Capillary Electrophoresis: Bioanalytical Applications

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DESCRIPTION (provided by applicant): The broad goals of this research are to develop capillary electrophoresis (CE) as an analytical technique to study the physicochemical parameters of the antibiotics vancomycin (Van), ristocetin (Ris), and teicoplanin (Tel). Its focus is on demonstrating principles and on developing useful bioanalytical procedures that can be applied to the analysis of receptor-ligand interactions and microscale reactions. The evolution of antibiotic resistant bacteria has made the study and development of Van-like antibiotics a critical area of research focus. Hence, studies probing the physicochemical parameters of these antibiotics via chemical modification are highly warranted. Two specific CE techniques will be utilized in this work: on-column microreactor techniques and affinity capillary electrophoresis (ACE). Techniques utilized in this study include chemical modification and characterization, chemical separation and identification of small molecules by CE utilizing both laser-induced fluorescence (LIF) and ultraviolet/visible (UV/VIS) detection schemes and high performance liquid chromatography (HPLC), and enzyme kinetics. The research will develop new bioanalytical techniques and will focus on examining small biomolecules involved in the prevention of disease and, hence, to the issue of public health. This understanding will strengthen the scientific base underlying the design, preparation, and application of CE towards a host of health-related problems. The specific aims of the research are to I. Utilize On-Column Microreactor Techniques and ACE in the Estimation of Binding Constants Between Antibiotics and Ligands. II. Determine the Charge and pl of Antibiotics Using On-Column Microreactor Techniques. III. Examine the Kinetics of Derivatization of Antibiotics Using Reactive Chemical Reagents. IV. Estimate Binding Constants of Linked Antibiotics to Peptides. V. Conduct Experimental Studies on D-Ala-D-Lac Terminus Peptide Binding to Van-Group Antibiotics. The proposed research will: Demonstrate the versatility of CE in determining physicochemical parameters of antibiotics; Demonstrate high-throughput derivatization of receptors and ligands coupled to ACE; Provide for rapid synthesis and accurate analysis of multiple drug targets simultaneously; Require reduced sample volumes compared to complementary analytical techniques, and; Reduce sample waste and disposal.