Carbohydrate Microarrays Fabricated by Using Derivatized Carbohydrates

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Derivatized carbohydrates, termed glycoligands, are carbohydrate moieties with functional tags prepared by chemical modification. Glycoligands provide more flexibility in the selection of array substrates and chemical-linking techniques for carbohydrate microarrays. Most importantly, the use of glycoligands in combination with properly functionalized surfaces allows for the site-specific immobilization of carbohydrates onto the substrates. With these technical features, it is possible to construct carbohydrate microarrays with control over the ways of presentation of carbohydrate moieties for molecular recognition. These characteristics are important for achieving the specificity or selectivity of carbohydrate-protein interactions that play importance roles in cell-cell communication, signaling, and modulation of immune responses (12,39,65). Microarray presentation of the native configurations of glycoepitopes is likely a challenging issue that requires substantial and relatively long-term collaborative efforts by carbohydrate researchers and microarray experts.

Specific technical considerations in exploring this approach may include (i) the feasibility of preparing carbohydrate derivatives; (ii) the spacer between the glycoligands and the slide surface should provide optimal presentation of glycans and prevent nonspecific binding of proteins; (iii) the suitability of materials comprising the chip, for example, a functionalized glass slide versus a metallic surface; and (iv) the availability of tagged carbohydrate ligands for desired chip substrates.

The Consortium for Functional Glycomics (www.functionalglycomics.org) (3) has provided remarkable support to this field, building a library of about 200 synthetic glycoligands, which represent the most typical terminations and core fragments of mammal glycoproteins and glycolipids. A similar set of biotinylated oligosaccharides (^180 in total) are also available in the Consortium (www.func-tionalglycomics.org) (4). The number of described and well-characterized 2-amino-pyridine derivatives of N-glycans (56-58) reaches several hundred; many of these derivatives are commercially available. With the advances being made in chemical and chemoenzymatic syntheses, increasing numbers of carbohydrate derivatives of known oligosaccharide sequences will become available for fabrication of carbohydrate microarrays.

1. Noncovalent Immobilization of Derivatized Carbohydrates in Microarrays Because of the small molecular size and hydrophilic nature, most oligosaccharides cannot be directly immobilized onto nitrocellulose or black polystyrene surfaces for microarray applications. The oligosaccharide probe can be modified with a tag or coupled to a larger carrier molecule for noncovalent immobilization. A research group led by Feizi has developed oligosaccharide microarrays by non-covalently immobilizing NGLs on nitrocellulose (25,44). The oligosaccharides were obtained by chemical or enzymatic methods by using glycoproteins, glycoli-pids, proteoglycans, polysaccharides, or whole organs, or from chemical synthesis. The chemical derivatives of the oligosaccharides were synthesized by reductive amination of the oligosaccharides to the amino phospholipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine or its anthracene-containing fluorescent analogue. The immobilization efficiency of the NGLs on nitrocellulose was found to be high irrespective of the size of carbohydrates. The carbohydrate-binding proteins were investigated with known monoclonal antibodies, the E- and L-selectins, a chemokine (RANTES), and a cytokine. Binding was detected by colorimetric ELISA-type methods. It was shown that carbohydrate-binding proteins could single out their ligands, not only in arrays of homogeneous, structurally defined oligosaccharides but also in an array of heterogeneous O-glycan fractions derived from brain glycoproteins. The unique feature of this carbohydrate microarray technology is that deconvolution strategies are included with mass spectrometry for further determining the sequences of ligand-positive components within mixtures.

Wong's group developed a method for fabricating oligosaccharide arrays, which is a noncovalent but site-specific immobilization. In essence, they applied aliphatic derivatives of monosaccharides and oligosaccharides onto a polystyrene 96-well microtiter plate (6). They found that the carbohydrates were efficiently immobilized when the saturated hydrocarbon chain was between 13 and 15 carbons in length. Several di- to hexasaccharides containing terminal galactose, glucose, and/or fucose residues were chemically modified with a C14-saturated hydrocarbon chain. Figure 9 illustrates the attachment of the modified carbohydrates to microtiter plate surfaces. All the sugars were stable after repeated washings and elicited the predicted binding signals with the lectins Ricinus B chain, Con A, and Tetragonolobus purpurea.

In addition, Wong and colleagues (6,21) reported that azide-derivatized forms of galactose and several azide-derivatized neutral and sialic acid containing di- to tetrasaccharides were immobilized onto aliphatic alkyne-coated plastic microtiter plate surfaces. These saccharides were immobilized on the surfaces by a 1,3-dipolar cycloaddition reaction between the azide and alkyne groups (Fig. 10). The noncova-lent attachment also allowed convenient characterization of the lipid-linked products by mass spectrometry, as well as the detection of lectin binding. Using Guanosine diphosphate (GDP)-fucose and a-1,3-fucosyltransferase, fucosylation of sialyl-N-acetyllactosamine was carried out within the wells, showing that the surface is well suited for the high-throughput identification of enzyme inhibitors.

2. Covalent Immobilization of Derivatized Carbohydrates in Microarrays Several other types of carbohydrate derivatives have been used for the fabrication of carbohydrate microarrays. Thiolated carbohydrate derivatives were immobilized on heterogeneous self-assembled monolayers that present maleimide end-functionalized and OH end-functionalized penta(ethylene glycol) chains on glass slides (31,47). The maleimide group provides an appropriate functionality that reacts efficiently with thiol-terminated glycoligands, whereas the penta(ethylene glycol) chains prevent the nonspecific adsorption of protein to the substrate. The penta(ethylene glycol) chain also works as a spacer arm to reduce the steric hindrance during protein binding to carbohydrates on the surface.

Figure 9 Carbohydrate microplate arrays prepared by the noncovalent immobilization of aliphatic alkyne-derivatized carbohydrates to microtiter plate surfaces. Carbohydrates can then be screened against a variety of biologically important substrates such as lectins and RNA.

S. nigra Lectin

S. nigra Lectin

Figure 10 Carbohydrate microplate arrays prepared by the noncovalent immobilization of azide-derivatized carbohydrates to microtiter plates via a 1,3-dipolar cycloaddition reaction between alkynes and azides. Carbohydrates displaying terminal azides can be captured on microtiter plate surfaces through a terminal alkyne attached to a long, aliphatic tether and screened directly on the microtiter plate surface.

Figure 10 Carbohydrate microplate arrays prepared by the noncovalent immobilization of azide-derivatized carbohydrates to microtiter plates via a 1,3-dipolar cycloaddition reaction between alkynes and azides. Carbohydrates displaying terminal azides can be captured on microtiter plate surfaces through a terminal alkyne attached to a long, aliphatic tether and screened directly on the microtiter plate surface.

Thiol-functionalized surface

Maleimide-derivated carbohydrate

Maleimide-derivated carbohydrate

Thiol-functionalized surface

Figure 11 Carbohydrate microarrays prepared by covalent immobilization of maleimide-derivatized carbohydrates onto a thiol-functionalized substrate surface.

In contrast to the above approach, Shin's group prepared carbohydrate microarrays by covalent immobilization of maleimide-derivatized carbohydrates to thiol-functionalized glass slides (45,46) (see Fig. 11). Lectin-binding experiments showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. The author also demonstrated the fabrication of carbohydrate microarrays that contained more diverse carbohydrate probes. Enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Lex.

Mrksich and coworkers (32) reported a chemical strategy for preparing carbohydrate arrays by the Diels-Alder-mediated immobilization of cyclopentadiene-derivatized carbohydrates to self-assembled monolayers that present benzoquinone and penta(ethylene glycol) groups (Fig. 12). Modification of the gold surface was initiated by immersing gold-coated glass slides into a mixture of alkanethiols with (1%) and without (99%) appended hydroquinone groups to produce self-assembled monolayers of hydroquinone and penta(ethylene glycol) groups. Chemical or electrochemical oxidation was then performed to convert hydroquinone to benzoquinone groups. Finally, the cyclopentadiene-derivatized monosaccharides were covalently immobilized on the gold surface through the Diels-Alder reaction. This reaction was found to be highly efficient and selective for the immobilization of carbohydrates on the surface. Carbohydrate arrays presenting 10 monosaccharides were then evaluated by profiling the binding specificities of several lectins. These arrays were also used to determine the inhibitory concentrations of soluble carbohydrates for lectins and to characterize the substrate specificity of p-1,4-galactosyltransferase.

Blixt et al. (3) constructed a diverse glycan microarray by using standard robotic microarray printing technology to couple amine-derivatized glycoligands to an N-hydroxysuccinimide (NHS)-functionalized slide. The array comprises 200 synthetic and natural glycan sequences representing major glycan structures of gly-coproteins and glycolipids. This array uses commercially available amine-reactive

Benzophenone-functionalized surface

Figure 12 Carbohydrate microarrays prepared by covalent immobilization of cyclopenta-diene-derivatized carbohydrates to a benzoquinone-functionalized substrate.

Benzophenone-functionalized surface

Figure 12 Carbohydrate microarrays prepared by covalent immobilization of cyclopenta-diene-derivatized carbohydrates to a benzoquinone-functionalized substrate.

NHS-functionalized glass slides, which allow rapid covalent coupling of amine-functionalized glycans or glycoconjugates. The fabricated glycan microarray has shown utility for profiling the specificity of a diverse range of glycan-binding proteins, including C-type lectins, siglecs, galectins, anti-carbohydrate antibodies, lectins from plants and microbes, and intact viruses.

A microarray substrate for covalent immobilization of aminophenyl-deriva-tized carbohydrates is commercially available (GlycoChip, Glycominds, Lod, Israel). This substrate is functionalized with an oligomer of 1,8-diamino-3,6-dioxaoctan. Schwarz et al. reported the application of this substrate to fabricate oligosaccharide microarrays by using p-aminophenyl-derivatized carbohydrates via a cyanurchlor-ide-activated linker (50). This approach allows the covalent attachment of glycans containing a terminal aliphatic amine by forming an amide bond under aqueous conditions at room temperature. The fabricated oligosaccharide microarray was used to analyze the glycan-binding antibody repertoire in a pool of affinity-purified IgG collected from a healthy human population. In addition, a novel anti-cellulose antibody was detected that binds specifically to |b4-linked saccharides with a preference for glucopyranose over galactopyranose residues with the oligosaccharide microarray.

The group led by Waldmann has prepared carbohydrate microarrays by using Staudinger reactions between phosphane-functionalized glass slides and azide-derivatized carbohydrate moieties (34). The glass slide surface was first functionalized with polyamidoamine (PAMAM) dendrimers bearing 64-aminofunctional groups with the purpose of maximizing potential reactive sites on the surface. The amino groups of the PAMAM-modified slide were then converted to terminal carboxylic acid groups by reacting with glutaric anhydride. The carboxylic acid of the dendrimer film was finally converted to a phosphane group by reacting with the 2-(diphenylphosphi-nyl)phenol. The phosphane group has a high reactive efficiency to azide-derivatized molecules. The azide-derivatized carbohydrate moieties were prepared by solid-phase synthesis using a safety-catch linker strategy. The azide-derivatized carbohydrates were found to be efficiently immobilized onto the phosphane-functionalized slide surface. A spot volume of a 0.25 nl sample arrayed on the phosphane-functionalized slide surface produced a spot size of 400 mM in diameter. A mannose-containing carbohydrate microarray was fabricated on this substrate. Carbohydrate-protein interactions were evaluated by incubating with Alexa647-labeled Con A. This shows that the immobilization of azide-derivatized carbohydrates via the Staudinger reaction is highly efficient and can be employed to detect biomolecular interactions.

Bovin's group reported a method principally different from all described above. Saccharides were immobilized inside droplets of a porous polymeric gel (26). Immobilization of amino-derivatized oligosaccharides was achieved by the formation of a covalent bond between the amino group and the growing polymer chain during photo-initiated polymerization in the presence of a cross-linking agent. The authors have demonstrated that a hydrogel carbohydrate microarray contains three different classes of glycomolecules, which are as follows: (i) oligo-saccharide derivatives bearing a primary amino group, (ii) oligosaccharide-poly-acrylamide conjugates bearing allyl groups, and (iii) oligosaccharide derivatives bearing 2-aminopyridine groups. All of the three types of oligosaccharide derivatives are readily subjected to covalent attachment in the same conditions during the radical process of gel formation. For hydrogel microarray manufacturing, the gel-forming monomers, that is, methacrylamide, methylenebisacrylamide, and oli-gosaccharide derivatives are printed onto hydrophobized glass and irradiated with UV light. The double bond of methylenebisacrylamide readily reacts with the amino group of the oligosaccharide derivatives at pH 10.5 giving rise to a Michael addition product. After polymerization, an array of individual 3D approximately 1-nl gel drops, 150 mm in diameter, and 25 mm in height was formed. The authors have shown that the 3D hydrogel provides high sensitivity in probing proteins due to the large amount of carbohydrates immobilized in the 3D hydrogel spots.

3. Affinity Immobilization of Derivatized Carbohydrates

Biotin-derivatized carbohydrates can be immobilized on a streptavidin-coated substrate through the affinity interaction of the streptavidin-biotin pair to create carbohydrate microarrays. Biotin-derivatized carbohydrates include carbohydrate ligands that are biotinylated via a short aliphatic spacer or at the peptide part of glycopeptides. Several commercially available streptavidin-coated microwell plates can be applied when biotin-derivatized carbohydrates are available, such as the streptavidin-coated 384-well plate with a well volume 25 ml (4,18) and a streptavidin-coated 192-spot slide format (27). The first was designed to be in maximal proximity to the traditional immu-nochemical assay using commercial streptavidin-coated black 384-well plates.

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