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Oyindasola Oyelaran, Joseph C. Manimala, and Jeffrey C. Gildersleeve, Laboratory of Medicinal Chemistry, Center for Cancer Research,
National Cancer Institute, Frederick, Maryland
doi: 10.1002/9780470048672.wecb023
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Article Contents
Biological Background Fabrication of Glycan Arrays Evaluation of Binding Applications of Glycan Arrays Conclusions
Glycan arrays are powerful tools for high throughput analysis of carbohydratemacromolecule interactions. A glycan array is composed of many different carbohydrate structures immobilized on a solid support in an orderly arrangement. This review describes the challenges and considerations in the development of a glycan array. Various array formats, immobilization techniques, and assay systems are discussed. In addition, several interesting applications of glycan array technology are described, such as assessing the specicities of carbohydrate-binding lectins and antibodies, proling antiglycan antibodies in serum as biomarkers of disease, and evaluating carbohydrate-dependent cell binding.
DNA and protein microarray technology has revolutionized the way scientists study complex biological processes. These arrays consist of thousands of nucleic acids or proteins immobilized on a solid support. The array format allows one to rapidly evaluate interactions with a large number of molecules simultaneously. For example, one can examine the expression proles of thousands of genes in a single experiment. Glycan arrays are an equally powerful technology for the evaluation of carbohydratemacromolecule interactions. Analogous to DNA and protein arrays, glycan arrays contain many different carbohydrates afxed to a solid support. This review will focus on development strategies, challenges, and applications of glycan arrays. Several other reviews have been published over the last few years (14) .
Biological Background
Carbohydrates, biopolymers composed of monosaccharide units, play a central role in a wide range of biological processes such as protein folding, inammation, and development. In addition, glycans undergo dramatic changes in expression during the onset and progression of many diseases such as rheumatoid arthritis and cancer. Unfortunately, progress toward dening the specic roles of most carbohydrates and understanding the relationships between structure and function has been frustratingly slow. Furthermore, efforts to exploit altered expression for therapeutic benet have only been successful in a limited number of cases. Molecular recognition is a fundamental element for both basic and applied carbohydrate research (see cross references:
Glycanglycan interactions, Glycanprotein interactions, and Sugarlectin interactions in cell adhesion). Many important biological processes involve specic interactions between a carbohydrate-binding protein (lectin) and a glycan. For example, one early stage of inammation involves interactions of selectins with carbohydrate ligands. Carbohydrateprotein interactions are also directly involved in diseases. For example, many pathogens such as the inuenza virus, E. histolytica , and H. pylori bind carbohydrates on the surface of host cells as a key step of infection (5). For many other carbohydrate-binding proteins, however, the biological functions are still unknown. For example, most lectins used routinely as research reagents are isolated from plants. Although used in the laboratory for many years, the biological roles of many of these proteins are not well understood. As a result, there has been signicant interest in identifying natural and unnatural ligands that can be used to modulate the activity of carbohydrate-binding proteins. Lectins and glycan-binding antibodies are also used extensively as research tools, diagnostics, and therapeutic agents. Information on the specicities of these proteins is critical for interpreting results and selecting the best clinical candidates. Although analysis of carbohydratemacromolecule interactions is crucial for glycobiology, it remains a challenging area of science. First, carbohydrates can be exceedingly difcult to obtain, especially in homogeneous form. With limited access to the ligands, one cannot easily assess recognition. Second, traditional methods used to evaluate carbohydrateprotein interactions such as monosaccharide and oligosaccharide inhibition studies, isothermal calorimetry (ITC), surface plasmon resonance (SPR), and enzyme-linked lectin assays (ELLAs) can be labor intensive or require large quantities of each carbohydrate. 1
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As a result, these methods are not well suited for high throughput evaluations. Finally, one must consider the issue of valency. In most cases, interactions between a single carbohydrate ligand and a single binding domain of a protein (referred to as monovalent binding) are very weak. The afnity of monovalent binding events is typically too low to withstand the washing involved in common biological assays such as enzyme-linked immunosorbent assays (ELISAs), Western blots, and immunohistochemical staining. However, most carbohydrate-binding proteins possess two or more binding sites or assemble into functional units with multiple binding sites. As a result, they can simultaneously bind two or more carbohydrate ligands (referred to as polyvalent binding) leading to a high overall afnity or avidity. Assays that probe interactions between carbohydrates and proteins must account for the unique aspects of polyvalent recognition. For example, the ligands should be presented in a polyvalent context. In addition, the spacing and orientation of ligands will affect the ability to form a polyvalent complex. As a result of the difculties mentioned above, researchers have been developing alternative methods to study carbohydrateprotein interactions. Array technology is specically designed for high throughput evaluations of molecular recognition events. Miniaturization facilitates the process by allowing minimal use of reagents and other hard-to-obtain components. In the following sections, the development and application of glycan array technology are described.
a single carbohydrate in each well (7). The miniaturized wells use a smaller amount of material than a microtiter plate but retain spatial separation of components (see Fig. 1b). A second format involves spotting components directly onto the slides. One of the key differences is that all the carbohydrates are in the same well (see Fig. 1c). Consequently, recognition of each component is compared under identical assay conditions and well-to-well variation is minimized. In addition, microarray printers used for producing DNA microarrays can be used to print very small features (50200-m spots) with high precision allowing for tens of thousands of spots on each slide. As a result, much smaller quantities of material are required and the total capacity is considerably higher than a microtiter plate. However, a microarray printer and high resolution scanner are required. A glass slide can also be modied to create 216 macrowells on a glass slide (see Fig. 1d). In this format, an entire array is printed in each well. Several three-dimensional (3-D) approaches to glycan arrays have also been published, including ber optic bead-based glycan arrays (see Fig. 1e) (8) and hydrogel arrays (9).
Array formats
Antecedents of modern glycan arrays can be traced back to the method of separating a complex glycolipid mixture using thin-layer chromatography, then probing the array with a carbohydrate-binding protein (6). Recently, most glycan arrays are created on either microtiter plates or glass microscope slides (see Fig. 1). The microtiter plate array format involves immobilizing carbohydrates in wells of 96-, 384-, or 1536-well microtiter plates (see Fig. 1a). Each carbohydrate component is spatially separated from other components within the plate. Two of the primary advantages of a microtiter plate format are cost and simplicity. Carbohydrates can be distributed into wells using multichannel pipettors, and assay results can be measured using standard plate readers. Thus, the equipment and supplies needed for the array are relatively inexpensive and common. However, microtiter plates generally require larger amounts of each carbohydrate and can accommodate a smaller total number of components per support unit. An alternative to the microtiter plate format is a glass microscope slide. Slides can come in various layouts. The array developed by Glycominds, Ltd. contains 200 microwells with 2
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Figure 1 Examples of formats that have been used for glycan arrays.
Cu(I)-catalyzed 1,3-dipolar cycloaddition (15); reductive amination reactions between underivatized oligosaccharides and immobilized aminolipids on microtiter plates (16); and urea formation between amino-derivatized sugars and tetradecyl isocyanate adsorbed on microtiter plates (17). Formation of hydrogel microarrays of amino-saccharides and polyacrylamide glycoconjugates has been reported. Here, glycans were printed on hydrophobic glass surfaces with polymerizable monomers, and then polymerization was induced by photoactivation to form 3-D gel elements comprising the glycans (4, 9). An elegant alternative approach for noncovalent interaction relies on uorousuorous interactions. A glycan array of monosaccharides and disaccharides bearing anomeric uorous tags was noncovalently immobilized on uorous-derivatized glass slides (19, 20). The attachment method is compatible with a wide range of functional groups and has been successfully used to probe carbohydrateprotein interactions. Glycan arrays have also been developed to take advantage of the noncovalent, but extraordinarily strong, biotinstreptavidin interaction. The Consortium for Functional Glycomics developed a glycan array consisting of biotinylated oligosaccharides and amino acid glycoconjugates immobilized on streptavidincoated 384-well microtiter plates (21, 22). Another biotinstreptavidin sandwich glycan microarray was produced by self-assembly of biotinylated alkylthiols on a gold substrate, followed by coating with streptavidin and printing of biotinylated polyacrylamide glycoconjugates (23). An alternative to noncovalent approaches is covalent attachment of ligands to the surface. Methods for covalent attachment can be broadly categorized as follows: 1) condensation with the aldehyde of a reducing sugar, 2) nucleophilic addition to or
displacement of a group on an activated surface or sugar, 3) cycloaddition, and 4) insertion reactions (see Fig. 2b). Some derivatization methods, for example, condensation and reductive amination, lead to ring opening of the reducing end sugar, whereas other methods result in indeterminate anomeric congurations, depending on the reaction conditions and point of attachment. The linker used to connect the sugar to the surface is an important consideration because it can affect recognition and accessibility of the ligands. In choosing an ideal linker, the issues to consider include the chemical nature and composition (hydrophobic vs. hydrophilic), stability, length, and exibility of the linker. Unmodied sugars have been attached to various hydrazideand amino-derivatized slides by condensation or by reductive amination, which proceeds by reduction of the intermediate Schiff base (see Fig. 2b.1). This strategy was used for immobilization of free oligosaccharides onto hydrazide-derivatized plates (24) and various unmodied carbohydrates (monosaccharides, oligosaccharides, and polysaccharides) on hydrazidecoated glass slides (25). Later, immobilization of free oligosaccharides on hydrazide-derivatized self-assembled monolayer (SAM) of gold-coated glass slides was also reported (26). The formation of oximes between reducing end aldehydes of sugars and amino-oxy groups has been exploited in the fabrication of glycans arrays. This method was explored for the immobilization of monosaccharides, disaccharides, and oligosaccharides on amino-oxy glass slides and successfully used for making a functional glycan array of oligosaccharides on aminooxyacetyl-functionalized glass slides (27). However, stronger signals were observed for hydrazide-coated slides in comparison with aminooxy-coated slides (25). Several glycans 3
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(a)
(b)
Figure 2 Immobilization strategies. (a) A schematic representation of noncovalent adsorption. The lipid tail of a neo-glycolipid adheres to a hydrophobic surface via noncovalent interactions. (b) Strategies used for covalent immobilization of glycans.
arrays have also been reported where the simple condensation of aldehydes and amines was used to covalently attach the saccharides. Deaminated heparin oligosaccharides bearing aldehyde groups were attached to amine-coated glass slides as Schiff bases without further reduction (28), while aldehyde and amino functionalized monosaccharides and disaccharides were immobilized on amine- and aldehyde-coated slides, respectively (29, 30). It should be noted that reductive amination results in opening of the reducing end sugar whereas condensation can lead to changes in anomeric conguration or mixtures of anomers at the reducing end. An alternative strategy used in the covalent attachment of glycans to solid supports is nucleophilic addition or displacement. In this method, the decision about where to install the nucleophile and the electrophileon the sugar or on the solid supportrests with the researcher, but could be inuenced by the ease of installation and availability of derivatized reaction partners. Glycan arrays involving the Michael addition of maleimide-linked carbohydrates and thiol-coated glass slides (see Fig. 2b.2) have been created and used to probe lectincarbohydrate interactions (31, 32). The transposition of the reacting functional groups has also been reported where glycans arrays were fabricated using thiol-linked sugars on maleimide-functionalized glass slides (33, 34) and on self-assembled monolayers presenting maleimide groups (35). Epoxide-opening reactions have also been used to covalently attach carbohydrates, glycoproteins, and neoglycoconjugates to glass slides (see Fig. 2b.3). One report demonstrated the feasibility of immobilizing hydrazide-functionalized small molecules, including monosaccharides, on epoxide-coated slides and further showed that hydrazide-containing ligands react more 4
rapidly with the epoxide-coated surface than thiols and amines (36). The use of epoxide-coated slides is relatively common for the fabrication of protein microarrays. One advantage of using epoxide-coated surfaces is the potential indiscriminate reactivity of epoxides to nucleophiles such as amines, thiols, and alcohols. However, it is important to note that the reactivity of these nucleophiles often depend on the pH conditions during printing. The development of glycan arrays of neoglycoconjugates and glycoproteins on epoxide-coated slides has been described (37, 38). In this report, neoglycoconjugates were synthesized by conjugating to lysine residues on serum albumin through activated carboxyl-bearing linkers, and then printed directly onto epoxide-functionalized glass slides. The facile reaction of amines with N-hydroxysuccinimide (NHS)-activated carboxyl groups, and the concomitant formation of a stable amide bond, has been exploited in the fabrication of glycan arrays (see Fig. 2b.4). This method has been used for covalent attachment of amine-presenting glycans ranging in size from monosaccharides to glycoproteins on NHS-activated slides (39, 40). In one report, oligosaccharides were derivatized with photocleavable amine-bearing linkers used to attach the ligands to a porous silicon surface presenting NHS groups (41). Oligosaccharides labeled with uorescent 2,6-diaminopyridine have also been printed on NHS-activated glass slides (42). An array of monosaccharides and oligosaccharides was created by the reaction of p-aminophenyl-glycosides with cyanochloride-activated linkers on wells of microtiter plates (7). In the fabrication of bead-based glycan arrays, glycopeptides and bovine serum albumin neoglycoproteins were conjugated to the carboxyl-presenting beads using water-soluble carbodiimides. The beads were spatially arrayed into microwells for
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screening with lectins (8) or assayed for lectin binding as a suspension (43). Covalent immobilization of glycans by cycloaddition (see Fig. 2b.5) and insertion reactions (see Fig. 2b.6 and 2b.7) has been used in the creation of arrays (4447). Cyclopentadiene-carbohydrates were arrayed on self-assembled monolayers alkanethiols presenting benzoquinione through penta(ethylene)glycol linkers (44). An efcient DielsAlder reaction between the modied glycan and the support and the reduction of nonspecic interactions by the use of the glycol linker are highlights of this report. One attachment method that portends the development of functional group-independent reactions where any underivatized molecule can be immobilized is the use of insertion reactions. In one report, various glycoproteins were printed on polymeric surfaces presenting aryltriuoromethyl diazirines. The surfaces were photoactivated resulting in formation of a covalent bond between the solid support and the printed ligand by carbene CH insertion (46). In an alternative formal insertion strategy, glycans arrays were created by printing underivatized carbohydrates on SAMs presenting phthalimide groups, which, on photoactivation, undergo hydrogen atom abstraction and recombination of radicals to form a covalent bond (47).
Glycopeptides and glycoproteins, synthesis of). This approach can provide both natural and unnatural structures that can be very useful for probing relationships between structure and function. Although there have been tremendous advances in our ability to synthesize carbohydrates both in solution and on solid phase, carbohydrate chemistry remains a challenging area of organic chemistry. Progress on the synthesis of carbohydrate libraries (4850), automated carbohydrate synthesis (51), and one-pot multicomponent synthetic strategies (41, 52, 53) will substantially aid glycan array development. Enzymes can be powerful tools for obtaining carbohydrates as well (54). Chemoenzymatic synthesis of oligosaccharides involves the use of glycosyltransferases or glycosidases for the regio- and steroselective formation (typically between unprotected reaction partners) or hydrolysis of glycosidic bonds, respectively. In some cases, one reaction partner is obtained by traditional solution or solid-phase chemical synthesis, followed by enzymatic glycosylation. In other cases, synthesis begins with enzymatic cleavage and continues with more traditional chemical synthesis methods.
Evaluation of Binding
Once a glycan array is constructed, one needs to develop a method to evaluate binding of proteins, cells, viruses, or other macromolecules to the array. The general process involves carrying out the assay, detecting signals on the array, and analyzing/processing the results. Although the overall process appears straightforward, assay development can be very challenging. Importantly, every step in the process from fabrication to assay conditions to detection must be successful to produce a signal. As a result, it can be difcult to determine which step or steps need to be optimized, especially when no signal is observed. Therefore, assay development typically requires systematic variation of many parameters. Additional considerations include sensitivity, signal-to-noise ratios, reproducibility, exibility, and dynamic range. The vast majority of array assays have used uorescence as the detection method due to the high sensitivity and the availability of detectors such as uorescent plate readers for microtiter plates and high resolution DNA microarray scanners for glass microarray slides. Therefore, the following discussion will focus on assay development coupled with uorescent detection.
Nonspecic adsorption
One important general consideration is nonspecic binding. Proteins and other macromolecules can adsorb to surfaces leading to high background signals and low signal-to-noise ratios. One common approach to prevent nonspecic adsorption is to cover or block any sticky surface areas with a noninterfering protein or polymer that is unrelated to the receptor or secondary reagent such as bovine serum albumin (BSA), which simply involves incubating the array with a solution of BSA in an appropriate buffer (e.g., 3% BSA in phosphate buffered saline). An alternative approach involves using modied surfaces that resist nonspecic adsorption of proteins such as uorous-coated slides (19, 20, 55). 5
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Table 1 Summary of glycan arrays Group or Company Consortium for Functional Glycomics Feizi, T. Gildersleeve, J. Glycominds Hsieh-Wilson, L. Koberstein, J. Ligler, F. Malayer, J. Melnyk, O. Mrksich, M. Pohl, N. Rubina, A. Ruhl, S. Schmidt, R. Seeberger, P. Shin, I. Sprenger, N. Number of Componentsa 264
Composition Oligosaccharides; neoglycoconjugates Neoglycolipids Neoglycoproteins; glycoproteins Oligosaccharides Oligosaccharides Oligosaccharides; polysaccharides Monosaccharides Lipopolysaccharides Polysaccharides Monosaccharides Mono-, disaccharides Oligosaccharides Neoglycoconjugates; glycoproteins Mono-, disaccharides Oligosaccharides Oligosaccharides Oligosaccharides; neoglycoconjugates; glycoproteins Neoglycolipids Anionic glycopolymers Polysaccharides; glycoproteins; glycosaminoglycans; neoglycoconjugates Neoglycolipids Polysaccharides; proteoglycans; neoglycoconjugates Oligosaccharides; Neoglycolipids Glycopeptides Oligosaccharides Oligosaccharides
Format(s) Plates; glass slides Glass slides 16-well glass slides 200 microwells 1 sugar/well Glass slides Glass slides Glass slides Glass slides Glass slides Glass slides Glass slides Glass slides Nitrocellulose membrane Glass slides Glass slides; Fiber optic Glass slides Glass slides
References [21, 22, 40, 64, 65, 68, 7072, 7476, 7881, 97, 98] [13, 67, 69, 73, 77] [37, 38] [7, 66, 85, 9093, 95] [29] [47] [82] [88] [12] [35, 44] [19, 20] [9] [96] [30] [8, 28, 34, 39, 5759, 83, 94] [25, 31, 32, 36] [46]
190 73 37 4 10 2 9 51 10 8 6 28 7 14 6 22 7
14 3 51
Wang, P. Willats, W.
Plates Black polystyrene slides Glass slides; Plates Fiber optic Glass slides Glass slides
4 23
[16] [11]
6 21 12 3 10
[14, 15, 17, 41, 45, 84, 86, 99, 100] [43] [26] [27]
of uorophore per molecule of receptor. With a limited number of uorophore molecules, this approach generally has the lowest sensitivity. In addition, covalent labeling of a protein can sometimes interfere with the functional properties of a protein. Nevertheless, this approach has been used successfully in many reports (19, 20, 3234, 42, 46, 5659).
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molecule of enzyme can generate many molecules of uorescent product, the signal is amplied. Moreover, the signal can be enhanced by increasing the length of time the substrate is incubated with the enzyme. In addition to enhanced sensitivity, use of a secondary reagent allows detection of receptors in the presence of complex mixtures of other proteins due to the specic binding between the primary and secondary proteins. For example, human serum contains a wide variety of different proteins and other components. With the aid of a secondary reagent, glycan-binding antibodies present in human serum can be proled as biomarkers of disease (see below). Naturally, this approach is limited to situations where a secondary reagent is available.
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Kelvin nanoprobe (63) are some of the tools that have been adopted for label-free assay systems.
also used to evaluate binding properties of the rat asialoglycoprotein receptor, Kupffer cell receptor, macrophage galactose lectin, and human scavenger receptor C-type lectin (71, 72). Based on their galactose-binding preferences, the lectins were classied into two categories: one group selective for Lewis antigens and the other showed broad selectivity for various Gal/GalNAc-containing residues. A glycan array consisting of LeX , SLeX , and their sulphated analogs was used to prole siglecs (73). This analysis revealed that the sulfate groups on the carbohydrate epitopes act as modulators of siglec binding. Through the use of a glycan array, mouse Siglec-F and human Siglec-8 (21) expressed on eosinophils were also shown to recognize 6 -sulfo-sialyl LeX (74). A polysaccharide microarray was used to study interactions between dextran polysaccharides and platelet-derived growth factor BB isoform (12). A chondroitin sulfate microarray was used as a screening tool to identify an antagonist for a therapeutically important proinammatory cytokine, tumor necrosis factor- (29). Other animal lectins proled with glycan arrays include galectin-1 (75), galectin-4 (76), mannose 6-phosphate receptor (77), dectin-2 (78), Manila clam lectin (79), and Langerin (67). Bacterial, viral, and microbial lectins are a third class of important proteins that have been screened on glycan arrays. One of the most interesting applications of glycan arrays involved proling hemagglutinins from pathogenic strains of the inuenza A virus including the pandemic 1918 strain (80, 81). Avian viruses were found to preferentially bind sialic acid with alpha 2-3 linkages while human viruses bound alpha 2-6 linkages. The difference in specicity provides a potential explanation for the variations in infectivity and pathogenicity. A group of bacterial toxins and cells, Salmonella typhimuriam , Listeria Monocytogenes , Escherichia coli , staphylococcal enterotoxin B, cholera toxin, and tetanus toxin, has also been proled on a glycan array (82). Several lectins with potent antiHIV activity such as cyanovirin, scytovirin, and microvirin have been evaluated on glycan arrays (58, 83). Another important application of glycan arrays is assessing the specicity of glycan-binding antibodies (37, 58, 8487). Carbohydrate-binding antibodies are important research tools, diagnostics, and therapeutic agents. Information on specicity is critical for the proper interpretation of results or selection of clinical candidates. For example, antibody 2G12 is known to neutralize a broad range HIV-1 isolates in a carbohydrate-dependent manner. Proling using a glycan array illustrated that the peptide backbone is not required for binding and that the antibody has a preference for Man1-2Man linkages (58, 84). A glycan array comprising the tumor antigen Globo H and its fragments was used to prole two monoclonal antibodies against Globo H and serum from breast cancer patients (86). This array showed that the fucose residue was essential for recognition by the monoclonal antibodies but not for the serum antibodies. In another glycan array study, various antibodies and lectins that have been used for decades to monitor the expression of the tumor-associated Tn antigen were found to cross-react with other human glycans (37). The results suggest that information on the expression of the Tn antigen used for the development of diagnostics and vaccines may be inaccurate. The antimalaria antibody MG96 was evaluated in an effort
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Conclusions
Glycan arrays have emerged as powerful tools for analysis of carbohydratemacromolecule interactions. As research tools, they are being used to identify ligands for glycan-binding proteins and evaluate specicity of carbohydrate-binding proteins. In a clinical setting, glycan arrays hold enormous potential for the development of diagnostic and therapeutic agents. For example, several studies have demonstrated the utility of glycan arrays for proling antiglycan antibodies in serum as diagnostic markers of disease. As the technology matures, the range of applications and capabilities will continue to expand and glycan array analysis is anticipated to become routine in chemistry, biology, and clinical laboratories.
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Cellcell recognition can be a complex process involving multiple interactions between different proteins and glycans. In some cases, the specic proteins involved are not known or are not functional on their own. In addition, cell binding can trigger signaling cascades or other responses. As a result, evaluation of binding of whole cells to glycans can be very useful and several reports have demonstrated successful screening of cells on glycan arrays. Examples include screening of E. coli strains (25, 94), chicken hepatocytes and CD4+ human T-cells (95), and Helicobacter pylori (96). Viruses and viral capsids have also been screened on glycan arrays (97, 98).
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Glycan-Glycan Interactions Glycan-Protein Interactions Glycopeptides and Glycoproteins, Synthesis of Isolation of Glycans Sugar-Lectin Interactions in Cell Adhesion
See Also
Glycan Synthesis, Key Reactions of Glycan Synthesis, Key Strategies for Glycan Synthesis, Protection and Deprotection Steps of
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WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.