Glycobiology Advance Access originally published online on September 12, 2006
Glycobiology 2006 16(12):21C-27C; doi:10.1093/glycob/cwl044
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COMMUNICATION |
Arraying glycomics: a novel bi-functional spacer for one-step microscale derivatization of free reducing glycans
Glycan Array Synthesis Core D, Consortium for Functional Glycomics, Department of Molecular Biology, The Scripps Research Institute, CB 248A 10550 N, Torrey Pines Road, La Jolla, CA 92037
1 To whom correspondence should be addressed; e-mail: olablixt{at}scripps.edu
Received on June 12, 2006; revised on August 25, 2006; accepted on August 30, 2006
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Abstract |
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 Top Abstract IntroductionResults and discussionMaterials and methodsSupplementary dataAcknowledgmentsConflict of interest statementReferences |
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Glycan array development is limited by the complexity of efficiently generating derivatives of free reducing glycans with primary amines or other functional groups. A novel bi-functional spacer with selective reactivity toward the free glycan and a second functionality, a primary amine, was synthesized. We demonstrated an efficient one-step derivatization of various glycans including naturally isolated N-glycans, O-glycans, milk oligosaccharides, and bacterial polysaccharides in microgram scale. No protecting group manipulations or activation of the anomeric center was required. To demonstrate its utility for glycan microarray fabrication, we compared glycans with different amine-spacers for incorporation onto an amine-reactive glass surface. Our study results revealed that glycans conjugated with this bi-functional linker were effectively printed and detected with various lectins and antibodies.
Key words: microarray / oligosaccharide / polysaccharide / reducing-end derivatization
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataAcknowledgmentsConflict of interest statementReferences |
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More than 50% of all proteins carry various glycan chains. Glycans are a large group of sugars with diverse structures present both inside and on the cell surface. The way they interact with proteins allows them to be involved in various biologically important events that underlie the development and function of multicellular organisms. Many of these interactions take place on the cell surface where sugars are linked to proteins and lipids. The interaction of glycans with proteins can be studied in various ways. Recently, we developed a new method using microarrays of immobilized glycan structures (Blixt et al., 2004
). The development of nucleotide and protein microarrays has revolutionized proteomics and pharmacogenomics. Microarray technology has become a key tool for new important discoveries highlighted in >3600 articles published in 2005 alone (Casey, 2005). However, the development of glycan microarrays has been very slow mostly because of difficulties in reliable immobilization of chemically and structurally diverse glycans and complex methods for their synthesis (Drickamer and Taylor, 2002). All glycans synthesized de novo chemically or chemo-enzymatically are subject to further derivatization and coupling to the reducing terminal sugar residue of an absorptive or reactive group required for printing on desired array surface (Ye and Wong, 2000; Blixt and Razi, 2004; Seeberger and Werz, 2005). Ultimately, the utility of glycan microarray depends on an appropriate match between the glycan structures it contains and the specificity of the glycan-binding protein (GBP) being analyzed (Paulson et al., 2006). The ideal array would have the entire glycome on a single chip allowing any GBP to be assessed. Thus, there is a need for a new and facile method to activate microgram quantities with the potential for derivatization of any free reducing glycan for direct printing or further conjugation.
The derivatization of free reducing glycans has mostly been done via reductive amination with various amine-containing compounds including proteins, glycolipids, and solid-supports (Baues and Gray, 1977; Matsumoto et al., 1982; Fukui et al., 2002). The recent work from Xia and others (2005) demonstrated a procedure to attach a 2,6-diaminopyridine (DAP) via reductive amination to obtain an aromatic amine for further functionalization. However, this anchoring technique suffers from poor reactivity, and the end result is an open-ring derivative on the penultimate structure where part of the structural integrity is lost. The limited reactivity can be overcome by substituting amino groups with hydrazide groups, which will lead to an increased nucleophilicity caused by the 
-effect (Seppälä and Mäkelä, 1989). Similarly, the N-hydroxylamine functional group has also been utilized for the attachment of free glycans as first demonstrated by Peri and others (1998) and later by others (Carrasco and Brown, 2003; Niikura et al., 2005). Alkyl N,O-substituted hydroxylamines demonstrated the ability to retain the cyclic nature of the saccharide reducing unit with high anomeric stereoselectivity and without the requirement of activation of the anomeric center (Peri et al., 1998). Recently, underivatized carbohydrates were immobilized on hydrazide- and aminooxy-coated glass slides (Lee and Shin, 2005; Vila-Perello et al., 2005; Zhou and Zhou, 2006) or onto a surface plasma resonance sensor surface. Despite increased reactivity due to the -effect, direct surface immobilization generally suffers from slow reaction rates and, consequently, requires high concentration (mM) of the carbohydrate ligand. Instead, our concept was to effectively derivatize free reducing glycans by using high concentrations of spacer and subsequently print them onto a reactive surface that requires much less glycans (µM).
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Results and discussion |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataAcknowledgmentsConflict of interest statementReferences |
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Taking advantage of the novel reactive chemical feature of hydroxylamines, we sought to develop a bi-functional spacer containing both the methyl N,O-hydroxylamine and a second amine functionality suitable for the attachment onto our established N-hydroxysuccinimide (NHS)-activated glycan array platform. N-Boc-protected 2-aminoethyl bromide (2) reacted with N-Boc-protected methyl N,O-hydroxylamine (4), which gave the intermediate Boc-protected bi-functional spacer in 54% yield. After deprotection with trifluoroacetic acid (TFA), the bi-functional spacer (5) was isolated in 87% yield in high purity (>95%) as estimated by nuclear magnetic resonance (NMR) spectroscopy (Figure 1A).
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Our new spacer reacted with various model compounds representing the reducing-end moiety of natural glycans both in analytical and in preparative scale. The N-acetylactosamine (LacNAc), where the reducing-end monosaccharide is N-acetylglucosamine (GlcNAc), was the model substrate for the corresponding chitobiose structure of N-glycans. For bacterial polysaccharides, where the reducing-end monosaccharide residue is 3-deoxy-manno-oct-2-ulosonic acid (Kdo), the model compound was N-acetylneuraminic acid (Neu5Ac) and the disaccharide, lactose, served as the model for milk- and ganglio-oligosaccharides.
Other derivatized glycans were prepared in 0.1–1 mg scale and identified by high-resolution mass spectrometry (HR-MS). Briefly, a given oligosaccharide and spacer 5 (excess) were each dissolved in aqueous buffer and incubated for 24–48 h. The derivatization was nearly quantitative (>95%). The excess spacer was completely removed from the glycoconjugate via a one-step purification process using a Carbograph column or size-exclusion chromatography to obtain the product with high recovery (80–95%). The thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) chromatogram, and MS profile of LacNAc derivative (6) and lactose derivative (14) confirmed >95% conversion of starting material to product with expected masses: m/z calculated for M+H was 456.2115 (found to be 456.2192) for compound 6 and m/z calculated for M+H was 415 (found to be 415) for compound 14 (Figure 2). The 1H-NMR spectra revealed that >95% of the conjugated LacNAc and lactose were ß-anomers (Supplementary Figures 2–5). The reaction with Neu5Ac was also nearly quantitative (m/z calculated for M+Na was 404.1645 but found to be 404.1684), but the 1H-NMR indicated a mixture of isomeric spacered products (16) (data not shown). However, these isomers are of minor importance for lectin recognition of larger polysaccharides. Moreover, small-scale derivatization and isolation of a biantennary N-glycan and the Galß1-3GalNAc disaccharide gave compounds 11 (m/z calculated for M+Na was 2043 but found to be 2043) and 12 (m/z calculated for M+H was 456.2115 but found to be 456.2189) (Figure 2), respectively.
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Next, we performed the conjugation of a bacterial core oligosaccharide from a phosphoethanolamine-deficient strain of Neisseria meningitidis (galE/lpt3 double mutant) (Cox et al., 2005
) to give structure 13. Although we did not obtain a good MS profile, printing and subsequent monoclonal antibody detection were successful. An attempted derivatization of a sulfated disaccharide did not show a correct MS profile. Nevertheless, the 1H-NMR spectrum indicated a coupling of the linker to the sulfated sugar (not shown). We assume that there was a degradation or fragmentation of derivatized product because the sulfated groups are very labile (Thanawiroon et al., 2004; Henriksen et al., 2006). Further optimizations and characterizations are in progress.
The stability of the derivatized sugars at different pH is of practical interest. Langenhan and others (Langenhan, Griffith, et al., 2005; Langenhan, Peters, et al., 2005) tested the chemical stability of N,O-dialkylamine derivatives at three different pH. Under neutral and basic pH, the neoglycoside was stable for 1 month, but a slow degradation was detected under acidic pH. To prove the stability of our spacered conjugates, we incubated the lactose conjugate (14) at neutral and acidic conditions at room temperature (RT) for 3 days followed by TLC and HPLC analysis. Chromatograms from samples incubated in deionized water (pH 5.5) and MES buffer (0.1 M, pH 6.0) did not show any degradation of derivatized product. After 3 days of incubation, there was 
10% degradation in the sample incubated with HCl (10 mM, pH 2.5). No degradation of conjugates was detected in print buffers (pH 8.5) or blocking buffers containing ethanolamine (pH 9.5) or during the storage of slides for at least 2 months (data not shown). The glycoconjugates were used for printing without further purifications.
Next, we compared the printing efficiency of the bi-functional spacer-derivatized glycans 6–13 with other commonly used amino derivatives such as 2-aminoethyl- (7), 4-aminophenyl- (8), 2,6-diaminopyridine (9), and aspargine (10) (Figure 2). All compounds were printed at equal conditions in a 2-fold dilution series (200–0.4 µM printing concentrations). We previously demonstrated that during these printing conditions, compound 7 is incorporated at saturated conditions at >50 µM printing concentrations (Blixt et al., 2004). The printed compounds were detected with biotinylated LacNAc-specific Ricinus communis agglutinin I (RCA I) and Neu5Ac2-6-specific Sambucus nigra agglutinin (SNA). The spacered disaccharide, Galß1-3GalNAc (12), was detected with Bauhinia purpurea lectin (BPL), which is specific to terminal ß-linked GalNAc. There was no binding when we used Amaranthus Caudatus lectin (ACL), which binds to -linked GalNAc, as we have previously confirmed (Alvarez et al., 2005). The N. meningitidis core oligosaccharide was detected, with a monoclonal antibody (IgG) (Cox et al., 2005).
As expected, compounds with a primary amine on an alkyl chain (6, 7, 11), or the amino acid asparagine (10), were printed with equal efficiency (Figure 3A and B). In contrast, the binding of primary aromatic amine (8) was less efficient and the DAP derivative (9) hardly bound at all. This is in sharp contrast to what was reported by Xia and others (2005). Moreover, the printed LacNAc derivatives were also stained with the Neu5Ac2-6-LacNAc-specific SNA lectin, which only bound to the spacered N-glycan containing the Neu5Ac2-6-LacNAc structure on one of the branches (image inset 11A and Figure 3C). Thus, the conjugation conditions used did not affect compounds with acid-labile Neu5Ac. Derivatized lactose (14) was well printed and detected with RCA I (not shown).
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In conclusion, the glycoconjugates synthesized with our new bi-functional spacer have several important advantages such as (i) a ring-closed derivative with preserved structural integrity, (ii) a reactive primary amine for efficient coupling onto amine-reactive glass slide or other supports, (iii) one-pot, one-step procedure, and (iv) stable conjugates. Despite a few limitations (both
- and ß-isomers of Neu5Ac and instability of sulfated disaccharide), the derivatization procedure we report will have the potential to further diversify and expand the structures for attachment onto amino-reactive microglass slides. Thus, in combination with recent developments of efficient isolation and purification of natural glycans, along with the increased availability of commercial glycans, our work will contribute significantly toward the ultimate goal of arraying both the human and bacterial glycomes. |
Materials and methods |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataAcknowledgmentsConflict of interest statementReferences |
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General methods
Compounds 8 (Blixt et al., 1999
), 9 (Xia et al., 2005), and 10 (Kajihara et al., 2005) were prepared as previously described. The Galß1-3GalNAc disaccharide was from Toronto Research Chemicals (North York, Ontario, Canada), and compound 13 was a generous gift from Dr. J. Richards and Frank St. Michael (National Research Council [NRC], Ottawa, Ontario, Canada). Plant lectins were from Vector Laboratories (Burlingame, CA). Silica gel (60 Å, 40–63 µm) was from EMD Chemicals (Gibbstown, NJ). All other chemicals were from Sigma-Aldrich (St. Louis, MO).
The reactions were monitored by TLC performed on Silica Gel 60F precoated TLC plates (EMD Chemicals). After development with appropriate eluents, the spots were visualized by UV light for nucleotides and/or dipping in 5% sulfuric acid in ethanol or a spraying with a solution of ninhydrin (0.5 M in dimethylsulfoxide), followed by charring to detect sugars. NMR spectra were recorded on Bruker DRX-500 and DRX-600 MHz instruments at 25°C and were referenced to H2O 
4.79 (1H in D2O), acetone ( 2.225), or MeOH 3.25 (1H in MeOD) or 49.0 (13C in MeOD). MS profiles were recorded with a liquid chromatography mass selective detector TOF (Agilent Technologies, Palo Alto, CA) using dihydroxybenzoic acid as matrix. Water was purified by NanoPure Infinity Ultrapure water system (Barnstead/Thermolyne, Dubuque, IL) and degassed by vacuum treatment before use.
Synthesis of compound 2
Ethanolamine (1) (40 mmol) and di-tertbutyl dicarbonate (32 mmol) were dissolved in CH2Cl2. Triethylamine (TEA) (40 mmol) was added, and the mixture was stirred for 4 h at room temperature under N2. The mixture was washed with Na2SO4 (0.1 M, 3x 200 mL) and brine (2x 200 mL). The organic layer was dried with anhydrous MgSO4 and filtered. The solvent was removed by rotary evaporation to yield the protected amine (1.3 g, 28%). The alcohol was dissolved in CH2Cl2 (45 mL), followed by adding MsCl (13.8 mmol) and TEA (17.9 mmol). The reaction mixture was stirred at room temperature for 45 min under N2. LiBr (138 mmol) in acetone (45 mL) was added, and the mixture was stirred for an additional 17 h. The solvents were removed by rotary evaporation, and the remaining residue was dissolved in ethyl acetate (EtOAc) (125 mL) and washed with H2O (2x 75 mL), saturated with NaCO3 (75 mL) and brine (75 mL). The solution was dried with anhydrous MgSO4 and filtered and concentrated by rotary evaporation. The product mixture was purified on a silica column (3 x 25 cm) and eluted with hexane : EtOAc (80:20). Appropriate fractions were collected and concentrated to give 2 (1.2 g, 64%), which was used without further purifications. Spectral data for compound 2 were as follows: 1H-NMR (500 Hz, CDCl3) (ppm) 5.04 (s, 1H, NH), 3.55 (dd, 4H, CH2), 1.51 (s, 9H, CH3).
Synthesis of compound 4
Methyl N,O-hydroxylamine (3) (40 mmol) and di-tertbutyl dicarbonate (32 mmol) were dissolved in CH2Cl2. TEA (40 mmol) was added, and the mixture was stirred for 4 h at room temperature under N2. The mixture was washed with 0.1 M Na2SO4 (3x 200 mL) and brine (2x 200 mL). The organic layer was dried with anhydrous MgSO4 and filtered. The solvent was removed by rotary evaporation to give 4 (3.0 g, 72%) and used without further purification. Spectral data for compound 4 were as follows: 1H-NMR (500 MHz, MeOD) (ppm) 3.18 (s, 3H), 1.54 (s, 9H).
Synthesis of compound 5
Compound 4 (7.2 mmol) was dissolved in N,N-dimethylformamide (DMF) (4.75 mL), and NaH (6.92 mmol) was added. The reaction mixture was stirred for 1 h at room temperature under N2. The mixture was cooled to 0°C, and compound 2 (5.8 mmol) was dissolved in DMF (5 mL). The mixture was stirred for 3 h on ice, followed by purification on a silica column (3 x 25 cm), and eluted with hexane : EtOAc (70:30). The appropriate fractions were collected and evaporated to give protected 5 (0.8 g, 54%). An aliquot of protected 5 (1.94 mmol) was dissolved in CH2Cl2 (2.5 mL), and TFA (9.68 mmol) was added. The reaction mixture was stirred at room temperature under N2 for 30 min. TLC confirmed quantitative deprotection to amine. Dowex 1 x 8 x 400 mesh (OH) was added (10 equiv.) to neutralize the TFA. The product solution was lyophilized and reconstituted in water, and any remaining precipitate was removed by centrifugation. The supernatant was lyophilized to yield 5 (0.15 g, 87%) as a white amorphous solid. Spectral data for compound 5 were as follows: 1H-NMR (500 MHz, D2O), (ppm): 2.99 (s, 3H, N-CH3), 3.27 (t, 2H, CH2), 4.35 (t, 2H, CH2) and 13C-NMR (500 MHz, MeOD), (ppm): 36.21, 38.91, 71.21 (Supplementary Figure 1, 1H-NMR compound 5). Electrospray ionization time-of-flight (ESI-TOF) high-accuracy MS m/z calculated for (M+H) was 91.087 but found to be 91.087.
General preparation of compounds 6, 11–14
Free reducing glycans (10–50 nmol) and spacer 5 (0.2–1.0 µmol) were dissolved in aqueous acetate buffer (0.1 M, pH 4.5–6.5, 20–200 µL) and incubated at 37°C for 24–48 h. To remove excess spacer and to desalt the sample, we purified the reaction mixture by Method A: neutral and charged oligosaccharides and polysaccharides were loaded on 0.5-mL Carbograph columns. Bound derivatized glycans were eluted with appropriate concentration of acetonitrile with or without TFA (Packer et al., 1998). Fractions were lyophilized, and the presence of synthesized product was proved by TLC and MS. Compounds were isolated in high yields (>90%) and high purity (>95%) and when possible verified by HPLC (see Method C). Lyophilized structures were used for printing without further purifications; or Method B—neutral and charged mono- and disaccharides were isolated by preparative TLC (ethyl acetate : acetic acid : methanol : water, 6:3:3:2, by volume) or size-exclusion chromatography (Sephadex G15, 0.8 x 120 cm, 5% n-BuOH). The obtained compounds were used for printing without further purifications; or Method C—the reaction mixture of 6 was loaded (100 µL injection volume, 1 mg/mL) onto an amino column (255 x 4.6 mm, 5 µm, Altima Amino, Altech, Deerfield, IL) conditioned in acetonitrile. Elution gradient (water : acetonitrile, 0–20%:100–80% over 20 min followed by isocratic water : acetonitrile 20:80 for 20 min) gave spacered products in >95% purity. For charged compounds, TFA (0.1%) was added to the gradient. Spectral data for compound 6 were as follows: selected 1H-NMR (600 MHz, D2O), (ppm)—4.48 (d, 1H, J = 7.8 Hz, Gal H-1), 4.26 (d, 1H, J = 9.6 Hz, GlcNAc H-1), 4.06–4.03 (2m, 2H, OCH2CH2NH2), 3.18–3.26 (2m, 2H, OCH2CH2NH2), 2.79 (s, 3H, NCH3), and 2.05 (s, 3H, NHCOCH3) (Supplementary Figure 3, 1H-NMR compound 6). ESI-TOF high-accuracy MS m/z calculated for (M+H) was 456.2115 but found to be 456.2192.
Printing of arrays
The glycan arrays were created by robotic contact printing of 0.6 nL of glycans linked to the different spacers in print buffer (300 mM phosphate, 0.005% Tween-20, pH 8.5) onto NHS-activated glass slides (Blixt et al., 2004). Each spacered structure (6–14) was printed at 10 different concentrations in 2-fold dilutions (200–0.4 µM), and each dilution was deposited 10 times, creating a 10 x 10 subgrid for each spacered compound. Immediately after the print, slides were placed in a chamber at 80% humidity for 30 min. The remaining NHS groups were blocked by immersing the slides in blocking buffer (50 mM ethanolamine in 50 mM borate buffer, pH 9.2) for 1 h. Slides were rinsed in water, dried under a stream of nitrogen, and stored in desiccator at room temperature before use.
Lectin staining
The spacer test arrays were analyzed with plant lectins and monoclonal antibody without any further surface modifications of the slides. Before incubation, the print area was bordered with a hydrophobic marker on the surface of the slides 20 min before incubation. Then the slides were washed with phosphate-buffered saline (PBS) for 2 min. The incubations followed a two-step procedure, in which the bound biotinylated GBP RCA I, SNA, and BPL (10 µg/mL) were each diluted with incubation buffer (PBS, 0.05% Tween-20). Alexa Fluor 488-conjugated streptavidin (0.4 mg/mL in PBS, 0.05% Tween-20) was used for detection. The samples (1 mL) were applied directly onto the surface and spread out over the entire print area bordered by the hydrophobic marker. The slides were incubated in a humidification chamber on a shaker for 1 h for each incubation step. In between incubations, the slides were washed by dipping four times each in (i) PBS, 0.05% Tween-20, (ii) PBS, and (iii) deionized water. Laser scanner imaging immediately followed the nitrogen-stream drying step.
Monoclonal antibody staining
Monoclonal antibody (lpt3-1) was diluted 1:5000 in incubation buffer (PBS, 0.05% Tween-20). Fluorescein isothiocyanate-conjugated goat anti-mouse IgG (10 µg/mL in PBS, 0.05% Tween-20) was used for detection in a secondary incubation, and the procedure followed the steps described above for lectin staining.
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Supplementary data |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataAcknowledgmentsConflict of interest statementReferences |
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Supplementary data are available at Glycobiology online (http://glycob.oxfordjournals.org/).
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Acknowledgments |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataAcknowledgmentsConflict of interest statementReferences |
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We thank Dr. J. Richards, Dr. Andrew Cox, and Frank St. Michael (NRC, Canada) for the generous gift of the N. meningitidis lpt3 core oligosaccharide, Margaret-Anne Gidney and Suzanne Lacelle (NRC, Canada) for providing the anti-lpt3-1 antibody, Drs. Michael McClelland and Gaelle Rondeau (Sydney Kimmel Cancer Center) for allowing us to use their laser scanner, Mrs. Daniela Vasiliu for synthetic support, and Ms. Jenesis Kam for final editing of the manuscript. This work was funded by NIGMS and The Consortium for Functional Glycomics GM62116.
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Conflict of interest statement |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataAcknowledgmentsConflict of interest statementReferences |
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None declared.
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Abbreviations |
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BPL, Bauhinia purpurea lectin; DAP, 2,6-diaminopyridine; EtOAc, ethyl acetate; GBP, glycan-binding protein; GlcNAc, N-acetylglucosamine; HPLC, high-pressure liquid chromatography; HR-MS, high-resolution mass spectrometry; LacNAc, N-acetylactosamine; Neu5Ac, N-acetylneuraminic acid; NHS, N-hydroxysuccinimide; NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; RCA I, Ricinus communis agglutinin I; SNA, Sambucus nigra agglutinin; TEA, triethylamine; TFA, trifluoroacetic acid; TLC, thin-layer chromatography
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataAcknowledgmentsConflict of interest statementReferences |
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