Synthesis And Biological Application Research Of Near-Infrared Heptamethine Cyanine Fluorescence Dyes With Aminophenol Substitution At The Central Position

With the development of life science, there is a growing requirement for high sensitive detection technologies in analysis. Fluorescence label is a convenient method. Heptamethine cyanine dyes employed as fluorescence labels and sensors of biomolecules in vivo have attracted immense interest because their spectra reach the near-infrared (NIR) region, where a biological matrix exhibits the least absorption and autofluorescence background. Much research work has been carried out.A new heptamethine indocyanine dye with good photostability and water-solubility was synthesized in this thesis. Then some new derivative dyes were obtained by an apparent S_nr1 reaction at the central chlorine atom of the parent dye. A great improvement in dyes' Stokes shift (from 20nm to over 140nm) is the most important innovation to the NIR heptamethine cyanine dyes.Two benzyl groups were introduced to the two N atoms at the 3H-indo rings of the new heptamethine cyanine dye respectively. The new parent dye was found better photostability, which indicates that N-ethyl group can improve photostability for its rigid and bulk structure. In additional, it is found that dyes in water solutions are easier to photo-bleach than in alcohol solutions. A reasonable mechanism was proposed to explain the phenomenon. The reaction condition of the parent dye was improved by replacing benzene by toluene as the solvent. The parent dye was purified by recrystalling in the mixed solvent of ethanol and acetone easily for derivative reactions.Two derivative dyes from the parent dyes were obtained by nucleophilic substitution reaction in which two primary aminophenol were employed as the nucleophilic reagent. Some particular spectral properties were found in the new dyes. They were found to exhibit a large Stokes shift. The influences on the spectral properties was investigated in different solvents, viscosity, pH media and dye concentration.The absorption and emission spectra of the synthesized squaraine dyes were investigated. Influences of solvent, pH and dye concentration were discussed.The NHS-carboxyl squaraine was prepared from the esterification of N-hydroxysuccinimide with carboxyl squaraine, and the NHS-carboxyl squaraine was used to conjugate with benzylamine and BSA in a pH9.5 bicarbonate buffer, respectively. The conjugates were separated and detected by ESI-MS and HPLC, and the result indicated that the dye could couple covalently to biomass containing free NH₂ group.