“Turn on” fluorescence response of monomethine cyanines caused by noncovalent binding to ct-DNA

Article abstract of DOI:10.1016/j.dyepig.2017.05.051

Monomethine cyanines have been extensively studied for their use as probes for nucleic acids among other biological systems. Four monomethine cyanine dyes were synthesized with various heterocyclic moieties including quinoline, benzoxazole, benzothiazole, and 3,3-dimethylindolenine adjoining benz[c,d]indol-1-ium, which was found to directly influence their optical and energy profiles. The dyes were characterized by ¹H and ¹³C NMR and HRMS. In this study the twisted conformation unique to monomethine cyanines was exploited in DNA binding studies where the benzoxazole containing sensor displayed up to 700-fold increase in fluorescence when bound to the DNA compared to the unbound form.

Full text of DOI:10.1016/j.dyepig.2017.05.051

Accepted Manuscript
“Turn on” fluorescence response of monomethine cyanines caused by noncovalent
binding to ct-DNA
Andrew Levitz, Cory Holder, Eduardo Soriano, Maged Henary
PII:
S0143-7208(17)30435-7
10.1016/j.dyepig.2017.05.051
DYPI 6013
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 1 March 2017
Revised Date: 25 April 2017
Accepted Date: 29 May 2017
Please cite this article as: Levitz A, Holder C, Soriano E, Henary M, “Turn on” fluorescence response
of monomethine cyanines caused by noncovalent binding to ct-DNA, Dyes and Pigments (2017), doi:
10.1016/j.dyepig.2017.05.051.
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ACCEPTED MANUSCRIPT
“Turn on” fluorescence response of monomethine cyanines caused by noncovalent binding
to ct-DNA
Andrew Levitzǂ, Cory Holder ǂ, Eduardo Soriano and Maged Henary*
Department of Chemistry, Georgia State University, Atlanta, GA 30302, USA
ǂ These authors contributed equally.
*To whom correspondence should be addressed.
*Corresponding Author:
Maged Henary, Ph.D.
100 Piedmont Ave. #315
Atlanta, GA 30303
Office: 404-413-5566; Fax: 404-413-5505
Email: mhenary1@gsu.edu
Abstract
Monomethine cyanines have been extensively studied for their use as probes for nucleic
acids among other biological systems. Four monomethine cyanine dyes were synthesized with
various heterocyclic moieties including quinoline, benzoxazole, benzothiazole, and 3,3-
dimethylindolenine adjoining benz[c,d]indol-1-ium, which was found to directly influence their
optical and energy profiles. The dyes were characterized by 1H and 13C NMR and HRMS. In this
study the twisted conformation unique to monomethine cyanines was exploited in DNA binding
studies where the benzoxazole containing sensor displayed up to 700-fold increase in
fluorescence when bound to the DNA compared to the unbound form.

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1. Introduction
In 1856 C.H.G. Williams heated quinolone with N-amyl lepidinium iodide in ammonia
producing a “magnificent blue color” referred to in latin as cyanos thus the first cyanine dye was
synthesized.1 This vast class of dyes shows absorption that covers a wider range of the electronic
spectrum than any other class of dyes, but due to their sensitivity to light the unimaginable value
of cyanines was not discovered until 20 years later when they were employed to increase the
sensitivity of the photographic plate. Since then cyanine dyes have been used in an incredible
amount of applications including bioimaging,2-4 solar cells,5,6 and data storage7 among other
applications 8-12
.
Cyanines are distinguished from other dyes in that they possess two nitrogen containing
heterocycles that are connected by a conjugated methine bridge. The delocalization of electrons
through this system gives them their characteristically high molar absorptivities and quantum
yields. Their names depend on the number of methine groups found in the bridge connecting the
two heterocycles. Monomethine dyes which include one methine group in the bridge tend to
absorb in the 400-500 nm range of the visible region.
Imaging of macromolecules such as DNA by staining with fluorescent compounds is of
great interest; therefore, expanding the options of available probes is vital to several areas of
research spanning from medical diagnostics to genomics. The synthesis of low cost, easy to
manipulate systems for fast analysis is required.13 Fluorescent detection has rapidly become one
of the most widely used techniques due to its sensitivity and noninvasiveness.14 Ethidium
bromide has commonly been used for the detection of DNA, however it has mutagenic effects
and poses other environmental concerns.15-17 On the other hand, cyanine dyes are sensitive, safe
and highly modifiable.18-20

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As described previously by our group, monomethine cyanines possess a valuable
characteristic of only displaying fluorescence when rotation around the methine bridge is
restricted.18,21 These dyes are the best non-convalent binding nucleic acid labels due to their high
binding constants, large molar absorptivities and quantum yields and high fluorescence signals
upon binding. Two commercially available monomethine cyanines are among the most popular
intercalating agents and have recently been used for DNA and chromatin imaging with super-
resolution fluorescence microscopy.22 While there is still much to explore regarding what effect
structure has on the photophysical properties, our lab has developed ways to redshift the
monomethine scaffold by altering the terminal heterocycles.18,21 Understanding how to redshift
the monomethine cyanine dyes allows for the enhancement of existing molecular probes
minimizing background absorption and fluorescence. While it is well known that monomethine
cyanines tend to bind DNA23-25 there is a lack of literature on what causes or prevents a cyanine
from binding to DNA. Calf thymus DNA (ct-DNA) is a heterogenous collection of linear
fragments of mammalian DNA that is readily available and therefore commonly used for in vitro
biological experimentation. Herein a series of monomethine cyanines that differ only in one
heterocycle were synthesized to investigate how these different heterocycles influence DNA
binding.
2. Results and Discussion
2.1 Synthesis
Toward correlating the various heterocyclic moieties and their ability to bind DNA, we
began to rationally design monomethine cyanines containing the benz[c,d]indole heterocycle on
one half of the dye. The other side possessed different heterocycles including 2-

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methylbenzothiazole, 2-methylbenzoxazole, 2-methylquinoline, or 3,3-dimethylindole,
heterocycles all with butyl side chains on the nitrogen. As shown in Table 1, the
physicochemical properties of these four compounds are all very similar with molecular weight
from 397-423 (without counterion) and polarizability from 76-80. They are all hydrophobic
molecules with LogP between 7 and 10. With the exception of the benzoxazole containing dye
they have similar topological polar surface area (TPSA). The oxygen atom typically raises the
TPSA, but there is no way to keep it in line with the other compounds without altering the
structure drastically. Due to the similarity of their physicochemical properties any change
observed in binding to DNA should be based on the 3D structure of each dye.
Table 1. Physicochemical properties of monomethine cyanines
Dye
MW
LogP
TPSA Polarizability
(amu)
397.54
413.60
407.58
423.62
(Å2)
7.68
8.25
8.69
9.23
5.479
1.674
1.165
1.752
76.21
77.39
78.36
80.37
OX
BTZ
Q
IN
*
Structures shown in Scheme 1 below
The asymmetrical red-shifted monomethine cyanine dyes were synthesized as shown in
Scheme 1. The key intermediate 4 was synthesized beginning with the alkylation of
benz[c,d]indol-2(1H)-one 1 by reflux with iodobutane in acetonitrile. The alkylated ketone 2 was
then converted to the thioketone 3 under reflux with phosphorous pentasulfide in pyridine before
methylation to a thioether with iodomethane creating the key precursor, quaternary ammonium
salt 4, which was used as one heterocycle for all the monomethine compounds. The other
heterocycle included 2-methylbenzothiazole, 2-methylbenzoxazole, 2,3,3-trimethylindole, and 2-
methylquinoline. These heterocycles were alkylated, respectively, with iodobutane in acetonitrile
to form quaternary ammonium salts 5. The two heterocycles 4 and 5 were condensed in
acetonitrile with triethylamine to afford final dyes 6.

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Scheme 1. Synthesis of monomethine cyanines
The condensation of the heterocycles to form monomethine cyanines begins with the
deprotonation of the methyl group at the 2 position of the heterocycle. This activated methylene
group of the various heterocyclic salts 5 attacks the 2 position of the other thioether heterocycle 4
and displaces the methyl sulfide moiety. Diethyl ether is added to the reaction mixture and red
crystals precipitate. The monomethine dyes 6 are then purified by dissolution in a minimal
amount of methanol and precipitation in diethyl ether to get an analytically pure sample.
1
Cyanines 6 were characterized by HRMS, H and 13C NMR and their optical properties were
investigated.
2.2 Optical Properties
Table 2. Energy values and optical properties of the monomethine cyanines
Dye
E.
E.
λmax
λmax
ε
HOMO (eV) LUMO (eV) (exp.)
(calc.) (M-1cm-1)
-8.31
-8.20
-6.06
-5.60
500
560
490
505
39,900
30,700
OX
BTZ
-7.99
-8.08
-5.96
-5.87
585
559
522
530
35,900
25,200
Q
IN
The optical properties of the monomethine cyanines were measured in ethanol. As shown
in Table 2 the absorption maxima range from 500 nm in the benzoxazole containing dye OX to

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585 nm with the increased conjugation of the quinoline system in Q. Their molar absorptivities
range from 25,000 M-1cm-1 in the dimethylindolenine compound IN to almost 40,000 M-1cm-1 in
the benzoxazole compound OX. Benzoxazole compounds are known to be brighter. In general,
the molar absorptivities are low for monomethine cyanines due to the benz[c,d]indole
heterocycle present on the other end of the dye, but it is this heterocycle that causes the
absorbance to be redshifted about 100 nm from where the dyes would generally absorb if they
were symmetrical.19 Scientists have attempted to approximate wave functions and energies for
atoms and ions since the late 1920s.26 Over the past 90 years there has been significant
improvements for predicting behaviors of these systems and more complex ones. The ability to
use theoretical calculations to predict optical properties of fluorophores, such as monomethine
dyes is a useful tool for screening compounds which can eliminate the need to synthesize them
first thus reducing harmful environmental impact. The ability to use theoretical calculations of
optical properties for fluorophores, such as monomethine dyes could be useful for the
development of viscosity detection fluorophores or bioimaging agents with desirable optical
profiles.18 Previously, we have shown using computational methods that when the dyes are
twisted out of plane, the HOMO orbitals are localized around the more conjugated
benz[c,d]indole heterocycle, preventing conjugation of the system.18 The dyes were therefore
modeled with a restricted dihedral angle to disallow rotation around the methine carbon. Their
geometries were optimized using Hartree-Fock dynamic functional theory (HF-DFT) hybrid
exchange-correlation functional and B3LYP/6-31G* basis set to predict the energy of the
compounds at ground state.27 The results from our previous paper were confirmed.18 The
predicted HOMO/LUMO energy gaps correspond to the experimentally observed absorption
maxima. The lowest energy gap was in dye Q at 2.16 eV which is corroborated by also having

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most bathochromic absorbance. Dye OX had the highest energy gap at 2.50 eV and displayed the
most hypsochromic absorbance. Again, the experimental results mostly agree with the trend of
the computationally predicted energy gaps showing that these computational methods can be
useful as a predictive tool for determining optical properties of the dyes. An example of the
predicted energy gaps is shown with dye OX in Figure 1. None of these compounds display
significant fluorescence unaccompanied in ethanol.
Figure 1. HOMO/LUMO diagrams of OX
2.3 DNA Binding
It was previously shown by our lab that only when the monomethine cyanine’s ability to
rotate around the methine bridge is restricted do the dyes relax by fluorescence.21 The dyes
generally do not fluoresce in free flowing solvent, but when the rotation around the methine
bridge is restricted such as in glycerol or when bound, fluorescence can be measured. The

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binding of these dyes to ct-DNA restricts this rotation and allows for the unique “turn-on”
fluorescence of these compounds.18 This property was exploited to determine how strongly each
heterocycle interacts with DNA compared to the others. Table 3 shows the binding constants and
emission maxima of the monomethine cyanines.
Table 3. Binding constants and λemission of monomethine cyanine dyes with various
heterocycles
Dye
λemission
(nm)
555
583
680
Kb
(M-1)
1.6 x 104
1.1 x 104
1.8 x 102
1.6 x 10-3
1.7 x 105
OX
BTZ
Q
686
525
IN
TOα
αPublished values λemission28 and Kb
29
As shown in Figure 2, dye OX displayed the greatest increase in fluorescence when in the
presence of ct-DNA with a 700-fold increase. The dye goes from non-fluorescent with no DNA
present to almost 700 fluorescence units with 20 µM DNA. This is due to both the binding
affinity of the dye (1.6 x 104 M-1) and the tendency of benzoxazole to decrease non-radiative
deactivation due to fast intersystem crossing.30 The benzothiazole containing dye BTZ shows a
similar binding affinity (1.1 x 104 M-1), but less fluorescence with only about a 40-fold increase.
The sulfur atom in benzothiazole can cause the opposite effect of the oxygen in benzoxazole due
to a lower singlet-triplet energy gap raising the opportunity for non-radiative relaxation.19 The
change in binding affinity is not very significant, but is likely due to the larger size of the sulfur
atom compared to oxygen. This dye is similar to the commercially available dye thiazole orange
(TO) which has a binding affinity in the order of 105 M-1.29 TO likely has higher affinity due to
steric hinderance of the butyl groups in our dyes as well as the larger size of the

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benz[c,d]indolium heterocycle compared to the quinoline in TO. These values are also on par
with similar monomethine cyanine dyes.31
Figure 2. Fluorescence spectrum of monomethine cyanines at constant dye concentration of 10
µM with increasing ct-DNA concentrations from 0-30 µM. The fluorometer reached saturation at
30 µM ct-DNA with dye OX.
Unlike the BTZ and OX compounds which showed excellent binding, the IN compound
containing a 3,3-dimethylindolenine heterocycle showed almost no binding with a binding
affinity of 1.6 x 10-3 M-1. This compound was 14 orders of magnitude lower than the
benzothiazole and benzoxazole compounds and any binding it may have shown is likely from the
benz[c,d]indole heterocycle on the other side of the dye. As shown in the 3D electrostatic
potential maps in Figure 3, dyes OX and BTZ on the left are planar. As we move to the right, the
size of the quinoline in Q starts to cause it to rotate slightly out of plane. While the heterocycles

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themselves in dye IN are flat, the dimethyl groups on the 3,3-dimethylindolenine are
perpendicular to the plane of the rest of the dye. Because all the physicochemical properties of
the dye have been kept similar and the only change is the dimethyl group, it can be concluded
that the perpendicular nature of the dimethyl groups provides steric hindrance that prevents this
binding. Our group has reported similar properties with the 3,3-dimethylindolenine preventing
duplex DNA binding but still allowing for binding to G-quadruplex DNA in other cyanine
classes.32
Figure 3. Electrostatic potential maps of monomethine cyanines
This finding is of utmost importance as the dimethyl functionality can be exploited to
prevent off site binding in the medicinal applications of these dyes. OX could be beneficial as a
molecular probe due to its redshifted nature over current commercially available DNA probes
(30 nm higher than TO). In addition, this dye should have higher fluorescence intensity than the
commercially available probes due to the exclusion of quinoline from the structure. Quinolines
are known to experience non-radiative return of decay. These compounds will continue to
become more useful as the fluorescence approaches the optical window (650-900 nm) and gets
further from the absorption of human tissues.

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3. Experimental
3.1 General Information
All chemicals and solvents were of American Chemical Society grade or HPLC purity
and were used as received. HPLC grade methanol and glycerol were purchased from Sigma-
Aldrich (Saint Louis, MO). All other chemicals were purchased from Fisher Scientific
(Pittsburgh, PA, USA) or Acros Organics. The reactions were followed using silica gel 60 F254
1
thin layer chromatography plates (Merck EMD Millipore, Darmstadt, Germany). The H NMR
and 13C NMR spectra were obtained using high quality Kontes NMR tubes (Kimble Chase,
Vineland, NJ) rated to 500 MHz and were recorded on a Bruker Avance (400 MHz) spectrometer
DMSO-d6. High-resolution accurate mass spectra (HRMS) were obtained at the Georgia State
University Mass Spectrometry Facility using a Waters Q-TOF micro (ESI-Q-TOF) mass
spectrometer. UV-Vis/NIR absorption spectra were recorded on a Varian Cary 50
spectrophotometer interfaced with Cary WinUV Scan Application v3.00 using VWR disposable
polystyrene cuvettes with a 1 cm pathlength. Laser Induced Fluorescence (LIF) emission spectra
were acquired using Shimadzu RF-5301 Spectroflurophotometer (Shimadzu Corporation
Analytical Instruments Division, Duisburg, F. R. Germany) interfaced to a PC with RF-5301PC
software using Sigma-Aldrich disposable polystyrene fluorimeter cuvettes with a 1 cm
pathlength. All spectral measurements were recorded at room temperature. The data analysis and
calculations were carried out using Microsoft Excel (Microsoft Corporation, Redmond, Wa).
3.2 Synthesis
The synthesis of monomethine cyanines was accomplished as reported previously by our
group.18,21
Compound 4 was previously synthesized by our group and others.18,21,33

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Each individual heterocycle was dissolved in acetonitrile and refluxed overnight with butyl
iodide to form quaternary ammonium salts 5.
Thioether 4 and each quaternary ammonium salt 5, respectively, were dissolved in acetonitrile
and triethlyamine was added to the solution. The reaction mixture was refluxed at 60 °C for 1 h
and monitored by UV-Vis. Upon cooling to room temperature, the corresponding dyes 6 were
precipitated by adding diethyl ether. The solid was collected by vacuum filtration and
triethylammonium salts were removed by washing with deionized water. The final dyes were
purified via precipitation from methanol with diethyl ether.
1-butyl-2-((3-butylbenzo[d]oxazol-2(3H)-ylidene)methyl) benzo[c,d]indol-1-ium iodide, OX:
Yield 60%; 1H NMR (400 MHz, DMSO-d6): δ ppm 0.96 (m, 6 H), 1.50 (m, 4 H), 1.83 (m, 4 H)
4.50 (t, J = 6.8 Hz, 2 H), 4.61 (t, J = 7.2 Hz, 2 H), 6.14 (s, 1 H), 7.62 (m, 4 H), 7.73 (t, J = 8.0
Hz, 1 H), 7.88 (d, J = 8.0 Hz, 1 H), 7.92 (d, J = 7.2 Hz, 1 H), 8.05 (t, J = 7.6 Hz, 1 H), 8.17 (d, J
= 8.0 Hz, 1 H), 9.22 (d, J = 7.2 Hz, 1 H); 13C NMR (100 MHz, DMSO-d6) δ ppm 14.1, 14.2,
19.8, 20.0, 30.3, 30.3, 43.6, 44.9, 75.4, 110.3, 112.4, 112.6, 122.9, 124.6, 126.5, 127.2, 128.4,
129.6, 130.4, 131.1, 132.9, 140.9, 146.9, 155.5, 161.3. HRMS m/z: calc. for C27H29N2O+
397.2274, obsd 397.2255.
1-butyl-2-((3-butylbenzo[d]thiazol-2(3H)-ylidene)methyl) benzo[c,d]indol-1-ium iodide, BTZ:
Yield 65%; 1H NMR (400 MHz, DMSO-d6): δ ppm 0.966 (m, 6 H), 1.53 (m, 4 H), 1.85 (m, 4 H)
4.40 (t, J = 7.2 Hz, 2 H), 4.76 (t, J = 7.6 Hz, 2 H), 6.46 (s, 1 H), 7.60 (m, 5 H), 7.90 (t, J = 7.6
Hz, 1 H), 8.08 (d, J = 8.4 Hz, 1 H), 8.35 (d, J = 8.0 Hz, 1 H), 9.34 (d, J = 7.2 Hz, 1 H); 13C NMR
(100 MHz, DMSO-d6) δ ppm 14.1, 14.2, 19.9, 20.1, 30.0, 30.2, 43.8, 47.7, 87.2, 109.6, 115.2,
122.5, 124.1, 124.8, 125.7, 127.0, 127.7, 128.2, 129.3, 129.7, 130.0, 132.8, 140.7, 141.3, 154.6,
164.9. HRMS m/z: calc. for C27H29N2S+ 413.2046, obsd 413.2039.

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1-butyl-2-((1-butylbenzo[cd]indol-2(1H)-ylidene)methyl) quinolin-1-ium iodide, Q: Yield 69%;
1H NMR (400 MHz, DMSO-d6): δ ppm 0.98 (m, 6 H), 1.48 (m, 2 H), 1.59 (q, J = 7.2 Hz, 2 H),
1.80 (m, 2 H), 1.94 (m, 2 H), 4.25 (t, J = 7.2 Hz, 2 H), 4.88 (t, J = 7.2 Hz, 2 H), 6.23 (s, 1 H),
7.33 (m, 1 H), 7.62 (m, 3 H), 7.83 (t, J = 7.6 Hz, 1 H), 8.10 (m, 2H), 8.23 (d, J = 8.0 Hz, 1 H),
8.32 (d, J = 7.2 Hz, 1 H), 8.39 (d, J = 8.0 Hz, 1 H), 8.67 (m, 2 H); 13C NMR (100 MHz, DMSO-
d6) δ ppm 14.0, 14.2, 19.8, 20.2, 30.0, 30.1, 43.3, 51.1, 92.5, 106.8, 118.7, 120.5, 124.3, 125.0,
125.8, 127.7, 128.0, 129.4, 129.9, 130.3, 130.4, 130.3, 130.4, 130.9, 134.6, 139.2, 142.0, 142.5,
+
152.9, 156.4. HRMS m/z: calc. for C29H31N2 407.2482, obsd 407.2463.
1-butyl-2-((1-butyl-3,3-dimethylindolin-2-ylidene)methyl)benzo[cd]indol-1-ium iodide, IN:
1
Yield 72%; mp 220-222 °C; H NMR (400 MHz, DMSO-d6): δ ppm 0.63 (t, J = 7.6 Hz, 3 H),
0.92 (t, J = 7.2 Hz, 3 H), 1.01 (m, 2 H), 1.40 (m, 2 H), 1.46 (m, 2 H), 1.60 (s, 6 H), 1.82 (m, 2
H), 4.19 (t, J = 6.4 Hz, 2 H), 4.50 (t, J = 7.2 Hz, 2 H), 7.43 (t, J = 7.2 Hz, 1 H), 7.57 (t, J = 7.2
13
Hz, 1 H), 7.74 (m, 4 H), 7.89 (m, 2 H), 8.01 (d, J = 7.6 Hz, 1 H) 8.36 (d, J = 8.0 Hz, 1 H); C
NMR (100 MHz, DMSO-d6) δ ppm 13.7, 14.1, 19.1, 19.9, 26.2, 29.7, 30.3, 44.1, 50.6, 52.1,
84.3, 111.5, 114.8, 123.6, 123.7, 124.3, 126.8, 128.9, 128.9, 129.7, 130.0, 130.5, 130.5, 133.2,
+
141.0, 141.1, 142.2, 156.3, 180.9. HRMS m/z: calc. for C30H35N2 423.2795, obsd 397.2773.
3.3 Computational Methods
The structure of each compound was first optimized using the HF-DFT method with the
hybrid exchange-correlation functional, B3LYP/6-31G* basis set using Spartan '14 (Irvine,
CA).34 The torsional angles from the quaternary nitrogen to the α-carbon on the alternate
heterocycle were restricted to 180° to get the calculated absorbance values, HOMO and LUMO
orbitals, physicochemical properties and electrostatic potential maps. The electrostatic potential

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maps and the calculated HOMO and LUMO orbitals were obtained using a restricted hybrid HF-
DFT, self-consistent field in vacuum performed with B3LYP/6-31G* basis set.
3.3 Stock Solutions
Stock solutions were prepared by weighing the solid of each individual compound on a 5-
digit analytical balance and adding solvent via class A volumetric pipette to make a 1.0 mM
solution. The vials were vortexed for 20 s and then sonicated for 5 min to ensure complete
dissolution. When not in use, the stock solutions were stored in the dark at 4 °C.
3.4 Method of Determining Absorbance and Fluorescence
Stock solutions were used to prepare five dilutions of dyes with concentrations ranging
from 5-25 µM using a class A volumetric pipette in order to maintain absorption between 0.1 and
1.0. The dye solutions were diluted ten-fold for fluorescence in order to minimize inner filter
effect. The absorption spectra of each sample were measured in duplicate from 400 to 750 nm.
The emission spectra of each sample were measured in duplicate with a 530 nm excitation
wavelength and slit widths of 5 nm for both excitation and emission. Emission spectra were
corrected by the developed method file.
3.5 DNA Binding Studies
A stock solution of each dye (1x10-4 M) and ct-DNA type 1 (7.5x10-3 M) were prepared
in ethanol and Tris-HCl buffer solution, respectively. Fluorescence titration with ct-DNA
concentrations (0-200 mM) were made by mixing 35 µL dye solution with Tris-HCl buffer
solution with and without ct-DNA to a total volume of 3500 µL in a fluorescence cuvette to
make working solutions of 10 µM. Fluorescence spectra were measured in duplicate with
excitation at 530 nm and slit widths of 5 nm for both excitation and emission.

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4. Conclusion
A series of monomethine cyanines were synthesized in good yield with red-shifted
absorbance properties in comparison to previously synthesized monomethine cyanine dyes.
Although the benz[c,d]indolium containing monomethine cyanine dyes in this report are non-
fluorescent in free flowing solvent, their fluorescence is “turned on” through the restriction of the
methine bridge caused by binding to DNA. Computational methods were shown to be useful as a
predictive tool for determining their optical properties. In keeping everything the same except for
a single heterocycle, binding affinity to ct-DNA of these heterocycles were able to be compared.
OX stood out as the best candidate as a starting point for building a new molecular probe due to
its strong binding affinity and 700-fold increase in fluorescence from binding. In addition, IN
with a 3,3-dimethylindolenine heterocycle showed almost no binding due to the steric hindrance
of the dimethyl group. This could be exploited to prevent offsite binding. Utilizing the described
techniques these dyes could be further developed as potential biological probes. Future studies
will investigate how different substitutions on the heterocycles could increase binding affinities
and increase optical activity to various biological targets.
Acknowledgements
MH, AL, CH and ES would like to thank the Department of Chemistry at Georgia State
University for their support and the funds provided for the Ph.D. dissertation of AL. MH wishes
to thank the Brains and Behavior Seed Grant, the Health Innovation Program Grant and Atlanta
Clinical & Translational Science Institute and the Georgia Research Alliance Grant for their
support.

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ACCEPTED MANUSCRIPT
A series of monomethine cyanines were synthesized in good yield with red-shifted absorbance
properties
Computational methods were shown to be useful as a predictive tool for determining their optical
properties
The dyes displayed “turn-on” fluorescence when bound to ct-DNA
One of these dyes, OX, displayed a 700-fold increase in fluorescence when bound to 20 µM ct-
DNA