Synthesis and Photophysical Characterization of Near-Ir Probes for Bioanalytical Applications.

Abstract

The research presented in this dissertation involves the synthesis and photophysical characterization of a series of tricarbocyanine dyes which can be used as fluorescent labels for bioanalytical applications. All of these dyes were synthesized to contain a heavy-atom modification to enhance k$\sb{\rm isc}$ through spin-orbit coupling and a functional group (isothiocyanate or succinimidyl ester) which is reactive towards primary amines. The spectroscopic characterization of the various chromophores was examined using absorbance and fluorescence measurements, time-correlated fluorescence, and flash photolysis. The dyes exhibit absorbance and fluorescence maxima in the near-IR (750-810 nm) with large $\varepsilon$ ($\sim$200,000 M$\sp{-1}$cm$\sp{-1})$ and $\Phi\sb{\rm f}\sim0.05$-0.15. Dyes which contain the heavy-atom modification possessed absorbance maxima which were similar (differences of only 1-2 nm) and similar fluorescence maxima (differences of only 1-2 nm), but have fluorescence lifetimes ($\tau\sb{\rm f})$ which were different and which were dependent upon the heavy-atom modification (I = 908 ps, F = 831 ps). The apparent inverse heavy-atom effect was studied by examining the triplet-state photophysics of these chromophores by laser-induced flash photolysis. Various photophysical constants were determined including the intersystem crossing rate (k$\sb{\rm isc}),$ internal conversion rate (k$\sb{\rm ic}),$ and $\Phi\sb{\rm t}.$ The results from these experiments indicate that k$\sb{\rm isc}$ is increasing with the heavier atom modification, but that k$\sb{\rm ic}$ decreasing possibly due to steric interactions of the heavy-atom modification with the chromophore restricting the vibrational interconversions of the dye. These dyes were examined as possible fluorescent labels for amino acid determination and DNA sequencing employing fluorescence detection. The experimental results with the capillary electrophoretic separation of dye-labeled amino acids indicated that in the presence of CH$\sb3$OH in the running buffer, the detection of the dye-labeled amino acids exhibited excellent signal-to-noise ratios corresponding to low mass detection limits for dye-labeled arginine (21 zmol). Additionally, these dyes were examined as possible dye labels for a Sanger DNA sequencing protocol employing near-IR fluorescence lifetime determination of dye-labeled dideoxynucleotides. Experimental results that these dyes efficiently react with an amine modified dideoxyguanosine triphosphate. The effect of the extension reaction conditions on the dye-labeled ddNTP was also examined.