Water-soluble 4-hydroxystyryl and 4-hydroxyphenyl-butadienyls dyes with switchable fluorescence

Article abstract of DOI:10.1016/j.dyepig.2019.107801

Fluorescent 4-hydroxystyryl dyes are useful for many biomedical and pharmaceutical assays, and other analyte sensing applications. However, these dyes often suffer from limited applicability in analytical and bioanalytical utilization due to insufficient water-solubility, high acid-ionization constants (pK_a) and short-wavelength absorption and emission. To solve these issues, a series of new, water-soluble 4-hydroxystyryl and longer-wavelength 4-hydroxyphenyl-butadienyl dyes were synthesized and the spectral and protolytic properties were studied. These new dyes contain electron-withdrawing substituents ortho to the triggering 4-hydroxyl group. The introduction of the cyano and formyl groups was found to decrease the pK_a and extend the pH-sensing region of these fluorophores. In respect of the molecular structure, these dyes exhibit either a “turn-on” activatable fluorescence, or a dual-fluorescence that enables ratiometric measurements. The 4-hydroxyphenyl-butadienyl dye was evaluated for fluorescence monitoring of drug delivery. These new dyes are promising fluorophores for acidity measurements and other sensing applications.

Full text of DOI:10.1016/j.dyepig.2019.107801

DyesandPigments172(2020)107801
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Water-soluble 4-hydroxystyryl and 4-hydroxyphenyl-butadienyls dyes with
switchable fluorescence
Kateryna Bondar, Maksym Bokan, Gary Gellerman, Leonid D. Patsenker⁎
Department of Chemical Sciences, Ariel University, Ariel, 40700, Israel
A R T I C L E I N F O
A B S T R A C T
Keywords:
Fluorescent 4-hydroxystyryl dyes are useful for many biomedical and pharmaceutical assays, and other analyte
sensing applications. However, these dyes often suffer from limited applicability in analytical and bioanalytical
utilization due to insufficient water-solubility, high acid-ionization constants (pKa) and short-wavelength ab-
sorption and emission. To solve these issues, a series of new, water-soluble 4-hydroxystyryl and longer-wave-
length 4-hydroxyphenyl-butadienyl dyes were synthesized and the spectral and protolytic properties were stu-
died. These new dyes contain electron-withdrawing substituents ortho to the triggering 4-hydroxyl group. The
introduction of the cyano and formyl groups was found to decrease the pKa and extend the pH-sensing region of
these fluorophores. In respect of the molecular structure, these dyes exhibit either a “turn-on” activatable
fluorescence, or a dual-fluorescence that enables ratiometric measurements. The 4-hydroxyphenyl-butadienyl
dye was evaluated for fluorescence monitoring of drug delivery. These new dyes are promising fluorophores for
acidity measurements and other sensing applications.
4-Hydroxystyryl
4-Hydroxyphenyl-1,3-butadienyl
Switchable fluorescence
Fluorescence ratiometry
Protolytic properties
Drug delivery monitoring
1. Introduction
group causes a decrease of the absorption band related to the non-
fluorescent “Off” form and an increase of the new, longer-wavelength
Fluorescent dyes sensitive to acidity [1], intracellular pH [2], pre-
sence of ions [3], drugs [4], enzymes [5], small molecules [6], and
high-molecular-weight analytes [7] are widely used in medical diag-
nosis and fluorescence-guided surgery [8], targeted drug delivery
monitoring [9], imaging [10], biomedical and pharmaceutical research
[11], environment monitoring [12], control of chemical processes [13],
and other sensing applications [14]. Many sensing dye molecules
comprise 4-hydroxystyryl fluorophore conjugated with a heterocyclic
terminal end-group in particular a quaternized indolenine moiety [15].
These 4-hydroxystyryl dyes were proposed for detection of fluoride
(F−) [16], sulfite (SO32−), bisulfite (HSO3−), bisulfate (HSO4−) [17],
sulfur dioxide (SO2) [18], hydrogen sulfide [19], thiophenols [20],
hydrazine [21], mercury (Hg2+) [22], and cysteine (Cys) in living cells
[23].
absorption and emission bands originated from the “On” form. In the
more acidic media, when the oxygen atom is protonated, the spectral
properties of 4-hydroxyl derivatives are similar to those for the O-
substituted “Off” forms [24,25]. Such an effect was found in the series
of many other dyes containing a conjugated hydroxylic group [26], e.g.
fluoresceins [4,27], coumarins [28], imidazolinones [29], and 1-hy-
droxyperylene bisimides [30].
The ability of a 4-hydroxyl group for protonation-deprotonation
leads to two main findings. First, the 4-hydroxystyryl dyes are able to
perform as analyte-sensitive probes (sensitive to the trigger group
cleavage) only within a certain pH range, which is above their pKa.
Second, due to the reversible protonation-deprotonation, these dyes can
act as pH indicators.
Obviously, the pH range required for both the analyte sensing and
pH probing applications can be extended by decreasing the dye pKa,
which can supposedly be achieved by introducing the electron-with-
drawing substituents conjugated with the 4-hydroxyl group.
Nevertheless, to the best of our knowledge, 4-hydroxystyryl dyes con-
taining electron-withdrawing groups have not been previously synthe-
sized and investigated as pH indicators or analyte-sensitive probes.
Importantly, many sensing applications in particular biomedical
The operative principle of these 4-hydroxystyryl dyes consists in
cleavage (sometimes addition) of an analyte-sensitive trigger group
attached to the 4-hydroxyl functionality, which results in a pronounced
change in the spectral properties due to the formation of negatively
charged phenolate anion [16,23] (Scheme 1). When the trigger sub-
stituent is a proton, the reversible protonation-deprotonation can des-
ignate the dye as a pH sensor. Analyte-mediated cleavage of the trigger
⁎ Corresponding author.
E-mail addresses: kateryna.bondar@msmail.ariel.ac.il (K. Bondar), maksymbo@ariel.ac.il (M. Bokan), garyg@ariel.ac.il (G. Gellerman),
leonidpa@ariel.ac.il (L.D. Patsenker).
https://doi.org/10.1016/j.dyepig.2019.107801
Received 18 July 2019; Received in revised form 12 August 2019; Accepted 13 August 2019
Availableonline14August2019
0143-7208/©2019ElsevierLtd.Allrightsreserved.

K. Bondar, et al.
DyesandPigments172(2020)107801
Scheme 1. Operative principle of analyte sensitive 4-hydroxystyryl dyes.
and pharmaceutical assays require water-soluble dyes or the dyes with
an adjusted hydrophilic-hydrophobic balance [31]. The improved
water-solubility helps to increase the dye concentration, reduce ag-
gregativity in aqueous solutions, increase the signal-to-noise ratio, and
avoid the non-specific interaction with high-molecular-weight analytes
in biological systems.
1260 Infinity (LC) 6120 quadruple (MS), column Agilent SB-C18,
1.8 mm, 2.1 × 50 mm, column temperature 50 °C, eluent water —
acetonitrile (CH3CN) + 0.1% formic acid.
HPLC purifications were carried out on an ECOM preparative
system, with dual UV detection at 230 nm and 254 nm. A Phenomenex
Gemini® 10 mm RP18 110 Å, LC 250 × 21.2 mm column was used. The
column was kept at 25 °C. Eluent A (0.1% TFA in water) and B (0.1%
TFA in CH3CN) were used. A typical elution was a gradient from 100%
A to 100% B over 35 min at a flow rate of 25 mL/min.
Finally, the dyes emitting within the most transparent red and near-
IR spectral range are beneficial for biomedical sensing applications,
fluorescence imaging, diagnostics, and drug delivery monitoring [32].
Presumably, the absorption and emission bands of 4-hydroxystyryls can
be red-shifted by introducing an additional double bond in the π-con-
jugated system to form 4-hydroxyphenyl-butadienyl dyes. However,
none of these structures have previously been reported.
Chemical reactions were monitored by TLC (Silica gel 60 F-254,
Merck) and by LC/MS. The purities of the dyes were determined by LC/
MS.
Absorption and fluorescence spectra were measured at 25 °C in
1 cm quartz cells at ~1 μM dye concentrations in 0.1 M buffer solutions
of different pH. Absorption spectra were recorded on a Jasco V-730
UV–Vis spectrophotometer and the fluorescence spectra were measured
on an Edinburgh Instruments FS5 spectrofluorometer. The recorded
fluorescence spectra were corrected using the wavelength-dependent
instrument sensitivity coefficients. Absorption and emission maxima
were determined with accuracy of 0.3 nm and 0.5 nm, respec-
tively, and rounded. The absorbances (A) of the samples and the re-
ference at the excitation wavelength (λEx) for the protonated dyes were
in general 0.04–0.08 measured in a 1-cm cell. λEx for the protonated
forms was 405 nm for St1–St4 and 430 nm for St5. λEx for the depro-
tonated forms was 500 nm for St1, St2, St4, St6, 470 nm for St3 and
560 nm for St5.
In this work, we synthesize and investigate a series of novel, water-
soluble of 2-(4-hydroxystyryl)- (n = 1) and 4-hydroxyphenyl-buta-
dienyl (n = 2) indolenine dyes containing electron-withdrawing cyano,
formyl and carboxyl groups ortho to the 4-hydroxyl function (Scheme
2). Dye St1 has previously been reported [18] while St2–St6 are first
synthesized and characterized in this research; the spectral and proto-
lytic properties are studied. In addition, dye St6 is tested as switchable
reporter for drug delivery monitoring.
2. Materials and methods
2.1. General information
Molar absorptivity (ε). The investigated dye (7–10 mg) was dis-
solved in buffer (50 mL), the stock solution was diluted to the dye
concentration cDye ~ 1 μM and the absorbance (A) in the absorption
band maximum was measured in a 5-cm standard quartz cell. The molar
absorptivities were calculated according to the Beer-Lambert law. The
molar absorptivity of each dye was independently measured three times
and the average value was taken. The reproducibility for determining
All chemicals were supplied by Alfa Aesar Israel. Solvents were
purchased from Bio-Lab Israel and used as is. Cell culture medium (CM)
was from Sigma-Aldrich. CM (500 mL) contained a RPMI-1640 standard
cell culture medium (435 mL), esterases containing fetal bovine serum
(50 mL), penicillin (10,000 U/mL, 5 mL), streptomycin (10 mg/mL,
5 mL), glutamine (200 mM, 5 mL), and phenol red indicator (5.3 mg/L).
TLC plates (Silica gel 60 F-254) were from Merck.
the molar absorptivity was within 200 M−1cm−1
.
1H NMR and 13C-NMR spectra were measured on a Bruker
(400 MHz) spectrometer in DMSO‑d6. TMS was used as an internal
standard.
Fluorescence quantum yields (ΦF) of the deprotonated dyes were
measured versus rhodamine B (FF = 71%) in ethanol as the reference
using excitation wavelength λex = 500 nm. The protonated dyes were
measured versus coumarine 153 (FF = 38%) in ethanol (λex = 405 nm
[33]. The absorbances of the samples and the references at the excita-
tion wavelength were 0.04–0.08 measured in a 1-cm cell. The absolute
High-resolution mass spectra (HRMS) were measured using an
Agilent QTOF mass-spectrometer in electrospray ionization (ESI) posi-
tive ion mode.
LC/MS analyses were performed using an Agilent Technologies
Scheme 2. Protonation-deprotonation of 2-(4-hydroxystyryl)- (n = 1) and 4-hydroxyphenyl-butadienyl (n = 2) indolenine dyes and resonance structures of the
deprotonated forms.
2

K. Bondar, et al.
DyesandPigments172(2020)107801
quantum yields were calculated according to Equation (1) [33].
(m, 2H), 4.65 (t, 2H), 2.83 (s, 3H), 2.64 (dd, J = 8.5, 4.5 Hz, 2H),
2.21–2.10 (m, J = 14.4, 7.1 Hz, 2H), 1.53 (s, 6H). MS m/z (El+)
C14H19NO3S+ calculated [M+H]+ 282.7, found: 282.9.
2
ΦF = ΦFRef × (F / FRef) × (ARef / A) × (nD(water) / nD(EtOH)2),
(1)
2-(4-Hydroxystyryl)-3,3-dimethyl-1-(3-sulfopropyl)-3H-in-
where ΦFRef is the quantum yield of the reference, FRef and F are the
areas (integral intensities) of the emission spectra (F = ∫ I(λ)dλ) of the
reference and the dye under examination, ARef and A are the absor-
bencies at the excitation wavelength of the reference and the dye under
examination, and nD(EtOH) and nD(water) are the refractive indices of
ethanol and water.
dolium (St1): To a solution of idolenine Ind (0.36 mmol, 100 mg) in
AcOH (2 mL), the 4-hydroxybenzaldehyde (0.43 mmol, 53 mg) and
AcONa (0.54 mmol, 44 mg) were added and refluxed for 30 min. The
reaction was monitored by TLC. After the reaction was completed, the
solution was diluted with Et2O (50 mL) and cooled to RT. The obtained
solid was filtered, dissolved in CH3CN—H2O (5:3, v.v.) and purified by
HPLC. Yield 33 mg (42%). 1H NMR (400 MHz, DMSO‑d6) δ 8.40 (d,
J = 16.0 Hz, 1H), 8.20 (d, J = 8.7 Hz, 2H), 7.94 (d, J = 7.2 Hz, 1H),
7.83 (dd, J = 7.2, 1.2 Hz, 1H), 7.69 (d, J = 16.0 Hz, 1H), 7.62–7.53 (m,
2H), 6.94 (d, J = 8.8 Hz, 2H), 4.79 (t, J = 8.0 Hz, 2H), 2.65 (t,
J = 6.0 Hz, 2H), 2.20–2.10 (m, 2H), 1.78 (s, 6H). 13C NMR (100 MHz,
DMSO‑d6) δ 174.73, 159.30, 154.79, 140.76, 140.03, 129.98, 129.98,
128.49, 127.62, 125.45, 122.88, 116.37, 116.37, 114.69, 106.17,
The quantum yield of each sample was independently measured 3–4
times and the average value was taken.
Brightness (B) was calculated as the molar absorptivity (ε) multi-
plied by the fluorescence quantum yields (ΦF) expressed in fractions of
a unit.
Acid-ionization constants (pKa) were determined from both the
absorption and emission titration curves using the non-linear fitting
method expressed by Equations (2) and (3) [34].
49.12, 48.51, 41.80, 26.14, 26.14, 23.14. HRMS m/z (El+
)
C
21H24NO4S+ calculated [M+H]+ 386.1426, found 386.1417.
(Amax A)
pKa (Abs) = pH log[
],
(A Amin
)
(2)
2-(3-Formyl-4-hydroxystyryl)-3,3-dimethyl-1-(3-sulfopropyl)-
3H-indolium (St2): Dye QCy7 (50 mg) was dissolved in 25% aq. NaOH
(2 mL) and kept at RT for 30 min. The obtained dark-purple solution
was purified by HPLC to give St2. Yield 15 mg (50%). 1H NMR
(400 MHz, DMSO‑d6) δ 8.26 (d, J = 15.0 Hz, 1H), 8.18 (d, J = 8.8 Hz,
2H), 7.66 (d, J = 7.4 Hz, 2H), 7.45 (t, J = 7.6 Hz, 1H), 6.45 (d,
J = 8.5 Hz, 1H), 4.51 (s, 2H), 2.59 (t, J = 6.4 Hz, 2H), 2.05 (d,
J = 1.4 Hz, 2H), 1.21 (s, 1H). 13C NMR (100 MHz, DMSO‑d6) δ 154.04,
153.94, 153.69, 142.04, 141.48, 128.59, 126.60, 126.25, 125.62,
122.56, 119.75, 112.58, 112.42, 62.78, 49.96, 47.43, 43.22, 29.17,
26.69, 23.74. HRMS m/z (El+) C22H24NO5S+ calculated [M+H]+
414.1375, found 414.1367.
where Amax and Amin are the absorbencies of the fully protonated and
the deprotonated dye forms; A is the absorbance at a certain pH for the
same dye concentration.
(Imax I)
pKa (Fl) = pH log[
],
(I Imin
)
(3)
where Imax and Imin are the fluorescence intensities of the fully proto-
nated and the deprotonated dye; I is the fluorescence intensity at a
certain pH for the same dye concentration.
The pKa of dyes St1–St6 were measured at 1 μM dye concentration
in buffer solutions of different pH. The following buffer solutions were
used: (a) KCl/HCl covering the pH range 1.0–2.0; (b) AcOH/AcONa for
the pH range 3.0–5.0; (c) NaH2PO4/Na2HPO4 for the pH range 6.0–8.0;
(d) NaHCO3/Na2CO3 for the pH range 9.2–10.8, (e) Na2HPO4/NaOH
for the pH range 11.0–11.9, and (f) KCl/NaOH for the pH range
12.0–14.0. All of these buffer solutions were prepared with the same
ionic strength (Ist = 0.1 M). The pH of the buffers was measured with a
glass electrode (HANNA HI-2211 pH-meter) at 25 °C. The stock solu-
tions of the compounds were prepared at 10 mM concentration in
DMSO. Then the appropriate aliquots of the stock solutions were taken
in 1-cm quartz cells equipped with screwed stoppers and diluted with
buffer solution so that the absorbance in the maximum was 0.05–0.08.
Each experiment was carried out in triplicate and the average values
were taken.
5-Formyl-2-hydroxybenzonitrile (4). The synthesis was per-
formed according to Ref. [36]. Yield 196 mg (40%). Purity: 89% (LC-
MS, 254 nm). MS m/z (El−) C8H4NO2− calculated [M-H]- 146.1, found:
145.8.
2-(3-Cyano-4-hydroxystyryl)-3,3-dimethyl-1-(3-sulfopropyl)-
3H-indolium (St3): The synthesis was performed according to Ref.
[36]. Yield 63 mg (42%). 1H NMR (400 MHz, DMSO‑d6) δ 8.58 (d,
J = 2.1 Hz, 4H), 8.51 (dd, J = 8.9, 2.2 Hz, 4H), 8.38 (d, J = 16.3 Hz,
4H), 7.99 (dd, J = 6.1, 2.7 Hz, 4H), 7.88–7.81 (m, 4H), 7.77 (d,
J = 16.3 Hz, 4H), 7.66–7.55 (m, 8H), 7.15 (d, J = 8.9 Hz, 4H), 5.72 (s,
1H), 4.90–4.75 (m, 9H), 2.73–2.63 (m, 9H), 2.23–2.10 (m, 8H), 1.77 (s,
25H), 1.22 (s, 4H). 13C NMR (100 MHz, DMSO‑d6) δ 152.00, 137.53,
136.34, 129.13, 128.97, 122.85, 116.93, 114.91, 111.15, 97.23, 46.97,
45.25, 39.92, 39.71, 39.50, 39.29, 39.08, 25.45, 24.37. HRMS m/z
(El+) C22H23N2O4S+ calculated [M+H]+ 411.1379, found 411.1377.
2-(3-Carboxy-4-hydroxystyryl)-3,3-dimethyl-1-(3-sulfopropyl)-
3H-indolium (St4): The solution of St3 (0.08 mmol, 34 mg) in 48% aq.
HBr (5 mL) was refluxed overnight and evaporated under low pressure.
The resulted solid was filtered, dissolved in CH3CN—H2O (5:3, v.v.)
and purified by HPLC. Yield 15 mg (42%). 1H NMR (400 MHz, DMSO) δ
8.68 (d, J = 2.0 Hz, 1H), 8.65 (dd, J = 4.0, 2.2 Hz, 1H), 8.52 (d,
J = 16.4 Hz, 1H), 8.02–7.97 (m, 1H), 7.87–7.84 (m, 1H), 7.78 (d,
J = 16.4 Hz, 1H), 7.64–7.56 (m, 2H), 7.13 (d, J = 8.7 Hz, 1H), 4.84 (t,
J = 8.0 Hz, 2H), 2.65 (d, J = 6.4 Hz, 2H), 2.21–2.12 (m, 2H), 1.80 (s,
6H). 13C NMR (100 MHz, DMSO‑d6) δ 181.79, 171.08, 165.32, 158.40,
158.02, 153.53, 143.76, 140.79, 136.94, 135.45, 129.04, 126.24,
122.94, 118.67, 114.95, 114.15, 113.88, 110.74, 52.01, 47.16, 45.27,
25.61, 24.58. HRMS m/z (El+) C22H24NO6S+ calculated [M+H]+
430.1324, found 430.1327.
pH sensing range was estimated as a pH region, where the ab-
sorption and fluorescence intensities change between 0.1 and 0.9 of the
normalized value [35].
Chemical stability and drug release rates were measured spectro-
photometrically and spectrofluorimetrically. For the chemical stability
measurements, the samples were incubated in PB pH 7.4 at 37 °C. For
the measurements of the drug release rates, the samples were incubated
in PB pH 7.4 and CM pH 7.4 at 37 °C. All the spectral measurements
were carried out at 25 °C. The absorption and fluorescence spectra for
both chemical stability and drug release experiments were measured at
~1 μM compound concentration in certain time period and the corre-
sponding graphs of the absorption and emission intensities vs. time
were obtained. The half-lives (τ1/2) were calculated from the obtained
exponential curves. Each experiment was carried out in triplicate and
the average values were taken.
4-Methoxybenzaldehyde (7): The synthesis was performed ac-
cording to Ref. [37]. Yield 2.15 g (63%). MS m/z (El+) C8H8O2 cal-
+
2.2. Synthesis
culated [M+H]+ 137.7, found: 137.1.
3-(2,3,3-Trimethyl-3H-1-indoliumyl)-1-propanesulfonate
(2)
3-(4-Methoxyphenyl)acrylaldehyde (9): The synthesis was car-
was synthesized according to Ref. [18]. Yield 5.4 g (89%). 1H NMR
ried out according to Ref. [38]. Yield 90 mg (15%). MS m/z (El+
)
10H10O2 calculated [M+H]+ 162.2, found: 162.1.
+
(400 MHz, DMSO‑d6, ppm): δ 8.05 (dd, J = 5.6, 3.2 Hz, 1H), 7.65–7.54
C
3

K. Bondar, et al.
DyesandPigments172(2020)107801
4-(4-Methoxyphenyl)buta-1,3-dienyl)-3,3-dimethyl-1-(3-sulfo-
propyl)-3H-indolium (St5-Me): Indolenine 2 (5 mmol, 0.145 g) was
dissolved in Ac2O (1.5 mL) and 4-methoxyphenylacrylaldehyde
49.12, 48.51, 41.80, 26.14, 26.14, 23.14. HRMS m/z (El+) C25H27N2O4S+
calculated [M-H]- 451.1692, found: 451.1694.
9
4-(3-Cyano-4-hydroxyphenyl)buta-1,3-dienyl)-1-(3-(mercapto-
trioxidanyl)propyl)-3,3-dimethyl-3H-indolium (St6): To a solution
of St2-Me (0.067 mmol, 30 mg) in dichloromethane (2 mL) was care-
(6 mmol, 0.1 g) was added. The reaction mixture was brought to the
reflux. Then, AcONa (7.5 mmol, 0.62 g) was added to the boiled mix-
ture was refluxed for additional 30 min. The reaction was monitored by
TLC. After the reaction was completed, the solution was diluted with
Et2O (25 mL) and cooled to RT. The obtained solid was filtered, dis-
solved in CH3CN—H2O (1:1, v.v.) and purified by HPLC. Yield 22 mg
(22%). 1H NMR (400 MHz, MeOH-d4) δ 8.34 (dd, J = 15.1, 10.9 Hz,
1H), 7.87 (d, J = 7.2 Hz, 1H), 7.76–7.70 (m, 3H), 7.67–7.55 (m, 3H),
7.42 (dd, J = 15.1, 10.9 Hz, 1H), 7.26 (d, J = 15.1 Hz, 1H), 7.03 (d,
J = 8.8 Hz, 2H), 4.73 (ABq, 2H), 3.88 (s, 3H), 3.04 (t, J = 6.5 Hz, 2H),
2.40–2.30 (m, 2H), 1.81 (s, 6H). 13C NMR (100 MHz, MeOH-d4) δ
175.69, 159.94, 154.08, 140.76, 140.03, 136.92, 129.44, 128.75,
128.75, 128.49, 127.63, 125.45, 122.88, 114.69, 114.58, 114.58,
108.61, 56.04, 49.12, 48.51, 41.80, 26.14, 26.14, 23.14. HRMS m/z
(El+) C24H28NO4S+ calculated [M+H]+ 427.1739, found: 426.1730.
4-(4-Hydroxyphenyl)buta-1,3-dienyl)-3,3-dimethyl-1-(3-sulfo-
propyl)-3H-indolium (St5): To a solution of St5-Me (0.122 mmol,
52 mg) in dichloromethane (5 mL) a solution of boron tribromide
(3 mmol, 300 μl) in dichloromethane (2 mL) was carefully added during
2 min. The reaction mixture was stirred at room temperature for
30 min, solvent evaporated and the residue purified by HPLC. Yield
45 mg (85%). 1H NMR (400 MHz, MeOH-d4) δ 8.92 (s, 1H), 8.33 (dd,
J = 15.0, 11.0 Hz, 1H), 7.84 (d, J = 7.3 Hz, 1H), 7.71 (d, 1H), 7.65 (d,
J = 8.6 Hz, 2H), 7.61–7.54 (m, 3H), 7.38 (dd, J = 15.1, 11.0 Hz, 1H),
7.22 (d, 1H), 6.88 (d, J = 8.7 Hz, 1H), 4.71 (ABq, 2H), 3.03 (t,
J = 6.5 Hz, 2H), 2.39–2.29 (m, 2H), 1.80 (s, 6H). 13C NMR (100 MHz,
MeOH-d4) δ 175.69, 158.47, 154.08, 140.76, 140.03, 136.92, 129.38,
129.38, 128.49, 128.39, 127.63, 125.45, 122.88, 116.38, 116.38,
114.69, 108.61, 49.12, 48.51, 41.80, 26.14, 26.14, 23.14. HRMS m/z
(El+) C23H26NO4S+ calculated [M+H]+ 412.1583, found 412.1581.
5-Formyl-2-methoxybenzonitrile (4): The synthesis was per-
fully added
a solution of boron tribromide in dichloromethane
(1.2 mmol, 1.2 mL) during 2 min. The reaction mixture was stirred at
room temperature for 30 min, solvent evaporated and the residue
purified by HPLC. Yield 24 mg (80%). 1H NMR (400 MHz, DMSO‑d6) δ
11.96 (s, 1H), 8.34 (dd, J = 15.2, 10.7 Hz, 1H), 7.98 (dd, J = 8.3,
4.7 Hz, 2H), 7.88–7.82 (m, 2H), 7.61 (ddd, J = 17.5, 13.4, 11.3 Hz,
3H), 7.42–7.30 (m, 2H), 7.13 (d, J = 8.8 Hz, 1H), 4.74–4.59 (m, 2H),
2.63 (t, J = 6.8 Hz, 2H), 2.14 (dd, J = 14.6, 6.9 Hz, 2H), 1.74 (s, 6H).
13C NMR (100 MHz, DMSO‑d6) δ 175.69, 161.81, 154.08, 140.76,
140.03, 137.09, 134.65, 132.71, 131.31, 128.64, 128.49, 125.45,
122.88, 115.38, 114.69, 114.69, 108.61, 94.20, 49.12, 48.51, 41.80,
26.14, 26.14, 23.14. HRMS m/z (El+) C24H25N2O4S+ calculated [M-
H]+ 437.1535, found: 437.1537.
2-(4-(4-(4-(4-(bis(2-chloroethyl)amino)phenyl)butanoyloxy)-3-
cyanophenyl)buta-1,3-dienyl)-3,3-dimethyl-1-(3-sulfopropyl)-3H-
indolium (St6-CLB): Chlorambucil (0.024 mmol, 7.3 mg) and DCC
(0.048 mmol, 10 mg) were stirred in NMP at RT for 10 min. Then St6
(0.012 mmol, 5 mg) and pyridine (2 μL) were sequentially added and
the reaction mixture was stirred for additional 1.5 h at RT. The reaction
was monitored by TLC. After completion of the reaction, the solution
was diluted with Et2O (25 mL), the obtained solid was filtered, dis-
solved in CH3CN—H2O (1:1, v.v.) and purified by HPLC. Yield 2 mg
(23%). 1H NMR (400 MHz, DMSO‑d6)
δ 8.43 (s, 4H), 7.57 (d,
J = 7.5 Hz, 4H), 7.48 (d, J = 7.2 Hz, 4H), 7.33 (d, J = 8.8 Hz, 4H), 7.17
(d, J = 7.5 Hz, 4H), 6.95–6.87 (m, 12H), 6.77 (d, J = 15.3 Hz, 4H),
6.67–6.55 (m, 12H), 4.51 (s, 8H), 3.70 (s, 16H), 3.55 (s, 16H), 3.16 (s,
8H), 2.63 (s, 8H), 2.53 (s, 8H), 2.29 (s, 3H), 2.25 (s, 3H), 1.90 (s, 7H),
1.44 (s, 24H). 13C NMR (100 MHz, DMSO‑d6) δ 175.69, 173.14, 156.49,
154.08, 146.56, 140.76, 140.03, 137.09, 135.02, 134.06, 132.73,
130.27, 130.27, 129.43, 128.64, 128.49, 125.45, 122.88, 120.53,
115.94, 115.22, 115.22, 114.69, 108.61, 102.71, 50.96, 50.96, 49.12,
48.51, 41.88, 41.88, 41.80, 35.85, 33.39, 26.14, 26.14, 24.39, 23.14.
HRMS m/z (El+) C38H42Cl2N3O5S+ calculated [M+H]+ 722.2222,
found: 723.2297.
formed according to Ref. [37]. Yield 12 mg (24%). MS m/z (El−
)
−
C9H7NO2 calculated [M-H]– 160.8, found: 160.1.
2-Methoxy-5-(3-oxoprop-1-enyl)benzonitrile (10): The synthesis
was carried out according to Ref. [38]. Yield 75 mg (30%). MS m/z
+
(El+) C11H9NO2 calculated [M-H]+ 187.2, found: 187.1.
4-(3-Cyano-4-methoxyphenyl)buta-1,3-dienyl)-1-(3-(mercapto-
trioxidanyl)propyl)-3,3-dimethyl-3H-indolium (St6-Me): Indolenine 2
(1.3 mmol, 0.370 g) was dissolved in AcOH (3 mL) and after addition of
benzonitrile 10 (6 mmol, 0.1 g) the reaction mixture was brought to the
reflux. Then, AcONa (7.5 mmol, 0.62 g) was added to the boiled mixture
and refluxed for additional 30 min. The reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was diluted with
Et2O (25 mL) and cooled to RT. The obtained precipitate was filtered,
dissolved in CH3CN—H2O (1:1, v.v.) and purified by HPLC. Yield
0.187 mg (21%). 1H NMR (400 MHz, DMSO‑d6) δ 8.35 (d, J = 15.1 Hz,
1H), 8.09 (s, 1H), 8.00 (s, 2H), 7.85 (s, 1H), 7.70 (s, 1H), 7.68 (s, 1H), 7.61
(s, 2H), 7.40 (d, J = 9.1 Hz, 1H), 4.67 (s, 2H), 4.00 (s, 4H), 2.64 (s, 2H),
2.14 (s, 2H), 1.74 (s, 5H). 13C NMR (100 MHz, DMSO‑d6) δ 175.69,
162.56, 154.08, 140.76, 140.03, 137.09, 134.05, 133.43, 131.04, 128.64,
128.49, 125.45, 122.88, 115.94, 114.69, 111.30, 108.61, 97.34, 56.79,
3. Results and discussion
3.1. Synthesis
Styryl dye St1 was previously reported in Ref. [18]. We synthesized
this dye in 42% yield by the condensation of 4-hydroxybenzaldehyde
(1) with the quaternized indolenine 2 (Scheme 3). The last one was
obtained by the quaternization of indolenine 3 with 1,3-propane sul-
tone.
The new dye St2 was synthesized by alkaline hydrolysis of cyanine
QCy7 (Scheme 4) similar to the method described in Ref. [39]. The
known procedure was improved by using 25% aq. NaOH instead of
100 mM phosphate buffer (pH 7.4), which allowed decreasing the re-
action time from 10 days to 30 min.
Scheme 3. Synthesis of dye St1.
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K. Bondar, et al.
DyesandPigments172(2020)107801
Scheme 4. Synthesis of 3-formyl-styryl dye St2.
Scheme 5. Synthesis of 3-cyano and 3-carboxy styryls St3 and St4.
Scheme 6. Synthesis of 4-hydroxyphenyl-butadienyl dyes St5-Me, St5, St6-Me, and St6.
O
Cl
Cl
OH
CN
O
CN
N
O
Cl
O
N
CLB
N
DCC, Pyridine / NMP
RT, 1.5 h
HO3S-(H2C)3
N
Cl
St6-CLB (23%)
O3S-(H2C)3
St6
Scheme 7. Synthesis of St6–CLB conjugate.
Attempts to synthesize the nitrile dye St3 starting from the for-
mylated dye St2 by using the procedure [36] have failed since St2
decomposed to indolenine 2 (37% yield) and a mixture of other, non-
identified products (Scheme 5). Therefore, dye St3 was synthesized by
condensation of indolenine 2 with 3-cyano-4-hydroxybenzaldehyde (4).
The last one was obtained by a reaction of dialdehyde 5 with hydro-
xylamine in DMSO. Aldehyde 4 was reacted with 2 in acetic anhydride
to form St3 in 63% yield (Scheme 5). The carboxylated dye St4 was
obtained in 62% yield by hydrolysis of cyano group in St3 under reflux
with 48% aq. HBr (Scheme 4).
Dyes St5 and St6 were synthesized starting from 4-hydro-
xybenzaldehydes 1 and 4 as shown in Scheme 6. By using methyl iodide
[37], a hydroxyl group in 1 and 4 was protected with a methyl to form
4-methoxybenzaldehydes 7 and 8 that were reacted with acetaldehyde
5

K. Bondar, et al.
DyesandPigments172(2020)107801
to give cinnamaldehydes 9 and 10, correspondingly. The last ones were
condensed with indolenine 2 to the methoxylated dyes St5-Me and St6-
Me, which were further demethylated with BBr3 to form the aimed
hydroxylic dyes St5 and St6, respectively, in good yields.
To examine the applicability of dye St6 as an “Off-On” switchable
reporter in drug delivery monitoring, this dye was bound to the antic-
ancer drug chlorambucil (CLB) acting as DNA alkylator [40]. Thus, CLB
was pre-activated with DCC and reacted with St6 to give the St6–CLB
conjugate in 23% yield. The reaction was carried out in mild conditions
at RT (NMP with a catalytic amount of pyridine) (Scheme 7). As a re-
sult, the drug was bound to the dye via a hydrolytically cleavable ester
bond, which is commonly used in the drug and prodrug delivery in
biological systems [41,42].
3.2. Spectral properties
Spectral properties of dyes St1–St6 were measured in 0.1 M buffer
solutions of different acidity. The absorption and emission maxima
(λmaxAb, λmaxFl), spectral half-widths (ω1/2Abs, ω1/2Fl), Stokes shifts
(ΔνSt), molar absorptivities (ε), fluorescence quantum yield (ΦF), and
brightness (B) of the protonated (OH) and deprotonated (O−) forms of
the dyes are summarized in Table 1.
Due to the presence of the sulfo group, all the investigated dyes
St1–St6 are well soluble in water and aqueous buffer solutions, which is
important for their practical applications. Thus, the absorption spectra
of these dyes do not exhibit any signs of aggregation: there is no ad-
ditional, aggregation band and the Beer–Lambert law is valid in the
investigated concentration range at least up to 10−5 M.
Dyes St1–St4 absorb in the blue-green and emit in the green-yellow
spectral region while the spectral bands of St5 and St6 are noticeably
red-shifted by about 33–74 nm for absorption (ΔλAb) and 83–107 nm
for emission (ΔλFl). As a result, St5 and St6 absorb in the green-yellow
and emit in the red region, which is more attractive for probing in
biological samples.
The introduction of the electron-withdrawing formyl (St2) and
cyano (St3, St6) groups in the ortho position to the hydroxyl func-
tionality (St1, St5) causes a blue-shift of the absorption maximum for
both protonated and deprotonated forms while a slight red-shift is ob-
served upon the introduction of the carboxylic group (St4). The dif-
ferent behavior of the carboxylic group is connected more likely with
the formation of an intramolecular H-bond with the 4-hydroxyl group;
the H-bonding with the water molecules might also be responsible for
this effect [43].
The molar absorptivities of the deprotonated forms of St1–St6 are
1.5–2.1-fold higher compared to those for the protonated forms while
for St5 and St6 this increase is less pronounced, 1.1–1.3 time. The
protonated forms of St2, St3 and St6 (R = CHO, CN) show no detect-
able fluorescence. At the same time, both protonated and deprotonated
forms of St1, St4 and St5 (R = H, COOH) are fluorescent and the
fluorescence quantum yields of the deprotonated dyes are 1.2–2.0 fold
higher compared to the protonated forms.
In general, the fluorescence quantum yields of previously reported
4-hydroxystyryl dyes measured in polar solvents are poor, which is
typical for any donor-acceptor systems. Nevertheless, these dyes were
proposed for many practical applications due to their extreme sensi-
tivity to analytes [16,18,23]. The quantum yield of St1 is in agreement
with recently reported data on the styryl dyes of similar structure [44].
Importantly, the introduction of the electron-withdrawing formyl,
cyano and carboxyl groups noticeably increases the quantum yield of
the deprotonated forms by factor of 2.1–3.6 (Table 1). In addition, the
extension of π-conjugated system (n = 2 versus n = 1) also increases the
quantum yield of St5 compared to St1 by factor of 4.9 and by factor of
5.3 for St6 versus St3. As a result, the quantum yield of the deproto-
nated form of St6 is about 17.5 fold higher compared to St1. For pro-
tonated form of St1 the quantum yield also increases upon the in-
troduction of the carboxylic group (dye St4) but this increase is not
6

K. Bondar, et al.
DyesandPigments172(2020)107801
Fig. 1. pH-Dependent absorption (a, c, e, g, i, k) and fluorescence (b, d, f, h, j, l) spectra of St1 (a, b), St2 (c, d), St3 (e, f), St4 (g, h), St5 (i, j), and St6 (k, l).
λ
Ex(protonated forms): 405 nm for St1–St4; 430 nm for St5. λEx(deprotonated forms): 500 nm for St1, St2, St4, and St6; 470 nm for St3 and 560 nm for St5.
7

K. Bondar, et al.
DyesandPigments172(2020)107801
Fig. 2. Titration curves for St1–St4 (a, b), St5 and St6 (c, d) in the protonated (dashed line) and deprotonated (solid line) forms measured spectrophotometically (a,
c) and spectrofluorimetrically (b, d).
Scheme 8. Hydrolytic cleavage of CLB in the non-
fluorescent St6–CLB conjugate to form fluorescent
dye St6 (deprotonated form).
pronounced (1.9 fold).
protonated and deprotonated forms were recorded under the excitation
in the corresponding absorption bands. Based on the obtained spec-
trophotometric and spectrofluorimetric titration curves, the acid-ioni-
zation constants (pKa) were calculated. The pKa values found from the
emission titration curves are very similar to those obtained spectro-
photometrically (the difference is only 0.03–0.17 pKa units), which
evidences that no pronounced structural changes occur with the dye
molecules in the excited state. In respect to the substituent R, the pKa
values change in a wide range between ~4.6 and ~9.9. The pKa in-
crease in the order St3 (n = 1, R = CN) < St6 (n = 2, R = CN) < St2
(n = 1, R = CHO) < St1 (n = 1, R = H) < St5 (n = 2, R = CN) <
St4 (n = 1, R = COOH) (Table 1). The introduction of the cyano group
decreases the acid-ionization constant by about 2.4 pKa units while the
extension of the π-conjugated chain (n = 2 vs. n = 1) slightly increases
it by ~0.9 pKa units. Although the carboxyl group is an electron-
withdrawing substituent, it increases pKa due to the intramolecular H-
bond formation. Such effect was discussed in Ref. [49] for salicylic acid
derivatives.
The deprotonated forms of St1–St6 emit with relatively small Stokes
shifts (ΔνSt ~ 850–3340 cm−1) while for the protonated forms of St1,
St4 and St5 ΔνSt noticeably increase by 5.3, 8.4 and 3.6 fold, respec-
tively. The Stokes shifts for 4-hydroxyphenyl-butadienyl dyes St5 and
St6 are a bit higher compared to 4-hydroxystyryls St1 and St3, which is
more likely due to the longer polymethine chain and increased con-
formational lability. The half-widths (ω1/2) of the absorption and
emission bands of the deprotonated forms are always narrower than
those for the protonated dyes.
In summary, the spectral properties of the deprotonated 4-hydro-
xystyryl and 4-hydroxyphenyl-butadienyl dyes containing the zwitter-
ionic fluorophore system, as compared to the positively charged pro-
tonated forms (Scheme 2), are very similar to those for the positively
charged cyanines and norcyanines and zwitterionic squaraines and
norsquaraines [45–47]. These deprotonated forms are brighter (have
higher ΦF and ε), exhibit shorter Stokes shifts, and their absorption and
emission spectral bands are red-shifted and narrower.
In addition, because the absorption bands of the dyes St1, St4 and
St5 lie in the substantially different spectral regions and the emission
maxima are also noticeably different, these dyes are potentially suitable
for the ratiometric fluorescence sensing measurements, that have cer-
tain advantages in particular for quantitative measurements in biolo-
gical systems [48].
Among all the investigated dyes, St6 seems most advantageous for
biomedical sensing applications. This is due to the deprotonated form of
this dye exhibits more pronounced brightness as compared to other
dyes, longer absorption and emission maxima compared to St1–St4 and
wider pH range (lower pKa) compared to the long-wavelength dye St5.
We also found that St6 has a reasonable chemical stability in PB pH
7.4 at 37 °C (Fig. S1). Therefore, we decided to evaluate St6 as the
switchable fluorescent reporter for the detection of drug release events.
3.3. Protolytic properties
The absorption and emission spectra of dyes St1–St6 versus pH were
measured in aqueous buffer solutions (Fig. 1) and the corresponding
titration curves were obtained (Fig. 2). The emission spectra of the
3.4. Monitoring of drug release
The applicability of dye St6 for monitoring of drug release was
8

K. Bondar, et al.
DyesandPigments172(2020)107801
Fig. 3. Time dependent absorption (a, c) and fluorescence (b, d) spectra and the corresponding cleavage profiles (e, f) of St6-CLB measured in PB (5 μM) pH 7.4 (a, c,
e) and CM (5 μM) pH 7.4 (b, d, f) at 25 °C (incubation at 37 °C).
tested in the example of anticancer drug chlorambucil (CLB). In a hy-
drolytic environment, the ester bond in the St6–CLB conjugate was
prone to cleavage releasing the drug molecule (Scheme 8). We in-
vestigated the spectral properties of St6–CLB and the CLB cleavage rate
in physiological conditions (37 °C), in PB pH 7.4 and esterase con-
taining cell culture medium pH 7.4 (CM).
emission spectra measured at the St6-CLB concentration ~1 μM and the
corresponding cleavage profiles are shown in Fig. 3. It can be seen that
in both media the absorption band at ~415 nm decreases over time and
a new, longer-wavelength band at ~536 nm increases (Fig. 3a and b).
Simultaneously, the fluorescence intensity dramatically increases sig-
naling the CLB release (Fig. 3c and d). Due to the presence of esterases,
hydrolytic cleavage in CM occurs about 45 times faster compared to PB.
The CLB release half-life (τ1/2) in CM is about 4 min while in PB it is τ1/
2–3 h (Fig. 3e and f). Because the cleavage in CM runs extremely fast,
even the initial absorption and emission spectra of St6–CLB in CM show
the presence of the free St6 formed upon the CLB release.
The free St6 is a pH-sensitive dye, which exists at pH 7.4 in the
fluorescent deprotonated form (Fig. 3). The absorption (~536 nm) and
emission (~653 nm) maxima in both PB and CM media are almost the
same. The fluorescence intensity of St6 in the protein containing CM is
about 1.5 times higher compared to PB. Supposedly, this is due to the
highly polar dye molecules in CM move from a polar aqueous media
into a less polar and more viscous protein phase resulting in the de-
crease of non-radiative relaxation and the increase of the fluorescence
quantum yield.
3.5. Chemical stability
The stability of dyes plays an important role for their practical use.
The stability of St6 was investigated upon incubation in PB pH 7.4 at
37 °C. The absorption and emission spectra were measured at 25 °C over
time (Figs. S1,a,b). The spectral bands related to the protonated form
were found to decrease very slowly, by ~20% during 7 days (Figs.
S1,c,d), which indicates that St6 is sufficiently stable for conventional
analytical and bioanalytical applications.
In contrast to the free St6, the CLB conjugated St6 in both solvent
systems behaves similar to the protonated St6: it absorbs at ~415 nm (a
~30-nm blue-shift compared to the protonated St6) and exhibits no
detectible fluorescence.
The CLB cleavage was investigated using spectrophotometric and
spectrofluorimetric methods. The time dependent absorption and
9

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DyesandPigments172(2020)107801
4. Conclusions
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St3 (R = CN), and St4 (R = COOH) and 3-R-4-hydroxyphenyl-buta-
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Dyes St2, St3 and St6 demonstrate “Off-On” activatable fluores-
cence; their brightness is higher compared to St1, St4 and St5 and the
pKa are lower, which makes these dyes beneficial for sensing applica-
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and St5 are fluorescent and therefore these dyes are potentially suitable
for the dual-wavelength ratiometric fluorescence measurements. The
new dyes can be used as acidity indicators within the physiological pH
range. Due to its activatable long-wavelength fluorescence, low pKa,
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Acknowledgements
This research was supported by the Israel Scientific Foundation
(ISF), project 810/18. Leonid Patsenker is thankful to the Center for
Absorption in Science of the Ministry of Immigrant Absorption of Israel
for the financial support under the KAMEA program. Gary Gellerman is
thankful to the Gale Foundation for the generous donation to support
this research.
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methylene-4H-pyran as a near infrared fluorescent sensor for lysozyme in urine
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2019.01.001.
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Appendix A. Supplementary data
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Lavis LD, Perrais D. Semisynthetic fluorescent pH sensors for imaging exocytosis
and endocytosis. Nat Commun 2017;8(1):1412. https://doi.org/10.1038/s41467-
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Supplementary data to this article can be found online at https://
doi.org/10.1016/j.dyepig.2019.107801.
[28] Yahaya I, Chemchem M, Aydıner B, Seferoğlu N, Erva Tepe F, Açık L, Seferoğlu Z.
Novel fluorescent coumarin-thiophene-derived schiff bases: synthesis, effects of
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