Distinguishing Hexose Isomers with Water Adduction to Lithiated Monosaccharides Matthew T. Campbell, Dazhe Chen, Gary L. Glish

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1 Distinguishing Hexose Isomers with Water Adduction to Lithiated Monosaccharides Matthew T. Campbell, Dazhe Chen, Gary L. Glish Introduction Elucidation of glycans is particularly challenging because the monosaccharide components have similar structures and several stereocenters. Aldohexoses such as glucose are monosaccharides with four chiral centers allowing for 16 diastereomers and other structural isomers such as fructose. Collision induced dissociation does not provide sufficient information to distinguish those isomers. Most methods for indentifying hexose isomers rely on lengthy chromatographic separations or ion mobility separations that often lack the resolving power to discriminate between some isomers. New methods have been developed to distinguish hexose isomers using mass spectrometry without a prior separation step. One method includes prior derivatization of the hexose epimers followed by subsequent CID to yield unique ratios of product ions. Other methods used the fixed ligand kinetic method which eliminates the need for a derivatization reaction, but it requires the addition of a divalent transition metal, a chiral reference molecule (typically an amino acid), and fixed ligand (usually a molecule with a fixed negative charge) which will all adduct with the hexose to form a tertameric complex. CID is used on the resulting complex and the relative

2 intensities of product ions can be used for distinguishing isomers. Here we present a method for distinguishing between biologically relevant hexose isomers (glucose, galactose, mannose, and fructose) by water adduction to the lithiated hexose ions in a quadrupole ion trap. Methods Solutions of 10 μm monosaccharide (D-glucose, D-mannose, D-galactose, D-talose, or D-Fructose shown in scheme 1) with 100 μm lithium acetate were prepared in 50/50 methanol/water. Solutions were analyzed on a Bruker Equire 3000 ion trap mass spectrometer via electrospray ionization. The lithiated monosaccharide was isolated, and a delay was applied before scanning the isolated lithiated ions from the trap to allow for controlled reaction times of water adduction. The total reaction time was measured from isolation of the lithiated monosaccharide (time = 0) to ejection of non-water adducted peak after the set delay. Signal intensity of the unreacted peak at m/z 187 (I 187 ) and the water adducted peak at m/z 205 (I 205 ) were used to study the kinetics of the water adduction reaction. Density functional theory was used to study the binding of lithium to D- glucose, D-mannose, D-galactose, and D-Talose. All computations were performed with the Gaussian 09 suite of programs. All optimizations and frequency

3 calculations were performed using the B3LYP/ G(d,p) level of theory. Because there are hundreds of possible conformations for any of the four hexoses, only the lowest energy conformation for both the α and β anomers were used as starting points. Results Scheme 1: Structures for all five hexoses studied The lithiated isomers of glucose, galactose, mannose, and fructose show no difference in MS/MS spectra after collision induced dissociation. Water adduction rates were measured for each monosaccharide. The signal intensity was measured for both the lithiated monosaccharide (I 187 ) and the corresponding water adducted peak (I 205 ) at reaction times of 11, 21,31,41,51, and 61 ms. Plotting ln ( I 187 ) I 187 +I 205

4 versus time yields a linear plot as expected for pseudo-first order reaction kinetics. The slope of the linear fit applied to each plot is a product of the reaction rate and an unknown concentration of water in the quadrupole ion trap. This slope, however, is constant across experiments done on multiple days, and can be used to distinguish between the four isomers studied (Figure 1). Figure 1: Reaction slope for each hexose Figure 2: Unreactive fraction for each hexose ESI of each hexose produces both reactive structures that forms a water adduct and a non-reactive structure that does not adduct water. After a sufficiently long delay time only the unreactive structure of the lithiated monosaccharides will be present at m/z 187. The final ratio of non-water adducted species (I 187 ) to the total lithiated hexose (I I 205 ) allows for identification of each isomer. These ratios are measured after allowing for reaction times greater than 1000 ms. Previous experiments have shown this to be more than sufficient time to completely deplete all the reactive ion structure(s).

5 Density functional theory was used to study the relative energies of several different lithiation sites for each hexose. After optimizing each hexose structure to the lowest energy conformation α and β anomer for D-glucose, D-galactose, D- mannose, and D-talose (8 structures total), a lithium cation was added. Several structures, each with a lithium cation in a different location relative to the hexose, were optimized. This resulted in several unique lithiation sites for each of the anomers studied. The coordination number for lithium was found for each of the resulting unique structures. In all structures lithium was found to bind bidentate, tridentate, or tetradetate with the oxygen atoms of the hexose. The number of oxygen atoms coordinated to the lithium cation dictates its Lewis acidity. The more hydroxyls bound the lithium, the more the charge is dispersed resulting in the cation being less Lewis acidic. Analogously, binding to fewer oxygen atoms retains the charge and the acidity of the cation. Higher acidity means there is a more favorable reaction between the lithium cation and water. Therefore, it is expected that hexoses that preferentially form bidentate structures over tridentate and tetradentate structures will be more reactive with water, and using this hypothesis the experimental data and theoretical calculations are in good agreement. Both anomers of glucose resulted in primarily bidentate structures. The optimized structures of lithium and galactose yielded mostly bidentate with some low energy tridentate structures (Figure 3). Mannose showed more lower energy

6 trivalent coordination structures, and talose showed almost exclusively tridentate and tetradentate structures. Figure 3: Relative energy for lithiated hexose structures. All energies are relative to the lowest energy structure for each respective hexose