Fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays

Abstract

A novel strategy for creating naturally derived glycan microarrays has been developed. Glycosylamines are prepared from free reducing glycans and stabilized by reaction with acryloyl chloride to generate a glycosylamide in which the reducing monosaccharide has a closed-ring structure. Ozonolysis of the protected glycan yields an active aldehyde, to which a bifunctional fluorescent linker is coupled by reductive amination. The fluorescent derivatives are easily coupled through a residual primary alkylamine to generate glycan microarrays. This strategy preserves structural features of glycans required for antibody recognition and allows development of natural arrays of fluorescent glycans in which the cyclic pyranose structure of the reducing-end sugar residue is retained.

Figure 1

Design of bifunctional fluorescent derivatization of free reducing glycans via a) the common reductive amination approach and b) a novel approach that retains the full ring structure mimicking natural glycoconjugate linkages.

Figure 2

The fluorescent derivatization of the free glycan LNFPIII: a) The conversion of LNFPIII (Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4Glc) to LNFPIII-Gly-AEAB according to the reaction scheme in Figure 1b; b) HPAEC-PAD analysis of starting material, intermediates, and final product of the reactions (from top to bottom): free reducing LNFPIII, the glycosylamine carbamate after ammonium bicarbonate treatment, the glycosylamine after desalting with carbograph, and the glycosylamide after reaction with acryloyl chloride; c) The HPLC profile on PGC of the final product mixture using fluorescence detection; d) The MALDI-TOF analysis of peaks 1 and 2 in c), which were collected from preparative HPLC separation.

Figure 3

Two strategies for generating TGLs for the production of glycan microarray starting from either purified free glycans or complex mixtures of glycans released from glycoconjugates from natural sources.

Figure 4

Structures of the defined glycans printed on the microarray. The symbol abbreviations for the glycans are indicated and are consistent with those used by the CFG.

Figure 5

Glycan array analysis of lectins binding to an array of 26 GGAEABs and the corresponding 26 GAEABs: Glycans from Figure 4 were prepared as open- (GAEABs as bars filled in blue) or closed-ring (GGAEABs as bars filled in red) derivatives and printed on NHS-slides as described in Methods. As described in the text with appropriate references, a) ConA recognizes high mannose-, hybrid-, and biantennary N-glycans; b) AAL recognizes Fuc-containing glycans; c) HPA binds to GalNAc residues; d) blood group H antibody recognizes Fucα1,2Galβ1-R; e) blood group A antibody recognizes type 2 chain GalNAcα1,3(Fucα1,2)Galβ1,4GlcNAcβ1-R but not type 1 chain GalNAcα1,3(Fucα1,2)Galβ1,2GlcNAcβ1-R (No. 16); f) blood group B antibody recognizes type 2 chain Galα1,3(Fucα1,2)Galβ1,4GlcNAcβ1-R; g) and h) anti-SLex 1 and 2, respectively, recognize NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1-R; i) IVIg is a product of pooled immunoglobulin G from thousands of individuals. Biotinylated lectins were detected by with cyanine 5-streptavidin (5 μg/mL). Binding of mouse antibodies (blood group antigen H, A, and B antibodies and anti-SLex antibodies) were detected by Alexa488 labeled goat anti-mouse IgM (5 μg/mL). Binding of Sandoglobulin IVIg was detected by Alexa488 labeled goat anti-human IgG (5 μg/mL).

Figure 6

The HPLC separation of GGAEABs prepared from a fraction of human milk oligosaccharides containing low molecular weight oligosaccharides: a) The PGC-HPLC purification of 8 major fractions; b) MALDI-TOF spectra of collected fractions from PGC-HPLC separation of human milk GGAEABs; All spectra showed a single molecular masses as different adducts: [M+H]+, [M+Na]+ and [M+2Na]+, that correspond to compositions of known milk oligosaccharides ; c) The second dimensional C18-HPLC profile of the collected fractions in a) indicating the presence of isomers in fractions 2,3, and 5.