Aza-substituted squaraines for the fluorescent detection of albumins

Article abstract of DOI:10.1016/j.dyepig.2010.11.005

Three benzothiazole substituted squaraines bearing hydroxyethylamino, aminoethylamino and 3-iodobenzylamino substituents on the cyclobutane unit have been synthesized and characterized. The structure of the hydroxyethylamino substituted analogue was deter

Full text of DOI:10.1016/j.dyepig.2010.11.005

Dyes and Pigments 90 (2011) 41e47
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Aza-substituted squaraines for the fluorescent detection of albumins
,
a
b
Kateryna D. Volkova a, Vladyslava B. Kovalska a , Mykhaylo Yu. Losytskyy , Lucinda V. Reis ,
*
Paulo F. Santos b, Paulo Almeida c, Daniel E. Lynch d, Sergiy M. Yarmoluk a
a Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnogo St., 03143 Kyiv, Ukraine
b Department of Chemistry and Centro de Química e Vila Real, Universidade de Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal
c Department of Chemistry and Unidade de I&D de Materiais Têxteis e Papeleiros, Universidade da Beira Interior, 6201-001 Covilhã, Portugal
d Exilica Limited, The Technocentre, Puma Way, Coventry CV1 2TT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Three benzothiazole substituted squaraines bearing hydroxyethylamino, aminoethylamino and 3-iodo-
benzylamino substituents on the cyclobutane unit have been synthesized and characterized. The
structure of the hydroxyethylamino substituted analogue was determined by X-ray crystallography. The
fluorescent properties of the new dyes plus those of seven previously reported aza-substituted squar-
aines were studied in both pure solution as well as in the presence of human, bovine and horse serum
albumins, and ovalbumin. All of the dyes demonstrated low fluorescence emission levels in pure solu-
tion, and fluorescence intensity increases of up to 400 times in the presence of albumins. Dyes with an
aminoethylamino and N,N-dimethylhydrazino substituents demonstrated good emission intensities
in complexes with albumins, with fluorescence quantum yields of the latter dye reaching 0.18.
The aminoethylamino substituted dye demonstrated a wide human serum albumin detection range
Received 18 June 2010
Received in revised form
3 November 2010
Accepted 8 November 2010
Available online 18 November 2010
Keywords:
Squaraine dyes
Fluorescent detection
X-ray analysis
(1.5 mg/mL to 20 mg/mL of protein). Studies of the dyes in the presence of model sodium dodecyl
Albumins detection
sulphate and bovine serum albumin e sodium dodecyl sulphate systems have shown that these
squaraines do not give a considerable fluorescent response in the presence of denatured proteins or
surfactant, with the exception of the 2-sulfoethylamino aza-substituted dye.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
fluorescent probes. Squaraine dyes have been reported as efficient
noncovalent labels for albumins [6], exhibiting high quantum yields
Albumins are the major plasma proteins circulating in the
bloodstream and are produced in the liver [1]. They play a signifi-
cant role in the maintenance of colloid osmotic pressure and the
binding of long-chain fatty acids, bile acids, bilirubin, haematin,
calcium and magnesium. They also act as carriers for nutritional
factors and drugs. Because serum albumin is a reliable prognostic
indicator for morbidity and mortality, liver disease, kidney disease
and malnutrition, analytical study methods are in high demand. Up
to date fluorometric methods have been proposed for the detection
and study of albumins. These have advantage over other methods
because of their high sensitivity, selectivity and convenience [2,3].
Currently, commercially available fluorescent dyes such as eosin
B and eosin Y [4], Albumin Blue [5] and Nano Orange are used for
protein determination in solution. Interest in squaraine compounds
has been recently renewed, due to their potential usefulness in a
large number of technologically relevant fields such as NIR-emitting
when bound to these proteins [7].
Previously, we studied a series of benzothiazole and benzosele-
nazole squaraines as fluorescent probes for protein detection. For
squaraine dyes with N-hexyl pendent groups, about a 100 to 540
fold fluorescence intensity increase upon albumin addition was
observed. Amongst the series of dyes examined the aza-substituted
compounds, possessing simple N-methylamino and N,N-dieth-
ylamino substituents on the central four-membered ring, displayed
pronounced enhancement of emission intensity, observed in the
presence of human (HSA) and bovine (BSA) serum albumins. Thus,
benzothiazole dye 6 with a N,N-diethylamino substituent on the
squaraine ring allows quantification of HSA in the range from
0.2
mg/mL to 500 mg/mL, which is comparable with commercially
used dyes such as Coomassie Brilliant Blue and Pyrogallol Red
Protein [8,9]. Since the presence of an amine moiety in the frame-
work of the dye embodies a diversity of possible specific interac-
tions between the dye and the protein, conditioning their mutual
affinity, it seemed worthwhile to study the effect that differently
substituted amine groups had on the fluorescent properties of aza-
substituted squarylium dyes in the presence of proteins.
* Corresponding author. Tel./fax: þ380 44 252 24 58.
E-mail address: v.b.kovalska@imbg.org.ua (V.B. Kovalska).
0143-7208/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2010.11.005

42
K.D. Volkova et al. / Dyes and Pigments 90 (2011) 41e47
As a continuation of these studies, we synthesized a range of new
(1H, d, J ¼ 8.2, ArH), 7.55e7.47 (2H, m, ArH), 7.39e7.30 (2H, m, ArH),
6.22 (1H, s, CH]C), 5.95 (1H, s, CH]C), 5.17 (1H, br s, NH or OH),
4.31e4.26 (4H, m, NCH2(CH2)4CH3), 3.68 (4H, br s, NHCH2CH2OH),
aza-substituted benzothiazole squaraine dyes (2, 3 and 10, Fig. 1)
and characterized them by IR, UV/Vis, 1H NMR, 13C NMR spectros-
copy and HRFABMS (High Resolution Fast Atom Bombardment Mass
Spectrometry). For dye 2, crystals were obtained and the structure
was solved and confirmed using X-ray crystallographic analysis.
A series of ten new, as well as previously reported [10], aza-
substituted benzothiazole and benzoselenazole squaraines (Table 1)
was studied as probes for the detection of various albumins. The
fluorescent properties of dyes when unbound and in the presence of
human (HSA), bovine (BSA) and horse (HRSA) serum albumin, and
ovalbumin (OVA) were characterized. The sensitivity of dyes to
denatured proteins e BSA/SDS mixture e and thus their applica-
bility for non-specific detection of proteins was also evaluated. The
dependence between dye molecule structure and its selectivity
towards certain protein was analyzed.
1.72 (4H, br s, NCH2CH2(CH2)3CH3), 1.39e1.29 (12H, m,
N
(CH2)2(CH2)3CH3), 0.85 (6H, br t, J ¼ 6.7, N(CH2)5CH3). 13C NMR
(100.62 MHz, DMSO-d6) d: 173.6, 163.7, 161.0, 159.4, 157.1, 155.7,
140.6, 127.8, 127.6, 127.5, 124.9, 124.4, 122.9, 122.6, 113.4, 113.0, 87.2,
86.3, 60.7, 46.3, 45.5, 30.9, 30.8, 27.2, 26.8, 25.7, 22.0, 21.9, 13.8.
HRFABMS (3-NBA): 588.2709 ([M-CF3SO3]þ, C34H42N3O2S2þ; calc.
588.2718).
2.1.2. 2-[2-(Aminoethylamino)-3-(3-hexyl-3H-benzothiazol-
2-ylidenemethyl)-4-oxocyclobut-2-enylidenemethyl]-3-
hexylbenzothiazol-3-ium trifluoromethanesulfonate (3)
To a solution of the O-methylsquaraine dye 1 [10] (0.20 g,
0.28 mmol) in anhydrous CH2Cl2 (100.0 mL), under N2 atmosphere,
was added 1,2-diaminoethane (0.021 mL, 0.31 mmol). The reaction
mixture was stirred at r.t. for 40 min. and then cooled in an ice-
bath. Upon addition of Et2O a solid precipitate, which was collected
by filtration under reduced pressure and was recrystallized from
CH2Cl2/MeOH/Et2O. Yield: 84%. Purple crystals. M.p. 202.0 ꢀC (dec.).
2. Materials and methods
2.1. Synthesis
UV/Vis (MeOH/CH2Cl2, 99/1) lmax (log 3): 654 (5.26). IR (KBr) nmax:
All reagents were purchased from Sigma-Aldrich and were used
without further purification. Solvents were of analytical grade. All
reactions were monitored by thin-layer chromatography (TLC)
using 0.25-mm Al-backed silica gel plates (Merck 60 F254). Melting
points (M.p.) were measured in open capillaries on a Büchi-530
melting-point apparatus and are uncorrected. IR spectra were
recorded on a Unicam Research Series FTIR spectrophotometer.
UV/Vis spectra were performed on a Spectronic Genesys 2PC
instrument. 1H and 13C NMR spectra were recorded on either
3480 w, 2929 w, 1633 w, 1562 w, 1511 w, 1427 s, 1357 m, 1238 s,
1155 m, 1133 m, 1027 m, 981 m, 827 w, 792 w, 748 w cmꢁ1. 1H NMR
(400.13, DMSO-d6)
d: 7.99 (1H, d, J ¼ 7.8, ArH), 7.94 (1H, d, J ¼ 7.8,
ArH), 7.71 (1H, d, J ¼ 8.2, ArH), 7.66 (1H, d, J ¼ 8.2, ArH), 7.56e7.49
(2H, m, ArH), 7.40e7.32 (2H, m, ArH), 6.18 (1H, s, CH]C), 5.91 (1H, s,
CH]C), 4.37 (2H, br s, NHCH2(CH2)4CH3), 4.28 (2H, br s,
NHCH2(CH2)4CH3), 3.67 (2H, t, J ¼ 5.8, NHCH2CH2NH2), 2.95 (2H, t,
J ¼ 5.8, NHCH2CH2NH2), 1.73 (4H, br s, NCH2CH2(CH2)3CH3),
1.39e1.29 (12H, m, N(CH2)2(CH2)3CH3), 0.85 (6H, t, J ¼ 6.6, N
Bruker ARX 300 or Bruker ARX 400 spectrometers;
d in ppm rela-
(CH2)5CH3). 13C NMR (100.62 MHz, DMSO-d6)
d: 173.6, 163.4, 161.1
tive to SiMe4 or to residual solvent signals. HRFABMS were obtained
on a Micromass AutoSpec M spectrometer, operating at 70 eV,
using a matrix of 3-nitrobenzyl alcohol (3-NBA).
159.8,157.1,155.5,140.6,127.8,127.7,127.5,125.4,125.0,124.6,122.9,
122.6, 122.2, 119.0, 115.8, 113.5, 113.2, 86.6, 86.2, 46.4, 45.7, 30.9,
30.8, 27.2, 26.9, 25.7, 21.9, 13.8. HRFABMS (3-NBA): 587.2877
([M-CF3SO3]þ, C34H43N4OSþ2 ; calc. 587.2878).
2.1.1. 3-Hexyl-2-[3-(3-hexyl-3H-benzothiazol-2-ylidenemethyl)-
2-(2-hydroxyethylamino)-4-oxocyclobut-2-enylidenemethyl]
benzothiazol-3-ium trifluoromethanesulfonate (2)
2.1.3. 3-Hexyl-2-[3-(3-hexyl-3H-benzothiazol-2-ylidenemethyl)-
2-(3-iodobenzylamino)-4-oxocyclobut-2-enylidenemethyl]
benzothiazol-3-ium iodide (10)
To a solution of the O-methylsquaraine dye 1 [10] (0.43 g,
0.61 mmol) in anhydrous CH2Cl2 (10.0 mL), under N2 atmosphere,
was added an excess of 2-aminoethanol (0.048 mL, 0.79 mmol). The
reaction mixture was stirred at r.t. for 30 min and then concentrated
by partial removal of the solvent under reduced pressure. Addition
of Et2O yielded a solid, which was collected by filtration under
reduced pressure and recrystallized from CH2Cl2/Et2O. Yield: 68%.
Purple crystals. M.p. 162.5e163.5 ꢀC. UV/Vis (MeOH/CH2Cl2, 99/1)
To a solution of the O-methylsquaraine dye 1 [10] (0.36 g,
0.50 mmol) and triethylamine (0.14 mL, 1.0 mmol) in anhydrous
CH2Cl2 (20.0 mL), under N2 atmosphere, was added an excess of
3-iodobenzylamine (0.33 mL, 2.5 mmol). The reaction mixture was
stirred at r.t. for 3 h and then washed with cold water. The organic
layer was dried over anhydrous Na2SO4 and the solvent
was removed under reduced pressure. The resulting residue was
dissolved in MeOH and to this solution was added an approxi-
mately equal volume of 14% aqueous KI. After about 2 h, the
precipitated dye was collected by filtration, washed with water and
lmax (log
1568w, 1457s, 1354w, 1254s, 1155 m, 1028 m, 983w, 748w. 1H NMR
(400.13 MHz, DMSO-d6) : 8.86 (1H, br s, NH or OH), 7.98 (1H, d,
3): 654 (5.30). IR (KBr) nmax: 3463w, 2928w, 2858w,1628w,
d
J ¼ 7.7, ArH), 7.92 (1H, d, J ¼ 7.7, ArH), 7.69 (1H, d, J ¼ 8.2, ArH), 7.64
CH2(CH2)4CH3
CH2(CH2)4CH3
N
N
O
O
S
S
S
S
Y
N
OMe
N
CH2(CH2)4CH3
CF3SO3
CH2(CH2)4CH3
X
1
2 Y = NHCH2CH2OH, X = CF3SO3
3 Y = NHCH2CH2NH2, X = CF3SO3
10 Y = NHCH2-3-I-C6H4, X = I
Fig. 1. Reagents and conditions. Dye 2: NH2CH2CH2OH, CH2Cl2, N2, r.t.; dye 3: NH2CH2CH2NH2, CH2Cl2, N2, r.t.; dye 10: 3-I-C6H4CH2NH2, Et3N, CH2Cl2, N2, r.t.

K.D. Volkova et al. / Dyes and Pigments 90 (2011) 41e47
43
Table 1
Chemical structures of studied aza-substituted squaraine dyes.
O
O
S
N
S
S
N
S
N+
N+
NH
OH
NH
F
F
O
S
F
F
O
S
F
O
F
O
NH2
O
O
2
3
O
O
S
N
S
S
N
S
N+
N+
HN
NH
I
I
I
10
11
O
O
Se
N
Se
S
S
N+
N+
N
N
N
I
I
4
6
O
O
Se
Se
N+
S
S
N+
N
N
NH
O
NH
O
O
S
O
S
O
O
5
8
O
O
Se
N
S
N
Se
N+
S
N+
NH
N
NH
N
9
7
recrystallized from CHCl3/MeOH/Et2O. Yield: 67%. Blue crystals.
M.p. 257.5 ꢀC (dec.). UV/Vis (MeOH/CH2Cl2, 99/1) lmax (log
): 657
(5.37). IR (KBr) nmax: 3130 w, 2962 w, 2920 w, 1634 w, 1562 m,
806 w, 755 w. 1H NMR (300.13 MHz, CDCl3/DMSO-d6)
d
: 9.18 (1H, t,
3
J ¼ 6.1, NH, exchanging with D2O), 7.90 (1H, d, J ¼ 7.8, ArH),
7.82e7.80 (2H, m, ArH), 7.69 (1H, d, J ¼ 7.8, ArH), 7.61 (1H, d,
J ¼ 8.3, ArH), 7.54e7.21 (7H, m, ArH), 6.32 (1H, s, CH]C), 5.64 (1H, s,
1458 s, 1431 s, 1353 m, 1238 s, 1178 m, 1155 m, 1134 m, 973 w,

44
K.D. Volkova et al. / Dyes and Pigments 90 (2011) 41e47
CH]C), 4.90 (2H, d, J ¼ 6.1, NHCH2Ph, collapses to s with D2O),
4.30 (2H, br t, J ¼ 7.0, NCH2(CH2)4CH3), 4.14 (2H, br t, J ¼ 6.5,
NCH2(CH2)4CH3), 1.81e1.78 (2H, m, NCH2(CH2)4CH3), 1.48e1.20
(14H, m, NCH2(CH2)4CH3), 0.91e0.82 (6H, m, N(CH2)5CH3). 13C NMR
each spectrum (S7 and SDCI respectively) was calculated. Further,
the fluorescence quantum yield of dye 7 in the presence of HSA was
calculated as 47
¼
4DCI ꢂ S7/SDCI
.
The dynamic range and lower detection limit of HAS detection
for benzothiazole squarilyum dye 3, was determined by the titra-
tion of a fixed concentration (5 ꢂ10ꢁ6 M) of the dye with the
increasing amounts of HSA. The constant of dye 3 binding to HSA
was estimated based on the aforementioned dependence of dye 3
(5 ꢂ10ꢁ6 M) fluorescence intensity on the HSA concentration
added (titration curve). The binding constant Kb is:
(75.47 MHz, CDCl3/DMSO-d6) d: 173.7, 163.4, 161.3, 159.6, 157.2,
155.5, 140.6, 140.4, 140.3, 136.2, 135.0, 130.6, 127.6, 127.4, 127.3,
125.6, 124.8, 124.3, 122.5, 122.2, 121.8, 113.1, 112.6, 95.0, 86.7, 86.3,
46.4, 45.8, 30.9, 30.8, 27.3, 26.7, 25.7, 21.9, 13.7. HRFABMS (3-NBA):
760.1856 ([M-I]þ, C39H43IN3OSþ2 ; calc. 760.1887).
2.1.4. X-ray crystallographic analysis
Single crystals were obtained by the slow evaporation of a dilute
solution of dye 2 in CHCl3. Diffraction data were collected on a Bruker
Cbd
Cfd ꢂ Cfp
Kb
¼
(1)
SMART APEX2 CCD diffractometer using synchrotron radiation
where Cbd, Cfd and Cfp are concentrations of bound dye, free dye and
free dye-binding sites of the protein respectively. If we consider, for
dye 3, one binding site per protein globule and then regard the total
protein concentrations Cp much exceeding the total dye concen-
trations Cd so that Cp z Cfp; and if the free dye fluorescence
intensity If is negligibly small as compared to the fluorescence
ꢀ
(l
¼ 0.7848 A). The structure was solved by direct methods SHELX97,
and refined by full-matrix least-squares calculations. Crystal data for
ꢁ
dye 2: C35H42F3N3O5S3, Mw ¼ 737.93, triclinic, Põ, Z ¼ 2, a ¼ 9.786(2),
ꢀ
b ¼ 10.538(2), c ¼ 18.370(4) A,
a
¼ 104.496(2),
b
¼ 94.051(3),
g
m
¼ 104.868(2)ꢀ, Dcalcd ¼ 1.397 g cmꢁ3, T ¼ 120(2) K, F(000) ¼ 776,
¼ 0.274 mmꢁ1, 7578 reflections were collected, 4800 unique
intensity in the presence of the protein IHSA so that Cbd/Cd ¼ IHSA
/
(Rint ¼ 0.0507), 2792 observed (I > 2
s (I)), 453 parameters,
Imax (Imax is the saturation dye fluorescence intensity upon HSA
R1 ¼0.0767, wR2 ¼ 0.2142. Crystallographic data (dye 2 (CCDC
754805) have been deposited at the CCDC,12 Union Road, Cambridge
CB2 1EZ, UK.
addition); then equation (1) could be transformed to:
Kb ꢂ Cp
IHSA ¼ Imax
ꢂ
(2)
1 þ Kb ꢂ Cp
2.2. Materials
Thus if we approximate the points of the titration curve corre-
sponding to HSA concentrations higher than 3 ꢂ 10ꢁ5 M (in HSA
globules) with equation (2), the value of Kb (together with this
of Imax) could be obtained as the approximation parameter. All
measurements were carried out at room temperature.
Dimethyl sulfoxide (DMSO) and 0.05 M TRISeHCl buffer (pH 8.0)
were used as solvents. DMSO, TRISeHCl, human serum albumin
(HSA), bovine serum albumin (BSA), horse serum albumin (HRSA),
ovalbumin (OVA) and SDS were purchased from SigmaeAldrich
(USA).
3. Results and discussion
2.3. Preparation of stock solutions of dyes and proteins
3.1. Synthesis of dyes
The 2 ꢂ10ꢁ3 M dye stock solutions were prepared by dilution of
the dye in DMSO. Stock solutions of proteins (HSA, BSA, HRSA, OVA)
and SDS were prepared by dissolving in 0.05 M TRISeHCl buffer
(pH 8.0). Proteins and SDS concentrations in stock solutions were
The aza-substituted squaraines 2, 3 and 10 were prepared
according to a previously established procedure [10,12] involving
the nucleophilic substitution of the methoxy group of 3-hexyl-2-
[3-(3-hexyl-3H-benzothiazol-2-ylidenemethyl)-2-methoxy-4-oxo-
cyclobut-2-enylidenemethyl]benzothiazol-3-ium trifluoromethane
sulfonate (dye 1), easily accessible by methylation of the unsub-
stituted analogue with methyl triflate, by the appropriate amine
(Fig. 1) [10]. Reaction with the weaker nucleophile 3-iodobenzyl-
amine to give compound 10 required catalysis with triethylamine.
The synthesis and structural characterization of squaraines 4, 5, 6, 7,
8, 9 and 11 has already been described [10].
equal to 3 mM and 0.05% w/w respectively.
2.4. Preparation of working solutions
Working solutions of free dyes were prepared by dilution of the
dye stock solution in 0.05 M TRISeHCl buffer. Working solutions of
the dyes in the presence of proteins were prepared by addition of
the dye stock solution to the protein stock solution. The concen-
trations of dye and proteins in working solutions amounted to
5 ꢂ10ꢁ6 M and 3 ꢂ10ꢁ6 M, respectively. All working solutions
were prepared immediately before the experiments.
3.2. X-ray crystallographic analysis of dye 2
2.5. Spectroscopic measurements
The X-ray crystal structure of dye 2 is shown in Fig. 2. In this
structure, the whole molecule is crystallographically unique
(Fig. 2a), caused by a disruption in the molecular symmetry due to
the substitution of the hydroxyethylamino group for one of the
squarate oxygen atoms. Another effect that this substitution has
had on the squaraine molecule, which also disrupts the symmetry,
is that the benzothiazole groups are pseudo-mirrored (cis confor-
mation), as opposed to being inverted/trans conformation (as in the
majority of symmetrical squaraines), to each other. The resultant
Absorption spectra were recorded on a Specord M40 spectro-
photometer (Carl Zeiss, Germany). Fluorescence excitation and
emission spectra were taken on a Cary Eclipse fluorescence spec-
trophotometer (Varian, Australia). Spectroscopic measurements
were performed in standard quartz cells (1 ꢂ1 cm). The quantum
yield value for dye 7 in the presence of HSA was determined using
3,30-diethylthiadicarbocyanine iodide solution in ethanol as the
reference (quantum yield value 4DCI ¼ 0.35) [11]. Namely, dye 7 in
the presence of HSA in buffer and 3,30-diethylthiadicarbocyanine
iodide in ethanol solutions were taken in such concentrations that
their optical density values were equal at 671 nm. Fluorescence of
both solutions was excited at this wavelength, and the area below
ꢀ
O/S distances (indicated in Fig. 2b) are 3.031(8) A (O1/S1) and
ꢀ
3.046(8) A (O1/S2). As with the majority of other squaraine dyes,
the squaraine unit in dye 2 is essentially flat with dihedral angles
between the two phenyl rings and the squarate ring being 1.0(6)
and 16.8(5)ꢀ, respectively. In terms of packing, dye 2 displays

K.D. Volkova et al. / Dyes and Pigments 90 (2011) 41e47
45
Fig. 2. X-ray crystallography of dye 2. A e ORTEP; B e strong hydrogen bonding associations and O/S interactions; C e weak CeH/O/S interactions between the dimers [C4/S1
ꢀ
ꢀ
ꢀ
3.854(8) A; C5/O1 3.337(8) A; C5/S2 3.744(8) A]; D e top-view (slipped-stack); E e side-view (slipped-stack), F- Hydrogen atoms have been removed for clarity.
a unique three-dimensional arrangement in that most squaraine
structures adopt slip-stacked charge-transfer column, with
benzothiazole/benzoselenazole dyes, the emission intensities are
higher for benzothiazole squaraines. The fluorescence intensity of
benzoselenazole squaraines does not exceed 1.4 a.u (dyes 4, 5 and 9).
The lowest emission intensity among the benzothiazole squaraines
was displayed by the dyes with aromatic substituents (3-iodo-
benzylamino (10) and anilino (11)) and a 2-sulfoethylamino frag-
ment (8) in the squaraine ring e If values for these dyes are extremely
low and do not exceed 1.2 a.u.
a
ꢀ
intermolecular distances between planes usually being ∼ 3.4e3.9 A,
the squaraine units in dye 2 form dimers that are then slip-stacked
ꢀ
as dimers (intermolecular distances > 3.4 A) (shown in Fig. 2d
and e). These dimers associate via weak CeH/O hydrogen-
bonding interactions (shown in Fig. 2c), which is interesting
because of the presence of the strong NeH and OeH hydrogen
bond donors that only associate with the sulfonate oxygen atoms
ꢀ
(shown in Fig. 2b) [Hydrogen bond distances: O2/O3 2.781(8) A;
3.4. Spectroscopic characterization of squaraine dyes
in the presence of various albumins
ꢀ
N3/O4 2.921(7) A], and not either the squarate oxygen atom or the
benzothiazole sulfur atoms.
The spectral-luminescent properties of the studied aza-
substituted squaraines in the presence of BSA (bovine serum
albumin), HSA (human serum albumin), HRSA (horse serum
albumin) and OVA (ovalbumin) are presented in Table 2. For all
squaraines, studied in the presence of albumins, noticeable spectral
changes were observed. For the majority of dyes, addition of the
proteins resulted in the shift of excitation and emission maxima
positions to the long-wavelength spectral region, of up to 35 nm. For
benzoselenazole dyes 5 (with 2-sulfoethylamino fragment) and 11
(with anilino group), excitation and emission maxima were bath-
ochromically shifted by up to 52 and 60 nm, respectively, relative to
the corresponding spectra of the free dyes. For squaraines 8 and 10,
3.3. Spectral-luminescent properties of free dyes in aqueous buffer
The fluorescent characteristics of a series of benzothiazole and
benzoselenazole aza-substituted squaraine dyes in aqueous buffer
are presented in Table 2. The excitation wavelengths of the studied
dyes in buffer reside in the range 651e695 nm, while the fluores-
cence emission maxima lie between 658 and 709 nm. Stokes shift
values observed for the free squaraines are small e between 6 nm
(dyes 2 and 3) and 18 nm (dye 9). All free squaraines demonstrated
low intrinsic fluorescence intensity values (If) e up to 17 a.u. (arbi-
trary units). It should be noted that in the pairs of structurally-related

46
K.D. Volkova et al. / Dyes and Pigments 90 (2011) 41e47
Table 2
Characteristics of fluorescence excitation/emission spectra of studied aza-substituted squaraine dyes solutions in buffer, as well as in the presence of albumins.
Dye In free form
In HSA presence
In BSA presence
In HRSA presence
BSA/If lem, nm HRSA, a.u.
In OVA presence
lex, nm lem, nm If, a.u. lex, nm lem, nm
I
HSA a.u.
I
HSA/If
lem, nm IBSA a.u.
I
I
I
HRSA/If lem, nm
I
OVA, a.u.
I
OVA/If
2
3
4
5
6
7
8
9
652
652
695
680
665
651
678
667
670
660
658
658
709
693
675
661
692
685
682
670
5.1
12
0.7
1
5.2
17
1.2
1.4
1
676
672
712
726
687
670
673
692
665
720
683
682
718
730
695
678
680
702
672
725
386
623
280
16
318
559
92
236
90
6.4
75.7
51.9
400
16.0
61.2
32.9
76.7
169
90.0
684
681
717
745
698
682
680
701
679
269
387
212
60
237
631
89
320
108
12
52.7 683
32.3 682
343
660
221
43
1113
827
187
205
21
67.3
55.0
316
43.0
214
48.6
156
146
21.0
682
680
714
734
696
681
680
700
677
276
408
257
16
358
429
47
275
83
36
54.1
34.0
367
16.0
68.8
25.2
39.2
196
83.0
51.4
303
718
60.0 739
45.6 699
37.1 685
74.2 680
229
108
703
680
10
11
0.7
9.14 721
17.1 722
3.6
5.14 721
lex (
lem) e maximum wavelength of fluorescence excitation (emission) spectrum, If (IBSA, IHSA, IHRSA, IOVA) e emission intensity of dye in free state (in presence of bovine serum
albumin, human serum albumin, horse serum albumin and albumin from chicken egg white respectively), a.u. d arbitrary units; concentrations of dyes and albumins in
working solutions were 5 ꢂ 10ꢁ6 M and 0.2 mg/ml respectively; (IBSA, IHSA, IHRSA, IOVA)/If e emission increasing of the dye in the presence of corresponding proteins.
spectral maxima were shifted to the short-wavelength region, by up
to 12 nm. The Stokes shift values for the dyes in the presence of the
albumins are in the range close to that of the free dyes, namely from
4 to 12 nm.
these dyes demonstrated a 25e56 fold increase in emission. The
fluorescence quantum yield of dye 7 in the presence of HSA was
estimated to be ca. 0.18. Due to these observations, benzothiazole
squaraines 3 and 7 could be proposed for further studies as dyes for
the detection and quantification of albumins.
The majority of the squaraines, studied in the presence of
albumins, demonstrated a good increase of emission intensity
value, up to 400 times (dye 4). But, because of the weak intrinsic
fluorescence of the dyes, the overall fluorescence intensity level of
the formed dye/albumin complexes could be characterized as from
low to medium. The lowest emission intensity values, in the pres-
ence of proteins, were observed for dyes bearing 2-sulfoethylamino
(5 and 8), m-iodobenzylamino (10) and anilino (11) substituents on
the squarate ring, ranging from 3.6 to 187 a.u. The benzothiazole
dye with a pendent diethylamino substituent (6) specifically forms
a bright complex with HRSA (emission intensity 1113 a.u.), while its
intensity in the presence of other proteins is 3e4 times lower. It
should be noted that its benzoselenazole analogue dye 4 did not
show a similar selectivity and displayed approximately the same
emission intensity in the presence of all of the studied albumins.
Benzothiazole dyes 3 and 7 possessing aminoethylamino and
N,N-dimethylhydrazino substituents, respectively, demonstrated
high emission intensities in the presence of albumins (up to 827 a.u.
for dye 7 in the presence of HRSA). Excitation profiles and emission
spectra of dye 7 are presented in Fig. 3. In complexes with proteins,
The dynamic range and lower detection limit of HSA detection
for benzothiazole squarylium dye 3 was determined via the titra-
tion of a fixed concentration of the dye (5 ꢂ10ꢁ6 M) with increasing
amounts of HSA. It was shown that the fluorescence intensity
enhancement relates to the protein concentration over the wide
range e 1.5 mg/mL to 20 mg/mL (Fig. 4). Based on the analysis of the
titration curve, the binding constant of dye 3 to HSA was estimated
to be Kb ¼ 2.5 ꢃ 0.5 ꢂ104 Mꢁ1; such a value lies in the range
104e106 Mꢁ1, which is characteristic of ligands binding with high-
affinity sites of HSA [13].
3.5. SDS and BSA/SDS mixture
Characteristics of excitation and fluorescence spectra of squar-
aine dyes studied in the presence of SDS and a BSA/SDS mixture are
presented in Table 3. For the majority of dyes in the presence of SDS
and the BSA/SDS mixture the excitation and emission maxima
positions were red-shifted by 4e25 nm, relative to that in the
simple dye buffer solution. Similarly as in the presence of albumins,
in the presence of SDS and the BSA/SDS mixture, benzoselenazole
540 560 580 600 620 640 660 680 700 720 740 760 780
dye 7
4000
3500
3000
600
500
400
300
200
100
0
600
500
400
300
200
100
0
5 6
1 - ex free (x10 times)
2 - em free (x10 times)
3 - ex BSA/SDS (x5 times)
4 - em BSA/SDS (x5 times)
5 - ex + HSA
6 - em + HSA
2500
2000
1500
1000
160
120
80
40
3 4
2
1
500
0
0
0
10
20
30
40
50
HSA, µg/ml
540 560 580 600 620 640 660 680 700 720 740 760 780
Wavelength, nm
0
5000
10000
15000
20000
25000
30000
(5 ꢂ10ꢁ6
) in the
HSA, µg/ml
Fig. 3. Profiles of excitation and fluorescence spectra of dye
7
unbound state in 0.05 M TRISeHCl buffer (pH 8.0), in presence of HSA and a BSA/SDS
mixture. The low-intensity spectra of free dye and dye in presence of BSA-SDS are
multiplied 10 times and 5 times respectively (a.u., arbitrary units). Proteins and SDS
Fig. 4. Dynamic range of HSA detection for benzothiazole squaraine dye 3. The fluo-
rescence intensity of the dye was plotted against increased amounts of protein. The
concentrations were 3
mM and 0.05% respectively.
results demonstrate a broad detection range from 1.5 mg/ml to 20 mg/ml.

K.D. Volkova et al. / Dyes and Pigments 90 (2011) 41e47
47
Table 3
albumins an increase in the emission intensity (up to 400
times) and shift of excitation and emission maxima of up to
60 nm occurred.
Spectral-luminescent characteristics of studied aza-substituted squaraine dyes
(5 ꢂ10ꢁ6 M) solutions in the presence of SDS and BSA/SDS mixture.
Dye In SDS presence
In BSA/SDS mixture presence
3. The aminoethylamino substituted dye 6 demonstrated the
strongest fluorescent response and bright emission in complex
with HRSA (emission intensity 1113 a.u.), while its fluorescence
intensity level in the presence of other albumins is 3e4 times
lower.
lex, nm lem, nm ISDS, a.u. ISDS/If lex, nm lem, nm IBSA/SDS, a.u. IBSA/SDS/If
2
3
4
5
6
7
8
9
665
672
702
721
686
688
668
687
673
680
713
727
700
675
677
695
675
725
45
13
8
8.82 665
1.08 671
11.4 707
2.00 726
7.50 687
1.41 670
676
680
717
730
700
677
680
696
678
729
154
36.5
51
50
97
54
30.2
3.04
72.9
50.0
18.7
3.18
1535
13.6
15.0
2
4. Benzothiazole dyes
3 and 7 with aminoethylamino and
39
24
327
5.3
11.4
2
N,N-dimethylhydrazino subsituents demonstrated good emis-
sion intensities in complexes with albumins (up to 827 a.u) and
sufficient increase in emission (up to 56 fold). The fluorescence
quantum yield of dye 7 in the presence of HSA was estimated to
be about 0.18. For dye 3 a wide HSA detection range (1.5 mg/mL
to 20 mg/mL of protein) has been shown.
5. The studied squaraine dyes demonstrated a weak fluorescent
response in the presence of SDS and a BSA/SDS mixture. The
exception is the benzothiazole dye with a 2-sulfoethylamino
substituent 8 that showed moderate fluorescence in the pres-
ence of SDS (347 a.u.), and bright emission (1842 a.u.) in the
presence of the BSA/SDS mixture.
273
671
3.79 684
11.4 667
2.86 717
1842
19
15
10 667
11 719
2
2.86
lex
(lem) e maximum wavelength of fluorescence excitation (emission) spectrum,
I
SDS (IBSA/SDS) e emission intensity of dye in presence of SDS (BSA/SDS mixture), a.u.
e arbitrary units; concentration of SDS in working solution and in BSA/SDS solution
was 0.05%; concentration of BSA was 0.2 mg/ml. ISDS/If, and IBSA/SDS/If e emission
increasing of the dye in the presence of correspondingly SDS and BSA/SDS.
dyes 5 and 11 demonstrated the largest shift of excitation and
emission maxima (37e59 nm). For benzothiazole squaraines 7 and
10, both excitation and emission maxima were shifted hyp-
sochromically by 3e15 nm. Thus, the excitation and emission
maxima of all the studied dyes were situated in the range
665e730 nm. The Stokes shifts for the dyes in the presence of SDS
and the BSA/SDS mixture were between 6 and 14 nm.
In the presence of SDS (0.05% w/w), which is five times lower
than its critical micelle concentration, the aza-substituted squar-
aines in this study increased their fluorescence intensity by up to
11.4 times. However, because of low intrinsic fluorescence intensity
of the studied squaraines, the emission intensity of aza-substituted
squaraines in the presence of SDS was quite low (about 2e45 a.u.).
The only exception to this was the benzothiazole dye 8 bearing
a 2-sulfoethylamino substituent on the squaraine ring for which
its fluorescence intensity value in the presence of SDS was ISDS
∼327 a.u.
For the majority of the studied squaraines, the emission inten-
sity enhancement in the BSA/SDS mixture, relative to that
measured for the pure SDS (IBSA/SDS/ISDS), was rather small (up to 6.3
times); with the exception being dye 5, which demonstrated an
increase in emission of ca. 25 times. The brightest fluorescence in
the presence of BSA/SDS (IBSA/SDS value of ∼1842 a.u.) was observed
for benzothiazole dye 8 having a 2-sulfoethylamino fragment,
while for its benzoselenazole analogue 5 the emission intensity in
the mixture with BSA/SDS was considerably lower e 50 a.u.
Acknowledgements
The authors are grateful to Fundação para a Ciência e a Tecno-
logia, Portugal, POCTI and FEDER for financial support (Project
POCTI/32915/QUI/00), and also thank the EPSRC National Crystal-
lography Service (Southampton University, England) and
Synchrotron Service (Newcastle University, England).
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1. Three novel aza-substituted benzothiazole squaraines 2, 3 and
10 were synthesized; their structures were characterized by
HRFABMS, IR, UV/Vis, 1H NMR, and 13C NMR spectroscopy.
Single crystals of dye 2 were obtained and the structure was
solved using X-ray crystallographic analysis.
2. A series of new and previously described aza-substituted
squaraine dyes was characterized in the presence of various
albumins and denatured proteins (BSA/SDS mixture) using
fluorescence spectroscopy. The studied dyes demonstrated
a weak intrinsic fluorescent emission, while in the presence of