Molecular structure and spectral properties of indolenine based norsquaraines versus squaraines

Article abstract of DOI:10.1016/j.dyepig.2018.12.007

Novel indolenine based norsquaraine dyes, wherein the oxygen of the squaric acid bridge was substituted with a barbituric or a dicyanomethylene group, were synthesized and their molecular structure, spectral and luminescent properties were compared to those of analogous squaraine dyes. The molecular structure was investigated using X-ray analysis, NMR spectroscopy and ab initio DFT B3LYP/6-311G (d, p) simulations. The calculated populations of possible conformers and the barriers of internal rotations were found to be in good agreement with the experimental data. Norsquaraines absorb and emitt light within the same long-wavelength spectral range as the corresponding squaraines but due to intramolecular H-bonds and increased conformational rigidity they were less sensitive to solvent polarity and the presence of protein (BSA).

Full text of DOI:10.1016/j.dyepig.2018.12.007

Dyes and Pigments 163 (2019) 318–329
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Molecular structure and spectral properties of indolenine based
norsquaraines versus squaraines
b
b,c
a
a
b
Olga S. Kolosova , Svitlana V. Shishkina , Vered Marks , Gary Gellerman , Iryna V. Hovor ,
b
d
a,∗
Anatoliy L. Tatarets , Ewald A. Terpetschnig , Leonid D. Patsenker
a
b
c
d
Department of Chemical Sciences, The Faculty of Natural Sciences, Ariel University, Ariel, 40700, Israel
SSI "Institute for Single Crystals" of the National Academy of Sciences of Ukraine, 60 Nauky Ave., Kharkov, 61001, Ukraine
V. N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv, 61022, Ukraine
SETA BioMedicals, 2014 Silver Court East, Urbana, IL, 61802, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Novel indolenine based norsquaraine dyes, wherein the oxygen of the squaric acid bridge was substituted with a
barbituric or a dicyanomethylene group, were synthesized and their molecular structure, spectral and lumi-
nescent properties were compared to those of analogous squaraine dyes. The molecular structure was in-
vestigated using X-ray analysis, NMR spectroscopy and ab initio DFT B3LYP/6-311G (d, p) simulations. The
calculated populations of possible conformers and the barriers of internal rotations were found to be in good
agreement with the experimental data. Norsquaraines absorb and emitt light within the same long-wavelength
spectral range as the corresponding squaraines but due to intramolecular H-bonds and increased conformational
rigidity they were less sensitive to solvent polarity and the presence of protein (BSA).
Norsquaraine
Squaraine
Molecular structure
Simulations
BSA-Complexes
Spectral properties
1
. Introduction
Squaraine dyes are a subclass of cyanines that contain a 3-ox-
be substituted with various ionic and other functional groups at the
indolenine nitrogens, which facilitate their solubility in hydrophobic or
hydrophilic media [25,26] and reaction with biomolecules [27].
Nevertheless, the molecular structure and spectral properties of
indolenine based squaraines containing hydrogen atoms at the in-
dolenine nitrogens, so-called norsquaraines, have not been sufficiently
investigated [28,29]. The simplest norsquaraine nor-SqO was synthe-
sized and its molecular structure was simulated by the semiempirical
methods AM1 and PM3 [30]. The calculations carried out in this pub-
lication predicted coexistence of very polar cis,syn-conformer labeled as
ISQ4 (C2v symmetry) as the major component and non-polar cen-
trosymmetric trans,anti-conformer ISQ6 (C2h symmetry) as the minor
ocyclobut-1-enolate (squarate) moiety in the polymethine chain [1–3].
Due to their excellent spectral characteristics such as the long-wave-
length absorption and emission, high molar absorptivity, high fluores-
cence quantum yields, and stability, squaraines are widely used as
fluorescent probes and labels for biomedical assays [4–6], clinical di-
agnostics [7,8], pharmaceutical research [9,10], as sensitive dyes in
electronics [11], sensitizers for photovoltaic cells [12] and sunlight
energy converters [13]. The molecular structure of these dyes was in-
vestigated using various experimental [14–17] and theoretical methods
1
[
[
18]. The spectral properties of squaraines [19], their complexes
20,21] and conjugates [22,23] with proteins were also thoroughly
component while the H NMR data evidenced the opposite conformer
population. The molecular geometry of nor-SqO investigated by X-ray
studied. Squaraine dyes comprising indolenine based terminal end
groups are of most interest in particular for biomedical applications due
to their increased brightness and photostability [24]. Squaraines may
analysis was affected by the interaction with two CHCl molecules ex-
3
isted in the unit cell, which did not allow to conclude on the anticipated
dominant conformer in solutions [31].
∗
Corresponding author.
E-mail addresses: kolosova@isc.kharkov.com (O.S. Kolosova), sveta@xray.isc.kharkov.com (S.V. Shishkina), veredma@ac.ariel.il (V. Marks),
garyg@ariel.ac.il (G. Gellerman), govor@isc.kharkov.com (I.V. Hovor), tatarets@isc.kharkov.com (A.L. Tatarets),
ewaldte@setabiomedicals.com (E.A. Terpetschnig), leonidpa@ariel.ac.il, leonid.patsenker@gmail.com (L.D. Patsenker).
https://doi.org/10.1016/j.dyepig.2018.12.007
Received 21 October 2018; Received in revised form 28 November 2018; Accepted 5 December 2018
Available online 10 December 2018
0143-7208/ © 2018 Elsevier Ltd. All rights reserved.

O.S. Kolosova et al.
Dyes and Pigments 163 (2019) 318–329
The PCM-PBE0/6-31 + G(d,p) calculations of nor-SqO [32] pre-
dicted that the conformer ISQ6 was 0.5 kcal/mol more stable than
ISQ4. Other conformers had much higher energies and had no effect on
these populations. It was demonstrated also that the absorption and
emission spectra of nor-SqO could be described as a combination of the
electronic spectra of these two forms.
measured on the Xcalibur-3 diffractometer (graphite monochromated
MoK radiation, CCD detector, ω-scanning, 2Θmax = 55°). The structure
α
was solved by direct method using SHELXTL package [35]. Positions of
the hydrogen atoms were located from electron density difference maps
and refined by “riding” model with Uiso = nUeq (n = 1.5 for methyl
hydrogens and n = 1.2 for other hydrogen atoms) of the carrier atom.
The hydrogen atoms at the indolenine nitrogens were refined using
A few substituted norsquaraines 1a–1f were also synthesized but
their molecular structures and fluorescence properties were not in-
vestigated [33]. Importantly, to the best of our knowledge, no other
norsquaraine derivatives were reported until now.
isotropic approximation. Full-matrix least-squares refinement against
2
F
in anisotropic approximation for non-hydrogen atoms using 4649
reflections was converged to wR
2
= 0.127 (R = 0.044 for 2936 re-
1
This work investigates the molecular structures and spectral prop-
erties of nor-SqO and its novel, first synthesized derivatives, where the
squaric oxygen is substituted with a barbituric (nor-SqB) and dicya-
nomethylene (nor-SqCN) group, and compares these properties with
those of conventional, previously reported squaraines SqO and SqCN
flections with F > 4σ(F), S = 0.972). The final atomic coordinates, and
crystallographic data for nor-SqO molecule have been deposited to the
Cambridge Crystallographic Data Center, 12 Union Road, CB2 1EZ, UK
(fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk) and are
available on request quoting the deposition numbers CCDC 998817).
Absorption spectra were recorded for the dye concentrations cDye
[
34] and a newly synthesized squaraine SqB.
∼
0.5 μM in phosphate buffer (PB) 7.4 and other solvent systems dis-
cussed below. All the absorption spectra were recorded in 1-cm quartz
cells at 25 °C using a PerkinElmer Lambda 35 UV/Vis spectrophotometer.
Absorption maxima were determined with an accuracy of ± 0.5 nm and
rounded off.
Molar absorptivity (ε). The investigated dye (7–10 mg) was dis-
solved in the solvent of interest (50 mL), the stock solution was diluted
to the dye concentration cDye ∼ 0.5 μM and the absorbance (A) in the
absorption band maximum was measured in a 5-cm standard quartz
cell. The molar absorptivity were calculated according to the
Beer—Lambert law. The molar absorptivityý of each dye was in-
dependently measured three times and the average value was taken.
The reproducibility for determining the molar absorptivity was
2
. Materials and methods
2.1. General information
Iodomethane and 2,3,3-trimethylindolenine were purchased from
−1
−1
Acros; 3,4-dihydroxy-3-cyclobutene-1,2-dione (squaric acid) was from
Aldrich; Bovine Serum Albumin (BSA, essentially fatty acid free), Silica
gel 60 for column chromatography and other materials were from
Merck and used as received. Phosphate buffer (PB) pH 7.4 (67 mM) was
within ± 2000 M cm
.
Emission spectra and quantum yields were measured for dye con-
centrations cDye ∼0.5 μM in PB pH 7.4 and other solvents listed below.
The excitation wavelength was 620 nm. The fluorescence measure-
ments were done in 1-cm standard quartz cells at 25 °C using a Varian
Cary Eclipse spectrofluorometer. The emission spectra were corrected
for wavelength-dependent instrument sensitivity. Emission maxima
were determined with an accuracy of ± 1.0 nm.
prepared by dissolving Na
in 1 L distilled water.
2
HPO
4
·2H
2
O (9.596 g) and KH
2
PO (1.743 g)
4
Mass spectra were measured by a BIFLEX III MALDI-TOF mass
spectrometer using 2,5-dihydroxybenzoic acid was taken as the matrix.
FAB mass spectra were recorded on a SELMI MI-1201E instrument using
Spectral characteristics of dyes in presence of BSA (dye—BSA
complexes) were measured at cDye = 0.5 μM and BSA concentration
3
-nitrobenzylalcohol (NBA) or glycerol as a matrix.
The C, H, N elemental analysis was performed by a EuroVector Euro
c
BSA = 90 μM (6 g/L).
EA 3000 EA-IRMS elemental analyzer.
Quantum chemical ab initio simulations were carried out using
1
13
H NMR and C NMR spectra were measured on a Varian Mercury-
Density Functional Theory (DFT) and B3LYP functional [36] with 6-
311G(d,p) basis sets [37] within the Gaussian 09 program [38]. The
vibrational frequencies were calculated and no imaginary frequencies
were found for the equilibrium geometries. The NPA charges were
calculated using the NBO pro6 program [39].
1
VX-200 ( H 200 MHz) spectrometer in DMSO‑d
6
using signal of re-
maining non-deuterated solvent as an internal standard (2.50 ppm for
1
13
DMSO). Selected spectra ( H 400 MHz and C 100 MHz) were recorded
III
in CDCl
3
using a 400 MHz Bruker Avance HD. BBI probe was equipped
with Z-axis gradients coils. TMS was used as an internal standard
Populations of the conformers (c, %) was calculated for 25 °C ac-
cording to the Boltzmann distribution.
(
(
0.00 ppm). A full assignment was done using 2D experiments: COSY
1 1 1 13
He H correlation), HMQC, HMBC ( He C short and long correla-
1
tion, respectfully) and J-resolved. H NOESY experiments employed
2.2. Synthesis
1
1
28 t increments with a dwell time of 83.2 μs and a mixing time of
1
.5 s. A recycle delay of 2.5 s was used in all NMR experiments.
General procedure. A mixture of quaternized or non-quaternized in-
dolenine (2 mmol) and squaric acid or its dicyanomethylene or barbi-
turic derivative was refluxed in 50 mL of a butanol—toluene mixture
(1:1, v/v). The solvent was removed and the residue was dried in a
X-Ray diffraction analysis. The dark-blue crystals with metallic
luster of nor-SqO (C26
H
24
N
2
2
O ) were obtained by recrystallization
from 1-propanol with 0.1% diethylamine. Crystals are monoclinic. At
2
93 K
a = 18.1805(8),
b = 6.4028(3),
c = 18.7430(8)
Å,
vacuum desiccator over P
2
O . The product was column purified (Silica
5
3
β = 107.185(5)°, V = 2084.4(2) Å , M
r
= 396.28, Z = 4, space group
gel 60, CHCl eMeOH, gradient).
3
3
−1
P2
1
/n, dcalc = 1.263 g/сm , μ(MoK
a
) = 0.080 mm , F(000) = 840.
2-(3,3-dimethyl-2,3-dihydro-1H-2-indolylidenmethyl)-4-(3,3-
dimethyl-3H-2-indoliumylmethylene)-3-oxo-1-cyclobuten-1-olate
Intensities of 20723 reflections (4757 independent, Rint = 0.025) were
319

O.S. Kolosova et al.
Dyes and Pigments 163 (2019) 318–329
Scheme 1. Synthesis of squaraines and norsquaraines.
1
(
nor-SqO): Yield 75%. Dark-blue solid. H NMR (400 MHz, CDCl
3
,
39%. Dark-green solid. H NMR (400 MHz, DMSO‑d
6
, ppm): δ 10.02
ppm): Major conformer: δ 12.84 (2H, NH, broad s), 7.27 (2H, arom-
ortho, d, J = 7.6 Hz), 7.25 (2H, arom-meta, m, second order), 7.13 (2H,
arom-ortho,d, J = 7.8 Hz), 7.08 (2H, arom-meta. ddd, J = 7.6, 7.5,
(2H, NH, s), 7.55 (2H, arom. H, d, J = 7.2 Hz), 7.42–7.36 (4H, arom. H,
m), 7.24 (2H, arom. H, t, J = 6.8 Hz), 6.45 (2H, CH, s), 3.58 (6H, NCH
3
,
+
s), 1.66 (12H, (CH
3
)
2
, s). FAB MS, m/z calcd. for [C32
H
30
30
N
4
O ]
4
+
0
1
.8 Hz), 5.48 (2H, CH, s), 1.453 (12H, CH
3
, s). Minor conformer: δ
534.23, found: 535.2 [M+H] . Anal. calcd. (%) for C32
H
N
4
O : C,
4
2.54 (2H, NH, broad s), 7.27 (2H, arom-ortho, d, J = 7.6 Hz), 7.25
71.89; H, 5.66; N, 10.48. Found: C, 71.85; H, 5.62; N, 10.50.
(
2H, arom-meta, m, second order), 7.13 (2H, arom-ortho,d, J = 7.8 Hz),
3-Dicyanomethylene-2-(3,3-dimethyl-2,3-dihydro-1H-2-in-
7
.08 (2H, arom-meta, ddd, J = 7.6, 7.5, 0.8 Hz), 5.52 (2H, CH, s), 1.450
dolylidenmethyl)-4-(3,3-dimethyl-3H-2-indoliumylmethylene)-1-
1
3
1
(
12H, CH
3
, s). C NMR (100 MHz, CDCl
83.42 (CO), 175.77 (CCNH), 175.14 (CHC(CO)
39.02 (arom, C), 128.29 (arom, CH), 123.20 (arom, CH), 122.35
), 26.65
). Minor conformer: 185.59 (CO), 180.31 (CO), 175.91(CCNH),
75.24 (CHC(CO) ), 141.86 (arom, C), 139.02 (arom, C), 128.24 (arom,
3
, ppm): Major conformer: δ
cyclobuten-1-olate (nor-SqCN): Yield 35%. Dark-green solid. H NMR
1
1
2
), 141.96 (arom, C),
(400 MHz, DMSO‑d , ppm): δ 12.00 (2H, NH, s), 7.54 (2H, arom. H, d,
6
J = 7.2 Hz), 7.39–7.33 (4H, arom. H, m), 7.19 (2H, arom. H, t,
1
3
(
arom, CH), 111.51 (arom, CH), 85.94 (CH), 48.70 (-C(CH
3
)
2
J = 6.8 Hz), 5.69 (2H, CH, s), 1.44 (12H, (CH
3
)
2
, s).
C NMR
(
CH
3
(100 MHz, CDCl , ppm): δ 178.58, 176.87, 163.00, 162.57, 141.34,
3
1
2
139.46, 128.56, 124.36, 122.81, 117.01, 111.95, 87.16, 49.68, 46.39,
+
CH), 123.26 (arom, CH), 122.48 (arom, CH), 111.36 (arom,C), 86.17
26.45. FAB MS, m/z calcd. for [C29
H
24
4
N
4
O] 444.20, found: 445.1 [M
+
(
CH), 48.70 (-C(CH
3
)
2
), 26.65 (CH
3
). MALDI-TOF MS, m/z calcd. for
+H] . Anal. calcd. (%) for C29
H
24
N O: C, 78.36; H, 5.44; N, 12.60.
+
+
+
[
[
C
26
H
24
N
2
O
2
]
396.2, found: 397.1 [M+H] , 419.0 [M+Na] , 434.9
Found: C, 78.32; H, 5.49; N, 12.65.
+
M+K] . Anal. calcd. (%) for C26
H
24
N
2
O
2
: C, 78.76; H, 6.10; N, 7.07.
3-Dicyanomethylene-2-(1,3,3-trimethyl-2,3-dihydro-1H-2-in-
dolylidenmethyl)-4-(1,3,3-trimethyl-3H-2-indoliumylmethylene)-
Found: C, 78.72; H, 6.12; N, 7.12.
1
2
-(1,3,3-trimethyl-2,3-dihydro-1H-2-indolylidenmethyl)-4-
1-cyclobuten-1-olate (SqCN): Yield 42%. Dark-green solid. H NMR
(
1,3,3-trimethyl-3H-2-indoliumylmethylene)-3-oxo-1-cyclobuten-
(400 MHz, DMSO‑d , ppm): δ 7.54 (2H, arom. H, d, J = 7.4 Hz),
6
1
1
-olate (SqO): Yield 78%. Dark-blue solid.
H
NMR (400 MHz,
7.47–7.37 (4H, arom. H, m), 7.32–7.18 (2H, arom. H, m), 6.30 (2H, CH,
13
DMSO‑d
6
, ppm): δ 7.51 (2H, arom. H, d, J = 7.2 Hz), 7.37–7.32 (4H,
s), 3.61 (6H, NCH
3
, s), 1.68 (12H, (CH
3
) , s). C NMR (100 MHz,
2
arom. H, m), 7.18–7.15 (2H, arom. H, m), 5.76 (2H, CH, s), 3.57 (6H,
CDCl , ppm): δ 173.48, 173.05, 167.94, 167.46, 142.79, 142.07,
3
1
NCH
3
, s), 1.69 (12H, (CH
3
)
2
, s). H NMR (400 MHz, CDCl
3
, ppm): δ 7.33
128.25, 124.86, 122.39, 119.23, 110.12, 89.21, 49.61, 40.88, 31.82,
+
(
2H, arom. H, m), 7.16–7.13 (2H, arom. H, t, J = 7.2 Hz), 7.00 (2H,
arom. H, d, J = 7.6 Hz), 5.91 (2H, CH, s), 3.56 (6H, NCH , s), 1.77
, ppm): δ 182.53 (CO),
), 143.20 (arom, C), 127.95
arom, C), 123.91 (arom, CH), 122.24 (arom, CH), 109.31 (arom, CH),
26.77. FAB MS, m/z calcd. for [C31
H
28
4
N
4
O] 472.23, found: 473.2 [M
+
3
+H] . Anal. calcd. (%) for C31
H
28
N O: C, 78.79; H, 5.97; N, 11.86.
1
3
(
12H, (CH
3
)
2
, s). C NMR (100 MHz, CDCl
3
Found: C, 78.81; H, 5.99; N, 11.85.
3. Results and discussion
3.1. Synthesis
1
80.31 (CCNCH
3
), 170.94 (CHC(CO)
2
(
9
9.23(arom, CH), 86.88 (CH), 49.38 (-C(CH
3
)
2
), 30.73(CH
3
), 27.22
424.22, found: 424.3
: C, 78.76; H,
+
(
CH
3
). FAB MS, m/z calcd. for [C28
H
28
N
2
O ]
2
+
+
[
M
], 425.3 [MH ]. Anal. calcd. (%) for C26
H
24
N
O
2 2
6
.10; N, 7.07. Found: C, 78.72; H, 6.12; N, 7.12.
The dyes in this research were chosen in such a way to compare
norsquaraines versus squaraines of similar structure, and to investigate
the effect of substitution in the central squaraine ring on the dye
structures and properties. Squaraine SqO and its derivatives, where the
squaric oxygen is substituted with a barbituric (SqB) and a dicyano-
methylene (SqCN) group, are known to be synthesized by condensation
of the quaternized indolenine 2a with the squaric acid 3a [40,41] or
squarates 3b and 3c, respectively [34,40,42] (Scheme 1). Analogous to
squaraine SqO, norsquaraine nor-SqO was obtained starting from in-
dolenine 2b and squaric acid 3a [33]. Following this approach, dyes
SqO, SqB, SqCN, nor-SqO and the new norsquaraines nor-SqB and
nor-SqCN were synthesized. The synthesis was carried out under reflux
in a butanol—toluene mixture (1:1, v/v) as proposed in Ref. [42].
2
-(3,3-dimethyl-2,3-dihydro-1H-2-indolylidenmethyl)-4-(3,3-
dimethyl-3H-2-indoliumylmethylene)-3-(2,4,6-trioxohexahydro-5-
pyrymidinylidene)-1-cyclobuten-1-olate (nor-SqB): Yield 16%.
1
Dark-green solid. H NMR (400 MHz, DMSO- d
6
, ppm): δ 12.19 (2H,
NH, s) 10.27 (2H, NH-barb, s), 7.53 (2H, arom. H, d, J = 7.6 Hz),
7
.39–7.33 (4H, arom. H, m), 7.18–7.15 (2H, arom. H, m), 6.63 (2H, CH,
+
s), 1.44 (12H, (CH
3
)
2
, s). FAB MS, m/z calcd. for [C30
H
26
26
N
4
O ]
4
+
5
06.20, found: 507.1 [M+H] . Anal. calcd. (%) for C30
H
N
4
O : C,
4
7
1.13; H, 5.17; N, 11.06. Found: C, 71.12; H, 5.13; N, 11.10.
-(1,3,3-trimethyl-2,3-dihydro-1H-2-indolylidenmethyl)-4-
1,3,3-trimethyl-3H-2-indoliumylmethylene)-3-(2,4,6-trioxohex-
ahydro-5-pyrymidinylidene)-1-cyclobuten-1-olate (SqB): Yield
2
(
320

O.S. Kolosova et al.
Dyes and Pigments 163 (2019) 318–329
Fig. 1. Internal rotation in the squaraine and norsquaraine molecules (a) and potentially possible NH and OH prototropic forms of norsquaraines (b).
3
.2. Molecular structure
existence of two halves of nor-SqO molecules (A and B) in the asym-
metric part of the unit cell, which is due to both molecules being lo-
cated in the special positions relatively to the center of symmetry that
coincides with the squaraine ring center. The analysis of the bond
lengths in SqO [43] and nor-SqO molecules evidences their mesoionic
structures (Table S2, the atom numbering is given in Fig. 3a and b). The
N1eC4 bond (1.343–1.354 Å) has ordinary character, which is con-
firmed by comparison of its value with “standard” mean value for N
Although nor-squaraines formally differ from squaraines only by the
presence of a hydrogen instead of an alkyl substituent at the indolenine
nitrogens, their electronic and spatial structures are quite different. Due
to internal rotation around the conjugated polymethine bonds (torsion
angles φ
1
, φ1′, φ , and φ2′, Fig. 1,a) the squaraine and norsquaraine
2
molecules can potentially exist in different conformational forms. These
conformers may have different planarity and different mutual orienta-
tion of both indolenine nitrogens relatively to each other (syn- and anti-
forms) and to the squaric oxygens. The indolenine terminal groups can
2
(3)–Csp bond (1.355 Å) [44]. The value of the C1eO1 bond is very
2
close to the mean value for Csp = O bond (1.210 Å) in SqO molecule
and it is slightly elongated in nor-SqO due to the formation of the
NeH⋯O intramolecular H-bond: NH form, H⋯O 1.87 Å, NeH⋯O 158°
in A and H⋯O 1.80 Å, NeH⋯O 157° in B. The C1eC2 bond has or-
dinary character in both dye molecules: the bond length
exist in the cis- or trans-form (rotation around φ and φ1′) relatively to
1
the central squaric moiety. Furthermore, norsquaraines as compared to
squaraines may potentially form two prototropic forms (Fig. 1,b), where
the hydrogen atom is located either at the indolenine nitrogen (NH
form) or the squaraine ring oxygen (OH form). In both prototropic
forms, one or two seven-membered quasicycles with an intramolecular
H-bond can be formed.
2
2
(
1.450–1.473 Å) is very close to the mean value for Csp –Csp bond
1.455 Å). Pronounced delocalization of the electron density is observed
(
within the C2eC3eC4 chain in both dyes: the C2eC3 and C3eC4 bond
lengths (1.371–1.405 Å) are in between the mean values for the
2
2
2
2
Csp = Csp (1.326 Å) and Csp –Csp (1.455 Å) bonds.
We investigated the molecular structures of norsquaraines nor-SqO,
nor-SqB and nor-SqCN versus squaraines SqO, SqB and SqCN using
experimental (X-ray and NMR) methods and the ab initio calculations
The analysis of non-bonded interactions in the SqO crystals reveals a
moderate intramolecular steric repulsion between the indolenine methyl
groups and squaric moiety: the H⋯C van der Waals radii sum [45] is
(
B3LYP/6-311G(d,p) method). The calculated total energies (E), re-
2
2
.87 Å as compared to the shortened intramolecular contacts
.54–2.87 Å (Fig. S1, Table S2). This repulsion is compensated by a
lative energies (ΔE) (differences in the energies of conformers), popu-
lations of the conformers (c) at 25 °C, calculated according to the
Boltzmann distribution, dipole moments (μ), and electron charges on
slight twisting of the indolenine and squaric moieties relatively to each
other (Fig. 3,b). Two parts of SqO molecule are nonequivalent due to
intermolecular interactions in crystals causing also a moderate non-pla-
narity of the molecules (the torsion angles are up to 17.1° and the di-
hedral angles are up to 25.3°). The participation of the methyl group of
part B in the CeH … π hydrogen bonding with squaric moiety of the
neighboring molecule (symmetry operation is 2–x, –y, 1–z; H⋯C
the indolenine nitrogens (qN1, qN2), squaric oxygen (q ) and/or sub-
O
stituted squaric oxygen (q
Supplementary data) while the most stable conformers are shown in
Table 1 and Fig. 2.
Z
) for some typical forms are given in Table S1
(
Prototropic forms. The ab initio calculations show that the most
stable prototropic form of the norsquaraine nor-SqO corresponds to the
centrosymmetric NH trans,anti-form, where both protonated indolenine
nitrogens form intermolecular H-bonds with two different squaraine
oxygens (Table 1). This form is 11.08 kcal/mol and 26.46 kcal/mol
more stable than the OH forms with one protonated squaric oxygen and
one protonated indolenine nitrogen, and 27.21 kcal/mol more stable
compared to the di-OH form (Table S1). Such a difference in the en-
ergies suggests that the NH form is the only prototropic form to be
realized. These simulations are in good agreement with our X-ray data
2
.62 Å, CeH⋯C 133°) causes a more pronounced twisting as compared
to part A (Fig. 3,b and S1).
In contrast to squaraine SqO, the H-bonding in norsquaraine nor-
SqO stabilizes the planar geometry (the torsion angle do not exceed 4.9°
and the dihedral angles 7.7°). It also causes the elongation of the
C1eO1 bond and equalization the C2eC3 and C3eC4 bonds. Thus, the
intramolecular and intermolecular steric interactions affect the planar
conformation and π-conjugation of squaraine SqO [43] in a greater
degree than that for norsquaraine nor-SqO.
(
Fig. 3,a) showing that nor-SqO in crystals exists in the theoretically
1
NMR study. The H NMR spectrum of SqO measured in DMSO‑d
6
predicted NH trans,anti-form.
1
(
Fig. S2,a) and CDCl
3
(Fig. S2,b) and the H NMR spectrum of nor-SqO
X-ray analysis was used also to compare the molecular structure of
nor-SqO and SqO in crystals. Both crystals did not contain solvent
molecules, which could affect the molecular conformation as reported
in Ref. [31]. Nevertheless, the molecular structure can be distorted by
intermolecular interactions in crystals. The obtained data show
in DMSO‑d
6
(Fig. 4,a) exhibit only one set of peaks. In contrast, the
1
13
room temperature H NMR and C NMR spectra of nor-SqO in CDCl
3
shows two sets of peaks (Fig. 4,b and c). A change in the solvents ratio
(
CDCl : DMSO‑d ) causes a change in the relative intensities of these
3
6
321

O.S. Kolosova et al.
Dyes and Pigments 163 (2019) 318–329
Table 1
The ab initio calculated total energies (E), relative energies (ΔE), populations of the conformers (c) at 25 °C, dipole moments (μ), and electron charges on the
indolenine nitrogens (qN1, qN2), squaric oxygen (q
O
) and/or substituted squaric oxygen atom (q ). The most stable conformers are underlined.
Z
Dye/Form
Conformation
E, a.u.
ΔE, kcal/mol (c, %)
μ, D
q
N1, qN2
q
Z,
q
O
Torsion angle
φ
1 2
, φ
φ1′, φ2′
nor-SqO
−1265.48611
1.35 (9.0%)
4.18
−0.544
−0.544
−0.745
−0.745
0.0, 0.0,
0.0, 0.0
NH cis,syn
nor-SqO
NH trans,anti
−1265.48827
0.00 (91.0%)
0.00
0.49
−0.543
−0.543
−0.674
−0.674
0.0, 0.0,
0.0, 0.0
SqO cis,syn(2)
−1344.09204
0.90 (16.75%)
−0.383
−0.635
−0.690
0.0, 0.0,
0.0, 0.0
−0.383
SqO trans,anti(1)
SqO trans,syn
−1344.09348
−1344.09074
−1679.23895
0.00 (79.04%)
1.72 (4.08%)
0.00 (100%)
0.00
1.39
7.85
−0.383
0.383
−0.662
−0.662
0.0, 0.0,
0.0, 0.0
−
−0.383
0.377
−0.660
−0.662
0.1, 0.1,
0.8, 0.3
−
nor-SqB
−0.538
−0.538
−0.360
−0.707
−1.1, 0.3,
−1.1, 0.3
NH cis,syn
SqB cis,syn(2)
SqB cis,anti
−1757.84196
0.00 (95%)
5.43
7.04
−0.373
−0.384
−0.641
−3.1, 6.6,
−3.1, 6.7
−
0.373
−1757.83912
1.78 (5%)
−0.373
0.373
−0.381
−0.640
−23.3, −24.3,
1.2, 4.8
−
nor-SqCN
−1414.07885
−1492.67819
0.00 (100%)
0.00 (100%)
10.21
10.12
−0.536
−0.536
−0.396
−0.713
0.0, 0.0,
0.0, 0.0
NH cis,syn
SqCN cis,syn
−0.372
−0.420
−0.650
−14.6, −20.9,
−14.6, −20.9
−0.372
two sets (Fig. 4,d). However, the addition of an equimolar amount of
one solvent to a dye dissolved in the other solvent has almost no effect
on the spectra evidencing that the above phenomenon is not connected
with the specific interaction between the dye and the solvent molecules
but is just due to the general solvent effect.
NH trans, anti has a cross peak between δ = 1.45 ppm (3-(CH
δ = 5.48 ppm (CH) and nor-SqO NH cis,syn has a cross peak between
δ = 1.45 ppm (3-(CH hydrogens) and δ = 5.52 ppm (CH) (Fig. S3,a).
The two conformers can be distinguished using C NMR spectro-
3
) ) and
2
)
3 2
1
3
scopy (Fig. 4,c and S4). The nor-SqO NH trans, anti has C symmetry
2
1
The two sets of peaks (Fig. 4,b) in the H NMR spectrum of nor-SqO
(Fig. 5); each squarate oxygen forms one intra-molecular H-bond with
in CDCl
3
belong to two conformers at a ratio of 2:1. Both conformers
the NH hydrogen and the chemical shift of the carbon is 183.42 ppm. In
have a symmetrical structure as evidenced by the presence of only one
contrast, nor-SqO NH cis,syn has a σ symmetry and thus the two
2
set of peaks for the methine CH, indolenine NH and 3-(CH
3
)
2
hydro-
oxygens are different. One oxygen forms two intramolecular H-bonds
and the chemical shift of the carbon is 185.59 ppm while the other
oxygen is lacking a H-bond and the chemical shift of the carbon is
observed at a higher field, δ = 180.31 ppm.
gens. These conformers have intramolecular hydrogen bonds with a low
field chemical shift of the NH hydrogen at δ = 12.84 ppm for the
trans,anti-form and δ = 12.54 ppm for the cis,syn-form. Only two
structures of the four possible symmetrical forms shown in Fig. 5, nor-
SqO NH trans, anti and nor-SqO NH cis,syn, have intramolecular hy-
drogen bonds.
1
15
1
The one bond coupling constants ( JN-H) between the N and H of
the two conformers were extracted from the 2D experiment HMBC
1
5
1
Ne H (Fig. S3,b) and their values are almost the same: 92 Hz and
1
1
Also the He H NOESY supports the coexistence of these two con-
94 Hz for the major and the minor conformers, respectively. These
values confirm that in solution the hydrogen atom is also bound to the
nitrogen atom as predicted by ab initio simulation.
formers, confirming that the hydrogens of the 3-(CH
3
) and the methine
2
CH groups in both conformers are in close proximity to each other and
therefore cross peaks are seen in the 2D-NOESY experiment. Nor-SqO
When the 1H NMR spectrum of nor-SqO in CDCl
(Fig. 4,b), is
3
322

O.S. Kolosova et al.
Dyes and Pigments 163 (2019) 318–329
Fig. 2. The most stable conformers of squaraine and norsquaraine molecules according to the ab initio simulations.
compared to the spectrum in DMSO‑d
6
, at room temperature, the latter
insufficient contribution of cis,syn-form, which is 1.35 kcal/mol less
stable (Table 1 and S1, Fig. 5). According to the Boltzmann distribution
the ratio between these forms at 25 °C is about 91% : 9%. This is in good
agreement with data reported in Ref. [32], where trans,anti-form was
found to be 0.5 kcal/mol less stable compared to cis,syn-form. The most
stable trans,anti-form of nor-SqO is realized in crystals (Fig. 3,a,
Table 1). However, due to the ΔE between these conformers is not
pronounced it is anticipated that trans,anti- and cis,syn-forms of nor-
SqO and SqO can coexist in solutions.
1
one shows only one set of broad peaks (Fig. 4,a). A series of H NMR
experiments at high temperatures (330–370 K) displays a narrowing of
some of the peaks as we increase the temperature. For example, the line
width of the methine hydrogen (CH) is decreasing from 4.86 Hz at
3
00 K to 0.8 Hz at 370 K (Fig. S5). These observations suggest that the
two conformers, NH trans, anti and NH cis,syn, are both present in
DMSO‑d and the equilibrium is rapid in the NMR time scale at room
6
temperature to be above the coalesce but not fully narrow. Further,
1
3
most of the C signals are broad at room temperature (Fig. S4) and the
carbonyl signal is very broad, so it disappears in the spectrum noise.
This equilibrium is obviously slow in the NMR time scale when the
Similar to nor-SqO, the squaraine molecule SqO can also exist in
different conformations (Table 1 and S1). The most stable conformer of
SqO is also centrosymmetric; it has a trans-orientation of indolenine
moieties and anti-orientation of indolenine nitrogens (Fig. 2). However,
the calculated energy of this form is only about 0.90 kcal/mol lower
compared to that of the cis,syn-form and 1.72 kcal/mol lower than that
compound is dissolved in CDCl to yield two sets of peaks.
3
Conformations. Ab initio simulations indicate that norsquaraine
nor-SqO exists mostly in the centrosymmetric trans,anti-form with an
323

O.S. Kolosova et al.
Dyes and Pigments 163 (2019) 318–329
Fig. 3. The nor-SqO (a) and SqO (b) structures according to the X-ray data.
of the trans,syn-form (Table 1). According to the Boltzmann distribution
the ratio between these forms at 25 °C is about 79%: 17%: 4%, re-
spectively. The contribution of other conformers is only minor
norsquaraine molecules. The calculations show that SqB, nor-SqB,
SqCN, and nor-SqCN exceptionally have a cis-orientation of indolenine
moieties and a syn-orientation of the indolenine nitrogens (Fig. 2).
1
(
< 0.2%). The most stable theoretically predicted trans, anti(1) con-
These simulations are in good agreement with the H NMR data ex-
former is realized in crystals (Fig. 3,b) [43].
hibiting only one set of peaks for these dyes. The nitrogen atoms are
located on the same side as the squaric oxygen. The exception is
squaraine SqB, where the nitrogen atoms are on the same side as the
barbituric moiety.
Planarity. Ab initio calculations show that both SqO and nor-SqO
molecules are flat (φ
1
, φ , φ1′, φ2' ≈ 0°). The planarity of nor-SqO was
2
found to be in good agreement with X-ray data (Fig. 3,a). However the
planarity of SqO in crystals (Fig. 3,b) is somewhat disrupted
In contrast to oxosquaraine SqO, barbituric (SqB) and especially
dicyanomethylene (SqCN) squaraines are less planar (see torsion angles
in Table 1). At the same time, norsquaraines nor-SqB and nor-SqCN as
well as nor-SqO are very flat.
(
φ
1
= +2.8°, φ = +7.0°, φ1' = −7.8°, φ2' = −17.1°, torsion angles
2
are designated in Fig. 1) [43]. The non-planarity of SqO in crystals is
rather due to the above mentioned inter-molecular interactions in the
crystal phase (Fig. S1, Table S2) than of intra-molecular sterical hin-
drance caused by the indolenine N-methyl groups.
Similar to nor-SqO, the intramolecular H-bonds between the in-
dolenine NH hydrogens and squaric acid oxygen are realized also in the
nor-SqB and nor-SqCN molecules. However, in contrast to nor-SqO,
these H-bonds are formed with the same squarate oxygen atom, so that
the molecules have a mirror-symmetrical structure. In the SqB and nor-
SqB the barbituric oxygens form additional H-bonds with the methine
hydrogens.
Substitution of squaric oxygen. Similar to nor-SqO, barbituric
(
nor-SqB) and dicyanomethylene (nor-SqCN) norsquaraines according
to ab initio calculations also exist entirely in the NH forms (Table 1 and
S1). Substitution of the squaric acid oxygen with these rather bulky
groups has a strong impact on the conformations of the squaraine and
Fig. 4. 1H NMR (a,b,d) and 13C NMR (c) spectra of norsquaraine nor-SqO in DMSO‑d
(a), CDCl
(b, c) and CDCl
: DMSO‑d 1:1 (d).
6
3
3
6
324

O.S. Kolosova et al.
Dyes and Pigments 163 (2019) 318–329
Fig. 5. Symmetrical forms of nor-SqO.
The intramolecular H-bonds facilitate flattening but also increase
the conformational rigidity of the norsquaraine molecules (Fig. 3,a) as
compared to squaraines (Fig. 3,b). Thus, the calculations show that the
with the barbituric moiety (SqB, nor-SqB) causes a 6–27 nm red-shift of
the absorption and emission bands (Table 2). Introduction of the di-
cyanomethylene group (SqCN, nor-SqCN) results in a more pro-
nounced red-shift (20–60 nm). Dyes SqB, nor-SqB, SqCN, nor-SqCN as
compared to SqO and nor-SqO exhibit an additional, short-wavelength
absorption band at 427–441 nm (barbituric) and 381–388 nm (dicya-
rotation barrier associated with torsion angle φ in the nor-SqO mo-
1
lecule is about 26.1 kcal/mol, whereas in the SqO molecule it is sub-
stantially less pronounced, 18.3 kcal/mol. The rotation barriers asso-
−
1
−1
ciated with torsion angle φ
mol, respectively).
2
are higher (33.6 kcal/mol and 24.0 kcal/
nomethylene) with molar absorptivity (ε) of up to 35,000 M cm
,
which makes them suitable for excitation within the blue/UV spectral
range (Fig. 6).
Electron density distribution. Both squaraine and norsquaraine
chromophores are neutral and have a zwitter-ionic structure. The elec-
tronic charge in all these molecules is delocalized mostly within the
polymethine chain (Table S3). The calculations exhibit a negative charge
The absorption and emission bands of norsquaraines are in the
long-wavelength range of 654–694 nm and 669–712 nm, respectively.
These bands are 11–28 nm red-shifted as compared to the corre-
sponding squaraines SqO, SqB and SqCN (Table 2), which is more
likely due to their more planar molecular structure. The main absorp-
tion spectra of squaraines and norsquaraines consist of an intense, long-
wavelength band and a less intense shoulder on the shorter-wavelength
slope of the main band (Fig. 6). This sholder is typical for cyanines and
squaraines and known to belong to a vibronic mode [19].
on the squaric oxygen (q ) and a less pronounced charge on the in-
O
dolenine nitrogens (qN1, qN2) and on the carbon of the substituted squaric
oxygens (q ) (Table 1). The positive charge is mostly on the squaric
carbonyl” carbons and on the indolenine carbons at the position 2.
The most stable conformers of squaraine SqO and norsquaraine nor-
SqO are centrosymmetric; q = q , qN1 = qN2 and therefore the mole-
cular dipole moments (μ) are about zero. While the negative charges on
the oxygens (q , q ) in SqO and nor-SqO are almost equal (−0.66 and
0.67, respectively), the charges on the nor-SqO nitrogens (qN1, qN2
Z
“
O
Z
While the absorption bands of the investigated squaraines and
O
Z
norsquaraines measured in CHCl do not exhibit any sign of aggrega-
3
−
)
tion, these dyes do strongly aggregate in aqueous media. The extinction
coefficients measured in CHCl significantly decrease in PB pH 7.4 —
are greatly increased as compared to SqO (−0.54 vs. −0.38).
3
Substitution of the oxygen atom in SqO and nor-SqO with barbituric
SqB and nor-SqB) and dicyanomethylene (SqCN and nor-SqCN) groups
DMSO (5:1, v/v) and a pronounced aggregation band appears on the
shorter-wavelength slope of the absorption band (Fig. 7,a–f). The ag-
gregation band of cyanines and squaraines is known to be located very
close to the vibronic shoulder [46]. Barbituric, dicyanomethylene
squaraines and norsquaraines exhibit more pronounced aggregation
than SqO and nor-SqO. The level of aggregation for the dye pairs SqO/
nor-SqO and SqCN/nor-SqCN seems very similar while for nor-SqB it
is higher compared to SqB.
(
causes a noticeable increase in the calculated dipole moments (μ) which
increases in the order of SqO (0 D) = nor-SqO (0 D) ≪ SqB (5.43
D) < nor-SqB (7.85 D) < SqCN (10.12 D) ≈ nor-SqCN (10.21 D).
Molecular structure of norsquaraines versus squaraines.
Squaraines and norsquaraines have similar molecular structure, elec-
tronic density distribution and dipole moments but norsquaraines have
an increased “local” polarity expressed by the increased electron
charges on the indolenine nitrogens as compared to those of squaraines.
In addition, norsquaraine molecules contain intramolecular H-bonds
and therefore they are conformationally more rigid and more planar.
It is worth mentioning that the investigated dyes have reduced so-
lubility not only in aqueous media but also in CHCl and DMSO. As a
3
1
3
result, we were unable to measure C NMR spectra of SqB and nor-SqB
in CDCl and DMSO.
The Stokes shifts (Δνst) for norsquaraines and squaraines in CHCl
increase in the order: oxo < dicyanomethylene < barbituric
Table 2), while the molecular polarity represented by the dipole mo-
3
3
3.3. Spectral properties
(
3
.3.1. Dyes free in solutions
ment increases in the order: oxo < barbituric < dicyanomethylene.
The increased Δνst of barbituric and dicyanomethylene derivatives as
compared to SqO and nor-SqO is more likely due to their more polar
structure. The substantially increased Δνst of SqB and nor-SqB as
compared to other derivatives is supposedly due to the change in the
intramolecular H-bonds between barbituric oxygens and methine hy-
drogens in the excited state. The intramolecular H-bonds cause a de-
crease in the molar absorptivities of barbituric derivatives compared to
other dyes. Interestingly, the Stokes shifts (Δνst) and the spectral band
The absorption and emission maxima (λmax), molar absorptivities
(
ε), quantum yields (Φ ), Stokes shifts (Δνst), and spectral band half-
F
widths (Δν1/2) of norsquaraine dyes measured free in solutions and after
non-covalent binding to protein (BSA), are summarized in Table 2.
The absorption spectra of norsquaraine nor-SqO and squaraine
SqO, measured in CHCl consist of only one long-wavelength band with
3
−1 −1 −1 −1
a molar absorptivityý ε = 218,000 M cm
and 219,000 M cm
,
respectively.
Substitution of squaric oxygen in both SqO and nor-SqO molecules
half-widths (Δν1/2) for nor-SqO in CHCl are higher than those of SqO
3
325

O.S. Kolosova et al.
Dyes and Pigments 163 (2019) 318–329
Table 2
Spectral characteristics of free dyes (cDye ∼ 0.5 μM) and their complexes with 90 μM BSA at Т = 25°С.
−
1
−1
Δνst, cm−1
Δν1/2 (Ab), cm−1
Δν1/2 (Em), cm−1
Dye
Media
λ
max (Ab), nm
λ
max (Em), nm
ε, M cm
F
Φ , %
CHCl
3
654
647
657
659
658
647
639
655
633
625
641
624
639
669
669
662
673
678
675
666
659
669
642
635
652
635
645
693
218,000
200,000
206,000
219,000
215,000
n/d
45
44
45
43
47
29
13
36
25
4
340
350
360
430
380
440
470
320
220
250
260
280
150
520
780
830
820
840
790
870
960
870
690
640
660
750
670
980
730
800
770
780
770
790
850
890
690
690
650
740
670
830
MeOH
DMF
nor-SqO
DMSO
3
DMSO—CHCl (1:1)
DMF—water (1:1)
PB pH 7.4—DMSO (5:1)
BSA-Complex PB pH 7.4
92,000
200,000
219,000
n/d
CHCl
3
MeOH
DMSO
n/d
15
3
SqO
PB pH 7.4—DMSO (5:1)
BSA-Complex PB pH 7.4
n/d
203,000
63,000
16,000
44
48
CHCl
3
4
41
a
a
a
a
a
PB pH 7.4—DMSO (5:1)
BSA-Complex PB pH 7.4
673
662
n/a
683
n/a
< 1
n/a
n/a
n/a
23,000
9
460
2270
980
4
60
15,000
nor-SqB
CHCl
3
641
27
618
26
633
22
694
669
646
657
712
n/aa
704
701
678
702
103,000
27,000
86,000
23,000
91,000
13,000
120,000
33,000
28,000
19,000
63,000
32,000
196,000
35,000
70,000
16,000
162,000
36,000
4
650
700
580
360
n/aa
290
380
400
420
1110
1410
1180
750
1380
1620
1540
730
4
PB pH 7.4—DMSO (5:1)
BSA-Complex PB pH 7.4
< 1
4
SqB
34
4
CHCl
3
28
3
88
PB pH 7.4—DMSO (5:1)
BSA-Complex PB pH 7.4
698
389
690
< 1
13
n/aa
1730
800
n/aa
nor-SqCN
830
3
94
CHCl
3
683
81
660
84
682
80
37
830
3
PB pH 7.4—DMSO (5:1)
BSA-Complex PB pH 7.4
< 1
50
2030
830
1510
850
SqCN
3
3
a
Not available because of the low solubility and/or high aggregation.
DMSO is substituted with a less polar solvent such as CHCl
For instance, the quantum yield of SqO in CHCl (Φ = 25%) is 8 fold
decreased in PB pH 7.4 — DMSO (5:1) (Φ = 3%) and 6 fold in MeOH
= 4%).
In contrast, the solvent polarity has a marginal effect on the
quantum yield of norsquaraines. The quantum yield of nor-SqO in
DMSO (Φ = 43%), DMF (Φ = 45%) and MeOH (Φ = 44%) is almost
the same as in the less polar solvent CHCl (Φ = 45%) and
DMSO—CHCl (1:1, v/v) (Φ = 47%) (Table 2). At the same time, the
of nor-SqO measured in DMSO or DMF decreases upon dilution with
is 43% in DMSO and 45% in DMF while it is only
9% in water—DMSO or water—DMF (1:1, v/v) and 13% in PB pH
3
(Table 2).
3
F
F
(
Φ
F
F
F
F
3
F
3
F
Φ
F
water. Thus, the Φ
F
2
7
.4—DMSO (5:1). The quantum yield decrease in aqueous media can be
attributed to the aggregation of nor-SqO molecules. This is evidenced
by the increase in the aggregation band at the short-wavelength slope of
the main absorption band (around 600 nm) and the decrease in the
main band (around 650 nm) (Fig. 8,a). Furthermore, upon increasing
the water ratio to more then about 40:1 (v/v), an additional aggrega-
tion band appears at the long-wavelength slope of the main absorption
band (at about 700 nm).
Fig. 6. Absorption and emission spectra of nor-SqO, nor-SqB and nor-SqCN
measured in CHCl .
3
while for the dye pairs nor-SqB/SqB and nor-SqCN/SqCN an opposite
correlation is observed. For norsquaraines Δν1/2(Ab) > Δν1/2(Em),
while for SqO they are almost the same and for SqB and SqCN Δν1/
In case of SqCN, nor-SqCN and especially SqB and nor-SqB, the
aggregation noticeably increases causing a dramatic decrease in the
2
(Ab) < Δν1/2(Em). Both Δνst and Δν1/2 increase, when CHCl is re-
3
placed with water, which is typical for polar dyes.
The quantum yields (Φ ) of norsquaraines nor-SqO and nor-SqB
measured in CHCl are 1.8 and 12 fold higher compared to SqO and
SqB while for nor-SqCN the Φ is surprisingly 1.3 times lower.
The quantum yields of squaraine dyes are known to be strongly af-
fected by the solvent polarity [47,48]. The Φ of SqO, SqB and SqCN
substantially increases, when a polar solvent such as water, alcohol or
quantum yields: Φ < 1% in PB pH 7.4 — DMSO (5:1, v/v) (Table 2).
F
1 13
As shown above, the H NMR and C NMR spectra of nor-SqO,
measured in CDCl as compared to DMSO‑d , contain two groups of
signals, which is due to coexistence of two conformers. However, the
F
3
3
6
F
absorption spectra measured in CHCl , MeOH, DMF, and DMSO contain
3
only one band (Fig. 8,b). This suggests that the above conformers have
similar spectral properties. The solvent causes a slight red-shift in the
F
326

O.S. Kolosova et al.
Dyes and Pigments 163 (2019) 318–329
Fig. 7. Absorption spectra of norsquaraines and squaraines in CHCl and PB pH 7.4 in presence of DMSO (5:1, v/v) and 90 μM BSA.
3
Fig. 8. Absorption spectra of nor-SqO (cdye = 0.5 μM) in PB 7.4 — DMSO mixtures (a) and in different solvents (b).
nor-SqO spectra and this shift increases in the order of PB–DMSO
5:1) < MeOH < CHCl < DMF < DMSO while the molar absorp-
tivity remain almost unchanged (Table 2).
classes, squaraines and norsquaraines, measured at cDye = 0.5 μM these
(
3
characteristics reach “saturation” when the concentration of BSA
reaches cBSA ∼ 90 μM and then do not change anymore. Therefore, the
spectral characteristics of the dye—BSA complexes presented in Table 2
were measured at cBSA = 90 μM.
3
.3.2. Non-covalent complexation with BSA
When compared to PB pH 7.4 upon complexation with BSA squar-
aines SqO and SqB — DMSO (5:1, v/v) exhibit an about 15/10 nm red-
shift of the absorption/emission bands and SqCN shows a 22/24 nm
red-shift. Norsquaraine nor-SqO in presence of BSA also shows about
the same red-shift (16/10 nm) as SqO while nor-SqB and nor-SqCN
exhibit ∼10 nm blue-shift, which is more likely due to their higher
polarity.
The absorption and emission spectra and the quantum yields of
squaraines measured in aqueous buffers are known to be substantially
affected by the presence of proteins in particular bovine serum albumin
(
BSA), which is due to the formation of dye—BSA complexes: there is a
moderate red-shift and noticeable increase in the quantum yield
42,49]. These effects have been explained by decrease in the en-
[
vironment polarity [46,50] and increase in the conformational rigidity
In general, the Stokes shifts of squaraines and norsquaraines (except
[
51] of dye molecules, when they move from water to protein phase.
SqCN) in aqueous BSA are shorter compared to CHCl , which is most
3
We found that the spectral properties of norsquaraines also do
likely due to the more viscous protein environment.
change in the presence of BSA (Fig. S6,a,b). For both investigated dye
327

O.S. Kolosova et al.
Dyes and Pigments 163 (2019) 318–329
The fluorescence intensities of squaraines measured in aqueous
grateful to Dr. Illia Serdiuk (Gdansk University) for the measurements
of mass-spectra and Dr. Hugo Gottlieb (Bar Ilan University, Israel) for
helpful discussion on NMR results.
media (PB pH 7.4 — DMSO, 5:1, v/v) dramatically increase in presence
of BSA (Fig. S6,b). The quantum yields (Φ ) increase by factors of
F
∼
15, > 34, and > 50, respectively (Table 2). It was difficult to de-
termine the accurate values of these increases for SqB and SqCN be-
cause of the extremely low Φ in PB pH 7.4 — DMSO (5:1, v/v). Im-
portantly, the Φ of norsquaraine nor-SqO increases only by factor of
Appendix A. Supplementary data
F
F
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.dyepig.2018.12.007.
2
.8 (as compared to 15 fold for SqO), while in the absence of BSA it is
higher compared to SqO (Table 2, Fig. S6,a). Thus, nor-SqO is less
sensitive to BSA than SqO, which can be attributed to its more rigid
structure. Nor-SqB and nor-SqCN demonstrate a substantially more
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