Photophysical characterization and BSA interaction of the direct ring carboxy functionalized unsymmetrical NIR cyanine dyes

Article abstract of DOI:10.1016/j.dyepig.2017.01.015

Novel near infrared (NIR) sensitive unsymmetrical cyanine dyes bearing direct –COOH functionalized indole ring were synthesized, characterized and subjected to photophysical investigations. These unsymmetrical cyanine dyes were then subjected to investigate their interaction with bovine serum albumin (BSA) as a model protein in Phosphate buffer solutions. Apart from NIR absorption and emission with high molar extinction coefficients they exhibit a blue shift in PBS solution owing to their enhanced dye aggregation. Interaction of these dyes with BSA leads to not only enhanced emission intensity but also bathochromically shifted absorption maximum due to formation of dye-BSA conjugate. These dyes bind strongly with BSA having about an order of magnitude higher binding constant as compared to the typical cyanine dyes. Amongst the unsymmetrical cyanine dyes investigated in this work one bearing substituents like Iodo and carboxylic acid in the terminal Indole rings (UCD-3) exhibited highest association with the BSA having very high binding constant of 1.01?×?10⁷?M^?1.

Full text of DOI:10.1016/j.dyepig.2017.01.015

Dyes and Pigments 140 (2017) 6e13
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Photophysical characterization and BSA interaction of the direct ring
carboxy functionalized unsymmetrical NIR cyanine dyes
*
**
Maryala Saikiran , Daisuke Sato, Shyam S. Pandey , Takeshi Ohta, Shuzi Hayase,
Tamaki Kato
***
Graduate School of Life Science and System Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-Ku, Kitakyushu-Shi, Fukuoka, 808-0196,
Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 September 2016
Received in revised form
Novel near infrared (NIR) sensitive unsymmetrical cyanine dyes bearing direct eCOOH functionalized
indole ring were synthesized, characterized and subjected to photophysical investigations. These un-
symmetrical cyanine dyes were then subjected to investigate their interaction with bovine serum al-
bumin (BSA) as a model protein in Phosphate buffer solutions. Apart from NIR absorption and emission
with high molar extinction coefficients they exhibit a blue shift in PBS solution owing to their enhanced
dye aggregation. Interaction of these dyes with BSA leads to not only enhanced emission intensity but
also bathochromically shifted absorption maximum due to formation of dye-BSA conjugate. These dyes
bind strongly with BSA having about an order of magnitude higher binding constant as compared to the
typical cyanine dyes. Amongst the unsymmetrical cyanine dyes investigated in this work one bearing
substituents like Iodo and carboxylic acid in the terminal Indole rings (UCD-3) exhibited highest asso-
22 December 2016
Accepted 9 January 2017
Available online 11 January 2017
7
ꢁ1
.
ciation with the BSA having very high binding constant of 1.01 ꢀ 10
M
©
2017 Elsevier Ltd. All rights reserved.
1
. Introduction
detected from deep tissues by commercially available imaging
modalities making them strong contender for the bio-imaging
applications [6e8]. Therefore, application of NIR sensitive organic
dyes in optical imaging and bio-diagnosis has emerged as potential
candidate due to its low energy radiation, non-invasive nature and
high sensitivity [9e11].
Biosensors having the fast, accurate and sensitive detection play
a dominant role and attracted huge interest of scientific community
in order to provide high quality of life by early disease diagnosis
[1,2]. Amongst various biosensors, one based on fluorescence
detection technique especially emitting fluorescence signals in the
near infra-red (NIR) region bear profound techno-scientific in-
terests. This is attributed to the fact that fluorescence signals in the
NIR (700 nm-900 nm) bear least auto-fluorescence associated with
background arising from the biological samples like blood, serum
and urine etc. providing relatively highly sensitive detection ca-
pabilities [3,4]. In this context, polymethene class of cyanine dyes
have attracted a great attention owing to their very high molar
extinction coefficients, small Stokes shift and tunability of optical
absorption and emission from visible to IR wavelength region by
judicious molecular design [5]. Their fluorescence can be readily
Cyanine dyes are a unique class of charged chromophores with
conjugated polymethene framework consisted of two quaternized
nitrogen-containing heterocyclic rings linked together with an in-
termediate polymethene bridge [12]. Cyanine dyes have been used
in varied applications such as fluorescent probes in luminescent
materials for labelling [13], analyte responsive fluorescent probes
[14] and in optoelectronics [15]. Wavelength tunable fluorescence
emission and good fluorescence quantum yield enable the cyanine
dyes to detect low concentrations of analytes [16]. Recent in-
vestigations on cyanine dyes have demonstrated that unsymmet-
rical cyanine dyes are more pronounced and gained much interest
due to their excellent nucleic acid staining properties [17]. Syn-
thetic versatility due to variable central methene units and avail-
ability of huge number of aromatic and heterocyclic terminal
functionalities, considerable quantum yield, good tissue penetra-
tion, lower noise due to auto fluorescence and possibility of
simultaneous multicolor and multi-target imaging enables the
*
Corresponding author.
*
*
* Corresponding author.
** Corresponding author.
E-mail addresses: msaikiran87@gmail.com (M. Saikiran), shyam@life.kyutech.ac.
jp (S.S. Pandey).
http://dx.doi.org/10.1016/j.dyepig.2017.01.015
0143-7208/© 2017 Elsevier Ltd. All rights reserved.

M. Saikiran et al. / Dyes and Pigments 140 (2017) 6e13
7
cyanine dyes for the utmost interest as fluorescent probes/labels
18,19].
This article describes the detailed synthesis and photophysical
2.3. Fluorescence quantum yield
[
Fluorescence emission quantum yield of dyes was estimated
based on the comparative method reported by Williams et al. [21],
which involves the use of well characterized standard samples with
characterization of representative unsymmetrical NIR cyanine dyes
bearing direct carboxyl functionalized indole ring. At the same time
other terminal indole ring were substituted with electron donating
eOH and electronic withdrawing eI groups. In order to explore
their potential application as fluorescent probes to sense protein in
phosphate buffer solution (PBS), these dyes were subjected to in-
vestigations pertaining to their interaction using Bovine serum al-
bumin (BSA) as a model protein. These newly designed dyes
bearing -COOH functionalized indole ring provide the capability of
covalent coupling with biomolecules such as peptides, oligonu-
cleotides and proteins to enhance their application potential as
florescence probes.
known
F
F values.
"
#
ꢀ
ꢁ
2
Grad
GradR
h
h
Q ¼ QR
(3)
2
R
R
where Q is the quantum yield of known (reference) sample, Grad is
the gradient obtained from the plot of integrated fluorescence in-
tensity vs. absorbance, Grad is the gradient of reference sample is
the refractive index of the solvent. The fluorescence emission
quantum yield assay was done by using different concentrations of
h
dyes ranging from 100 mM to 100 nm in the chloroform as solvent.
The electronic absorption and fluorescence emission spectra were
recorded. The emission spectra were measured using the corre-
sponding lmax of absorption spectra as the excitation wavelength.
2
. Experimental
Integrated fluorescence intensity (area under the peak) was ob-
tained and a graph between integrated fluorescence intensity vs.
absorbance were plotted to obtain straight line with Gradient (m).
2
.1. Materials and methods
All the chemicals for synthesis and photophysical characteriza-
tion are of analytical or spectroscopic grade and used as received.
Synthesized unsymmetrical cyanine dyes and dye intermediates
were analysed by MALDI-TOF/FAB-mass spectroscopy in positive
ion monitoring mode and nuclear magnetic resonance spectros-
copy (NMR 500 MHz for H NMR and 125 MHz for the C NMR) for
structural elucidation. Electronic absorption spectroscopic in-
vestigations in solution state were made using UV-visible-NIR
spectrophotometer (JASCO V-530 UV/VIS spectrophotometer). At
the same time, fluorescence emission spectrum was also recorded
using fluorescence emission spectroscopy (JASCO FP-6600
spectrophotometer).
2
.4. Synthesis of cyanine dyes and intermediates
Model NIR sensitive unsymmetrical cyanine dyes (UCD 1-3) has
1
13
been synthesized as per the Scheme 1 and reported literature
procedures.
2.4.1. Synthesis of substituted indole derivatives (2, 4, 5)
The derivatives 5-methoxy-2,3,3-trimethyl-3H-indole (2), 5-
iodo-2,3,3-trimethyl-3H-indole (4) and 2,3,3-trimethyl-3H-
indole-5-carboxylic acid (5) were Synthesized following the re-
ported methodology [22e24].
2
.4.2. Synthesis of 2,3,3-trimethyl-3H-indol-5-ol (3)
To a solution of compound 2 in dichloromethane (30 mL) at 0 C
was added dropwise 2 equivalents of 1 M BBr in dichloromethane
2
.2. BSA interactions
ꢂ
3
The protein (BSA)-dye interactions were conducted using
and the mixture was stirred at room temperature for 12 h. Upon the
completion of reaction as monitored by thin layer chromatography
phosphate buffer solution (PBS, 0.1 M at pH 7.4) and cyanine dye
solutions (2 M), prepared by the addition of 100 l of 0.1 M dye
solution in DMF to the various concentrations of PBS/BSA solutions
in the concentration range of (0e10 M). The final solutions were
stirred at room temperature before recording the respective elec-
tronic absorption and fluorescence emission spectra. To compare
the interactions between protein and dye, the apparent binding
constants (K ) were also calculated from fluorescent titrations of
a
dyes at 25 C. Considering BSA/dye association in 1:1 ratio, the
constant K
m
m
(
TLC), the reaction mixture was washed with saturated aqueous
solution of sodium bicarbonate and the organic extracts were dried
over Na SO , filtered, and evaporated to afford 3 as a brown solid in
9% yield [25].
m
2
4
7
2
.4.3. Synthesis of alkyl-3H-indolium iodides (1a-5a)
The derivatives 1-butyl-2,3,3-trimethyl-3H-indolium iodide
ꢂ
(1a),1-butyl-5-hydroxy-2,3,3-trimethyl-3H-indolium (3a),1-butyl-
a
was calculated using Equation (1) [20].
5
2
-iodo-2,3,3-trimethyl-3H-indolium (4a) and 1-butyl-5-carboxy-
,3,3-trimethyl-3H-indolium (5a) were synthesized by following
1
1
1
1
¼
þ K
(1)
the reported procedure [26e29].
ðF
x
ꢁ F0Þ
ðF
∞
ꢁ F0Þ
a
½BSAꢃ ðF
∞
ꢁ F0Þ
2
.4.4. Synthesis of hemi-cyanine dye (6-8)
In a round bottom flask one equivalent of corresponding alkyl-
H-indolium iodides (1a, 3a, 4a) and glutaconaldehyde dianil
where, F
0
, F
X ∞
, and F are the fluorescence intensities of dyes in the
absence, presence of BSA and at a concentration of complete
interaction, respectively, while [BSA] is the protein concentration.
Equation (1) can be modified as
3
monohydrochloride, along with catalytic amount of acetyl chloride
were dissolved in acetic anhydride. The reaction mixture was
refluxed at 140 C. After the completion of reaction as monitored by
TLC, the reaction mixture was poured on to crushed ice to precip-
itate the desired compound as black solid. This was filtered, dried
and purified by silica gel column chromatography (Ethyl acetate:
Hexane ¼ 1:1). The titled compound was obtained as red solid [30].
1-butyl-3,3-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetamido)hexa-
1,3,5-trien-1-yl)-3H-indolium chloride (6) was obtained in 60%
ꢂ
ðF
∞
ꢁ F Þ
1
0
¼
1 þ K
(2)
ðF
x
ꢁ F Þ
a
½BSAꢃ
0
The binding constant values (K
a
) for the interaction between the
BSA and cyanine dyes were calculated from the slopes of the cor-
responding plots between the (F )/(F ꢁ F ) as a function of
ꢁF
∞
0
X
0
ꢁ
1
[
BSA] as per the Equation (2).

8
M. Saikiran et al. / Dyes and Pigments 140 (2017) 6e13
Scheme 1. Synthesis of Unsymmetrical NIR Cyanine dyes.
þ
yield, MALDI-TOF mass (measured 414.33 [MþH] ; calculated
sodium sulphate and evaporated under reduced pressure. The
crude product was purified by silica gel column chromatography
(Chloroform: Methanol ¼ 9:1) to afford the respective unsym-
metrical cyanine dyes as blue green solid [31]. The FT-IR spectrum
of the dyes have been provided in supporting information
(Figs. S6eS8).
4
13.58), 1-butyl-5-hydroxy-3,3-dimethyl-2-((1E,3E,5E)-6-(N-phe-
nylacetamido)hexa-1,3,5-trien-1-yl)-3H-indolium chloride (7) ob-
tained in 26% yield, ESI-TOF mass (measured 429.2524 [M]
calculated 429.2537)1-butyl-5-iodo-3,3-dimethyl-2-((1E,3E,5E)-6-
N-phenyl-acetamido)hexa-1,3,5-trien-1-yl)-3H-indol-1-ium chlo-
þ
;
(
ride (8) obtained in 20% yield confirmed the successful synthesis of
intermediate hemi-cyanine's. The H NMR Spectra of these in-
termediates have been provided in supporting information
1-butyl-2-((1E,3E,5E)-7-((E)-1-butyl-5-carboxy-3,3-
1
dimethylindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-3,3-dimethyl-
3H-indolium chloride (UCD-1) was obtained in 10% yield. High
þ
(Figs. S3eS5).
resolution (HR)-FAB mass (measured 537.3453 [M] ; calculated
1
5
37.3476). H NMR (500 MHz CDCl
3
): 0.99 (6 H, t), 1.26e1.68 (8 H,
m), 1.71 (6 H, s), 1.78 (6 H, s), 3.65e4.10 (4H, t), 6.21e6.99 (7 H, m),
.45 (1 H, s), 7.49 (1 H, s), 7.81 (1 H, s), 7.91 (1 H, s), 8.05e8.13 (3 H,
2
.4.5. Synthesis of unsymmetrical cyanine dyes (UCD 1-3)
In a round bottom flask one equivalent of corresponding hemi
7
13
m). C NMR (125 MHz, DMSO:
166.92 (C-1), 146.65 (C-6), 145.67 (C-24),
C-5), 140.30 (C-7), 136.16 (C-15), 131.50 (4C, C-11,12,13,14),
127.60 (C-3, 22), 125.04 (C-21), 123.57 (C-20), 123.06 (C-2),
122.61 (C-23), 121.87 (C-4), 112.34 (C-16), 109.17 (C-10), 49.74
47.21 (C-18), 43.26 (C-29), 43.23 (C-25), 29.52 (C-30),
28.96 (C-26), 26.76 (C-33), 24.12 (C-34), 19.5 (C-27), 19.44 (C-
d180.59 (C-17),
d167.16 (C-9),
cyanine dye (6-8) was dissolved in 20 ml of ethanol. To the above
solution one equivalent of Compound 5a and 2 equivalents of so-
dium acetate were added. The reaction mixture was refluxed at
d
d
d
d142.01 (C-19),
d141.77
(
d
d
d
ꢂ
d
d
d
d
9
5
C. Upon the completion of reaction as monitored by TLC, the
d
d
d
d
d
solvent was evaporated under reduced pressure and the residue
was dissolved in chloroform and washed with water to remove
excess sodium acetate. The organic layer was dried over anhydrous
(C-8),
d
d
d
d
d
d
d
d
d

M. Saikiran et al. / Dyes and Pigments 140 (2017) 6e13
9
3
1
1
(
1),
694-s (C¼O), 1604-s (C¼ C), 1515-s (C¼ C (Ar)), 1417-s (CH
365-m (C-N), 1316-w (C-O), 833-w (P-substitution), 782-m 714-s
C-H (oop)).
-butyl-2-((1E,3E,5E)-7-((E)-1-butyl-5-carboxy-3,3-
d
13.78 (C-32),
d
13.56 (C-28). FTIR (KBr, cmꢁ1): 2958-m (OH),
bend),
2
1
dimethylindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-5-hydroxy-3,3-
dimethyl-3H-indolium chloride (UCD-2) obtained in 5% yield. (HR)-
ESI - TOF mass (measured 553.3415 [M] þ; calculated 553.3425). 1H
NMR (500 MHz CDCl3): 0.97 (6 H, t),1.12e1.58 (8 H, m), 1.65 (6 H, s),
1
.69 (6 H, s), 3.99 (2 H, m), 4.44 (2 H, m), 6.10e6.99 (7 H, m), 7.2 (3 H,
13
m), 7.99 (2 H, m), 8.33 (1 H, s). C NMR (125 MHz, DMSO:
C-17), 170.09 (C-9), 166.18 (C-1), 153.49 (C-6), 149.34 (C-24),
144.94 (C-19), 140.79 (C-5), 132.49 (C-15), 131.75 (C-
1,12,13,14), 129.92 (C-3, 22), 126.99 (C-7), 123.57 (C-21),
122.11 (C-20), 120.89 (C-2), 104.77 (C-23), 98.67 (C-4), 95.74
C-10), 93.05 (C-16), 58.26 (C-18), 48.73 (C-25), 44.34 (C-8),
42.38 (C-29), 29.69 (C-26), 28.22 (C-30), 25.54 (C-33), 24.80
20.41 (C-27), 20.16 (C-31), 15.29 (C-32), 14.55 (C-28).
d177.17
(
d
1
d
(
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
Fig. 1. Electronic absorption (solid line) and fluorescence emission (dashed line)
spectra of UCD 1-3 in DMF solution (5 mM).
(
C-34),
d
d
d
d
FTIR (KBr, cm-1): 2964-m (OH), 1695-m (C¼O),1605-s (C¼ C), 1521-
m (C¼ C (Ar)), 1417-m (CH2 bend), 1360-s (C-N), 1291-w (C-O
present in main
p-conjugated polymethene framework. The lmax of
(
carboxyl)), 1091-s (C-O), 831-w (p-substitution), 716-s (m-substi-
unsymmetrical cyanine dyes ranges from 764 to 774 nm with high
molar extinction coefficients (ε z 10 dm M cm ). This sharp
and intense light absorption in this class of dyes is associated with
tution), 658-w (C-H (oop)).
-butyl-2-((1E,3E,5E)-7-((E)-1-butyl-5-carboxy-3,3-
dimethylindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-5-iodo-3,3-
dimethyl-3H-indolium chloride (UCD-3) obtained in 11% yield. H
NMR (500 MHz CDCl ): 0.97 (6 H, t), 1.1e1.58 (8 H, m), 1.65 (6 H, s),
.69 (6 H, s), 4.0e4.10 (4H, m), 6.2e7.2 (7 H, m), 7.55 (1 H, s), 7.63
5
3
ꢁ1
ꢁ1
1
1
the
for each of dyes were measured slightly below (about 15e20 nm) to
the corresponding max of absorption spectrum as the excitation
pꢁ
p* electronic transitions. The fluorescence emission spectra
3
l
1
13
wavelength. For all of the unsymmetrical cyanine dyes one main
emission band can be observed which is ranging from 788 nm to
(
2 H, d), 7.99 (1 H, s), 8.09 (2 H, d). C NMR (125 MHz, DMSO:
176.20 (C-17), 173.70 (C-9), 168.09 (C-1), 154.23 (C-5), 147.10
C-24), 146.47 (C-19), 145.07 (C-6), 140.91 (C-15), 133.14 (C-14),
132.69 (C-13), 131.68 (C-12), 129.62 (C-11), 125.78 (C-7),
124.06 (C-3, 22), 123.70 (C-21), 122.37 (C-20), 121.87 (C-2),
119.29 (C-23), 113.63 (C-4), 112.14 (C-10), 86.92 (C-16), 69.18
63.35 (C-25), 48.29 (C-8), 44.65 (C-29), 29.89 (C-26),
29.87 (C-30), 27.98 (C-33), 24.44 (C-34), 20.36 (C-27), 20.13
14.35 (C-32), 14.20 (C-28). FTIR (KBr, cm ): 2958-m (OH),
d
(
d
d
d
d
d
d
d
798 nm, with a small Stokes shift of 23 and 24 nm. This small Stokes
d
d
d
d
d
d
shift represents the rigidity of the molecules without having any
conformational changes after the photoexcitation.
To investigate the interactions between the dyes and the bio-
molecules for imaging applications, Phosphate buffer solution
d
d
d
d
d
d
d
d
(
C-18),
d
d
d
d
(
PBS) has been most commonly used. Keeping this in mind, elec-
tronic absorption spectra of these dyes were also measured in the
.1 M PBS solution at pH 7.4 which has been shown in Fig. 2. It is
worth mentioning that the absorption spectra of cyanine dyes in
PBS exhibited slightly blue shifted max compared to that observed
in DMF. This behaviour of blue shifted max could be attributed to
d
d
d
d
d
ꢁ1
(
C-31),
d
d
0
1
699-m (C¼O), 1605-m (C¼ C), 1506-s (C¼ C (Ar)), 1415-s (CH
2
bend), 1360-s (C-N), 1309-w (C-O (carboxyl)), 816-w (p-substitu-
tion), 714-s (m-substitution), 649-m (C-H (oop)), 554-w (C-I).
l
l
the enhancement in aggregation of dye, promoted by the hydrogen
bonding between the dye molecules due to the presence of eCOOH
groups. It is well known that cyanine dyes exhibit dye aggregation
3
. Results and discussion
3.1. Photophysical characterization
owing to their flat molecular structure [32]. Apart from the main
p-
p
* electronic transition, cyanine and squaraine dyes also exhibit a
After the successful synthesis and purification these NIR sensi-
vibronic shoulder just before the main absorption peak and the
vibronic shoulder has been reported to be marker of molecular
aggregation [33]. Higher value of the ratio of absorbance for the
vibronic shoulder with respect to the absorbance corresponding to
tive dyes were subjected to photophysical investigations pertaining
to the electronic absorption and fluorescence emission spectros-
copy. Results thus obtained pertaining to the photophysical pa-
rameters have been summarized in Table 1. Fig. 1 exhibits the
solution state electronic absorption and fluorescence emission
spectra for the unsymmetrical cyanine dyes in the dime-
thylformamide (DMF) solution.
It can be observed that position of absorption maxima (lmax) for
the electronic absorption and fluorescence emission in DMF are not
much affected by the different substituents (hydroxyl and iodo)
the main
34,35].
A perusal of absorption spectra shown in Fig. 2 clearly corrob-
p-p* transition indicates an enhanced dye aggregation
[
orates that vibronic shoulders of the dyes in PBS are relatively more
pronounced as compared to that observed in the DMF (Fig. 1)
verifying the enhanced dye aggregation and this could be
Table 1
Spectral properties of dyes in DMF and 0.1 M PBS solution at pH 7.4.
Dye
DMF Solution
(max) Absorption
PBS Solution
(max) Absorption
Quantum Yield
(ε) (dm3
M
ꢁ1
ꢁ1
cm )
l
l
l
(max) Emission
Stoke Shift
5
UCD -1
UCD -2
UCD -3
764 nm
769 nm
774 nm
788 nm
792 nm
798 nm
24 nm
23 nm
24 nm
1.1 ꢀ 10
0.8 ꢀ 10
1.4 ꢀ 10
749 nm
753 nm
757 nm
0.327
0.196
0.649
5
5

10
M. Saikiran et al. / Dyes and Pigments 140 (2017) 6e13
protein. Fig. 3 depicts the absorption spectra of one of the repre-
sentative cyanine dyes (UCD-1) in the presence and absence of BSA.
It can be seen that there are two different sets of prominent elec-
tronic absorption bands in the wavelength region of
250 nme300 nm and 700 nm - to 800 nm associated with the
absorption of BSA and dye UCD-1, respectively. Increase in the BSA
concentration led to the gradual increase in intensity of absorption
between 250 nm and 300 nm, which was associated with electronic
absorption of the protein BSA.
However, there was random increase and decrease of absorption
intensity associated with dye in NIR region of 600 nme800 nm, a
behaviour similar to that observed by Pisoni et al. also [26]. At the
same time, random increase and decrease without any isobestic
point indicates that the equilibrium between the free and bound
protein is not simple in all the dyes under investigation. Interest-
ingly,
absence of BSA was found to be bathochromically shifted by
e10 nm in the presence of BSA. This could be attributed to the
lmax associated with UCD-1 which was around 750 nm in the
5
suppression of dye aggregation due to the interactions between the
protein and dye molecules. Similar type of behaviour has also been
observed for the other cyanine dyes (UCD-2 and UCD-3) and results
have been provided in the supporting information (Figs. S1eS2).
The fluorescence emission spectra of UCD-1 in the presence and
absence of BSA is shown in Fig. 4. It can be seen that dye exhibits
Fig. 2. Electronic absorption spectra for unsymmetrical cyanine dyes UCD 1-3 in 0.1 M
PBS.
increase in fluorescence intensity near the lmax along with slight
red shift of peak maxima upon the addition of increasing amounts
of BSA. The increase in fluorescence intensity could be attributed to
the possible interaction between the UCD-1 and BSA.
attributed to the blue-shifted lmax also. It is interesting to mention
here that amongst all of the unsymmetrical squaraine dyes used in
this work UCD-2 exhibits relatively enhanced dye aggregation due
to prominent vibronic shoulder which was visible even in the DMF
solution (Fig. 1) and becomes even more prominent in the PBS. This
could be attributed to the fact that apart from eCOOH functional
group which is present in all of the dyes, it bears eOH substituted at
the 5-position of the other indole ring which is expected to enhance
the hydrogen bond assisted enhanced dye aggregation.
This interaction may lead to the strengthening of the planar
configuration of the dye molecules especially in the excited state
ultimately enhancing the molecular rigidity after formation of
complex between UCD-1 and BSA. It seems quite probable that
eCOOH functionality of dye is expected to the hydrogen bond
assisted dye aggregate formation in PBS leading to the blue-shifted
emission maximum (Table 1). In the presence of BSA in the PBS this
aggregate formation is hampered due to the dye-BSA complex
formation leading to bathochromically shifted emission. For the
other dyes under investigation also electronic absorption and
fluorescence emission spectra in the presence and absence of BSA
were also measured and shown in the supporting information
Fluorescence quantum yield (f) is an important parameter to-
wards the development of fluorescent probes and is an indicator of
the capability of a fluorescent probe to convert absorbed photons in
to the emitted one in a particular environment. In combination
with molar extinction coefficient (ε) it signifies the strength of
fluorescence signal since product of the
brightness of fluorophore. A perusal of the
f
f
and ε determines the
calculated for different
(Figs. S1eS2).
Here one can argue that during the interaction of BSA with dyes
unsymmetrical cyanine dyes as shown in Table 1 indicates that they
exhibit fairly good values as compared to typical NIR cyanine dyes.
Amongst the dyes used in this work, it can be seen that eOH
substituted cyanine dye UCD-2 exhibits relatively hampered value
under investigation is there any detrimental effects like denatur-
ation of BSA with dyes. To have the in-depth insight about this we
have conducted the measurement of circular dichroism (CD) of the
BSA in the presence and absence of the dyes and obtained results of
the measurement have been shown in Fig. 5. The CD spectrum of
of
f (about 0.20) as compared to the unsubstituted UCD-1(0.33).
This could be attributed the fact that UCD-2 exhibits pronounced
aggregate formation and aggregate assisted non-radiative fluores-
BSA was taken by adjusting concentration of BSA to 5
while the CD spectrum of BSA-dye solutions were executed with
.2 M dye and 5 M BSA. It is well known that CD spectrum of BSA
typically consists of two negative bands appearing at 208 nm and
222 nm associated with the n/ * transition and negative Cotton
mM in PBS
cence decay leading to the hampered
.2. Interaction of dyes with bovine serum albumin
In order to avoid the purification steps in labelling of bio-
f.
0
m
m
3
p
effect, respectively [39,40]. BSA contains the distinct binding do-
mains for hydrophobic compounds. The specific interactions of dye
with BSA originates from the presence of two structurally selective
binding sites, namely site I and site II. The binding affinity offered
by site I is purely hydrophobic interactions where as the site II in-
volves a combination of hydrophobic, hydrogen bonding and
electrostatic interactions [41]. A perusal of Fig. 5 pertaining to the
CD spectra clearly reveals the persistence of both of negative bands
of BSA around 208 nm and 222 nm in the presence of all of the dyes
under investigation which depicts that there is no denaturation of
the BSA in the presence of dyes.
molecules with dyes towards the application of optical bio imaging,
non-covalent methods have been frequently used [36]. Patonay and
co-workers were among the first to study the noncovalent labelling
of the human serum albumin (HSA) with near-infrared dyes by
using high-performance liquid chromatography (HPLC) and ab-
sorption detection [37]. At the same time, bovine serum albumin
(
BSA) is a globular protein which has been prominently used as
protein model to investigate the interactions between dye and
protein owing to its high homology with HSA in the amino acidic
sequences [38]. Therefore, dyes used in this work have also been
subjected to investigate their interaction with the BSA as a model
BSA concentration dependence of the fluorescence intensity for
various unsymmetrical cyanine dyes have been shown in Fig. 6. A

M. Saikiran et al. / Dyes and Pigments 140 (2017) 6e13
11
Fig. 3. Electronic absorption spectra of UCD 1 in 0.1 M PBS at different concentrations of BSA for fixed dye concentration of 2 mM.
Fig. 4. Fluorescence emission spectra of UCD-1 with varying concentrations of BSA for a fixed dye concentration of 2 mM.
linear correlation between fluorescent intensity of dyes as a func-
tion of BSA concentration was observed. This increase in fluorescent
intensity along with red shift of lmax can be attributed to the non-
covalent interaction between dyes and protein due to the formation
of BSA-dye conjugates.
without causing any damage to its three dimensional conformation
which basically governs the protein function and activity. The
conformational changes in protein may occur at the point where
probe is located. As the dye concentration increases, there may be
more damage in its activity. Keeping this mind, a very low dye
It has been widely accepted that drugs or probes interact
differently with the protein under consideration and their ability to
bind depends on protein concentration [25]. It can be clearly seen
that ability to bind with fluorescent dyes utilized in this work were
enhanced upon the addition of increasing concentration of BSA. At
the same time, amongst the dyes utilized dye-BSA interaction
studies, UCD-3 was found to exhibit the highest binding affinity
with BSA. In order to successfully implement fluorescent dyes as a
probe, it is necessary that the dye should bind to the protein
concentration of (2
BSA interaction with all of the unsymmetrical NIR cyanine dyes.
Thanks to high ε and good of the dyes, it possible to investigate
the interaction satisfactorily even at lower dye concentration.
To compare the ability of binding and relative association of
mM) has been used for investigation of the dye-
f
dyes with BSA quantitatively, apparent binding constant (K
calculated using Equation (2) by plotting (F∞ ꢁ F )/(F ꢁ F
function of inverse of BSA concentration as shown in Fig. 7. The
value of K was calculated from the slopes of the this figure which
a
) was
0
X
0
) as
a

12
M. Saikiran et al. / Dyes and Pigments 140 (2017) 6e13
the presence of direct ring substituted hydrophilic carboxylic acid
eCOOH) functional group promoting hydrogen bonding with
(
binding sites of BSA.
As discussed earlier BSA possess distinct hydrophobic and hy-
drophilic active sites (Site I and Site-II). Upon interaction with the
ligands (drug or dyes) the relative hydrophobicity of ligands plays
an important role in controlling their interactions with the BSA
specifically with Site-I. A perusal of the molecular structures of the
dyes under investigation reveals that all of dyes possess nearly
same main p-framework, alkyl group and eCOOH functional group
apart from the varying substituents (-OH, H or eI) in the main ar-
omatic ring at the other terminal indole unit. Since other structural
factors are constant, UCD-3 exhibits relatively more hydrophobic
nature as compared to the UCD-2 bearing eOH group which is
responsible for its enhanced interaction with BSA ultimately lead-
ing to high binding ability. On the other hand enhanced aggregation
and competitive interactions due to presence of two functional
groups eOH and eCOOH on the two opposite ends of the chro-
mophore in the dye UCD-2 could be attributed to the least associ-
ation with the BSA.
Fig. 5. CD spectra of BSA and dye-BSA complexes at pH 7.4.
4. Conclusions
Direct ring carboxy functionalized NIR sensitive unsymmetrical
cyanine dyes having different substituents (-OH and -I) have been
successfully synthesized and characterized. These dyes were sub-
jected to the photophysical investigations in order to explore their
applicability as fluorescent probes. Interactions of these dyes with
BSA as a protein model suggested the formation of dye-BSA con-
jugates, which led to the enhancement in fluorescence intensity
along with bathochromic shift of emission maxima. Amongst the
various dyes utilized, one with mild electron withdrawing iodo
group (UCD-3) has been found to exhibit the highest affinity to-
wards the association with BSA. This high binding constant
observed in the case of UCD-3 could be associated with the pres-
ence of relatively bulkier iodo group along with the eCOOH group
directly attached with the indole ring.
Fig. 6. Plot of florescence emission intensity at peak maxima for cyanine dyes (UCD 1-
) as a function of BSA concentration. Dye concentration was constant (2 M) for each
of the dyes.
4
m
Acknowledgements
Authors would like to acknowledge the support from Prof. Shi-
geori Takenaka (Kyushu Institute of Technology, Tobata) in
obtaining Mass spectra and Jelli Venkata Prasad in obtaining H
NMR Spectra.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2017.01.015.
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