Characterization of a G-quartet-forming aptamer stationary phase for enantiomeric separations in capillary electrochromatography.

摘要

This dissertation describes the investigation of an aptamer stationary phase for enantioseparations of pharmaceutical compounds. Aptamers are oligonucleotides selected through combinatorial processes for high binding affinity towards a target compound. The DNA aptamer used in this research exhibits a sequence-specific G-quartet conformation that is proposed to demonstrate chiral selectivity towards non-target enantiomeric compounds. The G-quartet-forming aptamer was investigated as a chiral stationary phase (CSP) in open-tubular capillary electrochromatography (OTCEC) in which the aptamer was covalently attached to the inner surface of the capillary.; In order to maximize the surface area for aptamer attachment, the use of etched capillaries was investigated. The attachment of the aptamer to the inner capillary surface in open-tubular CEC requires a silanization step in which a solution of 3-aminopropyltriethoxysilane (APTES) was used. An investigation of aqueous and anhydrous APTES solutions in the silanization process was performed on capillary columns with unetched and etched inner surfaces. The etched columns showed no improvement in aptamer attachment or separations compared to unetched columns. The silanization using 10% aqueous APTES yielded the column with the highest efficiency and most promise for the separation of the β-blocker propranolol. The addition of 2-propanol in the mobile phase was found to enhance enantioseparations.; The porphyrin dye N-methylmesoporphynn IX (NMM) was investigated for use as an indicator of G-quartet formation on-column. The enhancement of NMM fluorescence in the presence of the G-quartet structure was confirmed by spectroscopic studies and applied to the detection of the G-quartet conformation on an aptamer-coated capillary column through laser-induced fluorescence (LIF) detection. A bare fused silica capillary and a capillary coated with a 15-mer that does not form a G-quartet were used as controls. The selective response of NMM fluorescence to the G-quartet conformation provides the first direct evidence of G-quartet formation in the aptamer stationary phase.; Chiral separation of the enantiomers of the drugs propranolol, ephedrine, chlorpheniramine, and brompheniramine was studied on an aptamer-coated column. Best results were obtained for propranolol. Partial separations of ephedrine and brompheniramine were also achieved.; A packing apparatus was developed for the fabrication of packed aptamer columns that could increase the stationary phase volume and enhance a nalyte-aptamer interaction, leading to improved chiral resolution.