Symmetric Meso-Chloro-Substituted Pentamethine Cyanine Dyes Containing Benzothiazolyl/Benzoselenazolyl Chromophores Novel Synthetic Approach and Studies on Photophysical Properties upon Interaction with bio-Objects

Article abstract of DOI:10.1007/s10895-015-1700-4

A series of symmetric pentamethine cyanine dyes derived from various N-substituted benzothiazolium/benzoselenazolium salts, and a conjugated bis-aniline derivative containing a chlorine atom at meso-position with respect to the polymethine chain, were synthesized using a novel improved synthetic approach under mild conditions at room temperature. The reaction procedure was held by grinding the starting compounds for relative short times. The novel method is reliable and highly reproducible. Some photophysical characteristics were recorded in various solvents, including absorption, and fluorescence quantum yields using Cy-5 as a reference. Additional studies on interactions with several bio-objects such as liposomes, DNA, and proteins have been investigated in the present work.

Full text of DOI:10.1007/s10895-015-1700-4

J Fluoresc
DOI 10.1007/s10895-015-1700-4
ORIGINAL ARTICLE
Symmetric Meso-Chloro-Substituted Pentamethine Cyanine Dyes
Containing Benzothiazolyl/Benzoselenazolyl
Chromophores Novel Synthetic Approach and Studies
on Photophysical Properties upon Interaction with bio-Objects
1
2
2,3
2
Atanas Kurutos & Olga Ryzhova & Valeriya Trusova & Galyna Gorbenko
&
1
1
Nikolay Gadjev & Todor Deligeorgiev
Received: 19 June 2015 /Accepted: 20 October 2015
Springer Science+Business Media New York 2015
#
.
.
.
Abstract A series of symmetric pentamethine cyanine dyes
derived from various N-substituted benzothiazolium/
benzoselenazolium salts, and a conjugated bis-aniline deriva-
tive containing a chlorine atom at meso-position with respect
to the polymethine chain, were synthesized using a novel im-
proved synthetic approach under mild conditions at
room temperature. The reaction procedure was held by
grinding the starting compounds for relative short times.
The novel method is reliable and highly reproducible.
Some photophysical characteristics were recorded in
various solvents, including absorption, and fluorescence
quantum yields using Cy-5 as a reference. Additional
studies on interactions with several bio-objects such as
liposomes, DNA, and proteins have been investigated in
the present work.
Fluorescent markers Liposomes DNA, proteins
H-aggregates
Introduction
Сyanine dyes, a wide class of fluorescent probes with unique
photophysical properties, have found numerous application in
different areas as optical imaging agents [1, 2], active ingre-
dients in semiconducting materials [3, 4], laser dyes [5], pho-
tographic sensitizers [6, 7], photopolymerization initiators [8,
9
], stains and fluorescent labels [10–13], to name only a few.
These compounds are of particular interest for biomedical
research and diagnostics due to their favorable spectral char-
acteristics, namely, longwave absorption and emission, large
extinction coefficient, high fluorescence quantum yield, etc.
Most of the cyanine dyes are photosensitive compounds
possessing two quaternized, nitrogen-containing, heterocyclic
structures, which are linked through a polymethine bridge
.
Keywords Pentamethine cyanine dyes
. .
-methylbenzothiazole 2-methylbenzoselenazole
2
[14]. Due to dual hydrophobic and cationic nature of these
dyes, which leads to strong interaction with polyanionic
DNA duplex, cyanines are mainly used as bright labels for
nucleic acids in microarray-based expression analysis [15],
DNA sequencing [16, 17] and DNA intercalation
bioanalytical assays [18]. It has been demonstrated that
mono-, tri- and pentamethine cyanine dyes are suitable for
quantitative detection of amyloid formation and protein label-
ing [19, 20]. Fluorescence intensity of cyanine dyes is greatly
increased upon their binding to nucleic acids or proteins as a
result of the rigidization of the fluorophores [19–22].
Furthermore, the utility of near infrared cyanine dyes demon-
strates unique hydrophobic characteristics in aqueous medium
that were exploited for target-specific pH probing [23, 24].
Moreover, it is known that the central polymethine chain of
*
*
Atanas Kurutos
ohtak@chem.uni-sofia.bg
Valeriya Trusova
valtrusova@yahoo.com
1
2
3
Sofia University BSt. Kliment Ohridski^, Faculty of Chemistry and
Pharmacy, 1, blv. J. Bourchier, 1164 Sofia, Bulgaria
Department of Nuclear and Medical Physics, V.N. Karazin Kharkiv
National University, 4 Svobody Sq., Kharkiv 61077, Ukraine
1
9-32 Geroyev Truda, Kharkiv 61044, Ukraine

J Fluoresc
cyanine dyes can be cleaved by reactive oxygen and nitrogen
species, which gives the impetus for cyanine use for in vivo
sensing of oxidative stress and monitoring the dynamics of
redox cycles in living cells [25]. In addition, cyanine dyes
showed unique properties as optical imaging probes for cancer
labeling [26].
Despite the essential utility and versatility of cyanines, sig-
nificant research efforts are being directed at the developing
and investigating new fluorescent compounds. The present
paper is aimed at the synthesis and characterization of a range
of symmetric pentamethine dyes differing in their structural
features and spectroscopic behaviour. More specifically, our
goals were: (i) to synthesize a series of symmetric
pentamethine fluorophores containing a benzothiazolic and
benzoselenazolic heterocycle with a meso-chlorine substituent
in the polymethine chain; (ii) to characterize their
photophysical properties; (iii) to explore possible biological
applications of cyanine analogues.
Scheme 1 Synthesis of 2,3-dimethyl benzothiazolium and 2,3-
dimethylbenzselenazolium salts 2a-2b
stored in a dessicator. The compounds 2a-2b were used with-
out further purification in the next reaction steps for obtaining
the pentamethine dyes. Their structures are evaluated on the
final products. Yields: 2,3-dimethylbenzothiazolium
methosulfate 2a = 87 %, 2,3-dimethylbenzselenazolium
methosulfate 2b = 83 %.
Preparation of N-Quaternary 2-Methyl Benzothiazolium
and 2-Methylbenzselenazolium Salts 2c-2g
Experimental
Materials
2-methylbenzoselenazole/2-methylbenzothiazole 1a,1b
(
5 mmol) and the corresponding alkylating agent R-X
(5.5 mmol) were mixed together in a 50 ml Erlenmeyer flask
with 10 ml 2-methoxyethanol and the mixture was refluxed
for 2 hours with stirring (Scheme 2). Upon completion, the
mixture was left to cool down to room temperature, followed
by addition of 20–30 ml diethyl ether to it, in order to precip-
itate the desired products. The quaternary salts were suction
filtered and stored in a dessicator. The compounds were used
without further purification in the next reaction steps for
obtaining the pentamethine dyes. Their structures were eval-
uated on the final products (Scheme 3). Chemical structures of
the quaternary benzazolium salts 2c-2g are presented in
Table 1.
All starting materials and solvents required for the synthesis of
the dyes were purchased from Sigma-Aldrich, Organica, Fluka,
Alfa-Aesar, Deutero GmBH. The structures of all intermediates
are evaluated on the final products. The target cyanine dyes were
recrystallized from methanol and characterized by H NMR
spectra recorded on a Brucker Avance III HD, 500 MHz in
1
DMSO-d at 25 °C. The chemical shifts were reported in ppm
6
in δ-values with respect to tetramethylsilane (TMS) as an internal
standard or the deuterated solvent peak. Coupling constants J are
expressed in Hz. The melting points were determined on a Kofler
apparatus and are uncorrected. Bovine heart cardiolipin (CL) and
1
-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) were
Synthesis of Pentamethine Cyanine Dyes AK5
by an Improved Method
from Avanti Polar Lipids (Alabaster, AL). Cattle spleen DNA
was from Reakhim (Russia), bovine serum albumin (BSA) and
Tris-HCl were obtained from Sigma. 4-(dimethylaminostyryl)-1-
dodecylpiridine n-toluenesulfonate (DSP-12) was purchased
from Zonde (Latvia). All other starting materials and solvents
were commercial products of analytical grade and were used
without further purification.
Substituted or unsubstituted 2-methylbenzothiazolium/2-
methylbenzoselenazolium quaternary salts 2a-2g (3 mmol)
and ((2-chloro-3-(phenylamino)allylidene)benzenaminium
chloride)) 3 (1.5 mmol), were mixed together with 5 ml
Preparation of Pentamethine dye Intermediates 2a, 2b
2
-methylbenzoselenazole 1a/2-methylbenzothiazole 1b
(
5 mmol) was dissolved in 10 ml acetone in an Erlenmeyer
flask, to which dimethyl sulfate (5.5 mmol) was added
dropwise. Upon completion of the addition, the resulting mix-
ture was stored in a dark, closed vessel at room temperature
for 48 hours (Scheme 1). The corresponding quaternary
benzazolium salt precipitate was collected on a Buchner and
Scheme 2 Synthesis of N-quaternary 2-methyl benzothiazolium and 2-
methylbenzselenazolium salts 2c-2g

J Fluoresc
(
500 MHz; DMSO d ) δ (ppm): 3.90 (s, 6 H, 2 x CH -N); 6.39
6
3
(d, J 13.0, 2 H, 2 x CH); 7.44 (m, 2 H, ArH.); 7.60 (m, 2 H,
ArH.); 7.80 (d, J 8.3; 2 H, ArH.); 8.03 (d, J 13.1, 2 H, 2 x CH);
8.06 (d, J 7.9, 2 H, ArH.);
AK5-2 2-((1E,3Z,5E)- 3-chloro-5-(3-methylbenzo[d] [1,
]selenazol-2(3 H)-ylidene)penta-1,3-dien-1-yl)-3-
3
methylbenzo[d] [1, 3]selenazol-3-ium iodide (Yield of crude
о
product 94 %, M.p. = 255–258 С); UV/VIS (methanol):
−1
−1
Scheme 3 Chemical structures of the quaternary benzazolium salts 2a-
λmax =655 nm, ε = 229.000 l.mol .cm , λem = 672 nm,
2
g
1
Q = 0.081; H NMR (500 MHz; DMSO d ) δ (ppm): 3.87 (s,
6
6
H, 2 x CH -N); 6.52 (d, J 12.9, 2 H, 2 x CH); 7.38 (m, 2 H,
3
ArH.); 7.57 (m, 2 H, ArH.); 7.75 (d, J 8.1; 2 H, ArH.); 8.00 (d,
J 12.8, 2 H, 2 x CH); 8.13 (dd, J 0.9, 7.9, 2 H, ArH.);
AK5-3 2-((1E,3Z,5E)- 3-chloro-5-(3-ethylbenzo[d]-
thiazol-2(3 H)-ylidene)penta-1,3-dien-1-yl)-3-ethylbenzo[d]-
thiazol-3-ium iodide (Yield of crude product 98 %,
methanol in a mortar with a pestle (Scheme 4). As soon as the
mixture becomes homogeneous, anhydrous sodium acetate
(
3 mmol) was added upon grinding at room temperature.
Usually, a dark blue color is spontaneously developed, which
indicates the formation of the target polymethine cyanine dye
о
M.p. = 251–253 С); UV/VIS (methanol): λmax =646 nm,
5
(Scheme 5). In addition to, the mixture crystalizes almost
−
1
−1
1
ε = 266.000 l.mol .cm , λem = 660 nm, Q = 0.10; H
NMR (500 MHz; DMSO d ) δ (ppm): 1.35 (t, J 7.1, 6 H, 2
straight forward. The reaction is also monitored by TLC.
Upon completion, (it usually takes few minutes) the product
was dissolved in methanol and transferred to a beaker contain-
ing 200 ml of an aqueous solution of potassium iodide. The
precipitated dye was suction filtered and air dried. All yield
calculations were made to the crude products. Their purifica-
tion was held by recrystallization from methanol. The struc-
tures of the pentamethine cyanine dyes AK5 were evaluated
6
x CH -CH ); 4.48 (q, J 6.9, 4 H, 2 x CH -CH -N); 6.40 (d, J
3
2
3
2
1
7
3.1, 2 H, 2 x CH); 7.46 (m, 2 H, ArH.); 7.60 (m, 2 H, ArH.);
.83 (d, J 8.3; 2 H, ArH.); 8.07 (d, J 13.0, 2 H, 2 x CH); 8.08
(d, J 7.5, 2 H, ArH.);
AK5-4 2-((1E,3Z,5E)- 3-chloro-5-(3-ethylbenzo[d] [1,
3
]selenazol-2(3 H)-ylidene)penta-1,3-dien-1-yl)-3-
1
ethylbenzo[d] [1, 3]selenazol-3-ium iodide (Yield of crude
by H NMR spectroscopy (see Experimental section).
о
product 95 %, M.p. = 241–243 С); UV/VIS (methanol):
−1
−1
λmax =657 nm, ε = 254.000 l.mol .cm , λem = 673 nm,
1
Analysis Methods and Equipment
Q = 0.07; H NMR (500 MHz; DMSO d
) δ (ppm): 1.34 (t, J
6
7
.2, 6 H, 2 x CH -CH ); 4.46 (q, J 7.1, 4 H, 2 x CH -CH -N);
3
2
3
2
1
H NMR spectra were recorded on a Bruker Avance III
00MHz instrument in DMSO-d6. Melting points were deter-
6.53 (d, J 12.8, 2 H, 2 x CH); 7.40 (m, 2 H, ArH.); 7.58 (m,
2 H, ArH.); 7.78 (d, J 8.3; 2 H, ArH.); 8.04 (d, J 12.8, 2 H, 2 x
CH); 8.15 (d, J 1.1, 7.9, 2 H, ArH.);
5
mined on a Köfler apparatus and are uncorrected.
AK5-1 2-((1E,3Z,5E)- 3-chloro-5-(3-methylbenzo[d]
thiazol-2(3 H)-ylidene)penta-1,3-dien-1-yl)-3-methylbenzo[-
d]thiazol-3-ium iodide (Yield of crude product 96 %,
AK5-6 2-((1E,3Z,5E)- 3-benzyl-5-(3-benzylbenzo[d]-
thiazol-2(3 H)-ylidene)-3-chloropenta-1,3-dien-1-yl)benzo[-
d]thiazol-3-ium iodide (Yield of crude product 99 %,
о
о
M.p. = 255–257 С); UV/VIS (methanol): λmax =645 nm,
M.p. = 224–227 С); UV/VIS (methanol): λmax =653 nm,
−1
-1,
1
−1
−1
1
ε = 204.000 l.mol .cm λem = 660 nm, Q = 0.10; H NMR
ε = 219.000 l.mol .cm , λem = 669 nm, Q = 0.13; H
NMR (500 MHz; DMSO d ) δ (ppm): 5.76 (s, 4 H, 2 x CH -
6
2
N); 6.45 (d, J 13.1, 2 H, 2 x CH); 7.25 (d, J 7.4, 4 H, ArH.); 7.33
d, J 7.2, 2 H, ArH.); 7.37–7.40 (m, 4 H, ArH.); 7.47 (m, 2 H,
ArH.); 7.58 (m, 2 H, ArH.); 7.83 (d, J 8.4; 2 H, ArH.); 8.06 (d, J
(
Table 1 Chemical structures of the quaternary benzazolium salts
Compound
Z
R
X
Yield (%)
m.p. (°C)
13.0, 2 H, 2 x CH); 8.12 (d, J 7.98, 2 H, ArH.);
AK5-8 2-((1E,3Z,5E)- 3-chloro-5-(3-(2-cyanoethyl)-
2
2
2
2
2
2
2
a
b
c
d
e
f
S
CH
3
CH
3
SO
4
87
83
74
67
63
91
72
143–145
benzo[d]thiazol-2(3 H)-ylidene)penta-1,3-dien-1-yl)-3-(2-
cyanoethyl)benzo[d]thiazol-3-ium iodide (Yield of crude
Se
S
*
2
C H
5
I
193–195
*
о
product 39 %, M.p. = 276–277 С); UV/VIS (methanol):
Se
S
−1
−1
λmax =652 nm, ε = 273.000 l.mol .cm , λem = 670 nm,
2
C H
2
C H
6
C H
4
4
5
CN
Br
248–249
*
1
Q = 0.08; H NMR (500 MHz; DMSO d ) δ (ppm): 3.12 (t, J,
6
OH
6
1
7
.5; 2 H, CH -CN); 4.83 (t, J 6.5, 4 H, 2 x CH -N); 6.58 (d, J
2 2
3.0, 2 H, 2 x CH); 7.48 (m, 2 H, ArH.); 7.62 (m, 2 H, ArH.);
.91 (d, J 8.3; 2 H, ArH.); 8.09 (dd, J 0.9, 7.9, 2 H, ArH.); 8.11
g
CH
2
224–227
*
Products 2b, 2d, 2f were found to be hygroscopic, so melting points
could not be determined
(d, J 12.9, 2 H, 2 x CH);

J Fluoresc
Scheme 4 Synthetic approach
for the synthesis of dyes AK5
AK5-9 2-((1E,3Z,5E)- 3-chloro-5-(3-(2-hydroxyethyl)-
benzo[d]thiazol-2(3 H)-ylidene)penta-1,3-dien-1-yl)-3-(2-
hydroxyethyl)benzo[d]thiazol-3-ium iodide (Yield of crude
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 ex-
truded through a 100 nm pore size polycarbonate filter
(Millipore, Bedford, USA). In this way, 3 types of lipid vesi-
cles containing PC and 11 or 67 mol% CL were prepared, with
the content of phosphate being identical for all liposome prep-
arations. Hereafter, liposomes containing 11 or 67 mol% CL
are denoted as CL11 or CL67, respectively. The phospholipid
concentration was determined according to the procedure of
Bartlett [27].
о
product 38 %, M.p. = 232–235 С); UV/VIS (methanol):
−1
−1
λmax =650 nm, ε = 278.000 l.mol .cm , λem = 666 nm,
1
Q = 0.10; H NMR (500 MHz; DMSO d ) δ (ppm): 3.83 (q, J,
6
5
5
7
.3; 4 H, CH -CH -OH); 4.51 (t, J 5.0, 4 H, 2 x CH -CH -N);
2 2 2 2
.14 (t, J 5.6, 2 H, OH-CH ); 6.50 (d, J 13.2, 2 H, 2 x CH);
2
.44 (m, 2 H, ArH.); 7.57 (m, 2 H, ArH.); 7.89 (d, J 8.4; 2 H,
ArH.) 8.02 (d, J 13.1, 2 H, 2 x CH); 8.06 (dd, J 0.9, 8.0, 2 H,
ArH.);
Preparation of Working Solution
Fluorescence Measurements
Stock solutions of cyanines were prepared immediately before
the absorption or fluorescence measurements by dissolving
the dyes in organic solvents. Stock solutions of DNA and
BSA were prepared in 10 mM Tris-HCl buffer, pH 7.4. The
concentrations of dyes, DNA and BSAwere determined spec-
trophotometrically using their molar absorptivities and ε260=
The absorption and fluorescence spectra of pentamethines in
organic solvents were recorded on a Cecil Aurius CE 3021
UV-Vis spectrophotometer and and Perkin Elmer LS45 spec-
trofluorimeter, respectively. The solvents used for the spectro-
scopic analyses were purchased form Macron Fine Chemicals
TM. Absorption and fluorescence spectra of penthamethine
dyes in the presence of liposomes, DNA and BSA were mea-
sured with CM2203 spectrophotometer at room temperature
using 10-mm path-length quartz cuvettes.
The measurements of the Forster resonance energy transfer
were performed using 4-(dimethylaminostyryl)-1-
dodecylpiridine n-toluenesulfonate as an energy donor, with
the excitation wavelength being 490 nm. Excitation and
emission slit widths were set at 10 nm. Fluorescence
intensity measured in the presence of pentamethine dyes
at the maximum of DSP-12 emission was corrected for
reabsorption and inner filter effects using the following
coefficients [28]:
3
‐1
‐1
4
‐1
‐1
6
.4×10 М cm , ε =4.25×10 М cm for DNA and BSA,
276
respectively. The molar absorptivities of the dyes are given in
Table 3. Working solution of cyanines were prepared by dilu-
tion of the dye stock solution in buffer. To determine the ab-
sorption of pentamethines in the presence of
biomacromolecules and model membranes, appropriate
amounts of the stock solution of DNA, BSA or liposomes
were added to each dye in buffer.
Lipid vesicles composed of PC and PC mixtures with CL
were prepared using the extrusion technique. A thin lipid film
À
ex ÁÀ
Á À
em ÁÀ
Á
A
ex
ex
a
−A0
em
em
a
0
1
−10−
A þ A
1−10
A
þ A
A
0
0
kcorr ¼ ꢀ
ꢁ
ꢀ
ꢁ
ð1Þ
ex
0
ex
em
em
a
−10−
ðA þA Þ
A
ex
1−10
−ðA þA Þ
em
0
a
0
1
0
ex
0
em
where A , A are the donor optical densities at the
0
excitation and emission wavelengths in the absence of
ex
a
em
a
acceptor, A , A are the acceptor optical densities at
Scheme 5 General structure of pentamethine cyanine dyes AK5
the excitation and emission wavelengths, respectively.

J Fluoresc
Quantum Yield Calculation
maximum (λ ); molar absorptivities (ε) and emission maximum
A
(λF). Absorption and fluorescence spectra of the dyes are typical
Quantum yields of the examined dyes were calculated using
Cy5 as a standard according to the formula:
for cyanines (Fig.1) with absorption maximum in DMSO in a
range from 652 to 660 nm (monomeric dye form) and emission
maxima from 670 to 682 nm depending on dye structure. A
small shoulder in the absorption spectra around 600 nm corre-
sponds to H-dimer formation [29]. It is well known that
photophysical properties of cyanine dyes are directly influenced
by the identity of the terminal heterocyclic groups and the length
of polymethine chain [30]. Replacement of benzoselenazolic
heterocycle instead of benzothiazolic one caused a 11 nm
bathochromic shift in absorption and fluorescent maxima posi-
tion for AK5-2 and AK5-4 in comparison with AK5-1 and AK5-
À
Á
−
As
2
Q 1−10
Sp
n
n
s
p
Q ¼ À
Á
⋅
ð1Þ
−
Ap
2
1−10
S
s
s
where Q is the standard quantum yield, S and S are the areas
s
p
s
under the fluorescence spectra of the dye and standard,
respectively, A and A are absorptions of the dye and
p
s
standard at a certain excitation wavelength, np and ns
are refractive indexes of the measured dye solution and stan-
dard, respectively.
3, respectively. Adding one methyl group to heterocyclic system
of AK5-3 and AK5-4, resulted in no significant change in max-
ima position. Introduction of a phenyl ring, cyano and hydroxy
group to AK5-6, AK5-8 and AK5-9 structure, respectively leads
to 5–8 nm shift in absorption maximum positions compared to
AK5-1. The Stocks shift of all compounds is approximately
equal to 15–20 nm, that are typical for this class of dyes [31].
Upon varying the organic solvent polarity from DMSO to chlo-
roform a 5–14 nm shift of absorption and fluorescence maximum
positions was observed without any significant change in spec-
trum shape. Presented in Table 4 are the fluorescence quantum
yield values of penthamethine cyanine dyes that were calculated
using Cy5 as an internal standard. The dye AK5-6, containing
phenyl ring displays the highest fluorescence quantum yield
which is comparable with that of Cy5 (0.28 in DMSO). For all
other studied dyes the quantum yields were in the range of 0.08–
0.24 depending on dye structure and the organic solvents they
were dissolved.
Results and Discussion
Synthesis and Structural Analysis of Pentamethine Dyes
A series of symmetric pentamethine cyanine dyes derived from
various N-quaternary benzothiazolium/benzoselenazolium
salts, and a conjugated bis-aniline derivative containing a chlo-
rine atom at meso-position with respect to the polymethine
chain, were synthesized using a novel synthetic approach using
mild conditions at room temperature. The products were isolat-
ed with moderate to excellent yields as illustrated in (Table 2).
Furthermore, the desired products obtained using the described
procedure were evaluated by melting point temperatures, and
1
H NMR spectroscopy (see section 2).
In an aqueous medium significant changes in cyanine spec-
tral behavior were observed. Figure 2 represents the absorp-
tion spectra of the examined cyanine dyes in buffer solution.
As judged from Fig. 2 and Table 5, the studied pentamethines,
except of AK5-6, are characterized by broad spectrum in the
range 500–700 nm with two well-defined peaks. The
bathochromic one (ranging from 640 nm to 655 nm) corre-
sponds to the absorption of monomeric dye species, while the
hypsochromic one (with maximum between 583 nm and
Spectroscopic Characterization of Pentamethine Dyes
in Different Organic Solvents and Buffer
As seen from Scheme 5, the cyanine dyes under investigation are
symmetric pentamethine dyes containing a benzothiazolic and
benzoselenazolic heterocycle with a meso-chlorine substituent
with respect to the polymethine chain. Presented in Table 3 are
the basic photophysical characteristics of the examined
pentamethines in different organic solutions: absorption
596 nm) is a hallmark of H-aggregates. The ratio of these
bands was found to depend on the dye chemical structure,
but generally the monomer maximum prevailed over that of
the aggregates. Furthermore, analysis of the absorption spectra
of AK5-1 and AK5-2 showed that three instead of two bands
are distinguished (low intensity maxima at 503 and 543 nm
are appeared), which were attributed to the dye H-aggregates,
H-dimers and monomers, respectively, starting from the left
peak. In turn, AK5-6 exhibited a somewhat different behavior.
More specifically, the absorption spectrum of this fluorophore
was blue-shifted relative to the other dyes with the maximum
centered around 542 nm. The monomer peak was virtually
unstructured and resembled the spectrum shoulder, indicating
Table 2 Chemical structures of the pentamethine cyanine dyes
Dye
X
R
Yield (%)
melting point
АК5-1
АК5-2
АК5-3
АК5-4
АК5-6
АК5-8
АК5-9
S
CH
3
96
94
98
95
99
39
38
255–257
255–258
251–253
241–243
224–227
276–277
232–235
Se
S
CH
3
2
C H
2
C H
6
C H
2
C H
2
C H
5
5
5
4
4
Se
S
CH
2
S
CN
S
OH

J Fluoresc
Fig. 1 Typical absorption spectra of pentamethine cyanine dyes in
different organic solvents. AK5-2 concentration was 0.25 μM
that H-aggregates represent the dominating fraction of the dye
molecules in buffer solution. It is known that the aggregation
properties of cyanines are controlled by the balance between
the van der Waals interactions, dispersion forces of the cya-
nine backbone, the forces of the alkyl chain of entropic and
hydrophobic nature together with H-bonding and the electro-
static interactions of the ionic groups [30]. It should be noted
that AK5-6 demonstrated the highest aggregation tendency.
To interpret the observed differences in the dye aggregation
behavior, Logarithmic Partition coefficients (LogP) were cal-
culated (Table 5). Lipophilicity, an important molecular pa-
rameter, characterizes the tendency of a molecule to distribute
between water and water-immiscible solvent [32–34]. This
parameter can be expressed by a volume or cavity term ac-
counting for hydrophobic and dispersion forces, and polarity
term determined by electrostatic interactions [32]. Having the
highest LogP value, AK5-6 with a phenyl ring in N-
substitution seems to be most hydrophobic, thereby displaying
most pronounced aggregation tendency among the dyes under
investigation.
Binding of Cyanine Dyes to Lipid Vesicles
Next step of the study was directed towards the evaluation of
possible biological applications of the examined cyanine dyes.
Table 4 Quantum yields of pentamethine cyanine dyes measured in
different solvents
Dye
MeOH
DMSO
3
CH CN
DMF
CHCl
3
AK5-1
AK5-2
AK5-3
AK5-4
AK5-6
AK5-8
AK5-9
0.10
0.08
0.10
0.07
0.13
0.08
0.10
0.19
0.18
0.18
0.24
0.28
0.24
0.21
0.12
0.08
0.11
0.08
0.09
0.09
0.10
0.09
0.15
0.15
0.12
0.17
0.14
0.17
0.17
0.30
0.19
0.25
0.22
0.08
0.11

J Fluoresc
a
Fig. 2 Absorption spectra of the dyes in buffer. Dye concentrations were
5
μM, 7,3 μM, 7.2 μM, 7.8 μM, 5.6 μM, 4.8 μM and 4 μM for AK5-1,
AK5-2, AK5-3, AK5-4, AK5-6, AK5-8 and AK5-9, respectively
b
Figure 3 shows the representative absorption spectra of
pentamethine cyanine dyes in the absence and presence of
CL67 lipid vesicles. The changes in these spectra upon lipid
addition can be summarized as follows:
i. absorbance of AK5-3, AK5-4, AK5-8 and AK5-9 de-
creases in the presence of liposomes. The absorption spec-
trum undergoes changes from ‘two-peak’ to ‘three-
peak’ form manifesting itself in the appearance of
the third well-defined hypsochromic band, suggest-
ing that in a lipid-bound state these dyes are repre-
sented by monomers, H-dimers and H-aggregates.
The magnitude of the absorbance decrease was more
pronounced for the monomeric species, resulting in
nearly equal contribution of different dye fractions
into the overall spectrum;
Fig. 3 Absorption spectra of AK5-6 (a) and AK5-2 (b) recorded in the
presence of liposomes. AK5-6 and AK5-2 concentrations were 4.5 μM
and 4.3 μM, respectively. Lipid concentration was 81.9 μM
aggregates are the dominating species of AK5-1 and
AK5-2 in a lipid-bound state;
ii. the monomeric band of AK5-1 and AK5-2 exhibites sig-
nificant decrease upon formation of dye-lipid complexes,
and the contribution of the aggregated species dominates
over the monomeric ones (Fig. 3b). This implies that H-
iii. association of AK5-6 with the lipid vesicles led to the
diminishing of the peak, attributed to the dye H-
aggregates (540 nm) and appearance of the new peak
around 660 nm, characteristic of the monomer absorption
Table 5 Absorption maxima of pentamethine cyanine dyes in buffer and in the presents of liposomes, DNA and BSA
b
Dye
Buffer*
In liposome
In DNA presence
In BSA presence
LogP
*
H
A
λ
M
A
H
A
M
A
H
A
M
A
H
A
M
A
λ
λ
λ
λ
λ
λ
λ
a
a
AK5-1
AK5-2
AK5-3
AK5-4
AK5-6
AK5-8
AK5-9
583, 503
592, 543
585
640
654
642
655
653
650
647
583, 521
649
644
652
664
660
660
654
504
640
654
642
655
653
648
647
589, 543
651
651
640
655
653
650
647
2.62
2.13
3.37
2.88
5.81
1.53
1.36
a
a
a
531 , 480
518
543 , 502
a
a
548, 594
585, 539
586
a
a
596
604 , 563
554
597, 566
542
a
542
542
542
594
592
603
597
600
601, 555
592, 540
592
*
a
H
M
λ , λ are absorption wavelength of aggregates maxima and monomeric dye form
A
A
The most intensive maximum, in the case that this maximum does not belong to dye monomers
Log P

J Fluoresc
(
Fig. 3a). The observed effect assumes that dye-lipid
arising from higher strength of interactions between the dye
and the macromolecule compared to the coupling between the
cyanine monomeric species in the aggregate. Additional argu-
ments in favor of dye disaggregation come from the appear-
ance of fluorescence spectra for AK5-6 in the range 630–
750 nm with the maximum at 675 nm (Fig. 4).
complexation breaks AK5-6 aggregates, and the dye ex-
periences transition into the monomeric state.
Furthermore, absorption spectra of AK5-6 measured at
increasing lipid concentration display isosbestic point,
indicating the equilibrium between the two main absorb-
ing species – monomers and H-aggregates.
Complexation of Pentamethine Cyanines to Bovine Serum
Albumin
Taken together, these results suggest that transfer of the
cyanine dyes into the lipid environment enhances their aggre-
gation propensity. The exception was only AK5-6 which
showed the opposite behavior. The aggregation of cyanine
dyes is a well-studied process. In an aqueous solution the
formation of cyanine aggregates occurs at high fluorophore
concentrations. Furthermore, such aggregates are usually un-
stable and easily destroyed upon heating. The situation chang-
es when the dye is transferred to the low-polarity environment
where cyanines self-associate easily even in diluted solutions
at room temperature [30]. The molecular basis for such phe-
nomenon lies in the fact that aggregation of the dyes in the low
polar solvents, in contrast to highly polar solvents, is con-
trolled not only by the dispersion forces (which govern the
stacking orientation of the dye monomers in the aggregate
structure), but also by electrostatic interactions [30]. It was
postulated that in solvents with high dielectric constant cya-
nines exist in the form of solvated ions [35]. Upon decreasing
the values of dielectric constant, cyanine dyes change their
functional state to so-called ‘contact ionic pairs’. Further low-
ering the dielectric constant results in close approaching of
these pairs with their concomitant association into the aggre-
gates, stabilized additionally by electrostatic interpair forces.
In the case of our systems, it is likely that lipid vesicles not
only provide the environment with reduced polarity, but also
serve as a template for dye oligomerization. It may be sup-
posed that strong electrostatic lipid-dye interactions ensure the
lipid bilayer surface association of the cyanines, increasing
thereby their local concentration and eventually promoting
the formation of stacked H-aggregates. The proposed scenario
is corroborated also by the observation that cyanine dyes were
non-fluorescent in the presence of liposomes (when excited
near the maximum of monomer adsorption 620 nm). This fact
represents the spectroscopic signature of aggregate formation
according to excitation coupling theory [36].
In the following, the spectral properties of the examined
pentamethine cyanine dyes were analyzed upon their binding
to bovine serum albumin. Absorption spectra of AK5-3, AK5-
6
, AK5-8 and AK5-9 in the presence of the protein were
featured by two main bands corresponding to monomers and
H-aggregates (Table 5). The position of monomer peak was
around 640–650 nm while aggregates were the most absorp-
tive at 586, 542, 600 and 592 nm, respectively. Furthermore,
BSA addition to the dye solution resulted in 1.3, 1.2, 1.2 and
1.1-fold decrease of monomer and aggregate absorption, re-
spectively, without any modification in the shape of the spec-
trum. In turn, association of AK5-1 and AK5-4 with BSAwas
followed by 1.9 and 2.3-fold lowering of the monomer absor-
bance, respectively, and 1.2-fold decrease in aggregate ab-
sorption. In addition, formation of BSA complexes with
AK5-1 and AK5-4 led to the appearance of the third blue-
shifted shoulder centered at 543 and 566 nm, reflecting the
formation of H-aggregates of higher order. Notably, the ab-
sorption of aggregate bands was slightly lower compared to
monomeric one, suggesting that in the presence of BSA the
concentration of dye monomers decreases and the concentra-
tion of aggregates increases, but the contribution of these frac-
tions into the overall spectral response is virtually equal. An
interesting behavior upon formation of dye-protein complexes
was revealed only for AK5-2 (Fig. 5). In the absence of BSA
the absorption spectra of this dye has two main bands at 654
(monomers) and 592 nm (H-dimers) coupled with a shoulder
at 543 nm (H-aggregates). The binding to BSA provoked 2.3-
µ
In contrast, the results obtained with AK5-6 suggest that
this dye reduces its aggregation propensity upon incorporation
into the lipid membranes. Presumably, upon exposure to more
hydrophobic lipid bilayer, highly organized plane-to-plane ar-
rangement, which is characterized by a broad H-band, is
disrupted, thereby favoring the randomized monomer arrange-
ment. Cyanine disaggregation as a result of dye association
with biological macromolecules such as DNA [37] and BSA
Fig. 4 Emission spectra of pentamethine cyanine dyes in CL67 lipid
bilayer. AK5-6 concentration was 1 μM
[31] was reported also in other studies and was interpreted as

J Fluoresc
characterized by two-leveled cooperativity [38]. At the first
level association of one monomer molecule with DNA groove
facilitates the binding of a second monomer. The origin of this
effect lies in unfavorable van der Waals interactions between
the planar dye and nonplanar DNA. Thus, association with
another monomer and dimer formation will be more optimal.
At the second level existing DNA-bound dimer stimulates
accumulation of other dimers or n-mers nearby. This phenom-
enon may be explained as follows. The formation of dye di-
mer in DNA groove is known to widen the groove. Such
preopening will induce the association of additional dye di-
mers or n-mers since the binding to the already perturbed
groove will require less energy penalty compared to unper-
turbed one.
Fig. 5 Absorption spectra of AK5-2 in the presence of bovine serum
albumin. AK5-2 concentration was 7 μM. BSA concentration was
6
.2 μM
fold drop in monomeric absorption, with almost fully
diminishing the dimeric band and 1.2- fold rise in H-
aggregate band. Moreover, the contribution of H-aggregate
band in the total spectrum was prevailing over the other dye
forms. This observation mirrors the ability of BSA to increase
the H-aggregation propensity of AK5-2, resulting in the dom-
ination of the aggregated dye fraction in protein-bound state.
Forster Resonance Energy Transfer Between DSP-12
and Cyanine Dyes
From the above results one can conclude that increase in the H-
aggregation propensity of pentamethine cyanine dyes under
study in the presence of biomacromolecules and lipid mem-
branes (except of the aforementioned particular cases), hinders
their application in fluorescence studies, since generally, plane-
to-plane H-aggregates are non-fluorescent [39]. Therefore, to get
further insights into the interactions of cyanine dyes with bio-
logical objects, Forster resonance energy transfer (FRET) be-
tween fluorescent probe DSP-12 as a donor and pentamethine
cyanines as acceptors was investigated in the presence of lipo-
some, DNA and BSA. As seen in Fig. 7, FRET between mem-
brane-, DNA- or BSA-bound DSP-12 and cyanine dyes mani-
fested itself in the progressive decrease of DSP-12 fluorescence
intensity and, accordingly, relative quantum yield, with increas-
ing concentration of pentamethines. The occurrence of FRET
can be considered as an additional argument in favor of
pentamethine ability to associate with DNA, BSA and model
membranes. Presented in Fig. 8a are the relative quantum yields
of DSP-12 plotted against the concentration of pentamethines
for different types of liposomes. As seen in Fig. 8, the efficiency
Association of the Cyanine Dyes with dsDNA
The complexation of pentamethine cyanine dyes with DNA
resulted in attenuation of the monomer absorption band
(
Fig. 6, Table 5). For AK5-1, AK5-2, AK5-3 and AK5-4 the
monomer peak almost disappeared and the shift of the aggre-
gate absorption towards the lower wavelengths was observed.
Furthermore, this blue-shifted band was found to increase
with DNA concentration. These findings suggest the enhance-
ment of dye aggregation in the presence of DNA. The role of
DNA as a template for cyanine aggregate growth was also
reported in other works [29, 31, 38]. It was postulated that
minor groove of nucleic acid provides a favorable environ-
ment for cyanine binding and self-assembly. Armitage et al.
suggested that DNA-templated aggregation of cyanines is
Fig. 7 Emission spectra of DSP-12 in CL11 liposomes recorded at vary-
ing concentration of the pentamethine cyanine dye AK5-6. Lipid concen-
tration was 81.9 μM. DSP-12 concentration was 0.4 μM
Fig. 6 Absorption spectra of AK5-1 in the presence of DNA. AK5-1
concentration was 4.5 μM. DNA concentration was 1·10− M b.p
5

J Fluoresc
a
observed tendency is most probably a result of high aggregation
propensity of AK5-6. The dye transition into aggregated form, is
likely to cause the overlapping of the aggregate peak with DSP-
1
2 emission spectrum, thereby complicating the interpretation of
FRET data.
In the case of AK5-3 and AK5-4 ambiguous effects were
observed: the initial increase in CL content up to 11 mol%
initiated 1.5-fold increase in energy transfer efficiency in com-
parison with the neat PC bilayer, while further increase in
anionic lipid concentration resulted in the drop of energy
transfer with the magnitude of this effect being most pro-
nounced for AK5-4. In the BSA-DSP12 system the largest
decrease in relative quantum yield of DSP-12 was observed
in the presence of pentamethine dyes AK5-2, AK5-4 and
AK5-6, whereas in DNA-containing mixtures marked in-
crease in FRET efficiency was caused by all studied dyes
except of AK5-2 and AK5-6.
b
In conclusion, the present study was focused on improved
synthesis and spectral characteristics of a series of
pentamethine cyanine dyes. The dyes under study were found
to aggregate in different solvents with the extent of aggrega-
tion being dependent on solvent polarity. Based on the results
of absorption studies, it was concluded that cyanine dyes are
capable of associating with nucleic acids, proteins and
biomembranes and this process is accompanied by the chang-
es in dye aggregation potential. The recovered pronounced
changes in spectral responses of AK5-1 – AK5-4 upon asso-
ciation with DNA allowed us to recommend these dyes for
sensitive detection of nucleic acids.
c
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