Novel Water-soluble Asymmetric Cy5 Dyes: Synthesis, Photo-stabilities and Fluorescent Labeling Protein

In the post-genome sequencing era,there has been more and more attention paid to protein analysis. Because of their large molar extinction coefficients,moderate-to-high fluorescence quantum yields and their spectra in the near-infrared( NIR) region,pentamethine cyanine( Cy5) dyes have received considerable attention and is considered as the main fluorescent labeling compounds for proteins. In order to improve detection sensitivity for proteins,a series of water-soluble asymmetric Cy5 dyes were synthesized and their spectral properties were tested in different solvents. The maximum absorption and emission wavelengths of these dyes in water are in the range from 647 nm to 665 nm. The fluorescence quantum yields( Φ) of these dyes are about 0. 1 in water. The photo-stabilities of these dyes in water were investigated as well. It is demonstrated that benzyl group and sulfo-group on N-position of cyanine dyes improved the photo-stability owing to their steric hindrance. The limit of detection( LOD) of succinimidyl ester of dye 4a( 4a-NHS) for BSA is 1. 2 × 10 -8 mol/L by HPLC with fluorescent director. Compared with UV detection,the sensitivity of detection for BSA is increased about 100 times by fluorescent labeling of dye 4a. Therefore,dye 4a could be used to improve potostability and detection sensitivity in protein analysis.