Analytica Chimica Acta 570 (2006) 214–223 Synthesis of water-soluble, ring-substituted squaraine dyes and their evaluation as fluorescent probes and labels Anatoliy L. Tatarets a , Irina A. Fedyunyayeva a , Tatyana S. Dyubko a , Yevgeniy A. Povrozin a , Andrey O. Doroshenko b , Ewald A. Terpetschnig c,d,∗ , Leonid D. Patsenker a,c a SSI “Institute for Single Crystals” of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001, Ukraine b Institute of Chemistry, V.N. Karazin National University, 4 Svobody Sq., Kharkov 61077, Ukraine c SETA BioMedicals, LLC, 2014 Silver Ct East, Urbana, IL 61801, United States d ISS, Inc., 1602 Newton Drive, Champaign, IL 61822, United States Received 15 December 2005; received in revised form 30 March 2006; accepted 7 April 2006 Available online 25 April 2006 Abstract A series of ring-substituted squaraines absorbing and emitting in the red and NIR spectral region was synthesized and their spectral and photophysical properties (quantum yields, fluorescence lifetimes) and photostabilities were measured and compared to Cy5, a commonly used fluorescent label. The absorption maxima in aqueous media were found to be between 628 and 667 nm and the emission maxima are between 642 and 685 nm. Squaraine dyes exhibit high extinction coefficients (163,000–265,000 M -1 cm -1 ) and lower quantum yields (2–7%) in aqueous buffer but high quantum yields (up to 45%) and long fluorescence lifetimes (up to 3.3 ns) in presence of BSA. Dicyanomethylene- and thio-substituted squaraines exhibit an additional absorption around 400 nm with extinction coefficients between 21,500 and 44,500 M -1 cm -1 . These dyes are excitable not only with red but also with blue diode lasers or light emitting diodes. Due to the favourable spectral and photophysical properties these dyes can be used as fluorescent probes and labels for intensity- and fluorescence lifetime-based biomedical applications. © 2006 Elsevier B.V. All rights reserved. Keywords: Probes; Labels; Squaraines; Fluorescence assay; Fluorescence lifetimes 1. Introduction Squaraines, a subclass of polymethine dyes, are among the promising classes of dyes that can be utilized to synthesize probes and labels for the red and NIR spectral region [1–10]. There are numerous publications on the synthesis of conven- tional squaraine dyes [1–5], but only a few describe the synthesis of mono-squaraine intermediates [3,5] and even less describe squaraine dyes with a derivatized central squaraine moiety [11,12]. Squaraine dyes also show potential as tracers in fluorescence lifetime (FLT) based applications. Surprisingly fluorescence lifetime is a parameter that has only been moderately explored for use in fluorescent assays, which is probably due to the lack of lifetime data that is available on various classes of fluorescent compounds and the limited number of long-wavelength fluo- ∗ Corresponding author. Tel.: +1 217 417 2160. E-mail address: ewaldte@juno.com (E.A. Terpetschnig). rophores that exhibit distinct lifetime changes upon interaction of the fluorescently labelled binding partner with its counterpart. In high throughput screening (HTS), where the assay robustness is of utmost importance, fluorescence lifetime promises great potential in helping to reduce the number of undesired, false results (both false positives and false negatives). Assay robust- ness can be defined as how a signal readout parameter tolerates assay interferences in a real screening experiment. Some of the key assay interferences are photo-bleaching, inner filter effects, quenching, light scattering and auto-fluorescence. Except for photobleaching all interferences either originate from the bio- logical sample or the compound libraries. FLT is ranked as the most robust homogeneous assay format. Fluorescence lifetime is unaffected by inner filter effect (including background absorption), light scattering and auto- fluorescence [13], all key interferences that can be caused by problem compound libraries. In this paper we are reporting on a new class of squaraine dyes – water-soluble, ring-substituted squaraines – investigate 0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.04.019