Synthesis of a Creatine-Coumarin Conjugate for Assessment of Creatine Transport

Undergraduate #14
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Session: 3
Room: Harding

Christopher Kelly - Bowie State University
Co-Author(s): Dr, Jacqueline Anita Smith, Bowie State University, Bowie, MD.

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Creatine (Cr) plays a central role in energy metabolism in tissues and organs with high energy demands such as the brain, and cardiac or skeletal muscle. Cr and its phosphorylated form phosphocreatine (PCr) constitute an energy shuttle that links ATP production in mitochondria, to intracellular sites of ATP consumption. Cells in these tissues, have very limited ability to synthesize Cr, and depend on Cr uptake across the cell membrane by a specialized Cr transporter (CrT-solute carrier SLC6A8). Inborn CrT defects result in intellectual disabilities, and CrT function anomalies are linked to heart disease (cardiac failure). On the other hand, Cr supplementation has been shown to have a beneficial effect in numerous in vitro and in vivo models, particularly in cases of oxidative stress. Intracellular Cr content is maintained within narrow limits. However, the molecular mechanisms regulating CrT function during health and disease are not understood. A limitation to studies of CrT structure/function relationships is that function quantification depends on the uptake of radioisotope labeled Cr, thus necessitating specialized facilities and processes for their use. The purpose of our research was to generate a fluorescent-labeled Cr analogue that could replace radioactive Cr as a tool for the assessment of CrT function. Cr fluorescent analogs were synthesized through Steglich esterification. Fluorescence characterization of Cr-Cou shows that excitation occurs at 370nm, while the maximum emission occurs at 398nm, indicating the suitability of Cr-Cou as a fluorescent probe. The ability of this newly created probe to serve as a surrogate measurement for CrT function is being tested using HEK293 cell cultures transfected with cDNA encoding the CrT protein. Cr-Cou uptake will be measured by quantification of intracellular fluorescence in HEK293 cells expressing CrT and compared with control cells. Binding to CrT protein will be quantified by measurement of cell membrane fluorescence. The specificity of these interactions will be further studied by competition assays using unlabeled Cr.

Funder Acknowledgement(s): HBCU-UP

Faculty Advisor: Dr. Lucia Santacruz, lsantacruzkozarinas@bowiestate.edu

Role: Synthesis, purification and initial fluorescence characterization of Creatine-Coumarin