FTIR characterisation of chondroitin sulfate E (CS-E) di-, tetra-, and hexasaccharide derivatives and their biotinylated or reducing conjugates

Abstract

Sulfated glycosaminoglycans (GAGs), namely, chondroitin sulfate (CS), dermatan sulfate, keratan sulfate, heparin, and heparan sulfate, are linear complex polysaccharides that are covalently attached to core proteins to form proteoglycans. They are present at the cell surface and in the extracellular matrix and play a key role in the regulation of cellular microenvironmental effectors. To better understand the biological functions of GAGs and particularly of CS-E (4,6-disulfated) at the molecular level, structurally well-defined oligosaccharides are necessary. Chemically synthesised biotinylated conjugates are useful to study the interactions with proteins at the intra- and extracellular levels. Herein, FTIR spectroscopy was used to characterise nine chondroitin oligosaccharides, including biotinylated or reducing CS-E di-, tetra- and hexasaccharides as well as their non-sulfated analogs. Spectral features characteristic of the vibrational modes of oligosaccharides (1640, 1626, 1565, 1418, 1375, and 1160 cm⁻¹), CS-E (1280–1200, 1134, 1065, 1034, 1000, 927, and 866–860, 815 cm⁻¹), and biotin (1681, 1460, 1425, and 792 cm⁻¹) were identified. FTIR spectroscopy was sensitive enough to reveal structural microheterogeneity, allowing distinguishing C-4 from C-6 sulfated isoforms. CS-E- and biotin-specific signatures were obtained via difference spectra. PCA plots revealed three distinct groups: biotinylated oligosaccharides, CS-E biotinylated oligosaccharides and CS-E reducing oligosaccharides. Furthermore, the first component clearly distinguished sulfated from non-sulfated forms, while component two tended to discriminate according to the chain length, exclusively for non-sulfated oligosaccharides. Identifying the spectral signatures of these oligosaccharides is an important step for future research on the monitoring of the internalisation of oligosaccharide- and cell-penetrating peptide-bound forms in drug-delivery studies.