gerfaud
authors.affiliation: Research A&C Galderma, Seminariegatan 21, SE-752 28 Uppsala, Sweden. Electronic address: Lars.Nord@galderma.com.
authors.forename: Lars I
authors.initials: LI
authors.lastname: Nord
authors.affiliation: Research A&C Galderma, Seminariegatan 21, SE-752 28 Uppsala, Sweden. Electronic address: Anders.Karlsson@galderma.com.
authors.forename: Anders
authors.initials: A
authors.lastname: Karlsson
authors.affiliation: Galderma R&D, Les Templiers, 2400 route des Colles BP 87, 06410 Biot Cedex, France. Electronic address: Jean-Guy.Boiteau@galderma.com.
authors.forename: Jean-Guy
authors.initials: JG
authors.lastname: Boiteau
authors.affiliation: Research A&C Galderma, Seminariegatan 21, SE-752 28 Uppsala, Sweden. Electronic address: Anne.HelanderKenne@galderma.com.
authors.forename: Anne Helander
authors.initials: AH
authors.lastname: Kenne
authors.affiliation: Department of Chemistry and Biotechnology, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden. Electronic address: Corine.sandstrom@slu.se.
authors.forename: Corine
authors.initials: C
authors.lastname: Sandström
mesh_terms: D002072:Butylene Glycols / Q000737:chemistry; D002810:Chondroitinases and Chondroitin Lyases / Q000378:metabolism; D006820:Hyaluronic Acid / Q000737:chemistry*; D020100:Hydrogels / Q000737:chemistry*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000737:chemistry; D011514:Proteus vulgaris / Q000201:enzymology
chemical_list: D002072:Butylene Glycols; D020100:Hydrogels; D009844:Oligosaccharides; D006820:Hyaluronic Acid; C014376:1,4-bis(2,3-epoxypropoxy)butane; D002810:Chondroitinases and Chondroitin Lyases
keywords: Cross-linked; Hyaluronic acid; Hydrogel; MS; NMR; Substitution