Yoshimi Kanie

Syntheses of C-3-Modified Sialylglycosides as Selective Inhibitors of Influenza Hemagglutinin and Neuraminidase

In an effort to develop new structures as inhibitors of both influenza virus proteins hemagglutinin and neuraminidase, a series of sialic acid derivatives, including those with one of the hydrogen atoms at the C-3 position replaced by either OH or F, were synthesized. The sialic acid derivative with a 3-eq-OH group was first synthesized by means of a new process and used as the key intermediate for further derivatization at the C-3 position. The stability of these compounds under acid- and sialidase-catalyzed hydrolysis conditions was studied, and the results showed that these compounds exhibit stronger resistance towards both conditions than their parent p-nitrophenyl α-sialoside. Further inhibition assay indicated that the 3-ax-OH or F derivatives 4, 5, and 24, the 4-epimer of 4, are effective specific inhibitors of the sialidases from Clostridium perfringens, among other bacterial sialidases tested. The 3-eq-OH derivative 3, however, showed little inhibition. The same tendency was observed for the inhibition of human influenza sialidases N1 and N2. Compounds 3−5 and sialic acid were then converted into the distealoylphosphatidylethanolamine conjugates. Of these liposome-like compounds, the ones from 4 and 5 showed potent and selective inhibitory activities against the hemagglutinin H3 subtype, but displayed resistance to the influenza virus neuraminidases N1 and N2.

Synthesis and enzymatic evaluation of five-membered iminocyclitols and a pseudodisaccharide

Described here are the synthesis of five-membered iminocyclitols with galacto-configuration and a pseudodisaccharide, and their inhibitory activities against beta-galactosyltransferase, beta-galactosidase and alpha-mannosidase.

Identification of genes required for neural-specific glycosylation using functional genomics.

Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, α1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific α1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating α1,3-fucosylation. Further analyses revealed that an RNA–binding protein, second mitotic wave missing (Swm), upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells.

Insight into the Regulation of Glycan Synthesis in Drosophila Chaoptin Based on Mass Spectrometry

Background A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown. Methodology/Principal Findings In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate α-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type. Conclusions/Significance These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins.

Electrophoretically mediated microscale reaction of glycosidases: kinetic analysis of some glycosidases at the nanoliter scale.

Capillary electrophoresis (CE) is one of the extremely important analytical techniques known for its high sensitivity and resolution. We have investigated electrophoretically mediated microanalysis (EMMA) for the assay of some native glycosidases. Under optimized conditions, the enzymatic reactions of alpha-glucosidase, beta-galactosidase and beta-N-acetylglucosaminidase were carried out, and the Michaelis constants were obtained. The current method may have advantages over traditional assay methods, especially in terms of the amount of enzyme and substrate required for a reaction.

Analysis of behavior of sodiated sugar hemiacetals under low-energy collision-induced dissociation conditions and application to investigating mutarotation and mechanism of a glycosidase

Analysis of anomericity is one of the most important issues in the structure elucidation of carbohydrates. Mass spectrometry (MS)-based methods are of particular interest and important to address the issue related to resolving anomericity of monosaccharide units in a glycan. However, direct analysis of hemiacetals has not been possible by MS because of the nonavailability of information regarding the gas-phase behavior of such ion species. We addressed this issue by using stage-discriminated energy-resolved mass spectrometry (ERMS) at the stages of MSn and MSn+1 and showed that such analysis can be made. This was achieved by proving that individual anomers can be identified and that the equilibrium of sodium adducted ion species of α- and β-anomers can be negated in the gas phase under collision-induced dissociation (CID) conditions. On the basis of these results, we could 1) observe the mutarotation of lactose and 2) speculate the hydrolysis mechanism of endo-glycosylceramidase by using mass spectrometry.

Comparative RP-HPLC for rapid identification of glycopeptides and application in off-line LC-MALDI-MS analysis.

Despite the increasing attention being paid to the functions of glycoproteins, their structural analysis is still difficult and hinders functional investigations. Structural analysis of post-translationally modified proteins is thought to be achieved using methods frequently utilized in proteomics research; however, the same methods cannot be used for glycosylated proteins. One of the difficulties associated with the physiochemical properties of glycopeptides and peptides is that the detection of the former is considerably more difficult, because of the existence of glycoforms that increase molecular weight and reduces quantities of individual species. Thus, difficulties are often faced in finding glycopeptide(s) by using MS when analyzing peaks (or fractions) obtained after proteolytic digestion and HPLC. One simple yet difficult solution to this problem would be to develop a purification method that provides better resolution. Our intention has been to address this issue by using a combination of conventional methods. We found that a method consisting of a combination of rough fractionation using a reverse-phase cartridge column under acidic conditions and comparative RP-HPLC, where the two chromatograms obtained using phosphate and borate buffers under basic conditions were compared, is effective for MS-based structural analysis. The applicability of the method in glycoprotein analysis was examined using various samples including ribonuclease B (RNase B), IgG1, ovalbumin (OVA), and asialo fetuin (ASF). The results suggest that the method is useful in the analysis of glycoproteins.

Multi-stage mass spectrometric information obtained by deconvolution of energy-resolved spectra acquired by triple-quadrupole mass spectrometry

Triple-quadrupole mass spectrometry (TQ-MS) provides the capability to carry out collision-induced dissociation (CID) and it offers advantages in quantification when connected with high-performance liquid chromatography through an electrospray ionization interface. However, although TQ-MS provides information on partial structures through the analysis of product ions obtained by CID experiments, the method only provides single-stage CID experiments, which limits the detailed structural information that can be obtained. Herein, a method of overcoming this limitation of TQ-MS is described. A spectrum obtained by energy-resolved mass spectrometry (ERMS) was used to deconvolute the fragmentation process, with a Galili-antigenic trisaccharide derivative being used as an example. A replot of the ERMS data showing the ratios of the product ions to the precursor ion resulted in a descriptive graph. Analysis of the sum of the ratios of individual product ions to the precursor ion at specific CID energies revealed that the members of a series of product ions were related to each other. The obtained relationships and the m/z values of the product ions provided information on the fragmentation process taking place during the dissociation, indicating that the ERMS spectrum obtained by TQ-MS contained equivalent information to that obtainable by multi-stage MS/MS (MS(n); n≥2). This method may allow users of triple-quadrupole mass spectrometers to obtain MS(n)-type information by performing a single ERMS experiment, which is even advantageous over quadrupole ion trap (QIT)-MS/MS because CID experiments on individual first-generation product ions are not required.

Analysis of pyridylaminated oligosaccharides using liquid chromatography–mass spectrometry with a monolithic capillary column

We examined the utility of a monolithic capillary column in the analysis of pyridylaminated oligosaccharides. Fluorescence detection and mass spectrometry were used to monitor a series of oligosaccharides. Although the total-ion chromatogram appeared similar to that obtained with fluorescence detection, the sensitivity of this technique was limited, especially in the case of smaller oligosaccharides. This limitation was overcome by applying selected ion current monitoring. Further, the capillary column also exhibited good reproducibility. We showed that the retention times obtained by using the monolithic capillary column could be converted into the standard data to enable comparison of the experimental data with the existing data. Furthermore, our studies revealed an important difference in the separation profile, i.e., the monolithic capillary column could resolve smaller oligosaccharides to a greater extent.

Addressing the glycan complexity by using mass spectrometry: In the pursuit of decoding glycologic

Biomolecules often contain carbohydrates. Such molecules, namely glycoconjugates, play central roles in cell adhesion, tumor migration, and attachment of pathogens. The conjugation of “natural products” with glycans is not a template-dependent process but is achieved by multiple enzymatic reactions. Because of the nature of the synthetic process, glycans exist as a complex mixture creating a glycoform and thus, the analysis becomes inevitably difficult. Mass spectrometry is one of the most informative, and thus important, tools for the structural analysis of glycans. The obvious advantage of mass spectrometry is the high sensitivity at low femtomole detection levels. A variety of ions can be obtained depending on the adducted cationic species. In order to obtain sequential information, the gas-phase dissociation reaction is used. Among the various techniques, collision-induced dissociation (CID) has been frequently used in the past. Although the technique is proven useful, there are cases that normal CID process is not suitable. This review highlights an emerging technique that focuses on the activation-energy difference between the isomeric glycans, and will complement the current methods. Furthermore, the possible source and the methodology for obtaining useful structural information are discussed.