UC Riverside

ABSTRACT OF THE DISSERTATION

Proton NMR Studies of the Interaction of Heparin-Derived Oligosaccharide with Biological Molecules and the Cis/Trans Isomerization of Amide Bonds in Peptides and Peptide/Peptoid Hybrids

By

Khanh Trong Nguyen

Doctor of Philosophy, Graduate Program in Chemistry

University of California, Riverside, March 2009

Professor Dallas L. Rabenstein, Chairperson

Two diverse topics in biological chemistry are the subject of this dissertation. In part I, the research focuses on heparin and the interaction of histidine-containing peptides with heparin. Heparin is a linear polysaccharide, which is formed through the linkage of variously sulfated uronic acid-(14)-D-glucosamine repeating disaccharide subunits by glycosidic bonds. The structure of heparin is not yet completely understood due to its extreme complexity. Consequently, details of the binding of peptides and proteins by heparin is not well understood. One approach is to use well-defined heparin-derived oligosaccharides with different structures as models to probe the question of specificity of binding.

Three heparin-derived tetrasaccharides, one hexasaccharide and one octasaccharide were isolated, purified and characterized. A structurally defined tetrasaccharide was then used as a model to study heparin binding by histidine-containing peptides and related molecules. The imidazolium group of all the molecules studied was found to bind site specifically to a binding pocket formed by an iduronic acid-glucosamine-iduronic acid trisaccharide sequence. Binding constants were determined for the complexes by NMR. Binding constants and relative binding affinities were also determined for the binding of the same molecules by intact heparin by isothermal titration calorimetry and heparin affinity chromatography, respectively.

In part II, the kinetics and thermodynamics of cis/trans isomerization of Xaa-sarcosine tertiary amide bonds in peptide/peptoid hybrids were studied. Xaa represents an amino acid and sarcosine is N-methyl glycine. Specifically, the affect of amino acid sequence on the kinetics and equilibria of cis/trans isomerization across Xaa-sarcosine peptide bonds in three series of peptide/peptoid hybrids having the sequences Ac-Cys-Sar-His-Xaa-(Ala)3-Cys-NH2, where Xaa is His, Gly, Lys, Phe, Asp and Glu, Ac-Cys-Sar-(Ala)x-His-(Ala)y-Cys-NH2, x = 0-4 and y = 4-0, and Ac-Cys-Sar-His-(Ala)3-Cys-NH2, Ac-Cys-His-Sar-(Ala)3-Cys-NH2, etc., were studied. The populations of the cis and trans isomers and the kinetics of cis/trans interconversion were found to depend on the amino acid preceding sarcosine.

The kinetics and equilibria of cis/trans isomerization across the Xaa-Xaa secondary amide bonds in a series of peptides of the sequence Ac-Cys-Xaa-Xaa-Cys-His-NH2, where Xaa is Ala, Tyr and Phe, were also studied. These peptides were studied as model peptides for oxido-reductase enzymes with the Cys-Xaa-Xaa-Cys motif. The effects of the type and position of the central Xaa residues on the kinetics and equilibria of cis/trans isomerization were examined. Rate and equilibrium constants for cis/trans isomerization together with thermodynamic parameters were determined for all the peptides and peptide/peptoid hybrids studied in both their dithiol and disulfide forms. Rate constants for interconversion between the cis and trans configurational isomers were determined by inversion-magnetization transfer NMR methods and equilibrium constants for the cis/trans isomerization were determined from resonance intensities.