Experimental and computational investigation of unsymmetrical cyanine dyes: Understanding torsionally responsive fluorogenic dyes

Article abstract of DOI:10.1021/ja070025z

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. Herei

Full text of DOI:10.1021/ja070025z

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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5710
J. AM. CHEM. SOC. 2007, 129, 5710-5718
10.1021/ja070025z CCC: $37.00 © 2007 American Chemical Society

Understanding Torsionally Responsive Fluorogenic Dyes
A R T I C L E S
Chart 1. Fluorinated and Nonfluorinated Cyanine Dyesa
a All dyes are iodide salts, except for S5-F8, for which the counterion is ethyl sulfate.
detection sensitivity often extending into the nanomolar con-
centration range.
allow us to study the effects of fluorine substitution on the
energetics of this twisting pathway and help explain the observed
substituent effects on the fluorescence quantum yield.
We are interested in using fluorogenic cyanine dyes as
components in DNA- and RNA-based biosensors. The goal of
the research described here was to synthesize and characterize
a series of TO analogues that have electron-withdrawing fluoro
or trifluoromethyl groups replacing hydrogen atoms on the
heterocycles. Fluorination of a symmetrical cyanine dye was
previously shown to improve the fluorescence properties of the
dye,12 and similar effects were expected for the unsymmetrical
dyes. While this was observed in some of the dyes, interesting
spectral shifts prompted us to perform calculations on the dyes.
The calculations show that the shifts may be attributed to the
effects of fluorine substitution on the frontier molecular orbitals.
The calculations also explore the origin of the observed effects
of the dyes’ local environment on the quantum yield. Fluores-
cence quenching in TO has been attributed to intramolecular
twisting in the excited state.13 This is consistent with the increase
in fluorescence quantum yield observed in environments that
rigidify the structure, such as in viscous solvents or upon binding
to biomolecules. Ab initio calculations, performed on a small
model cyanine, also implicate intramolecular twisting of the dye
as the dominant quenching mechanism in unconstrained envi-
ronments.14 Here, we use semiempirical methods to allow
computations on the full dye molecules. These semiempirical
calculations are consistent with the ab initio calculations, in that
they also find that intramolecular twisting is an efficient
quenching pathway. In addition, the semiempirical calculations
Results
Our previous work with symmetrical cyanine dyes such as
S5 and S5-F8 (Chart 1) demonstrated that fluorination of the
heterocycles can decrease aggregation and improve photosta-
bility,12 important parameters for fluorescence labels used in
bulk imaging and single molecule measurements. A series of
thiazole orange (TO) analogues bearing fluorine substituents
on the benzothiazole heterocycle or a trifluoromethyl group on
the quinoline heterocycle (Chart 1) was synthesized to inves-
tigate the effect of fluorination on unsymmetrical cyanines.
Spectroscopy. UV-vis absorption spectra were recorded for
each dye in methanol as well as in the presence of a large excess
of double-stranded DNA, where the dye is expected to be
intercalated (Figure 1). The absorption maxima for all five dyes
shift to the red upon binding to DNA, indicative of the lower
dielectric environment of the stacked base pairs (Table 1). In
addition, a distinct trend in the maxima is observed in both
cases: fluorination of the benzothiazole leads to a progressive
blue shift of the absorption band, whereas addition of a
trifluoromethyl group to the quinoline side of the dye causes a
red shift. A similar trend is observed in the emission data. (The
low fluorescence of the dyes in methanol necessitated the use
of 90% glycerol in water to observe fluorescence in the absence
of DNA.)
The electronic spectra of thiazole orange and its fluorinated
derivatives were modeled to help explain the trends described
above. Molecular structures were obtained using the SAM115
Hamiltonian in the AMPAC16 quantum chemistry package. The
ground electronic state was optimized using restricted Hartree-
(11) (a) Svanvik, N.; Westman, G.; Wang, D.; Kubista, M. Anal. Biochem. 2000,
281, 26-35. (b) Svanvik, N.; Nygren, J.; Westman, G.; Kubista, M. J.
Am. Chem. Soc. 2001, 123, 803-809.
(12) Renikuntla, B. R.; Rose, H. C.; Eldo, J.; Waggoner, A. S.; Armitage, B.
A. Org. Lett. 2004, 6, 909-912.
(13) Karunakaran, V.; Lustres, J. L. P.; Zhao, L.; Ernstring, N. P.; Seitz, O. J.
Am. Chem. Soc. 2006, 128, 2954-2962.
(14) (a) Hunt, P. A.; Robb, M. A. J. Am. Chem. Soc. 2005, 127, 5720-5726.
(b) Sanchez-Galvez, A.; Hunt, P.; Robb, M. A.; Olivucci, M.; Vreven, T.;
Schlegel, H. B. J. Am. Chem. Soc. 2000, 122, 2911-2924.
(15) Dewar, M. J. S.; Jie, C.; Yu, J. Tetrahedron 1993, 49, 5003-5038.
(16) AMPAC, 8, 1992-1994 Semichem, Inc. PO Box 1649 Shawnee, KS 66222.
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A R T I C L E S
Silva et al.
Table 2. Results from Modeling Electronic Spectra of TO and
Fluorinated Analogues
exptl
max (nm)
calcd
max(abs) (nm)
λ
max(Fl)
INDO ground
state (eV)
λ
λ
(nm)
h
dye
absa
Flb
SAM1c
INDOd
INDOe
HOMOf
LUMOg
∆
TO
TO-1F
511 528 476.0 476.5 489.0 -10.30 -4.47 5.83
509 529 476.1 471.4 483.3 -10.44 -4.57 5.87
TO-p2F 496 525 472.2 468.3 479.5 -10.51 -4.60 5.91
TO-4F
TO-CF3 527 549 479.1 482.5 496.4 -10.47 -4.69 5.78
492 523 473.2 463.0 475.7 -10.72 -4.79 5.93
b
a Experimental absorption maximum in DNA. Experimental emission
maximum in DNA. cExcitation energy from SAM1 for ground-state
d
optimized structure. Excitation energy from direct INDO/SCI for SAM1
ground-state optimized structure. eExcitation energy from direct INDO/SCI
f
for SAM1 excited-state optimized planar structure. INDO HOMO energy
for SAM1 ground-state optimized structure. gINDO LUMO energy for
SAM1 ground-state optimized structure. hHOMO-LUMO splitting from
INDO for ground-state optimized structure.
Figure 2. Correlation of predicted λmax with experimental λmax, in nm.
The dashed line is for absorption, and the solid line is for emission.
Figure 1. UV-vis absorption spectra of TO and fluorinated analogues
determined in methanol (top) and in the presence of double-stranded DNA
(bottom). Each spectrum was normalized at its wavelength of maximum
absorbance.
however INDO is only useful after the molecular geometry is
known. We therefore adopted a hybrid method in which
geometries are obtained from SAM1, while excitation energies
are obtained from a direct INDO/SCI18 method that includes
excitations between all molecular orbitals. The results in Table
2 and Figure 2 show that this hybrid method leads to good
correlation between experiment and theory for both absorption
(INDO excitation energy calculated at the SAM1 ground-state
geometry) and fluorescence (INDO excitation energy calculation
at the SAM1 excited-state geometry). This strong correlation
between experiment and theory indicates that the calculations
are useful for understanding the sign and relatiVe magnitude of
the effects of fluorine substitution. However, the absolute
magnitudes of the calculated substituent effects are not as
reliable, since a fit of experiment to theory yields a slope of
1.79 for absorption and 1.15 for emission. (The slope should
be 1.0 for perfect correlation.)
Table 1. Absorption and Emission Wavelengths (in nm) of
Thiazole Orange (TO) and Fluorinated Analogues
absorption
in MeOH
absorption
in DNA
emission in
emission in
DNA
dye
90% glycerol
TO
502
500
489
485
516
511
509
496
492
527
547
544
534
530
571
528
529
525
523
549
TO-1F
TO-p2F
TO-4F
TO-CF3
Fock (RHF) theory. The excited state was optimized using single
configuration interaction (SCI) theory including all excitations
between the 50 highest occupied molecular orbitals and 50
lowest unoccupied orbitals. While the optimized ground state
is planar, the excited state is highly twisted (as discussed further
below). To obtain an estimate of the Stokes’ shift in an
environment such as DNA that may prevent twisting, the
excited-state geometries were also optimized under the constraint
that the interplanar angle between the quinoline and benzothia-
zole groups is 0°. These planar excited-state geometries are used
below when making spectral predictions for dyes bound to DNA.
The vertical excitation energies predicted by SAM1 (Table
2) do not correlate well with experiment. More reliable
predictions of the excitation energy can be obtained from
Intermediate Neglect of Differential Overlap (INDO) theory;17
We next consider whether the substituent effects on the
excitation energy can be understood in terms of the HOMO-
LUMO gap. Table 2 lists the HOMO and LUMO energies and
the HOMO-LUMO gap obtained for the ground state geom-
etries. In Figure 3 the HOMO-LUMO gap is shown to be well
correlated with the predicted λmax. This correlation implies that
the observed spectral shifts can be understood in terms of the
effects of the electron-withdrawing substituents on the HOMO
(17) Ridley, J.; Zerner, M. C. Theor. Chim. Acta 1973, 32, 111-134.
(18) Tomlinson, A.; Yaron, D. J. Comput. Chem. 2003, 24, 1782-1788.
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Understanding Torsionally Responsive Fluorogenic Dyes
A R T I C L E S
Figure 3. Correlation of the HOMO-LUMO splitting with predicted
absorption λmax
.
Figure 5. Fluorescence spectra recorded for 1.0 µM TO-p2F in methanol
(dotted), 90% glycerol-water (dashed), and double-stranded calf thymus
(CT) DNA (solid). Spectra were corrected for differences in absorbance at
the excitation wavelength (450 nm).
Table 3. Fluorescence Quantum Yields (φf) Recorded in 90%
Glycerol and DNA
dye
φf (90% glycerol)
φf (DNA)
TO
0.027
0.029
0.038
0.051
0.017
0.110
0.091
0.092
0.040
0.030
TO-1F
TO-p2F
TO-4F
TO-CF3
Fluorogenicity. The effect of viscous solvent or double-
stranded DNA on dye fluorescence is shown in Figure 5 for
TO-p2F. Table 3 lists the fluorescence quantum yields for the
dyes in viscous solution and DNA. In solution, the quantum
yields are found to increase with fluorination of the benzothia-
zole but decrease when a trifluoromethyl group is present on
the quinoline. In contrast, the quantum yield decreases for all
fluorinated derivatives when bound to DNA. Repeating the
experiment at higher DNA concentration does not increase the
quantum yields, indicating that the trend shown in Table 3 is
not due to weaker binding of the fluorinated dyes to DNA.
Figure 4. HOMO and LUMO of TO obtained from SAM1 computations
on the ground-state structure. The molecules are drawn oriented as shown
in Chart 1.
The fluorescence enhancement exhibited by TO and its
analogues in viscous solution or DNA is due at least in part to
restriction of conformational freedom in the monomethine
bridge. As stated above, the excited-state geometries of these
dyes are highly twisted; attaining such a conformation within
the excited-state lifetime is hindered by viscous solvent or the
geometrical constraints imposed by intercalation into DNA. This
prompted additional calculations to better understand the
twisting of the dyes in the excited state.
and LUMO orbitals, which are plotted in Figure 4 for the
unsubstituted dye TO in a planar geometry. Inductive effects
from fluorine substitution may be expected to stabilize a
molecular orbital, with the decrease in energy being greater for
orbitals that have large populations on the site of substitution.
The HOMO is spread evenly between the benzothiazole and
quinoline ring systems, while the LUMO is mostly on the
quinoline. Inductive effects from fluorine substitution on the
benzothiazole thereby lower the HOMO energy more than the
LUMO energy. This leads to an increase in the HOMO-LUMO
gap, in agreement with the hypsochromic shifts seen experi-
mentally. On the other hand, inductive effects from CF3
substitution on the quinoline ring system lower the LUMO more
than the HOMO. This leads to a decrease in the HOMO-LUMO
gap, in agreement with the bathochromic shift seen experimen-
tally.
Figure 6 defines two interplanar angles φ1 and φ2 that we
will use to characterize the twisting motion. Twisting about φ1
was examined first (this choice will be justified below). Since
we do not currently have the ability to perform geometry
optimizations within our hybrid computational method, the
structures were obtained by optimizing the ground electronic
state using SAM1 theory with a constraint applied to φ1. The
INDO excitation energies and transition intensities are shown
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A R T I C L E S
Silva et al.
Figure 6. Definition of interplanar angles, φ1 and φ2, used to describe
twisting of TO.
Figure 7. Singlet excitation energies of TO as a function of the interplanar
angle φ1, holding φ2 fixed at 0° (angles defined in Figure 6). The radius of
the circles is proportional to the predicted absorption intensity. The excitation
energies for the S0-S1 transition are shown in blue, while energies for
transitions to higher states are shown in purple (S0-S2), violet (S0-S3),
magenta (S0-S4), and red (S0-S5).
Figure 8. HOMO and LUMO of TO obtained from SAM1 computations
on the 90° twisted structure. The molecules are drawn oriented as shown
in Chart 1.
interactions are taken into account, the excitation energy
associated with a HOMO-LUMO promotion, Eexc, is
as a function of φ1 in Figure 7 for the unsubstituted dye TO.
The lowest singlet excitation (blue circles) drops rapidly above
ca. 60° and becomes dark when fully twisted, with the optical
intensity shifting to higher energy excited states. This behavior,
which is also predicted for the substituted dyes, rationalizes the
low quantum yield of these dyes observed in solution. It also
helps rationalize the higher quantum yields observed in DNA,
since insertion into DNA likely hinders this geometric relaxation
such that the quantum yield is established by some other
mechanism. It is for this reason that, in the above prediction of
the Stokes’ shift in DNA, a planar constraint was applied while
optimizing the excited-state geometry.
Eexc ) ∆E0 - J + 2K
(1)
where ∆E0 is the difference of the HOMO and LUMO orbital
energies, J is the Coulomb repulsion integral, and K is the
exchange integral, defined as
Ji,j ) (ii|jj)
Ki,j ) (ij|ji)
(2)
(3)
using chemist’s notation.19 The contribution of the various terms
to the excitation energy of eq 1 is shown as a function of the
interplanar angle in Figure 9. The HOMO-LUMO gap does
decrease between 0° and 90°, but this accounts for only about
20% of the total drop in excitation energy. Since J has nearly
the same value at 0° and 90°, it does not contribute to the
difference in excitation energy between the planar and twisted
structures. (J does however vary at intermediate angles, in a
manner that decreases the barrier between the planar and twisted
conformations.) The drop in excitation energy at φ1 > 60° arises
primarily from K going to zero in the twisted structure. This is
related to the HOMO and LUMO being localized on different
ring systems, since the exchange interaction is small between
orbitals that occupy different spatial regions.
The drop in the excitation energy as the interplanar angle φ1
exceeds 60° is correlated with a loss of conjugation between
the benzothiazole and quinoline rings. This is reflected in the
bond orders on the methine bridge. The bond order for the
“single” bond (i.e., the bond connecting the bridge to the
benzothiazole in the resonance form shown in Chart 1) drops
from 1.33 at the planar structure to 0.97 at 90°, while the bond
order for the central “double bond” increases from 1.26 to 1.55.
The loss in conjugation is also reflected in the molecular orbitals.
In the planar geometry, both the HOMO and LUMO are
delocalized over the entire dye (Figure 4). However, in the
twisted geometry, the HOMO becomes localized to the quinoline
ring, and the LUMO becomes localized to the benzothiazole
ring (Figure 8).
We next consider the effects of fluorine substitution on the
above twisting mechanism. Figure 10 shows the excited-state
energy surfaces as a function of φ1 for the series of substituted
dyes. The excited-state surface is obtained as the sum of the
SAM1 ground-state energy and the INDO excitation energy.
The drop in excitation energy at 90° can be related to the
localization of these frontier orbitals. This requires, however,
going beyond just the orbital energies. When electron-electron
(19) Szabo, A.; Ostlund, N. S. Modern Quantum Chemistry: Introduction to
AdVanced Electronic Structure Theory; Dover Publications: 1996.
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Understanding Torsionally Responsive Fluorogenic Dyes
A R T I C L E S
to a maximum at about 60°. Above 60°, the sharp decrease in
excitation energy (Figure 7) dominates and the excited-state
surface decreases rapidly to a global, twisted minimum.
Above, the excited-state surfaces were obtained by optimizing
the ground state surface with constraints applied to φ1. In the
resulting structures, φ2 adopts a value very near 0°. However,
it is possible that the minimum energy path for twisting of the
dyes in the excited state involves some twisting about φ2 as
well. To explore this, Figure 11 shows a contour plot of the
excited-state potential energy surface of TO as a function of
both φ1 and φ2. The results show that the reaction path is
primarily along φ1, with similar results being obtained for the
substituted dyes. This supports the above use of φ1 to map out
the excited-state potential energy surfaces.
To further explore the nature of the twisting motion, we used
SAM1 to map out the reaction path between the planar and
twisted structures in the excited state. This was done using the
chain algorithm in AMPAC with the SAM1 Hamiltonian and
the SCI method including all excitations between the 50 highest
occupied molecular orbitals and 50 lowest unoccupied orbitals.
Table 4 shows the interplanar angles φ1 and φ2 at the predicted
local minima and transition states. For all dyes, the results
support the above assumption that the reaction path involves
mostly φ1. The local minima occur at φ1 of about 20° and φ2
near 0°. The transition states occur at intermediate values of φ1
with φ2 close to 0° (except for TO-CF3 where the local
minimum and transition state differ by only about 10° in φ1).
The predictions regarding the effects of fluorine substitution
on the activation barriers do not agree with those obtained above
and so are not in agreement with the experimental quantum
yields seen in solution. This is not too surprising given that, as
discussed above, SAM1/SCI does not correctly reproduce the
effects of fluorine substitution on the absorption maxima. We
therefore take these SAM1/SCI results as giving general support
to the assumption that the reaction coordinate for twisting is φ1
and that there is an activated crossing to a twisted structure.
They also confirm that SAM1/SCI does not give reliable results
for the effects of fluorine substitution on these dyes.
Willets et al.20 attributed the fluorogenic behavior of DCDHF
fluorophores (Scheme 1) to excited-state twisting motions that
have some similarities to that implicated here for TO. In both
DCDHF and TO, the excited-state surface exhibits a planar
minimum and a twisted minimum, with the twisted structure
decaying nonradiatively. However, in DCDHF, strong couplings
between the various interplanar angles (R, φ, and δ in Scheme
1) lead to a more complex situation than the essentially one-
dimensional mechanism put forward here for TO. In DCDHF,
the ground-state structure has (R,φ,δ) ) (1°,-39°,0°), and the
potential along φ is sufficiently weak that the room-temperature
population at φ ) -90° is about 10%. Excitation is then to a
Franck-Condon region near (1°,-39°,0°) which relaxes either
to a fluorescent planar configuration at (0°,-9°,1°) or to a
nonradiative twisted configuration at (0°,-42°,88°). The ex-
perimentally observed environmental sensitivity of the fluores-
cence properties of DCDHF is attributed to the effects of the
environment on this branching ratio.
Figure 9. Individual contributions of energy terms given in eq 1 to the
excited-state energy of TO as a function of the interplanar angle φ1, holding
φ2 fixed at 0°.
Figure 10. Excited-state energy surfaces as a function of φ1 for all dyes.
For clarity, the energies are referenced against the excited-state energy of
the planar structure, which is set to zero. Inset: expanded view of region
between 50 and 70°.
The potential surfaces have local minima at about 20° and show
activated crossing to a twisted structure. Similar barriers have
been observed computationally in trimethine cyanine, a small
molecule used as a computational model of symmetrical cyanine
dyes.14 The substituent effects on the activation barrier are
consistent with the trend in quantum yields measured in 90%
glycerol: the barrier increases with fluorination of the ben-
zothiazole heterocycle but decreases for the trifluoromethyl-
substituted dye. This indicates that activated crossing of this
barrier establishes the quantum yield.
The origin of the barrier in Figure 10 can be understood in
terms of the relative importance of the ground-state energy,
which increases as the molecule deviates from planarity, versus
the excitation energy, which decreases as the molecule deviates
from planarity. Near φ1 ) 0°, the excitation energy decreases
more rapidly than the ground-state energy increases, such that
the excited-state energy initially decreases with φ1. The increase
in the ground-state energy with φ1 dominates between 20° and
60°, leading to a gradual rise from a local minimum near 20°
Effect of Fluorination on Dye Aggregation and Photosta-
bility. As noted earlier, fluorination of a symmetrical cyanine
(20) (a) Willets, K. A.; Callis, P. R.; Moerner, W. E. J. Phys. Chem. B 2004,
108, 10465-10473. (b) Willets, K. A.; Ostroverkhova, O.; He, M.; Twieg,
R. J.; Moerner, W. E. J. Am. Chem. Soc. 2003, 125, 1174-1175.
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A R T I C L E S
Silva et al.
Figure 11. Contour plot of the excited-state potential energy surface of TO as a function of φ1 and φ2, obtained from the hybrid method. The dotted
contours are from 2.60 to 2.65 eV in steps of 0.005 eV. The solid contours begin at 2.65 eV with increments of 0.1 eV. The bold dotted line shows the lowest
energy path for the twisting motion. This path has a local maximum at (φ1, φ2) ) (0°,0°). Shallow local minima at about φ1 ) (20° are separated from deep
minima at φ1 ) (90° by a barrier at (60° (compare to Figure 10).
Table 4. Results from SAM1/SCI Calculations on the Reaction
Path between Planar and Twisted Structures in the Lowest Singlet
Excited Statea
c
d
dye
φf b
I (φ1
,
φ2)
TS (φ1
,
φ2
)
∆
E (eV)e
TO
0.027
0.029
0.038
0.051
0.017
(16°, -4°)
(19°, 3°)
(13°, 14°)
(7°, 6°)
(65°, 4°)
(53°, 3°)
(70°, 1°)
(49°, 0°)
(72°, 1°)
0.160
0.073
0.027
0.027
0.063
TO-1F
TO-p2F
TO-4F
TO-CF3
(64°, 3°)
a Shown are the experimental quantum yields, the interplanar angles at
both the nearly planar intermediate (I) and the transition state (TS), along
with the activation barrier. bExperimental quantum yield φf (90% glycerol).
cφ1 and φ2 at the local minima. dφ1 and φ2 at the transition state. eThe
activation energy for transition from the intermediate to the transition state.
Scheme 1
Figure 12. Plot of maximum UV-vis absorbance versus dye concentration
for TO and fluorinated analogues.
for the di- and tetrafluorinated dyes, demonstrating effective
suppression of aggregation by fluorination of the benzothiazole
side of the dye.
The use of unsymmetrical cyanine dyes as fluorescent stains
for nucleic acid detection requires that the dyes be photostable.21
To assess the photostability of the new fluorinated TO deriva-
tives, a photobleaching experiment for the dyes was performed
as described in the Supporting Information. The photostability
of 5 µM dyes was first tested in buffer solution containing 100
mM sodium chloride. Thiazole orange showed a pronounced
dye led to decreased dye aggregation and enhanced photosta-
bility.12 The effects of fluorination on these properties for the
unsymmetrical cyanine dyes are now presented.
Figure 12 illustrates the dependence of the visible absorbance
maximum on dye concentration. The slight downward curvature
evident in the data for TO, TO-1F, and TO-CF3 is characteristic
of dye aggregation. In contrast, the absorbance increases linearly
(21) Kanony, C.; A° kerman, B.; Tuite, E. J. Am. Chem. Soc. 2001, 123, 7985-
7995.
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5716 J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007

Understanding Torsionally Responsive Fluorogenic Dyes
A R T I C L E S
Table 6. DNA Duplex Stabilization by TO and Fluorinated
Analogues
dye
∆Tm (°C)
TO
22
22
21
15
14
TO-1F
TO-p2F
TO-4F
TO-CF3
DNA Binding. Finally, the effect of fluorination on DNA
binding behavior was studied by thermal denaturation experi-
ments. Dyes that bind with higher affinity to double-stranded
versus single-stranded DNA induce a stabilization of the former,
leading to an elevated melting temperature (Tm). For example,
[Poly(dA-dT)2], a double-helical DNA polymer consisting of
alternating A-T base pairs dissociates into single strands at Tm
) 42.4 °C under the conditions described in the Supporting
Information, but in the presence of TO at a ratio of one TO
per 2 DNA base pairs, the melting temperature increases by 22
°C. Repeating the experiment with the fluorinated dyes gives
the results shown in Table 6. All five of the TO analogues
significantly stabilize the DNA, with the mono- and difluorinated
dyes exhibiting similar Tm stabilizations as TO, while slightly
lower values are observed for TO-4F and TO-CF3.
Discussion
The effect of fluorination on the properties of the fluorogenic
dye thiazole orange was studied. Replacement of the benzothia-
zole heterocycle with mono-, para-di-, or tetrafluorobenzothia-
zole led to progressive reduction in aggregation and improve-
ment in photostability. These results are consistent with what
was observed previously for an octafluorinated analogue of the
symmetrical cyanine dye S5 (Chart 1). The enhanced photo-
stability of the fluorinated dyes could be due to lower efficiency
of singlet oxygen production. In addition, the fluorinated S5
dye exhibited significantly lower reactivity toward singlet
oxygen relative to the nonfluorinated dye,12 and a similar effect
could be operative for the unsymmetrical dyes. Moreover, these
substitutions did not substantially impact the DNA-binding
ability of the dyes, based on thermal denaturation experiments
with [Poly(dA-dT)]2 (Table 6), although the tetrafluorinated dye
TO-4F offered slightly less stabilization of the DNA duplex.
In terms of aggregation and photostability, substitution of a
trifluoromethyl group on the quinoline heterocycle (TO-CF3)
is comparable to monofluorination of the benzothiazole hetero-
cycle (TO-1F), although TO-CF3 offers less stabilization of
the [Poly(dA-dT)]2 duplex. Interestingly, TO-CF3 and TO-4F
exhibit not only less stabilization of [Poly(dA-dT)]2 but also
the lowest fluorescent quantum yields when bound to DNA.
One possible explanation for these results is that these two dyes
intercalate into the DNA helix in geometries that are less
restrictive to twisting in the excited state. For example, insertion
of only one of the heterocycles into the helix, while leaving
the other projecting into one of the grooves could lead to
fluorescence enhancements and duplex stabilizations that are
weaker than those for dyes that intercalate with both hetero-
cycles. A “half-intercalation” binding mode for other unsym-
metrical cyanines was also proposed previously by Yarmoluk
and co-workers,22 although verification requires high-resolution
structural studies.
Figure 13. Absorbance of 5.0 µM dye solutions irradiated with visible
light for 1 min time intervals in either aqueous buffer (A) or 100 µM base
pairs CT-DNA (B).
Table 5. Photobleaching of Cyanine Dyes in Aqueous Buffer and
in CT-DNA Solutions
dye
buffera
CT-DNAa
TO
55
48
22
12
43
29
26
16
08
26
TO-1F
TO-p2F
TO-4F
TO-CF3
a After 10 1-min irradiation intervals.
drop of its absorbance when it was irradiated with visible light
for 10 × 1 min intervals, leading to a 55% total decrease in
absorbance (Figure 13A and Table 5). The di- and tetrafluori-
nated analogues are considerably stabilized against photobleach-
ing, while the trifluoromethyl-substituted dye exhibits minimal
stabilization. No photobleaching was observed under the same
conditions for any of the dyes in pure methanol solution.
The photobleaching experiment was next repeated in the
presence of CT-DNA under conditions where the dyes are
completely bound. The results are shown in Figure 13B and
Table 5 and demonstrate that while all dyes are more photostable
when bound to DNA than in solution, TO-p2F and TO-4F are
once again more photostable than the other dyes.
9
J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007 5717

A R T I C L E S
Silva et al.
More dramatic differences among the dyes are observed in
the optical spectra. Fluorination of the benzothiazole leads to a
progressive hypsochromic shift of the absorbance and emission
maxima, while TO-CF3 exhibits a bathochromic shift. These
shifts can be attributed to the effects of fluorine substitution on
the frontier molecular orbitals. Inductive effects from fluorine
substitution stabilize both the HOMO and LUMO. However,
since the HOMO has a greater population density on the
benzothiazole ring than the LUMO, fluorine substitution on the
benzothiazole preferentially stabilizes the HOMO, leading to
the observed hypsochromic shift. The opposite is true for
trifluoromethyl substitution on the quinoline ring, which pref-
erentially stabilizes the LUMO leading to the observed batho-
chromic shift.
Clear differences are also observed in the fluorescence
quantum yields, where increasing fluorination of the benzothia-
zole leads to increasing quantum yield in glycerol, whereas
TO-CF3 exhibits the lowest quantum yield. This result is again
explained by calculations, which indicated that the lowest singlet
excited state undergoes an activated crossing from a planar
structure to a highly twisted structure. The crossing occurs at
an interplanar angle of approximately 60° for TO and the
fluorinated analogues. Structures having angles less than 60°
should be optically bright, but an activated crossing to twisted
structures having angles greater than 60° is expected to quench
the fluorescence. The activation energy for transition from the
planar to twisted conformations was calculated for the substi-
tuted dyes. The results indicate that the quantum yields observed
in glycerol can be rationalized in terms of the substituent effects
on this activation energy. This suggests that twisting through
the critical angle of 60° is the dominant quenching mechanism
in solution. Regarding the quantum yield in DNA, the observed
substituent effects follow a quite different trend, indicating that
the DNA environment prevents the dye from adopting a severely
twisted conformation, such that the quantum yield is established
by some other nonradiative relaxation process. These conclu-
sions are supported by recent ultrafast fluorescence experiments
on benzoxazole-based unsymmetrical cyanines reported by
Vauthey and co-workers where a rapid (<10 ps) nonradiative
relaxation of the dye excited state was observed in water, but
that component of the decay disappeared upon intercalation into
DNA.23
held in a planar conformation but rather that the dye be
rigidified. A striking example of this is provided by the binding
of malachite green to an RNA aptamer.25 This binding results
in a >1000-fold fluorescence enhancement,26 but a high-
resolution NMR structure of the dye-RNA aptamer complex
shows that the chromophore is decidedly nonplanar, with an
interplanar angle of 57°.27
Implications for Dye Design. Dyes for which the quantum
yield is sensitive to the local environment are useful for imaging
of biological and other systems. A particularly effective means
of achieving this environmental sensitivity is to use dyes that
quench due to intramolecular twisting, such as those studied
by Willets et al.22 or those described here. The twisting
mechanism presented in this work for fluorescence quenching
provides a framework in which to consider optimization of dyes
for sensing applications. A structural aspect that can be easily
altered is the length of the bridge between the heterocycles;
however for TO, it is only feasible to increase the length of
this bridge. We would expect this to increase the angle at which
the barrier occurs, since the total twist angle between the
heterocycles can be distributed among the various interplanar
angles of a longer bridge. Preliminary calculations support this
conclusion: replacing the methine bridge of TO with a
trimethine bridge results in approximate doubling of the barrier
height, and the barrier moves to about 80° (data not shown).
Thus, dyes with longer bridges are expected to have larger
fluorescence quantum yields in fluid solution, so the fluores-
cence enhancement observed upon binding to DNA or some
other molecule of interest will likely be smaller than that for
the analogous monomethine dye. Ongoing work is directed
toward investigating how other substituents on the heterocycles
or variations in the heterocycle itself influence the barrier height
and position.
Acknowledgment. Synthesis and spectroscopic experiments
were performed by G.L.S., while computational experiments
were performed by V.E. This work was supported by the
National Institutes of Health (1 R33 CA9754101-01 to B.A.A.)
and the National Science Foundation (Grant CCF-0330135 to
B.A.A. and CHE-0316759 to D.Y.). We also acknowledge the
CMU Center for Molecular Analysis, supported by NSF Grant
No. DBI-9729351, and the NMR facility, funded in part by NSF
Grant No. CHE-9808188.
These results raise an important but subtle point concerning
torsionally responsive dyes, including triphenylmethane dyes
such as malachite green and the dicyanodihydrofuran dyes
developed by Moerner and co-workers.20 Our calculations
indicate that there is a range of conformations over which these
dyes will remain fluorescent, provided twisting through the
critical angle is prevented by solvent viscosity or binding in a
constraining site, such as between two DNA base pairs.24 Thus,
large fluorescence enhancements do not require that the dye be
Supporting Information Available: Full description of dye
synthesis and characterization and experimental details for
spectroscopy, photobleaching, and DNA-binding experiments.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JA070025Z
(24) Spielmann, H. P.; Wemmer, D. E.; Jacobsen, J. P. Biochemistry 1995, 34,
8542-8553.
(25) Grate, D.; Wilson, C. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6131-6136.
(26) Babendure, J. R.; Adams, S. R.; Tsien, R. Y. J. Am. Chem. Soc. 2003,
125, 14716-14717.
(22) Yarmoluk, S. M.; Lukashov, S. S.; Ogul’chansky, T. Y.; Losytskyy, M.
Y.; Kornyushyna, O. S. Biopolymers 2001, 62, 219-227.
(23) Fu¨rstenburg, A.; Julliard, M. D.; Deligeorgiev, T. G.; Gadjev, N. I.; Vasilev,
A. A.; Vauthey, E. J. Am. Chem. Soc. 2006, 128, 7661-7669.
(27) Flinders, J.; DeFina, S. C.; Brackett, D. M.; Baugh, C.; Wilson, C.;
Dieckmann, T. ChemBioChem 2004, 5, 62-72.
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5718 J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007