Development of A Recycling Approach for Carbohydrate Derivatization with Aminooxy-based Labeling Reagents and Its Application to Purification and Identification of Oligosaccharides from Natural Source
Date Issued
2011
Date
2011
Author(s)
Wen, Hao-Yu
Abstract
Oligosaccharides with specific structures play a critical role in many biological functions such as cell-cell recognition, cytokine expressions and biomarkers. Many chemoselective ligation methods for carbohydrate have been developed to incorporate chromophores/fluorophores; however, most of them are often limited by slow reaction rate at neutral pH and non-reversibility of the modified compounds to their parental sugars for further studies.
In this study, we utilized the aminooxy-based labeling reagents, O-(2-naphthylmethoxy)amine, N-methyl-(2-naphthylmethoxy)amine and N-methyl-(9-anthrylmethoxy)
amine, to develop a reversible bioorthogonal glycoconjugation. Separation of these glycoconjugates was facilitated by easy detection of the fluorophore of naphthyl or anthryl groups. The naphthylmethyl oxime derivatives underwent a palladium-catalyzed hydrogenolysis, followed by deoximation with NBS, to regenerate their parental sugars.
The oxime ether derivative of saccharide bearing a free OH group at the C-5 position, underwnet the hydrogenolysis reaction to an N-hydroxy glycopyranosylamine intermediate,
which was labile to hydrolysis under acidic conditions to give directly the parental sugar. These results demonstrated that our approach allowed the separation and regeneration of many types of mono-, di-, and oligosaccharides. This oxime-based derivatization
method has several advantageous features including high chemoselectivity, high tolerance to di erent solvents, and high plasticity of chromogenic moiety.
Dendrobium huoshanense, a famous traditionally Chinese herb, has been utilized to prevent inflammation and deterioration of eyesight and to promote self-immunity.
The mucilage polysaccharide with acetyl groups from the stem of D. huoshanense showed to enhance the expression of granulocyte colony-stimulating factor (G-CSF), a
glycoprotein which can stimulate the mature of monogranulocytes via a NF- b-mediated
signal pathway in Raw 264.7 macrophages. A serious of experiment to determine the bioactive structure from polysaccharide mixture, including liquid chromatography,
matrix-assisted laser desorption/ionization-time of flight mass spectroscopy (MALDITOF MS), reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked
immunosorbent assay (ELISA), have been carried out. Our preliminary analyses from MS, RT-PCR and ELISA assays implied that a -1,4-oligomannoside containing seven
units of mannosides, with or without acetylation, was a possible smallest bioactive oligosaccharide for the production of G-CSF. The higher oligomannosides, e.g. up
to 12 DP exhibited even higher activity of G-CSF induction. Furthermore, separation of D. huoshanense oligosaccharide mixture by recycling derivatization with N-methyl-
(2-naphthylmethoxyl)amine was also successfully performed to obtain the portions of higher-molecular-weight oligosaccharide. This strategy will be a useful tool for the
isolation of the bioactive oligosaccharide from medicinal herbs.
Subjects
carbohydrate derivatization
oligosaccharide
polysaccharide
Dendrobium
SDGs
Type
thesis
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