Synthesis And Properties Of Water-soluble Squarylium Indocyanine Dyes

Fluorescence analysis technique can be fully applied in tissue cells with acceptable experiment results in the region of near-infrared. Cyanine dyes possess excellent spectral properties. Near-infrared cyanine dyes can be obtained by increasing length of conjugated methine chain in dye molecules. However, the photo-stability of cyanine dyes could be decreased when conjugated methine chain increases. The fluorescence probes in labeling the bio-substrates should be water-soluble because bio-substrates are dissolved in water and the organic solvents could be harmful to sensitive bio-substrates. In this paper, symmetrical and asymmetrical water-soluble squarylium indocyanine dyes were synthesized, the relationship between molecular structures and their spectral properties, especially in the aspect of the stability, was studied. Water-insoluble and chainlike pentamethine indocyanine dyes were synthesized for reference.In the process of synthesizing of asymmetrical water-soluble squarylium indocyanine dyes, it is found that the yields of solid synthesis method are 2 times of that of traditional two-step method. Solid synthesis method is more suitable to synthesize water-soluble asymmetric squarylium indocyanine dyes.The wavelengths of absorption and fluorescence emission maximum of the squarylium indocyanine dyes are from 628 to 672 nm. The Stokes shifts of the squarylium indocyanine dyes in the range of 10～17 nm are smaller than those of the chainlike pentamethine indocyanine dyes, which are in the range of 18～27 nm. Chainlike pentamethine indocyanine dyes display the absorption and fluorescence emission maximum from 646 to 687 nm. Water-insoluble symmetrical squarylium indocyanine dyes exhibit absorption and emission maximum from 621 to 658 nm. The Stokes shifts of these water-insoluble dyes with N-benzyl are in the range of 7～19 nm.All dyes synthesized in this paper show a high molar absorption coefficientsεat least 105 mol-1·cm-1·L and lower values of fluorescence quantum yields in the aqueous medium compared with that in organic solvents. The absorption spectra of these dyes exhibit negative solvatochromism in protonic solvents.The results of photo-stability tests demonstrate that photo-stability increases when shortening the length of alkyl group on N-position and introducing carboxyl groups on the alkyl group, squaric acid ring on conjugated methine chain or large benyzl ring in the N-substituents. Furthermore, the electron-withdrawing groups on the benzyl group improve the photo-stability compared with electron-donating groups. Photo-fading constant k of the squarylium indocyanine dye with N-carboxyl benzyl which is obtained with high purity only by recrystallizing is less than one-third of that of the squarylium indocyanine dyes with N-carboxyl pentyl that is commercially available. This lays the groundwork for subsequent bio-analytical applications of the dye with N-carboxyl benzyl.In cationic and non-ionic surface active agents, water-soluble squarylium indocyanine dyes exhibit a red shift on their absorption and emission maximum, a significant increase in their fluorescence yields, and an enhancement on photo-stabilities along with the formation of micelles especially to the dye with carboxyl. The interaction between non-ionic surfactant and the dyes is weaker than that between cationic surfactant and the dyes. Embeding model and adsorption model were presented in this paper to explain the behaviors of dyes in surface active agents.In aqueous solution with pH=2 and pH=12, absorption peak values of asymmetrical water-soluble sqarylium indocyanine dyes are reduced significantly. In aqueous solution with pH=4, aggregation of dyes molecules is apparent.The aggregation of water-insoluble symmetrical squarylium indocyanine dyes can be enhanced with the increasing of water content in DMF and DMSO-aqueous medium. Only the dye with carboxyl could be influenced by pH value and exhibit obvious aggregation in acidic medium. The photo-stabilities of water-insoluble dyes in DMF are better compared with that in methanol. By contrast, the water-insoluble squaraylium indocyanine dyes express better photo-stabilities than that of water-soluble squaraylium indocyanine dyes in DMF, and opposite in methanol.