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    Investigating the indentification and construction of therapeutic glycosaminoglycans

    Author
    Beaudet, Julie Michele
    View/Open
    167110_Beaudet_rpi_0185E_10110.pdf (4.700Mb)
    Other Contributors
    Linhardt, Robert J.; Dordick, Jonathan S.; McGown, Linda Baine; Dinolfo, Peter; Gilbert, Ryan;
    Date Issued
    2013-05
    Subject
    Biochemistry and biophysics
    Degree
    PhD;
    Terms of Use
    This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.;
    Metadata
    Show full item record
    URI
    https://hdl.handle.net/20.500.13015/867
    Abstract
    Using the heparin biosynthetic pathway as a model, three small-scale techniques were tested: milligram bench-top synthesis, channel microfluidics via SPR, and digital microfluidics. The target structure was a specific pentasaccharide which gives heparin its anticoagulant properties. Out of the three techniques, only the product synthesized using SPR was confirmed to have the desired sequence. This is likely due to the temperature control, detection sensitivity, and lack of manual handling provided by the instrument. However this technique is not ideal for testing many different potential targets. Therefore it is necessary to continue to improve the protocols used for the chemoenzymatic synthesis of GAGs and the techniques used to produce them. Using kinetic data regarding the binding affinity of physical characteristics in natural interactions when selecting and constructing potential therapeutic targets will expose new options for pharmaceuticals. Accurate and efficient microscale chemoenzymatic synthesis protocols will side in the screening of these options and eventual production.; Glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides. They are involved in scores of physiological interactions ranging from cell communication to lipid transport to blood anticoagulation. For this reason, they are prime candidates for pharmaceutical targets. However glycosaminoglycans can vary significantly in size and sulfonation so it is necessary to investigate binding interactions carefully when evaluating interactions for target GAG specifications. Protein-GAG interactions can be measured using surface plasmon resonance (SPR). SPR measures the interaction of a protein analyte with an immobilized ligand. A variety of experimental conditions exist to gather quantitative and qualitative interaction data.; Once kinetic data has been collected it can be used to appraise potential targets based on the size, sulfonation, and baseline kinetic rates. Targets can be graded using SPR or other analytical techniques. A popular technique in drug discovery is the use of microarrays. These arrays call for dozens of constructs to be evaluated at once. Pure chemical synthesis of GAGs is expensive in time and materials. Chemoenzymatic synthesis is a much more economical route but without a `glycomic code' comparable to that of genetics, enzymatic synthesis can be difficult to control.;
    Description
    May 2013; School of Science
    Department
    Biochemistry and Biophysics Program;
    Publisher
    Rensselaer Polytechnic Institute, Troy, NY
    Relationships
    Rensselaer Theses and Dissertations Online Collection;
    Access
    Restricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.;
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