Novel synthetic approach to near-infrared heptamethine cyanine dyes and spectroscopic characterization in presence of biological molecules

Article abstract of DOI:10.1016/j.jphotochem.2016.05.019

Two near-infrared symmetric heptamethine cyanine dyes, containing integrated chlorosubstituted cyclohexenyl ring were obtained via a novel synthetic approach, using mild conditions at room temperature. UV-vis and fluorescence properties of the dyes were e

Full text of DOI:10.1016/j.jphotochem.2016.05.019

Accepted Manuscript
Title: Novel synthetic approach to near-infrared heptamethine
cyanine dyes and spectroscopic characterization in presence of
biological molecules
Author: Atanas Kurutos Olga Ryzhova Ulyana Tarabara
Valeriya Trusova Galyna Gorbenko Nikolai Gadjev Todor
Deligeorgiev
PII:
S1010-6030(16)30077-6
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2016.05.019
JPC 10238
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Accepted date:
2-2-2016
26-5-2016
Please cite this article as: Atanas Kurutos, Olga Ryzhova, Ulyana Tarabara, Valeriya
Trusova, Galyna Gorbenko, Nikolai Gadjev, Todor Deligeorgiev, Novel synthetic
approachtonear-infraredheptamethinecyaninedyesandspectroscopiccharacterization
in presence of biological molecules, Journal of Photochemistry and Photobiology A:
Chemistry http://dx.doi.org/10.1016/j.jphotochem.2016.05.019
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Novel synthetic approach to Near-Infrared Heptamethine Cyanine Dyes and Spectroscopic
characterization in Presence of Biological Molecules
a*
b
b
b#
Atanas Kurutos ohtak@chem.uni‐sofia.bg, Olga Ryzhova , Ulyana Tarabara , Valeriya Trusova
b
a
a
valtrusova@yahoo.com,Galyna Gorbenko , Nikolai Gadjev , Todor Deligeorgiev
aSofia University “St. Kliment Ohridski”, Faculty of Chemistry and Pharmacy, 1, blv. J. Bourchier, 1164
Sofia, Bulgaria
bDepartment of Nuclear and Medical Physics, V.N. Karazin Kharkiv National University, 4 Svobody Sq.,
Kharkiv 61022, Ukraine
*Corresponding author: Tel: (+3592) 8161269; fax: (+3592) 9625438
#Tel: (+38057) 3438244; fax: (+38057) 7544746
1

Graphical abstract
2

Highlights





Novel synthetic approach to Near‐Infrared heptamethine cyanine dyes.
Evaluation of photophysical properties in organic solvents.
Association of the dyes AK7 in presence of dsDNA.
Binding of the dyes AK7 to Bovine Serum Albumine (BSA).
Partition of the dyes AK7 with lipid membranes (PC).
3

Abstract
Two near‐infrared symmetric heptamethine cyanine dyes, containing integrated chlorosubstituted
cyclohexenyl ring were obtained via a novel synthetic approach, using mild conditions at room
temperature. UV‐Vis and fluorescence properties of the dyes were evaluated in organic solutions
varying solvent polarity. Further spectral studies illustrating their application as potential labels for
biological molecules, were reported in the presence of deoxyribonucleic acid (DNA), bovine serum
albumin (BSA), and phospholipids (PC). The dyes undergo H‐type intramolecular complexation between
the two cyanine subunits when free in buffer. Absorption studies show that binding of cyanines to DNA,
BSA and lipid membranes leads to the red shift in the H‐aggregates peak with appearance of monomeric
and dimeric dye species pointing to the disaggregation of the dyes under study in the presence of
biological molecules. The fact that the decrease of liposome‐induced cyanine aggregation was more
pronounced in the zwitterionic phosphatidylcholine bilayer, suggests that the disruption of highly
organized molecular arrangements are most likely electrostatically‐controlled.
Keywords: Heptamethine cyanine dyes; H‐aggregates; DNA; Bovine serum albumin; Phospholipids.
4

1
. Introduction
Among a wide variety of fluorescent dyes currently used in biomedical research and industry,
considerable attention is given to near-infrared (NIR) cyanine probes, which have found
numerous analytical applications in enzymatic and immuno-assays [1, 2], drug displacement
studies [3], DNA sequencing [4] and cancer targeting applications[5], as optical imaging agents,
labels and stains [6-8], to name only a few. Undeniably, NIR cyanine dyes have been extensively
used due to their advantageous photophysical properties, namely absorption and fluorescence
spectrum in the NIR region (670-1100 nm). Another essential feature of cyanine dyes is very
high polarizability of the π-electron system along the polymethine group in the ground state,
which give rise to strong dispersion forces between two cyanine molecules in solution [9]. These
dispersion forces are thought to control the formation of extended aggregates of cyanines in
solutions [10-12]. Since cyanine dyes undergo self-organization, they have been found to be an
invaluable tool used to study molecular organization in proteomics [13], optics [14] and
nanoscience [15].
Depending on the angle of molecular slippage, α, self-association of cyanine dyes results in
the formation of molecular aggregates of different structures, known as H-and J-aggregates [16,
o
1
7]. Large molecular slippage (α<32 ) led to the self-organization of “brickwork” arrangements
o
(
J-aggregates), while small one (α>32 ) is characterized by “card-pack” structures (H-
aggregates). Dictated by the geometry of the molecules within the aggregate, H- and J-
aggregation patterns possess unique electronic and spectroscopic properties and can be easily
identified by spectroscopic methods. According to Kasha [18] exciton model for molecular
aggregates, the interactions between neighboring transition dipoles of tightly packed molecules
generates a splitting of the excited state into excitonic levels that are shared between all the
5

molecules within arrangement. When two dipoles are face-to-face (H-aggregates), the energy of
the allowed state corresponding to in phase transition dipoles is increased by repulsive
electrostatic interactions between the transition dipoles [18]. In contrast, the lower exciton level
is observed for molecules with end-to-end packing (J-aggregates), which is manifested in red
shift in the absorption spectrum.
Trends of cyanine dye aggregation are well described now, and it has been found that the
structure of the dye, particularly the presence of substituents in heterocyclic residues and the N-
alkyl chain length, directly dictates the type of aggregates formed [11, 19, 20]. The other factors
contributing to the formation of highly organized molecular arrangements are probe
concentration [21], solvent polarity [22], temperature [23], presence of polyelectrolytes [24, 25],
polymers [26], Langmuir-Blodgett films [19], etc. The self-association have been shown to be
inhibited also by high ionic strength since at high salt concentration the aggregates are more
energetically stable then the monomeric dye due to the formation of contact ion pairs between
the cationic dyes and counterions [10]. An additional agent affecting the formation of ordered
nanoassemblies of noncovalently coupled probes is surfactants [11, 27, 28]. Numerous studies
indicate that the addition of surfactants can provoke the aggregation of dyes, in certain cases
changing the morphology of arrangements [11, 28]. Recently, it was concluded that the presence
of biomolecules, such as proteins and nucleic acids, have a significant impact on aggregation
properties of organic dye molecules [15, 20, 21, 29-31]. Specifically, DNA was shown to serve
as a template for the growth of helical arrays of cyanine dyes [15, 21, 29]. The assembly of these
supramolecular structures occurs by a cooperative, chain-growth mechanism and is essentially
promoted by DNA [15, 29]. The role of proteins as scaffold for cyanine aggregation or
disaggregation was also described in a number of works [20, 31].
6

The aim of the present paper was threefold: i) to utilize a novel synthetic approach to NIR
heptamethine cyanine dyes containing integrated chloro-substituted cyclohexenyl ring (Figure 1),
ii) to investigate photophysical properties of the cyanines in organic solvents with varying
polarity and iii) to examine the impact of DNA, bovine serum albumin (BSA) and liposomes
(
PC) on aggregation properties of the examined dyes.
2
. Materials and methods
2.1. Materials
Unless otherwise stated, reagents and solvents used in the synthesis and analysis of the dyes
were obtained from Sigma-Aldrich, Organica Feinchemie GmbH Wolfen, Alfa-Aesar and
Deutero GmbH, as commercial products of analytical grade and used in the synthesis of all target
products without further purification. The solvents used for the spectrophotometric and
spectrofluorimetric analyses were supplied by Macron Fine Chemicals TM. Double stranded
deoxyribonucleic acid from salmon testes (DNA), bovine serum albumin (BSA), 2-Oleoyl-1-
palmitoyl-sn-glycero-3-phosphocholine (PC) and Tris-HCl were purchased from Sigma.
2
.2. Analysis methods and equipment
All products including dyes and intermediates were characterized using various spectroscopic
techniques. Recrystallization from methanol was carried out as a main method of purification in
order to obtain analytical samples of the target products. NMR spectra were recorded on Brucker
1
13
Avance III HD, 500 MHz instrument ( H-NMR 500 MHz, C-NMR 125 MHz) in DMSO-d at
6
o
2
5 C. The Coupling constants J were expressed in Hz. ESI mass spectra of both positive and
negative ionization modes were recorded on triple quadrupole mass spectrometer Agilent 6410
coupled with HPLC Agilent 1200 Series. Melting point temperatures were determined on a
Kofler bench and are uncorrected. UV-visible spectra of the dyes were recorded in various
7

solvents of spectroscopic grade on a Cecil Aurius CE 3021 UV-Vis spectrophotometer at room
temperature. The corresponding emission spectra were recorded on Perkin Elmer LS45
spectrofluorimeter at room temperature using 10-mm path-length quartz fluorescence cuvettes.
The dye stock solutions (1 mM) were prepared in DMSO, which were diluted with the
corresponding organic solvent. UV-visible spectra of heptamethine dyes in the presence of DNA,
BSA and liposomes were measured in the range 500-900 nm with Cecil Aurius CE 3021 UV-Vis
spectrophotometer at room temperature using 10-mm path-length quartz cuvettes.
2
.3. Synthesis of intermediates 2a, 2b
2
-methyl benzothiazole 1 (1.27 mL, 10 mmol) and a small excess of the corresponding
alkylating reagent R-Br (11 mmol) were dissolved in 2-methoxyethanol (2 mL). The reaction
mixture was heated at reflux for 2 hours. The reaction outcome was monitored by TLC. After the
reaction was completed, and the reaction mixture was cooled down to room temperature, N-
quaternary product 2 was precipitated with diethyl ether (20 mL). The precipitate formed was
filtered off, washed with diethyl ether, and stored in a desiccator (Scheme 1). Purification of the
benzothiazolium salts was held by recrystallization from methanol.
3
-isopentyl-2-methylbenzo[d]thiazol-3-ium bromide (2a): (pale brown solid), yield = 29 %, m.p.
o
1
=
190-193 C; H-NMR (DMSO-d
), 1.86 (1H, sept., CH), 3.27 ( 3H, s, CH
.82 (1H, m, ArH), 7.88-7.91 (1H, m, ArH), 8.31 (1H, d, J 8.5, ArH), 8.53 (1H, d, J 8.2, ArH);
6
, 500 MHz) δ/ppm: 1.00 (6H, d, J 6.6, 2 x CH
3
- CH), 1.71-
1
.76 (2H, m, CH
2
3
), 4.72-4.76 (2H, m, CH
2
-N), 7.78-
7
13C-NMR (DMSO-d
29.8; 141.0; 177.3;
, 125 MHz) δ/ppm: 17.4; 22.6; 26.0; 36.5; 48.4; 117.2; 125.3; 128.4; 129.6;
6
1
8

3
1
-benzyl-2-methylbenzo[d]thiazol-3-ium bromide (2b): (pale pink solid), yield = 72 %, m.p. =
o
o
o
1
90-193 C; m.p. = 246-249 C, lit. m.p. = 255-256 C [33]; H-NMR (DMSO-d
), 6.13 (2H, s, CH -N), 7.33-7.42 (5H, m, 5 x ArH), 7.78-7.86 (2H, m, 2
x ArH), 7.23 (1H, d, J 8.3, ArH), 8.55 (5H, m, 5 x ArH); C-NMR (DMSO-d
δ/ppm: 17.9; 52.4; 117.6; 125.4; 127.5; 127.5; 128.7; 129.0; 129.6; 129.7; 130.0; 133.3; 141.4;
6
, 500 MHz)
δ/ppm: 3.30 (3H, s, CH
3
2
1
3
6
, 125 MHz)
1
78.9;
2
.4. Synthesis of heptamethine cyanine dyes AK7 via novel method.
2
-methylbenzothiazolium salt 2 (3 mmol) and 2-chloro-3-((phenylamino)methylene)
cyclohex-1-en-1-yl)methylene) benzenaminium chloride 3 (1.5 mmol) were suspended in
methanol (5 mL) in a mortar, and grinded with a pestle at room temperature (Scheme 2),
followed by addition of sodium acetate (3 mmol) upon grinding. After 10 seconds a dark blue
color is developed, followed by the simultaneous crystallization of the reaction mixture. The
crude product was dissolved in 10 mL methanol and transferred to a beaker containing 100 mL
of an aqueous solution of potassium iodide. After 15 minutes the precipitated dyes were filtered
off and washed with diethyl ether. Purification of the heptamethine cyanine dyes was held by
recrystallization from methanol.
2
1
9
-((E)-2-((E)-2-chloro-3-((Z)-2-(3-isopentylbenzo[d]thiazol-2(3H)-ylidene)ethylidene)cyclohex-
-en-1-yl)vinyl)-3-isopentylbenzo[d]thiazol-3-ium iodide (AK7-5): (green crystals), yield =
o
1
0%, m.p. = 236-237 C; H-NMR (DMSO-d
); 1.78 (2H, sept., J 6.6, 2 x CH); 1.83-1.89 (2H, m, CH
); 4.40-4.48 (4H, m, 2 x CH -N); 6.45 (2H, d, J 12.1, 2 x CH); 7.40-7.46
6
, 500 MHz) δ/ppm: 1.00 (12H, d, J 6.6, 4 x CH
3
-
CH); 1.61 (4H, q, J 7.1, 2 x CH
2
2
); 2.64-
2.70 (4H, m, 2 x CH
2
2
(
2H, m, 2 x ArH.); 7.57-7.60 (2H, m, 2 x ArH.); 7.71-7.73 (2H, m, 2 x ArH.); 7.82 (2H, d, J
9

+
1
2.1, 2 x CH); 7.99 (2H, d, J 7.7, 2 x ArH.); ESI: m/z: Found [M+] 575.2 C34
2 2
H40ClN S
-
-
requires [M+] 575.23; Found [I ] 126.8 requires [I ] 126.91; UV/VIS (methanol): λmax = 795 nm,
-1
-1
-1
-1
ε = 189.500 M cm , λfl = 812 nm; (DMSO): λmax = 810 nm, ε = 138.800 M cm , λfl = 824 nm;
-1
-1
(
chloroform): λmax = 809 nm, ε = 230.600 M cm , λfl = 821 nm;
3
-benzyl-2-((E)-2-((E)-3-((Z)-2-(3-benzylbenzo[d]thiazol-2(3H)-ylidene)ethylidene)-2-
chlorocyclohex-1-en-1-yl)vinyl)benzo[d]thiazol-3-ium iodide (AK7-6): (green crystals), yield =
o
1
8
4% (greenish crystals), m.p. = 219-220 C; H-NMR (DMSO-d
6
, 500 MHz) δ/ppm: 1.73-1.78
(
2H, m, CH ); 2.53-2.57 (4H, m, 2 x CH ); 5.78 (4H, s, 2 x CH -N); 6.62 (2H, d, J 13.3, 2 x CH);
2
2
2
7.27-7.29 (4H, m, 4 x ArH.); 7.33 (2H, d, J 7.0, 2 x ArH.); 7.37-7.43 (6H, m, 6 x ArH.); 7.52-
7
.55 (2H, m, 2 x ArH.); 7.69-7.71 (2H, d, J 8.2, 2 x ArH.); 7.84 (2H, d, J 13.2, 2 x CH); 8.01
+
(2H, d, J 7.9, 2 x ArH.); ESI: m/z: Found [M+] 615.2 C38
H32ClN
2
S
2
requires [M+] 615.17;
-
-
-1
-1
Found [I ] 126.8 requires [I ] 126.91; UV/VIS (methanol): λmax = 802 nm, ε = 170.800 M cm ,
-1
-1
λ
fl = 824 nm; (DMSO): λmax = 817 nm, ε = 133.900 M cm , λfl = 834 nm; (chloroform): λmax =
-1
-1
8
15 nm, ε = 225.100 M cm , λfl = 833 nm;
2
.5. Study of DNA, BSA, and PC liposomes interactions.
Stock solutions (1 mM) of heptamethine cyanine dyes AK7 were prepared immediately
before the measurements by dissolving the dyes in DMSO. Stock solutions of DNA and BSA
were prepared in 10 mM Tris-EDTA buffer, pH 7.4. The concentrations of dyes, DNA and BSA
3
-
were determined spectrophotometrically using their molar absorptivities and ε260 = 6.4 × 10 M
1
-1
4
-1
-1
cm , ε276 = 4.25 × 10 M cm for DNA and BSA, respectively. Working solutions of the
heptamethines were prepared by dilution of the dye stock solution in buffer. To determine the
absorption of cyanine dyes in the presence of biomacromolecules and lipid membranes,
1
0

appropriate amounts of the stock solution of DNA, BSA or liposomes were added to each dye in
buffer.
Lipid vesicles composed of PC were prepared using the extrusion technique. A thin lipid film
was first formed from the lipid mixtures in chloroform by removing the solvent under a stream of
nitrogen. The dry lipid residues were subsequently hydrated with 20 mM HEPES, 0.1 mM
EDTA, pH 7.4 at room temperature to yield lipid concentration of 1 mM. Thereafter, lipid
suspension was extruded through a 100 nm pore size polycarbonate filter (Millipore, Bedford,
USA). In this way, lipid vesicles containing PC. The phospholipid concentration was determined
according to the procedure of Bartlett [32].
3
. Results and discussion
3
.1. Synthesis and structural analysis of AK7 dyes
Herein we report a novel synthetic approach for the synthesis of near-infrared symmetric
heptamethine cyanine dyes AK7-5 and AK7-6 [33], containing integrated chlorosubstituted
cyclohexenyl ring, under mild reaction conditions. The target cyanine dyes were obtained via a
simple environmentally green approach, involving a condensation reaction which proceeds
through grinding for few seconds the starting 2-methylbenzothiazolium quaternary salts 2 and 2-
chloro-3-((phenylamino)methylene)cyclohex-1-en-1-yl)methylene)benzenaminium chloride 3 in
a mortar. In order to exchange the anion of the target cyanine dyes, they were dissolved in a
minimum amount of methanol and transferred to a beaker containing an aqueous solution of
potassium iodide.
The main advantages of this novel synthetic approach are: i) Reactions for the synthesis of
the target heptamethine cyanine dyes were carried out at room temperature without need of any
heating sources. ii) a mild basic reagent, such as sodium acetate was used in order to generate
1
1

methylene group on the benzothiazolic chromophore. iii) The total amount of the solvent
involved in the synthesis is minimized to few mL, which is a semi-solid state approach. iv) The
condensation reaction yielding the target AK7 dyes proceeds within 10 seconds according to our
method which compared to reported methods [33, 34] the reaction time varies from 3.5 hours to
3
overnight. This accounts for over 10 times faster reaction. v) The target heptamethine cyanine
dyes were isolated with excellent yields.
1
The title heptamethine cyanine dyes AK7 were evaluated by melting point temperatures, H
NMR spectroscopy, and ESI mass spectrometry of both positive and negative ionization modes.
3
.2. Photophysical properties of the studies dyes AK7 in various solvents
The cyanines under study are symmetric heptamethine cyanine dyes, containing integrated
chloro-substituted cyclohexenyl ring. The dyes are cationic in nature due to the delocalized
positive charge of the chromophore. Spectroscopic characteristics of the two AK7 dyes in
organic solvents with different polarities are presented in Table 1.
Upon varying the solvent polarity from DMSO to chloroform a small shift of absorption and
fluorescence maximum positions was observed without almost any significant change in
spectrum shape as illustrated for dye AK7-5 (Figure 2). In the absorption spectra of the studied
heptamethine cyanine dyes, the band corresponding to the dye monomer form is observed in the
range 795-810 nm, and 802-817 nm for AK7-5 and AK7-6, respectively. Fluorescence spectra of
the dyes are typical for heptamethines with emission maxima around 812-824 nm and 824-834
nm for AK7-5 and AK7-6, respectively.
3.3. Quantum yield calculation
1
2

Quantum yields of the examined dyes were calculated using Indocyanine green (IR-125) as a
standard according to the formula:
The relative fluorescence quantum yield of the examined dyes AK7 were determined in
organic solvents with different polarities using the equation:
ꢋ
ꢆ ꢊ
ꢇ
ꢇ
ꢀꢁꢂꢃꢄ
ꢅ
ꢉ
ꢀꢁꢂꢈꢄ
ꢋ
ꢆ ꢊ
ꢈ
ꢈ
where, ꢀ is the fluorescence quantum yield, m is the slope obtained from the plot of the
ꢁ
integrated fluorescence intensity vs absorbance, n is the refractive index of the solvent used in
the measurement, and the superscripts s and x represent the reference and unknown parameters
respectively. Indocyanine green (IR-125) was used as a reference standard, which has a quantum
yield of 0.106 in DMSO [35]. The results depict lower relative quantum yields compared to
Indocyanine green (IR-125), which is most likely due to the rigidified polymethine chain.
3
.4. Photophysical studies of free dyes AK7 in buffer solutions
Figure 3 represents the absorption spectra of the examined cyanine dyes in buffer solution at
-6
different concentration ranging (1.0-20.0) × 10 M. In aqueous medium at the initial dye
concentration the absorption spectra of the heptamethines were blue-shifted by ~ 200 nm relative
to those in organic solvents, with the maximum being centered at 589 nm and 641 nm for AK7-5
and AK7-6, respectively. This implies that H-aggregates are the dominating species of the dyes
in a buffer solution. Increasing heptamethine concentration resulted in the change of the
absorption spectrum from ‘one-peak’ to ‘three-peak’ which is more pronounced for AK7-5,
indicating that these dyes are represented by monomers, H-dimers and higher class of H-
aggregates. It is known that the aggregation properties of cyanines are controlled by the balance
1
3

between the van der Waals interactions, dispersion forces within the cyanine backbone, the
forces of the alkyl chain of entropic and hydrophobic nature together with H-bonding and the
electrostatic interactions of the ionic groups [11]. The appearance of dimer and monomer peaks
in the absorption spectrum of dyes at the increasing heptamethine concentration is attributed to
the dual nature of these dyes. Initially, at low dye concentration in buffer solution the
polarizability and hydrophobicity of the dye molecules drive aggregation, overcoming the
Coulombic repulsion between the two dye molecules. Further increase in dye concentration
change the balance of forces, and electrostatic repulsion between heptamethine molecules led
presumably to the occurrence of dimeric and monomeric dye species.
-6
3
.5. Photophysical properties of free dyes at 1 × 10 M, and studies in presence of NaCl
Additional studies of the heptamethine cyanine dyes were performed at lower concentrations
-6
(
1 × 10 M) when free in buffer solution, and in presence of sodium chloride in order to examine
the behavior of the aggregates.
In the presence of salt, the absorption spectra of the dyes indicate mainly a decrease of the
maxima around 613-637 nm representing higher class of H-aggregates. Furthermore, a
bathochromic shift of 20 nm is observed for dye AK7-6, whereas in the case of AK7-5 almost no
significant changes can be observed. This suggests that the aggregation forces are greater in the
case where the dye contains pure aliphatic substituents on the nitrogen atoms of the two
chromophores. This red shift of the maxima is also followed by appearance of new peak at 711
nm. This phenomena indicates either disaggregation of the higher class of H-aggregates and
1
4

subsequently formation of H-dimers, or transition to different type such as J-aggregates, as a
consequence from the presence of salt.
3
.6. Asocciation of the dyes AK7 with dsDNA
Next step of the study was directed towards the evaluation of possible biological applications
of the examined cyanine dyes. Figure 5 shows the representative absorption spectra of AK7 dyes
in the absence and presence of dsDNA. The complexation of heptamethine cyanine dyes with
DNA resulted in attenuation of the intensity due to dilution of the sample concentration, and
almost no significant change in the shape of the spectra.
It is known, that depending on dye structure, cyanine dyes exhibit a variety of DNA binding
motifs including intercalation, minor groove binding and aggregation. The intercalation between
the base pairs is the predominant binding motif for the dyes with a single methine bridge at low
dye:base pair concentrations [36,37-39]. Groove binding becomes prevalent for cyanines with
more than one methine bridge [30, 40, 41]. Mikheikin et al. [40] demonstrates that trimethine
cyanine dyes bind to minor groove of DNA as monomers and dimers. The same binding mode
was also observed for tri- and penthamethines [41, 42].
Though, in the heptamethine cyanine dyes under study, the organization of the dye molecules
to higher class of H-aggregates most likely appears to be too bulky even for groove binding. In
addition to, the corresponding fluorescence studies depict no significant change in terms of
fluorescence enhancement (Figure 6), even at high ratios of [DNA]:[AK7] such a 80:1.
3
.7. Asocciation of the dyes AK7 with Bovine Serum Albumine (BSA)
In the following, the spectral properties of the examined heptamethine cyanine dyes were
analyzed upon their binding to bovine serum albumin. The interactions were monitored by
1
5

evaluating the changes in absorbance at a fixed concentration of dye with increasing protein
concentration (Figure 7). The changes in absorption spectrum at the presence of protein can be
summarized as follows: i) association of heptamethines with BSA led to the diminishing of the
peak, attributed to the dye H-aggregates (588 and 634 nm for AK7-5 and AK7-6, respectively)
and appearance of the new peaks around 811 and 818 nm, characteristic of the monomer
absorption. The observed effect assumes that dye-protein complexation breaks heptamethine
aggregates, and the dyes undergo the transition into the monomeric state. However, the
contribution of H-aggregate band in the total spectrum was still prevailing over the other dye
forms. ii) The reduction of H-aggregates peak is accompanied by red-shift of absorption
maximum from 588 nm to 645 nm and from 633 nm to 658 nm for AK7-5 and AK7-6,
respectively. iii) The formation of protein-dye complexes resulted in the broadening of H-
aggregates absorption spectrum, coupled with appearance of a shoulder around 708-715 nm.
Together with the absence of isosbestic point between aggregated and monomer dye forms, these
findings indicate that the heptamethine-protein interaction leads to the concomitant rupture of H-
aggregate, appearing in buffer solution and the subsequent formation of dimeric and monomeric
dye molecules. Most probably, the binding strength of heptamethines with BSA was strong
enough to cause the dissociation of aggregates with aggregation potential strongly reduced after
formation of the dye-BSA complexes. The disruption of plane-to-plane arrangements followed
by the formation of randomized monomers in the presence of BSA was reported also for
merocyanine 540 [43], and heptamethine cyanine dyes [20, 44] and was interpreted as arising
from higher strength of interactions between the dye and the BSA compared to the coupling
between the cyanine monomeric species in the aggregate. The possible explanation of
disassembly of H-aggregates in the presence of BSA in our study could lie in the strong
1
6

electrostatic interaction between cyanine dyes and protein. The isoelectric point of BSA is ~4.7
44, 45], so at the pH 7.4 this protein bears a negative electric charge. It can be seen from the
[
structure of AK7-5 and AK7-6 that this dyes are cationic in nature, therefore the binding of
heptamethines with BSA is likely to involve electrostatic attraction. In addition, crystal structure
of BSA shows that the regions of ligand-binding sites are located in hydrophobic cavities in BSA
subdomains, referred to as site I and site II according to terminology proposed by Sudlow et al.
[
46]. Since heptamethine cyanine dyes under study contain hydrophobic groups in their structure,
we can hypothesize that hydrophobic interactions along with electrostatic attractions serve as
driving forces to transfer the dye molecules from their aggregate states to the monomeric form.
Previously, Patonay and coworkers [47] characterized the hydrophobic binding pockets of the
albumin molecule and observed that the binding affinity of cyanine dyes is attributed to the
substitution of hydrophobic moieties at the heterocyclic ring of the cyanine dyes. Numerous
studies indicate that cyanine dyes generally exhibit a high specificity to the binding site II of
albumins [3, 20], so one can hypothesize that the same mode of interactions is realized in our
systems.
The disaggregation of the dyes in the presence of BSA and appearance of a monomeric peak
lead to multiple enhancement of the fluorescence signal at 818 nm and 824 nm respectively for
AK7-5 and AK7-6 upon excitation λex = 780 nm (Figure 8).
3
.8. Binding of cyanine dyes AK7 to lipid vesicles.
Cyanine dyes have been widely employed to trace the processes occurring in biological
assemblies such as micelles and vesicles, since their photophysical behavior is strongly depend
on the properties of the surrounding medium [22]. Therefore, to get further insights into the
1
7

interactions of cyanine dyes with biological objects, their behavior in lipid environment have
been studied. Presented in Figure 6 are the absorption spectra of the studied dyes in the presence
of liposomes. Association of heptamethines with the PC lipid vesicles (Figure 9) led to the
decrease of the absorption peak, attributed to the H-aggregates and appearance of two bands,
characteristic of the H-dimer and monomer absorption. The position of H-dimer peak was around
7
13-720 nm, while monomers were the most absorptive at 819-826 nm, respectively. These
findings suggest that upon transition to the lipid environment plane-to-plane arrangement is
disrupted, thereby favoring the randomized monomer arrangement. The results presented in
Figure 9, indicate that partition of heptamethine cyanine dyes into the liposomes is two-stage
cooperative process. Initially, only H-aggregates are present. At the first stage, the addition of the
liposomes in relatively low concentrations stimulates the dimer formation. The observed
dimerization is most probably arises from the decrease of polarity of the aggregates environment.
It is highly probable that the dispersion forces become incapable to govern the staking
orientation of the molecules in H-aggregates in low-polar solvents, so we observed the
accumulation of dimers in the polar region of lipid bilayer. At the second stage, increase in lipid
concentration promotes the appearance of monomeric dye form, and, finally, the contribution of
monomeric band in the total spectrum prevails over the other dye forms. The driving force of this
process involves, most likely, the hydrophobic interactions. One may suppose that it will be
energetically more favorable for hydrophobic moieties at the heterocyclic ring of heptamethines
dyes to penetrate dipper into the lipid bilayer orienting parallel to the lipid acyl chains. These
hydrophobic interactions led to further decrease of the polarity of lipid dye microenvironment,
since it is known that the polarity profile is change dramatically from polar to hydrophobic core
of liposomes [48].
1
8

Moreover, additional argument comes from the spectral response of a number of
environment-sensitive fluorophores [49-51]. It is noted that the changes in hydration extent may
considerably affect molecular organization of a lipid bilayer. In particular, increase of water
content in headgroup region was reported to modify the alignment of choline-phosphate dipole
2
and lateral packing of hydrocarbon chains [52]. In addition to, as follows from H-NMR
spectroscopic studies, the presence of PC bilayers alters the orientation and mobility of adjacent
choline headgroups [53, 54].
In terms of fluorescence the disaggregation of the dyes in the presence of phospholipids and
appearance of a strongly expressed monomeric peak (7.8 and 3.3 fold increase for AK7-5 and
AK7-6 respectively) lead to multiple enhancement of the fluorescence signal at 828 nm and 832
upon excitation at λex = 770 nm and 780 nm respectively (Figure 10).
3
. Conclusions
To summarize, the present study was focused on synthesis and spectral characteristics of two
near infrared heptamethine cyanine dyes, referred to here as AK7-5 and AK7-6. Based on the
results of absorption studies, it was concluded:
The free dyes in aqueous solutions exist mainly in the form of “card-pack” structures (H-
aggregates) with the absorption maximum being centered at 588 nm and 639 nm for AK7-5 and
AK7-6, respectively. The molecular distribution of the dye in buffer solution is dependent on
concentration.
Upon binding to DNA, heptamethines show almost no significant difference in terms of
disaggregation. Same observation applies to the fluorescence investigations on the studied dyes.
Heptamethine cyanine dyes form the complexes with BSA mainly in dimeric and monomeric
form, binding presumably to the II Sublow center. The possible explanation of dissagregation of
1
9

cyanine dyes under study lie in higher strength of interactions between the dye and the BSA
compared to the coupling between the cyanine monomeric species in the aggregate.
Association of heptamethines with the lipid vesicles led to the decrease of absorption peak,
attributed to the H-aggregates and appearance of two bands, characteristic of the H-dimer and
monomer absorption.
2
0

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