Synthesis, spectral properties, and detection limits of reactive squarainedyes, a new class of diode laser compatible fluorescent protein labels

We describe the synthesis and spectral characterization of two reactive lon g-wavelength fluorescence labels (Sq635-m and Sq635-b), having either one o r two N-hydroxysuccinimidyl esters. Both are squaraine derivatives and cons ist of a cyanine-type chromophore and a central squarate bridge. To improve water solubility, we introduced two sulfonic acid groups into the heterocy clic ring systems, and for covalent attachment to proteins, a reactive N-hy droxy-succinimide ester (NHS ester) was synthesized. The squaraine markers exhibit low quantum yields in water (phi = 0.15) and high quantum yields (p hi = 0.6-0.7) when bound to proteins. The absorption maxima at 635 nm in wa ter and at approximately 645 nm when bound to proteins allow excitation wit h commercially available diode lasers. The detection limit of a representat ive squaraine dye in blood was estimated to be half that of a commonly used fluorophore.