Synthesis of a new β-naphthothiazole monomethine cyanine dye for the detection of DNA in aqueous solution

Article abstract of DOI:10.1016/j.saa.2010.02.026

Novel monomethine cyanine dye (MC) derived from β-naphthothiazole and benzothiazole has been prepared and characterized by ¹H and ¹³C NMR, FTIR, ESIMS, elemental analyses, absorption and fluorescence spectroscopy. The dye was conveniently synthesized by the condensation of two sulfate heterocyclic quaternary salts. The interaction between calf thymus DNA (ct-DNA) in tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) aqueous buffer solution and MC has been studied with spectral fluorescence method. The binding constant value has been determined by fluorescence titration of MC with ct-DNA concentrations. The result obtained is consistent with an intercalative binding interaction between MC and ct-DNA. Compared with ethidium bromide (EB), MC showed a huge fluorescence enhancement upon mixing with ct-DNA.

Full text of DOI:10.1016/j.saa.2010.02.026

Spectrochimica Acta Part A 75 (2010) 1605–1609
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis of a new ␤-naphthothiazole monomethine cyanine dye for the
detection of DNA in aqueous solution
Reda M. El-Shishtawy∗, Abdullah M. Asiri, Salem A. Basaif, T. Rashad Sobahi
Faculty of Science, Chemistry Department, King Abdul-Aziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel monomethine cyanine dye (MC) derived from ␤-naphthothiazole and benzothiazole has
been prepared and characterized by 1H and 13C NMR, FTIR, ESIMS, elemental analyses, absorp-
tion and fluorescence spectroscopy. The dye was conveniently synthesized by the condensation of
two sulfate heterocyclic quaternary salts. The interaction between calf thymus DNA (ct-DNA) in
tris(hydroxymethyl)aminomethane–HCl (Tris–HCl) aqueous buffer solution and MC has been studied
with spectral fluorescence method. The binding constant value has been determined by fluorescence
titration of MC with ct-DNA concentrations. The result obtained is consistent with an intercalative bind-
ing interaction between MC and ct-DNA. Compared with ethidium bromide (EB), MC showed a huge
fluorescence enhancement upon mixing with ct-DNA.
Received 21 October 2009
Received in revised form 22 January 2010
Accepted 11 February 2010
Keywords:
Monomethine cyanine dyes
Ethidium bromide
DNA
Napthothiazole
Fluorescence enhancement
Intercalation
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
mers [9–12]. Cyanine dyes demonstrate a significant increase in
fluorescence intensity upon binding to DNA [13], and the resulting
Manipulation at the molecular level of individual chemical and
biological substances in solution is currently a challenging target
in physics, chemistry, and biology. In particular, direct visualiza-
tion of macromolecules such as DNA by staining with a suitable
fluorescent dye is of great interest. Therefore, exploring new flu-
orescent dyes and expanding the tool box of currently available
fluorescent probes for the monitoring of biological systems and
processes is a challenge crucial to several research areas spanning
from medical diagnostics to genomics and live cell metabolism [1].
In all these fields, new and more efficient analytical tools to char-
acterize nucleic acids, proteins, and cells and the production of
low cost, easy to manipulate systems for fast sample analysis and
large-scale screening are highly required. In this rapidly developing
context, fluorescent detection has become one the most exploited
techniques owing to its sensitivity and noninvasiveness [2]. Con-
ventionally, EB has been used for the detection of DNA, however its
mutagenic effects pose some environmental concerns [3–5].
On the other hand, cyanine dyes are sensitive and more safe
fluorescent probes and are widely used for the detection of nucleic
acids [6–8]. In this way femtomoles of nucleic acids may be detected
and selectively identified even in the presence of other biopoly-
high signal/noise ratio affords detection in a homogeneous medium
[14–18]. The huge enhancement in fluorescence upon binding to
DNA is believed to originate from the loss of mobility around the
methine bridge between the two heterocyclic moieties [19]. There
are three main modes of non-covalent interactions of these small
molecules with DNA, intercalation, minor groove binding and elec-
trostatic interaction of highly positively charged molecules with
nucleotide phosphate backbone [14,20].
The structural differences between biological molecules results
in a different modes of interaction with the same probe. Moreover,
size of aromatic moiety as well as bulkiness of attached substituents
and their ability to interact non-covalently with polynucleotide
could also control intercalation ability as well as orientation of
intercalated molecule [21,22]. Therefore, tailoring new molecular
probes for the detection of biological targets is being attractive for
many scientists. In a continuation of our interest in cyanine dyes
suitable for the detection of DNA [23], we report here the synthesis
of a new MC and its binding properties with ct-DNA.
2. Experimental
2.1. General
Ethidium bromide, calf thymus DNA of low molecular weight
(1254 g/mol base pairs) and other reagents were of the high-
est purity available, purchased from Sigma–Aldrich Company and
used as received. Solvents were of analytical grade. 1H and 13C
∗
Corresponding author. Permanent address: Department of Dyes, Dyeing and
Textile Auxiliaries, Textile Research Division, National Research Centre, El-Behouth
St. Dokki, Cairo, PO 12622, Egypt.
E-mail address: elshishtawy@hotmail.com (R.M. El-Shishtawy).
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.02.026

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R.M. El-Shishtawy et al. / Spectrochimica Acta Part A 75 (2010) 1605–1609
NMR spectra were recorded in DMSO-d6 solutions on a Bruker
Avance 600 MHz spectrometer. Infrared spectra were performed on
a PerkinElmer spectrum 100 FTIR spectrometer. ESI mass spectra
were recorded on a Bruker Esquire 6000. Elemental microanaly-
ses were performed at the Cairo University Microanalytical Center.
Melting points were determined in open capillary tubes in a Stuart
Scientific melting point apparatus SMP3 and are uncorrected.
2.2. Absorption spectra
Absorption measurements were carried out at the MC concen-
tration 1.3 × 10−6 in DMSO, 2 × 10−6 M in Tris–HCl buffer solution
(50 mM, pH 7.4) and the ct-DNA concentration 6 × 10−5 M. For
EB, the measurements were made at concentration 1 × 10−5 M
in DMSO, 2 × 10−6 M in buffer and the ct-DNA concentration
4 × 10−5 M. Absorption spectra were measured on a UV-1650 PC
Shimadzu spectrophotometer.
2.3. Fluorescence titration
Stock solutions of MC (1 × 10−5 M) and ct-DNA (5 × 10−4 M,
moles of base pairs per liter) were prepared in DMSO and Tris–HCl
buffer solution, respectively. Fluorescence titration with ct-DNA
concentrations (0–180 ␮M) were made by mixing 5 ml of MC solu-
tion with Tris–HCl buffer solution without and with ct-DNA up to
the meniscus of 25 ml volumetric flask for making the working solu-
tions of MC (2 × 10−6 M). Fluorescence spectra were measured on
a RF-5301 PC Shimadzu spectrofluorophotometer and are uncor-
rected.
Fig. 1. Ethidium bromide and monomethine cyanine dyes used.
plements the helical DNA minor groove, recognition units (H-bond
donors and acceptors) on the side of the molecule facing DNA,
cationic centre at terminals of the molecules to enhance electro-
static interactions with the negatively charged phosphate groups
on DNA, and hydrophobic character by an extended unfused hete-
rocyclic structure to allow optimization of the compound for DNA
minor groove interactions [24–26,21]. With this in mind, the syn-
thesis of MC (Fig. 1) was made for the detection of ct-DNA as an
alternative to the classical mutagenic and carcinogenic [23] EB stain
(Fig. 1).
Monomethine cyanine dyes are generally synthesized by the
reaction of two heterocyclic quaternary salts, one bearing a methyl
group and the other a thioalkyl as a good nucleofugic leaving group,
both in the 2-position in relation to the heteroaromatic ammonium
salt. Deprotonation of the first quaternary salt by a base such as tri-
ethyl amine or pyridine gives the methylene derivative base, which
acts as the nucleophilic reagent [27]. The synthetic route of MC is
shown in Scheme 1.
N-Alkylation of a mixture (1:1 molar ratio) of 2-methylnaphtho
[1,2-d]thiazole and 2-(methylthio)benzo[d]thiazole with dimethyl
sulfate at 130 ◦C afforded the corresponding methylate salts as a
viscous material, which was further used in the synthesis of MC
without purification. Treatment of the methylate salts with a pyri-
dine/triethylamine mixture (3:1 by volume) in MeOH at 100–105 ◦C
gave MC as a yellow product in 34% yield. The structure of MC was
evidenced by its 1H NMR, 13C NMR, FTIR, ESIMS, absorption and
fluorescence spectroscopy
2.4. Synthesis
2.4.1. (Z)-1-methyl-2-((3-methylbenzo[d]thiazol-2(3H)
ylidene)methyl)naphtho[1,2-d]thiazol-1-ium methylsulfate
A mixture of 2-(methylthio)benzo[d]thiazole (1.99 g, 10 mmol),
2-methylnaphtho[1,2-d]thiazole (1.8 g, 10 mmol), and dimethyl
sulfate (2.65 g, 21 mmol) was heated at 130 ◦C for 5 h until prac-
tically total conversion into the corresponding quaternary ammo-
nium salts was achieved. The resulting mixture of solid salts was
heated at 100–105 ◦C in dry MeOH/pyridine/triethyl amine solvent
mixture (60 ml, 2:3:1, v/v) for 9 h. The mixture was concentrated in
vacuo and the crude yellow product was filtered, washed with ace-
tone and air-dried. Recrystallization from DMF affords the analyti-
cal pure product as a yellow powder (1.6 g, 34%). Mp 304–305 ◦C; 1H
NMR (600 MHz, DMSO-d6): ı (ppm) 3.41 (3H, s, OCH3), 4.07 (3H, s,
N–CH3), 4.50 (3H, s, N–CH3), 6.82 (1H, s, H-␣), 7.48 (1H, t, J = 8.4 Hz,
H-5/), 7.67 (1H, t, 8.4 Hz, H-6/), 7.72 (1H, t, J = 7.8 Hz, H-6), 7.78 (1H,
t, J = 8.4 Hz, H-5), 7.87 (1H, d, J = 8.4 Hz, H-8), 8.05 (1H, d, J = 8.4 Hz, H-
7/), 8.16 (1H, d, J = 7.8 Hz, H-4/), 8.20 (1H, d, J = 7.8 Hz, H-7), 8.25 (1H,
d, J = 8.4 Hz, H-9), 8.83 (1H, d, J = 8.4 Hz, H-4); 13C NMR (150.87 MHz,
DMSO-d6): d (ppm) 33.99 (N–CH3), 39.97 (N–CH3), 40.08 (OCH3),
83.31 (C-␣), 113.63 (C-4/), 119.69 (C-6/), 121.81(C-3a/), 122.61
(C-9), 122.80 (C-9a), 123.39 (C-5/), 124.61 (C-3a), 126.49 (C-5),
124.68 (C-6), 126.97 (C-7), 127.43 (C-4), 128.46 (C-8), 129.54 (C-
7/), 133.55 (C-4a), 136.26 (C-7a), 140.73 (C-7a/), 161.17 (C-2/),
162.72 (C-2). IR ␯ (cm−1): 3065, 2935, 1531, 1472, 1438, 1280, 1227,
1127, 1165, 1055, 840, 807, 737. UV/vis (DMSO): ꢀmax = 445 nm and
εmax = 60,000 M−1 cm−1. MS (ESI): M+; found: 361.0, C21H17N2S2
requires 361.0. Anal. Calc. For C22H20N2O4S3.1.5H2O: C; 52.88, H;
4.64, N; 5.61. Found. C; 52.42, H; 4.95, N; 6.19%.
3.2. Absorption and fluorescence measurements
It is known that cyanine dyes tend to self-aggregate in solution
[28,29], and since this process can influence the course of dye bind-
ing to a given substrate such as ct-DNA, a dilute solution of the dye
in the range of 10−6 M has to be used. The absorption and fluores-
cence spectra of free MC in DMSO (1.3 × 10−6 M), Tris–HCl buffer
(pH 7.4) (2 × 10−6 M) and in Tris–HCl buffer (2 × 10−6 M) containing
ct-DNA (6 × 10−5 M) are presented in Fig. 2.
3. Results and discussion
3.1. Synthesis
The absorption spectrum of MC (Fig. 2) in DMSO in the visible
region exhibits one absorption band (with a maximum at 446 nm)
and a shoulder at about 422 nm. The spectrum of MC in buffer solu-
tion in the visible region has a similar shape, and its maximum is
The classical structural criteria of small molecules for optimum
DNA fit and interaction have to meet: a crescent shape that com-

R.M. El-Shishtawy et al. / Spectrochimica Acta Part A 75 (2010) 1605–1609
1607
[19]. Increasing the polarity of the solvent from DMSO to aque-
ous medium has led to a blue shift in the absorption spectra. This
shift is most likely explained as follows; since MC is ionic dye,
which exhibits a polar character in the ground state, hence the
solvent molecules are oriented in such a way as required by the
polar character of the MC molecule. During the transition, which
occurs within a very small time interval, only the electrons have
the time to change position. The excited MC molecules, in which
the electric dipole has been weakened and has been reoriented,
are now within a solvent cage that is no longer adopted to the
electronic requirements of the excited molecule, since the solvent
cage is suitable for the electronic distribution in the ground state
molecule. Thus, a polar solvent creates a stabilizing solvent cage
around this ionic dye in the ground state, but a destabilizing sol-
vent cage for the excited state. The transition energy increases with
increasing solvent polarity. An increase in solvent polarity results
in a blue shift of the charge transfer band. In this context, hydro-
gen bonds created between water molecules in aqueous medium
and MC molecules can reduce the delocalization of the lone pair
of electrons of hetrocyclic nitrogen, which result in the observed
solvatochromic behavior.
The fluorescence spectrum of MC (Fig. 2) in DMSO exhibits a
weak fluorescence band with a maximum at 503 nm, which is blue
shifted to 493 and 487 nm in buffer and buffer solution containing
ct-DNA, respectively. This blue shift can be explained as above, in
which the aqueous solvent stabilize the ground state and destabi-
lize the excited state of MC molecules. It is worth noting that, the
fluorescence of MC is largely enhanced upon binding with ct-DNA
when compared with its fluorescence in the free state in both DMSO
and buffer solutions. This fluorescence enhancement is attributable
to the fact that on photoexcitation a lack of free rotation around the
internuclear bridge makes isomerisation around the methine bond
impossible [19,30], and subsequently nonradiative deactivation of
the excited state is not possible causing the dye to fluoresce.
For comparison, the absorption and fluorescence spectra of EB
(Fig. 3) were similarly measured in DMSO (1 × 10−5 M) and Tris–HCl
buffer (pH 7.4) (2 × 10−6 M) and in Tris–HCl buffer (2 × 10−6 M)
containing ct-DNA (4 × 10−5 M) and summarized in Table 1.
As can be seen, a similar spectral shape of absorption is also
obtained for EB in DMSO, Tris–HCl buffer and in Tris–HCl buffer
containing ct-DNA. This indicates that the observed spectrum cor-
responds to the absorption of the monomer dye molecules. Also, the
blue shift observed in the absorption as well as in the fluorescence
can be rationalized as in the case of MC since EB dye is also ionic
with a positive charge as in the case of MC. However, the enhance-
ment of the fluorescent intensity of EB was far weak relative to the
Scheme 1. Synthesis of monomethine cyanine dye.
blue shifted by 6 nm and its shoulder is also blue shifted by about
3 nm when compared to the maximum of the dye in DMSO. On
the other hand, the absorption spectrum of MC in buffer solution
containing ct-DNA in the visible region has also a similar shape
with those of DMSO and buffer and its maximum is the same as in
buffer, whereas its shoulder appears at about the same as in DMSO.
This spectral similarity of MC in the above different media sug-
gests that the observed spectrum corresponds to the absorption
of the monomer dye molecules and the shoulders correspond to
different vibronic transitions to the same electronic excited state
Fig. 3. Absorption and fluorescence spectra of EtBr dye in DMSO solution, in Tris–HCl
buffer and in the presence of ct-DNA solution. The dye concentration used for the
measurements was 1 × 10−5 M in DMSO, 2 × 10−6 M in buffer and DNA concentration
was 4 × 10−5 M.
Fig. 2. Absorption and fluorescence spectra of MC dye in DMSO solution, in Tris–HCl
buffer and in the presence of ct-DNA solution. The dye concentration used for the
measurements was 1.3 × 10−6 M for DMSO, 2 × 10−6 M for buffer and DNA concen-
tration was.

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R.M. El-Shishtawy et al. / Spectrochimica Acta Part A 75 (2010) 1605–1609
Table 1
Spectroscopic data of the investigated dyes.
a
b
c
d
Dye
ε (DMSO)
ꢀmax
(DMSO)
(nm)
ε (DNA)
ꢀmax (DNA) (nm)
ꢀex (nm)
Fem (DMSO) (nm)
Fem DNA (nm)
Kb (M−1
)
FE (%)e
(M−1 cm−1
)
(M−1 cm1)
MC
EB
62,230
5830
446
535
62,500
6650
440
490
440
490
503
628
487
597
1.01 × 104
1.50 × 105
1914
26
a
Maximum wavelength of absorption of MC dye in Tris–HCl buffer solution containing ct-DNA.
Excitation wavelength.
Emmision wavelength.
b
c
d
e
Data of Kb for EB is taken from Ref. [15].
FE is the calculated fluorescence enhancement percent at the same ct-DNA base pairs/dye ratio (20) for both EB and MC dyes using this equation: %FE = ((I − I0)/I0) × 100
where I0 and I are the fluorescence intensity in presence and absence of DNA, respectively.
case of MC as shown in Figs. 2 and 3 and Table 1. This result suggests
the suitability of using MC as a fluorescent probe for the detection
of ct-DNA in aqueous solution.
Eq. (6) leads to Eq. (7).
CDye
Cb
1
=
− 1
(7)
Cct-DNAKb
Upon MC binding with ct-DNA during the fluorescence titration,
the fluorescence intensity I increases until it reaches its maximum
value Imax when all CDye get completely bound with ct-DNA, assum-
ing that there is only one mechanism of the dye ct-DNA interaction
in this case and the intrinsic fluorescence of the free dye is negligi-
ble. Hence, the fraction of the bound dye as follows;
3.3. Fluorescence titration of MC with ct-DNA and binding
constant
It has been reported that the binding constant of probes which
bind to DNA by intercalation mode via hydrophobic, van der Waals
and electrostatic forces usually do not exceed 107 M−1. For exam-
ple Kb values of acridine orange [31,32], EB [31,15] and thiazole
orange [33] are equal to 3.1 × 104, 1.5 × 105 and 106 M−1, respec-
tively. However, complexes of groove-binding molecules such as
netropsin and distamycin A stabilized by hydrogen bonds may have
the binding constants values as large as 108–109 M−1 [34]. Since MC
chemical structure (Fig. 1) reveals possibility for a crescent shape,
hence it was of interest to find out whether MC probe is complexing
with ct-DNA by intercalation or groove mode. For this purpose, fluo-
rescence titration of MC (2 × 10−6 M) was conducted using different
concentrations of ct-DNA.
Cb
I
=
(8)
CDye
Imax
Thus, Eq. (7) can be rewritten in terms of the fluorescence inten-
sity to give Eq. (9)
1
Imax
=
− 1
(9)
(10)
(11)
Cct-DNAKb
I
Eq. (9) can have the form of Eq. (10).
Imax
1
= 1 +
The binding of MC with ct-DNA can be represented by the fol-
lowing equation [34];
I
C
ct-DNAKb
Dividing Eq. (10) by Imax leads to Eq. (11).
[Dye] + [DNA] → [Dye · DNA]
1
I
1
1
=
+
C
C
C
b
f
bs
Imax
Cct-DNAImaxKb
A plot of 1/I versus 1/Cct-DNA is expected to be linear with a slope
Cb
Cf Cbs
Kb
=
(1)
of 1/ImaxKb and an intercept of 1/Imax. As can be seen in Fig. 4 the lin-
ear fitting of Eq. (11) between the inverse of ct-DNA concentration
and the inverse of the fluorescence intensity holds indeed and the
estimated Kb value was equal to1.01 × 104 M−1 as shown in Table 1.
It is indicated (Table 1) that the binding constant Kb of MC is approx.
15-times lower of Kb of EB. On other hand, fluorescence enhance-
ment is 74-times higher. It means that the ability of visualization
of ct-DNA using MC dye is 74/15 = 4.933. Also, Kb value of MC sug-
gests that MC forms a stable complex with ct-DNA and the order
of 104 M−1 for Kb indicates that MC can be considered as a small
molecule that interacts with ct-DNA by intercalation via a non-
crescent shape (Fig. 1). A similar intercalation binding constants
where Cb is the concentration of the MC molecules bound to ct-DNA,
Cf the free MC concentration, Cbs the concentration of the available
binding site of ct-DNA. We consider the number of available ct-DNA
binding sites to be equal to the number of base pairs in the ct-DNA
strand which are unoccupied by dye molecules. If CDye is the total
concentration of MC in solution and Cct-DNA (ct-DNA concentration)
significantly higher than CDye, we can consider Cb to be equal to
Cct-DNA and then we can get Eqs. (2) and (3).
Cf = CDye − Cb
Cbs = Cct-DNA
(2)
(3)
Using Eqs. (1)–(3) leads to Eq. (4).
Cb
Kb
=
(4)
(5)
(6)
[CDye − Cb]Cct-DNA
Eq. (4) can be rewritten to give Eq. (5).
[CDye − Cb]Cct-DNA
1
Kb
=
Cb
Dividing Eq. (5) by Cct-DNA leads to Eq. (6).
[CDye − Cb]Cct-DNA
1
Fig. 4. Fluorescence titration of MC dye solution (2 × 10−6 M) as a function of the
=
Cct-DNAKb
Cb
DNA concentrations.

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Acknowledgments
The authors wish to express their gratitude to the Deanship of
Scientific Research, King Abdul Aziz University (Project 428/163/␧)
for funding this study.
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