Published on Web 04/06/2007
Experimental and Computational Investigation of
Unsymmetrical Cyanine Dyes: Understanding Torsionally
Responsive Fluorogenic Dyes
Gloria L. Silva, Volkan Ediz, David Yaron,* and Bruce A. Armitage*
Contribution from the Department of Chemistry, Carnegie Mellon UniVersity,
4400 Fifth AVenue, Pittsburgh, PennsylVania 15213
Received January 11, 2007; E-mail: yaron@cmu.edu; army@andrew.cmu.edu
Abstract: Unsymmetrical cyanine dyes are widely used in biomolecular detection due to their fluorogenic
behavior, whereby fluorescence quantum yields can be very low in fluid solution but are significantly
enhanced in conformationally restricted environments. Herein we describe a series of fluorinated analogues
of the dye thiazole orange that exhibit improved fluorescence quantum yields and photostabilities. In addition,
computational studies on these dyes revealed that twisting about the monomethine bridge beyond an
interplanar angle of 60° leads to a dark state that decays nonradiatively to the ground state, accounting for
the observed fluorogenic behavior. The effects of position and number of fluorine substituents correlate
with both observed quantum yield and calculated activation energy for twisting beyond this critical angle.
yields.5,6 In fluid solutions, these dyes typically exhibit φf <
Introduction
0.001, i.e., more than 100-fold lower than their symmetrical
counterparts. In contrast, viscous solvents such as glycerol
promote substantial (>102-fold) enhancements in φf for unsym-
metrical dyes. Thus, unsymmetrical cyanines can act as fluo-
rogenic sensors that report on the local viscosity of their
environment.
Thiazole orange and many closely related dyes are widely
used as stains for nucleic acids, allowing detection of DNA and
RNA in gels, flow cytometers, or microscopes.4 Most of these
dyes bind to nucleic acids by intercalating between base pairs6,7
(or presumably between individual bases in single-stranded
nucleic acids). Nucleobases are stacked on one or both faces of
the dye in these complexes, effectively raising the viscosity of
the dye’s local environment and leading to large increases in
fluorescence. The strong fluorescence of nucleic-acid-bound dye
and weak fluorescence of unbound dye result in effective
staining for imaging and detection.
In addition to their uses as soluble intercalating probes,
unsymmetrical cyanines have been conjugated to a variety of
molecules, including peptides,8 proteins,9 DNA,10 and DNA
analogues such as peptide nucleic acid (PNA).11 In these
contexts, the dyes often exhibit substantially greater fluorescence
than the free dye.11b Nevertheless, binding of the dye-conjugated
probe to a target molecule results in increased fluorescence, with
The cyanine class of organic dyes has been studied for over
150 years and continues to be the focus of considerable interest
in chemistry, biology, and biotechnology.1 Most research and
applications have involved symmetrical cyanine dyes, where
equal heterocycles are linked by a polymethine bridge. The
widely used fluorescent labels Cy3 and Cy5 are examples of
symmetrical cyanines, which typically exhibit large molar
extinction coefficients (ꢀmax > 105 M-1 cm-1) and at least
moderate fluorescence quantum yields (φf > 0.1). The develop-
ment of efficient strategies for synthesizing these dyes in forms
that allow facile conjugation to biomolecules2 has spurred their
use in a range of applications, including cell microscopy, flow
cytometry, “gene-chip” microarrays, and single molecule spec-
troscopy.
The unsymmetrical cyanine dyes3 do not enjoy as long a
history as their symmetrical counterparts, but they have been
used extensively in sensing applications, particularly in detection
of nucleic acids.4 This class of dyes features two different
heterocycles linked by a mono- or polymethine bridge. For
example, thiazole orange (TO, Chart 1) has benzothiazole and
quinoline heterocycles connected by a monomethine group.
Different heterocycles and bridge lengths allow tuning of the
excitation and emission wavelengths of these dyes throughout
the visible spectrum. A variety of substituents can be added to
the two heterocyclic nitrogens, allowing further diversification
of the dye structure.
(5) Rye, H. S.; Yue, S.; Wemmer, D. E.; Quesada, M. A.; Haugland, R. P.;
Mathies, R. A.; Glazer, A. N. Nucleic Acids Res. 1992, 20, 2803-2812.
(6) Netzel, T. L.; Nafisi, K.; Zhao, M.; Lenhard, J. R.; Johnson, I. J. Phys.
Chem. 1995, 99, 17936-17947.
(7) Nygren, J.; Svanvik, N.; Kubista, M. Biopolymers 1998, 46, 39-51.
(8) Carreon, J. R.; Mahon, K. P. J.; Kelley, S. O. Org. Lett. 2004, 6, 517-
519.
(9) Babendure, J.; Liddell, P. A.; Bash, R.; LoVullo, D.; Schiefer, T. K.;
Williams, M.; Daniel, D. C.; Thompson, M.; Taguchi, A. K. W.; Lohr, D.;
Woodbury, N. W. Anal. Biochem. 2003, 317, 1-11.
(10) Ishiguro, T.; Saitoh, J.; Yawata, H.; Otsuka, M.; Inoue, T.; Sugiura, Y.
Nucleic Acids Res. 1996, 24, 4992-4997.
The most useful property of unsymmetrical cyanine dyes is
the environmental sensitivity of their fluorescence quantum
(1) Mishra, A.; Behera, R. K.; Behera, P. K.; Mishra, B. K.; Behera, G. B.
Chem. ReV. 2000, 100, 1973-2011.
(2) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.; Waggoner,
A. S. Bioconjugate Chem. 1993, 4, 105-111.
(3) Armitage, B. A. Top. Curr. Chem. 2005, 253, 55-76.
(4) Lee, L. G.; Chen, C.; Liu, L. A. Cytometry 1986, 7, 508-517.
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J. AM. CHEM. SOC. 2007, 129, 5710-5718
10.1021/ja070025z CCC: $37.00 © 2007 American Chemical Society