Synthesis of chloro-substituted analogs of Thiazole orange - Fluorophores for flow cytometric analyses

Article abstract of DOI:10.1016/j.jphotobiol.2011.03.012

Synthesis, absorption and fluorescence properties of a series of asymmetrical monomethine cyanine dyes, chloro-containing analogs of Thiazole orange, are reported. Their staining ability was studied by flow cytometry. The saturating concentrations of each dye that gives a stable staining intensity have been determined. The ability of dyes B9, B11, B13 to stain live macrophages and apoptotic splenocytes was investigated. Positive signal in nucleus of adherent macrophages detected by fluorescent microscopy showed good specificity of B9, B11 and B13 dyes for DNA. In apoptotic assay cells positive for Annexin V were stained more brightly with the dyes B9, B11 and B13 than with propidium iodide. Despite that B13 showed high DNA selectivity it induces apoptosis of splenocytes and it is not suitable for detection of dead cells. The other synthesized chloro-containing analogs of Thiazole orange B9 and B11 can be successfully used for flow cytometric analyses of DNA content in live cells and for analyses of cell apoptosis.

Full text of DOI:10.1016/j.jphotobiol.2011.03.012

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Journal of Photochemistry and Photobiology B: Biology 103 (2011) 215–221
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Synthesis of chloro-substituted analogs of Thiazole orange – Fluorophores
for flow cytometric analyses
b
b
b
a
S. Kaloyanova a, , I. Ivanova , A. Tchorbanov , P. Dimitrova , T. Deligeorgiev
⇑
a Department of Applied Organic Chemistry, Faculty of Chemistry, University of Sofia, 1164 Sofia, Bulgaria
b Department of Immunology, Stefan Angelov Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis, absorption and fluorescence properties of a series of asymmetrical monomethine cyanine dyes,
chloro-containing analogs of Thiazole orange, are reported. Their staining ability was studied by flow
cytometry. The saturating concentrations of each dye that gives a stable staining intensity have been
determined. The ability of dyes B9, B11, B13 to stain live macrophages and apoptotic splenocytes was
investigated. Positive signal in nucleus of adherent macrophages detected by fluorescent microscopy
showed good specificity of B9, B11 and B13 dyes for DNA. In apoptotic assay cells positive for Annexin
V were stained more brightly with the dyes B9, B11 and B13 than with propidium iodide. Despite that
B13 showed high DNA selectivity it induces apoptosis of splenocytes and it is not suitable for detection
of dead cells. The other synthesized chloro-containing analogs of Thiazole orange B9 and B11 can be suc-
cessfully used for flow cytometric analyses of DNA content in live cells and for analyses of cell apoptosis.
Ó 2011 Elsevier B.V. All rights reserved.
Received 24 November 2010
Received in revised form 17 February 2011
Accepted 15 March 2011
Available online 8 April 2011
Keywords:
Thiazole orange analogs
DNA-binding dye
Apoptosis
Flow cytometry
Fluorescence
1. Introduction
(FCM) remains the methodology of choice to rapidly study the
apoptotic cascades in relation to cell type and time [9].
Optical probes for labeling biomolecules have been widely used
in science and in clinical analysis. Due to their excellent staining
properties cyanine dyes have been utilized in biomedical screening
techniques [1,2] as fluorescent probes in real-time PCR and flow
cytometry. They have also been used for DNA fragment sizing, to
detect nucleic acids by fluorescent microscopy and for visualiza-
tion in gel electrophoresis [3–8]. An advantage of cyanine dyes is
that by synthesizing structural modifications of the chromophore
moiety, different fluorescent labeling reagents can be designed,
which absorb and fluoresce light in the visible and near infrared re-
gion of the spectrum. This structural versatility permits control
over factors such as the solubility of the dye and its selectivity to
the labeled material.
Apoptosis, or programmed cell death, is a general mechanism
for removal of unwanted cells from the immune system. The char-
acteristic morphological signs of apoptosis are blebbing, loss of cell
membrane asymmetry and attachment, cell shrinkage, nuclear
fragmentation, chromatin condensation and DNA cleavage. All
these events are the final result of complex biochemical cascades
involving endonuclease activation, expression of caspases and of
surface apoptotic markers. Techniques designed to identify, quan-
tify and characterize apoptosis are numerous, but flow cytometry
One of the early and characteristic events in apoptosis is DNA
cleavage. In flow cytometry, DNA fragments can be detected by
various DNA dyes [10]. The dyes used in apoptotic assay must ful-
fill several general requirements: (a) the dyes should be specific for
nucleic acids; (b) the dyes should have a strong degree of fluores-
cence enhancement upon binding to the particular nuclear target;
(c) the dyes should not be toxic for the cell; (d) the dyes should be
excluded by live cells but not by apoptotic cells.
DNA dyes can be used also for determination of DNA content in
live cells. Ethidium bromide and propidium iodide (PI) were among
the first dyes used for DNA content determination. They fluoresce
after non covalent association with the nuclear target. Neither dye
demonstrates selectivity to the nucleotide composition or DNA
strand [11]. In order to impose correct DNA quantitation on a
per-cell basis, the dyes must fulfill other criteria than the dyes used
for detection of apoptosis: (a) the dyes should have DNA selectiv-
ity; (b) after cell staining, the fluorescence emission of the dyes
should be proportional to the cellular DNA content; (c) the dyes
should easily enter the cell.
In the present study, we report the synthesis of a series of
chloro-containing analogs of the commercial monomethine cya-
nine dye Thiazole orange. In the design of the structures of the dyes
we aimed to obtain more hydrophobic molecules to ensure better
permeability through the cell membrane and high affinity to DNA.
The presence of a hydroxyethyl moiety affects the dyes solubility
in polar solvents. This could increase dye fluorescence in
⇑
Corresponding author. Address: 1 James Bourchier Ave, Sofia 1164, Faculty of
Chemistry, University of Sofia, Bulgaria. Tel.: +359 2 8161270; fax: +359 2 9625438.
E-mail address: ohtsk@chem.uni-sofia.bg (S. Kaloyanova).
1011-1344/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2011.03.012

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S. Kaloyanova et al. / Journal of Photochemistry and Photobiology B: Biology 103 (2011) 215–221
hydrophobic environment and will allow their use at very low con-
centrations and in combination with other dyes or dye-labelled
antibodies for multicolor flow cytometric analyses. The abilities
of the dyes to stain live and apoptotic cells have been studied. Dyes
with less permeability could be used for apoptotic assay while
these with permeability into live cells could be used for detection
of DNA content.
2.1.3. Synthesis of intermediates 5a–5d [15]
Intermediate 4a–4e (0.005 mol) was dissolved in trichloroethyl-
ene, dimethyl sulfate (DMS, 0.0055 mol) was added and the reac-
tion mixture was refluxed for 30 min. After cooling the product
5a–5d precipitated as solid and was isolated by filtration. The solid
was washed with diethyl ether. Yields 70–94%.
2.1.4. Synthesis of intermediate 7 [16]
Dimethyl sulfate (0.01 mol) was added dropwise to 4-methyl-
quinoline 6 (0.01 mol) by stirring and cooling of the reaction mix-
ture. The precipitate 7 was diluted with diethyl ether and isolated
by filtration. The solid was washed with diethyl ether. Yield 96%.
2. Materials and methods
2.1. Synthesis of starting materials and cyanine dyes
All starting materials and solvents were commercial products
from Sigma–Aldrich (Germany), except compounds 2, 4a–4d,
5a–5d, 7 and 8 which were synthesized by procedures described
elsewhere (2 [12]; 3a–3e [13]; 4a–4d [14]; 5a–5d[15]; 7, 8 [16]).
Analytical samples of the reaction products were obtained by
recrystallization in ethanol.
All products were characterized and/or compared with reported
data. 1H NMR spectra were obtained on a Brucker Avance II
600 MHz instrument in DMSO-d6 as solvent. Elemental analyses
were performed on a Vario III instrument. Melting points were
determined on a Köfler apparatus and are uncorrected.
2.1.5. Synthesis of intermediate 8 [16]
2-Bromoethanol (0.011 mol) and 4-methylquinoline 6 (0.01
mol) were mixed together and heated at 140 °C by stirring for
40 min. After cooling of the reaction mixture the product precipi-
tates as oil, which has been dissolved in 5 ml methanol and diluted
with 100 ml diethyl ether. The mixture was kept in refrigerator for
24 h and the product was isolated by filtration. Yield 73%.
2.1.6. Synthesis of dyes B9–B15
In a reaction vessel equipped with magnetic stirrer equimolar
amounts of intermediates 5a–5d (0.001 mol) and 6 or 7 (0.001
mol) were suspended or dissolved in 10 ml ethanol. N-diisopropyl-
ethylamine (0.002 mol) was added dropwise and the reaction mix-
ture was stirred at room temperature for 1 h to 1.5 h. The progress
of the reactions was monitored by TLC (Merck F 254 silica gel;
dichloromethane: methanol: acetic acid 84:15:1). The resulting
precipitate was filtered off, washed with diethyl ether and air-
dried. Dyes B9–B15 were recrystallized from ethanol.
2.1.1. Synthesis of intermediates 3a–3d
The starting materials 1a–1d (0.005 mol) and potassium
O-ethyldithiocarbonate 2 (0.0125 mol) were mixed in a 25 ml
Erlenmeyer flask with 5–10 ml of the appropriate solvent (PEG-
400 or glycerol). The reaction mixture was microwave irradiated
at 80 Watt for time periods in the range 12–18 min (Table 1).
The products (3a–3d) were precipitated by dilution with water
(50 ml), acidified to pH 3–4 with CH3COOH and isolated by
filtration.
2.2. Absorption and fluorescence measurements
Absorption spectra to determine the extinction coefficients of
the dyes were obtained on a Unicam UV 500 spectrophotometer
in methanol (10À5 mol/l). Absorption spectra of dye solutions in
10 mM Tris, 0.5 mM EDTA buffer, pH 7.4 (TE buffer), free and after
binding to dsDNA (salmon sperm, Sigma–Aldrich), were scanned
on a CECIL Aurius 3021 spectrophotometer. Fluorescence spectra
of the dye solutions free and after binding to dsDNA (salmon
sperm, Sigma–Aldrich) were scanned on a PerkinElmer LS45 fluo-
rescence spectrometer in TE buffer at room temperature using
2.1.2. Synthesis of intermediates 4a–4e [14]
Intermediate 3a–3d (0.005 mol) was dissolved in acetone,
N-ethyldiisopropylamine (0.005 mol) and methyl iodide (0.005
mol) were added. The reaction mixture was sonicated at a
frequency of 40 kHz at room temperature for 5–10 min (TLC
monitoring, dichloromethane:n-heptane, 4:1). The product was
precipitated by dilution with water (50 ml) and isolated by filtra-
tion. Yields 79–95%.
Table 1
Starting compounds, reaction conditions and isolated yields for products 3a–3d.
Starting compound
Product
Irradiation time (min)
2
Power (W)
45
Yield (%)
82
Melting point (°C)
259–261 °C/262–265 °C [20]
NH2
Cl
Cl
S
N
3a
3b
SH
SH
1a
Cl
5
30
79
189–191/191–193 °C [20]
NH2
S
N
Cl
1b
Cl
Cl
Cl
3
4
40
40
78
95
239–241/242 °C [21]
NH 2
Cl
S
N
1c
3c
SH
Cl
NH2
Cl
210–212/212–216 °C [22]
Cl
Cl
S
N
3d
SH
1d
Cl
Cl

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S. Kaloyanova et al. / Journal of Photochemistry and Photobiology B: Biology 103 (2011) 215–221
217
10 mm path-length quartz cuvettes. Dye stock solutions (1 mM)
were prepared in DMSO and these were diluted with TE buffer to
a final dye concentration of 1 Â 10À7 M. A stock solution of salmon
(1 Â 10À11 M) or with PI (50
lg/ml). After 4 times washing with
PBS solution, slides with cells were let to dry, cover with GLC
mounting medium (Sacura Finetek, Tokyo, Japan) and examined
by fluorescent microscope (BM-180 T/PL, Boeco, Hamburg, Ger-
many) at magnification 1 Â 100.
sperm dsDNA in TE buffer with a concentration of 50 lg/ml was
prepared and an appropriate amount was added to each dye solu-
tion in TE buffer. The final dsDNA concentration was 9.54 Â 10À6
M
b.p., yielding a molar ratio of dye – dsDNA bp of 1:100. The mix-
tures were incubated at room temperature for 15 min before spec-
troscopic measurement.
2.6. Apoptosis
Freshly isolated splenocytes at a concentration of 2 Â 106 cells/
ml in RPMI-1640 were incubated with 2 lM cyclophosphamide
2.3. Cell isolation
(Sigma–Aldrich, Germany) for 4 h at 37 °C, 5% CO2. The cells (un-
treated (control cells) or treated with apoptotic agent) were then
Peritoneal cells were isolated by washing peritoneal cavity of
ICR mice with 10 ml sterile phosphate-buffered saline (PBS; Lonza,
Basel, Switzerland). After centrifugation at 1200g cells were re-sus-
pended at a concentration of 2 Â 106 cells/ml in RPMI-1640 med-
ium (Lonza) containing 5% fetal calf serum (FCS; Sigma–Aldrich)
and were let to adhere in 6 well plates (TPP, Trasadingen, Switzer-
land) with cover slides for 1 h at 37 °C, 5% CO2. The nonaherent cells
were removed by two times washing with warm RPMI-1640 med-
ium and adherent cells were used for immunefluorescent analyses.
Spleens were removed from ICR mice, placed in petri dishes
with 5 ml RPMI-1640 medium and smashed through the cell stai-
ner (BD Falcon, Germany). The resultant cell suspension was col-
lected and washed with PBS. Erythrocytes were lysed in buffer
containing 155 mM NH4Cl, 10 mM KHCO3, 2 mM EDTA (pH 7.4).
After washing twice with PBS, splenocytes were counted and re-
suspended at a concentration of 2 Â 106 cells/ml in PBS containing
2% FCS and were used for flow cytometry analyses and apoptotic
assay.
collected, washed and re-suspended in 500
l
l of 1Â binding buffer
(e-Biosciences, Vienna, Austria). Propidium iodide (PI; 5 ll) or B9,
B11, B13 dyes (5
l
l; 1 Â 10À9 M) and annexin V-PerCP-eFluor710
(5
ll; eBiosciences, Vienna, Austria) were added to the cells for
5 min, at room temperature, in the dark. Control samples were un-
stained or single stained with the dyes. Cells were analyzed for
apoptosis within 15 min using flowcytometry.
3. Results and discussion
3.1. Synthesis and structural analysis of dyes D1–D16
Monomethine cyanine dyes are generally synthesized by the
reaction of two heterocyclic quaternary salts, bearing respectively
an alkylthio and a methyl group [17]. Intermediates 5a–5d were
prepared according to the reported procedures [13–15] (Scheme 1).
Recently, we simplified the procedure for the preparation of
2-mercaptoheterocycles suitable as starting compounds for
monomethine cyanine dye synthesis. Substituted 2-merca-
ptobenzothiazoles were obtained in excellent yields by cyclization
under microwave irradiation using o-haloanilines and potassium
O-ethyldithiocarbonate as starting materials (Scheme 1 and Table
1). The reaction was carried out using low power microwaves for
short reaction times, without use of any catalysts and in glycerol
or PEG 400, which are confirmed as eco-friendly solvents [18,19].
Intermediates 7 and 8 were synthesized by quaternization of
4-methylquinoline (6) under solvent-free conditions with DMS
and bromoethanol using modified procedures [16] (Scheme 2).
The intermediates 5a–5d, 7 and 8 are hygroscopic and their
structures were confirmed along with the dyes’ structures by 1H
NMR and elemental analyses.
2.4. Flow cytometry
Freshly isolated splenocytes at a concentration of 2 Â 105 in
100 ll of 2% FCS/PBS solution were incubated for 15 min at room
temperature, in the dark with 100 ll of appropriately diluted dyes
(dilutions ranged from 10À6 M to 10À13 M). After washing 4 times
in 2% FCS/PBS solution the cells were analysed in a flow cytometer
(BD™LSR II) using FCS Express™ Diva Software (Beckton and
Dickinson, NJ, USA). For detecting the emission of the dyes the fol-
lowing detection channels were used: FL-1 (Ex = 488 nm; Em = 530
nm), FL-2 (Ex = 480; Em = 578 nm) 4 and FL-3 (Ex = 490 nm; Em =
675 nm).
2.5. Immunofluorescence
The series of monomethine cyanine dyes were synthesized in
good to high yields with a high purity by condensation of quater-
nary 2-methylthiochlorobenzothiazolium salts 5a–5d with quater-
nary 4-methylqunolinium salts 7 and 8 in the presence of a basic
Adherent peritoneal macrophages were washed with PBS and
incubated for 15 min at room temperature, in the dark with dyes
Scheme 1. Synthesis of intermediates 5a–5d.
Scheme 2. Synthesis of intermediates 7 and 8.

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agent (N-diisopropylethylamine) in ethanol, at room temperature
(Scheme 3). The dyes obtained were isolated as crude products
by direct filtration, which were then dissolved in ethanol (or meth-
anol) and isolated as iodides upon dilution with KI/H2O solution.
Scheme 3. Synthesis of dyes B9–B15.
Table 2
Reaction yields, melting points and elemental analysis of the dyes examined.
Dye Molecular formula
Mw
Yield/reported yield
(%)
Melting point/reported M. p.
(°C)
Elemental analysis
C% calc./
found
H% calc./
found
N% calc./
found
S% calc./
found
B9
C
C
C
C
C
C
C
19H16ClIN2SÁH2O
20H18ClIN2OSÁH2O
19H16ClIN2S
484.78 62/54 [23]
514.81 80/31 [24]
466.77 78.5/not reported [23]
514.81 93
>300
47.07/47.49
3.74/3.95
3.92/4.13
3.46/3.13
3.92/4.39
4.00/4.01
4.16/4.00
3.92/4.15
5.78/5.77
5.44/6.05
6.00/5.58
5.44/5.31
5.56/5.37
5.25/5.12
5.44/5.01
6.61/6.96
6.22/6.84
6.87/6.80
6.23/6.20
12.74/12.31
12.02/12.47
6.23/6.21
B10
B11
B12
B13
B14
B15
296–298 decomp./302–304 [23] 46.66/47.12
>300
294–297 decomp.
>300
>300
>300
48.89/48.49
46.66/46.14
47.72/47.18
47.28/46.88
46.66/46.20
20H20ClIN2O2ÁH2O
20H20Cl2N2O5S2ÁH2O 503.42 43
21H22Cl2N2O6S2ÁH2O 533.45 51
20H20ClIN2O2ÁH2O
514.81 77
Table 3
Structures and 1H NMR analysis of dyes B9–B15.
Dye Dye structure
1H NMR (600 MHz, DMSO-d6, d(ppm))
B9
3.95 t (3H, N+CH3); 4.18 t (3H, NCH3); 6.85 s (1H, ArH); 7.30 d (1H, ArH); 7.60 dd (1H, ArH); 7.70 d (1H, ArH);
7.79 t (1H, ArH); 8.00–8.06 m (2H, ArH); 8.12 d (1H, ArH); 8.65 d (1H, ArH); 8.77 d (1H, ArH);
Cl
S
N
C
H
N
CH3
CH3
I
B10
3.78 d (2H, NCH2); 3.91 s (3H, NCH3); 4.63 t (2H, CH2OH); 5.07 t (1H, OH); 6.83 s (1H, CH); 7.32 d (1H, ArH);
7.57 dd (1H, ArH); 7.67–7.73 m (2H, ArH); 7.92 t (1H, ArH); 8.10–8.15 m (2H, ArH); 8.52 d (1H, ArH); 8.74 d
(1H, ArH);
Cl
S
C
H
N
N
N
CH2CH2OH
N
CH3
I
I
I
B11
B12
B13
3.97 t (3H, N+CH3); 4.21 t (3H, NCH3); 6.91 s (1H, ArH); 7.38 d (1H, ArH); 7.43 dd (1H, ArH); 7.79 d (1H, ArH);
7.91 t (1H, ArH); 8.03–8.06 m (2H, ArH); 8.09 d (1H, ArH); 8.70 d (1H, ArH); 8.82 d (1H, ArH);
S
C
H
CH3
N
CH3
Cl
Cl
3.85 d (2H, NCH2); 3.97 s (3H, NCH3); 4.69 t (2H, CH2OH); 5.17 t (1H, OH); 6.90 s (1H, CH); 7.38–7.44 m (1H,
ArH); 7.78 t (1H, ArH); 7.90–8.03 m (2H, ArH); 8.20 d (1H, ArH); 8.58 d (2H, ArH); 8.79 d (1H, ArH); 8.83 d
(1H, ArH);
S
C
H
CH2CH2OH
N
CH3
3.94 t (3H, N+CH3); 4.23 t (3H, NCH3); 6.89 s (1H, CH); 7.37 d (1H, ArH); 7.82 t (1H, ArH); 8.07 m (2H, ArH);
8.12 d (1H, ArH); 8.30 s (2H, ArH); 8.73 d (1H, ArH);
Cl
Cl
S
C
H
N
N
CH3
N
CH3
CH3SO4
B14
B15
3.85 d (2H, NCH2); 3.94 s (3H, NCH3); 4.73 t (2H, CH2OH); 5.15 t (1H, OH); 6.87 s (1H, CH); 7.38 d (1H, ArH);
7.78 t (1H, ArH); 7.81–8.04 m (2H, ArH); 8.20–8.29 m (2H, ArH); 8.64 d (1H, ArH); 8.80 d (1H, ArH);
Cl
Cl
S
C
H
CH2CH2OH
N
CH3
I
3.85 d (2H, NCH2); 3.99 s (3H, NCH3); 4.75 t (2H, CH2OH); 5.18 t (1H, OH); 6.90 s (1H, CH); 7.48 dd (2H, ArH);
7.54 t (1H, ArH); 7.79 d (1H, ArH); 7.81 t (1H, ArH); 8.00 t (1H, ArH); 8.02 d (1H, ArH); 8.61 d (1H, ArH); 8.82
d (1H, ArH);
Cl
S
N
C
N CH2CH2OH
H
CH3
I