Energy-resolved structural details obtained from gangliosides.

Gangliosides, a family of glycosphingolipids (GSLs) that comprise sialic acid residue(s), are an important class of molecules that exist on the outer surface of the plasma membrane. To assess the functions of a particular series of gangliosides that play important roles in brain functions, their structures and localizations need to be investigated. We studied the structures of these gangliosides by collision-induced dissociation using quadrupole ion-trap mass spectrometry. The dissociation processes were investigated in detail based on energy-resolved mass spectrometry using sodiated molecules. The decision of utilization of the positive mode was based on the assumption that it was the generally applicable method for GSLs, including neutral ones. In this investigation, sialic acid residues were esterified to stabilize the linkages and to generate multiple fragment ions for successful structural investigations. A detailed analysis of a series of sodiated species of gangliosides based on energy-resolved mass spectrometry revealed that the GM1-equivelent fragments generated from the precursor ions under low energy CID conditions had the structural characteristics of their individual precursors. It was suggested that this information will be useful in determining the structures of their precursor gangliosides.

Discrimination of 16 structural isomers of fucosyl galactoside based on energy-resolved mass spectrometry.

Glycans, a family of compounds often attached to proteins and ceramides, are diverse molecules involved in a wide range of biological functions. Their structural analysis is necessary and is often carried out at the microscale level. Methods based on mass spectrometry are therefore used, although they do not provide information regarding isomeric structures often found in glycan structures. If one finds "factors" characteristic of a certain isomer, this information can be used to elucidate an unknown oligosaccharide sequence. One potential technique is to use energy-resolved mass spectrometry (ERMS) that has been used to distinguish a pair of isomeric compounds. Thus, compounds in a combinatorial library might be effectively used for this purpose. We analyzed a set of 16 isomeric disaccharides, the structures of which consisted of all possible combinations of anomeric configurations and interglycosidic linkage positions. All of the compounds were distinguished based on ERMS where normal collision-induced dissociation could distinguish only seven compounds. Furthermore, it was shown that alpha-glycosidic linkages of fucose were more reactive than the beta-isomers and the secondary glycosides were more reactive than the primary glycosides.

Anomeric information obtained from a series of synthetic trisaccharides using energy resolved mass spectra.

The majority of structural investigations of oligosaccharides based on mass spectrometry use naturally occurring oligosaccharides, which do not allow extracting any common feature associated with anomeric structures and linkage positions. In order to address the issue to find such characteristics possibly contained in oligosaccharide structure, a synthetic combinatorial trisaccharide library was analyzed. The trisaccharides used in the analysis consisted of L-fucose, D-galactose and D-glucose, in which individual glycosidic linkages existed in either alpha- or beta-anomers. The analysis of energy-resolved mass spectra (ERMS) and the scattered plot analysis of some parameters obtained from ERMS for a series of trisaccharides revealed that lower activation energy was required for the dissociation of alpha-glycosides of these sugars compared to those of the corresponding beta-anomers. It is suggested that this finding may be useful in structural analysis of natural oligosaccharides.

Synthesis of a library of fucopyranosyl-galactopyranosides consisting of a complete set of anomeric configurations and linkage positions.

A library composed of a complete set of fucopyranosyl-galactopyranosides was synthesized. A perbenzylated phenylthio fucopyranoside and a series of tri-O-benzyl-galactopyranosyl fluorides having single hydroxyl groups at the 2-, 3-, 4-, and 6-positions were used as the glycosyl donor and glycosyl acceptors, respectively. The chosen set of functionalities at the anomeric centers enabled rapid access to the oligosaccharides based on chemoselective activation. The first coupling reaction was achieved by the action of dimethyl(methylthio)sulfonium trifluoromethanesulfonate (DMTST). The resulting disaccharide fluoride was readily activated by hafnocene bistrifluoromethanesulfonate [Cp2Hf(OTf)2] and glycosidated with n-octanol.

Ion-trap mass spectrometry unveils the presence of isomeric oligosaccharides in an analyte: stage-discriminated correlation of energy-resolved mass spectrometry.

Mass spectrometry, especially tandem mass spectrometry, has been widely used in the field of analytical sciences for handling biological and chemical samples. The technique resolves molecular and fragment ions based on the mass to charge ratio. Energy-resolved mass spectrometry (ERMS) further provides an activation energy-related factor in the dissociation reaction. Therefore, it is a very powerful technique that can discriminate isomeric compounds. Despite the power of ERMS, useful information cannot be obtained when an analyte contains structural isomers. Carbohydrates carry multiple chiral centers, thus oligomers of monosaccharides can form a vast number of structural isomers. We decided to use such species in our endeavors to establish a method of identifying the 'purity' of an analyte solely based on mass spectrometry. In the present paper, we describe a stage-discriminated spectral correlation of ERMS, which not only enables identification of the presence of contaminants in an analyte, but also provides information regarding the 'purity' of fragment ions.

High-yielding and controlled dissociation of glycosides producing B- and C-ion species under collision-induced dissociation MS/MS conditions and use in structural determination.

Collision-induced dissociation (CID) in mass spectrometry is a powerful technique with which to understand gas-phase chemical reactions. A mass spectrometer is used to carry out the reaction, isolation, and analysis. On the other hand, structural analysis of glycan structures is of extreme importance in the analysis of biomolecules, such as glycoproteins and glycolipids. In the analysis of glycan structures based on CID, certain ion species, including B-/Y-, C-/Z-, and A-/X-ions, are produced. Among these ions, we are interested in C-ion species that carry a glycosyl oxygen atom at the anomeric center and that possibly provide information regarding anomeric configuration. A method for generating C-ion species when necessary is thus considered to be important; however, none is currently available. In this study, synthetic glycosides carrying a series of aglycons were analyzed with the aim of identifying suitable glycosides with which to produce C-ions to be used in the structural determination of oligosaccharides. The results showed a 4-aminobutyl group was an excellent candidate. Furthermore, the use of C-ion species obtained in this manner in the structural characterization of a ganglioside, GM3, is described. The type of glycoside is believed to be valuable not only in structural analysis but also in biological investigation, because of the existing amino functionality that has been proven to be useful by enabling the generation of conjugates with other molecules and materials.

A method of orthogonal oligosaccharide synthesis leading to a combinatorial library based on stationary solid-phase reaction.

A new, efficient synthesis of oligosaccharides, which involves solid-phase reactions without mixing in combination with an orthogonal-glycosylation strategy, is described. Despite a great deal of biological interest, the combinatorial chemistry of oligosaccharides is an extremely difficult subject. The problems include 1) lengthy synthetic protocols required for the synthesis and 2) the variety of glycosylation conditions necessary for individual reactions. These issues were addressed and solved by using the orthogonal-coupling protocol and the application of a temperature gradient to provide appropriate conditions for individual reactions. Furthermore, we succeeded in carrying out solid-phase reactions with neither mechanical mixing nor flow. In this report, the synthesis of a series of trisaccharides, namely, alpha/beta-L-Fuc-(1-->6)-alpha/beta-D-Gal-(1-->2/3/4/6)-alpha/beta-D-Glc-octyl, is reported to demonstrate the eligibility of the synthetic method in combinatorial chemistry.