Dyes and Pigments 192 (2021) 109405

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Dyes and Pigments

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On the chemical reactivity of tricyanofuran(TCF)-based near-infrared

fluorescent redox probes – Effects of glutathione on the probe response and
product fluorescence
Przemysław Siarkiewicz a, Radosław Michalski b, Adam Sikora b, Renata Smulik-Izydorczyk b,
Marcin Szala a, Aleksandra Grzelakowska a, Julia Modrzejewska a, Asha Bailey c, Jacek E. Nycz e,
Balaraman Kalyanaraman c, d, Jan Grzegorz Malecki e, Jacek Zielonka c, d, **,
Radosław Podsiadły a, *
a
Institute of Polymer and Dye Technology, Faculty of Chemistry, Lodz University of Technology, Stefanowskiego 12/16, 90-924, Lodz, Poland
b
Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924, Lodz, Poland
c
Department of Biophysics, Poland
d
Cancer Center Redox & Bioenergetics Shared Resource, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, United States
e
Institute of Chemistry, University of Silesia in Katowice, Ul. Szkolna 9, Katowice, 40-007, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Recent research towards the development of the redox probes for in vivo applications focuses on near-infrared
Redox probes fluorescent sensors and tricyanofuran-based fluorophores gained popularity thanks to their favorable spectral
Tricyanofuran-based probes properties. The tricyanofuran-based boronate probe (TCF-BA) has been proposed for specific fluorescent
Fluorescence
detection of selected biological oxidants in vitro and in vivo. Here, we report the detailed chemical reactivity of
Peroxynitrite
Thiol adducts
TCF-BA toward hydrogen peroxide, hypochlorite, and peroxynitrite in the presence and absence of glutathione, a
major small molecule biothiol present intracellularly at millimolar concentrations. We demonstrate that, at the
physiologically relevant concentration of glutathione, the TCF-BA probe forms an adduct, resulting in decreased
reactivity of the probe toward the oxidants tested. Only peroxynitrite efficiently oxidizes TCF-BA in the presence
of GSH. Furthermore, the fluorescent phenolic oxidation product, TCF-OH, also reacts with glutathione, which
results in a decreased fluorescence intensity. This observation suggests that the results reported with TCF-based
probes may be affected by the changes in intracellular glutathione, in addition to the desired analyte. We also
report a modified probe (TCF-BA-2) derived from 1-naphthalene boronic acid, which has similar reactivity to­
ward peroxynitrite. Although the TCF-BA-2 probe also reacts with glutathione, the absorption spectrum of its
oxidation product, TCF–OH–2, is not influenced by glutathione and, therefore, can be applied for real-time
monitoring of peroxynitrite formation in biological systems.

1. Introduction autofluorescence and cell photodamage [1,2], long wavelength red or

near infrared (NIR) fluorogenic chemical probes are desired for in vivo
Fluorescence microscopy and imaging are indispensable in studies of applications. In the last decade, numerous probes containing the tri­
cell function and intracellular analytes in vitro and in vivo. To provide cyanofuran (TCF) ring (see Scheme 2) have been synthesized and
deeper tissue light penetration and decrease background characterized [3–15] with the aim of detecting a specific analyte in vivo.

* Corresponding author.
** Corresponding author. Institute of Polymer and Dye Technology, Faculty of Chemistry, Lodz University of Technology, Stefanowskiego 12/16, 90-924, Lodz,
Poland.
E-mail addresses: przemyslaw.siarkiewicz@dokt.p.lodz.pl (P. Siarkiewicz), radoslaw.michalski@p.lodz.pl (R. Michalski), adam.sikora@p.lodz.pl (A. Sikora),
renata.smulik-izydorczyk@p.lodz.pl (R. Smulik-Izydorczyk), marcin.szala@p.lodz.pl (M. Szala), aleksandra.grzelakowska@p.lodz.pl (A. Grzelakowska), julia.
modrzejewska@dokt.p.lodz.pl (J. Modrzejewska), baileyaa1211@gmail.com (A. Bailey), jacek.nycz@us.edu.pl (J.E. Nycz), balarama@mcw.edu
(B. Kalyanaraman), gmalecki@us.edu.pl (J.G. Malecki), jzielonk@mcw.edu (J. Zielonka), radoslaw.podsiadly@p.lodz.pl (R. Podsiadły).

https://doi.org/10.1016/j.dyepig.2021.109405
Received 28 January 2021; Received in revised form 7 April 2021; Accepted 18 April 2021
Available online 1 May 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.
P. Siarkiewicz et al.
The p-amino [6–8] or several p-hydroxyl [8–15] derivatives have been
applied as fluorescent sensors for a range of analytes in cells, including
carbon monoxide (CO) [16], nitroxyl (HNO) [12], selected oxidants
[3–5,17,18], hydrazine [19], albumin [6,20], biothiols [11,13,15,21,
22], F− [23], K+ [7], and several metal ions: Cu2+ [10,24], Hg2+ [25]
and palladium (Pd0) [26]. Also, TCF-based probes have been designed to
monitor cellular pH [9] and enzymatic activity of NADPH-quinone
oxidoreductase (NQO1) [14], alkaline phosphatase [27], and carbox­
ylesterase 2 [28].
The p-phenyl-boronic acid pinacol ester linked via a double bound to
the TCF ring (TCF-BE, Scheme 2) has been proposed for detection of
selected reactive oxygen species (ROS), including H2O2 [4,17], HOCl [5]
or ONOO− [3], via oxidation to the TCF-OH red fluorescent product.
However, none of mentioned works have reported competing for
oxidation of TCF-BE between ROS. Interestingly, ROS are generated
upon one- or two-electron reduction of molecular oxygen to superoxide
radical anion (O•− 2 ) and hydrogen peroxide (H2O2), respectively. Sub­
sequent reactions of those species may result in the formation of, e.g.,
peroxynitrite (ONOO− ) [29], hydroxyl radical (•OH), or hypochlorous
acid (HOCl). At physiological conditions, intracellular ROS levels are
regulated by antioxidant enzymes and non-enzymatic antioxidants, such
as superoxide dismutases (SOD), peroxiredoxins (Prx), glutathione
peroxidases, catalase (CAT), glutathione (GSH), or ascorbate. The
increased production of ROS or the defective defense mechanisms leads
to oxidative stress, which is widely believed to be cytotoxic. However,
O•−
2 and H2O2 generated at low levels may be involved in
redox-signaling mechanisms, e.g., promoting cancer cell proliferation
[30–34]. It was shown that the occurrence, growth, and metastasis of
tumors, and even the apoptosis, necrosis, and autophagy of tumor cells,
are all closely related to ROS production [35]. ROS also have an impact
on the tumor microenvironment. At moderate concentrations, ROS
activate cancer cell survival signaling and immune evasion, whereas at
high concentrations, ROS can cause cancer cell apoptosis [36].
Recently, we reported that tumor growth is accompanied by
increased ROS formation and revealed differences in oxidant formation
in the inner and outer sections of tumor tissue, respectively, demon­
strating tumor redox heterogeneity [37]. Several factors—such as the
type and maturity of the tumor, the biological and metabolic environ­
ment, and the location, identity, and level of ROS—may determine
whether ROS act as tumor-promoting or tumor-inhibiting agents [38]. It
was also shown that there is no linear relationship between antioxidant
supplementation and cancers prevention, with the clinical studies
yielding mixed results [39–41]. In this context, to leverage ROS in
effective anti-tumor strategies, it is important to provide noninvasive
tools to image and quantify ROS levels in tumor tissue and its
microenvironment.
The basic chemical properties of ROS and their reactivity toward
cellular components result in a short lifetime of those species and pre­
vent from them from significantly accumulating. Low steady-state con­
centrations and unfavorable spectroscopic properties make direct
detection of ROS in cells practically impossible. The use of chemical or
genetically encoded probes that produce easily detectable oxidation
products has been proposed and widely applied to overcome those
limitations. Furthermore, to identify ROS in biological systems, the use
of probes that are selective and/or form species-specific products has
been recommended [42].
Fluorogenic and luminogenic boronates have been recently devel­
oped as tools for detection of selected ROS and reactive nitrogen species
(RNS) in cell-free systems and cultured cells in vitro and in animals in vivo
[2,4,37,42–47]. The boronate-based probes are oxidized by H2O2 [1,45,
47–49], peroxymonocarbonate (HCO−4 ) [50,51], selected amino acid
hydroperoxides (AA-OOH) and protein hydroperoxides (Pr-OOH) [52],
HOCl [2,47,49,53–56], and ONOO− [3,4,49,53–55], to the same major
phenolic product. These oxidants differ, however, in their reaction ki­
netics with boronate-based probes [47,49,53]. ONOO− is the fastest
reported oxidant (k ~ 106 M− 1s− 1 [47,49,55]) in comparison with H2O2
2
Dyes and Pigments 192 (2021) 109405
(k ~ 1 M− 1s− 1 [47,49]), AA-OOH (k ~ 10 M− 1s− 1 [52]), and HOCl (k ~
104 M− 1s− 1 [47,49]).
In addition to fast reaction kinetics, another advantage of boronic
probes (of the general structure depicted as Ar–B(OH)2 in Scheme 1) as
tools for ONOO− detection is their specific reaction mechanism with this
oxidant (Scheme 1). The major, phenolic product Ar-OH (~85–99%
yield), common for all listed oxidants, is formed via the heterolytic
cleavage of the O–O bond in the anionic adduct. However, homolytic
cleavage of the peroxide bond results in the formation of an Ar–B
(OH)2O•− radical anion, which further fragments to a phenyl-type
radical (Ar•) and nitrogen dioxide (•NO2). Stable, non-radical products
Ar-NO2 and Ar–H are formed in the reaction of Ar• with •NO2 or
hydrogen donor, respectively [57]. Although the yield of the minor
product(s) is relatively low (~1–15%), their formation may serve as a
unique footprint, providing a diagnostic marker for ONOO− formation.
This is a significant advantage over the use of the nitrotyrosine marker,
as the latter can be also formed via ONOO− -independent pathways.
Changing the phenyl ring in Ar–B(OH)2 for the appropriate fluorophore
(e.g., coumarin) or luciferin backbone results in a fluorogenic probe
(coumarin boronic acid, CBA [55,58]) or a bioluminogenic probe
(luciferin boronic acid, LBA [59]), since in both cases an identical
oxidative conversion occurs, and ONOO− -specific products were
detected.
Here, we present the results of the detailed investigation of the
products formed during oxidation of TCF-BE by different biologically
relevant oxidants and identify the products of nitration and chlorination
of the oxidation product, TCF-OH, to help interpret the in vivo fluores­
cence results obtained with this probe and identify the oxidants
involved. Furthermore, we show that the presence of GSH significantly
affects the probe performance in the detection of the oxidants studied.
The chemical mechanisms beyond the observed effects of GSH are
described. Our investigations brought about a novel probe derived from
1-naphthalene boronic acid TCF-BA-2 (Scheme 2). The colorimetric
response of this probe is not influenced by GSH and, therefore, can be
applied in cell culture or other systems compatible with spectrophoto­
metric detection.
2. Material and methods
2.1. Materials
3-Hydroxy-3-methyl-2-butanone, 2-hydroxy-4′ -(2-hydroxyethoxy)-
2-methylpropiophenone (EtxPhMP), malononitrile, lithium, benzalde­
hyde (4-FPh-H), 4-hydroxybenzaldehyde (4-FPh-OH), 4-nitrobenzalde­
hyde (4-FPh-NO2), 4-hydroxy-3-nitrobenzaldehyde (4-FPh-OH-3-
NO2), 4-hydroxy-3-chlorobenzaldehyde (4-FPh-OH-3-Cl), 4-formylphe­
nylboronic acid pinacol ester (4-FPh-BE), 4-formylphenylboronic acid
(4-FPh-BA), 4-hydroxy-1-naphthaldehyde (4-FNaph-OH), 1-naphthal­
dehyde (4-FNaph-H), and Pd(dppf)Cl2 were purchased from Sigma­
–Aldrich (Poznan, Poland). 4-Formyl-naphthalene-1-boronic acid (4-
FNaph-BA) was purchased from Combi-Blocks (San Diego, CA). Sol­
vents used for syntheses were reagent grade. (Z)-1-[N-[3-aminopropyl]-
N-[4-(3-aminopropylammonio)butyl]-amino]diazen-1-ium-1,2-diolate
(Spermine-NONOate) and (Z)-1-[N-(3-aminopropyl)-N-(n-propyl)
amino]diazen-1-ium-1,2-diolate (PAPA-NONOate) were purchased
from Cayman Chemical (Ann Arbor, MI). Hypoxanthine (HX), xanthine
oxidase (XO), GSH, CAT, bovine SOD, and penta(carboxymethyl)-
diethylenetriamine (DTPA) were purchased from Sigma-Aldrich (Mil­
waukee, WI).
2.2. Synthesis
All synthesized compounds containing the TCF ring were purified by
column chromatography using silica gel (0.063–200 mm, 70–230
mesh). 1H nuclear magnetic resonance (NMR) and 13C NMR spectra
were recorded on Bruker AVANCE 400 spectrometer at 400 MHz (1H) or
P. Siarkiewicz et al.
Scheme 1. The mechanism of ONOO− -induced oxidation of arylboronate compounds Ar–B(OH)2 [49,57].
Scheme 2. Synthesized compounds containing the TCF ring.
100 MHz (13C), respectively. Compounds were dissolved in DMSO‑d6,
and tetramethylsilane (TMS) was added as an internal reference for 1H
NMR and 13C NMR spectra. Chemical shifts (δ) are reported in ppm, and
coupling constant J values in hertz (Hz). The crystallographic data of
compounds TCF-NO2 and TCF-H are shown in the Supplementary In­
formation (Figures S1 and S2, Tables S1 and S2). 2-Dicyanomethylene-3-
cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) was synthesized accord­
ing to the procedure described elsewhere [60] and used for subsequent
syntheses without further purification. Scheme 3 shows in condensed
form the synthetic pathways. For detailed synthetic procedures, melting
points and also NMR and HRMS spectra of synthesized compounds the
reader is referred to the Supplementary Information of this article.
3
Dyes and Pigments 192 (2021) 109405
2.3. General preparation of dyes and chemicals used in analyses
The stock solutions of oxidants (HOCl, and H2O2) were prepared
freshly before each experiment and theirs concentrations were deter­
mined by spectrophotometry, using appropriate molar extinction coef­
ficient (ε292 = 350 M− 1cm− 1 in 0.1 M NaOH for HOCl and ε240 = 43.6
M− 1cm− 1 in water for H2O2) [45]. Peroxynitrite was synthesized in re­
action of 0.6 M nitrite with 0.7 M hydrogen peroxide at pH 13 [45].
Excess H2O2 was removed by passage through a column of MnO2 and the
solution was frozen at − 20 ◦ C. The liquid over the frozen solid was
collected, aliquoted into 1.5 mL tubes and stored at − 80 ◦ C. The con­
centration of peroxynitrite was determined spectrally at 302 nm (ε302 =
1.7 × 103 M− 1cm− 1), after dilution in 0.1 M NaOH to ~10 mM con­
centration, immediately prior to each experiment. During high perfor­
mance liquid chromatography (HPLC) analyses it was noticed that
TCF-BE (pinacolate ester) undergoes fast hydrolysis to the boronic
acid form (TCF-BA) upon dilution in the aqueous phosphate buffer.
Therefore, although we added TCF-BE to the investigated mixtures,
TCF-BA is the species that is tested.
2.4. Spectroscopic studies
Absorption and fluorescence spectra were recorded using a Jasco UV-
VIS-NIR V–670 spectrophotometer (Jasco, Japan) and a FLS-920 spec­
trofluorometer (Edinburgh Instruments, UK), respectively. The fluores­
cence quantum yield of the dye (ΦDYE) was calculated from the equation,
GDYE ⋅η2DYE
ΦDYE = ΦST (1)
GST ⋅η2ST
where the subscripts ST and DYE denote standard and tested fluorescent
dye, respectively; Φ is the fluorescence quantum yield; G is the gradient
from the plot of integrated fluorescence intensity versus absorbance; and
P. Siarkiewicz et al.
Scheme 3. Synthetic pathways and reagents used to obtain the (A) TCF-BE, TCF-BA probe and TCF-OH, TCF-H, TCF-NO2, TCF-OH-3-NO2, TCF-OH-3-Cl dyes, (B)
TCF-BA-2, and TCF-H-2, TCF-OH-2 dyes, (C) TCF-Etx dye. The description of synthetic procedures is detailed in Supplementary Information.
η is the refractive index of the solvent. The reference systems used were
rhodamine 101 in ethanol (ΦST = 100% [61]) or 4-methylumbelliferone
in water (ΦST = 35.6% [62]).
The dissociation constants of the adducts were determined spectro­
scopically by measuring the absorption spectra of probes or dyes in
aqueous solutions (pH 7.4) at different concentrations of GSH.
2.5. Reaction with singlet oxygen and enzymatically generated ROS/RNS
All solutions were prepared using deionized water (Millipore Milli-Q
system). Due to the poor solubility of probes in water, all experiments
were performed with the addition of acetonitrile (MeCN) at up to 5% (v/
v). •NO was generated from thermal decomposition of PAPA-NONOate.
•
NO fluxes were determined from the rate of decomposition of the
donor, measured following the decrease of its characteristic absorbance
at 250 nm (ε = 8.1 × 103 M− 1cm− 1). At a temperature of 25 ◦ C, 50 μM of
PAPA-NONOate produced 1.08 μM/min •NO flux in solutions containing
a phosphate buffer (pH 7.4, 50 mM) and DTPA (100 μM). The flux of O•− 2
was produced by the XO-catalyzed oxidation of hypoxanthine in the
presence of CAT and determined by monitoring the cytochrome c(Fe3+)
reduction reflected in an increase in absorbance at 550 nm (using a
difference in the values of the extinction coefficients between reduced
and oxidized cytochrome of 2.1 × 104 M− 1 cm− 1). 70 μU/mL of XO in
the presence of 1 mM HX and 100 U/mL CAT produced 0.3 μM/min flux
of O•−
2 . H2O2 flux was generated from the same flux of O2 in the absence
•−
of CAT and presence of SOD (100 μg/mL). ONOO− flux was produced
from co-generated fluxes of O•−
2 and NO in a phosphate buffer (pH 7.4,
•
4
Dyes and Pigments 192 (2021) 109405
50 mM) with DTPA (100 μM), HX/XO (1 mM/70 μU/mL), PAPA-
NONOate (50 μM), and CAT (100 U/mL). This system produced 1.08
μM/min flux of ONOO− .
The reactivity of the TCF-BA probe and TCF-OH dye toward 1O2 was
investigated according to a protocol described previously [21]. Briefly, a
solution comprising the TCF-BA probe (10 μM) or TCF-OH dye (10 μM),
sensitizer - Rose Bengal (10 μM), and CAT (100 U/mL) in a phosphate
buffer (pH 7.4, 50 mM) was placed in a quartz cell and illuminated with
visible light using a 200-W xenon lamp at room temperature with
continuous bubbling with oxygen. The illuminating light was passed
through a water filter and a 400 nm cutoff filter.
2.6. Pulse radiolysis study
Pulse radiolysis experiments were carried out with high-energy (8
MeV) 7 ns electron pulses generated from an ELU-6 linear electron
accelerator [46]. The aqueous solution of potassium thiocyanate (0.01
M) saturated with nitrous oxide was used to determine the dose absor­
− 7
bed per pulse, assuming G[(SCN)•− 2 ] = 6.2 × 10 mol/J, and
3 − 1 − 1
ε[(SCN)2 ] = 7.6 × 10 M cm (G represents the radiation chemical
•−
yield, and ε is the molar absorption coefficient at 475 nm). The dose
delivered per pulse was within the range of 20 Gy.
ONOO− was generated by irradiation of aerated solutions containing
10 mM nitrite, 1 M methanol, TCF-BA or TCF-BA-2 probe (10–40 μM),
and a phosphate buffer (pH 7.4, 50 mM). Irradiation produces nearly
equal amounts of •NO and O•− 2 radicals from the primary products of
water radiolysis (e−aq, •OH, H•) via reactions 1–7:
P. Siarkiewicz et al. Dyes and Pigments 192 (2021) 109405

e−aq + NO−2 → NO•2−

2 (reaction 1, k1 = 4 × 109 M− 1s− 1) oxidation product, TCF-OH, reacts with GSH, resulting in a decrease in
fluorescence intensity [3]. Addition of thiols to the vinyl bond of the
NO•2− + H2PO−4 → •NO + HPO2− − 8 TCF-based probe was also explored as a basis for cellular GSH mea­
2 4 + OH (reaction 2, k2 = 1.5 × 10
M− 1s− 1) surements [13]. No studies have been, however, reported on the per­
formance of TCF-based probes for other analytes in the presence of GSH.
OH + CH3OH → •CH2OH + H2O (reaction 3, k3 = 9.7 × 108 M− 1s− 1) To clarify the oxidation chemistry of TCF-BA probe in GSH-free solution
•

and at physiologically relevant GSH concentrations, we firstly synthe­

H• + CH3OH → •CH2OH + H2 (reaction 4, k4 = 2.6 × 106 M− 1s− 1)
•
CH2OH + O2 → •O2CH2OH (reaction 5, k5 = 4.9 × 109 M− 1s− 1)
O2CH2OH + HPO2−
• •−
4 → O2 + CH2O + H2PO4 (reaction 6, k6 = 2 × 10
6
− 1 − 1
M s )
O•− • − 9 − 1 − 1
2 + NO → ONOO (reaction 7, k7 = 4-7 × 10 M s )
The product build-up kinetics were monitored at 585 and 630 nm for
TCF-BA and TCF-BA-2, respectively. The recorded kinetic traces were
fitted to a function corresponding to the first order product formation.
Results from the fitting of at least three kinetic traces were averaged to
determine the observed first-order rate constant. The second order rate
constants were obtained from the slopes of the plots of the observed first-
order rate constants versus the concentration of the studied probe.
sized TCF-BA probe, its major oxidation product (the TCF-OH dye), and
the anticipated minor products of its reactions with ONOO− (TCF-NO2,
TCF-H, TCF-OH-3-NO2) and with HOCl (TCF-OH-3-Cl). Scheme 3 pre­
sents all of the synthetic pathways in a condensed form. TCF-derived
compounds were synthesized by modifying the published method [60]
using TCF and the appropriate benzaldehyde as the starting materials.
The results of our investigation (see section 3.3) encouraged us to design
and synthesize a novel probe, TCF-BA-2, and its oxidation product, the
TCF-OH-2 dye. As previously discussed, these compounds were syn­
thesized via a simple condensation of TCF with 4-formylnaphthalene-1-­
boronic acid or 4-formyl-1-naphthol, respectively. To clarify the
reactivity of TCF-derived compounds with GSH, we also synthesized
TCF-Etx dye, an analogue lacking the styryl unit (Scheme 3).
3.2. Spectroscopic characterization of TCF-derived compounds

2.7. Stopped-flow measurements The spectroscopic characterization of probes and dyes in aqueous
solution at physiological pH is mandatory for the correct interpretation
2.7.1. Kinetics of TCF-BA reaction with HOCl of the signals obtained when using the fluorogenic probes in cell culture
The rate constant of the reaction of TCF-BA with HOCl was deter­ models. Therefore, we determined the absorption and fluorescence
mined using an Applied Photophysics SX 20 stopped flow spectropho­ emission spectra of the synthesized compounds in aqueous solutions
tometer equipped with an absorption detector (Surrey, UK). HOCl containing a phosphate buffer (pH 7.4, 50 mM) and acetonitrile (20%).
(0.4–2.0 mM) was rapidly mixed with the solution containing TCF-BA The results of spectroscopic characterization are summarized in Table 1.
(40 μM), phosphate buffer (pH 7.4, 100 mM), and MeCN (10%). The From these data, one can conclude that the TCF-BA probe can serve not
thermostated cell (25 ◦ C) with a 10-mm optical pathway was used for only as a fluorogenic probe but also as a naked-eye colorimetric sensor,
kinetic measurements. The kinetic traces were analyzed in the same way since a replacement of the boronate group by hydroxyl group causes a
as described above for pulse radiolysis experiments. large bathochromic (almost 190 nm) and hyperchromic effect (ε587 =
1.85 × 105 M− 1cm− 1). The nitrated and chlorinated dyes, TCF-OH-3-
2.7.2. Kinetics of the reaction between the TCF-BA-SG adduct and NO2 and TCF-OH-3-Cl, exhibit absorption and emission bands located in
ONOO− the same region as the TCF-OH dye. This implicates the necessity of the
The reaction mixture contained 0–25 μM TCF-BA probe, 1 mM GSH, high-performance liquid chromatography (HPLC) analyses to identify
5% MeCN, and a phosphate buffer (pH 7.4, 50 mM) with 100 μM DTPA. the specific products formed in the reaction between the TCF-BA probe
After 1 h of incubation, the CBA probe (10 μM) and ONOO− (5 μM) were and ONOO− or HOCl (see the next section).
added. Spectra were collected 15 min after adding ONOO− with the aid
of a Varian Cary Eclipse spectrofluorometer equipped with a plate
reader accessory (Agilent Technologies, Santa Clara, CA). The solutions
were excited at 332 nm, and the emitted light intensity was measured at Table 1
470 nm (PMT voltage = 670 V, emission/excitation slit = 5 nm). Each Absorption and fluorescence emission properties of studied compounds in
point represents the average value of four samples. The error bars aqueous solution containing a phosphate buffer (pH 7.4, 50 mM) and MeCN
represent standard deviations. The rate constant was determined using (20%).
the competition kinetic approach described by the following equation: Absorption Emission
( )
1 1 1 kTCF− BA− SG [TCF − BA − SG] λmax/nm (ε/103 λmax/nm (ε/103 λex λem Φ (%)
= + × (2) M− 1cm− 1) M− 1cm− 1) (nm) (nm)
Fi F0 F0 kCBA [CBA] i
TCF-BA 407 (12.7) – –
The rate constant of the CBA reaction with ONOO− is equal to k = TCF-OH 452, 448 (48.5)c, 587 607 0.052a
1.1 × 106 M− 1s− 1 [55]. 587 (185.0)c 588 (181.0)d
TCF-H 400 (16.8) – –
TCF-NO2 389 (36.3) – –
3. Results TCF-OH- 539 (64.3) c
414 (60.0) , 539 607 0.069a
3-NO2 537 (203.0)d
3.1. Synthesis of TCF-derived compounds TCF-OH- 585 (54.0) 438 (44.0)c, 585 614 0.094a
3-Cl 584 (234.0)d
TCF-BA- 310 (21.0),
Several papers described the syntheses of fluorogenic boronate – –
2 440 (7.6)
probes derived from p-phenyl-boronic acid ester linked via double TCF-OH- 629 (141.0) c
512 (52.3) , 629 650 0.025a
bound with the appropriate strong-electron acceptor unit, e.g., the TCF 2 632 (101.0)d
ring. The TCF-based probe (TCF-BE) has been proposed for selective TCF-Etx 386 (31.5) 386 470 0.052b
detection of H2O2 [4,17], HOCl [5], or ONOO− [3]. Here, we performed a
Rodamine 101 in ethanol (ΦST = 1.0 [61]) used as the standard.
detailed stoichiometric, kinetic and product analysis studies to define b
4-methylumbelliferone in water (ΦST = 0.356 [62]) used as the standard.
the selectivity of the probe and its potential applicability as a redox c
in DMSO/water (1/5 v/v) at pH = 2 from Ref. [9].
probe in biological systems. It has been also recently reported that the d
in DMSO/water (1/5 v/v) at pH = 10 from Ref. [9].

5
P. Siarkiewicz et al.
3.3. Oxidation of the TCF-BA probe by biologically relevant oxidants in
GSH-free solution
After determining the spectroscopic properties of the TCF-BA probe
and TCF-OH dye, we examined the reactivity of these two TCF-derived
compounds toward several ROS/RNS in GSH-free systems. Analyses of
fluorescence and absorption spectra of TCF-BA and TCF-OH in the
presence of O•– 2 , H2O2, NO, and ONOO fluxes (Figs. S3–S5) showed
• −
that TCF-OH dye is consumed in the presence of singlet oxygen or
ONOO− , while the TCF-BA probe responds primarily to ONOO− and, to
a smaller extent, to H2O2 or •NO-generating system. Therefore, we
decided to identify and quantify the products formed in the reaction
between ONOO− and TCF-BA or TCF-OH. A bolus addition of ONOO− to
the solution of the TCF-BA probe led to the appearance of a red color and
fluorescence (Fig. 1) due to the formation of TCF-OH [3].
Next, we investigated the stoichiometry and products of the reaction
between the probe and ONOO− (Fig. 2). HPLC analyses confirmed that
ONOO− oxidized the TCF-BA probe to the TCF-OH dye. Analysis of the
data in Fig. 2 revealed that complete consumption of the probe required
a slight excess of ONOO− . It is well known that ONOO− oxidizes boronic
compounds, giving corresponding phenol as the major, non-radical end
product (~85–99% yield) [45,47,49,53,57]. The minor, radical
pathway leads to a phenyl-type radical, nitrogen dioxide (•NO2), and
stable products formed from them (~1–15% yield) (Scheme 1) [45,47,
49,53,57,63]. Oxidation of TCF-BA by ONOO− resulted in the formation
TCF-OH in an almost quantitative (99%) yield. Additionally, it is evident
that TCF-OH was consumed when an excess of ONOO− was present in
the reaction mixture. In order to clarify the reactions that take place
between ONOO− and the TCF-BA probe or TCF-OH product, we syn­
thesized the expected stable end-products formed in the radical minor
pathway, TCF-NO2 (the anticipated product of the recombination of
6
Dyes and Pigments 192 (2021) 109405
TCF-Ph• phenyl-type radical and •NO2) and TCF-H (TCF-Ph• reduction
product); nitrated the TCF-OH dye, TCF-OH-3-NO2; and performed
HPLC analyses of the reaction mixture (Fig. 2A). HPLC analyses showed
that the TCF-NO2 and TCF-H compounds in the reaction between
TCF-BA and ONOO− are formed in a yield of below 1%. Moreover, our
experiments with 2-methyl-2-nitroso-propane as the spin trap did not
show any electron paramagnetic resonance (EPR) signal of a phenyl
radical adduct, while under the same conditions, we observed an intense
EPR spectrum of a phenyl radical spin adduct when acetylphenylboronic
acid was used instead of TCF-BA (Fig. S6), in agreement with previous
report [57]. Instead of TCF-NO2 and TCF-H, we identified
TCF-OH-3-NO2 as a minor product formed in the reaction mixture,
which we attributed to the nitration of the phenolic product formed. We
have confirmed that TCF-OH undergoes nitration to TCF-OH-3-NO2 in
the presence of ONOO− (Fig. 2).
Next, we investigated the reaction between the TCF-BA probe and
HOCl. The stoichiometric analysis of this reaction is shown in Fig. 3B.
Similar to ONOO− , HOCl oxidized the TCF-BA probe, forming TCF-OH
at 1:1 stoichiometry in a 100% yield. As in the case of ONOO− , the
amount of the TCF-OH product decreased when an excess of the oxidant
was added. The disappearance of TCF-OH in a reaction mixture can be
attributed to the chlorination reaction leading to the formation of
chlorinated derivative(s). This is supported by the detection of a new
HPLC peak assigned to TCF-OH-3-Cl (Fig. 3A) of the same retention time
as the synthesized authentic standard and of the product formed when
TCF-OH was mixed with HOCl. Chlorinated products formed from the
boronate-derived phenols [45,47] or from hydroethidine [64,65] can be
used as specific markers for HOCl.
Because the second-order rate constant of the reaction of arylboronic
acids with H2O2 is of the order of 1 M− 1s− 1 [47,49], the stoichiometric
analysis of the reaction between the boronic probe and H2O2 requires
Fig. 1. (A) Absorption and (B) emission
spectra of the TCF-BA probe (100 μM)
before and after mixing it with ONOO−
in a phosphate buffer (pH 7.4, 50 mM)
containing MeCN (50%) and DTPA (10
μM). Absorption and emission spectra
were recorded after 5 × and 50 × dilu­
tion of reaction mixture, respectively.
The final concentration of the TCF-BA
probe was 20 μM (absorption) and 2 μM
(emission, λexc 550 nm). (C) Absorption
spectra of the TCF-BA probe (25 μM)
and TCF-OH dye (30 μM). (D) Emission
spectrum of the TCF-OH dye (2 μM) in a
phosphate buffer (pH 7.4, 50 mM) con­
taining of MeCN (50%) (λexc 550 nm).
P. Siarkiewicz et al. Dyes and Pigments 192 (2021) 109405

Fig. 2. Reaction between the TCF-BA probe and ONOO− in an aqueous solution containing a phosphate buffer (pH 7.4, 50 mM), DTPA (10 μM), and MeCN (5%). (A)
HPLC chromatograms of the standards (10 μM) and reaction mixtures of the TCF-BA probe (20 μM) and TCF-OH dye (20 μM) with ONOO− . The traces were collected
using an absorption detector set at 440 nm. (B) HPLC-based titration of the TCF-BA probe (20 μM) with ONOO− .

Fig. 3. Reaction between the TCF-BA probe and HOCl in an aqueous solution containing a phosphate buffer (pH 7.4, 50 mM), DTPA (10 μM), and MeCN (5%). (A)
HPLC chromatograms of the standards and reaction mixtures of the TCF-BA probe (20 μM) and TCF-OH dye (20 μM) with HOCl. The traces were collected using an
absorption detector set at 440 nm. (B) HPLC-based titration of the TCF-BA probe (20 μM) with HOCl.

the reaction mixture to have a long (≥24 h) incubation time, especially
for low concentrations of H2O2, to ensure completion of the reaction.
Tests carried out under analogous conditions as for HOCl and ONOO− (i.
e., for a 20 μM concentration of the probe in a phosphate buffer with 5%
of MeCN) showed the instability of TCF-BA during a 24-h incubation
period. Since it was difficult to precisely determine the concentration of
the residual boronic probe, we repeated experiments with a higher
concentration of the probe in an aqueous solution containing a phos­
phate buffer and MeCN (50%). Under those conditions, we observed that
30% of the TCF-BA undergoes spontaneous decomposition, but an
excess of H2O2 still was required for complete consumption of the probe
(Fig. 4). H2O2 converted TCF-BA to TCF-OH as the sole detectable
product (Fig. 4A).
Finally, the rate constants of the TCF-BA probe reactions with
selected biological oxidants (ONOO− , HOCl, and H2O2) were
determined. Using time-resolved techniques, pulse radiolysis, and
stopped-flow, we determined the second-order rate constants for the
reaction of the TCF-BA probe with ONOO− (Fig. 5A) and HOCl (Fig. 5B),
respectively. Because the reaction of boronates with H2O2 is relatively
slow [45,47,49], time resolution of regular spectrophotometers is suf­
ficient to monitor the reaction kinetics of TCF-BA with H2O2 (Fig. 5C).
Table 1 summarizes the results of the kinetic experiments. ONOO− ox­
idizes the TCF-BA probe with a higher rate constant of (1.8 ± 0.3) × 106
M− 1s− 1 than HOCl of (2.4 ± 0.1) × 104 M− 1s− 1 or H2O2 of (1.9 ± 0.4)
M− 1s− 1. These rate constants are typical for aromatic boronic acids [47,
49,54]. The spectroscopic (Fig. S7) and kinetic (Fig. S8) experiments
also showed that TCF-OH reacts with H2O2 with a rate constant of (5.21
± 0.04) × 10− 2 M− 1s− 1, which is 40 times lower than that for the re­
action between H2O2 and TCF-BA. However, it can be observed that,
upon the reaction of TCF-BA with an excess of H2O2 (100-fold excess),

7
P. Siarkiewicz et al. Dyes and Pigments 192 (2021) 109405

Fig. 4. Reaction between the TCF-BA probe and H2O2 in an aqueous solution containing a phosphate buffer (pH 7.4, 50 mM), DTPA (10 μM), and MeCN (50%). (A)
HPLC chromatograms of the standards and reaction mixtures of the TCF-BA probe with H2O2. (B) HPLC-based titration of the TCF-BA probe (100 μM) with H2O2. The
traces were collected using an absorption detector set at 440 nm.

Fig. 5. Kinetics of the reaction between TCF-BA and studied oxidants. The pseudo-first order rate constants were obtained by fitting the build-up of the signal
recorded at 585 nm. (A) The dependence of pseudo-first order rate constants of the reaction of TCF-BA with ONOO− on the TCF-BA concentration. The pseudo-first
order rate constants were obtained by fitting the build-up of the signal at 585 nm obtained by pulse radiolysis of aqueous solution containing TCF-BA (10–30 μM), 10
mM NaNO2, 1 M CH3OH, and a phosphate buffer (pH 7.4, 50 mM). The optical length of the cuvette was 1 cm, and the solutions received a radiation dose of 20 Gy.
(B) The dependence of the pseudo-first order rate constants of the reaction of TCF-BA probe with HOCl on the initial concentration of HOCl. (C) The dependence of
pseudo-first order rate constants of the TCF-BA reaction with H2O2 on the initial concentration of H2O2. Solutions consisted of TCF-BA (20 μM), a phosphate buffer
(pH 7.4, 50 mM), DTPA (100 μM), MeCN (10%), and H2O2 (200–400 μM).

TCF-OH was formed and subsequently underwent consumption
(Fig. S7).
3.4. Oxidation of the probe in the presence of GSH
The use of a fluorogenic probe (e.g., TCF-BA) for the determination of
biologically important oxidants (ONOO− , HOCl, H2O2) also requires the
recognition of the reactivity of the probe and the resulting fluorescent
product (e.g., TCF-OH) with tripeptide GSH (L-γ-glutamyl-L-cysteinyl-
glycine), which serves as a cellular low molecular antioxidant and a
substrate for antioxidant enzymes (glutathione peroxidase and gluta­
redoxin). Based on our experience developing probes for thiols [66–69],
recent report on the TCF-OH fluorescence decrease in the presence of
GSH, and taking into account high intracellular concentration (1–10
mM) of GSH, we anticipated that GSH may form a Michael adduct with
tricyanofuran-based probes and dyes (e.g., TCF-BA and TCF-OH). The
electron-withdrawing group (e.g., TCF) conjugated to the carbon–carbon
double (C–– C) bond is a suitable site to attach nucleophiles, for example,
GSH. Such reactions are usually catalazed by presence of base in solu­
tions [13,70–72]. Therefore, we determined whether GSH interacts with
the TCF-BA probe and TCF-OH product. The spectrophotometric
measurements (Fig. 6A and B) revealed that, as the concentration of GSH
in the solution increases, the intensity of the absorption bands of the
TCF-BA probe and TCF-OH dye decreases. Moreover, a new absorption
band with a maximum at 339 nm appears. In order to confirm that GSH
forms an adduct to the double bound, we synthesized a TCF-Etx dye (see
Scheme 2). Because TCF-Etx, an analogue without a styryl unit, behaved
in a different way (Fig. 6F), we conclude that the studied TCF-BA probe
and TCF-OH dye might react with GSH via a Michael addition to the
double bond, yielding two diastereoisomers of GSH adduct TCF-SG (R)
and TCF-SG (S) (Fig. 6E). Such reactivity was utilized recently in
GSH-sensing probe developed by Peng Hou and co-workers [13].
Based on UV–vis absorption spectra of TCF-BA and TCF-OH recorded
at different GSH concentrations (Fig. 6A and B), the dissociation con­
stants Kdis for TCF-BA-SG and TCF-OH-SG were estimated at 5.6 × 10− 5
and 5.6 × 10− 4, respectively (Fig. S9). The comparison of these disso­
ciation constants allows the conclusion that the oxidation of the boronic
acid group in the TCF-BA-SG adduct at submillimolar concentrations of
GSH may result in partial release the TCF-OH fluorescent dye.
The formation of the TCF-BA-SG and TCF-OH-SG adducts may not
only affect the probe fluorescence response but also its reactivity toward
oxidants. Therefore, we decided to study the effect of GSH on TCF-BA

8
P. Siarkiewicz et al. Dyes and Pigments 192 (2021) 109405

Fig. 6. Electronic absorption spectra of TCF-derived probes (A) TCF-BA (25 μM) and (C) TCF-BA-2 (20 μM), and dyes (B) TCF-OH (8 μM), (D) TCF-OH-2 (6 μM), and
(F) TCF-Etx (4 μM) before and after mixing them with GSH in a phosphate buffer (pH 7.4, 50 mM) containing of MeCN (5%) and DTPA (10 μM). (E) Stereoisomers
formed in the reaction of TCF-BA with GSH.

oxidation by ONOO− . For comparison, we also measured colorimetric
response of TCF-BA-SG after addition of HOCl or H2O2 under the same
conditions. Only bolus addition of ONOO− led to the appearance of a
slight red color due to the formation of TCF-OH (Fig. S12B). A lower
intensity of color is caused by the subsequent reaction of TCF-OH with
GSH. The lack of response of the probe to HOCl and H2O2 could be
explained by direct scavenging of those oxidants by GSH. HPLC analyses
of the reaction mixture of TCF-BOR with ONOO− in the presence of GSH
(Fig. 7A) show that GSH did not significantly affect the consumption of
the probe by ONOO− [46]. However, it is evident from Fig. 7B that GSH
inhibits the consumption the TCF-OH dye by ONOO− . Using the
competition method, we determined the second-order rate constants for
the reaction between ONOO− and the TCF-BA-SG adduct. The adduct is
oxidized by ONOO− with a lower rate constant (6.2 ± 0.6) × 105 M− 1s− 1

Fig. 7. (A) Loss of the TCF-BA probe

concentration in the reaction of 20 μM
TCF-BA with 10 μM ONOO− . (B) Loss of
the TCF-OH dye concentration in the
reaction of 20 μM TCF-OH with 10 μM
ONOO− . Reactions were performed in
an aqueous solution containing 50 mM
PB (pH 7.4), 100 μM DTPA, 10% MeCN,
and without or with the addition of 0.4
mM GSH. The concentrations of TCF-BA
and TCF-OH were determined based on
the HPLC measurements. The traces
were collected using an absorption de­
tector set at 440 nm. Data are the
means ± standard deviation of three
independent experiments.

9
P. Siarkiewicz et al.
Fig. 8. The dependence of the reciprocal of fluorescence intensity monitored at
470 nm on the [TCF-BA-SG]/[CBA] ratio.
(Fig. 8, Table 2) than the TCF-BA probe, but the rate constant still is high
enough to outcompete other pathways of ONOO− decay, including
self-decay and scavenging by GSH. Collectively, these results demon­
strate that, at physiologically relevant GSH concentrations, the TCF-BA
probe may undergo oxidation to TCF-OH only in a reaction with
ONOO− .
Because the signal intensity from the TCF-BA probe depends on the
amount of GSH (Fig. 6), we decided to modify the structure of the probe
in an attempt to minimize the effect of GSH. The reaction between GSH
and hydroxyl bearing styryl compound is more probable for phenol state
than phenolate. Because of that we searched for analogical dye which
has much lower pKa and a good solubility in a aqueous solution con­
taining a small amount of organic solvent (e.g. ~ 10% MeCN). The latter
requirement resulted in the resignation from the synthesis of the probe
based on the TCF–OH–3-NO2 dye. TCF-OH-2 with naphthalene moiety
fulfilled all the requirements, i.e. pK = 5.3 [9] and sufficient solubility in
our system. We synthesized the TCF-BA-2 probe and TCF-OH-2 dye
(Scheme 2), and, like before, we spectroscopically characterized those
compounds and measured the reactivity of the TCF-BA-2 probe toward
ONOO− . The spectroscopic studies (Table 1, Fig. 9C and D) revealed that
replacing the benzene ring with a naphthalene moiety led to the
red-shift of the absorption and fluorescence emission bands of
TCF-OH-2 in comparison with TCF-OH. After adding ONOO− to the
solution of TCF-BA-2, we observed the solution become an intense blue
color due to the formation of TCF-OH-2 dye (Fig. 9A and Fig. S13A). The
lower value of the Φfl of TCF-OH-2, in comparison with TCF-OH, but
high value of the extinction coefficient suggest that TCF-BA-2 may
perform better as a colorimetric probe than as a fluorogenic sensor.
As for the TCF-BA probe, we determined the second-order rate
constants for the reaction of ONOO− with TCF-BA-2 (Table 1, Fig. 10)
Table 2
Determined values of the second-order rate constants for the reaction of TCF-BA
with different oxidants and for the reaction of the TCF-BA-SG adduct and TCF-
BA-2 with ONOO− at pH 7.4
ONOO− HOCl H2O2
− 1 − 1
k (M s ) at 25 C ◦
TCF-BA (1.8 ± 0.3) × 106a (2.4 ± 0.1) × 104b (1.9 ± 0.4)c
TCF-BA-SG (6.2 ± 0.6) × 105b
TCF-BA-2 (6.0 ± 0.2) × 105a
a
Pulse radiolysis.
b
Stopped flow.
c
Regular spectrophotometer.
10
Dyes and Pigments 192 (2021) 109405
and tested the possibility of the formation of a specific nitro-naphthalene
product, anticipated for the potential minor, radical pathway of the
reaction between TCF-BA-2 and ONOO− . The TCF-BA-2 probe reacts
with ONOO− with a rate constant of (6.0 ± 0.2) × 105 M− 1s− 1, and HPLC
analyses (Fig. 11) showed formation of the TCF-OH-2 dye as a sole re­
action product. Moreover, similar to the results observed for TCF-BA,
the EPR spin trapping experiment with 2-methyl-2-nitroso-propane
(Fig. S6, trace c) did not produce detectable levels of the spin adduct.
This indicates that the reactions of both the TCF-BA and TCF-BA-2
probes with ONOO− proceed predominantly via the non-radical
pathway, resulting in the formation of the corresponding phenolic
products.
We also determined whether TCF-BA-2 and TCF-OH-2 can form
corresponding adducts with GSH. Spectrophotometric analyses (Fig. 6C)
showed that the TCF-BA-2 probe forms an adduct with GSH (Kdis = 7.1
× 10− 5), whereas the absorption band of TCF-OH-2 dye did not change
with an increased concentration of GSH (Fig. 6D). Like in the case of
TCF-BA, in the presence of GSH only ONOO− is able to induce the
colorimetric response of the TCF-BA-2 probe (Fig. S13B).
Finally, we compared oxidation of the TCF-BA and TCF-BA-2 probes
in an enzymatic HX/XO/spermine-NONOate system, which produced a
flux of ONOO− in situ (Figs. S14 and S15), and tested the effect of the
presence of GSH on the signal detected. Again, it can be clearly seen in
Fig. 12 that the TCF-BA probe response is attenuated in the presence of
GSH whereas the colorimetric monitoring of the oxidation of the TCF-
BA-2 probe by ONOO− is not affected by GSH. The lower fluorescent
intensity observed in an enzymatic system with GSH is caused by the
formation of the TCF-OH-SG adduct.
4. Discussion
Identification and quantification of ROS/RNS is extremely impor­
tant, since these species play a crucial role in many pathophysiological
processes. For example, depending on their concentrations, ROS may
show antitumor activity or may initiate cancer proliferation, angiogen­
esis, immune evasion, and tumor growth and metastasis. Understanding
the mechanisms of ROS/RNS formation and localization may allow the
development of novel, effective redox-active drugs that inhibit tumor
growth and prevent metastasis. A commonly used method for detecting
ROS/RNS in vitro and in vivo is the bioluminescence (BLI) or fluorescence
imaging (FLI). BLI and FLI are based on suitable probes that, upon re­
action with an oxidant, release a luciferase bioluminescent substrate or a
highly fluorescent compound, respectively. The appearance of the
bioluminescence or fluorescence signal and its intensity reflect the
presence and amount of the oxidant, respectively. However, interpre­
tation of the BLI or FLI signal is not trivial, and several factors should be
considered. First, a significant challenge is to develop a selective probe
that reacts with the oxidant with a 1:1 stoichiometry. Second, the rate
constant of the reaction of the oxidant of interest with the probe should
be high enough to enable a sufficient accumulation of the detectable
oxidation product, in competition with the oxidant’s reaction with other
cellular targets (e.g., GSH, thiols, CO2, Prx, heme-containing proteins
[73,74]). Third, for in vivo applications, emission at far-red and/or at
NIR wavelengths, which are minimally absorbed by tissue, is preferred.
For these reasons, the most promising luminogenic and fluorogenic
probes for the detection of several non-radical ROS/RNS, produced
under inflammatory conditions (i.e., H2O2, HOCl, ONOO− ), are based on
the oxidative conversion of arylboronic acids or esters [75]. Masking of
hydroxyl or amino group(s) of a fluorophore or firefly luciferin de­
rivatives by boronate or boronobenzyl moieties turns off the fluores­
cence [46,48,53–55,58] or luminescence [59], respectively. The
reaction of boronic/boronate group with H2O2, HOCl, and ONOO−
proceeds with a 1:1 stoichiometry and uncages the hydroxyl group and
thus turns on the fluorescence/luminescence [46,48,53–55,58,59].
Under physiological pH, boronate probes are oxidized by HOCl and
ONOO− at a significantly higher rate than is the case of H2O2 [47,49,55].
P. Siarkiewicz et al. Dyes and Pigments 192 (2021) 109405

Fig. 9. (A) Absorption and (B) emission

spectra of probe TCF-BA-2 (100 μM)
before and after mixing it with ONOO−
in a phosphate buffer (pH 7.4, 50 mM)
containing of MeCN (50%) and DTPA
(10 μM). Absorption and emission
spectra were recorded after 10 × and
50 × dilution of reaction mixture,
respectively. The final concentration of
the TCF-BA-2 probe was 18 μM (ab­
sorption) and 2 μM (emission, λexc 625
nm). (C) Absorption spectra of the TCF-
BA-2 probe (10 μM) and TCF-OH-2 dye
(6 μM). (D) Emission spectrum of the
TCF-OH-2 dye (2 μM) in a phosphate
buffer (pH 7.4, 50 mM) containing of
MeCN (20%) (λexc 625 nm).

Fig. 10. Kinetics of the reaction be­

tween TCF-BA-2 and ONOO− . (A) Ki­
netic traces of TCF-OH-2 build-up were
observed at 630 nm. (B) The depen­
dence of pseudo-first-order rate con­
stants of the reaction of TCF-BA-2 with
ONOO− on the TCF-BA-2 concentration.
The pseudo-first-order rate constants
were obtained by fitting the build-up of
the signal at 585 nm obtained by pulse
radiolysis of aqueous solution contain­
ing TCF-BA-2 (20–40 μM), 10 mM
NaNO2, 1 M CH3OH, and a phosphate
buffer (pH 7.4, 50 mM). The optical
length of the cuvette was 1 cm, and so­
lutions received a radiation dose of 20
Gy.

Several boronate probes were synthesized and characterized as far-red
or NIR fluorogenic probes for H2O2, HOCl, or ONOO− [75].
TCF-BA was developed as a selective probe for H2O2 [4,17], HOCl
[5], or ONOO− [3]. The data presented here show that this probe can be
oxidized by all three oxidants with a 1:1 stoichiometry in GSH-free so­
lutions. HPLC analyses confirmed formation of TCF-OH dye as a sole
oxidation product. The anticipated phenyl radical-derived products
(TCF-NO2, TCF-H) were not detected. Spin trapping experiments also
confirm that ONOO− -induced TCF-BA oxidation proceeds without
radical intermediates. This makes the TCF-BA probe similar to fluores­
cein boronate (Fl-B) [47], which produced fluorescein in a 99% yield in
the reaction with ONOO− [76–78].
In the case of excess of ONOO− and HOCl, we detect nitrated (TCF-
OH-3-NO2) and chlorinated (TCF-OH-3-Cl) products, respectively.

11
P. Siarkiewicz et al. Dyes and Pigments 192 (2021) 109405

Fig. 11. The reaction between the TCF-BA-2 probe and ONOO− in an aqueous solution containing a phosphate buffer (pH 7.4, 50 mM), DTPA (10 μM), and MeCN
(5%). (A) HPLC chromatograms of the standards (10 μM) and reaction mixtures of the TCF-BA-2 probe (20 μM) and TCF-OH-2 dye (20 μM) with ONOO− . The traces
were collected using an absorption detector set at 440 nm. (B) HPLC-based titration of the TCF-BA-2 probe (20 μM) with ONOO− .

Fig. 12. Oxidation of TCF-BA and TCF-

BA-2 probes by ONOO− generated from
co-generated O•–
2 from HX/XO, and NO
•

from spermine-NONOate. (A) The real-

time recorded fluorescent signal of
TCF-OH dye formed during TCF-BA
probe oxidation by ONOO− . (λexc 535
nm) (B) The real-time recorded absorp­
tion signal of TCF-OH-2 dye formed
during TCF-BA-2 probe oxidation by
ONOO− . Reactions were conducted in a
phosphate buffer (pH 7.4, 50 mM) con­
taining DTPA (100 μM), HX/XO and
spermine-NONOate (black) and with the
addition of 400 μM of GSH (red).

These compounds have similar spectroscopic characteristics as TCF-OH, accordingly adjusted.

which means that HPLC or liquid chromatography–mass spectrometry
(LC-MS) should be used for their detection and identification. Among
the oxidants tested, the fastest colorimetric and fluorescence response of
TCF-BA was observed for ONOO− . Like the boronates previously tested
[47,49,53,55], TCF-BA reacts with ONOO− with a rate constant several
orders of magnitude greater than for H2O2 or HOCl (Table 1). The high
rate constant of the reaction of the TCF-BA probe with ONOO− should
enable the detection of ONOO− in the presence of its physiological
scavengers (e.g., CO2 kONOO ~1 × 104 M− 1s− 1 [79], GSH kONOO = 1.36
× 103 M− 1s− 1 [80], and albumin kONOO = 8.31 × 103 M− 1s− 1 [81]), with
the caveat that signal intensity may depend on cellular nucleophile
levels (GSH and other biothiols). We demonstrated that thiols form
Michael adduct with both TCF-BA and TCF-OH. Therefore, the quanti­
fication of ONOO− with the aid of the TCF-BA probe in cell culture
should be done using HPLC or LC-MS to take into account effects of GSH
and other nucleophiles on the signal intensity from TCF-OH. These re­
sults also suggest that other chemical probes built on a fluorophore
similar to TCF-OH may also be reactive towards GSH, and thus, such a
possibility should be experimentally tested and data interpretation
We have also identified a TCF-based boronate probe containing the
naphthalene moiety, TCF-BA-2, whose oxidative response is indepen­
dent of GSH levels. TCF-BA-2 is oxidized to the appropriate 1-naphthol-
derived dye (TCF-OH-2) with a lower pKa (pKa = 5.3 [9]) than TCF-OH
dye (pKa = 7.6 [9]). An alternative approach based on chlorination of
the phenyl ring, resulting in a lower pKa value of the OH group in the
TCF-OH fluorophore, has been previously proposed to prevent its re­
action with GSH [11]. A negative charge of ionized hydroxyl group O−
can delocalize within chromophore. Thus, diminished partial positive
charges at the carbon atoms of the central double bond prevent forma­
tion of adducts with GSH. We propose to use the TCF-BA-2 probe as a
highly sensitive colorimetric probe for ONOO− in the presence or
absence of GSH.
In summary, we have characterized chemical reactivity of a TCF-
based boronate redox probe, TCF-BA, toward ONOO− , HOCl, and
H2O2, and identified reactions (Scheme 4) affecting the fluorescent
response of the probe. The red fluorescent product TCF-OH is formed in
the reaction of TCF-BA with all three oxidants. In the case of ONOO−
and HOCl, TCF-OH may undergo subsequent reactions to form nitrated

12
P. Siarkiewicz et al.
Scheme 4. Oxidative conversion of the studied TCF-BA and TCF-BA-2 probes and subsequent reaction.
TCF-OH-3-NO2 and chlorinated product TCF-OH-3-Cl, respectively. We
demonstrated that both the TCF-BA probe and TCF-OH dye react with
GSH to form TCF-BA-SG and TCF-OH-SG adducts, which affects the
chemical reactivity and colorimetric and fluorescence response of the
probe. Although the TCF-BA-2 probe also reacts with GSH, the absorp­
tion spectrum of its oxidation product, TCF-OH-2, is not influenced by
GSH and, therefore, can be applied for real-time monitoring of ONOO−
formation in the presence of GSH.
Author contributions
Przemysław Siarkiewicz: Investigation, Methodology, Formal anal­
ysis. Radosław Michalski: Methodology, Formal analysis, Funding
acquisition. Adam Sikora: Methodology, Investigation, Formal analysis,
Funding acquisition. Renata Smulik-Izydorczyk: Methodology, Investi­
gation, Formal analysis. Marcin Szala: Methodology, Investigation.
Aleksandra Grzelakowska: Methodology, Investigation. Julia Mod­
rzejewska: Investigation. Asha Bailey: Investigation. Jacek E. Nycz:
Methodology, Investigation. Balaraman Kalyanaraman: Funding
13
Dyes and Pigments 192 (2021) 109405
acquisition, Writing – review & editing. Jan Grzegorz Malecki: Meth­
odology, Investigation. Jacek Zielonka: Conceptualization, Investiga­
tion, Methodology, Formal analysis, Resources, Writing – review &
editing, Supervision, Funding acquisition. Radosław Podsiadły:
Conceptualization, Investigation, Methodology, Resources, Writing –
review & editing, Funding acquisition, Supervision. All authors
reviewed the results and approved the final version of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the Polish National Science Centre
within the SONATA BIS 6 program (grant no. 2016/22/E/ST4/00549 to
R.P.), the Polish National Science Centre within the SONATA BIS
P. Siarkiewicz et al.
program (grant no. 2015/18/E/ST4/00235 to A.S.), the Polish National
Science Centre within the SONATA program (grant no. 2018/31/D/
ST4/03494 to R.M.), Institutional Research Grant IRG #16-183-31 from
the American Cancer Society and the MCW Cancer Center to J.Z., and
NIH NCI (grant no. R01 CA208648) to B.K.
This work was supported by the National Center for Research and
Development (Warsaw, Poland) within the grant InterChemMed
(POWR.03.02.00–00-I029/16).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2021.109405.
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