Cyanine Dyes For Biological Fluorescent Labeling

In the post-genome sequencing era, protein analysis has been paid more and more attention. Fluorescence detection was readily applied in high performance liquid chromatography (HPLC) or electrophoresis techniques, especially the differential in-gel electrophoresis (DIGE), enabling qualitative and quantitative analysis of proteins. For proteomics, the improvement on the detection sensitivity of proteins is an imperative task. Cyanine dyes have found widespread use as fluorescent labels for biomolecules, particularly for fluorescent labeling proteins. However, two common problems encountered by cyanine dyes are their susceptibility to form nonfluorescent aggregates and their tendency to undergo photobleaching. In this thesis, cyanine dyes based on 2,3,3-trimethyl-3H-indolium are synthesized and their corresponding properties and biological applications are studied.In order to improve the protein labeling effect, a series of water-soluble trimethine 3H-indocyanine (Cy3) dyes were synthesized. The limit of detection of the asymmetric dye II e for BSA decreased to 4.8×10-10 mol L-1. Two methods, high performance liquid chromatography and gel electrophoresis (SDS-PAGE), were used to evaluate the influence of different dyes'structures on protein labeling. The results indicate the asymmetric dyes can benefit protein labeling, which possess the carboxyl group on the hydrophobic side of the dyes to synthesize the NHS ester and sulfo-groups on the other side to provide the dyes with necessary water-solubility.Four novel water-soluble asymmetric Cy5 dyes were synthesized because their spectra reach the near-infrared (NIR) region, where a biological matrix exhibits the least absorption and autofluorescence background. It is demonstrated that the benzyl group and sulfo-group on the N-position of 3H-indolium pentamethine cyanine dyes improved the photostability in aqueous solutions. In addition, the limit of detection of dye IIIj for BSA was 1.2×10-8 mol L-1 about 100-fold lower than that by UV detection. Compared to commercial fluorescein isothiocyanate (FITC), more proteins with low concentrations could be labeled, resulting in improved detection sensitivity for protein analysis. All results demonstrated that asymmetric dye IIIj possesses better photostability, which can be a good fluorescent labeling reagent for protein labeling.Focus on the poor photostability of pentamethine cyanine dyes, a series of water-soluble squarylium cyanine dyes were synthesized, including two novel asymmetric dyes. The result is shown that their photostabilities of squarylium cyanine dyes reported here are better than that of pentamethine cyanine dyes. The effect of different N-substituents on the photostability of the dyes can be placed in the order:p-carboxylbenzyl group> benzyl group> ethyl group > carboxylpentanyl group. Therefore, the benzyl group on the N-position of 3H-indolium pentamethine cyanine dyes improved the photostability in aqueous solutions, and reduced reactivity toward singlet oxygen, and the electron-withdrawing group on the benzyl group on nitrogen atom of cyanine dyes is good for improving photostability.Finally, focus on the problem of aggregation, a novel polyfluorinated cyanine dye was synthesized and its reaction conditions were optimized to imcrease the yields of intermediates and product. Compared with the nonfluorinated analogue, the novel dye exhibits significantly reduced aggregation in aqueous media, enhanced fluorescence quantum yield, greater resistance to photobleaching.