Linkage Position in Oligosaccharides by Fast Atom Bombardment Ionization, Collision-Activated Dissociation, Tandem Mass Spectrometry and Molecular Modeling. L-Fucosyl_p-(α 1 →X)-D-N-acetyl-D-glucosaminyl_p-(β1 →3)-D-galactosyl_D-(β1-O-methyl) Where X = 3, 4, or 6

Fast atom bombardment mass spectra (FABMS), and collision-activated dissociation (CAD) studies of three synthetic, linkage-isomeric trisaccharides were conducted to assess whether ion patterns could distinguish among the three possible different linkage positions of the terminal fucose in the following otherwise identical structures: L-fucosyl_p-(α1→3)-D-N-acetylglucosaminyl_p-(β1→3)-galactosyl_p-(β1-O-methyl) (F3) L-fucosyl_p-(α1→4)-D-N-acetylglucosaminyl_p-(β1→3)-galactosyl_p-(β1-O-methyl) (F4) L-fucosyl_p-(α1→6)-D-N-acetylglucosaminyl_p-(β1→3)-galactosyl_p-(β1-O-methyl) (F6) FABMS, low-energy CAD spectra were recorded for the protonated molecular ions as well as for major fragment ions occurring in the FABMS spectra. The rationale was that differences in internal, nonbonding free energy and entropic terms such as degrees of freedom of motion near minimum energy conformations could allow threshold CAD experiments to distinguish among the three structures, resulting in a statistically higher percentage of bond cleavage for more sterically hindered bonds. Structures with more freedom of motion would more readily dissipate energy absorbed from collision events due to lowered probability of populating the reaction coordinate for glycosidic bond cleavage. Repeatability was ensured by choosing a standard survival percentage for the collided ion in one compound and comparing CAD spectra for the remaining compounds at the same instrument settings, Chem-X and MMP2 (version 1985) molecular modeling programs were used to calculate minimum energy structures for the fucosyl→GlcNAc moieties and freedom of motion volumes near these minima among the three trisaccharides. Volumes of conformational freedom within 50 kcal above the minimum corresponded to density of states near the transition-state energy for bond cleavage. Observed CAD stability levels of molecular ions and observed intensity differences in product ions resulting from collision-activated glycosidic cleavages of fucosyl-GlcNAc bonds gave strong indications that steric factors in the positions of fucose linkage to GlcNAc in the three trisaccharides contribute to bond stability during CAD experiments in the relationship 6 > 4 > 3. This approach may provide another line of evidence for assignment of linkage positions in microgram quantities of intact saccharides where suitable standards are available.