Introduction

Oligosaccharides are involved in many biological processes, such as inflammation, immune response, and viral infections [1, 2]. In addition, functional oligosaccharides show positive effects on human health, both in the prevention and treatment of chronic diseases [3]. A variety of approaches for oligosaccharide synthesis have been reported that include: solid-phase based synthesis [4], the one-step synthesis [5], the two-stage activation procedure [6], enzyme assisted synthesis [7], and the one-pot sequential synthesis [8]. Among these strategies, the one-pot sequential synthesis is advantageous because it can be used to synthesize oligosaccharide libraries very efficiently with automated control [9]. This approach integrates several glycosylation steps into a single synthetic operation based on the differences of reactivity of various glycosyl donors. Glycosyl donors are sequentially added to the reaction system in the order of reduced reactivity (most reactive first), until the least reactive glycosyl donor is placed at the reducing end [9,10,11]. Glycosyl phosphates play important roles in oligosaccharide synthesis as they serve as great glycosyl donor because the phosphate moiety is an excellent leaving group [12, 13]. Therefore, it is very interesting to understand the effects of linkage stereochemistry, the geometry of the leaving groups, and protecting groups on the stability of the glycosyl phosphate linkage so that more effective glycosyl donors can be developed and synthesized.

The study of carbohydrate biology has seen enormous growth in the past decade, and as a result, there is an increasing need for improved analytical characterization [14,15,16,17]. Mass spectrometry has been used to effectively distinguish glycosyl phosphate (GP x ) isomers and offers the possibility of examining details of their reactivity/stability. The influence of linkage stereochemistry, leaving group geometry, and protecting groups on the stability of a series of GP x has been evaluated by in-source threshold fragmentation electrospray ionization (ESI) mass spectrometry (MS) of the sodium cationized complexes of these GP x [18]. However, in-source fragmentation of [GP x +Na]+ is influenced by many factors, including spray conditions, analyte concentration, and pH. Moreover, the fragment ions observed are typically accompanied by product ions derived from glycosylation and/or hydrolysis that occurs in solution. Thus, results are not generally interpreted straightforwardly.

Collision-induced dissociation (CID) is a powerful activation method commonly employed in mass spectrometry analyses to elucidate structural information, including isomer differentiation and characterization of ion fragmentation behavior and mechanisms [19]. In the case of carbohydrates, CID offers the possibility to assign details of carbohydrate structure such as sugar sequence and linkage positions [20,21,22,23,24,25,26,27]. McLafferty rearrangements are common molecular rearrangement reactions observed in mass spectrometry [28]. In the case of carbohydrates, most of the observed glycan rearrangements appear to be linked to the presence of a proton [29]. However, McLafferty-type, even-electron rearrangements have been observed for both deprotonated and metal cationized systems [30, 31]. Indeed alkali metal cationization has been widely used to characterize structure and investigate the CID behavior of carbohydrates [32,33,34,35]. The choice of alkali metal cation does influence the CID behavior. The smaller alkali metal cations (i.e., Li+ and Na+) are generally chosen as they bind more tightly and produce richer CID mass spectra, which facilitate structural elucidation and isomer differentiation [36,37,38,39,40,41]. The larger alkali metal cations often bind to carbohydrates weakly enough that only simple noncovalent bond cleavage occurs such that their CID provides almost no direct structural information [42, 43]. As isomeric carbohydrate complexes generally exhibit highly parallel fragmentation pathways, the energy dependence of the CID behavior has been found to provide additional information that is often key to their differentiation [44,45,46]. CID-based survival yield analysis was initially developed as a tool to quantify the distribution of precursor ion internal energies to explain fragmentation patterns that are observed in tandem mass spectrometry experiments [47, 48]. Survival yield analysis has since been used as a method to correlate conditions in the mass spectrometer to the energetics of sample ions. These studies have developed energy-dependent models for understanding molecular decompositions that occur upon CID [49,50,51].

Recently, survival yield analyses have been used to examine and compare the energetics of dissociation for noncovalently bound complexes as well as to the activated cleavage of covalent bonds. In particular, survival yield analyses have been employed to examine the disruption of noncovalent interactions of macrocyclic polyethers with alkali metal cations [52], ligand binding modes to duplex and triplex DNA [53], the analysis and structural differentiation of seven isomers with the chemical formula C9H11NO2 [49], and have also been used to determine the relative N-glycosidic bond stabilities of protonated and sodium cationized DNA and RNA nucleosides [54,55,56,57,58]. Correlation between collision energy and dissociation of precursor ions has been semi-quantified in terms of EC50 or CID50%, the rf excitation energy (voltage) that results in 50% dissociation of the precursor ion [49, 52,53,54,55,56,57,58,59,60].

In this study, we investigate the stability of a series of sodium cationized glycosyl phosphates, [GP x +Na]+, via survival yield analyses based on their energy-resolved collision-induced dissociation (ER-CID) behavior. The GP x examined are shown in Figure 1. GP1α and GP1β are 2,3,4,6-tetra-O-benzyl-α- and β-D-glucosyl dibutyl phosphate, respectively, which are isomers with the chemical formula C42H53O9P. GP2α and GP2β are 2-O-benzyl-4,6-O-benzylidene-α- and β-D-glucosyl dibutyl phosphate, respectively, which are isomers with the chemical formula C28H39O8P and carry a 4,6-O-benzylidene acetyl cyclic protecting group, but lack a 3-O-benzyl substituent. For convenient comparison, GP3α and GP3β are used to represent 2,3-di-O-benzyl-4,6-O-benzylidene-α- and β-D-mannopyranosyl dibutyl phosphate, respectively, whereas GP4α and GP4β are 2,3-di-O-benzyl-4,6-O-benzylidene-α- and β-D-glucosyl dibutyl phosphate, respectively. GP3α, GP3β, GP4α, and GP4β are isomers with the chemical formula C35H45O9P, which carry the same 4,6-O-benzylidene acetyl cyclic protecting group as GP2α and GP2β. A four parameter logistic dynamic fitting model is employed to extract CID50% values. Trends in the CID50% values are examined to elucidate the effects of linkage stereochemistry, the geometry of the leaving group, and protecting groups on the stability of the glycosyl phosphate linkage and thus the efficiency of these species to serve as glycosyl donors for carbohydrate synthesis, and are found to provide insight into the mechanism by which these sodium cationized GP x complexes dissociate.

Figure 1
figure 1

Structures of the eight glycosyl phosphates (GP x ) examined in this work, GP1α, GP1β, GP2α, GP2β, GP3α, GP3β, GP4α, and GP4β. The GPα versus GPβ isomers differ only in the C1-C2 configuration, cis versus trans. GP1α and GP1β, possess two O-benzyl substituents (at C4 and C6) rather than a cyclic protecting group, GP2α and GP2β instead lack a 3 BnO substituent, whereas the 2 BnO substituent of GP3α and GP3β is axial rather than equatorial, as highlighted in the figure

Full size image

Experimental Procedure and Survival Yield Analysis

Chemicals

The eight glycosyl and mannopyranosyl dibutyl phosphates used in this work were synthesized using synthetic procedures described previously [61,62,63,64]. Methanol (MeOH, HPLC grade) was purchased from EMD Millipore Co., Billerica, MA, USA. DI water (HPLC grade) was purchased from EMD Chemicals Inc., Gibbstown, NJ, USA. Sodium acetate (NaOAc) was purchase from Sigma-Aldrich, St. Louis, MO, USA.

Mass Spectrometry

ER-CID experiments of sodium cationized GP x were performed using a Bruker amaZon ETD quadrupole ion trap mass spectrometer (QIT MS) (Bruker Daltonics, Bremen, Germany). Sample solutions were prepared by dissolving the GP x in a 50%:50% MeOH:H2O solution to a concentration of ~10 μM to which a sodium acetate solution was then added to produce a final concentration of 0.1 mM NaOAc for direct infusion electrospray ionization (ESI). The sodium cationized GP x complexes were generated using an Apollo ESI source at a flow rate of 3 μL/min. The N2 drying gas temperature was set at 200 °C and a flow rate of 3 L/min. The N2 nebulizer gas was set at 0.69 bar. The ESI probe capillary voltage was set at −4.5 kV with the end plate offset set at –500 V. Helium was used as the cooling and collision gas at a stagnation pressure in the ion trap of ~1 mTorr. The number of trapped ions was kept constant (2 × 105) using the ion charge control (ICC) in all experiments. The low-mass cutoff for the ER-CID experiments was set to 27%, corresponding to a qz value of 0.25. The rf excitation amplitude was varied from 0 V to the amplitude required to produce complete fragmentation of the precursor glycosyl phosphate complex at a step size of 0.01 V. An activation time of 40 ms was used for all CID experiments. Data analysis was performed using Compass Data Analysis 4.0 software (Bruker Daltonics, Bremen, Germany).

Survival Yield Analysis

Survival yield analysis is a robust technique to study the relative stabilities of various ions when judiciously applied [65,66,67,68]. In order to elucidate the relative stabilities of eight [GP x +Na]+ complexes, survival yield analyses of these complexes were performed by calculating and comparing the survival yields based on the ER-CID experimental results as a function of rf excitation amplitude. The survival yield of each [GP x +Na]+ complex was determined based on the ratio of the intensity of the precursor glycosyl phosphate complex (I p ) to the total ion intensity as shown in Equation 1,

$$ \mathrm{Survival}\kern0.5em \mathrm{yield}={I}_p/\left({I}_p+\sum_i{I}_{f_i}\right) $$
(1)

where \( \sum_i{I}_{f_i} \) is the total fragment ion intensity. The survival yield of [GP x +Na]+ was plotted as a function of rf excitation amplitude to generate the survival yield curve. The CID50% value, which represents the rf excitation amplitude required to produce 50% dissociation of precursor glycosyl phosphate complex was determined by four parameter logistic dynamic fitting as shown in Equation 2,

$$ \mathrm{Survival}\kern0.5em \mathrm{Yield}=\min +\frac{\max -\min }{1+{\left({\mathrm{rf}}_{\mathrm{EA}}/{\mathrm{CID}}_{50\%}\right)}^{\mathrm{CID}\mathrm{slope}}} $$
(2)

where max and min are the maximum (1) and minimum (0) values of the survival yield, rfEA is the rf excitation amplitude applied, and CIDslope is the slope of the declining region of the survival yield curve. Because the fragmentation of the [GP x +Na]+ complexes is dominated by glycosidic bond cleavage, the CID50% values of [GP x +Na]+ provide the relative glycosidic bond stabilities of these complexes. Data analysis was performed using SigmaPlot 10.0 (Systat Software, Inc., San Jose, CA, USA).

Results

CID Fragmentation Pathways of [GPx+Na]+

The CID mass spectra of eight sodium cationized GP x at an rf excitation amplitude that results in approximately 50% dissociation are compared in Figure 2. The m/z values of the precursor [GP x +Na]+ complexes and their CID products are given in the figure and summarized in Supplementary Table S1 in the Electronic Supplementary Material, whereas survival/fragment yields for each [GP x +Na]+ complex across the entire range of rfEA values measured are shown in Supplementary Figure S1. For all eight sodium cationized glycosyl phosphates examined, the primary fragmentation pathway across the entire range of rfEA values observed involves cleavage of the glycosidic bond with the sodium cation retained by either the sugar or phosphate moieties as summarized in the Reactions 3 and 4, respectively (see Supplementary Figure S1).

$$ {\left[{\mathrm{GP}}_x+\mathrm{Na}\right]}^{+}\overset{n\kern0.5em He}{\to }{\left[{\mathrm{GP}}_x-\mathrm{DBPA}+\mathrm{Na}\right]}^{+}+\mathrm{DBPA} $$
(3)
$$ {\left[{\mathrm{GP}}_x+\mathrm{Na}\right]}^{+}\overset{n\kern0.5em He}{\to }\ {\left[\mathrm{DBPA}+\mathrm{Na}\right]}^{+}+\left({\mathrm{GP}}_x-\mathrm{DBPA}\right) $$
(4)
Figure 2
figure 2

Energy-resolved CID mass spectra of eight sodium cationized glycosyl phosphates [GP x +Na]+, where x = 1α, 1β, 2α, 2β, 3α, 3β, 4α, and 4β. The precursor ion, [GP x +Na]+ is indicated by (○). The major CID product ions result from cleavage of the glycosyl phosphate linkage with the sodium cation retained by the sugar moiety corresponding to loss of 210 Da from the precursor ion [GP x –DBPA+Na]+ (reaction 3, ), or retained by the phosphate moiety [DBPA+Na]+, and observed at m/z = 233.2 Da (reaction 4, ). Minor fragmentation pathways are also labeled with symbols with color coding as defined in the text for reactions (5)–(11)

Full size image

Reaction 3 is the primary CID pathway for the [GP x +Na]+ complexes of GP1α, GP1β, GP3α, GP3β, GP4α, and GP4β and observed as a loss of 210 Da () from the precursor glycosyl phosphate complex (loss of DBPA) as the sodium cation is preferentially retained by the sugar moiety. In contrast, loss of neutral DBPA from [GP2α+Na]+ and [GP2β+Na]+ is only a very minor dissociation pathway. Instead the dibutyl phosphoric acid moiety preferentially retains the sodium cation upon CID of the [GP2α+Na]+ and [GP2β+Na]+ complexes, Reaction 4 (). Reaction 4 is also a minor CID pathway for GP3α, GP3β, GP4α, and GP4β at elevated values of rfEA. It should be noted that GP1α, GP1β, GP3α, GP3β, GP4α, and GP4β all possess a 3-O-benzyl protecting group, whereas both GP2α and GP2β possess neither a 3-hydroxyl substituent nor a 3-O-benzyl protecting group, suggesting that the configuration at the C3 position plays a key role in determining which fragment retains the sodium cation upon dissociation of the glycosidic bond.

Sequential dissociation of the sodium cationized glycoside, ([GP x −DBPA+Na]+, () resulting in a further loss of 91 Da, is also observed in the CID of sodium cationized GP1α, GP1β, GP3α, GP3β, GP4α, and GP4β, Reaction 5 (), and is the most abundant minor product observed for these complexes (see Supplementary Figure S1). This may be deduced as a loss of one of the benzyl protecting groups (Bn) as a tropylium radical.

$$ {\left[{\mathrm{GP}}_x+\mathrm{Na}\right]}^{+}\overset{n\kern0.5em He}{\to }{\left[{\mathrm{GP}}_x-\mathrm{DBPA}-\mathrm{Bn}+\mathrm{Na}\right]}^{+}+\mathrm{DBPA}+\mathrm{Bn} $$
(5)

GP1α and GP1β uniquely exhibit two minor fragmentation pathways that involve loss of 90 Da from the ionic product of Reaction (3) and 420 Da from the precursor complex, Reactions 6 () and 7 (). The loss of 90 Da likely involves elimination of C7H6. However, the 420 Da loss pathway is not obviously assigned, but does correspond to a mass loss equivalent to that of four benzyl protecting groups and butene (4Bn+B).

$$ {\left[{\mathrm{GP}}_x+\mathrm{Na}\right]}^{+}\overset{n\kern0.5em He}{\to }\ {\left[{\mathrm{GP}}_x-\mathrm{DBPA}-{\mathrm{C}}_7{\mathrm{H}}_6+\mathrm{Na}\right]}^{+}+\mathrm{DBPA}+{\mathrm{C}}_7{\mathrm{H}}_6 $$
(6)
$$ {\left[{\mathrm{GP}}_x+\mathrm{Na}\right]}^{+}\overset{n\kern0.5em He}{\to }{\left[{\mathrm{GP}}_x-4\mathrm{Bn}-\mathrm{B}+\mathrm{Na}\right]}^{+}+4\mathrm{Bn}+\mathrm{B} $$
(7)

At elevated rf excitation amplitudes, another minor dissociation pathway involving the loss of 56 Da, and corresponding to loss of butene (B) is observed in the CID of the GP2α, GP3β, and GP4α complexes, Reaction 8 (). Loss of 136 Da, corresponding to loss of monobutyl phosphoric acid (MBPA), is also observed as a minor dissociation pathway in the CID of the GP1α, GP1β, GP3β, and GP4α complexes, Reaction 9 ()

$$ {\left[{\mathrm{GP}}_x+\mathrm{Na}\right]}^{+}\overset{n\kern0.5em He}{\to }\ {\left[{\mathrm{GP}}_x-\mathrm{B}+\mathrm{Na}\right]}^{+}+\mathrm{B} $$
(8)
$$ {\left[{\mathrm{GP}}_x+\mathrm{Na}\right]}^{+}\overset{n\kern0.5em He}{\to }{\left[{\mathrm{GP}}_x-\mathrm{MBPA}+\mathrm{Na}\right]}^{+}+\mathrm{MBPA} $$
(9)

Elimination of PhCH=O (neutral loss of 106 Da) is observed as minor CID product for GP2α, GP2β, and GP4α at high rfEA values, Reaction 10 ().

$$ {\left[{\mathrm{GP}}_x+\mathrm{Na}\right]}^{+}\overset{n\kern0.5em He}{\to }{\left[{\mathrm{GP}}_x-\mathrm{PhCH}=\mathrm{O}+\mathrm{Na}\right]}^{+}+\mathrm{PhCH}=\mathrm{O} $$
(10)

The peak deduced as [DBPA+W+Na]+ is only observed in the mass spectra of GP2α and GP2β, Reaction 11 (). This product appears to be the result of adduction of a background water molecule (W) to the primary CID product [DBPA+Na]+, which is stabilized by collisions with the He present in the trap.

$$ {\left[{\mathrm{GP}}_x+\mathrm{Na}\right]}^{+}\overset{n\kern0.5em He}{\to }{\left[\mathrm{DBPA}+\mathrm{W}+\mathrm{Na}\right]}^{+}+\left({\mathrm{GP}}_x-\mathrm{DBPA}-\mathrm{W}\right) $$
(11)

Discussion

Mode of Sodium Cation Binding in the [GPx+Na]+ Complexes

Although the exact modes of sodium cation binding in the [GP x +Na]+ complexes are not known, the CID behavior observed suggests that Na+ favorably binds to the sugar and phosphate moieties. What is not entirely clear is whether binding to both moieties occurs in the same conformer(s), or a mixture of conformers involving different modes of sodium cation binding are present in the experiments. All eight [GP x +Na]+ complexes exhibit Reaction 3 with the sodium cation preferentially retained by the sugar moiety for all but the complexes to GP2α and GP2β. Six of the [GP x +Na]+ complexes exhibit Reaction 4 with the sodium cation preferentially retained by the phosphate moiety only for GP2α and GP2β. These results clearly suggest that the 3 BnO substituent must be present and involved in the binding for the sodium cation to preferentially retain the sugar moiety upon CID. Given the geometry of these GP x it seems unlikely that the mode of Na+ binding could simultaneously involve both the phosphate moiety and the 3 BnO substituent, thus suggesting that a mixture of conformers involving two different modes of sodium cation binding (one involving the phosphate moiety with possible additional chelation interactions with the sugar, and another involving only chelation interactions with the sugar) are present for all complexes except those of GP1α and GP1β. Reaction 4 is not observed for GP1α and GP1β, suggesting that the 4- and 6- BnO substituents also contribute to enhanced binding to the sugar moiety, and do so more effectively than the cyclic protecting group, such that binding to the phosphate moiety is not competitive for these species. Numerous studies in the literature have found that sodium cations preferentially bind via multiple chelation interactions to hetero atoms [54,55,56,57,58, 69,70,71,72,73], and in particular oxygen atoms, and π-systems of organic and biologically relevant ligands [73,74,75,76,77,78,79,80]. Thus, the sugar moiety of each of these glycosyl phosphates offers multiple oxygen and π-donors for binding, whereas the phosphate moiety offers the oxo and alkoxy oxygen atoms for binding. Therefore, a mixture of conformers bound via multiple chelation interactions with the oxygen and π donors of the sugar moiety may be of similar stability and present in the experiments for GP1α, GP1β, GP3α, GP3β, GP4α, and GP4β. A very minor population of phosphate bound species appears to be present for these complexes. For the complexes to GP2α and GP2β, it appears that binding to the phosphate moiety is preferred and likely involves the phosphate oxo oxygen, one or possibly two of the alkoxy oxygen atoms [73], and possibly the oxygen atom of the sugar ring.

Survival Yield Analyses of [GPx+Na]+

Effects of C1-C2 Configuration, Cis versus Trans

Survival yield curves of the eight [GP x +Na]+ complexes are compared in Figure 3. Each anomeric pair is individually compared in parts a–d to readily elucidate the effects of the C1−C2 configuration on the stability of the glycosyl phosphate linkage. It should be noted that the GPα exhibit a cis configuration at C1–C2 and the GPβ exhibit a trans configuration at C1–C2 for all GP x except GP3α and GP3β where the opposite is true. The CID50% values of these complexes determined by four parameter logistic dynamic fitting are shown in Figure 3 and listed in Table 1 for comparison. Because the major fragmentation pathway observed in all cases involves glycosidic bond cleavage and accounts for >95% of the observed dissociation at all values of rfEA, the CID50% values provide a measure of the relative glycosidic bond stabilities. Overall, the CID50% values of the 1,2-cis [GP x +Na]+ exceed those of the 1,2-trans [GP x +Na]+, indicating that the glycosidic bonds of 1,2-cis [GP x +Na]+ are more stable than those of the 1,2-trans [GP x +Na]+. Differences in the CID50% values for the 1,2-cis and 1,2-trans anomeric pairs are smallest for GP1α and GP1β, which lack a cyclic protection group (0.064 V). Absence of the C3 BnO substituent also leads to a relatively small difference in the CID50% values (0.094 V) for the 1,2-cis versus 1,2-trans isomers. In contrast, larger differences in the CID50% values for the 1,2-cis versus 1,2-trans isomers are observed when both a cyclic protecting group and C3 BnO substituent are present, with a larger difference (0.324 V) found when the C2 BnO substituent is axial, and a somewhat smaller difference (0.310 V) when C2 BnO is equatorial. It has also been reported that it is the trans configuration between the substituents at C1–C2 and the resulting anchimeric assistance that controls the stereospecific CID behavior [14, 81, 82]. The present results clearly indicate that the C1–C2 configuration plays a major role in determining the stability/reactivity of glycosyl phosphate stereoisomers and that syn elimination from 1,2-trans [GP x +Na]+ is more favorable (less energetic) than anti elimination from 1,2-cis [GP x +Na]+. Although changes in the C1−C2 configuration alter the energetics for glycosidic bond cleavage, they do not alter the preferred reaction pathway for any of the isomeric pairs. Thus the mode of sodium cation does not appear to be significantly influenced by the C1−C2 configuration, which suggests that the C2 BnO substituent is likely not involved (or not a significant contributor) to the binding of the sodium cation.

Figure 3
figure 3

Survival yield curves for energy-resolved CID of eight sodium cationized glycosyl phosphates, [GP x +Na]+. Each anomeric pair is individually compared to facilitate elucidation of the effects of the C1-C2 linkage geometry and leaving group stereochemistry on the stability of the glycosyl phosphate linkage, (parts a–d)

Full size image
Table 1 Summary of the CID50% Values of [GP x +Na]+ and Experimental Relative Abundances of Sodium Cationized Ions Formed Upon Glycosidic Bond Cleavage
Full size table

D-Mannopyranosyl versus D-Glucosyl Dibutyl Phosphate

Survival yield curves of the sodium cationized complexes of GP3α, GP3β, GP4α, and GP4β are overlaid in Figure 4a. These GP x are also anomeric pairs that differ only in the orientation of the C2 BnO substituent and C1−C2 configuration. The 1,2-trans isomers of these complexes exhibit very similar CID50% values, whereas a modest change in the CID50% values is observed for the 1,2-cis isomers. Thus, the orientation of the C2 BnO substituent exerts a negligible effect on the glycosidic bond stabilities of sodium cationized GP3α and GP4β, and a minor influence on those of GP4α and GP3β. In contrast, the C1–C2 configuration plays a major role in determining the stability/reactivity of glycosyl phosphate stereoisomers, consistent with the conclusion of the previous section.

Figure 4
figure 4

Survival yield curves for energy-resolved CID of eight sodium cationized glycosyl phosphates, [GP x +Na]+. Overlays of the survival yield curves of the anomeric pair GP4α and GP4β with each of the other anomeric pairs are shown (parts a–c) are shown to facilitate elucidation of the effects of the orientation of the C2 BnO substituent (GP3α and GP3β), the presence of the C3 BnO substituent (GP2α and GP2β), and the cyclic protecting group (GP1α and GP1β) on the glycosidic bond stabilities of these complexes

Full size image

Effects of the 3-O-Benzyl Substituent

The survival yield curves of the sodium cationized GP2α, GP2β, GP4α, and GP4β are compared in Figure 4b. These complexes differ only in the absence or presence of the C3 BnO substituent and the C1−C2 configuration. As discussed above, the C3 BnO substituent is key to determining whether the sugar or phosphate moiety retains the sodium cation upon CID. As can be seen in the figure, the C3 BnO substituent has a more significant effect on the dissociation energetics of the 1,2-trans (lowers the activation barrier) than the 1,2-cis complexes (increases the activation barrier). Thus, the 3-O-benzyl substituent is critical to the mode of binding, whereas its influence on glycosidic bond stability depends on the C1−C2 configuration.

Effects of Cyclic Protecting Groups

The survival yield curves of sodium cationized GP1α, GP1β, GP4α, and GP4β are compared in Figure 4c to elucidate the effect of the cyclic protecting group at the C4 and C6 positions on the glycosidic bond stabilities of [GP x +Na]+. These complexes are chosen for comparison to examine the effects of the C4 and C6 BnO substituents of GP1α and GP1β versus the cyclic protecting group at the C4 and C6 positions of GP4α and GP4β on glycosidic bond stability. Previous studies using in-source fragmentation showed that a greater cone voltage is required to dissociate the glycosidic bonds of sodium cationized glycosyl phosphates that possess a cyclic protecting group [18]. Figure 4c shows that the sodium cationized forms of a 1,2-cis anomer carrying a cyclic protecting group, [GP4α+Na]+ (CID50% = 0.795 V), is more stable than [GP1α+Na]+ (which does not possess a cyclic protecting group, CID50% = 0.732 V), which suggests that the cyclic protecting group at the C4 and C6 enhances the stability of the anomer bearing a 1,2-cis configuration. This result is in agreement with the in-source threshold fragmentation studies [18]. For the sodium cationized forms of 1,2-trans isomers with a cyclic protecting group at the C4 and C6 positions, [GP4β+Na]+ (CID50% = 0.471 V), are more active than that of [GP1β+Na]+ (which does not possess a cyclic protecting group, CID50% = 0.668 V). Thus the effect of the cyclic protecting group on glycosidic bond stability is also correlated with the C1−C2 configuration.

Effects of the Mode of Sodium Cation Binding

Although the exact modes of sodium cation binding in the [GP x +Na]+ complexes are not known, they do not appear to be significantly influenced by the C1−C2 configuration as the same dominant CID pathway is observed for each anomeric pair. Instead, the C3 BnO substituent appears to be key to determining the preferred mode of sodium cation binding. For the complexes of GP1α, GP1β, GP3α , GP3β , GP4α, and GP4β, the cation binds to the sugar moiety such that the sodium cation is remote from the glycosidic bond cleavage process. Thus, the sodium cation is not expected to exert a significant impact of the dissociation energetics, and primarily serves as a charge carrier to enable this reaction to be observed mass spectrometrically. In contrast, for the complexes of GP2α and GP2β, where binding involves the phosphate moiety, it appears that binding of the sodium cation activates the glycosidic bond of the 1,2-cis isomer and stabilizes the glycosidic bond of the 1,2-trans isomer. This is consistent with expectations based on the mechanisms proposed below and will be revisited in the mechanistic discussion of the next section.

Mechanism of Glycosidic Bond Cleavage

The lower CID50% values determined for 1,2-trans [GP x +Na]+ suggest that a plausible mechanism for cleavage of the glycosidic bond involves syn elimination via a McLafferty-type rearrangement as shown in Figure 5 pathway A for anomers bearing a trans configuration at C1–C2. During the unimolecular dissociation process, based on a McLafferty-type rearrangement, H2 is transferred to the dibutyl phosphate moiety via a six-membered ring transition state, which leads to the loss of neutral dibutyl phosphoric acid. When R2 = BnO, the substituent is involved in the binding and provides an additional chelation interaction with the sodium cation. Therefore, neutral dibutyl phosphoric acid loss becomes the dominant fragmentation pathway observed for anomers of GP1, GP3, and GP4 (reaction 3, ). When R2 = H, the absence of the C3 BnO substituent leads to preferential binding to the phosphate moiety, and thus sodium cationized dibutyl phosphoric acid, [DBPA+Na]+, becomes the dominant ionic product observed for both GP2 anomers (Reaction 4, ). The fragmentation of 1,2-cis anomers likely involves the formation of an oxocarbenium ion intermediate as shown in Figure 5 pathway B [18]. The formation of an oxocarbenium ion transition state has been assumed as the rate-determining step of the one-pot sequential synthesis [9, 18]. Elongation of the glycosidic bond leads to formation of an oxocarbenium ion intermediate. Transfer of the H2 atom to the dibutyl phosphate moiety then produces the observed products. The ability of the 1,2-cis anomers to stabilize the transition state for dissociation via hydrogen bonding in the six-membered ring formed reduces the activation energy relative to the 1,2-trans anomers, consistent with the results of the survival yield analyses that find pathway B requires more energy than pathway A in the gas phase. Likewise, binding of Na+ to the phosphate moiety would stabilize the oxocarbenium ion transition state (TS) of the 1,2-cis anomers and activate the glycosidic bond, whereas Na+ binding to the phosphate moiety should weaken the hydrogen-bonding interaction and destabilize the TS for 1,2-trans species in accord with the experimental observations..

Figure 5
figure 5

Proposed mechanisms for glycosidic bond cleavage of 1,2-trans and 1,2-cis sodium cationized glycosyl phosphates, [GP x +Na]+

Full size image

CID50% versus Dissociation Onsets

The trends in the CID50% values extracted from the ER-CID measurements performed here are used to assess the relative glycosidic bond stabilities of eight [GP x +Na]+ complexes. However, one might arbitrarily chose a different level of dissociation to make these assessments. Most often, the threshold or onset energy (or rf excitation energy) would be of interest. A detailed examination of Supplementary Figure S1 shows that 1,2-trans [GP x +Na]+ isomers begin to undergo glycosidic bond cleavage at lower values of the rf excitation energy than the corresponding 1,2-cis isomers, consistent with the trends in the CID50% values. Likewise, the dissociation behavior of GP3α versus GP4β and GP3β versus GP4α are highly parallel over the entire range of excitation energies whether examining the survival yields of Figure 4 or the product yields of Supplementary Figure S1. Analogous comparisons hold for GP4x versus GP2x and GP1x such that the value chosen for comparison is somewhat arbitrary for the [GP x +Na]+ complexes examined here.

Conclusions

The relative glycosidic bond stabilities of eight [GP x +Na]+ complexes have been examined via survival yield analyses based on ER-CID experiments performed in a QIT MS. Present results indicate that the C1-C2 configuration is a dominant factor influencing the stability of glycosyl phosphate linkage, such that 1,2-cis isomers, corresponding to a trans orientation of the leaving groups, are found to be more stable than the corresponding 1,2-trans isomers where the leaving groups are syn. Thus, the glycosyl phosphates having the leaving groups in a syn orientation (the 1,2-trans isomers) make better glycosyl donors than those with the leaving groups anti (the 1,2-cis isomers). These behaviors suggest that a plausible mechanism for the glycosidic bond cleavage of 1,2-trans [GP x +Na]+ involves syn elimination via a McLafferty-type rearrangement, whereas the dissociation of 1,2-cis [GP x +Na]+ likely involves the formation of an oxocarbenium ion intermediate. The fragmentation behaviors indicate that the cyclic protecting groups stabilize the glycosidic bond of the 1,2-cis anomers, but is activating for the 1,2-trans anomers. The fragmentation behaviors also suggest that the C3 BnO substituent provides additional stabilization to the sodium cation, alters the fragment that retains Na+ upon dissociation, and leads to the loss of neutral dibutyl phosphoric acid, Reaction 3 (). The corresponding GP3 and GP4 complexes exhibit similar CID50% values, suggesting that the orientation of the C2 BnO substituent does not significantly impact the glycosidic bond stabilities.