Water Soluble 3H-Indocyanine Fluorescence Labeling Reagents For Biological Analysis

With the development of life science, there is a growing requirement for high sensitive detection technologies in analysis. Fluorescence label is a convenient method. Indocyanines with larger extinction coefficients (105 L/mol-cm), good fluorescent properties and fluorescence enhancement combining with biomass have been used as a new generation of fluorescent label compouds in biological analysis. However, photostability of these dyes has been a problem. In this paper the substituents on the nitrogen atoms in the heterocyclic rings of these indocyanines were designed as p-carboxybenzyl groups, and series novel straight chain polymethine indocyanines 1-3 were synthesized. Introduction of rigid rings into the methine chain resulted in novel 3H-indocyanines 4 and 5.They contained at least.one H-carboxybenzyl group on the nitrogen atoms in the heterocyclic rings. The sulfonate and carboxyl groups made them become water soluble, and the carboxyl group was employed as a linker for covalently linking to biomolecules in their biological applications.They have been synthesized by condensation of their intermediates(quaternary salt of indolenine) with corresponding condensating agents, respectively. The intermediates were prepared by the quaternization of 2,3,3-trimethylindoleninium-5-sulfonate with p-(chloromethyl)benzoic acid, 6-bromohexanoic acid or ethyl iodide. The 2,3,3-trimethyl-3H-indoleninium-5-sulfonate was from p-aminobenzoic acid. The dyes were separated, purified, and confirmed by 'HNMR and ESI-MS.The absorption and emission maxima of the novel dyes are between 540 nm and 850 nm,molar extinction is above 10~5 L/mol-cm. Compared to those of the corresponding straight pentamethine and heptamethine indocyanines, the absorption and emission maxima of these dyes become blue shift while the squaric ring was introduced into the methine chain, and red shift while the introduction of six-membered ring. The absorption and emission wavelength of these dyes were shifted with the changing of solvents. Within 4