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A rapid and quantitative method for solid-phase methyl esterification of carboxy groups of various sialylated oligosaccharides has been established. The method employed a triazene derivative, 3-methyl-1-p-tolyltriazene, for facile derivatization of oligosaccharides immobilized onto general solid supports such as Affi-Gel Hz and gold colloidal nanoparticles in a multiwell plate. The workflow protocol was optimized for the solid-phase processing of captured sialylated/unsialylated oligosaccharides separated from crude sample mixtures by chemical ligation. From tryptic and/or PNGase F-digest mixtures of glycoproteins, purification by chemoselective immobilization, esterification and recovery were achieved in the same well of the filter plate within three hours when used in conjunction with "glycoblotting technology" (S.-I. Nishimura, K. Niikura, M. Kurogochi, T. Matsushita, M. Fumoto, H. Hinou, R. Kamitani, H. Nakagawa, K. Deguchi, N. Miura, K. Monde, H. Kondo, High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry: Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 91-96). The recovered materials were directly applicable to subsequent characterization by mass spectrometric techniques such as MALDI-TOF for large-scale glycomics of both neutral and sialylated oligosaccharides. On-bead/on-gold nanoparticle derivatization of glycans containing sialic acids allowed rapid and quantitative glycoform profiling by MALDI-TOF MS with reflector and positive ion mode. In addition to its simplicity and speed, the method eliminates the use of unfavorable halogenated solvents such as chloroform and dichloromethane or volatile solvents such as diethyl ether and hexane, resulting in a practical and green chemical method for automated robotic adaptation.

Glycans are expected to be one of the potential signal molecules for controlling drug targeting/delivery or long-term circulation of biopharmaceuticals. However, the effect of the carbohydrates of artificially glycosylated derivatives on in vivo dynamic distribution profiles after intravenous injection of model animals remains unclear due to the lack of standardized methodology and a suitable platform. We report herein an efficient and versatile method for the preparation of multifunctional quantum dots (QDs) displaying common synthetic glycosides with excellent solubility and long-term stability in aqueous solution without loss of quantum yields. Combined use of an aminooxy-terminated thiol derivative, 11,11'-dithio bis[undec-11-yl 12-(aminooxyacetyl)amino hexa(ethyleneglycol)], and a phosphorylcholine derivative, 11-mercaptoundecylphosphorylcholine, provided QDs with novel functions for the chemical ligation of ketone-functionalized compounds and the prevention of nonspecific protein adsorption concurrently. In vivo near-infrared (NIR) fluorescence imaging of phosphorylcholine self-assembled monolayer (SAM)-coated QDs displaying various simple sugars (glyco-PC-QDs) after administration into the tail vein of the mouse revealed that distinct long-term delocalization over 2 h can be achieved in cases of QDs modified with α-sialic acid residue (Neu5Ac-PC-QDs) and control PC-QDs, while QDs bearing other common sugars, such as α-glucose (Glc-PC-QDs), α-mannose (Man-PC-QDs), α-fucose (Fuc-PC-QDs), lactose (Lac-PC-QDs), β-glucuronic acid (GlcA-PC-QDs), N-acetyl-β-D-glucosamine (GlcNAc-PC-QDs), and N-acetyl-β-D-galactosamine (GalNAc-PC-QDs) residues, accumulated rapidly (5-10 min) in the liver. Sequential enzymatic modifications of GlcNAc-PC-QDs gave Galβ1,4GlcNAc-PC-QDs (LacNAc-PC-QDs), Galβ1,4(Fucα1,3)GlcNAc-PC-QDs (Le(x)-PC-QDs), Neu5Acα2,3Galβ1,4GlcNAc-PC-QDs (sialyl LacNAc-PC-QDs), and Neu5Acα2,3Galβ1,4(Fucα1,3)GlcNAc-PC-QDs (sialyl Le(x)-PC-QDs) in quantitative yield as monitored by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses. Live animal imaging uncovered for the first time that Le(x)-PC-QDs also distributed rapidly in the liver after intravenous injection and almost quenched over 1 h in similar profiles to those of LacNAc-PC-QDs and Lac-PC-QDs. On the other hand, sialyl LacNAc-PC-QDs and sialyl Le(x)-PC-QDs were still retained stably in the whole body after 2 h, while they showed significantly different in vivo dynamics in the tissue distribution, suggesting that structure/sequence of the neighboring sugar residues in the individual sialyl oligosaccharides might influence the final organ-specific distribution. The present results clearly visualize the evidence of an essential role of the terminal sialic acid residue(s) for achieving prolonged in vivo lifetime and biodistribution of various glyco-PC-QDs as a novel class of functional platforms for nanomaterial-based drug targeting/delivery. A standardized protocol using multifunctional PC-QDs should facilitate live...

Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hydrazide-polymers/solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing O-glycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers.

A simple and efficient assay for glycosyltransferase activity on gold colloidal nanoparticles (GCNPs) by using laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) is demonstrated by the enzymatic synthesis of the Lewis X trisaccharide on GCNPs containing GlcNAc residues. GCNPs containing multivalent sugars were well dispersed in aqueous solution and proved to be excellent acceptor substrates for the glycosyltransferase reaction. Direct LDI-TOF MS analysis of these GCNPs provided the ion peaks of the sugar derivatives, chemisorbed through S--Au linkages onto the GCNPs, even in the presence of contaminants such as proteins and salts. Thus, it enabled the rapid and direct detection of the enzymatic reaction on the GCNPs by subjecting a small amount (0.15 muL) of the reaction mixture to MS analysis without purification. Subsequent MS/MS analyses (LDI-LIFT-TOF/TOF method) of the product-carrying GCNPs enabled the structures of the sugar derivatives that had been constructed on the GCNPs by enzymatic glycosylation to be determined. A quantitative inhibition assay for glycosyltransferase by using LDI-TOF MS analysis on the GCNPs was demonstrated by using uridine 5'-diphosphate (UDP) as the inhibitor. This simple assay was then applied to the detection of the enzymatic activity of a crude cell extract of Escherichia coli, which produces Neisseria meningitidis beta-1,4-galactosyltransferase (beta-1,4-GalT). In this case, the GCNPs were roughly purified by means of ultrafiltration to remove the buffer and detergents before MS analysis. That the GCNPs are dissolved in solution in the reaction medium but are solid in the purification process is greatly advantageous for the simple and efficient detection of enzymatic activity in crude biological samples. Thus, GCNPs containing a variety of biomolecules may become a versatile and efficient tool for the rapid and direct monitoring of metabolism (metabolomics) in living cells when combined with LDI-TOF MS analysis.

The first paragraph (144 words).Glycosphingolipids (GSLs) synthesized in Golgi apparatus by sequential transfer of sugar residues to a ceramide lipid anchor are ubiquitously distributing on vertebrate plasma membranes. Standardized method allowing for high throughput structural profiling and functional characterization of living cell surface GSLs is of growing importance because they function as crucial signal transduction molecules in various processes of dynamic cellular recognitions. However, methods are not available for amplification of GSLs, while the genomic scale PCR amplification permits large-scale mammalian genomics and proteomics. Here we communicate such an approach to a novel "omics", namely glycosphingolipidomics based on the glycoblotting method.The method, which involves selective ozonolysis of the C-C double bond in ceramide moiety and subsequent enrichment of generated GSL-aldehydes by chemical ligation using aminooxy-functionalized gold nanoparticle (aoGNP) should be of widespread utility for structural identification and functional characterization of whole GSLs present in the living cells/tissues. 3 Main text (1424 words).Cell surface GSLs exhibit various and crucial functions in cell growth, differentiation, adhesion, and malignant alteration 1 . It has been documented that dramatic changes in GSL composition and metabolism are strongly associated with oncogenic transformation 2,3 . Therefore, identification of disease-related structural alteration of cell surface GSLs will become a key to develop diagnostic biomarkers and therapeutic anticancer vaccines. However, purification and structural characterization of cellular GSLs have not been routinely possible to date, typically requiring tedious and time-consuming extraction/purification steps of GSLs of interest from extremely complex mixtures before analysis. In general, protocols for the isolation of GSLs would vary depending upon the analytical methods as well as chemical properties of individual GSLs and would need specialized expertise at step-by-step/case-by-case handling for separation. These crucial problems in the enrichment of whole GSLs make it difficult to achieve high throughput structural and functional analyses of biologically importantGSLs. In addition, we should pay attention to the fact that cell surface GSLs often exhibit specific biological functions through dynamic mechanism such as clustering or To illustrate the new method, crude lipids fraction 24 of C57BL/6JJcl adult (7 weeks) and embryonic (13.5 days) mouse brains were subjected directly to "ozonolysis" and "glycoblotting" protocol. Reaction of GSL-aldehydes with aoGNPs proceeded smoothly under mild condition without any special reagent. After simple purificationGSLs transferred onto aoGNPs (GSLs-GNPs) were employed directly for further structural characterization. The merit of this method is evident because whole procedure from GSLs enrichment to mass spectrometry-based structural profiling can be performed within 3~4 hours by employing approximately 100 mg (wet w...

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An affinity labeling reagent, uridine 5'-(6-amino-{2-[(7-bromomethyl-2-naphthyl)methoxycarbonylmethoxy]ethoxy}acetyl-6-deoxy-alpha-D-galactopyranosyl) diphosphate (1a), was designed on the basis of 3D docking simulation and synthesized to investigate the functional role of Trp310 residue located in the small loop near the active site of human recombinant galactosyltransferase (betaGalT-1). Mass spectrometric analysis revealed that the Trp310 residue of betaGalT1 can be selectively modified with the naphthylmethyl group of compound 1a at the C-3 position of the indole ring. This result motivated us to synthesize novel uridine-5'-diphosphogalactose (UDP-Gal) analogues as candidates for mechanism-based inhibitors for betaGalT-1. We found that uridine 5'-(6-O-[10-(2-naphthyl)-3,6,9-trioxadecanyl]-alpha-d-galactopyranosyl) diphosphate (2) is the strongest inhibitor (K(i) = 1.86 microM) against UDP-Gal (Km = 4.91 microM) among compounds reported previously. A cold spray ionization time-of-flight mass spectrometry study demonstrated that the complex of this inhibitor and betaGalT-1 cannot interact with an acceptor substrate in the presence of Mn2+.