The broad, long term objectives of this proposal are two fold. First, it is proposed to distinguish between two possible mechanisms for the hydrolysis of oligosaccharides by lysozyme, such that this enzyme can be better understood. Second, it is proposed to develop synthetic receptors for monosaccharides to be used in both aqueous and nonaqueous media.
The specific aims are: 1) Synthesis of a deuterium labeled disaccharide mimic in which the deuteriums possess different chemical fates depending upon the site of disaccharide mimic protonation in specific acid hydrolysis, 2) Synthesis of a similar mimic which yields different products depending upon exo or endocyclic acetal cleavage, 3) Incorporate these same saccharide mimics into the natural substrate for lysozyme so as to determine the site of Glu-35 general acid protonation, 4) Contrast the rate of alpha to beta anomerization catalyzed by lysozyme to the rate of (18)O incorporation as a probe of exo or endocyclic mutarotation pathways, 4) Complete the binding studies of a polyazacleft host with cyclohexanepolyol guests, 5) Deter-mine differences in internal enthalpy and entropy of the guests and correlate the differences to host selectivities, 6) Measure the strength of intramolecular hydrogen bonds in cyclohexane diols and triols and correlate the hydrogen bond strengths to host selectivities and binding constants, 7) Synthesize second generation saccharide receptors that either retain the intramolecular hydrogen bonds, or make both a donor and acceptor H-bond to the sugar alcohols, 8) Develop a water soluble host for glucose with boronic acid esters and a reversible Fe(II)-Fe(III) couple, and 9), covalently attach this host to an Pt electrode. The health relatedness of the project derives from three important goals. The first is to gain greater insight into the mechanism of acetal hydrolysis used by lysozyme such that glycosyl transfers catalyzed by enzymes can be better understood. The second benefit derives from an increased understanding of the energetics of complexation of saccharides in lipophilic media such that transport systems can be rationally designed. Finally, the benefits of an aqueous glucose binding host derive from the possibility of attaching the host to an electrode. Electrochemical responses would then be modulated by sugar absorption, and thus sensitive to blood sugar levels. The methods to be used involve 1H and 2H NMR analysis of the products derived from hydrolysis of the deuterium labeled disaccharide mimic. The binding studies will use variable temperature 1H NMR titrations. The internal entropy and enthalpy differences between the guests will be determined by known conformational analysis techniques that use variable temperature 1H, 13C NMR and FT-IR. The electrode development will be performed with currently used deposition methods and voltametric and amperometric techniques.
| Pallan, Pradeep S; Marshall, William S; Harp, Joel et al. (2005) Crystal structure of a luteoviral RNA pseudoknot and model for a minimal ribosomal frameshifting motif. Biochemistry 44:11315-22 |
