Analysis of trinitrophenylated adenosine and inosine using capillary electrophoresis-laser induced fluorescence detection and gamma-cyclodextrin

Thesis (M.S.) University of Alaska Fairbanks, 2016Adenosine (Ado) and adenine ribonucleotides are essential in cell metabolism and energy production, cellular signaling, and DNA and RNA synthesis. The biosynthesis of these molecules takes place in both the intracellular and extracellular space via transphosphorylation reactions catalyzed by several distinct kinase enzymes like adenylate kinase. Several analytical detection methodologies have been developed to monitor these molecules in biological tissue, including both liquid chromatography (LC) and capillary electrophoresis (CE) techniques. However, many of these methodologies are limited by separation resolution and sample injection volume requirements. This thesis presents a novel capillary electrophoresis-laser induced fluorescence detection (CE-LIF) method with high separation power to analyze Ado and Inosine (Ino), a metabolite of Ado, by derivatization with 2,4,6-trinitrobenzenesulfonic acid to form fluorescent trinitrophenylated complexes of Ado (TNP-Ado) and Ino (TNP-Ino). The development and validation of the CE-LIF method, optimization of the trinitrophenylation reaction, and fluorescence enhancement of TNP-Ado and TNP-Ino with γ-cyclodextrin will be discussed. Detection limits were 1.6 μM for Ado and 4 μM for Ino in rat brain tissue. Large-volume sample stacking (LVSS) was employed to further enhance the sensitivity of the CE-LIF method, with detections limits of 310 nM and 159 nm for Ado and Ino, respectively. The CE-LIF method offers promise for the analysis of Ado, Ino and potentially other adenine ribonucleotides in small volume generating biological experiments like in vivo microdialysis and single cell metabolomics.Chapter 1: Introduction -- 1.1 Biological Significance of Adenosine and Adenine Ribonucleotides -- 1.2 Current Methodologies to Detect Adenosine and Adenine Ribonucleotides -- 1.3 Summary of Research Aims -- 1.4 References -- Chapter 2: Analysis of Trinitrophenylated Adenosine and Inosine using Capillary Electrophoresis-Laser Induced Fluorescence Detection and γ-Cyclodextrin -- 2.1 Abstract -- 2.2 Introduction -- 2.3 Methods -- 2.3.1 Safety Considerations -- 2.3.2 Chemicals and Reagents -- 2.3.3 Preparation of TNP-Ado and TNP-Ino Standards -- 2.3.4 1H, HMQC and ROESY NMR Spectra -- 2.3.5 CE-LIF Analysis and LVSS Studies -- 2.3.6 Trinitrophenylation Reactions -- 2.3.7 Biological Sample Preparation -- 2.3.8 Statistical Analysis -- 2.4 Results and Discussion -- 2.4.1 CE-LIF Optimization -- 2.4.2 Determination of γ-CD Association Constants -- 2.4.3 Structural Determination of TNP-Ado and γ-CD Inclusion Complex -- 2.4.4 Trinitrophenylation Reaction Kinetics Monitored by CE-LIF -- 2.4.5 CE-LIF Method Validation -- 2.4.6 Analysis of Adenosine and Inosine in Rat Brain Tissue -- 2.4.7 Large-Volume Sample Stacking of TNP-Ado and TNP-Ino -- 2.5 Conclusions -- 2.6 Acknowledgments -- 2.7 References -- Chapter 3: Conclusion -- 3.1 Overview -- 3.2 Future Directions -- Appendix A Supporting Information for Chapter 2 -- Appendix B Trinitrophenylation and Capillary Electrophoresis Analysis of Adenine Ribonucleotides, N6-Cyclohexyladenosine, Guanosine, Cytidine, and Uridine