Elsevier

Carbohydrate Polymers

Volume 136, 20 January 2016, Pages 1348-1357
Carbohydrate Polymers

Determination of substitution positions in hyaluronic acid hydrogels using NMR and MS based methods

https://doi.org/10.1016/j.carbpol.2015.09.112Get rights and content
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Highlights

  • NMR and MS methods to determine linker position in HA hydrogels were developed.

  • Oligosaccharides of different sizes were obtained by enzymatic degradation.

  • Substitution occur at all four available hydroxyl groups of the HA repeating unit.

  • Linker distribution among the four positions was obtained by NMR and MS.

  • Distribution of substitution positions depend on the size of the oligosaccharides.

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.

Abbreviations

HA
hyaluronic acid/hyaluronan
BDDE
1,4-butanediol diglycidyl ether
BDPE
1,4-butanediol di-(propan-2,3-diolyl)ether
DCM
dichloromethane
THF
tetrahydrofurane
TOCSY
total correlation spectroscopy
HSQC
heteronuclear single quantum correlation
NOESY
nuclear Overhauser effect spectroscopy
HMBC
heteronuclear multiple bond correlation
VCD
Vibrational circular dichroism

Keywords

Hyaluronic acid
Hydrogel
Cross-linked
Substitution
NMR
MS

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