The diversity of stereochemical isomers present in glycans and glycoconjugates poses af ormidable challenge for comprehensive structural analysis.T ypically,s ophisticated mass spectrometry (MS)-based techniques are used in combination with chromatography or ion-mobility separation. However,c oexisting structurally similar isomers often render an unambiguous identification impossible.O ther powerful techniques such as gas-phase infrared (IR) spectroscopyh ave been limited to smaller glycans,since conformational flexibility and thermal activation during the measurement result in poor spectral resolution. This limitation can be overcome by using cold-ion spectroscopy. The vibrational fingerprints of cold oligosaccharide ions exhibit aw ealth of well-resolved absorption features that are diagnostic for minute structural variations.T he unprecedented resolution of cold-ion spectroscopy coupled with tandem MS may render this the key technology to unravel complex glycomes.Carbohydrates are ubiquitous in nature,and are historically associated with their prominent roles as structural scaffolds and energy sources within the cell. However,s horter chains, often referred to as oligosaccharides or glycans,a re also essential in numerous biological signaling processes.[1] The field of glycomics,w hich aims to comprehensively elucidate the structure and functions of glycans,i sc urrently profiting from technical breakthroughs in automated chemical synthesis and analysis but remains largely underexplored when compared to genomics and proteomics.T he inherent structural diversity of oligosaccharides creates major challenges for progress in glycobiology.Whereas DNAand proteins are exclusively assembled in alinear and template-driven fashion, glycans are non-template derived, branched, and exhibit complex stereo-and regiochemistry.Exploring the structure, shape,and resulting functions of oligosaccharides is difficult.Detailed structural analyses of glycans typically involve liquid chromatography (LC) and/or mass spectrometry (MS)-based techniques that require only small amounts of sample. [2] In addition to the molecular composition, sophisticated MS techniques such as sequential mass spectrometry (MS n )o r chemical derivatization can yield information about the sequence and connectivity of the constituting monosaccharide building blocks.[3] Ion-mobility mass spectrometry (IM-MS) has proven ap owerful tool to rapidly separate and identify the connectivity and configurational isomers of carbohydrates. [4] Combining MS and infrared (IR) spectroscopy provides av ery sensitive alternative by interrogating the vibrational modes of isolated molecules in the controlled environment of the gas phase.E xploring the characteristic absorption bands of molecules is ar outine means to deduce structural information concerning functional groups,hydrogen-bonding patterns,a nd preferred molecular conformations.A ss uch, gas-phase IR spectroscopy is aw ell-established tool for the structural analysis of peptides,proteins,and small mol...
Fucose is an essential deoxysugar that is found in a wide range of biologically relevant glycans and glycoconjugates. A recurring problem in mass spectrometric analyses of fucosylated glycans is the intramolecular migration of fucose units, which can lead to erroneous sequence assignments. This migration reaction is typically assigned to activation during collision-induced dissociation (CID) in tandem mass spectrometry (MS). In this work, we utilized cold-ion spectroscopy and show for the first time that fucose migration is not limited to fragments obtained in tandem MS and can also be observed in intact glycan ions. This observation suggests a possible low-energy barrier for this transfer reaction and generalizes fucose migration to an issue that may universally occur in any type of mass spectrometry experiment.
Nature excels at breaking down glycans into their components, typically via enzymatic acid–base catalysis to achieve selective cleavage of the glycosidic bond. Noting the importance of proton transfer in the active site of many of these enzymes, we describe a sequestered proton reagent for acid-catalyzed glycan sequencing (PRAGS) that derivatizes the reducing terminus of glycans with a pyridine moiety possessing moderate proton affinity. Gas-phase collisional activation of PRAGS-derivatized glycans predominately generates C1–O glycosidic bond cleavages retaining the charge on the reducing terminus. The resulting systematic PRAGS-directed deconstruction of the glycan can be analyzed to extract glycan composition and sequence. Glycans are also highly susceptible to dissociation by free radicals, mainly reactive oxygen species, which inspired our development of a free radical activated glycan sequencing (FRAGS) reagent, which combines a free radical precursor with a pyridine moiety that can be coupled to the reducing terminus of target glycans. Collisional activation of FRAGS-derivatized glycans generates a free radical that reacts to yield abundant cross-ring cleavages, glycosidic bond cleavages, and combinations of these types of cleavages with retention of charge at the reducing terminus. Branched sites are identified with the FRAGS reagent by the specific fragmentation patterns that are observed only at these locations. Mechanisms of dissociation as well as application of the reagents for both linear and highly branched glycan structure analysis are investigated and discussed. The approach developed here for glycan structure analysis offers unique advantages compared to earlier studies employing mass spectrometry for this purpose.
The stereoselective formation of 1,2‐cis‐glycosidic bonds is challenging. However, 1,2‐cis‐selectivity can be induced by remote participation of C4 or C6 ester groups. Reactions involving remote participation are believed to proceed via a key ionic intermediate, the glycosyl cation. Although mechanistic pathways were postulated many years ago, the structure of the reaction intermediates remained elusive owing to their short‐lived nature. Herein, we unravel the structure of glycosyl cations involved in remote participation reactions via cryogenic vibrational spectroscopy and first principles theory. Acetyl groups at C4 ensure α‐selective galactosylations by forming a covalent bond to the anomeric carbon in dioxolenium‐type ions. Unexpectedly, also benzyl ether protecting groups can engage in remote participation and promote the stereoselective formation of 1,2‐cis‐glycosidic bonds.
We designed and synthesized a methylated free radical activated glycan sequencing reagent (Me-FRAGS) for eliminating mass spectrometric glycan rearrangement.
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