Synthesis, characterization and protein-association of dicyanomethylene squaraine dyes

Article abstract of DOI:10.1016/j.dyepig.2017.07.070

We prepared several new dicyanomethylene squaraine dyes derived from indolenine, benzothiazole and benzoselenazole, with different substituent groups. All the obtained dyes display intense and narrow absorption bands and bright fluorescence emission in the Vis/NIR region (665–716 nm) when dissolved in organic solvents, especially for the unsymmetrical dyes containing sulphur and selenium atoms. The use of synthesized squaraine dyes as fluorescent probes for detection of bovine serum albumin (BSA) and human serum albumin (HSA) was explored in Tris-HCl buffer solutions. We observed that all the dyes are non-emissive in buffer solution but become fluorescent in the presence of BSA or HSA proteins, with the emission intensity increasing proportionally to the protein concentration (0.00–1.22 μM). The most pronounced effect was observed for the symmetrical squaraine having two moieties of indolenine.

Full text of DOI:10.1016/j.dyepig.2017.07.070

Accepted Manuscript
Synthesis, characterization and protein-association of dicyanomethylene squaraine
dyes
Tiago D. Martins, Marlene L. Pacheco, Renato E. Boto, Paulo Almeida, José Paulo S.
Farinha, Lucinda V. Reis
PII:
S0143-7208(17)30617-4
10.1016/j.dyepig.2017.07.070
DYPI 6159
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 19 May 2017
Revised Date: 23 July 2017
Accepted Date: 31 July 2017
Please cite this article as: Martins TD, Pacheco ML, Boto RE, Almeida P, Farinha JoséPauloS, Reis
LV, Synthesis, characterization and protein-association of dicyanomethylene squaraine dyes, Dyes and
Pigments (2017), doi: 10.1016/j.dyepig.2017.07.070.
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Synthesis, characterization and protein-association of
dicyanomethylene squaraine dyes
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Tiago D. Martinsa,b, Marlene L. Pachecoa, Renato E. Botoa, Paulo Almeidac, José
Paulo S. Farinhab, Lucinda V. Reisa, *
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aChemistry Department and CQ-VR, UTAD, Quinta de Prados, 5001-801 Vila Real,
Portugal
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bCQFM-Centro de Química-Física Molecular and IN-Institute of Nanoscience and
Nanotechnology, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av.
Rovisco Pais, 1049-001 Lisboa, Portugal
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cCICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av. Infante
D. Henrique, 6201-506 Covilhã, Portugal
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*Corresponding author
Lucinda V. Reis
Department of Chemistry and CQ-VR; School of Life and Environmental Sciences
University of Trás-os-Montes e Alto Douro (UTAD); Quinta de Prados; 5001-801 Vila
Real; Portugal.
Tel.: +351 259350225; Fax: +351 259350480; E-mail address: lucinda.reis@utad.pt
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Abstract
We prepared several new dicyanomethylene squaraine dyes derived from
indolenine, benzothiazole and benzoselenazole, with different substituent groups. All
the obtained dyes display intense and narrow absorption bands and bright fluorescence
emission in the Vis/NIR region (665-716 nm) when dissolved in organic solvents,
especially for the unsymmetrical dyes containing sulphur and selenium atoms.
The use of synthesized squaraine dyes as fluorescent probes for detection of
bovine serum albumin (BSA) and human serum albumin (HSA) was explored in Tris-
HCl buffer solutions. We observed that all the dyes are non-emissive in buffer solution
but become fluorescent in the presence of BSA or HSA proteins, with the emission
intensity increasing proportionally to the protein concentration (0.00-1.22µM). The
most pronounced effect was observed for the symmetrical squaraine having two
moieties of indolenine.
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Keywords
Dicyanomethylene squaraine dyes, Fluorescent probes, BSA, HSA
1. Introduction
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Fluorescence is now a well-established tool in many areas, with new
fluorescence-based assays and techniques continuously appearing in the literature,
especially in the biological and biomedical fields. The many advantages of
fluorescence-based methods led to their application in flow cytometry, medical
diagnosis, DNA sequencing, forensics, genetic analysis, etc. Fluorescence probes are
also at the core of advanced laser scanning microscopy methods, used in the real-time
imaging of live cells, [1] as markers to track cellular events with minimal disturbance
on the cellular environment, [2–4] and in super-resolution methods. [5] This increasing
plethora of applications has fostered the search for new fluorescent probes with
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improved photostability, higher brightness, and new sensing functionalities. Fluorescent
dyes with their absorption and fluorescence shifted to the red/near infrared (NIR) are
especially useful in the biomedical field. [6] In this spectral region there is less light
scattering, light absorption and autofluorescence by biological tissues [7] and higher
tissue penetration. [8] Also, dyes with red-shifted absorption can use less expensive
lasers (He:Ne or red diode lasers).
Not only fluorescence methods allow the imaging, detection and quantification
with much higher sensibility than absorbance and other optical methods, but also some
fluorescent probes are extremely sensitive to their environment and can thus be used as
probes and sensors in many different applications. [9]
Squaraines are a class of dyes with intense and sharp absorption bands at long-
wavelengths, and also sharp and intense fluorescence bands. [2,3] These properties are
of great interest for several biological and biomedical applications, such as fluorescent
detection of toxic substances biomolecules, etc. [10] In particular, squaraines have been
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described to form stable conjugates with proteins through hydrophobic binding sites,
with these conjugates enhancing squaraine fluorescence emission and thus allowing
protein detection. [11,12]
Studies by Terpetschnig et al. [13] showed that symmetrical squaraine
indolenine derivatives have higher photostability than benzoselenazole and
benzothiazole derivatives, which rapidly fade under excitation light. Results by Tatarets
et al. [14] further showed that dicyanomethylene squaraines present higher extinction
coefficients and fluorescence quantum yields, longer fluorescence lifetimes and better
photostability than conventional polymethine squaraines dyes, making these potentially
more useful for fluorescence lifetime-based biomedical applications. Volkova et al. [15]
showed that the fluorescence intensity of squaraine dyes derivatives from benzothiazole and
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benzoselenazole increased significantly upon protein binding and therefore can be used
as protein detection fluorescent probes.
Albumins are a family of globular proteins, with serum albumins being some of
the most studied proteins due to their properties and high biological relevance. Serum
albumins like BSA and HSA are synthesized on the liver and are the most common type
of protein found on blood. With good water-solubility, its main function consists on the
maintenance of the osmotic pressure in blood and transport of substances like calcium,
magnesium, other proteins, fatty acids and even some drugs. [16–18]
Here we describe the synthesis and spectroscopic characterization of new
dicyanomethylene squaraine dyes derived from indolenine, benzothiazole and
benzoselenazole, as well as the evaluation of their potential use as fluorescent probes for
BSA and HSA proteins.
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2. Results and discussion
2.1 Synthesis of squaraine dyes (7a-f)
Symmetrical (7a) and unsymmetrical (7b-f) dicyanomethylene squaraine
derivatives were successfully synthesized by a multistep reaction (Scheme 1). Although
the synthesis of the symmetrical squaraine 7a has been previously described by Tatarets
et al. [11], the preparation of the unsymmetrical squaraines (7b-f) is presented for the
first time. In the first stage, dibutylsquarate 4, obtained by refluxing squaric acid (3,4-
dihydroxycyclobut-3-en-1,2-dione) (3) in n-BuOH [12], reacted with 1,2,3,3-
tetramethylindolium iodide (2a), which was obtained by alkylation of the 2,3,3-
trimethylindolenine (1a) with an excess of iodomethane, to afford the monosubstituted
intermediate 5. The later, after reaction with malonitrile and trimethylamine in ethanol
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at room temperature gave the key intermediate 6. The desired 3-dicyanomethylene-
substituted cyanine dyes 7a-f were easily obtained by a base-catalysed condensation
reaction, under reflux in a n-butanol/toluene (1:1 v/v) mixture, between the intermediate
6 and the appropriate quaternary salts 2a-f which were synthesized as previously
described in literature. [13–15]
All synthesized dye structures were confirmed by standard spectroscopic
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methods (1H NMR, C NMR and HRMS spectrometry) being the assignments of the
carbons based on DEPT 135 spectra (Supporting Information). We note that the 1H and
13C spectra of 7c show peaks corresponding to an additional alkyl chain resulting from
the esterification of the carboxylic acid group present in the salt 2c with the n-butanol
reaction solvent.
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Scheme 1 – Synthesis of dicyanomethylene squaraine dyes 7a-7f. Conditions: i)
acetonitrile, reflux; ii) n-butanol, reflux; iii) ethanol, Et3N, rt; iv) ethanol,
malonitrile/Et3N, rt; v) n-butanol/toluene (1:1, v/v), reflux.
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2.2. Photochemical properties
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The absorption and fluorescence properties of the new squaraine dyes were
studied in methanol and chloroform solutions (Figure 1, Table 1). Although all
squaraines have and indolenine core structure, with similar and high extinction
coefficients (in the range of ca. 1 × 105 – 2 × 105 M-1 cm-1, typical of π-π* transitions)
and maximum absorption wavelengths in the range 665-700 nm, the benzothiazole and
benzoselenazole derivatives (7b-7f) present higher fluorescence intensities, probably
due to the sulphur or selenium atoms in the dyes structure. A solvatochromic shift (18-
23 nm) is observed between the more polar (methanol) and less polar (chloroform)
solvents. [16] The symmetrical indolenine derivative 7a, with no heteroatom
substitution in the X position, features the lowest maximum absorption wavelength (665
nm) and the highest molar absorptivity, both in methanol and in chloroform. On the
other hand, this derivative features the lowest fluorescence quantum yield in both
solvents. The highest fluorescence quantum yield in methanol is observed for 7e, which
contains an iodine substituent at the Y position. In chloroform, all samples show a
fluorescence quantum yield of 60%, except 7a which has only 30%. Our
dicyanomethylene squaraines presents high emission efficiency, as previously reported
by Sameiro et al.. [5] However, analogous dicyanomethylene squaraines studied by
Pisoni et al.. [4] having N-hexyl chains in both aromatic moieties (indolenine and
benzothiazole), showed lower fluorescence quantum yields than 7d. This effect is
probably due to the shorter N-alkyl chain on the indolenine moiety of 7d.
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The internal heavy atom effect by iodine is not observed, possibly due to the
rigidity of the molecule. However, no increase in the fluorescence intensity is observed
either. The observed fluorescence quantum yields seem to be related to the symmetry of
the molecules, with the lowest values observed for the fully symmetrical derivative.
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This hints at the possible formation of non-emissive aggregates with large absorptivity,
which decrease the fluorescence quantum yield.
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We also measured the fluorescence lifetimes of all derivatives (Table S1,
Supporting information), with laser excitation at λexc = 640 nm and collecting the
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emission at the wavelength of maximum emission, λem = 687 - 716 nm. The
fluorescence decays are mono-exponential and faster in methanol than in chloroform (in
methanol the decays have a short component of ca. 50 ps which weighs less than 1% to
the decay and is probably a fitting artefact). The symmetrical indolenine derivative 7a,
presented a faster decay in both solvents than the benzothiazole and benzoselenazole
derivatives (7b-7f). The largest difference in the fluorescence lifetimes, both in
methanol and chloroform, is observed between the symmetrical derivative and 7b-f,
with 7a presenting the lowest lifetime. Again the predominating effect seems to be the
symmetry. The possible formation of non-emissive aggregates can be an explanation for
the decrease in lifetimes.
A
B
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Figure 1 – Absorption (thick lines) and fluorescence spectra (thin lines) of
dicyanomethylene squaraines (7a-f) in methanol (A) and chloroform (B).
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Table 1 – Photophysical data from UV/vis absorption and fluorescence emission of
dicyanomethylene squaraines (7a-f) in methanol and chloroform.
Methanol
Dye
λabs (nm)
665
ε (M-1.cm-1)
196347
106986
142291
162172
185262
181848
λem (nm)
687
Stokes’ shift (nm)
ΦF
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0.05 ± 0.01
0.16 ± 0.01
0.19 ± 0.01
0.20 ± .002
0.29 ± 0.01
0.26 ± 0.02
7a
7b
7c
7d
7e
7f
666
691
669
695
668
694
677
703
677
702
Chloroform
λem (nm)
700
Dye
λabs (nm)
683
ε (M-1.cm-1)
210781
163676
187668
193517
198535
195303
Stokes’ shift (nm)
ΦF
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0.33 ± 0.01
0.58 ± 0.01
0.56 ± 0.03
0.59 ± 0.03
0.59 ± 0.01
0.56 ± 0.02
7a
7b
7c
7d
7e
7f
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708
689
709
688
707
700
716
698
715
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2.3. Dye-protein association
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The dicyanomethylene squaraine dyes (7a-f) showed high emission efficiencies
in organic solvents (higher in chloroform than in methanol) but no emission in water,
due to their very poor solubility. This led us to further experiment their ability to bind to
albumins (BSA and HSA) in Tris-HCl buffer. Previous studies by Volkova et al. [25]
using several squaraine dyes, among which some dicyanomethylene squaraine
derivatives, showed considerably higher emission in the presence of BSA. Using a 5.0
µM stock solution of the squaraine dyes (7a-f) we added BSA and HSA concentrations
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in the range 0.00 – 1.22 µM, in Tris-HCl buffer (with final dye concentration in the
range of 1.3 to 0.8 µM).
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The free dyes in buffer solution exhibited near-zero fluorescence intensity,
probably due to the formation of non-emissive aggregates. [20,21] However, their
emission increased significantly with the addition of either BSA or HSA (Figure 2 and
3). This observation confirms the dyes ability to interact with the albumin proteins. This
noncovalent interactions have previously been described to involve hydrophobic,
electrostatic and hydrogen bonding interactions, [2] and depend strongly on the
structures of the dye and the protein as discussed below.
The symmetrical squaraine 7a presented higher fluorescence intensity than the
non-symmetrical dyes (7b-f) for all BSA concentrations (Figure 2), in spite of its lower
fluorescence quantum yield in both methanol and the less polar chloroform. This means
that 7a has a stronger interaction to BSA, which is in agreement with the previous
report of Terpetsching et al. [6], where the indolenine moiety is clearly favourable for
protein labelling in aqueous environment.
The unsymmetrical dyes derived from benzothiazole and benzoselenazole (7b-f),
yielded much lower fluorescence intensities, although they also increase with protein
concentration. Although these dyes have high quantum yields in both methanol and
chloroform, their very low water solubility strongly limits their application for protein
detection.
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7a
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Wavelength (nm)
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7f
7e
0.03
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Wavelength (nm)
Wavelength (nm)
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Figure 2 – Fluorescence spectra of the dicyanomethylene squaraine dyes (7a-f) in Tris-
HCl buffer pH = 8.0, with increasing concentrations of BSA (0.00, 0.21, 0.25, 0.29,
0.33, 0.37, 0.41, 0.48, 0.55, 0.61, 0.67, 0.73, 0.79, 0.94, 1.08, 1.20 µM). Curves
corrected for 1 µM dye concentration and normalized to the intensity obtained for 7a
with 1.2 µM BSA.
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Among the four dyes derived from benzothiazole (7b-e), 7c and 7e stand out
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hydrogen bonding which increases the solubility in water, possibly increasing the
interaction with proteins, 7e has a heavy atom (iodine) on the benzothiazole moiety,
hindering the dye solubility and thus, its protein labelling ability.
A similar effect was observed on the HSA assays (Figure 3). The dyes 7a and 7c
showed much lower fluorescence intensity in the presence of HSA than in BSA assays,
but still higher than the other dyes. Some of the dyes with lower fluorescence intensity
in the BSA assays presented a small increase in the HSA assays (7b, d, f) in spite of the
low solubility of the substituents groups, suggesting that those groups have stronger
interaction with HSA than the groups in 7a and 7c.
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Wavelength (nm)
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Figure 3 – Fluorescence spectra of the dicyanomethylene squaraine dyes (7a-f) in Tris-
HCl buffer pH = 8.0, with increasing concentrations of HSA (0.00, 0.21, 0.25, 0.33,
0.41, 0.48, 0.55, 0.61, 0.80, 0.96, 1.10, 1.22 µM). Curves corrected for 1 µM dye
concentration and normalized to the intensity obtained for 7a with 1.2 µM BSA (Figure
2).
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An increase in fluorescence emission upon addition of protein was observed
for all dyes, especially for 7a, which presented an increase of up to 309 times in BSA (I0
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= 0.003 to I0.79 = 1.00) and 101 times in HSA (I0 = 0.006 to I1.22 = 0.590). Squaraine 7c
also shows a considerable increase of up to 30 times in BSA (I0 = 0.010 to I1.20 = 0.301)
and 80 times in HSA (I0 = 0.002 to I1.10 = 0.161). The poorest result was obtained for
squaraine 7e, which presented an increase factor of only up to 2 and 5 for BSA and
HSA, respectively. The fluorescence intensity increases proportionally to the protein
concentration (Figure 4), with a linear correlation observed for a protein concentration
interval of 0.21 to 0.80 µM, for all the squaraines in both proteins.
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0.2
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0.25
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BSA concentration (µM)
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7b
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7d
7e
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1.25
HSA concentration (µM)
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Figure 4 – Fluorescence emission intensity of the dicyanomethylene squaraine dyes
(7a-f) as a function of BSA (A) or HSA (B) concentration in Tris-HCl buffer, pH = 8.0.
Values corrected for 1 µM dye concentration and normalized to the intensity obtained
for 7a with 1.2 µM BSA.
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3. Experimental Section
3.1. Materials and methods
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All solvents and reagents (i.e. 2,3,3-trimethylindolenine (1a), 2-
methylbenzothiazole (1b), 2-methylbenzoselenazole (1f) and squaric acid (3) were
commercially acquired, analytically pure, and used as received without further
purification. 1,2,3,4-Tetramethylindolin-1-ium iodide (2a) [13], 3-ethyl-2-
methylbenzothiazol-3-ium iodide (2b) [13], 3-(4-carboxybutyl)-2-methylbenzothiazol-
3-ium bromide (2c) [14], 3-hexyl-2-methylbenzothiazol-3-ium iodide (2d) [13], 3-
hexyl-6-iodo-2-methylbenzothiazol-3-ium
iodide
(2e)
[15],
3-hexyl-2-
methylbenzoselenazol-3-ium iodide (2f) [13], 3,4-dibutoxycyclobut-3-en-1,2-dione (4)
[12], 3-butoxy-4-(1,3,3-trimethyl-2,3-dihydro-1H-2-indolylidenmethyl)-3-cyclobutene-
1,2-dione (5) [22], triethylammonium 3-(dicyanomethylene)-4-oxo-2-[(1,3,3,-
trimethylindolin-2-ylidene)methyl]cyclobut-1-en-1-olate (6) [11] were prepared
according to the literature procedure.
The reactions were followed by thin layer chromatography (TLC), using
aluminium plates with 0.25 mm of silica gel (Merck 60 F254). Melting points (M.p.)
were determined in a hot plate binocular microscope apparatus (URA Technic,
Portugal) and were not corrected. The IR spectra were recorded on a UNICAM
Research Series FTIR spectrophotometer and iS50 Thermo Scientific Nicolet FTIR,
using KBr pellets; νmax in cm−1. 1H and 13C NMR spectra were recorded at 298.15 K, on
a NMR Brücker Avance III 400 spectrometer operating at 9.4 T, observing 1H at 400.13
MHz or on a NMR Brücker Avance III 600 spectrometer operating at 14.09 T,
observing 1H at 600.13 and 13C at 150.91 MHz. Solutions were prepared in CDCl3 or in
DMSO-d6. The chemical shifts are expressed as δ (ppm) relative to tetramethylsilane
(internal standard) or to residual solvent signals and J values are given in hertz (Hz).
Splittings were described as s (singlet), d (doublet), t (triplet), qt (quintet) or
combinations of the above or m (multiplet). The assignments of the carbons were made
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based on DEPT 135 spectra. High resolution electrospray ionization time-of-flight mass
spectra (HRESI-TOFMS) were recorded using a microTOF (focus) Brücker Daltonics
spectrometer (C.A.C.T.I. at the University of Vigo).
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3.2. Synthesis of squaraine dyes
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3.2.1
Synthesis
of
3-dicyanomethylene-4-[(1,3,3-trimethyl-3H-indol-1-ium-2-
yl)methylene)]-2-[(1,3,3-trimethylindolin-2-ylidene)methyl]-cyclobut-1-en-1-olate (7a).
The title dye was prepared by a similar method to that described in literature[11], from a
solution of 6 (500 mg, 1.2 mmol) and 2a (360 mg, 1.2 mmol) in a mixture (1:1 v/v) of
n-butanol/toluene (10 mL) heated under reflux.
The mixture was kept under stirring for 5 days and the reaction was monitored
by TLC (silica gel plates, CH2Cl2/MeOH 5%). After reaction was completed, the
mixture was cooled on an ice bath to allow the product to precipitate by the addition of
methanol. The resulting residue was filtered under reduced pressure and recrystallized
with methanol. Red crystals were recoiled and dried under reduced pressure. Yield:
12%; M.p. 290-292 ºC (dec.); IR (KBr): 3047 (w, ArC-H), 3018 (w, ArC-H), 2927 (w,
CH), 2193 (m, CN), 2172 (m, CN), 1496 (s), 1480 (s), 1456 (s), 1432 (s), 1348 (m),
1285 (s), 1268 (s), 1246 (s), 1223 (s), 1211 (s), 1155 (m), 1107 (s), 1080 (s), 1044 (m),
1018 (m), 969 (m), 921 (m), 864 (m), 823 (m), 801 (m), 740 (m), 675 (w) cm-1; H-
1
NMR (600 MHz, DMSO-d6, ppm) δ: 7.54 (2H, d, J = 7.2 Hz, Ar-H), 7.41-7.39 (4H, m,
Ar-H), 7.27-7.23 (2H, m, Ar-H), 6.29 (2H, s, C=CH), 3.59 (6H, s, NCH3), 1.67 (12H, s,
C(CH3)2; 13C-NMR (150.90 MHz, DMSO-d6, ppm) δ: 173.1, 172.6, 166.7, 165.1, 142.9,
142.1, 128.6 (ArC-H), 125.2 (ArC-H), 122.7 (ArC-H), 119.0 (CN), 111.6 (ArC-H), 88.5
(C=CH), 49.5 (C(CH3)2), 31.8 (NCH3), 26.4 (C(CH3)2).
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3.2.2
Synthesis
of
3-dicyanomethylene-4-[(1,3,3-trimethyl-3H-indol-1-ium-2-
yl)methylene)]-2-[(3-ethylbenzothiazol-2-ylidene)methyl]-cyclobut-1-en-1-olate
(7b).
This dye was synthesized analogously to 7a, after refluxing a mixture of 6 (300 mg, 0.72
mmol) and 2b (220 mg, 0.72 mmol), in a mixture (1:1 v/v) of n-butanol/toluene (10
mL) for 48 h. After recrystallization in methanol, brown crystals were recoiled and
dried under reduced pressure. Yield: 10%; M.p. >295 ºC; IV (KBr): 3075 (w, ArC-H),
2918 (w, CH), 2193 (m, CN), 2176 (w, CN) 1720 (w), 1622 (w), 1515 (s, C=C), 1463
(s), 1429 (m), 1363 (w), 1309 (m), 1276 (s), 1265 (s), 1245 (m), 1224 (s), 1212 (s),
1158 (m), 1111 (s), 1078 (m), 984 (m), 830 (m) cm-1; H-NMR (600 MHz, CDCl3,
1
ppm) δ: 7.66 (1H, d, J = 7.8 Hz, Ar-H), 7.50 (1H, t, J = 7.5 Hz, Ar-H), 7.36-7.31 (4H,
m, Ar-H), 7.15 (1H, t, J = 7.5 Hz, Ar-H), 7.00 (1H, d, J = 7.8 Hz, Ar-H), 6.58 (1H, s,
C=CH), 6.30 (1H, s, C=CH), 4.30 (2H, q, J = 7.2 Hz, NCH2CH3), 3.54 (3H, s, NCH3),
1.74 (6H, s, C(CH3)2), 1.52 (3H, t, J = 7.2 Hz, NCH2CH3; 13C-NMR (150.90 MHz,
CDCl3, ppm) δ: 173.9, 170.4, 167.0, 165.4, 163.7, 162.2, 142.8, 141.7, 140.2, 128.8,
127.9 (ArC-H), 127.9 (ArC-H), 125.4 (ArC-H), 123.8 (ArC-H), 122.6 (ArC-H), 122.1
(ArC-H), 119.1 (CN), 118.8 (CN), 112.3 (ArC-H), 109.2 (ArC-H), 88.1 (C=CH) 87.9
(C=CH), 48.7 (C(CH3)2), 42.2 (NCH2), 30.9 (NCH3), 27.0 (C(CH3)2), 12.6 (CH3);
EMAR-ESI-TOF m/z: 476.16623 [M]+ (C29H24N4OS calc. 476.16653).
3.2.3.
Synthesis
of
3-dicyanomethylene-4-[(1,3,3-trimethyl-3H-indol-1-ium-2-
yl)methylene)]-2-[(3-(4-oxo(butoxy)butyl)benzotiazol-2-ylidene)methyl]-cyclobut-1-en-
1-olate (7c). This dye was synthesized analogously to 7a after refluxing a mixture of 6
(150 mg, 0.36 mmol) and 2c (120 mg, 0.36 mmol), in a mixture (1:1 v/v) of n-
butanol/toluene (10 mL) for 20 h. After recrystallization in methanol, green crystals
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were recoiled and dried under reduced pressure. Yield: 26%; M.p. 238-241 ºC; IV
(KBr): 3066 (w, ArC-H), 2929 (w, CH), 2194 (w, CN), 2177 (w, CN), 1728 (w, C=O),
1517 (m, C=C), 1458 (s), 1359 (w), 1308 (m), 1279 (s), 1265 (s), 1241 (s), 1226 (s),
1156 (m), 1115 (m), 1081 (m), 1045 (w), 1019 (w), 977 (w), 931 (w), 869 (w), 813 (w)
cm-1; 1H-NMR (400 MHz, DMSO-d6, ppm) δ: 8.11 (1H, d, J = 7.6 Hz, Ar-H), 7.85 (1H,
d, J = 8.4 Hz, Ar-H), 7.58 (1H, t, J = 7.8 Hz, Ar-H), 7.47 (1H, d, J = 6.8 Hz, Ar-H),
7.44 (1H, t, J = 7.6 Hz, Ar-H), 7.33 (1H, t, J = 7.6 Hz, Ar-H), 7.25 (1H, d, J = 8.0 Hz,
Ar-H), 7.13 (1H, t, J = 7.6 Hz, Ar-H), 6.40 (1H, s, C=CH), 6.04 (1H, s, C=CH), 4.35
(2H, t, J = 7.4 Hz, NCH2(CH2)3COO(CH2)3CH3), 3.97 (2H, t, J = 6.6 Hz,
N(CH2)4COOCH2(CH2)2 CH3), 3.45 (3H, s, NCH3), 2.36 (2H, t, J = 7.2 Hz,
N(CH2)3CH2COO(CH2)3CH3), 1.79 (2H, qt, NCH2CH2(CH2)2COO(CH2)3CH3), 1.64
(2H, qt, N(CH2)2CH2CH2COO(CH2)3CH3), 1.64 (6H, s, C(CH3)2), 1.50 (2H, qt,
N(CH2)4COO CH2CH2CH2CH3), 1.27 (2H, sext, N(CH2)4COO(CH2)2CH2CH3), 0.84
13
(3H, t, J = 7.4 Hz, N(CH2)4COO(CH2)3CH3); C-NMR (150.90 MHz, CDCl3, ppm) δ:
210.8, 173.8, 173.3, 173.0, 170.4, 166.7, 165.3, 163.7, 162.2, 142.8, 141.6, 140.5,
128.5, 127.9 (ArC-H), 125.4 (ArC-H), 123.8 (ArC-H), 122.5 (ArC-H), 122.1 (ArC-H),
119.0 (CN), 118.7 (CN), 112.5 (ArC-H), 109.2 (ArC-H), 88.1 (C=CH), 88.1 (C=CH),
64.4 (OCH2), 48.7 (C(CH3)2), 46.8 (NCH2), 33.4 (CH2), 31.7 N(CH3), 30.6 (CH2), 27.0
(CH2), 26.9 (C(CH3)2), 22.0 (CH2), 19.1 (CH2), 13.7 (CH3); EMAR-ESI-TOF m/z:
604.25047 [M]+ (C36H36N4O3S calc. 604.25026).
3.2.4.
Synthesis
of
3-dicyanomethylene-4-[(1,3,3-trimethyl-3H-indol-1-ium-2-
yl)methylene)]-2-[(3-hexylbenzothiazol-2-ylidene)methyl]-cyclobut-1-en-1-olate (7d).
This dye was synthesized analogous to 7a after refluxing a mixture of 6 (300 mg, 0.72
mmol) and 2d (260 mg, 0.72 mmol), in a mixture (1:1 v/v) of n-butanol/toluene (10
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mL) for 24 h. After recrystallization in methanol, brown crystals were recoiled and
dried under reduced pressure. Yield: 11%; M.p. 285-288 ºC (dec.); IV (KBr): 3054 (w,
ArC-H), 2926 (w, CH), 2194 (s, CN), 2174 (m, CN), 1513 (s, C=C), 1475 (s), 1458 (s),
1432 (s), 1377 (m), 1359 (m), 1304 (s), 1265 (s), 1238 (s), 1223 (s), 1154 (s), 1111 (s),
1
1082 (s), 1021 (m), 981 (s), 931 (m), 855 (m), 814 (m), 745 (m), 673 (w) cm-1; H-
NMR (400 MHz, DMSO-d6, ppm) δ: 8.11 (1H, d, J = 8.4 Hz, Ar-H), 7.86 (1H, d, J =
8.4 Hz, Ar-H), 7.60 (1H, t, J = 7.8 Hz, Ar-H), 7.48 (1H, d, J = 6.6 Hz, Ar-H), 7.46 (1H,
t, J = 7.2 Hz, Ar-H), 7.33 (1H, t, J = 8.1 Hz, Ar-H), 7.25 (1H, d, J = 7.8 Hz, Ar-H), 7.14
(1H, t, J = 7.5 Hz, Ar-H), 6.43 (1H, s, C=CH), 6.06 (1H, s, C=CH), 4.33 (2H, t, J = 7.5
Hz, NCH2(CH2)4CH3), 3.46 (3H, s, NCH3), 1.76 (2H, qt, NCH2CH2 (CH2)3CH3), 1.64
(6H, s, C(CH3)2), 1.42 (2H, qt, N(CH2)2CH2(CH2)2CH3), 1.34-1.26 (4H, m,
13
N(CH2)3(CH2)2CH3), 0.86 (3H, t, J = 7.2 Hz, N(CH2)5CH3); C-NMR (150.90 MHz,
DMSO-d6, ppm) δ: 173.5, 169.0, 165.5, 164.1, 163.5, 160.8, 143.3, 141.5, 141.0, 128.8,
128.6 (Ar-CH), 128.4 (Ar-CH), 126.2 (Ar-CH), 123.9 (Ar-CH), 123.80 (Ar-CH), 122.5
(Ar-CH), 119.0 (CN), 118.9 (CN), 114.6 (Ar-CH), 110.3 (Ar-CH), 88.6 (C=CH), 87.0
(C=CH), 48.5 (C(CH3)2), 47.6 (NCH2), 31.3 (CH2), 30.9 (NCH3), 27.8 (CH2), 26.9
(C(CH3)2), 26.1 (CH2), 22.4 (CH2), 14.3 (CH3); EMAR-ESI-TOF m/z: 532.22922 [M]+
(C33H32N4OS calc. 532.22913).
3.2.5.
Synthesis
of
3-dicyanomethylene-4-[(1,3,3-trimethyl-3H-indol-1-ium-2-
yl)methylene)]-2-[(6-iodo-3-hexylbenzothiazol-2-ylidene)methyl]-cyclobut-1-en-1-olate
(7e). This dye was synthesized analogously to 7a, after refluxing a mixture of 6 (260 mg,
0.61 mmol) and 2e (310 mg, 0.0.61 mmol), in a mixture (1:1 v/v) of n-butanol/toluene
(10 mL) for 48 h. After recrystallization in methanol, brown crystals were recoiled and
dried under reduced pressure. Yield: 25%; M.p. 260 ºC (dec.); IV (KBr): 3055 (w, ArC-
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H), 2926 (w, CH), 2197 (m, CN), 2177 (m, CN), 1513 (s, C=C), 1474 (s), 1454 (s),
1434 (s), 1314 (m), 1264 (s), 1224 (s), 1154 (s), 1110 (s), 1080 (s), 977 (m), 928 (m),
830 (m), 814 (m), 771 (m), 679 (w) cm-1; 1H-NMR (400 MHz, DMSO-d6, ppm) δ: 8.47
(1H, d, J = 1.6 Hz, Ar-H), 7.87 (1H, dd, J = 8.0 Hz, J = 1.6 Hz, Ar-H), 7.63 (1H, d, J =
8.8 Hz, Ar-H), 7.50 (1H, d, J = 7.2 Hz, Ar-H), 7.36 (1H, t, J = 7.6 Hz, Ar-H), 7.30 (1H,
d, J = 7.6 Hz, Ar-H), 7.17 (1H, t, J = 7.6 Hz, Ar-H), 6.35 (1H, s, C=CH), 6.09 (1H, s,
C=CH), 4.26 (2H, t, J = 7.6 Hz, NCH2(CH2)4CH3), 3.49 (3H, s, NCH3), 1.73 (2H, qt,
NCH2CH2 (CH2)3CH3), 1.65 (6H, s, C(CH3)2), 1.40 (2H, qt, N(CH2)2CH2(CH2)2CH3),
1.34-1.24 (4H, m, N(CH2)3(CH2)2CH3), 0.86 (3H, t, J = 7.0 Hz, N(CH2)5CH3); EMAR-
ESI-TOF m/z: 658.12552 [M]+ (C33H31IN4OS calc. 658.12578).
3.2.6
Synthesis
of
3-dicyanomethylene-4-[(1,3,3-trimethyl-3H-indol-1-ium-2-
yl)methylene)]-2-[(3-hexylbenzoselenazol-2-ylidene)methyl]-cyclobut-1-en-1-olate (7f).
This dye was synthesized analogously to 7a, after refluxing a mixture of 6 (300 mg, 0.72
mmol) and 2f (290 mg, 0.72 mmol), in a mixture (1:1 v/v) of n-butanol/toluene (10 mL)
for 48 h. After recrystallization in methanol, red crystals were recoiled and dried under
reduced pressure. Yield: 26%; M.p. 270-273 ºC (dec.); IV (KBr): 3053 (w, ArC-H),
2926 (w, CH), 2191 (m, CN), 2173 (w, CN), 1519 (m, C=C), 1449 (s), 1306 (m), 1292
(m), 1261 (s), 1225 (s), 1113 (s), 1089 (m), 984 (m), 929 (w), 812 (w), 746 (m) cm-1;
1H-NMR (600 MHz, DMSO-d6, ppm) δ: 8.10 (1H, d, J = 7.8 Hz, Ar-H), 7.76 (1H, d, J
= 8.4 Hz, Ar-H), 7.54 (1H, t, J = 7.8 Hz, Ar-H), 7.47 (1H, d, J = 7.2 Hz, Ar-H), 7.36
(1H, t, J =7.8 Hz, Ar-H), 7.33 (1H, t, J = 7.8 Hz, Ar-H), 7.26 (1H, d, J = 7.8 Hz, Ar-H),
7.14 (1H, t, J = 7.5 Hz, Ar-H), 6.53 (1H, s, C=CH), 6.05 (1H, s, C=CH), 4.28 (2H, t, J =
7.5 Hz, NCH2(CH2)4CH3), 3.45 (3H, s, NCH3), 1.72 (2H, qt, NCH2CH2(CH2)3CH3),
1.62 (6H, s, C(CH3)2), 1.42 (2H, qt, N(CH2)2CH2(CH2)2 CH3), 1.32-1.25 (4H, m,
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N(CH2)3(CH2)2CH3), 0.85 (3H, t, J = 6.9 Hz, N(CH2)5CH3); C-NMR δ (150.90 MHz,
DMSO-d6, ppm): 174.2, 169.3, 168.3, 165.6, 164.5, 160.6, 143.2, 142.5, 141.5, 130.2,
128.4 (Ar-CH), 128.4 (Ar-CH), 126.8 (Ar-CH), 125.9 (Ar-CH), 123.9 (Ar-CH), 122.5
(Ar-CH), 118.8 (CN), 118.8 (CN), 116.0 (Ar-CH), 110.4 (Ar-CH), 92.3 (C=CH), 87.1
(C=CH), 48.6 (C(CH3)2), 48.2 (NCH2), 31.3 (CH2), 31.0 (NCH3), 27.7 (CH2), 26.8
(C(CH3)2), 26.01 (CH2), 22.4 (CH2), 14.3 (CH3); EMAR-ESI-TOF m/z: 580.17369 [M]+
(C33H32N4OSe calc. 580.17380).
3.3. Preparation of working solutions of dyes and proteins
The working solutions were prepared in a similar way to described by Volkova
et al.. [15] Each dye was dissolved in anhydrous dimethylformamide (DMF) to obtain a
solution of 2 x 10-3 M and then diluted in methanol and chloroform to obtain working
solutions of 5 x 10-6 M. The DMF solutions were diluted in 0.05 M Tris-HCl buffer
solution (pH 8.0) and then the protein solution (0.2 mg of protein/mL of 0.05 M Tris-
HCl buffer, pH 8.0) was added to achieve the desired concentration of protein. The first
sample has a ratio of 1:1499 (DMF:Tris-buffer, with a dye concentration of 1.3 x 10-6 M
and 0.07% DMF), and the last sample a ratio of 1:2490 (DMF:Tris-buffer, with a dye
concentration of 8.0 x 10-7 M) for BSA assays, and 1:2520 (DMF:Tris-buffer, with a
dye concentration of 7.9 x 10-7 M) for HSA assays, corresponding to 0.04% DMF. All
working solutions were prepared immediately before the experiments start.
3.4. UV-Visible absorption
The steady-state absorption spectra were recorded with the use of a JASCO V-
660 spectrophotometer at room temperature and in the spectral range of 500-750 nm,
using a UV quartz cuvette (1 cm path length).
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3.5. Fluorescence quantum yield
The fluorescence spectra were recorded on a Horiba Jobin Yvon Fluorolog 3-22
spectrofluorimeter with a xenon lamp of 450 W, using quartz cells (l = 1 cm), excitation
and emission slits with 2 nm bandwidth, integration time of 1 s/point, maximum
sensitivity, and right angle mode. The emission spectra were obtained with excitation
wavelength λex = 615 nm.
The quantum yield was determined according to the following equation:
ꢂ(ꢃ)ꢄꢅꢆꢇꢈꢉ ꢊꢃ
ꢍꢋꢉꢌ
ꢎꢏ
ꢁ
Φꢀ =
x
x
ꢄꢅꢆꢇꢈꢉ x Φꢋꢉꢌ
ꢎꢏ
ꢍꢄꢅꢆꢇꢈꢉ
ꢂ(ꢃ)ꢋꢉꢌ ꢊꢃ
ꢁ
ꢋꢉꢌ
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432
433
434
where F(λ)sample is the emission intensity of the sample, F(λ)ref is the emission intensity
of the reference, Asample is the absorbance of the sample, Aref is the absorbance of the
reference, n2sample is the refractive index of the sample, n2ref is the refractive index of the
reference, and Φref is the quantum yield of the reference. The reference used was 4-[(3-
hexylbenzothiazol-3-ium-2-yl)methylidene]-2-[(3-hexyl-3H-benzothiazol-2-
ylidene)methyl]-3-oxocyclobut-1-en-1-olate [29]
.
Fluorescence intensity decay curves with picosecond resolution were obtained
by the single photon timing technique using a Spectra-Physics Vanguard 2000-HM532
mode locked, DPSS green laser, sinchronously pumping Coherent 702 cavity dumped
DCM dye laser, delivering 8 ps (620-700nm) pulses at a repetition rate of 3.8 MHz. The
intensity decay measurements were made by alternate collection of excitation and decay
curves, using an emission polarizer set at the magic angle. The excitation profile was
recorded at the excitation wavelength with a scattering suspension. For the decays, a
cut-off filter was used to remove all excitation light. The emission signal passed through
a depolarizer, a Jobin Yvon HR-320 monochromator with a grating of 100 lines/mm,
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and was detected with a Hamamatsu 2809U-01 microchannel plate photomultiplier
(MCP-PT). The instrument response had effective FWHM of 35 ps. The fluorescence
intensity decay curves were collected at the wavelength of maximum intensity of each
sample and analysed using home-made non-linear least-square reconvolution software
based on the Marquard algorithm. [30] The quality of the fit was evaluated by the
reduced χ2, the weight residuals and the autocorrelation of the residuals.
3.6. Protein assays
Due to the high insolubility of the dyes in water/buffer solution, the solutions for
protein assays were prepared by mixing a DMF solution of the dye in the buffer. To this
solution, the protein stock solution was added and the fluorescence intensity measured
using right angle geometry, 7 nm slits, λex = 630 nm, and integration time 0.5 s
4. Conclusions
Several new dicyanomethylene squaraines that have indolenine, benzothiazole
and benzoselenazole moieties with different substituent groups were successfully
synthesized. The dyes containing benzothiazole and benzoselenazole moieties have high
absorption and emission efficiencies at wavelengths in the red/NIR (665-716 nm), with
extinction coefficients of ca. 105 M-1 cm-1 and fluorescence quantum yields of up to
59% in chloroform. On the other hand, the high insolubility of these squaraines in water
resulted in no detectable absorption and emission in aqueous buffer solution. However,
in the presence of BSA or HSA, the fluorescence intensity of all the squaraine dyes
increases proportionally to the concentration of both BSA and HSA, suggesting a strong
interaction with these proteins. This increase was more significant for the symmetrical
squaraine 7a with both proteins.
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We acknowledge the European Investment Funds by FEDER/COMPETE/POCI
under projects POCI-01-0145-FEDER-007491 (CICS-UBI) and Funds by FCT -
Portuguese Foundation for Science and Technology, under the projects PTDC/CTM-
POL/3698/2014 (IST), RECI/QEQ-QIN/0189/2012 (IST), UID/NAN/50024/2013 (IST)
and Pest-OE/QUI/UI0616/2014 (CQ-VR). Financial support was also received from
FCT and from European Funds (PRODER/COMPETE) under the projects M-ERA-
NET-0004/2015-PAIRED and project UID/Multi/00709/2013 (CICS-UBI) and co-
financed by FEDER, under the Partnership Agreement PT2020.
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Appendix A. Supplementary data
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[3] Welder F, Paul B, Nakazumi H, Yagi S, Colyer CL. Symmetric and asymmetric
squarylium dyes as noncovalent protein labels: a study by fluorimetry and
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0232(03)00367-2.
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