Synthesis, Optical Properties And Biological Labeling Of Asymmetric Water-Soluble Indocyanine Dyes

Objective:Indocyanine dyes had many advantages, such as:wide spectral range (550nm-1200nm), high fluorescence quantum yield and high sensitivity detection etc. They are widely applied in fluorescent probes, protein labeling, DNA sequencing and cell staining etc now. We designed adding a non-ionic water-soluble lamb-like PEG ether chain in indol "N" bit to get new water-soluble asymmetric indolecyanine dyes that had better properties. Such as:higher yield, better water solubility and better spectral properties etc.Methods:We generally got indocyanine dyes through three steps, first:We synthesized the basic heterocyclic by the reaction of Fisher; what’s more:We synthesized the quaternary ammonium salt from a basic heterocyclic and a alkylating agent; last:We synthesized the target product from a condensing agent and two active methyl quaternaries. We synthesized a new water-soluble asymmetric indole pentamethine cyanine dye Cy5-671and a new water-soluble asymmetric indole cyanine dye Cy3-567from sulfonic acid phenyl hydrazine, methyl chloride and polyethylene three glycol etc; We synthesized a pentamethine indole cyanine dye Cy5-668from sulfonic acid phenyl hydrazine and bromomethylbenzoic acid etc; We tested optical properties of products with UV-Vis spectrophotometer and fluorescence spectrophotometer; We also marked bovine serum albumin or goat anti-mouse IGg with activated ester of dyes; While natures of Cy5-671were campared with Cy5-668’s.Results:We synthesized a dozen intermediate and eventually got three indocyanine dyes. Their structures were confirmed by’HNMR and MS. The maximum absorption wavelength, extinction coefficient, the maximum emission wavelength and the fluorescence quantum yield of Cy5-671were657nm,1.6×105/M-1cm-1,671nm and0.24respectively. The maximum absorption wavelength, extinction coefficient, the maximum emission wavelength and the fluorescence quantum yield of Cy5-668were650nm,1.4×105/M-1cm-1,668nm and0.10respectively. The maximum absorption wavelength, extinction coefficient, the maximum emission wavelength and the fluorescence quantum yield of Cy3-567were559nm,0.8×105/M-1cm-1,567nm and0.08respectively. light stability of Cy5-671was significantly higher than Cy5-668’s; D/P of Cy3-567,Cy5-671and Cy5-668were1.59,1.96and1.57respectively. Conclusions:The new water-soluble asymmetric indole pentamethine cyanine dyes Cy5-671had a non-ionic water-soluble lamb-like PEG ether chain in indol "N" bit. It had better water solubility, easy separation and purification, higher yield, better spectral properties and higher fluorescence quantum yield and a better D/P in favor of the practical application comparing with Cy5-668.