Halogen-containing thiazole orange analogues - New fluorogenic DNA stains

Article abstract of DOI:10.3762/bjoc.13.283

Novel asymmetric monomeric monomethine cyanine dyes 5a-d, which are analogues of the commercial dsDNA fluorescence binder thiazole orange (TO), have been synthesized. The synthesis was achieved by using a simple, efficient and environmetally benign synthetic procedure to obtain these cationic dyes in good to excellent yields. Interactions of the new derivatives of TO with dsDNA have been investigated by absorption and fluorescence spectroscopy. The longest wavelength absorption bands in the UV-vis spectra of the target compounds are in the range of 509-519 nm and these are characterized by high molar absorptivities (63000-91480 L·mol^-1·cm^-1). All investigated dyes from the series are either not fluorescent or their fluorescence is quite low, but they become strongly fluorescent after binding to dsDNA. The influence of the substituents attached to the chromophores was investigated by combination of spectroscopic (UV-vis and fluorescence spectroscopy) and theoretical (DFT and TDDFT calculations) methods.

Full text of DOI:10.3762/bjoc.13.283

Halogen-containing thiazole orange analogues – new
fluorogenic DNA stains
Aleksey A. Vasilev*1, Meglena I. Kandinska2, Stanimir S. Stoyanov2,
Stanislava B. Yordanova2, David Sucunza3, Juan J. Vaquero3, Obis D. Castaño3,
Stanislav Baluschev4 and Silvia E. Angelova*3,5
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2017, 13, 2902–2914.
1Department of Pharmaceutical and Applied Organic Chemistry,
doi:10.3762/bjoc.13.283
Faculty of Chemistry and Pharmacy, Sofia University “St. Kliment
Ohridski”, 1 James Bourchier Blvd., 1164 Sofia, Bulgaria,
Received: 01 August 2017
Accepted: 15 December 2017
Published: 28 December 2017
2Department of Organic Chemistry and Pharmacognosy, Faculty of
Chemistry and Pharmacy, Sofia University “St. Kliment Ohridski”, 1
James Bourchier Blvd., 1164 Sofia, Bulgaria, 3Departments of
Organic and Physical Chemistry, University of Alcala, 28871-Alcala
de Henares, Madrid, Spain, 4Max Planck Institute for Polymer
Research, Ackermannweg 10, 55128 Mainz, Germany and 5Institute
of Organic Chemistry with Centre of Phytochemisty, Bulgarian
Academy of Sciences, 1113 Sofia, Bulgaria (permanent address)
Associate Editor: K. N. Allen
© 2017 Vasilev et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Aleksey A. Vasilev* - ohtavv@chem.uni-sofia.bg; Silvia E. Angelova* -
sea@orgchm.bas.bg
*
Corresponding author
Keywords:
cyanine dyes; DFT calculations; green synthesis; nucleic acids;
thiazole orange
Abstract
Novel asymmetric monomeric monomethine cyanine dyes 5a–d, which are analogues of the commercial dsDNA fluorescence
binder thiazole orange (TO), have been synthesized. The synthesis was achieved by using a simple, efficient and environmetally
benign synthetic procedure to obtain these cationic dyes in good to excellent yields. Interactions of the new derivatives of TO with
dsDNA have been investigated by absorption and fluorescence spectroscopy. The longest wavelength absorption bands in the
UV–vis spectra of the target compounds are in the range of 509–519 nm and these are characterized by high molar absorptivities
(
63000–91480 L·mol−1·cm−1). All investigated dyes from the series are either not fluorescent or their fluorescence is quite low, but
they become strongly fluorescent after binding to dsDNA. The influence of the substituents attached to the chromophores was in-
vestigated by combination of spectroscopic (UV–vis and fluorescence spectroscopy) and theoretical (DFT and TDDFT calcula-
tions) methods.
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Introduction
Since the discovery by Lee and co-workers [1,2] that the old PNA [18,19,21,31]) strands have been constructed by several
photographic dye thiazole orange, TO, (Scheme 1) has excel- research groups (the Krull, Kubista, Seitz and Wagenknecht
lent properties as a fluorogenic noncovalent DNA or RNA groups). The continued scientific and commercial interest in the
binder, many representatives of this class of dyes have been de- preparation and application of cyanine dyes as bio-probes
veloped [3-7].
[37,38] and our research in this area [39-41] has led us to search
for novel representatives of this interesting family of com-
pounds. The main goal of the study described here was to inves-
tigate the influence of different halogen atoms (connected
directly to the chromophore or as side groups) on the molar
absorptivity and the fluorescence intensity of a series of new
TO analogues. Photophysical properties of a series of TO ana-
logues that have fluoro- or trifluoromethyl groups connected to
the heterocyclic end groups of the chromophores have been
studied by Armitage and co-workers [42,43]. According to
these studies the fluorination of the symmetrical [42] and un-
symmetrical [43] cyanine dyes results in reduced aggregation
and significantly improved the photostability and photophysi-
Scheme 1: Chemical structures of TO and SYBR Green I – commer-
cial monomethine fluorescent dsDNA binders.
Thiazole orange does not fluoresce in the free state in solution. cal properties of the dyes.
Fluorescence appears when the rotation about the monomethine
bridge between the two heterocyclic moieties is somehow The authors [43] used an interesting modification of Brooker’s
limited [5-7]. Such a restriction occurs when TO derivatives method (Scheme 2) for the synthesis of the fluorinated TO ana-
bind to nucleic acids by intercalation between the base pairs logues but they obtained the target compound in very low
[
6,8] or presumably between individual bases in single-stranded overall yield. Generally, symmetric or unsymmetric monome-
nucleic acids and in both cases a dramatic increase of the fluo- thine cyanine dyes can be synthesized by the method of Brooker
rescence is observed. The valuable properties as nucleic acid et al. [44,45] using a condensation reaction between 2-methyl-
stains have made these dyes an irreplaceable tool in the active thiobenzothiazolium salts with 1-alkyl-4-methylquinolinium
and developing area of bioanalytical chemical research [9,10]. salts.
Cyanine dyes have found various bioanalytical applications as
soluble DNA intercalators (e.g., in living cell imaging [11] and Although Brooker’s synthetic approach usually provides high
flow cytometry [12,13], DNA fragment sizing [14,15], as reaction yields and has very low costs, the method suffers from
reporter groups in hybridization probes [16-23], single DNA serious disadvantages. One of these is the possibility of inter-
molecule fluorescence microscopy [24,25], gel staining [26], in change of the alkyl groups at the thioalkyl unit and nitrogen
real-time PCR analysis [27,28]) because of their excellent atom in the quaternized 2-alkylthio starting materials, which
staining properties. Unsymmetrical cyanines have been conju- leads to unexpected reaction products [45,46]. The other major
gated to a variety of molecules, including peptides [29], pro- disadvantage is the evolution of methylmercaptan – a toxic
teins [30], DNA [17], and DNA analogues such as peptide pollutant with a very unpleasant odor.
nucleic acid (PNA) [16,31]. TO-based chromophores assem-
Results and Discussion
bled as a structural scaffold inside nucleic acids (TO-tethered
nucleic acids) have attracted considerable attention [32]. Synthesis of intermediates and dyes
Hybridization-sensitive fluorescent probes in which TO is teth- On the basis of Green Chemistry concepts [47-49] and with the
ered to a nucleic acid: DNA [22,23,33-36], RNA [20,36] or aim of avoiding the disadvantages of Brooker’s method and
Scheme 2: Synthesis of the monofluoro-substituted dye TO-1F. Reagents, conditions and yields: (i) sodium methanethiolate (NaSCH3), 7 h, reflux;
(ii) methyl iodide, overnight, overall yield for (i) and (ii) 9%; (iii) 5-fluoro-2,3-dimethylbenzothiazolium iodide and triethylamine, rt, overnight, yield 24%.
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reducing the adverse effects of the synthesis, we report here a with the environmentally more benign and less harmful ethanol
simple, environmentally more benign (in comparison the significantly decreases the formation of blue-colored self-con-
Brooker’s method), and efficient synthetic procedure. The densation byproducts (probably a chromophore constructed
method started with the quaternization of all starting materials from quinolone and quinolinium end groups) and subsequently
(
Scheme 3), which was performed in a sealed tube under an increases the yield and the purity of the crude products of
argon atmosphere to prevent possible side reactions like oxida- interest. As mentioned previously, prolonged sonication does
tion of the corresponding N-containing heterocylic intermedi- not increase the reaction yields, probably due to the promotion
ates or self-condensation of the resulting quaternary products. of side reactions.
Carrying out the reaction in bulk without solvents allows scale-
up of the quaternary salt synthesis and prevents possible envi- Therefore, the synthesis of the dyes 5a–d was achieved accord-
ronmental pollution. The known synthetic procedure [50,51] for ing to procedure C1. In addition, our observation that lower
the target dyes (Scheme 3) was modified to obtain the model amounts of the blue-colored byproduct were obtained in ethanol
compound TO-7Cl. The condensation of 2-methylbenzothia- solution is probably related to the polarity and the basicity of
zolium salt 2a and 4,7-dichloroquinolinium methyl sulfate 4a the medium. We observed that filtration of the final dyes fol-
was conducted in water, methanol or ethanol by stirring the lowed by washing the precipitate with ethanol dissolves signifi-
reaction mixture at room temperature or by ultrasound irradia- cant amounts of the byproduct, which was not observed in
tion for not more than 40 minutes.
water or methanol. The subsequent recrystallization of the prod-
uct from ethanol leads to the isolation of pure (analytical grade)
Prolonged sonication causes heating of the samples in the ultra- final dyes (TO-7Cl and 5a–d). Furthermore, the use of Hünig’s
sonic bath and this makes external cooling difficult. In addition, base (N,N-diisopropylethylamine, DIPEA) is essential for the
the ultrasound irradiation promotes side reactions and thus leads success of the reaction because of the CH-acidity of the benzyl
to slightly lower reaction yields when compared to the room fragment, which otherwise leads to the possibility of side reac-
temperature conditions. It is well known that water is one of the tions if trimethylamine or other less hindered basic reagents are
most useful green solvents in organic synthesis. The reaction in employed.
water as a solvent provided moderate yields (procedures B1 and
B2), but the subsequent scale-up of these reactions could be Photophysical properties
favorable on using intermediates with better water solubility. Absorption
The best reaction yields for TO-7Cl were obtained on using The longest wavelength absorption maxima of the studied dyes
procedure C1 (Experimental part) with ethanol as the reaction are in the region 502–519 nm. The corresponding molar absorp-
medium. It is worth noting that replacement of the methanol tivities are in the range of 70000–92000 L·mol–1·cm–1 (Figure 1
Scheme 3: Synthesis of new halogen-containing analogues of TO. Reagents, conditions and yields: (i) DMS, 100 °C, 2 h, 2a: 81%, 2b: 74%; (ii) DMS
or benzyl bromide, 100 °C, 1 h, 4b: 97%, 4c: 93%; (iii) EtOH, DIPEA, 2.5 h, rt, TO-7Cl: 67%, 5a: 71%, 5b: 68%, 5c: 72%, 5d: 77%.
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Table 1: Absorption and emission properties of studied dyes in the absence and presence of dsDNA, calculated fluorescence enhancement factor
(
) and relative fluorescence quantum yield in presence of dsDNA with respect to TO.
Dye
Chemical structure
λmax (εmax)a
λemc
c
d
TO
502 (86100)
510 (61100)
520 (50500)
549
529
535
458
325
1
TO-7Cl
509 (63000)
513 (91480)
519 (87520)
513 (88400)
519 (70120)
550
620
627
600
602
0.62
5a
5b
5c
5d
522 (89600)
535 (83200)
520 (84900)
528 (62100)
540
550
538
550
454
1.28
0.29
0.82
0.16
38
367
41
aWavelength of maximum absorbance (nm) and molar absorptivity (L·mol–1·cm–1) in methanol. bWavelength of maximum absorbance (nm) and molar
absorptivity (L·mol−1·cm−1) in TE buffer in presence of dsDNA. cWavelength of maximum fluorescence emission (nm) in absence (λem) and presence
(
) of dsDNA. Excitation wavelength in all cases corresponds to the absorption maximum of the dye–dsDNA complex; dThe fluorescence quan-
tum yields in presence of DNA were measured by a gradient method (integrated fluorescence intensity against absorbance) using TO as a reference.
All measurements were performed under the same experimental conditions (dsDNA concentration, optical setup, etc.). These are relative values, and
should be discussed only in terms of comparison with TO.
and Table 1). The shape of the absorption bands is typical of bathochromic shift of the longest wavelength absorption
TO-based chromophores [47-51], with a well pronounced maximum in all new compounds when compared to TO. The
shoulder on the shorter wavelength slope, and it is not sensitive presence of a benzyl substituent in compounds 5a–d doesn’t
to the type of substituent in the series, thus suggesting similar have significant effect on the maxima position, slightly shifting
transition type and geometry of the Franck–Condon excited the transition further towards lower energies when compared to
state.
TO-7Cl (in the case of 5a the red shift is 4 nm and can be attri-
buted solely to this effect). The bathochromic shift is more pro-
It can be seen from the results in Table 1 that the replacement of nounced in 5b and 5d, which bear the stronger electron-with-
a hydrogen atom with an electron-withdrawing group at the C-7 drawing CF3 group, when compared to the chloro-substituted
position in the quinoline moiety of the chromophore leads to a analogues. It is also noteworthy that the presence of the Br sub-
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Beilstein J. Org. Chem. 2017, 13, 2902–2914.
fluorescence intensity is observed, even in equimolar ratios of
dye/DNA base pairs (Table 1, Figure 3). A strong hypso-
chromic shift accompanied by a dramatic change in the band
shape is observed in the fluorescence maxima of the free dyes
in TE buffer upon addition of dsDNA. The fluorescence
maxima of dye–dsDNA complexes suggest an increased rela-
tive intensity of the 0–0 transition, which further confirms the
intercalation of the ligands in all cases studied.
Figure 1: Absorption spectra of the studied compounds in MeOH
c = 1 × 10−5 M).
(
stituent in the C-5 position of the benzothiazole system does not
influence the positions of the absorption maxima (cf. 5a vs 5c
and 5b vs 5d) but does lead to a decrease in the molar absorp-
tivity (up to 20% from 5b to 5d).
The addition of dsDNA to the dye solutions in TE buffer led to
clear bathochromic (in the range 6 to 10 nm) and hypohromic
shifts and this is illustrated as an example for 5b in Figure 2.
Such a spectral change is generally accepted as evidence for the
intercalation of dye molecules into DNA [39-41].
Figure 3: Fluorescence spectra of dye 5a (c = 5 × 10–7 M) in TE buffer
and in the presence of increasing concentrations of dsDNA
(c = 0–1.4 × 10−4 M).
The measured enhancement in the fluorescence intensity upon
binding to dsDNA at constant dye concentration (5 × 10−7 M)
and increasing dsDNA concentration (from 0 to 1.4 × 10−4 M)
is shown in Figure 3. Comparison of the spectroscopic titration
data for the series of dyes revealed that 5a has the lowest initial
intrinsic fluorescence on the one hand and the highest
fluorescence intensity in presence of dsDNA on the other,
which determines the highest fluorescence enhancement factor
of 454 for this compound. The latter value (454) is practically
identical to that of the reference TO (458), but the fluorescence
quantum yield
measured for 5a is twice as high as that
of TO-7Cl, which can be ascribed to the effect of the benzyl
substituent. While the chloro substituent itself leads to a de-
crease of the relative fluorescent quantum yield (62% for
TO-7Cl compared to TO), the benzyl group in 5a increases it
two fold, making 5a almost 30% brighter than TO under the
same conditions (Table 1). In contrast, the dyes bearing tri-
fluoromethyl groups in the 7 position of the quinoline moiety
show weak emission even in presence of host molecules. This
can be attributed to the strong electron-withdrawing effect of
the CF3 group, which change the pathways for deactivation of
the excited state [43]. The replacement of the hydrogen atom at
Figure 2: Absorption spectra of dye 5b (c = 5 × 10−7 M) in TE buffer,
in the absence and presence of dsDNA (cDNA = 1.4 × 10−4 M).
Emission
All dyes have a negligible intrinsic fluorescence in TE buffer, the C-5 position in the benzothiazole side of the molecules with
but upon addition of dsDNA a considerable enhancement in a bromo substituent does not have any significant influence on
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the fluorescence of dyes 5c and 5d, which is similar to its effect All dyes from the series demonstrate much higher photosta-
on the absorption spectra and is in contrast to the by Armitage bility upon irradiation at 532 nm by 80 mW continuous wave
et al. observed influence of the replacement of hydrogen with frequency doubled DPSS (diode pumped solid state) laser,
fluorine atom in the benzothiazole ring [43].
(Figures S21–S24, Supporting Information File 1).
Photostability
Computational studies
The photostability of all dyes in the series was evaluated in Optimized structures of the preferred conformers of the cationic
acetonitrile with irradiation at 254 nm with a mercury lamp in fragments of all dyes with the counterion I− (in the preffered
equal intervals of two minutes and the results were compared position) are presented in Figure 5 with the respective atom
with those obtained for the commercial dye TO. All dyes show color scheme.
similar photostability to TO except for dye 5c, which has a
much higher photostability than the commercial dye (Figure 4). The electronic reorganizations upon excitation were probed
using time-dependent density functional theory (TDDFT) calcu-
lations. TDPBE0 (time-dependent density functional theory
calculations using Perdew–Burke–Ernzerhof exchange-correla-
tion functional) calculations with the 6–311+G(2d,p) basis set
for all atoms except I and with the Stuttgart-Dresden SDD
effective core potential (ECP) basis set for I predict two bands
for all compounds in the range of 400–500 nm. The calculated
optical parameters such as the absorption maximum (λmax),
oscillator strength (f) and frontier orbital energy levels are pro-
vided in Table 2. The calculated λmax for the dyes are in the
following order: TO (452 nm) < TO-7Cl (460 nm) = 5a
(
460 nm) < 5c (468 nm) < 5b (468 nm) < 5d (481 nm). The
bathochromic shift of the longest wavelength maximum upon
substitution in the quinolone moiety is consistent with the ex-
perimentally observed behavior (TO-7Cl compared to TO).
There is no difference in the predicted maxima for TO-7Cl and
Figure 4: Photostability of dyes TO-7Cl and 5a–d in acetonitrile in the
concentration range of 1 × 10−5 M.
5
a, so the experimentally observed 4 nm red shift upon replace-
Figure 5: B3LYP optimized structures.
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Table 2: TDDFT/PBE0 excitation energies (eV), wavelengths (nm) (in parentheses), oscillator strength f, HOMO and LUMO energies and energy
differences (HOMO–LUMO gap) (eV) in methanol for compounds 5a–d, TO and TO-7Cl in methanol.
Compound
HOMO→LUMO
HOMO-3→LUMO
Excitation energy
f
Excitation energy
(wavelength)
f
HOMO
LUMO
HLG
(wavelength)
TO
2.74 (452)
2.70 (460)
2.70 (460)
2.64 (470)
2.65 (468)
2.58 (481)
0.777
0.647
0.595
0.327
0.403
0.218
2.90 (428)
2.82 (439)
2.83 (437)
2.77 (447)
2.78 (446)
2.73 (454)
0.137
0.310
0.471
0.703
0.714
0.862
−5.99
−6.03
−6.04
−6.08
−6.10
−6.11
−2.72
−2.80
−2.81
−2.90
−2.89
−2.98
3.27
3.23
3.24
3.18
3.21
3.14
TO-7Cl
5a
5b
5c
5d
ment of the methyl by a benzyl group in the same moiety is not Conclusion
manifested in the theoretical calculations. Theoretically calcu- Two novel cationic compounds 4b and 4c and four asymmetric
lated absorption maximа for the couples 5a/5c and 5b/5d differ monomeric monomethine cyanine dyes containing halogen sub-
by 8 nm and 11 nm, respectively.
stituents (5a–d) were synthesized by an environmentally more
benign procedure. The properties of the dyes as fluorescence
The first excited states are determined by HOMO (highest occu- probes for dsDNA detection were investigated and compared
pied molecular orbital)→LUMO (lowest unoccupied molecular with those of the commercial dye TO. In TE buffer solution the
orbital) transitions with oscillator strengths in the range of dyes from the series have negligible fluorescence themselves,
0
.218–0.777. The oscillator strengths of the HOMO-1 and but they form strongly fluorescent dye–dsDNA complexes.
HOMO-2 to LUMO transitions are very small (negligible), so Apart from dyes 5b and 5d, all other dyes demonstrated photo-
the transitions seen in the absorption spectra in the range of physical properties similar to those of TO. One of the new ana-
4
37–454 nm are actually HOMO-3→LUMO transitions. The logues (5a) shows higher fluorescence in presence of dsDNA in
oscillator strengths of HOMO-3→LUMO transitions are in the comparison with TO and this property can be used to increase
range of 0.137–0.862 and for dyes 5b–d these transitions are the sensitivity and precision in the appropriate bioanalytical
predicted to be more intense than the HOMO–LUMO ones. The methods. Higher photochemical stability was demonstrated by
HOMO–LUMO gaps are similar and range from 3.14 eV to dye 5c and this had very low fluorescence in the free state.
3
.27 eV for the compounds in methanol. The frontier orbital Computational tools were used in an attempt to explain the
energy difference for TO is the highest, thus confirming the optical properties of the studied chromophores. The results are
lower absorption wavelength of TO. In compounds 5a–d the encouraging and warrant further investigations into the newly
HOMO was found to be populated over the entire molecular synthesized dyes as DNA fluorogenic binders. A further study
system except for the benzyl fragment and the substituents in of several aspects (including quantum chemical calculations and
position 7 in the quinolone moiety. The LUMO was found to be experimental studies on the DNA binding mechanism) should
delocalized over the quinolinium and, to a lesser extent, the be carried out on these dyes in order to exploit in details their
benzothiazolium fragments (Figure 6).
promising properties.
Experimental
The simulated with Doppler broadening with a full width at
half-maximum (fwhm) of 0.25 eV and a height proportional to Computational details
the oscillator strength for each transition spectra (Figure 7) are The molecular ground state geometries of the cationic frag-
consistent with those previously reported for cyanine dye mono- ments of TO, TO-7Cl, 5a–d with I− counterions were fully op-
mers [52] and explain the inherent band structure of the mono- timized using the Becke three-parameter exchange functional
mer’s experimental spectrum with one maximum and a shoulder with the Lee–Yang–Parr correction (B3LYP) functional
at shorter wavelength.
[53,54]. We adopted the 6–31+G(d,p) [55-57] basis set for C,
H, O, N, F, Cl, and Br and the SDD [58] effective core poten-
The bathochromic shift observed for compounds 5b and 5d tial (ECP) basis set basis set for I. C1 symmetry was assumed
bearing the stronger electron-withdrawing CF3 group) is not for all systems and default convergence criteria were used; local
(
surprising in view of the higher negative electron density of the minima were verified by establishing that the Hessians had zero
cationic species located in this fragment (electron density, negative eigenvalues. The structures with the counterion were
mapped with electrostatic potential for 5a and 5b, Figure 8). optimized in methanol by means of the integral equation
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Beilstein J. Org. Chem. 2017, 13, 2902–2914.
Figure 6: Graphical representation of the frontier orbitals (isodensity plot, isovalue = 0.02 a.u.).
formalism polarizable continuum model (IEFPCM) [59]. included in TDDFT calculations. All calculations were per-
TDPBE0 calculations were performed to compute the 20 lowest formed using Gaussian 09 [60]. The PyMOL molecular
excited states of each structure [6–311+G(2d,p) basis set graphics system was used to generate the molecular graphics
for all atoms except I]. The effects of surroundings were images [61].
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Beilstein J. Org. Chem. 2017, 13, 2902–2914.
Figure 7: Simulated TDPBE0 spectra in methanol.
ysis was performed on a gas chromatograph HP 6890 GC
System Plus coupled to a mass selective detector HP 5973
MSD. The mass selective detector operated in electron impact
ionization mode at 70 eV electron energy, the ion source tem-
perature was set to 230 °C, and the quadrupole temperature was
1
5 °C. The mass scan range was 30–750 amu. Compounds 2a
and 4a were synthesized by methods described in the literature
51,62].
[
Figure 8: Electron density (isovalue = 0.002) mapped with electro-
static potential (color scheme: green for negative surface map values
and blue for the positive ones).
Synthesis
5-Bromo-2,3-dimethylbenzo[d]thiazolium methyl
sulfate (2b)
General
5-Bromo-2-methylbenzothiazole (1b, 1 mmol) and dimethyl
All solvents used in the present work were commercially avail- sulfate (DMS, 1.2 mmol) were mixed in a 50 mL sealed tube
able (HPLC grade). The starting materials 1a, 1b, 3a, and 3b equipped with a magnetic stirrer. The tube was flushed with
were commercially available and were used as supplied. argon for 5 min and was sealed. After 2 h of vigorous stirring at
Melting points were determined on a Kofler apparatus and are 100 °C the reaction mixture was cooled to room temperature
uncorrected. NMR spectra were obtained on a Bruker Avance and methanol (10 mL) was added. The mixture was heated
III 500 DRX 600 MHz spectrometer in DMSO-d6. UV–vis again until the product had fully dissolved and then cooled
spectra were measured on a Unicam 530 UV–vis spectropho- down to room temperature. Diethyl ether (30 mL) was added
tometer and the fluorescence spectra on a Cary Eclipse fluores- and the resulting precipitate was filtered off and dried in a
cence spectrophotometer (Varian, Australia). The GC–MS anal- desiccator. Yield: 74%; mp 80–83 °C; 1H NMR (500 MHz,
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Beilstein J. Org. Chem. 2017, 13, 2902–2914.
DMSO-d6) δ 3.16 (s, 3H, CH3), 4.18 (s, 3H, NCH3), 7.99 (d, Ar-H), 8.53 (d, 3JHH = 7.2 Hz, 1H, Ar-H), 8.79 (d, 3JHH = 9.27
3
JHH = 8.7 Hz, 1H Ar-H), 8.36 (d, 3JHH = 8.7 Hz, 1H, Ar-H), Hz, 1H, Ar-H) ppm; 13C NMR DEPT (135 MHz, DMSO-d6) δ
.65 (s, 1H, Ar-H) ppm. 34.5 (CH3) 42.9 (CH3), 88.9 (CH), 108.2 (CH), 113.6 (CH),
18.2 (CH), 123.4 (CH), 125.2 (CH), 127.3 (CH), 128.10 (CH),
128.70 (CH), 145.8 (CH) ppm; IR (nujol) νmax: 1601, 1490,
,7-Dichloroquinoline (3a, 1 mmol) and benzyl bromide 1468, 1367, 1321, 1203, 1160, 1115, 915, 880, 765, 725, 625
1.2 mmol) were mixed in a 50 mL sealed tube equipped with a cm−1; GC–MS m/z: 324 (100%, [M+] − CH3); elemental analy-
8
1
1
-Benzyl-4,7-dichloroquinolinium bromide (4b)
4
(
magnetic stirrer. The tube was flushed with argon for 5 min and sis (%) for C19H16ClIN2S Mw = 466.77, calcd for N, 6.00;
was sealed. After 1 h of vigorous stirring at 100 °C the reaction found: 5.57.
mixture was cooled to room temperature and acetone (10 mL)
was added. The mixture was stirred at room temperature and Procedures B1 and B2: 2a (1 mmol) and 4a (1 mmol) were
diethyl ether (30 mL) was added. The resulting precipitate was finely ground together and the mixture was transferred to a
filtered off and dried in a desiccator. Yield: 97%. The com- 50 mL reaction flask. Distilled water (15 mL) and N,N-diiso-
pound is hygroscopic and unstable and its chemical structure propylethylamine (DIPEA, 2.2 mmol) were added. The reac-
was confirmed from the structures of the final dyes.
tion mixture was vigorously stirred for 2.5 h at room tempera-
ture (B1) or sonicated for 40 min (B2). Saturated aqueous
1-Benzyl-4-chloro-7-(trifluoromethyl)quinolinium bro- potassium iodide (5 mL) was added and the resulting precipi-
mide (4c)
tate was filtered off and washed with methanol (30 mL), cold
4
-Chloro-7-(trifluoromethyl)quinoline (3b, 1 mmol) and benzyl water (30 mL), diethyl ether (10 mL) and dried in a vacuum
bromide (1.2 mmol) were mixed in a 50 mL sealed tube desiccator. The dye was purified by two recrystallizations from
equipped with a magnetic stirrer. The reaction and the work-up methanol. Yield B1: 40%; yield B2: 31%.
were performed in the same way as in the procedure above.
Yield: 93%. The compound is hygroscopic and unstable and its Procedures C1 and C2: 2a (1 mmol) and 4a (1 mmol) were
chemical structure was confirmed from the structure of the final finely ground together in a mortar and the mixture was trans-
dye.
ferred to a 50 mL Erlenmeyer flask. Ethanol (15 mL) and N,N-
diisopropylethylamine (DIPEA, 2.2 mmol) were added. The
7-Chloro-1-methyl-4-((3-methylbenzo[d]thiazol-
reaction mixture was vigorously stirred for 2.5 h at room tem-
2(3H)-ylidene)methyl)quinolin-1-ium iodide (TO-7Cl) perature (C1) or ultra-sonicated for 40 min (C2). Diethyl ether
Procedures A1 and A2: 2,3-Dimethylbenzo[d]thiazolium (20 mL) was added and the resulting precipitate was filtered off
methyl sulfate (2a, 1 mmol) and the appropriate 4,7-dichloro-1- and dissolved in boiling ethanol (60 mL). To the hot solution
methylquinolinium methyl sulfate (4a, 1 mmol) were finely was added saturated aqueous potassium iodide (5 mL). The
ground together in a mortar and the mixture was transferred to a mixture was cooled to room temperature and the resulting pre-
5
0 mL Erlenmeyer flask equipped with a magnetic stirrer and a cipitate was filtered off and washed with ethanol (20 mL) and
reflux condenser. Methanol (15 mL) and N,N-diisopropylethyl- diethyl ether (20 mL) and dried in a vacuum desiccator. The dye
amine (DIPEA, 2.2 mmol) were added. The reaction mixture was purified by two recrystallizations from ethanol. Yield C1:
was vigorously stirred for 2.5 h at room temperature (A1) or 67%; yield C2: 59%.
sonicated (A2) for 40 min. Diethyl ether (20 mL) was added
and the resulting precipitate was filtered off and dissolved in hot Synthesis of monocationic monomethine cyanine
methanol (50 mL). To the hot solution was added saturated dyes 5a–d
aqueous potassium iodide (5 mL). The mixture was cooled to 2,3-Dimethylbenzo[d]thiazolium methyl sulfate (2a, 1 mmol) or
room temperature the precipitate was filtered off, washed with 5-bromo-2,3-dimethylbenzo[d]thiazolium methyl sulfate (2b)
methanol (3 × 10 mL) and diethyl ether (10 mL) and dried in a and the appropriate 4-chloro-substituted quinolinium intermedi-
vacuum desiccator. The dye was purified by three recrystalliza- ate 4a or 4b (1 mmol) were synthesized and purified according
tions from methanol. Yield from reaction at room temperature to procedure C1. The dyes were purified by multiple recrystal-
(
Procedure A1): 43% yield from reaction with ultrasound (Pro- lizations from ethanol.
cedure A2) 41%; mp 281–283 °C; 1H NMR (500 MHz, DMSO-
d6) δ 4.01 (s, 3H, NCH3), 4.11 (s, 3H, NCH3), 6.87 (s, 1H, CH), 2-((1-Benzyl-7-chloroquinolin-4(1H)-ylidene)methyl)-3-
7
.27 (d, 3JHH = 7.2 Hz, 1H, Ar-H), 7.42 (ddd, 3JHH = 7.9 Hz, methylbenzo[d]thiazol-3-ium iodide (5a): Yield 71%; mp
JHH = 7.2 Hz, 4JHH = 0.7 Hz, 1H, Ar-H), 7.61 (ddd, 3JHH = 8.4 288–290 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.13 (s, 3H,
Hz, 3JHH = 7.3 Hz, 4JHH = 1.1 Hz, 1H, Ar-H), 7.74–7.78 (m, NCH3), 5.94 (s, 2H, NCH2Ph), 7.05 (s, 1H, CH), 7.33–7.35 (m,
H, Ar-H), 8.04 (d, 3JHH = 7.2 Hz, 1H, Ar-H); 8.09 (s, 1H, 3H, Ar-H), 7.39–7.42 (m, 2H, Ar-H), 7.45 (d, 3JHH = 6.9 Hz,
3
2
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Beilstein J. Org. Chem. 2017, 13, 2902–2914.
1
7
H, Ar-H), 7.52 (dd, 3JHH = 7.0 Hz, 4JHH = 0.9 Hz, 1H, Ar-H), 2-((1-Benzyl-7-(trifluoromethyl)quinolin-4(1H)-ylidene)-
.69 (dd, 1H, Ar-H), 7.91 (d, 3JHH = 7.5 Hz, 1H, Ar-H), 7.92 methyl)-5-bromo-3-methylbenzo[d]thiazol-3-ium iodide
(
d, 3JHH = 8.1 Hz, 1H, Ar-H), 8.15 (d, 3JHH = 7.8 Hz, 1H, (5d): Yield 77%; mp = 298 °C dec.; 1H NMR (500 MHz,
Ar-H), 8.22 (s, 1H, Ar-H), 8.78 (d, 3JHH = 6.9 Hz, 1H, Ar-H), DMSO-d6) δ 4.02 (s, 3H, NCH3), 4.09 (s, 2H, NCH2), 5. 98 (s,
.00 (d, 3JHH = 8.9 Hz, 1H, Ar-H) ppm; 13C NMR DEPT (135 1H, CH), 6.71 (s, 1H, Ar-H), 7.04 (s, 1H, Ar-H), 7.34–7.36 (m,
MHz, DMSO-d6) δ 33.8, 56.0, 89.5, 107.6, 113.2, 122.6, 123.9, 3H, Ar-H), 7.39–7.42 (m, 1H, Ar-H), 7.47 (d, 3JHH = 7.3 Hz,
9
1
1
1
24.7, 126.1, 127.2, 127.7, 127.9, 128.5, 134.7, 136.5, 139.8, 1H, Ar-H), 7.65–7.69 (m, 1H, Ar-H), 7.95 (d, 3JHH = 8.5 Hz,
44.9 ppm; IR (nujol) νmax: 1602, 1495, 1450, 1370, 1315, 1H, Ar-H), 8.08 (d, 3JHH = 8.4 Hz, 1H, Ar-H), 8.16–8.19 (m,
110, 974, 780, 750, 725 cm−1; GC–MS (m/z): 324 (100%, 2H, CH), 8.27 (s, 1H, Ar-H), 8.86 (d, 3JHH = 7.4 Hz, 1H,
[
M+] − CH2Ph); elemental analysis (%) for C25H20ClIN2S Mw Ar-H), 9.03 (d, 3JHH = 8.8 Hz, 1H, Ar-H) ppm; 13C NMR
542.86, calcd for N 5.16; found: 5.53. DEPT (135 MHz, DMSO-d6) δ 35.00 (CH3), 57.4 (CH2), 84.1
CH), 90.7 (CH), 109.3 (CH), 117.0 (CH), 117.3 (CH), 125.2
-((1-Benzyl-7-(trifluoromethyl)quinolin-4(1H)-ylidene)- (CH), 125.6 (CH), 126.1 (CH), 126.4 (CH), 127.3 (CH), 128.1
methyl)-3-methylbenzo[d]thiazol-3-ium iodide (5b): Yield (CH), 128.9 (CH), 129.6 (CH), 146.4 (CH) ppm; IR (nujol)
8%; mp 284–285 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.13 νmax: 1602, 1515, 1460, 1455, 1365, 1325, 1220, 1090, 875,
s, 3H, NCH3), 5.94 (s, 2H, NCH2Ph), 7.06 (s, 1H, CH), 805, 790, 740, 725, 560, 530, 401 cm−1; GC–MS (m/z): 438
=
(
2
6
(
7
1
.34–7.36 (m, 3H, Ar-H), 7.39–7.43 (m, 2H, Ar-H), 7.49 (d, (100%, [M+] – CH2Ph); elemental analysis (%) for
H, 3JHH = 6.5 Hz, Ar-H), 7.52 (dd, J3HH = 6.8 Hz, 4JHH = 1.0 C26H19BrF3IN2S Mw = 655.31, calcd for N, 4.27; found: 4.52.
Hz, 1H, Ar-H), 7.69 (dd, J3HH = 6.5 Hz, 4JHH = 0.7 Hz, 1H,
Ar-H), 7.88–7.90 (m, 2H, Ar-H), 7.91 (d, 3JHH = 7.1 Hz, 1H,
Ar-H), 8.15 (d, 3JHH = 7.1 Hz, 1H, Ar-H), 8.22 (s, 1H, Ar-H),
Supporting Information
8
.77 (d, 3JHH = 6.4 Hz, 1H, Ar-H), 9.00 (d, 3JHH = 7.7 Hz, 1H,
Supporting Information File 1
Ar-H) ppm; 13C NMR DEPT (135 MHz, DMSO-d6) δ 34.9
Characterisation data for the compounds: NMR and
GS–MS spectra; validation of the theoretical computations;
photostabilities at 532 nm.
(
(
(
(
CH3) 57.1 (CH2), 90.6 (CH), 108.7 (CH), 114.3 (CH), 116.4
CH), 116.5 (CH), 122.4 (CH), 122.3 (CH), 123.6 (CH), 125.8
CH), 127.3 (CH), 128.3 (CH), 128.8 (CH), 129.0 (CH), 129.6
CH), 146.0 (CH) ppm; IR (nujol) νmax: 1602, 1503, 1450,
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-13-283-S1.pdf]
1
7
455, 1380, 1310, 1210, 1155, 1120, 1095, 860, 790, 745, 735,
20, 545 cm−1; GC–MS (m/z): 358 (100%, [M+] − CH2Ph);
elemental analysis (%) for C26H20F3IN2S Mw = 576.42, calcd Acknowledgements
for N, 4.86; found: 5.20.
The theoretical calculations were performed on the High Perfor-
mance Computing Cluster installed at IOCCP – BAS with the
-((1-Benzyl-7-chloroquinolin-4(1H)-ylidene)methyl)-5- financial support of the Bulgarian Scientific Fund under Project
bromo-3-methylbenzo[d]thiazol-3-ium iodide (5c): Yield “MADARA” (RNF01/0110, contract N_DO02-52/2008). The
2%; mp = 279–281 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.03 work was supported by the programs: “Ayudas para Estancias
s, 3H, NCH3), 5.89 (s, 2H, NCH2), 6.94 (s, 1H, CH), 7.33 (d, de Científicos y Tecnólogos Extranjeros en la Universidad de
JHH = 7.4 Hz, 2H, Ar-H), 7.36 (d, 3JHH = 7.2 Hz, 1H, Ar-H), Alcalá”, “Materials Networking” (Twining-692146), Projects
.41 (dd, J3HH = 7.5 Hz, 3JHH = 1.6 Hz, 2H, Ar-H), 7.43 (s, 1H, from the Spanish Ministerio de Economía y Competitividad
Ar-H), 7.60 (dd, 3JHH = 8.4 Hz, 3JHH = 1.5 Hz, 1H, Ar-H), 7.72 (MINECO): CTQ2016-80600-P, CTQ2014-52488-R, and Proj-
dd, 3JHH = 9.1 Hz, 4JHH = 1.7 Hz, 1H, Ar-H), 8.04 (d, 3JHH = ect from the University of Alcalá CCG2016/EXP-076.
.4 Hz, 1H, Ar-H), 8.09 (d, 3JHH = 8.0 Hz, 1H, Ar-H, 8.093 (d,
JHH = 7.1 Hz, 1H, Ar-H), 8.79 (d, 3JHH = 7.3 Hz, 1H, Ar-H), ORCID® iDs
2
7
(
3
7
(
8
3
8
.83 (d, 3JHH = 9.2 Hz, 1H, Ar-H) ppm; 13C NMR DEPT (135
Aleksey A. Vasilev - https://orcid.org/0000-0003-2199-5644
Stanimir S. Stoyanov - https://orcid.org/0000-0002-7830-1538
Silvia E. Angelova - https://orcid.org/0000-0003-4717-8028
MHz, DMSO-d6) δ 34.8 (CH3), 57.4 (CH2), 89.9 (CH), 108.8
(
(
(
CH), 116.6 (CH), 118.3 (CH), 125.0 (CH), 125.1 (CH), 127.2
CH), 127.5 (CH), 127.9 (CH), 128.6 (CH), 128.8 (CH), 128.8
CH), 129.6 (CH), 145.9 (CH) ppm; IR (nujol) νmax: 1600, References
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M+] – CH2Ph); elemental analysis (%) for C25H19BrClIN2S
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2912

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