Cyanine dyes are widely used as fluorescent labels in many biomedical applications, such as cancer imaging, enzyme assay, nucleic acid detection, and biomolecular labeling. This is due to their excellent spectral properties, including narrow absorption bands, large molar absorption coefficients and an intense π-π* absorption which can be easily tuned by structural modifications in the chromophore moiety. Among these dyes, pentamethine cyanine (Cy5) fluorophores which emit in the NIR region can be used to effectively reduce the background signal arising from auto-fluorescence of the biological matrix and light scattering. For this reason, pentamethine dyes have been employed as donor/acceptor groups in FRET-based fluorescent probe to study biological processes in vitro and in vivo.

Having these facts in mind and considering our interest in the synthesis of FRET-labelled peptide substrates for in vitro validation of a nanoconstructed drug delivery system, we report herein the synthesis and characterization of a pentamethine cyanine dye. Its structure contains two sulfonic acid groups to increase its aqueous solubility and a carboxylic acid group, which can be activated to form succinimidyl ester and thus be conjugated to a specific peptide. The pentamethine dye preparation was performed by traditional solution phase chemistry and the desired compound was characterized by NMR, UV/Vis absorption and fluorescence spectroscopy.