Glucosamine sulfoforms: Solid-phase synthesis, mass spectrometry analysis and biological profiling

Runhui Liu, Purdue University

Abstract

Sulfated α-glucosamine derivatives (GlcN sulfoforms) bearing hydroxyethyl linkers were prepared by solid-phase synthesis, establishing the feasibility of generating sulfate esters on resin-immobilized carbohydrates. An orthogonally protected GlcN scaffold was loaded onto a trityl-PS resin and subjected to regioselective deprotection and sulfation conditions, which was optimized with the aid of on-resin IR and Raman analysis. The sulfoforms were cleaved from the resin using mild Lewis acid conditions without affecting the O- or N-sulfate esters, and purified by reversed-phase high-performance liquid chromatography (HPLC) with retention of Bu4N+ as the sulfate counterion. The α-GlcN sulfoforms and their 4-O-benzyl ethers were examined by electrospray ionization tandem mass spectrometry (ESI-MS/MS) with daughter ions produced by collision-induced dissociation (CID). The benzylated α-GlcN sulfoforms were suitable models of internal glucosamines within heparan sulfate, and had significant differences in parent ion stabilities and fragmentation rates as a function of sulfate position. Mass fragmentation spectra of GlcN sulfoforms showed characteristic patterns with strong correlation to the position of both free hydroxyl groups and sulfate esters. Fragmentation analysis could readily distinguish GlcN sulfoforms bearing the relative rare 3-O -sulfate from isomers with the more common 6-O-sulfate. The solid-phase synthesis strategy was also optimized for the preparation of mono- and disaccharide GlcN sulfoforms bearing an aminoethyl linker, to facilitate the formation of glycoconjugates for biological studies. This second-generation solid-phase synthesis included an optimized cleavage condition from the trityl-PS resin and eliminated redundant ion exchange operations, which improved the efficiency and yields of GlcN derivatives with multiple-sulfates. GlcN and GlcN(1→4)Glc sulfoforms were prepared by second-generation solid-phase synthesis, converted to amine-terminated hexaethyleneglycol conjugates, and tested for their binding affinity to FGF2 and L-selectin using microarray-based screening. Our results indicate that even monosaccharide GlcN sulfoforms can exhibit significant differences in binding affinity. The affinities of sulfoforms linked in 1,4-linked fashion to β-glucoside were not significantly higher than that of monosaccharides, but GlcN sulfoforms linked to a 6-O -sulfo-β-glucoside appeared to have higher affinity for L-selectin and FGF2. Lastly, a GlcN(α1→4)Glc disaccharide scaffold bearing 5 orthogonal protecting groups was synthesized for generating GlcN(α1→4)Glc-6S sulfoforms, for screening against L-selectin, FGF2, and other heparin-binding proteins.

Degree

Ph.D.

Advisors

Wei, Purdue University.

Subject Area

Organic chemistry

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