Glucose Spectroscopy in the Gas Phase with Cryogenic Ion Vibrational SpectroscopySTEVE 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

1) ESI sugar solution with ~1mM NaCl

2) Trap ions and attach D2 tag

5) Acquire MS of detagged ions

3) Remove untagged ions from TOF path

4) Excite vibrational modes and remove tag

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)

5% β1 (+504cm-1)

All calculations were done at the cam-B3LYP/6-311+G(d,p) level

3500 3550 3600 3650 3700012345

(Na-

Glu

cose

)+

3500 3550 3600 3650 3700012345

Cal

cula

ted

3500 3550 3600 3650 3700012345

Ove

rlay

Frequency (cm-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…

3300 3350 3400 3450 3500 3550 3600 3650 37000.0

0.5

G6P

-

3300 3350 3400 3450 3500 3550 3600 3650 37000.0

0.5

1.0

(Na-

Glu

cose

)+

Frequency (cm-1)

… But it’s Still Complicated

3300 3350 3400 3450 3500 3550 3600 3650 37000.0

0.5

G6P

-

3300 3350 3400 3450 3500 3550 3600 3650 37000

250

500

Alp

ha

3300 3350 3400 3450 3500 3550 3600 3650 37000

250

500

Bet

a

3300 3350 3400 3450 3500 3550 3600 3650 37000

250

500

Bes

t M

atch

Frequency (cm-1)

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

Brett Marsh

Jon Voss

Etienne Garand

Jia Zhou

Erin Duffy