Cell penetrating, mitochondria targeting multiply charged DABCO-cyanine dyes

Article abstract of DOI:10.1016/j.dyepig.2018.05.035

We have prepared new series of lipophilic cyanine dyes equipped with several cationic quaternary ammonium moieties. This was achieved by introducing DABCO within the structures. Furthermore, to investigate the influence of the additional cationic charges on the cellular uptake, DABCO was quaternized with methyl group or alkyl piperidine cation. Spectroscopic and isothermal titration calorimetry studies as well as MTT assay and subcellular localization using confocal laser scanning microscopy revealed that some of the presented dyes combine low anti-proliferative effect with efficient cellular uptake, high affinity towards ds-DNA/RNA and remarkable fluorescent marking of mitochondria.

Full text of DOI:10.1016/j.dyepig.2018.05.035

Accepted Manuscript
Cell penetrating, mitochondria targeting multiply charged DABCO-cyanine dyes
Atanas Kurutos, Iva Orehovec, Dijana Saftić, Lucija Horvat, Ivo Crnolatac, Ivo
Piantanida, Todor Deligeorgiev
PII:
S0143-7208(18)30322-X
10.1016/j.dyepig.2018.05.035
DYPI 6764
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 9 February 2018
Revised Date: 16 May 2018
Accepted Date: 16 May 2018
Please cite this article as: Kurutos A, Orehovec I, Saftić D, Horvat L, Crnolatac I, Piantanida I,
Deligeorgiev T, Cell penetrating, mitochondria targeting multiply charged DABCO-cyanine dyes, Dyes
and Pigments (2018), doi: 10.1016/j.dyepig.2018.05.035.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Cell penetrating, mitochondria targeting multiply charged DABCO-cyanine dyes
Atanas Kurutosa†, Iva Orehovecb†, Dijana Saftićb, Lucija Horvatc, Ivo Crnolatacb*, Ivo
Piantanidab*, Todor Deligeorgievd
a Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of
Sciences, Acad. G. Bonchev str., bl. 9, 1113 Sofia, Bulgaria
b Laboratory for Biomolecular Interactions and Spectroscopy, Division of Organic Chemistry
and Biochemistry, Ruđer Bošković Institute, Bijenička c. 54, 10000 Zagreb, Croatia
c Laboratory for Molecular Plant Biology and Biotechnology Division of Molecular Biology,
Ruđer Bošković Institute, Bijenička c. 54, 10000 Zagreb, Croatia
d Department of Pharmaceutical and Applied Organic Chemistry, Faculty of Chemistry,
University of Sofia, 1164 Sofia, Bulgaria
*corresponding author:
E-mail: icrnolat@irb.hr; tel: +38514571326; fax: +38514680195
E-mail: pianta@irb.hr; tel: +38514571326; fax: +38514680195
† Authors share equal contribution to the first position.
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1. Introduction
N
N
O
N
N
N
N
N
B1
N
O
N
I
I
I
Br
N
Br
I
I
A
N
O
N
N
S
N
I
N
N
N
I
I
I
I
I
C
B2
Figure 1 Structures of the studied DABCO-cyanine dyes
In recent years, structurally diversified low molecular weight derivatives that are able to
recognize and delineate tiny structural distinctions of DNA/RNA have attracted increasing
interest, due to their various potential applications in many areas [1-5]. Therefore, the design
and discovery of new low molecular weight molecules targeting DNA/RNA has been a
subject of extensive studies. The underlying noncovalent interactions involved in these
processes of specific recognition mainly combine few different binding modes, among which
intercalation is highly represented [6]. Within this context, aromatic cationic dyes with the
ability to penetrate through the cell membranes are of great interest. The tendency of such
lipophilic cationic dyes to preferentially retain in the mitochondrial space makes them
important group of fluorescent probes for the study of mitochondrial lipid bilayer, membrane-
permeability and specific staining of these organelles [7-9]. Shortcomings of such dyes are
most commonly related to effects on mitochondrial respiration (electron transport chain
activity), (photo)chemical stability (photobleaching), selectivity and high toxicity [10-14].
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Although beneficial for the photo-chemotherapeutic applications, the singlet oxygen, O2 (1∆g),
as a reactive intermediate generated from energy transfer causes severe mitochondrial
dysfunction [15]. Consequently, to overcome problems of probing the morphology and
functions of mitochondria, the development of novel dyes with improved photochemical
features is still a huge challenge. In that sense, 1,4-diazabicyclo[2.2.2]octane (DABCO) has
been shown to be an efficient electron transfer quencher able to quench the singlet oxygen,
stabilize fluorescence and reduce photobleaching effect [16]. Moreover, the DABCO-based
species, with the ability to have two positive charges (with both nitrogen atoms reacted), have
higher fixed charge density than other commonly used cationic antimicrobial agents. This
increases their ability to specifically target mitochondria due to their high membrane potential
∆ψmito = 150-180 mV [8].
Encouraged by our previous research efforts in design of specific mitochondrial fluorescent
probes based on benzoxazolium and benzothiazolium derived dicationic monomethine
cyanine dyes [17-19], we have prepared new series of lipophilic cyanine dyes equipped with
several cationic quaternary ammonium moieties. This was achieved by introducing DABCO
within the structures. Furthermore, to investigate the influence of the additional cationic
charges to the mitochondrial uptake, DABCO was quaternized with methyl group or alkyl
piperidine cation. Spectroscopic and isothermal titration calorimetry studies as well as MTT
assay and subcellular localization using confocal laser scanning microscopy revealed the
presented dyes combine very low cytotoxicity with efficient cellular uptake and remarkable
fluorescent marking of mitochondria.
2. Materials and methods
2.1. Materials
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Unless otherwise stated, all starting materials and solvents required for the synthesis of the
DABCO cyanine dyes were purchased from Sigma-Aldrich, Organica Feinchemie GmbH
Wolfen, Fluka, Alfa-Aesar, TCI Europe, Deutero GmbH, and used without any further
purification. The solvents used for the spectroscopic analyses were purchased form Macron
Fine Chemicals TM. All other starting materials and solvents were commercial products of
analytical grade and were used without further purification.
2.2 Synthesis and spectroscopic analysis of the dyes
2.3. Analysis methods and equipment
All products were characterized using various spectroscopic techniques. The progress of the
reactions was monitored employing TLC (Merck F 254 silica gel; chloroform: methanol:
acetic acid - 80:15:5). Recrystallization from methanol yielded analytical samples of the title
1
cyanines. H-NMR and APT-NMR spectra of the compounds were recorded on a Brucker
Avance III 500 MHz instrument in DMSO-d6 at room temperature. The chemical shifts were
shown in ppm downfield from trimrthylsilane (TMS), or using the residual dimethyl sulfoxide
1
signal (d = 2.50 in H NMR, d = 39.51 in 13C NMR) as an internal standard. Coupling
constants J are expressed in Hz. The following abbreviations are used to describe spin
multiplicity in 1H-NMR: s = singlet, d = doublet, t= triplet, q = quartet, dd = doublet of
doublets, ddd = doublet of doublets of doublets, m = multiplet. The structures of all
intermediates were also evaluated on the target cyanine dyes. Melting point temperatures were
evaluated on a Kofler bench and are uncorrected. Absorption and steady state fluorescence
spectra of the unbound dyes were recorded in methanol solutions at room temperature, using
10-mm path-length quartz cuvettes on a Cecil Aurius CE 3021 UV-Vis spectrophotometer
and Perkin Elmer LS45 fluorescence spectrometer (fixed slits 10–10 nm) at room
temperature.
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2.4. Synthetic approach to the DABCO monomethine cyanines and intermediate products
2.4.1. Preparation of intermediates 2a-2c
1,4-diazabicyclo[2.2.2]octane DABCO (1.12 g, 10 mmol) were suspended in 30 mL diethyl
ether, and methyl iodide 1a (0.94 mL, 15 mmol) or butyl iodide 1b (1.70 mL, 15 mmol) were
added dropwise to it over 15 minutes with stirring. A white precipitate was formed, and the
reaction mixture was further stirred for 30 minutes at room temperature and filtered under
vacuum (Scheme 1). The products 2a and 2b were found to be highly hygroscopic, hence they
were stored in a desiccator.
1-methyl-1,4-diazabicyclo[2.2.2]octan-1-ium iodide (2а); yield of crude product = 87 % -
(white powder), m.p. =. 213-215 oC, lit. m.p. = 220 oC [20-23]; 1H-NMR (500 MHz, DMSO-
13
d6, δ/ppm): 3.01 (s, 3H, CH3-N), 2.98 (m, 6H, 3
×
CH2), 3.28 (m, 6H, 3
×
CH2); C-NMR (125
MHz, DMSO-d6, δ/ppm): 44.68, 50.77, 50.81, 50.84, 53.21, 53.23, 53.26;
1-butyl-1,4-diazabicyclo[2.2.2]octan-1-ium iodide (2b) [21]; yield of crude product = 83 % -
o
1
(white powder), m.p. = 36-37 C; H-NMR (500 MHz, DMSO-d6, δ/ppm): 0.93 (t, 3H, JH-H
7.4, CH3-CH2), 1.30 (sext., 2H, JH-H 7.34, -CH2-CH2-CH3), 1.65 (m, 2H, CH2), 3.02 (t, 6H, JH-
7.7, 3×CH2), 3.20 (m, 2H, CH2), 3.29 (t, 6H, JH-H 7.1, 3×
CH2); 13C-NMR (125 MHz,
H
DMSO-d6, δ/ppm): 13.5, 19.25, 23.03, 44.64, 51.42, 51.45, 51.47, 62.96, 62.98, 63.01;
1,4-diazabicyclo[2.2.2]octane 1 (5.61g, 50 mmol) and 1-(3-bromopropyl)-1-methylpiperidin-
1-ium bromide 2c (3.01 g, 10 mmol) [24] were dissolved in 50 mL methanol, and the reaction
vessel was stored in dark place at room temperature for 1 week. Subsequently, the methyl
alcohol was evaporated to 10-15 mL and 50 mL of acetone was added to it. The reaction
mixture was placed in a freezer for 12 hours, after which the precipitated product 2c was
filtered and stored in a desiccator (Scheme 1).
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1-(3-(1-methylpiperidin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octan-1-ium bromide (2c);
1
yield = 79% - (white powder), m.p. > 300 oC; H NMR (DMSO-d6, 500 MHz) δ/ppm: 1.54
(2H, m, CH2), 1.80 (4H, m, 2
×
CH2), 2.26 (2H, m, CH2), 3.05 (6H, t, JH-H 7.34, 3×CH2), 3.10
13
(3H, s, CH3-N), 3.33 (2H, m, CH2), 3.42 (12H, m, 6
×
CH2); C NMR (DMSO-d6, 125 MHz)
δ/ppm: 15.29, 19.29, 20.53, 44.61, 48.06, 51.76, 58.20, 59.90, 60.28;
2.4.2. Preparation of intermediates 3a, 3b
Products 3a and 3b (Scheme 1) and N-quaternary intermediate chromophores required for
their synthesis of the target polycationic DABCO cyanine dyes, were obtained via methods
described in the literature [25-33].
2.4.3. Synthesis of the tetracationic dye A
In a reaction vessel equipped with magnetic stirrer, (E)-2-((1-(3-iodopropyl)quinolin-4(1H)-
ylidene)methyl)-3-methylbenzo[d]oxazol-3-ium iodide 3a (1 g, 1.75 mmol) and 1-(3-(1-
methylpiperidin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octan-1-ium bromide 3c ( 3.62 g,
8.75 mmol) were dissolved in 20 mL 2-methoxyethanol, and the reaction mixture was heated
о
at 135 C for 3 hours with stirring. After cooling down to room temperature, 20 mL of
methanol and 5 mL water were added to the crude product. Precipitated A dye was suction
filtered and washed with diethyl ether (Scheme 1). The tetracationic product was purified by
triple recrystallization from methanol.
2.4.4. Synthesis of the tricationic B1, B2, and C cyanine dyes
(E)-2-((1-(3-iodopropyl)quinolin-4(1H)-ylidene)methyl)-3-methylbenzo[d]oxazol-3-ium
iodide 3a (1.14g, 2 mmol) or 2-((1-(3-iodopropyl)pyridin-4(1H)-ylidene)methyl)-3-
methylbenzo[d]thiazol-3-ium iodide 3b (1.07g, 2 mmol) and mono-N-quaternary derivative of
1,4-Diazabicyclo[2.2.2]octane 2a (1.52g, 6 mmol) or 2b (1.78g, 6 mmol) were suspended in 5
mL 2-methoxyethanol, and the reaction mixture was heated to reflux for 40 minutes with
6

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stirring. After cooling down to room temperature, 3 mL of N,N-dimethylformamide were
added, and the reaction vessel was placed in freezer for 2 hours. Precipitated DABCO dyes
were suction filtered and washed with diethyl ether (Scheme 1). The tri-cationic products
were purified by recrystallization from methanol. Chemical structures, yields and melting
point temperatures are given in Table 1.
Scheme 1. Synthetic approach to the DABCO cyanine dyes.
Table 1. Chemical structures, yields and melting point temperatures of DABCO dyes.
Dye
A
X
Heterocycle
R
A
Yield (%) a
m.p. (оC) b
O
quinoline
Br
40
249-251
O
quinoline
I
92
213-214
B1
CH3
CH3
C4C9
S
pyridine
I
I
55
87
258-260
210-212
B2
C
O
quinoline
a yield of pure product after recrystallization. b all ϑ values are uncorrected.
(E)-1-(3-(4-((3-methylbenzo[d]oxazol-3-ium-2-yl)methylene)quinolin-1(4H)-yl)propyl)-4-(3-
(1-methylpiperidin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1,4-diium
dibromide
о
1
diiodide (A); yield of crude product = 40 % - (orange solid), m.p. = 249-251 С; H-NMR
(500 MHz, DMSO-d6, δ/ppm): 1.57 (m., 2H, CH2), 1.81 (m, 4H, 2
CH2), 3.07 (s, 3H, CH3-N), 3.38 (m, 4H, 2 CH2), 3.59 (m., 2H, CH2), 3.80 (m., 2H, CH2),
3.90 (s, 3H, CH3-N), 3.97 (s, 8H, 4 CH2), 4.67 (t, 2H, JH-H 6.92, CH2), 6.35 (s, 1H, CH), 7.42
×CH2), 2.32 (m., 6H,
3
×
×
×
(td, 1H, JH-H 1.0, 8.1, ArH), 7.52 (td, 1H, JH-H 0.8, 7.8, ArH), 7.69 (d, 1H, JH-H 7.82, ArH),
7

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7.76 (m, 1H, ArH), 7.83 (d, 1H, JH-H 7.82, ArH), 7.97 (d, 1H, JH-H 7.35, ArH), 8.03 (d, 1H, JH-
8.01, ArH), 8.23 (d, 1H, JH-H 8.76, ArH), 8.51 (d, 1H, JH-H 7.35, ArH), 8.82 (d, 1H, JH-H
H
8.19, ArH); APT-NMR(75 MHz, DMSO-d6, δ/ppm): 15.59, 19.27, 20.52, 22.09, 30.65,
48.13, 50.40, 50.72, 50.78, 60.31, 60.41, 74.56, 109.13, 110.94, 117.91, 123.39, 124.53,
126.07, 126.28, 126.53, 131.34, 133.46, 137.19, 143.50, 146.11, 150.09, 161.64; UV/VIS
(methanol, 1×
10-5 mol dm-3): λmax = 482 nm, λfl = 565 nm;
(E)-1-methyl-4-(3-(4-((3-methylbenzo[d]oxazol-3-ium-2-yl)methylene)quinolin-1(4H)-
yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1,4-diium iodide (B1); yield of crude product = 92
% - (orange solid), m.p. = 213-214 оС; 1H-NMR (500 MHz, DMSO-d6, δ/ppm): 2.33 (m, 2H,
CH2), 3.27 (s, 3H, CH3-N), 3.71 (m, 2H, CH2), 3.88 (m, 15H, 6×CH2 + CH3), 4.65 (t, 2H, JH-H
7.15, CH2), 6.34 (s, 1H, CH), 7.42 (m, 1H, ArH), 7.51 (m, 1H, ArH), 7.68 (d, 1H, J 7.95,
ArH), 7.76 (m, 1H, ArH), 7.82 (d, 1H, JH-H 8.07, ArH), 7.97 (d, 1H, JH-H 7.34, ArH), 8.00 (m,
1H, ArH), 8.17 (d, 1H, JH-H 8.80, ArH), 8.45 (d, 1H, JH-H 7.46, ArH), 8.81 (d, 1H, JH-H 8.44,
ArH); APT-NMR (75 MHz, DMSO-d6, δ/ppm): 19.84, 21.98, 30.67, 50.42, 50.65, 52.43,
60.51, 74.58, 109.14, 110.94, 117.86, 123.44, 124.57, 126.11, 126.33, 126.56, 129.78, 131.37,
133.45, 137.15, 143.54, 146.12, 150.10, 161.66; UV/VIS (methanol, 1× =
10-5 mol dm-3): λmax
482 nm, λfl = 566 nm;
1-methyl-4-(3-(4-((3-methylbenzo[d]thiazol-3-ium-2-yl)methylene)pyridin-1(4H)-yl)propyl)-
1,4-diazabicyclo[2.2.2]octane-1,4-diium iodide (B2); yield of crude product = 55 % - (yellow
solid), M.p. = 258-260 оС; 1H-NMR (500 MHz, DMSO-d6, δ/ppm): 2.341 (m, 2H, CH2), 3.28
(m, 7H, 2
×
CH2 + CH3-N), 3.56 (m, 2H, CH2), 3.76 (s, 3H, CH3-N), 3.88 (s, 8H, 4
×
CH2), 4.31
ArH),
(t, 2H, JH-H 7.16, CH2), 6.32 (s, 1H, CH), 7.34 (m, 1H, ArH), 7.45 (d, JH-H 7.25, 2H, 2
×
7.55 (m, 1H, ArH), 7.65 (d, 1H, JH-H 7.91, ArH), 7.95 (dd, 1H, JH-H 0.6, 7.8, ArH), 8.36 (d,
1H, JH-H 7.06, ArH); APT-NMR (75 MHz, DMSO-d6, δ/ppm): 23.27, 33.03, 44.63, 50.60,
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51.51, 52.41, 54.21, 60.25, 89.79, 112.30, 122.64, 123.37, 123.77, 127.96, 140.49, 141.27,
150.56, 157.66; UV/VIS (methanol, 1×
10-5 mol dm-3): λmax = 453 nm, λfl = 487 nm;
(E)-1-butyl-4-(3-(4-((3-methylbenzo[d]oxazol-3-ium-2-yl)methylene)quinolin-1(4H)-
yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1,4-diium iodide (C); yield of crude product = 87 %
- (orange solid), m.p. = 210-212 оС; 1H-NMR (500 MHz, DMSO-d6, δ/ppm): 0.94 (t, 3H, JH-H
7.30, CH3-CH2), 1.33 (quint., 2H, JH-H 7.25, CH2), 1.67 (m, 2H, CH2), 2.33 (m, 4H, 2
×CH2),
3.24 (m, 2H, CH2), 3.50 (m, 2H, CH2), 3.71 (m, 2H, CH2), 3.87 (m, 11H, 4 CH2+CH3), 4.64
×
(t, 2H, JH-H 6.84, CH2), 6.35 (s, 1H, CH), 7.42 (m, 1H, ArH), 7.52 (m, 1H, ArH), 7.69 (d, 1H,
JH-H 7.72, ArH), 7.76 (m, 1H, ArH), 7.82 (d, 1H, JH-H 8.01, ArH), 7.97 (d, 1H, JH-H 7.35,
ArH), 8.03 (d, 1H, JH-H 7.25, ArH), 8.19 (d, 1H, JH-H 8.76, ArH), 8.45 (d, 1H, JH-H 7.35, ArH),
8.82 (d, 1H, JH-H 8.48, ArH); APT-NMR (75 MHz, DMSO-d6, δ/ppm): 13.47, 18.99, 22.0,
23.31, 30.66, 50.41, 50.75, 60.47, 63.24, 74.56, 109.13, 110.96, 117.88, 123.41, 124.54,
126.08, 126.29, 126.29, 126.54, 131.35, 133.43, 137.17, 143.50, 146.10, 150.09, 161.64;
UV/VIS (methanol, 1×
10-5 mol dm-3): λmax = 482 nm, λfl = 565 nm;
2.5 Study of DNA/RNA interactions
2.5.1 UV/Visible Spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy
The UV/Vis spectra were recorded on a Varian Cary 100 Bio spectrophotometer;
fluorescence spectra on a Varian Cary Eclipse fluorescence spectrophotometer and CD
º
spectra were collected with a Jasco J-810 spectropolarimeter at 25 C using 1 cm path quartz
cuvettes.
The polynucleotides: p(dAdT)2, p(dGdC)2, p(rA-rU), and calf thymus (ct)-DNA (Sigma-
Aldrich, St. Louis, USA) were dissolved in sodium cacodylate buffer, I = 0.05 mol dm-3,
pH=7, ct-DNA was additionally sonicated and filtered through a 0.45 µm filter. Aqueous
solutions of compounds were buffered to pH=7 (sodium cacodylate buffer, I = 0.05 mol dm−3).
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The polynucleotide concentration was determined as the concentration of phosphates by UV
absorption [34]. Spectrophotometric titrations were performed at pH = 7.0 (sodium cacodylate
buffer, I = 0.05 mol dm-3) by adding portions of polynucleotide solution into the solution of
the studied compound. Thermodynamic equilibrium is reached within a minute of
polynucleotide addition to dye and 2-3 min incubation time was maintained through all the
experiments.
In fluorescence spectroscopy experiments the excitation wavelength above 450 nm (λexc
= 482 nm; λexc (B2) = 452 nm; excitation slits 5 nm) was used to avoid the possible
(A; B1; C)
inner filter effect caused by increasing absorbance of the polynucleotide. The emission spectra
were collected in the range λem= 490 – 650 nm and λem= 460 – 650 nm respectively, slits were
set to 5 nm in both cases. The CD experiments were performed by adding aliquots of the
aqueous solutions of the compounds into the buffered solution of polynucleotide. The titration
data was processed using Scatchard equation by fitting fluorescence data with Origin 7.0
software package.
2.5.2 Thermal denaturation experiments
Thermal melting curves for p(dAdT)2, p(rA-rU) and ct-DNA and their complexes with
studied compounds were determined by following the change in the absorption at 260 nm as a
function of temperature. The absorbance of the ligands was subtracted from each curve and
the absorbance scale was normalized. Tm values are the midpoints of the transition curves,
determined from the maximum of the first derivative and checked graphically by the tangent
method. The ∆Tm values were calculated subtracting Tm of the free nucleic acid from Tm of
the complex. The ∆Tm values (with the instrumental error ± 0.5 °C) reported are the average
of at least duplicate measurements.
2.5.3 Isothermal titration calorimetry (ITC)
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Titrations were performed on MicroCal VP-ITC (Malvern Panalytical, UK), the
polynucleotide solutions (ct-DNA, p(dAdT)2, p(dGdC)2 prepared as in Section 2.1.1. The
compounds were prepared from dimethyl sulfoxide (DMSO) stock solutions, dissolving them in
50 mM sodium cacodylate buffer, pH 7.0, resulting in c = 100×10−6 mol dm−3 of the dyes and 1%
DMSO in the buffer. To avoid buffer mismatch, 1% DMSO was added to the polynucleotide
solution. Origin 7.0 software, supplied by the manufacturer was used for data analysis. The
reference cell was filled with ultrapure water. In the experiments, one aliquot of 2 µL five aliquots
of 5 µL and 24 aliquots of 10 µL of the compound A, B1, B2, or C were injected from a rotating
syringe (220 rpm) into the isothermal cell, equilibrated at 25.0 °C, containing 1.4406 mL of the
polynucleotide (c = 30×10−6 mol dm−3).
The spacing between each injection was in the range 240–300 s. The initial delay before the
first injection was 2000 s in all experiments. All solutions used in ITC experiments were degassed
prior to use under vacuum (0.64 bar, 10 min) to eliminate air bubbles.
Microcalorimetric experiment directly gave three parameters: reaction enthalpy change (∆rH),
binding constant (Ks) and stoichiometric ratio (N), number of dye molecules bound per
polynucleotide. The value of ∆rG was calculated from the binding constant (∆rG= −RT ln K) and
the reaction entropy change was calculated from the binding enthalpy and Gibbs energy (∆rS=
(∆rH− ∆rG)/T).
2.6 Biological assays
2.6.1 Subculturing of cells
Adherent human cell lines (H460, non-small cell lung cancer) growing as monolayers on
the surface of T-25 flasks (Sigma) were used. Dulbecco's Modified Eagle's medium (DMEM)
(Sigma-Aldrich) containing amino acids and vitamins, as well as additional supplementary
components were used for cell growth (complete medium). The primary cell culture media
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spent was removed and discarded from the culture flask. Cells were rinsed with 5 ml of the
pre-warmed phosphate-buffered saline (PBS) to remove protease inhibitors, and 0.1% trypsin
solution was added to detach the cells from the substrate. Flask was further placed back in an
incubator at 37 ºC. The progress of the enzyme treatment was checked with an inverted phase
contrast microscope. As the cells have rounded up and detach from the surface, 5 ml of
growth medium was added to the cell suspension and cells were vigorously washed.
Suspended cells were further centrifuged (100×g for 5 minutes) and trypsin containing
medium was replaced with the fresh complete medium. Cells were further counted using
Neubauer chamber under inverted phase contrast microscope. Considering the required
dilutions, the amount of the cells in fresh medium was transferred to new flasks.
2.6.2 Anti-proliferative activity evaluation by MTT assay
The test was performed using H460 cells. Tested compounds, (A, B1, B2, and C) were
prepared as stock solutions (4
3) were prepared in DMEM medium accordingly. Cells were seeded in a 96 micro well flat
104 cells/l and incubated overnight allowing them to
×
10-2 mol dm-3) in DMSO, working solutions (10-4-10-8 mol dm-
bottom plates at the concentration 1
×
attach to the plate surface, before exposure to the compounds. After 72 h incubation with
tested compounds, growth medium was discarded and 5 mg ml-1 of MTT was added. After 4h
incubation at 37°C water insoluble MTT-formazan crystals were dissolved in DMSO.
Absorbance was measured at 570 nm on a microplate reader (Multiskan EX, Thermo Fisher
Scientific). Control cells were grown under the same conditions, with the absence of dyes.
The experiments were performed in tetraplicates. The IG50 value, defined as compound
concentration leading to cellular viability reduction by 50%, compared to the control was
calculated and used as a comparison parameter.
2.6.3 Confocal Laser Scanning Microscopy (CLSM)
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Leica SP8X FLIM was used for fluorescence experiments in live-cell imaging (H460
cells). The images were processed in LAS X Leica Microsystems software packages. The
cells were seeded into 4-chamber 35mm glass bottom petri dishes (Cellvis, Mountain View,
USA) and incubated overnight allowing them to attach to the glass bottom of the petri dish.
Three hours prior to cell imaging, the dyes were added to the medium (c = 10-6 mol dm−3).
3. Results and Discussion
3.1 Synthesis and structural analysis of DABCO cyanine dyes
A series of 1,4-diazabicyclo[2.2.2]octane derived unsymmetrical tri- and tetra-cationic
cyanine dyes was obtained in moderate to high (40-92%) yields by heating to reflux the two
main components for 2-3 hours in a minimum amount of 2-methoxyethanol. The DABCO
moieties were obtained by mono N-quaternization using an excess of the corresponding
alkylating agent 1a-1c (either 1.5 equivalents of methyl iodide / butyl iodide or 2-fold excess
of 1-(3-bromopropyl)-1-methylpiperidin-1-ium bromide). The reactions were implemented at
room temperature, yielding the products 2a-2c (79-87% yield). The former derivatives were
subsequently attached to a monomethine dye scaffold containing a good leaving group-iodide,
aiming to introduce multicationic nature on cyanine dyes. The chemical structures of the 1,4-
1
diazabicyclo[2.2.2]octane derivatives and the target DABCO dyes, were elucidated by H-
NMR, 13C-NMR, APT-NMR spectroscopy and melting point temperatures. In the present
work, our synthetic strategy was focused on the introduction of multiple positive charges on
the main scaffold of oxazole yellow and thiazole orange derivatives in order to enhance the
affinity towards polynucleotide structures. Another variation on the chromophore possessing a
methyl group at para-position with respect to the N-quaternary nitrogen atom (pyridine or
quinoline moiety) was directed by both electronic and steric properties. Namely, the
formation of H-aggregates due to pi-stacking of the aromatic system, which can subsequently
13

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lead to alternation with respect to the binding mode (intercalation as monomer or
accumulation in the hydrophobic grooves of the double stranded polynucleotide helixes as
dimer). Hence, this alternation could potentially express selectivity towards certain nucleic
acid sequences.
3.2 Interactions of A, B1, B2 and C dyes with ds-DNA and ds-RNA.
The UV/vis spectra of the dyes aqueous solutions are proportional to their concentration
o
up to 0.01 mM concentrations and solution thermal stability upon heating to 95 C was
excellent. Due to the problems with aggregation at higher conncentrations, UV/vis titration
experiments were not used to calculate binding parameters and are not presented. Molar
extinction coefficients are given in Table 2.
Table 2. Absorption maxima and molar extinction coefficients of the dyes
Dye
λ
max (nm)
482
ε
(dm-3 mol-1 cm-1)
8.55×104
A
B1
B2
C
482
7.49×104
9.17×104
8.22×104
482
452
Dye solutions in buffer exhibit negligible fluorescence emission, however upon addition
of DNA/RNA the emission intensity increases dramatically, as shown in the figure 3 and
supp. info. The increase of the fluorescence intensity upon planarization and rigidification of
carbocyanines, due to DNA/RNA binding is well documented and studied phenomena [35,
36]. In short, by binding with biomacromolecules, cyanine dye loses the freedom of torsional
motion around the methine bridge which is the main mode of nonradiative decay of the free
dye molecule in the excited state. Thus, instead of losing absorbed energy through such
nonradiative process, the bound dye shows increased emission quantum yield [37, 38].
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250
200
150
100
50
-8
[B1] = 7.65×10 mol dm
Na cacodylate buffer, pH7
p(dGdC)2 titration
-3
0
500
525
550
575
600
625
650
Wavelength (nm)
Figure 2. Changes in fluorescence spectrum of B1 (c = 7.65×10-8 mol dm−3) upon titration with p(dGdC)2
Therefore, the intensity of fluorescence response upon DNA/RNA binding depends on
number of different factors such as: the dye structure (absorption, Table 2), but even more so
on the mode of binding (intercalation, groove binding or electrostatic interaction), magnitude
of the interaction (represented by Kd or Ks), the differences between DNA/RNA secondary
motifs, groove geometry (Table S3, Supp. info) and the local micro-environment at the
binding site.
700
600
500
ct-DNA
400
p(rA-rU)
300
p(dAdT)2
p(dGdC)2
200
100
0
A
B1
B2
C
Figure 3. The increase of the fluorescence intensity upon addition of the polynucleotide (ct-DNA, pApU,
p(dAdT)2 and p(dGdC)2) to the dye solutions (c = 1
10-7 mol dm−3 in 50 mM Na cacodylate, pH7). The increase
×
was calculated by subtraction of the intrinsic dye fluorescence intensity from the maximal measured intensity of
the formed complex [bound dye/polynucleotide], upon saturation of the binding sites.
As a result, the prediction of the fluorescence response is never straightforward and
correlating it individually to any one of these factors is not possible.
15

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To further evaluate the mode of interaction and gain more structural information on the
resulting dye-polynucleotide complex, we used CD spectropolarimetry (Figure 4, supp. info.),
sensitive method for the elucidation of conformational changes in the secondary structure of
polynucleotides [39]. Also, the dye-DNA/RNA complex often shows induced (I)CD band
coinciding with the UV/Vis absorption band of the dye, which in this case is far from the CD
bands of DNA/RNA. The appearance, sign and magnitude of such bands provides information
on the binding mode (intercalation, groove binding, agglomeration, etc.) [40].
8
6
4
2
a
b
4
dye B1
dye
C
2
0
0
-2
-4
-6
-8
-10
-12
p(rA-rU)
r = 0.1
r = 0.2
r = 0.3
r = 0.4
r = 0.5
r = 0.6
p(dAdT)2
-2
-4
-6
r = 0.08
r = 0.16
r = 0.23
r = 0.31
250
300
350
400
450
500
550
250
300
350
400
450
500
550
Wavelength (nm)
Wavelength (nm)
Figure 4. CD titration of a) p(dAdT)2 (c = 2× ×
10-5 mol dm−3), b) p(rA-rU) (c = 2 10-5 mol dm−3), with a) dye B1;
b) dye C at molar ratios r = [compound] / [nucleotide] (pH 7.0, buffer sodium cacodylate, I = 0.05 mol dm−3).
Considering that the DNA/RNA CD bands (230-290 nm) coincide with the absorption
bands of the dyes (supp. info), the changes in those bands, induced by the addition of dyes
could appear as a result of the changes in the DNA/RNA secondary structure or as a ICD
signal of a complex. Therefore the mode of binding could only be elucidated from the
appearance of ICD bands at 450-550 nm. Addition of A, B1, B2 and C dyes to ds-DNA
resulted in negative ICD band within 450-550 nm range, characteristic for the intercalative
binding mode (Figure 4a, Supp. Info). At higher ratios r (r > 0.3) dye is in the excess with
respect to the intercalative binding sites, according to the “neighbour exclusion principle”
[41]. Thus the saturation of the intercalative binding sites could be observed even at lower
ratios, represented by the absence of changes in the ICD bands. On the other hand, upon
addition of dyes to ds-RNA solution, binding event represented by bisignate CD signals at
16

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450-550 nm can be observed (Figure 4b, Supp. info), which indicates excitonic coupling of
dye aggregates [35]. After meticulous examination it can be observed that at lower ratios (r =
0.1), there is only slight positive ICD signal, the bisignate ICD appears only after more dye
has been added. This strongly signifies two different binding modes; first occuring at lower
ratios is followed by dimerization or higher scale aggregation within the ds-RNA groove,
most probably major groove, due to the unfavoured geometry of the minor groove (Table S3,
Supp. Info).
Although, binding parameters (stability constant Ks; stoichiometric binding ratio N) can
be calculated from the fluorimetric titration data, we used calorimetric titrations (ITC) to
evaluate binding of the dyes with DNA/RNA. Above mentioned binding parameters along
with the thermodynamic properties of the binding process (∆rH, ∆rG, and -T∆rS) are presented
in the Table 4. Calorimetric titration of ds-DNA/RNA sequences (mixed sequence-ctDNA,
alternating AT and GC, homopolymer rArU sequences) with the studied compounds revealed
comparable stability constants (Ks), with somewhat higher affinity for DNA/RNA binding of
A (Figure 5).
ctDNA
p(dAdT)2
1.0 107
×
p(dGdC)2
p(rA-rU)
8.0 106
×
6.0 106
×
4.0 106
×
2.0 106
×
0
A B1 B2 C
A B1 B2 C
A B1 B2 C
A B1 B2 C
Figure 5. The stability constants (Ks) obtained by ITC, using specific models and Origin 7.5 software
The binding isotherms (Figure 6, supp. info), indicate that in the case of binding of B1 and
C to p(dAdT)2 there are two independent binding sites. The first binding process, that occurs
17

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when the dye molecules are scarce (lower N ratios) is characterised by high enthalpic
contribution to the free Gibbs energy ∆rG and adverse entropic contribution (Table 4). Such
combination usually indicates intercalative binding [42]. The interaction that drives
intercalation, π-π stacking results in high enthalpic contribution, while loss of freedom of
motion results in decrease of enthropy and therefore works against free energy.
Figure 6. Final figure of the ITC titration of p(dAdT)2 (c = 3.00×10-5 mol dm−3)) with C (c = 1×10-4 mol dm−3) in
50 mM Na-cacodylate buffer, pH 7.00 at 25 °C. Raw data above, calculated binding isotherm bellow.
The entropic contribution to ∆rG is related to non-specific hydrophobic interaction,
displacement of water molecules hydrophobic pockets, such as DNA/RNA grooves [43]. So
the second binding incident has thermodynamic signature of groove binding. The enthropic
contribution drives binding process with RNA sequence. With the exception of compound A
which again shows two independent binding sites, with second one showing all the signs of
groove binding and first has a thermodynamic signature of intercalation.
Table 4 Binding parameters and thermodynamic properties of the binding process from ITC titrations
[Cell]
N
Ks
∆rH
∆rG
-T∆rS
N₂
Ks₂
∆rH
₂
∆rG₂
-T∆rS₂
(mol dm-3)
(sites) (mol-1dm3) (kJ/mol) (kJ/mol) (kJ/mol) (sites) (mol dm-3) (kJ/mol) (kJ/mol) (kJ/mol)
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A-p(dAdT)2
A-ctDNA
3.14×10-5
3.14×10-5
3.15×10-5
3.83×10-5
0.15
0.213
0.181
0.139
7.46×106
1.17×106
7.87×106
1.54×107
-9.69
-14.5
-17.6
-256
-39.3
-34.6
-39.4
-41.1
-29.6
-20.2
-21.8
215
-
-
-
-
-
-
-
-
-
-
A-p(dGdC)2
A-p(rA-rU)
0.1
1.62×107
321
-41.2
-362
[Cell]
N
Ks
∆rH
∆rG
-T∆rS
N₂
Ks₂
∆rH
₂
∆rG₂
-T∆rS₂
(mol dm-3)
(sites) (mol-1dm3) (kJ/mol) (kJ/mol) (kJ/mol) (sites) (mol dm-3) (kJ/mol) (kJ/mol) (kJ/mol)
B1- p(dAdT)2)
B1-ctDNA
2.20×10-5
3.40×10-5
3.40×10-5
3.30×10-5
0.034
0.127
0.208
0.434
4.08×105
1.19×106
4.17×106
7.09×106
-82.4
-13.2
-15.5
-9.89
-32
50.3
-21.5
-22.3
-29.2
0.244
1.79×106
-2.63
-35.7
-33.1
-34.7
-37.8
-39.1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B1- p(dGdC)2
B1-p(rA-rU)
[Cell]
N
Ks
∆rH
∆rG
-T∆rS
N₂
Ks₂
∆rH
₂
∆rG₂
-T∆rS₂
(mol dm-3)
(sites) (mol-1dm3) (kJ/mol) (kJ/mol) (kJ/mol) (sites) (mol dm-3) (kJ/mol) (kJ/mol) (kJ/mol)
B2- p(dAdT)2
B2-ctDNA
3.60×10-5
3.00×10-5
3.00×10-5
7.40×10-5
0.17
0.087
0.179
0.117
3.32×106
2.38×106
1.02×106
1.06×106
-14.8
-55.1
-24.7
-19.4
-37.3
-36.4
-34.3
-34.4
-22.4
18.7
-9.62
-15
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B2- p(dGdC)2
B2-p(rA-rU)
[Cell]
N
Ks
∆rH
∆rG
-T∆rS
N₂
Ks₂
∆rH
₂
∆rG₂
-T∆rS₂
(mol dm-3)
(sites) (mol-1dm3) (kJ/mol) (kJ/mol) (kJ/mol) (sites) (mol dm-3) (kJ/mol) (kJ/mol) (kJ/mol)
C- p(dAdT)2
C-ctDNA
3.00×10-5
2.45×10-5
3.00×10-5
3.60×10-5
0.037
0.141
0.163
0.297
1.10×106
1.38×106
3.97×106
3.73×106
-67.2
-21
-34.5
-35.1
-37.7
-37.5
32.7
-14
0.159
3.46×106
-0.733
-37.4
-36.6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
C- p(dGdC)2
C-p(rA-rU)
-23.8
-13.8
-13.9
-23.7
3.3 Biological activity, cellular uptake and distribution
We tested A, B1, B2 and C for their antiproliferative activity against human tumour cell
line H460 (Table 5). The compounds showed considerable antiproliferative effect after 3 days
of incubation at concentrations exceeding 10-5 mol dm−3 (Table 5). Therefore, to assess their
subcellular localisation in H460 cell line, we used “safe” 10-6 mol×dm−3 concentration for the
confocal microscopy live cell imaging experiment. After initial screening, we performed co-
localisation study to confirm the cellular accumulation of the dyes. We used MitoTracker™
Deep Red FM (MitoTracker, ThermoFisher Scientific) as a mitochondrial probe and Hoechst
34580 (Hoechst, Sigma Aldrich) for the staining of nuclei.
Table 5 IG50 values, compound concentrations leading to cellular viability reduction by 50%, compared to the
control.
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Dye
A
B1
B2
C
68.6 ± 2.75
125 ± 25.2
23 ± 2.19
72.5 ± 8.39
IG50 ± SD (µM)
Cells were incubated with the compounds for 2h prior to confocal microscopy, allowing
enough time to enter the cell and reach their preferred subcellular location. Considering their
structural similarity, relatively similar interaction with the DNA/RNA and similar
antiproliferative activity, their faith after cell access is remarkably different. The A and B1
dyes enter the cell and target mitochondria, showing excellent denotation of those organelles,
B2 marks nucleoli, while C seems not able to cross the cellular membrane or its fluorescence
response is somehow quenched (Figure 7). The co-localization with the commercially
available probes was used to verify these claims. MitoTracker (c = 200 nM) was added 30
min prior to visualisation, Hoechst was added 10 min before confocal microscopy. To excite
the compounds A, B1 and B2 we used 482 nm laser line, and the emission was collected with
a band pass detector 498 – 535 nm. Compound C was excited at 452 nm and emission was set
to 499 – 547 nm. The excitation wavelength for the MitoTracker was set to 647 nm, emission
was captured at 670 – 712 nm and Hoechst was excited at 405 nm, emission at 420 – 469 nm.
Co-localization of the compounds and the probes was confirmed by overlaying the
photomicrographs of the A, B1 vs. MitoTracker channels of the same specimen and B2 vs.
Hoechst 33342 (Figure 8, 9, 10).
A
B1
B2
C
Figure 7. Confocal photomicrographs of live H460 cells after incubation with 1×10-6 mol dm−3 of A, B1, B2
and C dyes.
20

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A
Figure 8. Confocal photomicrographs of live H460 cells after incubation with 1×10-6 mol dm−3 of A and
0.2×10-6 mol dm−3 MitoTracker probe, LEFT: A channel, green; RIGHT: MitoTracker channel, red; MIDDLE:
Overlay of the two channels.
B1
Figure 9. Confocal photomicrographs of live H460 cells after incubation with 1×10-6 mol dm−3 of B1 and
0.2×10-6 mol dm−3 MitoTracker probe, LEFT: B1 channel, green; RIGHT: MitoTracker channel, red; MIDDLE:
Overlay of the two channels.
B2
Figure 10. Confocal photomicrographs of live H460 cells after incubation with 1×10-6 mol dm−3 of B2 and
0.5×10-6 mol dm−3 Hoechst probe, LEFT: B2 channel, green; RIGHT: Hoechst channel, blue; MIDDLE:
Overlay of the two channels.
4. Conclusions
The A, B1, B2 and C dyes bind with high affinity to ds-DNA/RNA. The dyes
predominantly intercalate into ds-DNAs, but also exhibit certain secondary binding event with
alternating AT sequences. From CD titrations assessment we can conclude that the ds-RNA
binding site could be major groove, where dyes form aggregates. As mentioned in the
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discussion, fluorimetric response of the dye-polynucleotide complexes depends on the number
of parameters, and is therefore too complex to control and predict. The compound A shows
highest fluorescence increase upon interaction with the DNA/RNA. Even though they interact
strongly with biomacromolecules, their influence on the proliferation of cells is not so
prominent. Only at high concentrations (10-5 M or higher), after 3 days the compounds
hamper the proliferation to 50% compared to the control. With the exception of C, the dyes
enter the cell and accumulate within the mitochondrial space (A and B1) or in the nucleoli
(B2). Noteworthy, compound B2 also shows highest antiproliferation potential, as its
subcellular target seems to be DNA rich nucleus. Brilliant fluorescence denotation of the
mitochondria even at low concentrations of A and B1 dyes, absence of photobleaching effect
and relative safety for the cell homeostasis, marks this two dyes as promising fluorescent
markers for live cell imaging. Our previous live cell fluorescent imaging dyes [18, 19] were
also based on oxazole yellow or thiazole orange scaffold bearing two positive charges. We
added multiple charges to increase the water solubility and selectively accumulate the
compounds in the mitochondria, while the other substituents were designed to keep the impact
on cell proliferation minimal. Accumulation of compounds, with such high charge density, in
intracellular space is not common and will be further explored in comparative studies.
Acknowledgements
AK is grateful for the financial support by the National Science Fund of Bulgarian Ministry of
Education and Science under grant - contract № DМ09/5 from 17.12.2016. IO, DS. IC and IP
gratefully acknowledge financial support of Croatian Science Foundation Grant No 1477:
DNA/RNA-MolSense. The authors extend their gratitude to the Laboratory for mitochondrial
bioenergetics and diabetes staff, Division of Molecular Medicine, Ruđer Bošković Institute
for their help and kind donations of the MitoTracker and Hoechst dyes.
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ACCEPTED MANUSCRIPT
Highlights
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New DABCO-cyanine dyes dyes bind with high affinity to ds-DNA/RNA.
Relatively low anti-proliferation effect.
Fluorescence emission increase upon binding ds-DNA/RNA
Fluorescence emission in the live cells
A and B1 co-localize with the MitoTracker® dye.