| dc.description.abstract | The focuses of this dissertation are an examination and advancement of chemistries for the fluorogenic derivatization of the analysis of proquiniodal species. The term proquinoidal species specifically refers to a group of biologically active compounds that easily may be oxidized to the corresponding quinones or imino-quinones. Catechols (Cs), catecholamines (CAs), and 5-hydroxyindoles (5-HIs) are classes of biogenic amines that affect neuroregulatory functions such as mood and appetite. While the determination of these classes of substances has been the accomplished by a variety of techniques over the past several decades, a recent approach has been their conversation to a fluorescent product by use of the fluorogenic reagents benzylamime (BA) or diphenylethylenediamine (DPE). In the first section of this work, a critical examination of these derivatization chemistries is undertaken through product isolation, derivatization kinetics investigations, and yield determination for three substances that represent each analyte class. While not investigated in detail, it is recognized that 3-nitrotyrosine residues in peptides and proteins (a post-translational modification that has been associated with various age related pathologies such as atherosclerosis, neuropathies and others conditions), when reduced to the corresponding 3-aminotyrosine residues, undergo an analogous derivatization reaction to the catechol class. Application of the described derivatization reaction to such substances is an obvious and valuable extension of the present findings. Results obtained from the initial finding indicated that improvements regarding the physical-chemical properties of the DPE and/or BA regents would be highly useful in future applications directed toward the analysis of 3-nitrotyrosine residues, particularly as regards solubility and selective isolation of the formed product. The approach described herein relies on the reduction of the 3-NY residue to the corresponding 3-aminotyrosine (3-AY), with subsequent derivatization by either BA or DPE. Enhancement of the existing derivatization reagents is explored through chemical modification of the reagents through the attachment of various substituents for the purpose of enhanced isolation and/or detection, increasing reagent aqueous solubility, maximization of single production formation, and allowing for the incorporation of stable isotopes. Recognizing that samples of biological origin are present as a complex mixture, further to the alluded to selectivity achieved via reagent design, significant improvements of chromatographic peak capacity were additionally sought. Efforts toward this goal included the fabrication of an Extreme Ultra-Pressure Liquid Chromatography (XUPLC) system, which utilized long columns packed with sub-2µm particles (75 µm id x 0.5-2.0 m L; packed with 1.9 µm BEH C18 particles) necessitating operation at 30,000 psi. An overall goal was the integration of the results obtained from these two differing but complimentary research areas. The derivatization investigations revealed the derivatization reactions to be complete with 2 hours, with yields for Cs of no less than 70% when either reagent, BA or DPE, was utilized. Further reagent design and evaluation efforts were directed toward the characterization of BA and DPE that was elaborated to possess a sulfonic acid moiety, which provided for enhanced aqueous solubility and a selective sample preparation handle via strong anion exchange solid phase extraction. An alternate approach was the elaboration of each base regent to the dimethylamino-analog, again with the motivation being enhanced aqueous solubility but now potentially better electrospray ionization properties for mass spectrometry. Efforts in the XUPLC area revealed the expected gain in resolution and concurrent increased peak capacity. In summary, this dissertation describes advances in the utilization of existing reagents, the preparation and characterization of new reagent analogs and describes an advanced chromatographic system. All of these aspects are expected to be of high valuable in the analytical chemistry of catechol determinations and the related area of 3-nitrotyrosine residues present in peptides and proteins. | |
