Structure property analysis of pentamethine indocyanine dyes: Identification of a new dye for life science applications

A collection of nine pentamethine indocyanine dyes was synthesized, and the photophysical characteristics relevant to applications in cell biology and single molecule detection were analyzed in detail. Substituents at the aromatic system covering the auxochromic series and substitutions in the polymethine chain were investigated with respect to absorption and emission spectra, fluorescence lifetimes, fluorescence quantum yields, and fluorescence autocorrelations. Substitutions in the polymethine chain increased the nonradiative energy dissipation of the excited singlet state and decreased the fluorescence quantum yield, relative to the unsubstituted compound. For substituents at the aromatic rings the fluorescence quantum yield negatively correlates with the position of the substituents in the auxochromic series -SO3-, -H, -F, -CH3. Compounds with sulfonic acid groups or halogen atoms attached to the indolenine systems had the highest fluorescence quantum yields. The compound S0387 had nearly 70% of the quantum yield of Cy5 and comparable photostability. The free carboxylic acid of S0387 was attached to peptides in high yield and purity by established procedures of solid-phase synthesis. The dye-labeled peptides did not aggregate or bind to tissue culture cells and proteins unspecifically. The indocyanine dye S0387 is therefore an attractive new fluorophore for in vitro and cell-based detection of receptor ligand interaction at nanomolar concentrations by flow cytometry, fluorescence correlation spectroscopy, and laser scanning microscopy.