The Glycan Microarray Story from Construction to Applications

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The Glycan Microarray Story from Construction to Applications Ji Young Hyun, Jaeyoung Pai, and Injae Shin* National Creative Research Initiative Center for Biofunctional Molecules, Department of Chemistry, Yonsei University, Seoul 03722, Korea CONSPECTUS: Not only are glycan-mediated binding processes in cells and organisms essential for a wide range of physiological processes, but they are also implicated in various pathological processes. As a result, elucidation of glycan-associated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to utilize glycan microarrays in efforts aimed at the rapid analysis of glycan-associated recognition events. Because they contain a number of glycans immobilized in a dense and orderly manner on a solid surface, glycan microarrays enable multiple parallel analyses of glycan−protein binding events while utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this Account, we summarize our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces are described. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan−protein interactions quantitatively, detect pathogens, and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses. Owing to these efforts, it is now possible to use glycan microarrays to expand the understanding of roles played by glycans and glycan binding proteins in biological systems.

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INTRODUCTION Carbohydrates, which are present mainly in the form of glycoconjugates (e.g., glycoproteins, glycosphingolipids, and proteoglycans) on the surface of or inside cells (Figure 1), are involved in a wealth of physiological and pathological processes through association with glycan-binding proteins (GBPs).1−4 For instance, by binding to GBPs cell surface glycans play key roles in cell communication, cell adhesion, and signaling. Additionally, recognition of pathogenic glycans by lectins, expressed on the immune cell surface, elicits immune responses to pathogens including viruses, bacteria, and yeast.5,6 Moreover, glycan− protein interactions are involved in pathological processes, such as infections caused by pathogens (e.g., toxins, bacteria, and viruses)7,8 and tumor metastasis.9 Also, carbohydrate−carbohydrate interactions are known to mediate diverse biological processes.10,11 As a consequence, functional studies of glycans and GBPs in biological systems, particularly those that focus on glycan-associated recognition events, have the potential of providing deep insight into various pathophysiological processes and designing efficacious therapeutic agents and diagnostic tools. In 2002, we and others were the first to describe carbohydrate microarrays, which contain a high density of various glycans attached to a solid surface.12−15 In addition, we and others © 2017 American Chemical Society

showed that these microarrays could be used for rapidly assessing glycan-mediated binding events. Observations made in these studies demonstrated that this microarray technology enables simultaneous analysis of multiple glycan−protein interactions using only small amounts of carbohydrate samples. Because carbohydrates immobilized on a solid surface are displayed in a multivalent manner (Figure 2), they interact strongly through a cluster effect with proteins that normally have low binding affinities to monovalent carbohydrates. As a result, they can be utilized for sensitive and rapid assessment of the nature and consequences of glycan-associated recognition events. Over the intervening years, this technique has become one of the leading tools for functional studies of glycans and GBPs. In this Account, we summarize progress that has been made in studies by our group on the creation of carbohydrate microarrays and their applications to rapid analysis of glycan−protein interactions, quantitative determination of binding affinities associated with glycan−protein interactions, simple assessment of substrate specificity of enzymes, detection of pathogens, and identification of functional glycans that stimulate cellular responses. Received: January 22, 2017 Published: March 17, 2017 1069

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modified forms and are prepared by chemical or enzymatic synthesis as well as generated by cleavage of natural glycoproteins and glycolipids. We have developed several immobilization techniques to create glycan microarrays. One approach relies on the reaction of a maleimide group with a thiol group at pH ≈ 7.0, a process that has been widely utilized for the preparation of versatile bioconjugates.20,21 The reaction takes place selectively even when other functional groups, such as amines, alcohols, and carboxylates, are present. In an initial effort, this ligation reaction was employed to attach maleimide-linked carbohydrates, prepared by solution-phase synthesis, to thiol-derivatized glass slides (Figure 3).22 This immobilization method was successfully Figure 1. Major classes of glycans in mammalian cells. Glycoproteins are glycoconjugates in which diverse glycans are conjugated to a serine/ threonine (O-linked glycoproteins) or an asparagine residue (N-linked glycoproteins) of the polypeptide backbone. Glycosphingolipids contain mono- or oligosaccharides that are linked to ceramides. Proteoglycans are glycoconjugates in which glycosaminoglycans are attached to a serine/threonine residue of the polypeptide backbone via a xylose moiety. Glc, glucose; GlcNAc, N-acetylglucosamine; Gal, galactose; GalNAc, N-acetylgalactosamine; Man, mannose; Fuc, fucose; NeuNAc, N-acetylneuraminic acid; Xyl, xylose. Figure 3. Construction of glycan microarrays by the reaction of maleimide-linked glycans with a thiol-derivatized surface.

used to construct microarrays to assess glycan binding to plant lectins. Because thiol groups present on glass slides used in the immobilization approach described above undergo relatively facile air oxidation, other immobilization protocols have been devised. For example, we developed an immobilization method that relies on the reaction of a hydrazide group in a conjugated glycan with an epoxide moiety on a modified glass slide (Figure 4A).23−25 The required hydrazide-linked glycans are readily produced by solid-phase synthesis, and the epoxidederivatized glass slides are prepared by immersing amine-coated glass slides into a solution of poly(ethylene glycol) diglycidyl ether. The hydrophilic poly(ethylene glycol) tether is inserted between the epoxide group and the solid surface to abrogate nonselective protein binding to the immobilized glycans. Because both hydrazide-conjugated glycans and epoxidederivatized glass slides are relatively stable, they can be stored for several months. Notably, hydrazide groups in the conjugates react more rapidly with surface epoxides at pH < 6 than do amine- or thiol-linked counterparts (Figure 4B). Glycan microarrays prepared by using this immobilization technique have been utilized to evaluate the glycan binding properties of lectins and the acceptor specificities of glycosyltransferases (see Figure 12). In addition, microarrays that contain neoglycopeptides with various valences and different spatial arrangements of the sugar ligands were constructed by using this immobilization method in order to investigate the density-dependent binding of glycans by lectins (Figure 4C).26 Furthermore, we have demonstrated that this immobilization strategy can be used to prepare microarrays containing various peptides.27,28 Microscope glass slides used in the microarrays are normally first cleaned with piranha solutions (concentrated sulfuric acid/ hydrogen peroxide = 3:1) and then treated with alkoxysilaneappended aldehydes, carboxylic acids, or amines. The resulting slides are then subjected to glycan attachment or are further modified to introduce functional groups needed for attachment of glycans. N-Hydroxysuccinimide (NHS) ester is most widely utilized for this purpose because it readily reacts with amine and

Figure 2. Glycans immobilized on a microarray (top) are displayed in a multivalent manner like cell surface glycans (bottom). Therefore, glycans on the microarrays bind strongly to proteins as a result of a cluster effect even though they have low binding affinities to monovalent glycans.

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IMMOBILIZATION OF GLYCANS Glycan microarrays are prepared by attaching unmodified or functionalized glycans to appropriately derivatized solid surfaces. Techniques employed for efficient immobilization of glycans on a solid surface are key to the successful use of glycan microarrays. A number of immobilization strategies have been reported over the past decade.16−19 Microscope glass slides have been most extensively employed as the surface material because they are easily manipulated, inexpensive, and compatible with optical detection systems. In addition, gold and a nitrocellulose membrane have also been utilized as surface materials. Glycans required for microarray construction are used in either free or 1070

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Figure 4. (A) Construction of glycan microarray by immobilizing hydrazide-linked glycans on an epoxide-coated surface. (B) (a) A mixture of FucNHNH2 and GlcNAc-SH (1:1) was printed on the epoxide-coated surface at pH 3−10. The resulting microarrays were probed with Cy5-AA (Argiope aurantia lectin, blue line) or Cy3-WGA (wheat germ agglutinin, red line). (b) A mixture of Fuc-NHNH2 and B-NH2 (1:0, 1:1 and 1:4) was printed on the epoxide-coated surface at pH 3−10. The microarrays were then probed with Cy5-AA (black line, 1:0 mixture; blue line, 1:1 mixture; red line, 1:4 mixture). Reproduced with slight modification from ref 23. Copyright 2005 John Wiley & Sons. (C) Construction of microarrays containing multivalent neoglycopeptides by attaching C-terminal hydrazide-conjugated neoglycopeptides, prepared by solid-phase synthesis, on an epoxide-coated surface.

Figure 5. One-step surface modification of a glass slide with NHS ester. (A) The proposed mechanism of acid-catalyzed silylation of silanols on the glass surface. (B) Construction of glycan microarrays using the NHS ester-derivatized glass slide prepared by one-step surface modification. 1071

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Figure 6. (A) Construction of glycan microarrays by immobilizing free glycans on (upper) hydrazide- and (lower) aminooxy-coated surfaces. (B) (a) Unmodified fucose was separately immobilized on (red) hydrazide- and (blue) aminooxy-coated surfaces for different times. The microarrays were probed with Cy5-AA. (b) Unmodified N,N′-diacetylchitobiose was separately immobilized on (red) hydrazide- and (blue) aminooxy-coated surfaces for different times. The microarrays were probed with Cy3-WGA. Reproduced with slight modification from ref 30. Copyright 2005 American Chemical Society.

strategy requires functional group-conjugated glycans, which are normally synthesized by using multistep routes. To circumvent the time-consuming and labor-intensive synthesis of modified glycans, we developed a method for covalent, sitespecific, and size-independent immobilization of free sugars.30,31 For this immobilization, reactions of aminooxy or hydrazide groups with free glycans, which are widely employed for the preparation of versatile glycoconjugates, were used. Our results showed that free glycans, including simple carbohydrates, oligosaccharides, and polysaccharides, are attached to both aminooxy- and hydrazide-modified glass surfaces (Figure 6A). Interestingly, the hydrazide-based immobilization process is more efficient than the method employing aminooxy containing surfaces (Figure 6B). Because reactions of free glycans with hydrazide groups generate predominantly cyclic adducts with β-configurations at the anomeric positions but reactions of free glycans with aminooxy groups produce mainly acyclic adducts, the different immobilization efficiency may be a consequence of the different nature (cyclic in hydrazide surfaces verse acyclic in aminooxy surfaces) of linkages of the anomeric position.

hydrazide groups linked to glycans. To avoid the need for laborious surface modification, we developed an efficient, onestep process involving acid-mediated reaction of NHS ester functionalized bis-methallylsilanes with glass surface silanol groups (Figure 5).29 In this protocol, piranha solution-treated glass slides are immersed into a solution of the NHS ester containing bis-methallylsilane derivatives 1a or 1b in the presence of Sc(OTf)3 or TfOH as an acid catalyst. The resulting NHS estercoated glass slides are then used for attachment of amine- or hydrazide-appended sugars. It was found that (1) the length of tether between Si and NHS ester groups (1a versus 1b) has little influence on the efficiency of surface modification and protein binding and (2) TfOH is superior to Sc(OTf)3 as a catalyst for the key Si−O−Si bond forming process. Importantly, this surface modification procedure should be applicable to the preparation of various functional group-derivatized glass surfaces for microarray construction. The generally utilized approach for creation of glycan microarrays involves site-specific and covalent attachment of modified sugars to appropriately derivatized surfaces. However, this 1072

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Figure 7. Other immobilization techniques used to prepare glycan microarrays. (A) Noncovalent and site-specific attachment, (B) covalent and sitenonspecific attachment, and (C) covalent and site-specific attachment.

In addition to the techniques developed in our laboratory, alternatives have been devised in parallel by other researchers. These include noncovalent and site-specific immobilization of fluorous glycans,32 neoglycolipids,14 or oligonucleotide-linked glycans (Figure 7A),33 covalent and site-nonspecific immobilization of free glycans on photolabile-group-coated surfaces (Figure 7B),34 and covalent and site-specific immobilization using cycloaddition (Diels−Alder reactions35 and click chemistry36) or amine coupling reactions (Figure 7C).37 Because many immobilization techniques have been developed to date, practical issues, such as availability and ease of preparation and stability of glycan probes and modified surfaces, should be considered when fabricating glycan microarrays.

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APPLICATIONS During the time period following the first report in 2002,12−15 great strides have been made in applications of glycan microarrays. The most extensive use of this technology has been in the rapid profiling of glycan binding properties of proteins, including plant and animal lectins, antibodies, and growth factors. In these assays, carbohydrate microarrays are incubated with fluorophorelabeled GBPs (Figure 8A), fluorophore-labeled secondary reagents that interact with the GBP (Figure 8B), or a tag (e.g., biotin, His tag) conjugated to the GBP (Figure 8C). The fluorescence intensity of the bound protein is then quantified using a high-resolution fluorescence microarray scanner. A number of glycan−protein interactions taking place on the microarrays have been rapidly analyzed using this approach. In addition, surface materials including glass, gold, and membrane do not interfere with this detection method. However, protein modification with fluorescent dyes (e.g., fluorescein, Cy3, or Cy5) or a tag sometimes results in protein denaturation or interference with binding to glycans. In addition to the fluorescence detection method, surface plasmon resonance (SPR) imaging38 and mass spectrometry (MS) techniques39 are employed as labelfree detection methods in glycan microarrays. Plant lectins have been utilized widely as biological research tools for the molecular understanding of the glyco-code.40

Figure 8. Fluorescent detection of bound proteins on glycan microarrays. Glycan microarrays are incubated with (A) a fluorophore-labeled protein, (B) an unlabeled protein followed by treatment with a fluorophore-labeled antibody, and (C) a tag-labeled (e.g., biotin or His tag) protein followed by treatment with a fluorophore-labeled secondary reagent. The fluorescence intensity of the bound protein is normally measured using a microarray scanner. His tag refers to at least six histidine (His) residues inserted mostly at the N- or C-terminus of the protein.

This time- and cost-effective immobilization technique, which does not require modified glycans, is especially suited to studies conducted by biologists who lack organic synthesis experience. 1073

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Figure 9. Fluorescence images of glycan microarrays containing 58 glycans probed with (A) Cy5-AA, (B) FITC-ConA (concanavalin A), (C) Cy3-BS-I (Bandeiraea simplicifolia-I), and (D) Cy3-RCA120 (Ricinus communis agglutinin I). Center-to-center spot distance, 250 μm; spotting volume, 1 nL. Glycans in the box are used for construction of glycan microarrays. Reproduced from ref 31. Copyright 2009 American Chemical Society.

Because interactions of mammalian lectins with specific glycans are involved in a wide range of important biological processes, glycan microarrays have been employed to determine the glycan binding patterns of mammalian lectins.43 We employed this approach in an investigation of the glycan binding property of the animal lectin SIGN-R1.44 At present, collections of lectin binding specificity data are available at the Web site of The Consortium for Functional Glycomics (www.functionalglycomics. org). In addition to plant and animal lectins, binding specificities of GBPs, including antibodies, pathogenic and viral lectins, and growth factors, have been determined using glycan microarrays by others.45−48 Importantly, this microarray technology has great potential to identify new GBPs.49 Glycan microarrays can also be utilized to examine glycan binding properties of whole pathogens and to detect pathogens as part of diagnoses.30,31,50 Many pathogenic bacteria express specific lectins on pili. Moreover, their pathogenic properties are caused by the initial binding of pathogens to host cell surface glycans through specific glycan−protein interactions.7 In order to demonstrate that glycan microarrays can be applied to detect pathogens, we incubated a microarray containing 58 glycans with Escherichia coli ORN178 strain, which was stained with the fluorescent dye SYTO 83 (Figure 10).31 Because the ORN178 strain expresses the mannose-binding protein, FimH, on pili, microspots containing mannose epitopes exhibited fluorescence. The glycan microarrays have also been employed to detect harmful pathogens in blood samples and to analyze glycan binding properties of viruses by other researchers.50−53 Although glycan microarrays can be applied to profile whole cells, optimization of experimental conditions employed for this purpose is needed in order for applications of this approach to become widespread. The microarray technology has been employed to determine the quantitative binding affinities of proteins to glycans. In an initial study, we applied glycan microarrays to determine concentrations (IC50) of soluble inhibitors to inhibit 50% of protein binding to glycans (Figure 11A).22 In this assay, a series of preincubated mixtures of the fluorophore-labeled lectin and an inhibitor are applied to glycan microarrays. The IC50 values of the inhibitors are then determined by measuring the fluorescence intensities of bound proteins that remain on the microarrays after washing. In a later study, we utilized glycan microarrays to determine dissociation constants (Kd values) for complexes of

Figure 10. (A) Schematic overview of detection of live pathogenic bacteria using glycan microarrays. (B) Glycan microarrays prepared by immobilizing 58 unmodified glycans shown in Figure 9 on the hydrazide-derivatized surface were incubated with SYTO 83-labeled E. coli ORN178. Shown are the fluorescence image of (a) the treated glycan microarrays and (b) its enlarged images. Center-to-center spot distance, 250 μm; spotting volume, 1 nL. Reproduced with modification from ref 31. Copyright 2009 American Chemical Society.

In addition, plant lectins have potential as therapeutic and diagnostic agents because cancer cells overexpress specific glycans on their surface, which are detected by using the lectins. Also, plant lectins promote apoptotic cancer cell death by binding to surface glycans.41,42 Because of their biological and biomedical significance,40−42 plant lectins have been subjected to extensive glycan microarray-based investigations aimed at elucidating their binding specificities. For example, we utilized glycan microarrays containing 58 glycans (mono-, di-, oligo-, and polysaccharides) to analyze binding properties of several plant lectins (Figure 9).31 1074

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Figure 11. (A) Determination of concentrations (IC50) of α-GlcNAc-OMe to inhibit 50% of WGA binding to (a) α-GlcNAc and (b) α-GalNAc attached to the microarray. Reproduced with slight modification from ref 22. Copyright 2004 American Chemical Society. (B) Determination of dissociation constants (Kd) between A. aurantia lectin (AA) and (a) Fucα1,2Gal and (b) fucose using carbohydrate microarrays. Reproduced with slight modification from ref 31. Copyright 2009 American Chemical Society.

require cumbersome and lengthy protection and deprotection steps. For efficient use of glycosyltransferases as synthetic catalysts, detailed knowledge about their acceptor specificities is needed. We have used the microarray technology to evaluate the acceptor specificities of β-1,4-galactosyltransferase (β-1,4-GalT). In this approach, β-1,4-GalT and UDP-Gal are applied to the glycan microarray. The glycans that are substrates for β-1,4-GalT undergo galactosylation to yield products which can be detected using the Gal-binding lectin RCA120 (Figure 12).24 It was found that among 20 glycans on the microarray only α-GlcNAc and β-GlcNAc are converted to galactosylated products, Galβ1,4GlcNAc-α (LacNAc-α), and Galβ1,4GlcNAc-β (LacNAc-β), respectively. In addition, β-GlcNAc is a better substrate for this enzyme than is α-GlcNAc, an observation that was confirmed by conducting enzymatic reactions in solutions. Other research groups evaluated substrate specificities of several glycosyltransferases using glycan microarrays.39,55 When microarrays containing large glycan libraries are employed, glycosyltransferase specificities can be evaluated in greater detail, and new reactions promoted by these enzymes can be probed. As described above, enzymatic glycosylation processes serve as alternatives to chemical methods for the synthesis of complex carbohydrates. To examine the usefulness of enzymatic transformations of glycans on microarrays, we prepared sialyl Lex from GlcNAc on a microarray by using three glycosyltransferasecatalyzed reactions.24 Specifically, sialyl Lex tetrasaccharide was prepared by consecutive treatment of immobilized GlcNAc with β-1,4-GalT/UDP-Gal, α-2,3-sialyltransferase (α-2,3-SialT)/ CMP-NeuAc and α-1,3-fucosyltransferase (α-1,3-FucT)/GDPFuc (Figure 13). Probing the enzyme-treated sugars on the microarray with the anti-sialyl Lex antibody showed that sialyl Lex is successfully generated from GlcNAc in these processes. Diverse complex oligosacchrides were also prepared on microarrays by using a similar approach by others.56 Most uses of glycan microarrays have been for elucidation of lectin binding properties. In a novel application, we employed the microarray technology for rapid screening of functional glycans that elicit cellular responses by interacting with cell-surface lectins. In this effort, microarrays containing 31 glycans were incubated with mammalian cells expressing SIGN-R1 on their

Figure 12. Glycan microarrays are incubated with β-1,4-GalT in the presence of UDP-Gal. The enzyme-treated microarrays are incubated with RCA120 to detect the transferred Gal residue to glycans on the microarray. Center-to-center spot distance, 250 μm; spotting volume, 1 nL. Highlighted by the box, both α- and β-GlcNAc are substrates for this enzyme, and β-GlcNAc is a better substrate than α-GlcNAc.

proteins and surface-immobilized glycans (Figure 11B).24,31 In this method, glycan microarrays are incubated with various concentrations of fluorophore-labeled proteins. Apparent dissociation constants for glycan−protein interactions are determined by measuring fluorescence intensities of proteins remaining on the microarrays after washing. Notably, the apparent Kd values obtained using this approach are similar to those arising from SPR experiments.24 The findings demonstrate that microarrays are powerful tools to measure multiple dissociation constants in a single experiment.26,54 Glycosyltransferases are biocatalysts that are involved in the biosynthesis of complex oligosaccharides. These enzymes can be utilized in laboratory preparations of these biomolecules because, unlike chemical glycosylation procedures, their use does not 1075

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Figure 13. Enzymatic synthesis of sialyl Lex from GlcNAc on microarray by using three glycosyltransferases (β-1,4-GalT, α-2,3-SialT, and α-1,3-FucT).

microarray technology can be used to identify glycans that stimulate the cell-surface lectin-mediated cellular response in a high-throughput manner.

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CONCLUSION AND OUTLOOK Because they have been extensively employed for rapidly profiling glycan binding patterns of many types of proteins over the past decade, glycan microarrays have become powerful and transformative tools in biological research. In this Account, we highlighted our efforts aimed at designing methods for construction of glycan microarrays and at determining their applications in studies of various glycan-mediated biological processes. Particular attention was given to the use of this technology for simple analysis of the binding properties of a diverse array of binding partners such as plant and animal lectins, antibodies, cells, and pathogens. In addition, we described the use of these microarrays for screening glycans that promote cell surface lectin associated cellular responses. Other research groups have also contributed to the development and applications of glycan microarrays. The combined results of these studies have led to a much more comprehensive understanding of glycans and GBPs in biological systems. For example, determination of glycan binding properties of malectin by using glycan microarrays have paved the way for studies of its roles in cells.49 The future of glycan microarrays is full of opportunities. Nonetheless, more research is needed to optimize the potential of this technology. For example, unlike DNA microarrays, which contain the entire genomes of organisms, the glycan microarrays utilized to date contain only a limited number of glycans compared to those present in nature. This limitation would be overcome if a great effort were made to prepare large glycan libraries using improved chemical or (chemo)enzymatic synthesis as well as to develop better methods for isolation of glycans from natural glycoconjugates. Another shortcoming stems from the fact that glycan microarrays have been used thus far in a small number of laboratories. As this microarray technology becomes more familiar to members of the broad scientific community, novel and unanticipated applications will surely arise and will yield a wealth of new information on biological functions of glycans and their binding proteins.

Figure 14. Glycan microarray-based method for screening of glycans that stimulate cellular responses by binding to cell-surface lectins. Unmodified glycans are attached to the hydrazide-coated surface to construct carbohydrate microarrays. Lectin expressing cells, pretreated with a ROS probe, PF1, are applied to carbohydrate microarrays. The fluorescence intensity of each spot is measured to identify functional glycans. Center-to-center spot distance, 350 μm; spotting volume, 2.6 nL. Reproduced with modification with permission from ref 44. Copyright 2016 Royal Society of Chemistry.

surface that were pretreated with a fluorescent probe, PF1 (Figure 14).57 This probe selectively detects reactive oxygen species (ROS).44 Because binding of glycan ligands to SIGN-R1 on cell surfaces triggers a cellular response that leads to generation of ROS, the resulting fluorescence signal emanating from PF1 is directly related to glycan binding to cell-surface SIGN-R1. Importantly, the differential response of cells to different glycans can be quantitatively determined by using this ROS probe. The results of the effort showed that cells that adhere to glycans on the microarrays generate ROS, as reflected in an increase in fluorescence signals induced by the ROS probe. As expected, levels of ROS in bound cells are attenuated when a ROS scavenger or an NADPH oxidase inhibitor is present. The pattern of ROS production in carbohydrate microarrays was found to be similar to that arising from traditional cell assays. The findings suggest that carbohydrate microarrays can be employed for rapid screening of glycans that enhance the cell-surface lectinassociated cellular response. Because most animal lectins function as signal transducers after binding to glycans, the carbohydrate

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 1076

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(12) Park, S.; Shin, I. Fabrication of carbohydrate chips for studying protein-carbohydrate interactions. Angew. Chem., Int. Ed. 2002, 41, 3180−3182. (13) Wang, D. N.; Liu, S. Y.; Trummer, B. J.; Deng, C.; Wang, A. L. Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells. Nat. Biotechnol. 2002, 20, 275−281. (14) Fukui, S.; Feizi, T.; Galustian, C.; Lawson, A. M.; Chai, W. G. Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nat. Biotechnol. 2002, 20, 1011−1017. (15) Willats, W. G. T.; Rasmussen, S. E.; Kristensen, T.; Mikkelsen, J. D.; Knox, J. P. Sugar-coated microarrays: A novel slide surface for the high-throughput analysis of glycans. Proteomics 2002, 2, 1666−1671. (16) Park, S.; Lee, M. R.; Shin, I. Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes. Chem. Commun. 2008, 4389−4399. (17) Park, S.; Gildersleeve, J. C.; Blixt, O.; Shin, I. Carbohydrate microarrays. Chem. Soc. Rev. 2013, 42, 4310−4326. (18) Rillahan, C. D.; Paulson, J. C. Glycan Microarrays for Decoding the Glycome. Annu. Rev. Biochem. 2011, 80, 797−823. (19) Shin, I.; Park, S.; Lee, M. R. Carbohydrate microarrays: An advanced technology for functional studies of glycans. Chem. - Eur. J. 2005, 11, 2894−2901. (20) Shin, I.; Jung, H. J.; Lee, M. R. Chemoselective ligation of maleimidosugars to peptides/protein for the preparation of neoglycopeptides/neoglycoprotein. Tetrahedron Lett. 2001, 42, 1325− 1328. (21) Shin, I.; Jung, H.-J.; Cho, J. W. Chemoselective Ligation of Acetylated 1-Maleimidosugars to Peptides for the Preparation of Neoglycopeptides. Bull. Korean Chem. Soc. 2000, 21, 845−846. (22) Park, S.; Lee, M. R.; Pyo, S. J.; Shin, I. Carbohydrate chips for studying high-throughput carbohydrate-protein interactions. J. Am. Chem. Soc. 2004, 126, 4812−4819. (23) Lee, M. R.; Shin, I. Fabrication of chemical microarrays by efficient immobilization of hydrazide-linked substances on epoxidecoated glass surfaces. Angew. Chem., Int. Ed. 2005, 44, 2881−2884. (24) Park, S.; Shin, I. Carbohydrate microarrays for assaying galactosyltransferase activity. Org. Lett. 2007, 9, 1675−1678. (25) Park, S.; Lee, M. R.; Shin, I. Fabrication of carbohydrate chips and their use to probe protein-carbohydrate interactions. Nat. Protoc. 2007, 2, 2747−2758. (26) Tian, X.; Pai, J.; Shin, I. Analysis of Density-Dependent Binding of Glycans by Lectins Using Carbohydrate Microarrays. Chem. - Asian J. 2012, 7, 2052−2060. (27) Pai, J.; Yoon, T.; Kim, N. D.; Lee, I. S.; Yu, J.; Shin, I. HighThroughput Profiling of Peptide-RNA Interactions Using Peptide Microarrays. J. Am. Chem. Soc. 2012, 134, 19287−19296. (28) Pai, J.; Hyun, S.; Hyun, J. Y.; Park, S. H.; Kim, W. J.; Bae, S. H.; Kim, N. K.; Yu, J.; Shin, I. Screening of Pre-miRNA-155 Binding Peptides for Apoptosis inducing Activity Using Peptide Microarrays. J. Am. Chem. Soc. 2016, 138, 857−867. (29) Park, S.; Pai, J.; Han, E. H.; Jun, C. H.; Shin, I. One-Step, AcidMediated Method for Modification of Glass Surfaces with NHydroxysuccinimide Esters and Its Application to the Construction of Microarrays for Studies of Biomolecular Interactions. Bioconjugate Chem. 2010, 21, 1246−1253. (30) Lee, M.; Shin, I. Facile preparation of carbohydrate microarrays by site-specific, covalent immobilization of unmodified carbohydrates on hydrazide-coated glass slides. Org. Lett. 2005, 7, 4269−4272. (31) Park, S.; Lee, M. R.; Shin, I. Construction of Carbohydrate Microarrays by Using One-Step, Direct Immobilizations of Diverse Unmodified Glycans on Solid Surfaces. Bioconjugate Chem. 2009, 20, 155−162. (32) Ko, K. S.; Jaipuri, F. A.; Pohl, N. L. Fluorous-based carbohydrate microarrays. J. Am. Chem. Soc. 2005, 127, 13162−13163. (33) Chevolot, Y.; Bouillon, C.; Vidal, S.; Morvan, F.; Meyer, A.; Cloarec, J. P.; Jochum, A.; Praly, J. P.; Vasseur, J. J.; Souteyrand, E. DNA-

Injae Shin: 0000-0001-6397-0416 Notes

The authors declare no competing financial interest. Biographies Ji Young Hyun was born in Kyunggi-do, Korea. She received her B.S. degree in Chemistry in 2013 from Yonsei University. She began her Ph.D. with Prof. Injae Shin in 2013. Her current research includes synthesis of various glycans, construction of glycan microarrays for functional studies of glycans, and development of new methods to target cancer and pathogens. Jaeyoung Pai was born in Seoul, Korea. He received his B.S. degree in chemistry in 2007 from Yonsei University. His Ph.D. degree was awarded in chemistry in 2016 at Yonsei University under the guidance of Prof. Injae Shin. During this period, he conducted research on identification of bioactive peptides using peptide microarrays and functional studies of glycans using synthetic carbohydrates and glycan microarrays. Injae Shin is a director of Center for Biofunctional Molecules and an Underwood Distinguished Professor at Yonsei University. He received his Ph.D. in 1995 in chemistry with Prof. Hung-wen Liu at University of Minnesota and then completed a postdoctoral fellowship in 1998 with Prof. Peter Schultz at University of California at Berkeley. Since 1998 in the Department of Chemistry of Yonsei University, he has conducted functional studies of glycans using chemical tools including glycan microarrays as well as developing many bioactive molecules that are used for biological and biomedical studies.

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ACKNOWLEDGMENTS I.S. thanks all the researchers who have contributed to the creation of this story. This work was supported financially by a grant from National Creative Research Initiative (Grant 20100018272, I.S.) program.

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