Non-cytotoxic photostable monomethine cyanine platforms: Combined paradigm of nucleic acid staining and in vivo imaging

Article abstract of DOI:10.1016/j.jphotochem.2020.112598

Cyanine based chemosensing platforms have successfully been employed over the past couple of decades in various fields of biomolecular sciences. Still a substantial number of recent advances and improvements on this class of compounds are reported in the art. This paper presents our latest work on the improved synthetic approach, study on photophysical properties, and biosensing applicability of monomethine cyanine dyes. The series of mono-, di- and tricationic dyes showed up to 5-fold enhanced resistance against photobleaching compared to the commercially available Thiazole Orange (TO). The title compounds were studied as potential molecular probes for the detection of deoxyribonucleic acid, demonstrating their capacity as excellent fluorescent labeling agents. Depending on the dye chemical structure, current Cl-TO compounds exhibit up to 834-fold enhanced fluorescence emission and form stable complexes with Calf Thymus-DNA. The calculated binding constants were found to be higher than several conventional fluorogenic dyes for nucleic acid detection. All studied derivatives appeared as less cytotoxic than the Thiazole Orange. IC50 concentrations in human fibroblasts MRC5 cell line were calculated up to 50 μM for the synthesized Cl-TO dyes, and 0.5 μM for the parental Thiazole Orange. Two of the dyes were found very competent in post-electrophoretic visualization of DNA. As demonstrated by the agarose gel electrophoresis, the staining efficiency and detection limits of the dyes were comparable to the widely used Ethidium Bromide. The tricationic dye revealed great potential for cell cycle analysis in G1, S and G2 phases. The chlorinated TO derivatives readily stain human cells in vivo, while they can effectively be applied for eukaryotic and microbial cell staining.

Full text of DOI:10.1016/j.jphotochem.2020.112598

JournalofPhotochemistry&PhotobiologyA:Chemistry397(2020)112598
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Non-cytotoxic photostable monomethine cyanine platforms: Combined
paradigm of nucleic acid staining and in vivo imaging
Atanas Kurutosa,*, Tatjana Ilic-Tomicb, Fadhil S. Kamounahc, Aleksey A. Vasilevd,
Jasmina Nikodinovic-Runicb,*
a Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev str., bl. 9, 1113 Sofia, Bulgaria
b Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000 Belgrade, Serbia
c University of Copenhagen, Department of Chemistry, Universitetsparken 5, DK-2100 Copenhagen, Denmark
d Department of Pharmaceutical and Applied Organic Chemistry, Faculty of Chemistry and Pharmacy, Sofia University, 1164 Sofia, Bulgaria
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cyanine dyes
Fluorescent microscopy
Microbial cell staining
Nucleic acid detection
Non-Cytotoxic molecular probes
Cyanine based chemosensing platforms have successfully been employed over the past couple of decades in
various fields of biomolecular sciences. Still a substantial number of recent advances and improvements on this
class of compounds are reported in the art. This paper presents our latest work on the improved synthetic
approach, study on photophysical properties, and biosensing applicability of monomethine cyanine dyes. The
series of mono-, di- and tricationic dyes showed up to 5-fold enhanced resistance against photobleaching
compared to the commercially available Thiazole Orange (TO). The title compounds were studied as potential
molecular probes for the detection of deoxyribonucleic acid, demonstrating their capacity as excellent fluor-
escent labeling agents. Depending on the dye chemical structure, current Cl-TO compounds exhibit up to 834-
fold enhanced fluorescence emission and form stable complexes with Calf Thymus-DNA. The calculated binding
constants were found to be higher than several conventional fluorogenic dyes for nucleic acid detection. All
studied derivatives appeared as less cytotoxic than the Thiazole Orange. IC50 concentrations in human fibro-
blasts MRC5 cell line were calculated up to 50 μM for the synthesized Cl-TO dyes, and 0.5 μM for the parental
Thiazole Orange. Two of the dyes were found very competent in post-electrophoretic visualization of DNA. As
demonstrated by the agarose gel electrophoresis, the staining efficiency and detection limits of the dyes were
comparable to the widely used Ethidium Bromide. The tricationic dye revealed great potential for cell cycle
analysis in G1, S and G2 phases. The chlorinated TO derivatives readily stain human cells in vivo, while they can
effectively be applied for eukaryotic and microbial cell staining.
1. Introduction
Among numerous spectroscopic techniques, fluorescence-based
methods are commonly employed for highly accurate detection of nu-
Due to their excellent photophysical properties and great bio-
compatibility, the group of cyanine dyes has undeniably acquired the
popularity of a robust chemosensing platform for the visualization of
various biologically important processes. Biomolecular imaging tech-
nologies including fluorescent labelling [1–5], tumour cell line markers
[6–8], super-resolution imaging [9], enzymatic and immune-assays
[10], nucleic acid sequencing [11,12], purification and sizing of
deoxyribonucleic acid fragments [13], optical sensing [14,15], detec-
tion of proteins [16], interactions with lipids [17], detection of reactive
oxygen species [18], drug delivery [19], and theranostic [20,21] are
only few examples on recent advances and topics of this class of organic
compounds.
cleic acids. Intrinsically non-emittive in aqueous media, cyanines ac-
quire strong fluorescence response when forced immobilization of the
dye backbone occurs within the target binding sites of DNA or RNA.
This is due to loss of mobility around the methine bond joining the two
heterocyclic chromophores [22]. The nonradiative coupled photo-
isomerization and the torsional motion between the benzothiazolium
and the quinolinium moieties account for reported quantum yield as
low as 0.0002 in aqueous solutions [23]. Over the past decade, the
continuous and rapid development of new devices and instrumentarium
for the detection of biologically important samples appealed to the
design of specific labelling small molecules. High molar extinction
coefficients, enhanced fluorescence emission response, resistance
⁎ Corresponding authors.
E-mail addresses: akouroutos@gmail.com (A. Kurutos), jasmina.nikodinovic@imgge.bg.ac.rs (J. Nikodinovic-Runic).
https://doi.org/10.1016/j.jphotochem.2020.112598
Received 28 October 2019; Received in revised form 24 April 2020; Accepted 25 April 2020
Availableonline05May2020
1010-6030/©2020ElsevierB.V.Allrightsreserved.

A. Kurutos, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry397(2020)112598
and used as received. The high-performance liquid chromatography
(HPLC) grade solvents used for the spectroscopic analyses were pur-
chased form Sigma-Aldrich, Scharlau Chemicals, and Macron Fine
Chemicals TM. Deuterated solvents were purchased from Eurisotop and
used as received. All other starting materials and solvents were com-
mercial products of analytical grade. Purification of the target cyanines
was achieved by repeated recrystallization from absolute ethanol.
Analytical thin-layer chromatography (TLC) was performed on Merck
silica gel 60 F254 0.2 mm-thick precoated TLC plates using chloroform :
methanol : acetic acid (86:13:1 v/v) as a mobile phase. Spots were vi-
sualised under VILBER Ultraviolet Lamp BVL-6.LC Dual Wavelength
254/365 nm and power of 2 × 6 W. The identity of the compounds was
confirmed by various spectroscopic techniques including NMR and
mass spectrometry. 1H-NMR and 13C-NMR spectra were recorded using
5 mm tubes at 500.13 MHz and 125.77 MHz, respectively, in DMSO-d6
at 25 °C on a Bruker Ultrashield Plus 500 spectrometer. The signals
corresponding to the residual protons of DMSO-d6 at 2.50 ppm were
used as an internal reference. All chemical shifts (δ) are quoted in ppm
with accuracy of 0.01 ppm. Coupling constants J are expressed in Hz
and determined with accuracy of 0.1 Hz. The following abbreviations
are quoted to describe spin multiplicity in 1H-NMR: ss = singlet, d =
doublet, t = triplet, q = quartet, dd = doublet of doublets, ddd =
doublet of doublets of doublets, m = multiplet. HRMS LC/MS were
recorded on a Dionex Acclaim RSLC 120 C18 2.2 μm 120 Å 2.1 × 50
mm column maintained at 40 °C carried out on a Bruker MicrOTOF-QII-
system with ESI-source with nebulizer 1.2 bar, dry gas 8.0 L/min, dry
temperature 200 °C, capillary 4500 V and plate offset -500 V.
Fig. 1. Molecular representation of the parental nucleic acid stain “Thiazole
Orange – TO” and current Cl-TO monomethine cyanine derivatives under study.
against photobleaching, negligible spectral overlap with target biomo-
lecules, binding selectivity, strong complexation with DNA, and low
cytotoxicity are key factors to the successful design of new fluorogenic
probes.
Earlier, Shank and co-workers examined the effect of halogen atoms
to the monomethine scaffold, where an increased brightness and pho-
tostabilizing effect were addressed [24]. Further reports also revealed
advantages upon addition of a strong electron acceptor (e.g. a CF3
group) to the quinolinium moiety [25]. Similarly, the introduction of a
CN group to the cyanine backbone improves the photophysical prop-
erties and decreases the susceptibility of the chromophore towards
singlet oxygen species [26]. The α-CN-TO dye proved to exhibit su-
perior photostability and brightness, however it was found to be non-
fluorescent in the presence of dsDNA. This modified cyanine was un-
suitable for binding nucleic acids due to significantly twisted geometry.
Previously, we also reported halogen-containing Thiazole Orange de-
rivatives, whose optical properties were found promising for con-
tinuous investigation [27]. The aim of our extended study is to design a
series with improved photophysical characteristics, good biocompat-
ibility, and increased labelling capacity (Fig. 1).
2.2. Synthesis of intermediate products
Intermediates 2c, 3a-3c, 5d and 6a-6d were obtained by reported
protocols [28–33].
To achieve our goal, current efforts were focused on: i) improve the
synthetic protocol for the preparation of monomethine cyanine scaf-
fold; ii) prepare TO analogues carrying single or multiple positive
charges; iii) evaluate the photophysical characteristics in both organic
media and buffer solutions; iv) study the resistance against photo-
bleaching; v) examine the in vivo cytotoxicity; vi) apply the newly
synthesized dyes to cell cycle analysis and last but not least vii) de-
monstrate bioimaging applications of current fluorogenic compounds.
2.3. Synthesis of the target Cl-TO monomethine cyanine dyes
A mixture of N-quaternary-2-methylbenzothiazolium salt 3a-3c (1
mmol) and the corresponding 4,7-dichloroquinolinium derivative 6a-
6d (1 mmol) were suspended in ethanol / dichloromethane (15 mL, 3:2
v/v). An excess of N,N-diisopropylethylamine (2 equiv., 0.35 mL, 2
mmol) was added dropwise over 5 min, and the resulting mixture was
vigorously stirred under argon atmosphere for 3 h at room temperature.
A spontaneous advent of red colour was observed followed by the
formation of thick precipitate (Scheme 1). The crude product was col-
lected by filtration, washed with diethyl ether (3 × 20 mL) and dried
under reduced pressure, affording the monomethine cyanine dyes as
red solids. Repeated recrystallization from absolute ethanol yielded the
analytical samples of the target Cl-TO dyes.
2. Materials and methods
2.1. Analysis methods and equipment
All chemicals and solvents required for the synthesis of the dyes
were purchased from Sigma-Aldrich, Alfa-Aesar, Acros Organics, Merck
Scheme 1. Improved synthetic approach to the fluorogenic Cl-TO monomethine cyanine dyes.
2

A. Kurutos, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry397(2020)112598
2-((7-chloro-1-methylquinolin-4(1
H)-ylidene)methyl)-3-methyl-
2-((7-chloro-1-(3-(4-(dimethylamino)pyridin-1-ium-1-yl)propyl)
quinolin-4(1 H)-ylidene) methyl)-3-(3-(pyridin-1-ium-1-yl)propyl)
benzo[d]thiazol-3-ium iodide (Cl-TO-6) – (Figs. S16−18 / Supporting
Information), red solid, yield = 37 %; 1H-NMR (500 MHz, DMSO-d6) δ
9.13 (d, J = 6.1 Hz, 2 H), 8.82 (d, J = 9.1 Hz, 1 H), 8.61 (dd, J = 12.0,
7.6 Hz, 2 H), 8.31 (d, J = 7.2 Hz, 2 H), 8.23 (d, J = 1.9 Hz, 1 H), 8.16
(t, J = 7.0 Hz, 1 H), 8.12 (d, J = 7.9 Hz, 1 H), 7.90 (d, J = 8.4 Hz, 1 H),
7.79 (d, J = 8.7 Hz, 1 H), 7.67 (t, J = 7.8 Hz, 1 H), 7.49 (t, J = 7.6 Hz,
1 H), 7.40 (d, J = 7.2 Hz, 1 H), 7.05 (d, J = 7.2 Hz, 2 H), 6.90 (s, 1 H),
4.94 – 4.88 (m, 2 H), 4.84 (t, J = 7.6 Hz, 2 H), 4.65 (t, J = 7.5 Hz, 2 H),
4.37 (t, J = 7.4 Hz, 2 H), 3.18 (s, 6 H), 2.38 (dt, J = 11.7, 5.6 Hz, 4 H);
13C-NMR (126 MHz, DMSO-d6) δ 160.49, 155.85, 148.46, 145.69,
144.78, 144.69, 141.90, 139.76, 138.48, 138.02, 128.38, 128.11,
126.98, 125.03, 124.17, 123.14, 122.95, 117.25, 113.30, 108.37,
107.78, 88.40, 57.89, 54.01, 51.18, 43.01, 29.75, 28.76; HR ESP MS:
benzo[d]thiazol-3-ium iodide (Cl-TO-1 (TO-7Cl / [27]) – (Figs. S1−3 /
Supporting Information), red solid, yield = 45 %; 1H-NMR (500 MHz,
DMSO-d6) δ 8.82 (d, J = 9.3 Hz, 1 H), 8.54 (d, J = 7.2 Hz, 1 H), 8.13
(d, J = 2.1 Hz, 1 H), 8.06 (dd, J = 8.0, 1.2 Hz, 1 H), 7.80 (d, J = 8.3
Hz, 1 H), 7.78 (dd, J = 9.1, 2.1 Hz, 1 H), 7.63 (ddd, J = 8.5, 7.3, 1.2
Hz, 1 H), 7.46 – 7.41 (m, 1 H), 7.32 (d, J = 7.2 Hz, 1 H), 6.91 (s, 1 H),
4.12 (s, 3 H), 4.03 (s, 3 H); 13C-NMR (126 MHz, DMSO-d6) δ 160.51,
148.01, 145.37, 140.44, 138.98, 138.03, 128.25, 127.65, 126.89,
124.71, 124.00, 122.93, 122.68, 117.78, 113.20, 107.79, 88.40, 42.40,
33.98; HR ESP MS: m/z: Found 339.07457 [M+] C19H16ClN2S+; Re-
quires [M+] 339.0717;
3-butyl-2-((7-chloro-1-methylquinolin-4(1
H)-ylidene)methyl)
benzo[d]thiazol-3-ium iodide (Cl-TO-2) – (Figs. S4−6 / Supporting
Information), red solid, yield = 61 %; 1H-NMR (500 MHz, DMSO-d6) δ
8.82 (d, J = 9.3 Hz, 1 H), 8.54 (d, J = 7.2 Hz, 1 H), 8.13 (d, J = 2.1 Hz,
1 H), 8.07 (dd, J = 8.0, 1.2 Hz, 1 H), 7.81−7.78 (m, 2 H), 7.63 (ddd, J
= 8.5, 7.3, 1.2 Hz, 1 H), 7.46 – 7.43 (m, 1 H), 7.36 (d, J = 7.2 Hz, 1 H),
6.92 (s, 1 H), 4.66 (t, J = 7.4 Hz, 2 H), 4.13 (s, 3 H), 1.80−1.74 (dq, J
= 9.9, 7.4 Hz, 2 H), 1.49−1.42 (m, 2 H), 0.93 (t, J = 7.4 Hz, 3 H); 13C-
NMR (126 MHz, DMSO-d6) δ 160.51, 148.01, 145.37, 140.44, 138.98,
138.03, 128.25, 127.65, 126.89, 124.71, 124.00, 122.93, 122.68,
117.78, 113.20, 107.79, 88.40, 42.40, 33.98.; HR ESP MS: m/z: Found
381.11886 [M+] C22H22ClN2S+; Requires [M+] 381.1187;
m/z: Found 848.05542 [M+]
848.0542;
C ; Requires [M+]
35H37ClI2N5S+
2.4. Photophysical study of Cl-TO cyanines in organic solvent
The UV–vis spectra were recorded at 25 °C using 10-mm path-length
quartz cells on a JASCO V-570 UV–vis-NIR double beam spectro-
photometer, equipped with a thermostatic cell holder (Huber MPC-K6
thermostat with precision 1 °C). Steady state fluorescence measure-
ments were performed at room temperature using 10-mm path-length
quartz cells on a JASCO FP6600 fluorimeter. Analysis of scientific data
and spectral processing were conducted using OriginPro 2019 graphing
and analysis software, v. 9.6.0172 (OriginLab Corporation). The pho-
tochemical stability was studied in acetonitrile solutions by irradiation
at 254 nm in equal intervals of 5 min. The measured absorbance was
expressed in % relative to the original intensity.
3-butyl-2-((7-chloro-1-ethylquinolin-4(1 H) ylidene)methyl)benzo
[d]thiazol-3-ium iodide (Cl-TO-3)
– (Figs. S7−9 / Supporting
Information), red solid, yield = 52 %; 1H-NMR (500 MHz, DMSO-d6) δ
8.76 (d, J = 9.2 Hz, 1 H), 8.59 (d, J = 7.3 Hz, 1 H), 8.22 (d, J = 2.1 Hz,
1 H), 8.07 (dd, J = 7.9, 1.2 Hz, 1 H), 7.81 (d, J = 8.3 Hz, 1 H), 7.77
(dd, J = 9.0, 2.0 Hz, 1 H), 7.62 (ddd, J = 8.4, 7.2, 1.2 Hz, 1 H), 7.47 –
7.41 (m, 1 H), 7.36 (d, J = 7.3 Hz, 1 H), 6.91 (s, 1 H), 4.66 (t, J = 7.3
Hz, 2 H), 4.61 (q, J = 7.2 Hz, 2 H), 1.84 – 1.70 (m, 2 H), 1.44 (t, J =
7.2 Hz, 5 H), 0.93 (t, J = 7.3 Hz, 3 H); 13C-NMR (126 MHz, DMSO-d6) δ
160.11, 148.08, 144.26, 139.92, 138.31, 137.81, 128.34, 127.88,
126.97, 124.80, 124.08, 123.03, 122.91, 117.28, 113.32, 108.25,
88.15, 49.37, 45.71, 29.28, 19.41, 14.63, 13.79; HR ESP MS: m/z:
Found 395.13447 [M+] C23H24ClN2S+; Requires [M+] 395.1343;
2-((1-benzyl-7-chloroquinolin-4(1 H)-ylidene)methyl)-3-butylbenzo
2.5. UV–vis and spectrofluorimetric interactions with calfthymus-DNA
Calf Thymus-deoxyribonucleic acid – Type I fibres (Calf Thymus-
DNA) was obtained from Sigma-Aldrich. Tris hydrochloride (Molecular
Biology
Grade,
Ultrapure,
Thermo
Scientific)
and
Ethylenediaminetetraacetic acid (Cell Culture Reagent) were purchased
from Alfa Aesar. For the nucleic acid interactions, Calf Thymus-DNA
solution was prepared by dissolution in Tris-EDTA buffer to a final
concentration of 1.8658 × 10−2 M, omitting sonication. The aqueous
solutions were buffered to pH 7.4 (10 mM Tris−HCl, 0.5 mM EDTA).
The DNA concentration per nucleotide can be determined by absorption
spectroscopy using the molar absorption coefficient at 258 nm (ε = 6.6
× 103 mol-1 dm3 cm-1) [34]. Dye stock solutions (1 mM) were freshly
prepared in DMSO and further diluted with buffer to the appropriate
working concentrations. The spectrophotometric and the spectro-
fluorimetric titrations were performed by adding small aliquots of the
deoxyribonucleic acid solution into the solution of the studied mono-
methine cyanine dyes. All samples were equilibrated at 25 °C for 2 min
prior to scanning. The fluorescence emission spectra were recorded at
[d]thiazol-3-ium iodide (Cl-TO-4)
– (Figs. S10−12 / Supporting
Information), red solid, yield = 68 %; 1H-NMR (500 MHz, DMSO-d6) δ
8.78 (d, J = 9.3 Hz, 1 H), 8.71 (d, J = 7.4 Hz, 1 H), 8.12 (dd, J = 8.0,
1.2 Hz, 1 H), 8.04 (d, J = 2.1 Hz, 1 H), 7.87 (d, J = 8.4 Hz, 1 H), 7.72
(dd, J = 9.1, 2.0 Hz, 1 H), 7.66 (ddd, J = 8.5, 7.3, 1.2 Hz, 1 H), 7.54 –
7.46 (m, 1 H), 7.45 (d, J = 7.3 Hz, 1 H), 7.40 (tt, J = 8.0, 1.5 Hz, 2 H),
7.37 – 7.26 (m, 3 H), 6.98 (s, 1 H), 5.86 (s, 2 H), 4.72 (d, J = 14.6 Hz, 2
H), 1.79 (d, J = 30.1 Hz, 2 H), 1.46 (d, J = 37.6 Hz, 2 H), 0.93 (d, J =
14.7 Hz, 3 H); 13C-NMR (126 MHz, DMSO-d6) δ 160.86, 148.10,
145.02, 139.93, 138.16, 137.95, 135.25, 129.06, 129.01, 128.46,
128.24, 127.92, 126.92, 126.64, 125.09, 124.34, 123.13, 123.04,
123.00, 117.77, 113.61, 107.94, 88.93, 56.52, 45.91, 29.40, 19.41,
13.78; HR ESP MS: m/z: Found 457.15078 [M+] C28H26ClN2S+
;
Requires [M+] 457.1500;
excitation to the longest wavelength absorption maxima (λabs =
2-((1-benzyl-7-chloroquinolin-4(1
H)-ylidene)methyl)-3-(3-(pyr-
508−514 nm). The data from the spectrofluorimetric titrations were
processed by the Scatchard equation in order to evaluate the binding
constants (Ks) of the cyanine dyes to Calf Thymus DNA [35,36]. Fitting
of the data revealed good correlation, with regression coefficients
R2 > 0.998.
idin-1-ium-1-yl)propyl) benzo[d]thiazol-3-ium iodide (Cl-TO-5) – (Figs.
S13−15 / Supporting Information), red solid, yield = 44 %; 1H-NMR
(500 MHz, DMSO-d6) δ 9.14 – 9.10 (m, 2 H), 8.80 (dd, J = 10.9, 8.2 Hz,
2 H), 8.60 (tt, J = 7.7, 1.4 Hz, 1 H), 8.19 – 8.11 (m, 3 H), 8.08 (d, J =
2.0 Hz, 1 H), 7.92 (d, J = 8.4 Hz, 1 H), 7.73 (dd, J = 9.0, 2.0 Hz, 1 H),
7.68 (ddd, J = 8.4, 7.3, 1.3 Hz, 1 H), 7.50 (dd, J = 10.4, 7.5 Hz, 2 H),
7.41 (t, J = 7.3 Hz, 2 H), 7.38 – 7.27 (m, 3 H), 6.93 (s, 1 H), 5.90 (s, 2
H), 4.98 – 4.81 (m, 4 H), 2.60 – 2.51 (m, 2 H); 13C-NMR (126 MHz,
DMSO-d6) δ 160.88, 148.46, 145.70, 145.31, 144.81, 139.78, 138.14,
138.10, 135.24, 135.21, 129.10, 128.45, 128.33, 128.12, 126.95,
126.69, 125.17, 124.36, 123.20, 123.08, 117.83, 113.42, 108.28,
88.90, 57.94, 56.66, 43.12, 28.81; HR ESP MS: m/z: Found 648.07448
[M+] C32H28ClIN3S+; Requires [M+] 648.0732;
2.6. Agarose gel electrophoresis
For the DNA samples, high molecular weight genomic DNA isolated
from Pseudomonas aeruginosa, plasmid DNA (pUC18) isolated from
Escherichia coli and DNA size standards Hyperladder I (Bioline), were
loaded onto 1 % (w/v) agarose gel and then electrophoresed at 120 V
for 45 min in TBE (Tris-Boric acid) buffer. The gel was stained with
either Cl-TO-4 or Cl-TO-6 dyes (2 μM solution in TBE buffer) and the
3

A. Kurutos, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry397(2020)112598
gels were visualized using ChemiDoc imager system (Bio-Rad, USA)
applying SYBR green filter.
Jose, USA), under 100× magnification. For Candida biofilm staining C.
albicans ATCC 10,231 cells were harvested from overnight grown cul-
tures (Sabouraud broth, 180 rpm, 30 °C) by centrifugation (5000×g, 5
min, 4 °C), washed twice with sterile phosphate-buffered saline (PBS;
Sigma-Aldrich, Munich, Germany), and resuspended in RPMI 1640
medium (Sigma-Aldrich) containing 2 % glucose (w/v) at a con-
centration of 2 × 106 cells/mL. C. albicans suspension was incubated
for 48 h at 37 °C without shaking, on a surface of glass cover slips (pre-
treated with serum for 2 h) in leaning position to allow biofilm at air-
liquid interfaces to form. The biofilm was stained with 10 μM of Cl-TO-
4 in PBS at room temperature for 30 min in the dark. Biofilm growth
was observed by Cl-TO-4 staining of adherent cells, under a fluores-
cence microscope (Olympus BX51, Applied Imaging Corp., San Jose,
CA, United States) under 40 × magnification.
2.7. Cytotoxicity assay
The cytotoxicity (anti-proliferative activity) of the compounds was
measured using the methods described previously [37]. MRC5 cells
(human lung fibroblast), HCT116 (human colon carcinoma), A549
(human lung carcinoma), MDA-MB-231 (human breast cancer) were all
obtained from ATCC and plated in a 96-well flat-bottom plate at a
concentration of 1 × 104 cells per well, grown in humidified atmo-
sphere of 95 % air and 5 % CO2 at 37 °C, and maintained as monolayer
cultures in RPMI-1640 medium. After 24 h of incubation, the media
containing increasing concentrations of each tested compound (0.1–100
μM) were added to the cells. Control cultures received the vehicle sol-
vent DMSO and blank wells contained 200 μL of growth medium. After
48 h of treatment, cells proliferation was determined using 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction
assay. Cell proliferation was determined from the absorbance at 540 nm
on Tecan Infinite 200 Pro multiplate reader (Tecan Group, Männedorf,
Switzerland). The MTT assay was performed twice in quadruplicate and
the results were presented as percentage of the control (untreated cells)
that was arbitrarily set to 100 %. The cell viability rate (%) was cal-
culated: (OD of the treated group/OD control group) × 100.
3. Results and discussion
3.1. Synthesis of intermediate products and target Cl-TO cyanine dyes
The preparation of intermediates 3a-3c is based on reported pro-
cedures for the N-quaternization of hetaryl compounds, using an excess
of the alkylating agent 2 (methyl iodide - 2a, butyl iodide - 2b or 1-(3-
iodopropyl)pyridin-1-ium iodide - 2c) and heating to reflux for 2 h in 2-
methoxyethanol serving as the reaction media. All operations were
carried out under argon atmosphere in order to hamper possible oxi-
dation or formation of side products. The 2-methylbenzothiazolium
salts 3a-3c were obtained in moderate yields varying between 61–72 %.
These compounds were found highly hygroscopic, therefore structural
identification was achieved on the final dyes. Similarly, derivatives 6a-
6d were isolated in good to excellent yields (71–88 %) via direct
melting for 20 min at 120 °C of the bulk 4,7-dichloroquinoline 4 and a
slight excess of the corresponding alkylating reagent 5 (methyl iodide -
5a, ethyl iodide - 5b, benzyl bromide - 5c or 4-(dimethylamino)-1-(3-
iodopropyl)pyridin-1-ium iodide - 5d) under solvent-free reaction
conditions. Due to instability of the quinolinium salts, they were pre-
pared and used immediately in the synthesis of the cyanine dyes.
The design of current molecular probes for nucleic acid detection
relies on a well-established core of monomethine cyanines. The series of
six dyes under investigation represent unsymmetrical mono-, di- and
tricationic molecular structures prepared by a condensation reaction
between N-quaternary benzothiazolium chromophores and chlorine-
containing quinolinium salts. The target Cl-TO dyes were obtained
suggesting a modified environmentally benign concept as opposed to
the classical Brooker’s method for cyanine dye synthesis, which features
numerous drawbacks (e.g. evolution of toxic methane thiol, relatively
poor yields, and complex crude product mixtures among others)
[38,39]. Previously we reported 3 different reaction procedures for the
preparation of TO derivatives using polar protic solvents and sonication
(Procedure “A” - methanol, Procedure “B” - water, and Procedure “C” -
ethanol) [27]. Among those, both approaches “A” and “B” promoted
significant amount of blue coloured by-product. Although ethanol in
procedure “C” appears to reduce the quinoline self-condensation, it
does not completely hamper the formation. As opposed to previous
protocol, our revised synthetic method makes use of a mixture of
ethanol (EtOH) : dichloromethane (DCM) at a 3:2 v/v ratio, which was
found to improve the reaction yield and afford higher purity of the
target monomethines. The formation of bis-quinoline by-product was
vanished by current protocol. The reaction proceeds at room tempera-
ture employing the sterically hindered N,N-diisopropylethylamine
(DIPEA -Hünigs base). Recrystallization from absolute ethanol afforded
the analytical grade samples of the title dyes. Structural elucidation was
held by 1H-NMR, 13C-NMR spectroscopy, and high-resolution mass
spectrometry (ESI+).
2.8. Fluorescence microscopy
MRC5 cell staining. Upon being plated on microscopic slides (1.5 ×
105 cells/slide), human cells were fixed in 4 % paraformaldehyde in
PBS (Phosphate-buffered saline) for 20 min at room temperature (RT),
washed 3 times with PBS and incubated in 0.25, 0.5 and 5 μM cyanine
dyes solutions, for 30 min at room temperature in the dark. Cells were
rinsed with PBS, covered with a glass coverslip and analysed. Nuclei
were stained with 0.1 mg/mL diamino phenylindole (DAPI; Sigma-
Aldrich). The entry and intracellular distribution of tested cyanine dyes
were analysed under the fluorescence microscope (Olympus BX51,
Applied Imaging Corp., San Jose, USA), under 100 × magnification.
2.9. Flow cytometry analysis of the cell cycle
Fixed and RNAse A treated MRC5 cell line was stained with Cl-TO-4
and Cl-TO-6 (1μM solution in PBS) or with propidium iodide (PI;
Sigma-Aldrich) (10 μg/mL). The cell cycle phases were analysed by
Partec CyFlow Space flow cytometer (Sysmex Partec, Görlitz, Germany)
using FL-1 detector (FITC, 525 nm Band Pass Filter) and FL-2 detector
(PE and PI, 575 nm Band Pass Filter). The stained cells could be de-
tected in S, G1 and G2 phases.
2.10. Microbial cells staining
Overnight Candida albicans (ATCC 10,231) or Saccharomyces cere-
visiae (baker’s yeast from local health food store) cultures were diluted
to OD600 = 0.6 and incubated for 3 h at 30 °C. After completion of the
incubation period, cells were diluted to 1 × 107 cells/mL, washed twice
with PBS, fixed with paraformaldehyde solution (4 %, v/v) and stained
with 20 μM of Cl-TO-4 or Cl-TO-6 in PBS at RT for 30 min in the dark.
Cells were visualized using a fluorescence microscope (Olympus BX51,
Applied Imaging Corp., San Jose, USA), under 100 × magnification.
Pseudomonas aeruginosa (ATCC 10,145) and Staphylococcus aureus
(ATCC 43,300) strains were grown at 180 rpm and 37 °C in Luria-
Bertani broth. Overnight cultures were diluted to OD600 = 0.1 and
incubated for 2 h at 37 °C. Microbial cells were harvested by cen-
trifugation, washed twice with PBS, fixed with paraformaldehyde so-
lution (4 %, v/v) and stained with 20 μM of Cl-TO-4 or Cl-TO-6 in PBS
at room temperature for 30 min in the dark. Cells were visualized using
a fluorescence microscope (Olympus BX51, Applied Imaging Corp., San
The design of the Cl-TO series was inspired by previous report on
halogenated cyanines, which revealed promising photophysical char-
acteristics. Our efforts were focused to continue the design of the
4

A. Kurutos, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry397(2020)112598
Table 1
Optical characteristics of the monomethine cyanine dyes in organic media.
a
b
Compound
λabs
ε
λfl
Kobs
*
t1/2 *
(min)
(nm)
(Lmol−1 cm−1
)
(nm)
(min −1)(× 10−
2
)
Thiazole Orange
Cl-TO-1
Cl-TO-2
Cl-TO-3
Cl-TO-4
502
509
510
511
514
512
513
68.500
79.500
82.000
81.400
81.900
80.700
82.000
538
548
548
550
553
551
552
17.20
14.00
9.56
8.75
6.70
3.84
2.95
4.9
8.1
12.6
12.4
13.9
21.6
23.3
Cl-TO-5
Cl-TO-6
a
UV–vis absorption maximum.
excitation wavelength = λabs
b
.
* evaluated in acetonitrile.
chlorinated analogues and examine their photophysical properties and
staining ability. Initially, minor variations were introduced on the dyes
Cl-TO-1 [27], Cl-TO-2, Cl-TO-3, and Cl-TO-4 in order to perform a
systematic study on the interactions with DNA. In the case of Cl-TO-5
and Cl-TO-6, incorporation of multiple positive charges aimed to im-
prove water solubility and increase the binding constants between the
dye molecules and the double-stranded helix [40–43].
3.2. Evaluation of optical properties of the Cl-TO series in organic media
The photophysical characteristics of the title cyanines were eval-
uated in methanol using UV–vis and steady state spectrofluorimetric
techniques. The well-defined longest wavelength absorption maxima
(λabs) of the Cl-TO dyes were registered between 509−514 nm, the
fluorescence emission maxima (λfl) were allocated around 548−553
nm, while the corresponding molar extinction coefficients (ε) were
evaluated from 79.500 to 82.000 mol dm−3 cm-1. The optical char-
acteristics are summarized in Table 1.
In comparison to the parental Thiazole Orange, the introduction of a
chlorine atom to the C-7 position to the quinolinium chromophore re-
sulted in a small bathochromic shift (Δλ =7 nm for Cl-TO-1), which fits
to the Woodward-Fieser empirical rules [44,45]. Further variations of
the N-alkyl substituents of Cl-TO-2, Cl-TO-3 and Cl-TO-6 barely af-
fected the position of the maxima. The presence of a benzyl group was
found to apply additional minor red shift in the cases of the cyanines Cl-
TO-4 and Cl-TO-5. Similarly, the fluorescence emission maxima were
shifted towards the lower energies by approximately 10−15 nm.
The ability of a chromophore to resist against photooxidative de-
struction is a highly desired property. Singlet oxygen produced in situ by
irradiation of the dyes can be added to the conjugated system, leading
Fig. 2. Photobleaching decay curve of the studied cyanine dyes (panel A), and
pseudo-first order linear representation of ln A0/At against the irradiation time
(panel B).
discoloration of the solutions. The photodegradation of the dyes obeys a
pseudo-first order kinetics. The corresponding correlation coefficients
of the tendency lines were evaluated between 0.97−0.99. In order to
obtain further information on the parameters of the photodegradation
process, the following kinetic relationship was used :
to
a
disruption of the conjugation, causing photobleaching.
Photobleaching is related to the kinetic stability of the dye molecules in
solution under atmospheric oxygen and continuous exposure to UV-ir-
radiation. According to several reports, intersystem crossing yields are
rather poor for cyanine dyes. However, this class of organic molecules is
known to rapidly react with singlet oxygen [46]. Therefore, such kind
of processes are very important and should not be underestimated when
designing new chromophores.
For the purpose of our current study, we used the commercial
product Thiazole Orange (tosylate) without attempting further mod-
ifications. We intended to do a direct comparison of the newly syn-
thesized dyes to the conventional nucleic acid stain. Methanol and
acetonitrile are the most frequently employed solvents for “forced de-
gradation studies” [47,48]. Acetonitrile was used to perform the pho-
tostability tests in order to avoid hydrogen abstraction, or any other
photo catalytic reactions related to the nature of the polar protic al-
cohol. Fig. 2A illustrates the comparative resistance to photooxidative
destruction of the dyes by continuous irradiation (over 5-minute in-
tervals) with a strong UV-light. The position of the longest wavelength
absorption maxima remained unchanged until reaching complete
dC
dt
r = −
= Kobs
C
(1)
where, C represents the concentration of the cyanine dye and Kobs is the
observed first-order rate constant [49,50]. As shown in Eq. 2, the
concentration-time dependence was formed from the integration of the
previous expression.
C
Ct
ln( 0 ) = Kobs 1/2
t
(2)
According to the Lambert-Beer law, the absorbance is proportional to
the concentration. Substituting the concentration C variable with that
of the absorbance A at maximal wavelength, and bearing in mind the
unchanged spectral shape upon irradiation, Eq. 3 can be derived [51]:
A
At
ln( 0 ) = Kobs 1/2
t
(3)
The plot of ln(A0/At) against the irradiation time (Fig. 2B) shows the
5

A. Kurutos, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry397(2020)112598
angular coefficient, which equals the rate constant Kobs of the photo-
degradation process. The half-life (t1/2) of the title monomethines was
found up to 5 times greater than the commercially available Thiazole
Orange (t1/2 = 23.3 min for Cl-TO-6, t1/2 = 4.9 min in the case of TO).
Early works of Armitage and co-workers also suggest, that halogenated
Thiazole Orange derived dyes (fluorine containing analogues) exhibit
increased oxidation potential and are less reactive towards singlet
oxygen [52]. With respect to the counterion, several articles report
enhanced photostabilization of the I− over that of the TsO− (tosylate)
counter ion [53]. Perhaps attraction between the π-bonds of the ben-
zene ring in the tosylate anion and the π -bonds of the cyanine dye are
responsible for energy transfer between the ion pair causing photo-
bleaching. On the other hand, small hard anions are prone to have a
quenching effect on the singlet oxygen formed during the photo-
dynamic process [54–56]. Previously we conducted a direct comparison
between a synthesized TO cyanine and its chlorinated analogue (both
dyes carrying iodide counter ion). The results proved comparable
photostability between the two dyes [29]. Superior resistance to the
photodegradation was observed on the addition of extra charges to the
molecular structure. The 4-fold enhanced photostability for Cl-TO-5
and 5-fold in the case of Cl-TO-6 (t1/2 = 21.6 min and t1/2 = 23.3 min
respectively), might be attributed to the increased number of inorganic
counterions consuming the formed singlet oxygen. The two multi-
cationic cyanines reach complete discoloration after about 90 min of
irradiation with UV-light.
3.3. UV–vis and fluorescence titrations of the free Cl-TO monomethine
cyanine dyes and association to Calf Thymus-DNA in buffer solutions
Concentration dependent studies of the free dyes were held in
aqueous buffer media to monitor the spectral shape, allocate the ab-
sorption maxima (λabs), and evaluate the molar absorptivities (ε). All
experiments were performed at a micromolar range. The UV–vis spectra
of the buffered solutions revealed a good linear correlation, with re-
gression coefficients R2 > 0.98. The Beer-Lambert law was examined
and found to be valid over the concentration range of 1−12 μM, where
no systematic deviation could be observed (Fig. S19). Therefore, the
possibility of self-association between the dye molecules was neglected.
The Cl-TO compounds were characterized by a typical broad single-
peak absorption band ranging approximately between 400−580 nm.
The longest wavelength absorption maxima of the title dyes were re-
gistered between 508 nm and 514 nm, while the molar absorptivities
were evaluated to be 67.900-74.100 L mol−1 cm−1. The position of the
Fig. 3. Spectrophotometric titration between Calf Thymus-DNA and Cl-TO-4
(panel A), and fluorometric titration of the Cl-TO-5 dye (panel B). Shown in
inset to the right is the binding isotherm processed by the Scatchard equation at
538 nm.
acids were studied in TE aqueous buffer solutions (10 mM Tris−HCl /
0.5 mM EDTA, pH 7.4). The titrations revealed homogeneous changes
to the absorbance bands. The increase of the Calf Thymus-DNA con-
centration resulted in a continuous decrease of the absorption intensity
(Fig. 3A and Fig. S20). The attenuation of the absorption maxima (up to
39 % hypochromism) was accompanied by a moderate bathochromic
shift (Δλ = +7 nm) until saturation of the binding was reached. This
λ
abs were almost identical to those observed in methanol. The evaluated
optical characteristics of the monomethine cyanine dyes are summar-
ized in Table 2.
In order to evaluate the affinity of the synthesized molecules to-
wards DNA, we conducted spectrophotometric and fluorimetric titra-
tions. The interactions of the Cl-TO monomethine cyanines with nucleic
Table 2
Optical characteristics of the cyanine dyes in the presence of Calf Thymus-DNA in TE buffer solutions (10 mM Tris−HCl / 0.5 mM EDTA), pH 7.4, T=25 °C.
Ks (×106)
log Ks
n
error
a
b
c
Compound
λabs (nm)
λfl (nm)
ε
ΔI
calc.
(Lmol−1 cm−1
)
Thiazole Orange
Cl-TO-1
Cl-TO-2
Cl-TO-3
Cl-TO-4
502
508
509
510
513
511
514
526
534
536
536
540
538
539
63.000
74.100
67.900
68.300
70.400
69.600
72.900
252
211
270
150
261
834
499
1.55
2.22
2.16
1.26
6.32
4.56
4.76
6.19
6.35
6.34
6.10
6.80
6.66
6.68
0.25 0.04
0.24 0.02
0.17 0.02
0.25 0.06
0.20 0.04
0.18 0.02
0.15 0.01
Cl-TO-5
Cl-TO-6
a
UV–vis absorption maximum of the free dyes.
Fluorescence emission maximum upon excitation at λabs
Changes in the fluorescence signal (ΔI calc. = I / I0, where I is the maximum fluorescence emission intensity of the dye-DNA complex, while I0 represents the
b
c
.
fluorescence emission intensity of the free dye.
6

A. Kurutos, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry397(2020)112598
observation presumably accounts for strong interaction of the mono-
methines with the double stranded helix. Similar red shift of 8 nm was
previously reported for the intercalating agent Thiazole Orange by
Prodhomme et. al. [57].
Cyanine dyes have a strong tendency to form H- or J-type of self-
associates due to π-stacking. This phenomenon is favored when per-
forming studies in highly viscous solvents or in the presence of hy-
drophobic host molecules with appropriate cavity size [58–61]. The
formation of self-associates is typically followed by the appearance of
new absorption bands in the UV–vis spectrum. The spectral shape of the
Cl-TO compounds remained unaltered in the presence of DNA which led
us to assume, that the title cyanines are bound as monomers. Usually,
pronounced decrease in the absorption intensity and bathochromically
shifted λmax is an evidence for the “intercalation” of a small molecule
between the DNA base pairs [62–66]. On the other hand, the hydro-
phobic pockets of the beta-helix are relatively wide and deep to ac-
commodate small molecules (DNA width =11.7 Å – major groove, 5.7
Å minor groove / depth =8.5 Å – major groove, 7.5 Å – minor [67]).
Groove binding of a single dye molecule is also not an unknown aspect.
Monomeric “groove binders” typically resemble crescent shape. Clas-
sical examples involve the Hoechst33258, BOXTO, netropsin, dis-
tamycin, berenil, and the furamidine derivative DB293, which strongly
bind pure AT-rich sequences [68–74]. Since both, the “intercalation”
and the latter “groove binding” motifs could potentially exhibit similar
spectral behavior, the spectrophotometric data is insufficient to rule out
any of them.
In parallel to the UV–vis measurements, we performed spectro-
fluorimetric studies in order to assess the fluorescent labelling ability of
the newly synthesised dyes. The experimental part was carried out in a
similar way to the spectrophotometric experiments. An excess of the
polynucleotides was applied over that of the monomethine chromo-
phores. Intrinsically non-emittive in aqueous solution, the Cl-TO cya-
nines acquired strong fluorescence signal after incubation with Calf
Thymus-DNA. The addition of increasing amounts of DNA, resulted in
the continuous increase of the fluorescence emission signal intensity till
saturation of binding was reached. The I/I0 ratios were evaluated be-
tween 211–270 for the monocationic analogues Cl-TO-1, Cl-TO-2, and
Cl-TO-4. On the other hand, the di- and tricationic Cl-TO-5 and Cl-TO-
6 cyanines exhibited approximately 2–3 times greater increase of the
fluorescence emission response compared to the parental Thiazole
Orange. Shown in Fig. 3B is the titration of the Cl-TO-5 with Calf
Thymus-DNA, which revealed an 834-fold signal enhancement. The
experimental data were processed using the Scatchard equation for the
evaluation of the binding sites n, and to calculate the stability constants
Ks. The fitting of the data showed good correlation, with regression
coefficients R2 > 0.998. The binding parameters are summarized in
Table 2. The spectrofluorimetric titrations gave ratios n between
0.15−0.25. Following the nearest neighbour exclusion principle, n
values of 0.25 represent saturation of the available intercalative
binding sites of DNA as reported for Thiazole Orange [75–77]. De-
pending on the dye chemical structure, the binding constants of the Cl-
Fig. 4. Agarose gel electrophoresis (1%, w/v) of Hyperladder I standard DNA
(Bioline) stained with Cl-TO-4 and Cl-TO-6 in comparison to commercial EtBr
(Ethidium
kb.html.
Bromide)
staining (https://www.bioline.com/hyperladder-1
Table 3
Cytotoxic effects of the dyes on various cancer cell lines expressed as the IC50
values (μM).
IC50a, μM
MRC5
normal
HCT116
colon
A549
lung
MDA-MB-231
breast
TO analogues to the double-stranded helix vary between 1.26 × 106
-
Thiazole Orange
Cl-TO-1
0.5
2
0.8
2
1.5
2
2
2
6.32 × 106. The calculated Ks values were found to be higher than
several reported DNA fluorescent stains, such as phenanthridine,
fluorene and TO derived probes [78–81]. Comparable fluorescence re-
sponse and binding constants were observed for the monocationic dyes
Cl-TO-1 and Cl-TO-2. Apparently, varying the substituent on the ben-
zothiazole only (from N-methyl to the longer N-butyl) did not affect the
binding parameters. However, a minor alternation of the alkyl side-
chain on the quinolinium heterocycle (form N-methyl to N-ethyl in the
case of Cl-TO-3) was responsible for decreasing both ΔI and Ks. In-
triguingly, the N-benzyl substituted monocationic Cl-TO-4 bound to
Calf Thymus-DNA, revealed similar fluorescence response to Cl-TO-1
and Cl-TO-2, but the binding constant was significantly larger (Ks =
6.32 × 106). The Ks values of the multicationic Cl-TO-5 and Cl-TO-6
cyanines were found to be greater than the single charged molecules
Cl-TO-2
2
2
2
2
Cl-TO-3
Cl-TO-4
Cl-TO-5
Cl-TO-6
2.5
1.8
20
50
2
2
20
55
2
2
40
80
2
2
40
80
a
IC50 – the concentration inhibiting 50 % of cell growth after treatment
with the test compounds. Values were determined as quadruplicate of three
independent experiments with SD values between.1–3 %.
(except Cl-TO-4). Approximately 2–3 times higher Ks were evaluated
for the di- and tricationic compounds compared to the dyes featuring
pure alkyl sidechains.
7

A. Kurutos, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry397(2020)112598
Fig. 5. MRC5 cells stained with the Cl-TO derivatives and Thiazole Orange coupled with CellTracker Red CMTPX (fluorescent dye suited for whole-cell detection).
Fixed cells were stained using 1 μM dyes for 30 min and analyzed under fluorescent microscope (green: FITC channel for cyanine dyes and red: Texas Red channel for
red tracker dye; 100 × magnification).
3.4. Agarose gel electrophoresis
both Cl-TO derivatives were comparable to that of widely used
Ethidium Bromide (EtBr), as 20 ng of 200 bp as well as 15 ng of 1500 bp
long DNA fragments were clearly observable after gel electrophoresis
using Cl-TO and EtBr staining.
The monomethines Cl-TO-4 and Cl-TO-6 were also used in staining
a variety of DNA molecules after their separation by agarose gel elec-
trophoresis (Fig. 4 and Fig. S22).
These two dyes were very competent in post-electrophoretic vi-
sualization of both types of double-stranded DNA, plasmid (pUC18)
DNA that is a small supercoiled and circular and bacterial genomic DNA
of high molecular weight. The staining efficiency and detection limits of
3.5. Cytotoxicity assay
Nucleic acid targeting compounds usually appear to be highly cy-
totoxic. Such action mode is often observed in several classes of anti-
8

A. Kurutos, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry397(2020)112598
Fig. 6. Flow cytometric analysis of cell cycle distribution in
MRC5 cells stained with 1 μM of Cl-TO-4 or Cl-TO-6 and
propidium iodide (PI; 10 μg/mL) for 30 min. Cell cycle ana-
lysis was performed on an equal number of MRC5 cells (106).
The cells were ethanol fixed, RNase A treated and stained. The
stained cells could be detected in G1, S and G2 phases with %
of cell population given in table form. FACS analysis was done
by Partec CyFlow Space flow cytometer.
cancer agents. In the present study we have evaluated the anti-
proliferative activity of Thiazole Orange and its six chlorinated deri-
vatives (Cl-TO-1, Cl-TO-2, Cl-TO-3, Cl-TO-4, Cl-TO-5 and Cl-TO-6)
against normal human lung fibroblasts (MRC5) and a panel of three
human cancer cell lines included non-small cell lung carcinoma (A549),
colon carcinoma (HCT116) and breast cancer (MDA-MB231). The IC50
concentration values of the monomethine cyanine dyes obtained after
48 h cell exposure are presented in Table 3.
The halogenated cyanines exhibited cytotoxic activity in the con-
centration range from 1.8–80 μM on all cell lines. All newly synthesised
compounds were found to be less cytotoxic against healthy human fi-
broblasts (MRC5) in comparison to the TO from 3.6 to 100 times. The
lowest cytotoxicity was exhibited by Cl-TO-6 followed by Cl-TO-5
across all tested cell lines. The monocationic derivatives were com-
parably cytotoxic to the TO with IC50 values in the range from 1.8 to
2.5 μM. In comparison, IC50 values of 21 μM for A549 and 18 μM for
MRC5 cells are reported in the literature for the conventional hepta-
methine cyanine IR-780 [82].
heterochromatin, part of the chromosomes, which is a firmly packed
and considered genetically inactive [83].
3.7. Flow cytometry analysis of the cell cycle
Cyanines Cl-TO-4 and Cl-TO-6 were used in flow cytometry analysis
of cell cycle using MRC5 cell line at 1 μM or with PI (10 μg/mL) (Fig. 6).
The monomethine cyanine Cl-TO-6 manifested great potential to be
used for evaluation of the number of cells in G1, S and G2 phases with
comparable efficiency with widely used PI (Propidium Iodide) [84].
However, Cl-TO-4 was not suitable for this purpose, indicating differ-
ential behaviour towards living non-permeabilized cells.
3.8. Microbial cells staining
Cl-TO-4 appeared to localize predominantly with cell material,
while Cl-TO-6 was dispersed (Fig. S24). The two dyes were examined
for their ability to stain wide selection of microbial cells, as well
(Fig. 7). The monomethine derivative Cl-TO-4 was able to stain all
microbial cells (C. albicans, S. cerevisiae, P. aeruginosa and S. aureus)
more efficiently than Cl-TO-6, although microbial cell selection in-
cluded Gram-positive, Gram-negative, fungal and eukaryotic re-
presentatives with distinct differences in cell membrane and envelope.
Efficient fluorescent staining of microbial cells is of great importance
for their counting and sorting using flow cytometry approach [85–88].
Cl-TO-4 dye could also be successfully used to stain C. albicans biofilms
preformed on glass cover slips (Fig. S25). Notably, TO is also suitable
for similar type of microbial staining (Fig. 7), however given its higher
cytotoxicity, Cl-TO dyes are deemed as more suitable for this applica-
tion.
3.6. Fluorescence microscopy
Moderate to low cytotoxicity of these dyes could be their advantage
in applications as cellular fluorescent probes. All Cl-TO derivatives were
suitable for cell staining (1 μM solution, 30 min) and visualization
under fluorescent microscope (Fig. 5). Concentration of 1 μM was
chosen according to their cytotoxic effects on treated human cell lines
(Table 3), however lower concentrations such as 0.25 and 0.5 μM
provided stable staining intensity (Fig. S23). From counterstaining with
CellTracker Red probe, which distributes throughout cell cytoplasm,
the preferential specificity of Cl-TO dyes to nuclei was demonstrated
although they are also present in the cytoplasm. The observed dis-
tribution could be due to the differential preference between DNA and
RNA molecules. Cl-TO-2 and Cl-TO-3 are highly selective towards the
nucleus with Cl-TO-3 stained the chromosomes efficiently in MRC5 cell
lines. The careful study of these images revealed that some areas of
nuclei were more compactly stained with Cl-TO-1, Cl-TO-2, Cl-TO-5
and Cl-TO-6. These may be the regions of condensed chromatin,
4. Conclusions
In summary, this paper reports the synthesis of monomethine cya-
nine series suggesting an advanced synthetic protocol. Elucidation of all
chemical structures was achieved employing NMR spectroscopy and
high-resolution mass spectrometry (ESI+). The title compounds
9

A. Kurutos, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry397(2020)112598
Fig. 7. Microbial cells stained with Cl-TO-4, Cl-TO-6 and TO visualized under fluorescent microscope (FITC channel; 100 × magnification). Candida albicans,
Saccharomyces cerevisiae, Pseudomonas aeruginosa and Staphylococcus aureus cells were fixed and stained with 20 μM dyes.
manifested improved resistance against photobleaching, and higher
molar extinction coefficients compared to the commercially available
Thiazole Orange. Up to 5-fold increased photostability was observed for
the Cl-TO series as opposed to the conventional fluorescent probe. The
title compounds were screened as fluorescent markers for nucleic acid
detection. The UV–vis and spectrofluorimetric studies provided clear
evidence for strong interactions with biomacromolecules at micromolar
concentrations. Significant fluorescence enhancement was observed
among all chlorinated monomethines - up to 834-fold increase of the
fluorescence emission signal. The observed differences in the ΔI and the
Ks magnitudes were found to be dependent on the dye chemical
structure. The multicationic monomethines exhibited greater fluores-
cence enhancement and higher stability constants compared to the
single charged TO analogues. The results from the spectroscopic titra-
tions allowed as to recommend the current non-cytotoxic compounds as
good potential candidates for DNA staining. Agarose gel electrophoresis
showed that the staining efficiency and detection limits of the tested Cl-
TO derivatives is comparable to the widely used Ethidium Bromide
(EtBr). The results from the flow cytometry revealed great potential for
the Cl-TO-6 dye used in G1, S and G2 phases of the cell cycle analysis.
The applicability of the synthesized fluorogenic dyes for staining both
eukaryotic and microbial cells was also demonstrated. The experi-
mental findings further advance our knowledge on future design and
development of fluorogenic platforms for biological applications.
Authorship statement
All persons who meet authorship criteria are listed as authors, and
all authors certify that they have
participated sufficiently in the work to take public responsibility for
the content, including participation in
the concept, design, analysis, writing, or revision of the manuscript.
Furthermore, each author certifies that
this material or similar material has not been and will not be sub-
mitted to or published in any other
publication before its appearance in the Journal of Photochemistry
and Photobiology A: Chemistry.
10

A. Kurutos, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry397(2020)112598
Authorship contributions
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Please indicate the specific contributions made by each author (list
the authors’ initials followed by their
surnames, e.g., Y.L. Cheung). The name of each author must appear
at least once in each of the three
categories below.
Category 1
Conception and design of study: A. Kurutos, J. Nikodinovic-Runic;
acquisition of data: A. Kurutos, T. Ilic-Tomic, F. S. Kamounah, A. A.
Vasilev, J. Nikodinovic-Runic;
analysis and/or interpretation of data: A. Kurutos, T. Ilic-Tomic, J.
Nikodinovic-Runic;
Category 2
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Drafting the manuscript: A. Kurutos, J. Nikodinovic-Runic;
revising the manuscript critically for important intellectual content:
A. Kurutos, J. Nikodinovic-Runic;
Category 3
Approval of the version of the manuscript to be published (the
names of all authors must be listed): A. Kurutos, T. Ilic-Tomic, F. S.
Kamounah, A. A. Vasilev, J. Nikodinovic-Runic.
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The financial support by the Bulgarian National Science Fund
(BNSF) under grant – “Novel monomethine cyanine dyes as potential
non-covalent biomarkers: synthesis, evaluation, and studies on photo-
physical properties” contract № DM09/5 from 17.12.2016 is gratefully
acknowledged by AK for the preparation of the compounds Cl-TO-1, Cl-
TO-2, Cl-TO-3, Cl-TO-4, the photophysical studies and the interactions
of all dyes with DNA. This research has also been financially supported
by the Ministry of Education, Science and Technological Development
of the Republic of Serbia, under Grant No. 173048. AV gratefully ac-
knowledge the Bulgarian National Science Fund (BNSF) project SOFIa
(КP-06-H39/11) from 09.12.2019 for the support in preparation of dyes
Cl-TO-5 and Cl-TO-6.
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