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nh-coch2-o-nh2

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Figure 4 Chemical procedure for the preparation of aminooxyacetyl-functionalized glass slides and the immobilization of underivatized oligosaccharides. The aminooxyacetyl groups react selectively with the carbonyl group at the reducing end of carbohydrates via an irreversible condensation while the penta(ethylene glycol) groups serve as spacer arms and prevent the nonspecific adsorption of protein to the monolayer.

NH-COCH-O-NH-Boc HCl/CH3COOH

NH-COCHrO-NH2

nh-coch2-o-nh2

nh-coch2-o-nh2

Aminooxyacetyl-functionalized glass slides were prepared in four steps starting with a (3-glycidyloxypropyl)trimethoxysilane (GPTS)-functionalized glass slide. The synthesis of the functionalized glass slide is presented in Fig. 4. The glycidyl group of the GPTS monolayer was treated with diamino-poly(ethylene glycol) (PEG), resulting in a PEG monolayer end-functionalized with an amine. The amine groups were then coupled to the carboxyl groups of an N-Boc-Aoa-OH that was activated with a hydroxyl succimide group. Free aminooxyacetyl groups were then obtained upon treatment of the glass slide with HCl/acetic acid in order to remove the Boc- group. The aminooxyacetyl groups on the slide surface reacted with formyl groups at the reducing ends of the oligosaccharides to form oxime bonds. In contrast to reductive amination, the sugar structure was preserved after coupling; equilibrium between the closed-ring and the open-ring forms might occur at the surface of the support. This chemical-linking technique reported requires only a few modification steps on the surface, allowing for the functional chips to be created in a timely manner making it an attractive method for preparing carbohydrate microarrays in individual laboratories.

The poly(ethylene glycol) layer on the glass slides provides essentially complete resistance to unwanted protein adsorption and other nonspecific interactions at the surface and ensures that only specific interactions between soluble proteins and immobilized ligands occur. The poly(ethylene glycol) containing carbohydrate microarrays showed lower background signal even without commonly used blocking procedures such as treating the substrate with bovine serum albumin or other blocking proteins to passivate the surface. This excellent control over unwanted adsorption has also been reported with monolayers presenting oligo(ethylene glycol) groups (9,30). The poly(ethylene glycol) also functions as a spacer between the carbohydrate and the substrate, which is expected to increase the accessibility of proteins to the binding site of the carbohydrates.

Since the carbohydrates are chemically linked to the aminooxy-functiona-lized substrate surface through the reducing end, the carbohydrate moieties are immobilized in a well-defined orientation (i.e., site-specific immobilization). The main advantages of an aminooxy-functionalized substrate for the fabrication of carbohydrate microarrays include (i) the ease of formation of oxime bonding under mild conditions between the underivatized carbohydrates and the slide surface; (ii) good stability of the oxime bonding under a wide range of pH; (iii) the monosaccharides are in a ring-closed format which will not affect protein binding in an irrelevant manner, allowing for a more accurate evaluation of the protein-binding function of the carbohydrate.

To demonstrate the utility of this chemistry for the immobilization of carbohydrates and the use of the arrayed carbohydrates for parallel determination of protein-carbohydrate interactions, Zhou et al. (73) printed 10 oligosaccharides on the aminooxyacetyl-functionalized glass slide. The arrayed substrates were kept in a humidified chamber at room temperature overnight, washed with water, and dried. These conditions permitted near quantitative immobilization using minimal quantities of carbohydrate conjugates. After incubation and washing away the unbound oligosaccharide, the remaining aminooxyacetyl groups on the substrate were inactivated by treatment of the glass slides with succinic anhydride [10 mM in dimethylformamide (DMF)] overnight followed by rinsing with DMF to remove physically adsorbed succinic anhydride.

To investigate the carbohydrate-protein-binding properties of the fabricated carbohydrate microarrays, identical arrays were treated separately with three biotin-labeled lectins [with a concentration of 2 mM in phosphate-buffered saline/ tween (PBST)] for 2 h, and then washed with PBST. Detection of the bound ana-lyte was subsequently achieved by incubating the microarray with Cy3-streptavidin at a final concentration of 5 mg/ml, and then imaged with a confocal array scanner. Figure 5 shows the results of the analyte characterization on the carbohydrate microarray. As expected, the oligosaccharides were found to bind to their specific lectin proteins. For example, the carbohydrate array probed with Con A showed significant fluorescence intensity in the spots arrayed with mannose, glucose, and N-acetylglucosamine (GlcNAc) (Fig. 5A). Analysis of the fluorescence intensity further reveals that the binding of Con A to the oligosaccharides is in the order of mannose > glucose > GlcNAc. The affinity binding difference of the arrayed oli-gosaccharides is consistent with solution-phase assays. A weak signal was obtained in the spots that were arrayed with maltooligosaccharide which has 4-10 units of a-glucose. This could be the result of an inefficient immobilization of the sugar on the substrate due to the reducing activity of the formyl groups of the

Mannose Glucose G1cNAc Cellobiose

Maltooligosaccharide Rhamnose a-Methyl-mannoside Lactose Galactose Fucose

Figure 5 Fluorescence image of oligosaccharide microarrays spotted with 10 oligosaccharide probes with identical carbohydrate chips that were separately incubated with each of 3 biotin-labeled lectins (0.1 mg/ml in PBST) for 1 h, washed with PBST 3 times for 5 min each, and stained with 5 mg/ml of Cy3-strptavidin and evaluated by confocal fluorescence microscopy. Fluorescence images of oligosaccharide microarrays probed with (A) concanavali A (Con A), (B) LoTus Tetragonolobus (LT), and (C) Erythrina Gristagalli (EC).

maltooligosaccharide. In addition, no signal was observed in the spots arrayed with methyl-a-mannoside in which the C1 position was substituted with a methyl group. This result suggests that methyl-a-mannoside could not be immobilized on the glass surface. Probing the microarrays with the two other lectins also gave the expected results: Lotus tetragonolobus bound to the spots presenting a-fucose (Fig. 5B), whereas Erythrina cristagalli bound only to spots presenting lactose and galactose (Fig. 5C). Nonspecific adsorption was not observed on the spots arrayed with cello-biose and rhamnose and essentially no fluorescence was obtained from surfaces without carbohydrates. The weak signal obtained from the glucose spots when the microarray was probed with L. tetragonolobus may be caused by the weak cross-reaction of the lectin L. tetragonolobus. However, this weak cross-interaction that gave less than 8% of signal compared with the specific interaction would not affect the rapid determination of the presence or absence of specific carbohydrate epitopes.

Overall, these results demonstrated that the binding of lectins with the prepared carbohydrate microarrays are specific and multiple-analytic characterization can be achieved on the aminooxyacetyl-functionalized slide with good selectivity. Furthermore, periodate oxidation of the immobilized oligosaccharides with NaIO4 resulted in the loss of lectin binding. These experiments verify that the fabricated carbohydrate microarray is well suited for the selective identification of carbohydrate-binding proteins.

Figure 6 shows the dose-response curves of lectins applied to the fabricated oligosaccharide microarrays. It was apparent that an increase in lectin concentration resulted in a corresponding increase in the fluorescence emitted from the arrayed spots, and saturation of affinities was obtained at high concentration. The calculated limit of detection (LOD, the concentration which gives a fluorescent signal higher than the background+three standard deviation units) was determined to be approximately 0.008 mg/ml for Con A, which is lower than the microtiter plate assay developed by Hatakeyama.

Figure 5 Fluorescence image of oligosaccharide microarrays spotted with 10 oligosaccharide probes with identical carbohydrate chips that were separately incubated with each of 3 biotin-labeled lectins (0.1 mg/ml in PBST) for 1 h, washed with PBST 3 times for 5 min each, and stained with 5 mg/ml of Cy3-strptavidin and evaluated by confocal fluorescence microscopy. Fluorescence images of oligosaccharide microarrays probed with (A) concanavali A (Con A), (B) LoTus Tetragonolobus (LT), and (C) Erythrina Gristagalli (EC).

Lectin conc. (ig/ml)

Figure 6 Dose-response binding curve of lectins to the arrayed oligosaccharides. Con A binding on mannose (▲), glucose (♦), and GlcNAc (O); EC on lactose (x) and galactose (□); LT on fucose (•). Each data point represents the average value of the mean signaliSD (standard deviation) of 18 replicate spots from 2 slides.

Lectin conc. (ig/ml)

Figure 6 Dose-response binding curve of lectins to the arrayed oligosaccharides. Con A binding on mannose (▲), glucose (♦), and GlcNAc (O); EC on lactose (x) and galactose (□); LT on fucose (•). Each data point represents the average value of the mean signaliSD (standard deviation) of 18 replicate spots from 2 slides.

Carbohydrate arrays have the characteristics required for quantitative assays of multiple protein-carbohydrate interactions with minimal quantities of reagents. To assess the utilization of carbohydrate microarrays fabricated on aminooxyace-tyl-functionalized slides for quantitative assays, a-methyl mannose was applied to inhibit Con A binding to the immobilized mannose spots. A series of mixtures containing biotin-labeled Con A (2 mM in PBST) and a-methyl mannose (0-4 mM in PBST) was applied on the microarray surface and incubated for 1 h at 25° C. The substrates were rinsed with PBST, stained with 5 mg/ml of Cy3-streptavidin, and analyzed with a fluorescence scanner to quantify the amount of bound Con A on the spots of mannose and glucose. The amount of lectin that bound to the chips for each concentration of soluble ligand (i.e., a-methyl mannose) is shown in Fig. 7. The IC50 (concentration of inhibitor required to prevent 50% of lectin binding to array spot) was determined using a-methyl mannose as an inhibitor of Con A binding to glucose and mannose. The results verified that the microarray spots of mannose (IC50 = 60 mM) competed more effectively with the soluble carbohydrate for Con A than that of glucose (IC50 = 23 mM). The relative binding affinities of these carbohydrates for Con A is consistent with those obtained in previous studies (31).

In a similar method, Lee and Shin (36) have developed hydrazide-coated glass slides to immobilize a wide range of carbohydrates including mono-, di-, and oligosaccharides in a simple, efficient, and chemoselective fashion. Preliminary protein-binding experiments show that carbohydrate microarrays prepared by this method are suitable for the high-throughput analysis of carbohydrate-protein interactions.

The advantages of the above two methods for covalent immobilization of underivatized carbohydrates rely on the ease of formation and on the good

Log (a-methyl mannose) (|M)

Figure 7 Quantitative inhibition assays in oligosaccharide microarray. (A) Determination of the concentration of soluble a-methyl mannose to inhibit 50% of Con A binding to spotted mannose. (B) Determination of the concentration of a-methyl mannose to inhibit 50% of Con A binding to spotted glucose. Each data point represents the mean±SD for 10 spots from 2 independent experiments.

Figure 7 Quantitative inhibition assays in oligosaccharide microarray. (A) Determination of the concentration of soluble a-methyl mannose to inhibit 50% of Con A binding to spotted mannose. (B) Determination of the concentration of a-methyl mannose to inhibit 50% of Con A binding to spotted glucose. Each data point represents the mean±SD for 10 spots from 2 independent experiments.

stability of the oxime linkage and hydrazide linkage for oligosaccharides up to pH 9. However, reactions of aminooxy or hydrazide groups with free carbohydrates are slow when the carbohydrate MW increases because of the reducing activity of the aldehyde group of the carbohydrates. To improve the immobilization of larger carbohydrates, such as polysaccharides, on the aminooxy- and hydrazide-functionalized slide substrates, Zhou et al. have been investigating the utilization of microwave radiation energy to facilitate the fabrication of carbohydrate micro-arrays on a 3D polymer film bearing aminooxy- and hydrazide-functional groups (Zhou X., personal communication). Using microwave radiation to facilitate the reaction, oligosaccharides and polysaccharides can be covalently linked on the aminooxy- and hydrazide-functionalized surface within minutes.

Carroll et al. developed a method for covalent immobilization of underiva-tized mono-, oligo-, and polysaccharides onto glass substrates functionalized with self-assembled photoactive phthalimide chromophores (7). Upon exposure to UV light, the photoactive aromatic carbonyls presumably react with the C-H groups of the sugars by hydrogen abstraction followed by radical recombination to form a covalent bond (Fig. 8). Immobilization of unmodified carbohydrates by this approach was demonstrated to be much less dependent on the MWs of the spotted carbohydrates compared to a nitrocellulose-coated slide. Furthermore, for oligosaccharides the grafting efficiency was shown to be much higher than nitrocellulose. However, the method of photocoupling, which is expected to target any CH- group on the sugar rings with varying specificity depending on the structure of the ring, may interfere with the protein-binding specificity of monosaccharides. Wang et al. recently used this approach to generate a glycan array containing a large panel of synthetic carbohydrates and characterized their antigenic reactivities with pathogen-specific antibodies (64). As described in Section I, this investigation led to the discovery of a highly specific carbohydrate moiety of B. anthracis spores.

Figure 8 Covalent immobilization of underivatized carbohydrates on a phthalimide-func-tionalized slide surface by photo-immobilization.

Figure 8 Covalent immobilization of underivatized carbohydrates on a phthalimide-func-tionalized slide surface by photo-immobilization.

Another type of photoactive microarray platform, based on dextran-coated glass slides (PhotoChips from CSEM, Switzerland) was reported by Sprenger's group (2). The dextran-based polymer OptoDex is functionalized with aryl-trifluoromethyl-diazirine groups. On illumination, aryl-trifluoromethyl-diazirine groups form reactive carbenes, which can undergo a variety of reactions with a vicinal molecule that result in covalent bond formation including insertion into a and p bonds, addition of a nucleo-phile or electrophile and hydrogen abstraction. The authors have demonstrated that this substrate immobilizes polysaccharides and glycoligands. However, since the aryl-trifluoromethyl-diazirine-functionalized surface can react with any type of bio-molecule, this type of array substrate is not suitable for preparing carbohydrate micro-arrays with unpurified carbohydrate extractions from cells or plants. Precaution must be made when applying this platform for serological studies since antidextran natural antibodies are frequently detected in human circulation (33,67).

The above slide surfaces and linking techniques provide the feasibility to fabricate microarrays of carbohydrates by using underivatized carbohydrate moieties. These methods are especially useful when working with complex oligosac-charides isolated from natural sources and when derivatized carbohydrates are not available. In many cases, the glycoepitopes contained in these microarrays are reactive toward appropriate antibodies, lectins, or other carbohydrate-binding partners of defined carbohydrate-binding specificities. However, these methods of saccharide immobilization are not expected to be site specific. Instead, the carbohydrates are attached without control of the orientation of saccharide display. A given saccharide spotted may, thus, present a glycoepitope in a spectrum of different configurations, although one or a few might be predominant. Thus, in order to further characterize the fine specificity of carbohydrate binding, especially the orientation effect of epitope display, one may want to explore the technologies described in the subsequent sections.

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