Biomimetic Reagents for the Selective Free Radical and Acid–Base Chemistry of Glycans: Application to Glycan Structure Determination by Mass Spectrometry

Abstract

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.

Radical mediated dissection of oligosaccharides

“…This contrasts with the previous results of Beauchamp, which required examination of two distinct molecules to observe proton and radical initiated dissociation chemistry [24]. Photoactivation of iodoaniline labeled maltoheptaose with 266 nm photons yields loss of iodine almost exclusively, generating radical saccharide (see Supporting information (SI)).…”

“…Third, unlike ECD and ETD, singly charged ions can also be analyzed because generation of radicals does not involve charge reduction. Recently, Beauchamp and co-workers utilized collisional activation of TEMPO based radical precursors to generate radicals in covalently modified oligosaccharides [24]. Glycoside bond cleavage as well as two types of cross-ring fragments, 1,5 X and 0,2 X, were observed in this study, suggesting that radical chemistry may be advantageous for the study of oligosaccharides.…”

Formation of sugar radical cations from collision-induced dissociation of non-covalent complexes with S-nitroso thiyl radical precursors

“…Thus the radical is located on a carbon atom and the charge is located on an oxygen. This is consistent with the lower bond dissociation energy of the C-H versus O-H bonds in sugars [11], and the fact that the only sites for protonation in sugars are the O atoms [20]. Thus, the type of radical cation formed in our method is distinct to the canonical structures of radical cations of sugars formed via EI/MS [2].…”

“…In contrast, the use of gas phase radical ion chemistry to direct the fragmentation of sugars has lagged behind. Exceptions include the use of electron capture dissociation (ECD) and electron transfer dissociation (ETD), which result in radical-directed cleavages in oligosaccharides [5]; formation of radical ions by electron detachment dissociation (EDD) and the related negative electron transfer dissociation (NETD) method [6] or photo-activated electron detachment [7]; use of Siu's method [8] to form radical anions by dissociation of a ternary metal complex containing the carbohydrate [9]; and extension of Beachamp's free radical initiated peptide sequencing (FRIPS) method [10] to oligosaccharides [11].…”

Collisional Activation Dissociation Mass Spectrometry Studies of Oligosaccharides Conjugated with Na+-Encapsulated Dibenzo-18-Crown-6 Ether

“…Maltoheptaose is a good linear glycan model that has been studied by many other researchers. 23,24 Singly protonated maltoheptaose shows only B series glycosidic bond cleavage products as main fragments. In contrast, the CAD spectrum of singly sodiated maltoheptaose yields abundant Figure 4.…”