## LSU Historical Dissertations and Theses

#### Title

Studies in Fructofuranose Chemistry.

1991

Dissertation

#### Degree Name

Doctor of Philosophy (PhD)

#### First Advisor

Ezzat S. Younathan

#### Abstract

The glycolytic pathway enzyme phosphofructokinase (E.C. 2.7.1.11) has as its substrate and activator two different carbohydrate phosphate esters. Both are derivatives of D-fructose in the furanose ring form. This dissertation is divided into two chapters: the first deals with the conformational specificity of the active site and the second the structure of the activator. The compound 2,5-anhydro-3,4-O-(1,2-ethanediyl)-D-mannitol was designed to test the active site. The synthesis utilized a phase-transfer cyclo-dialkylation of a vicinal diol to yield a trans-fused 2,5,8-trioxabicyclo (4.3.0) nonane system. The final product was shown to exist in a locked conformation in solution by temperature dependent n.m.r. experiments which showed no line shape change. The data indicate that the five-membered ring is locked by the trans-fused six-membered 1,4-dioxane ring into a twist $\sp4$T$\sb3$ conformation. A single crystal X-ray study was carried out. The crystalline product exists in an ideal twist conformation with a pseudorotation angle of 0$\sp\circ$amplitude of 47.2$\sp\circ$in agreement with the n.m.r. results. The compound, as the monophosphate, is intended to verify the linear plot of substrate efficacy index versus $\beta$-$\sp4$T$\sb3$ concentration observed with several ketose 6-phosphates. Chapter two describes detailed n.m.r. studies of the activator molecule, D-fructofuranose 2,6-bisphosphate, which allowed the unequivocal assignment of all the proton, carbon and phosphorus resonances. Several unexpected chemical shift values and coupling constants were obtained. The usual near-gauche orientations of C-1 and C-3 to P-2, obtained by molecular mechanics calculations, can explain their small vicinal coupling constants in contrast to the expected larger value seen for C-5 to P-6. Reduction did not affect the n.m.r. spectrum substantiating that C-2 is phosphorylated. Oxidation yielded an unstable intermediate which decomposed by a beta elimination mechanism involving the phosphate group on C-6. These data establish unequivocally the $\sp1$H, $\sp{13}$C and $\sp{31}$P assignment and explain the observed anomalous shifts. Moreover, they establish that the activator of fructose 6-phosphate 1-kinase is the $\beta$-anomer of the $\sp4$T$\sb3$ conformer of D-fructose 2,6-bisphosphate. The conclusion from both investigations is that both the active site and the activation site of phosphofructokinase seem to prefer the $\sp4$T$\sb3$ conformer of these fructofuranose ligands.

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