Steve Kregel Garand Group-UW Madison 6/17/2014 Glucose Spectroscopy in the Gas Phase with Cryogenic Ion Vibrational Spectroscopy Steve Kregel Garand Group-UW Madison 6/17/2014

Background and Motivation Sugar mixtures extracted from glycoproteins are very complex All hexoses yield the same information via mass spectrometry Typically separated with HPLC to determine isomeric ratio Slow Requires significant molecular modifications Gas phase spectroscopy could potentially identify isomeric mixtures without separation Alley, William R. Jr.; Mann, Benjamin F.; Novotny, Milos V.; Chemical Reviews 2013, 113 2668-2732

Instrumental Setup-CIVS 3) Remove untagged ions from TOF path 1) ESI sugar solution with ~1mM NaCl ISOMER BURNING LASER! 2) Trap ions and attach D2 tag 4) Excite vibrational modes and remove tag 5) Acquire MS of detagged ions

Glucose Background Anomeric ratio is 44% α and 56% β in solution phase Interconverts through ring opening deprotonation mechanism at C1 Requires solvent to interconvert α and β anomers must be considered distinct species in gas phase Multiple Na+ binding sites possible - yields a distribution of conformers Fishman, P.H. and Bailey, J. Martyn. American Journal of Physiology Vol. 226, No.4 April 1974

Experimental and Calculated Spectrum of Sodiated Glucose 72% α0 (+0cm-1) 9% α1 (+717cm-1) 9% α2 (+731cm-1) 5% β0 (+370cm-1) Spectrum possibly saturated Isomer Burning to deconvolute Many Conformers present, will be hard to do with multiple sugars All calculations were done at the cam-B3LYP/6-311+G(d,p) level 5% β1 (+504cm-1)

Glucose-6-Phosphate Background Addition of a phosphate group greatly expands the PES Anomeric conversion IS possible in gas phase due to the dual acid- base nature of the phosphate group α and β G6P cannot be considered separately Naturally charged, requires no Na+ atom to become an ion Phosphate group can move to lowest energy position before cryogenic trap Implies that only a single conformer should be present

Phosphorylation Simplifies the Spectrum… Lack of shoulders, fewer peaks

… But it’s Still Complicated α-G6P (+0 cm-1) β-G6P (+324 cm-1) Best Match (+1555 cm-1)

Conclusions Gas phase spectroscopy has the potential to identify mixtures of hexoses Multiple (Na-Glucose)+ conformers complicate the spectrum Could lead to difficulty in creating a hexose spectral library G6P is likely not in its low energy conformation

Acknowledgements Etienne Garand Brett Marsh Erin Duffy Jon Voss Jia Zhou