Free Radical Biology and Medicine 179 (2022) 34–46

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Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

Water-soluble cationic boronate probe based on coumarin imidazolium

scaffold: Synthesis, characterization, and application to cellular
peroxynitrite detection
Aleksandra Grzelakowska a, *, Julia Modrzejewska a, Jolanta Kolińska a, Marcin Szala a,
Monika Zielonka b, Karolina Dębowska c, Małgorzata Zakłos-Szyda d, Adam Sikora c,
Jacek Zielonka b, **, Radosław Podsiadły a, ***
a
Institute of Polymer and Dye Technology, Faculty of Chemistry, Lodz University of Technology, Stefanowskiego 16, 90-537, Lodz, Poland
b
Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, United States
c
Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology, Żeromskiego 116, 90-924, Lodz, Poland
d
Institute of Molecular and Industrial Biotechnology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 2/22, 90-537, Lodz,
Poland

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

Keywords: Peroxynitrite (ONOO− ) has been implicated in numerous pathologies associated with an inflammatory compo­
Water-soluble fluorescent probe nent, but its selective and sensitive detection in biological settings remains a challenge. Here, the development of
Coumarin-based probe a new water-soluble and cationic boronate probe based on a coumarin-imidazolium scaffold (CI-Bz-BA) for the
Mitochondria-targeted two-photon fluorescent
fluorescent detection of ONOO− in cells is reported. The chemical reactivity of the CI-Bz-BA probe toward
probe
Redox probe
selected oxidants known to react with the boronate moiety was characterized, and the suitability of the probe for
Peroxynitrite the direct detection of ONOO− in cell-free and cellular system is reported. Oxidation of the probe results in the
formation of the primary hydroxybenzyl product (CI-Bz-OH), followed by the spontaneous elimination of the
quinone methide moiety to produce the secondary phenol (CI–OH), which is accompanied by a red shift in the
fluorescence emission band from 405 nm to 481 nm. CI-Bz-BA reacts with ONOO− stoichiometrically with a rate
constant of ~1 × 106 M-1s-1 to form, in addition to the major phenolic product CI–OH, the minor nitrated
product CI-Bz-NO2, which is not formed by other oxidants tested or via myeloperoxidase-catalyzed oxidation/
nitration. Both CI–OH and CI-Bz-NO2 products were also formed in the presence of cogenerated fluxes of nitric
oxide and superoxide radical anion produced during decomposition of a SIN-1 donor. Using RAW 264.7 cells, we
demonstrate the ability of the probe to report endogenously produced ONOO− via fluorescence measurements,
including plate reader real time monitoring and two-photon fluorescence imaging. Liquid chromatography/mass
spectrometry analyses of cell extracts and media confirmed the formation of both CI–OH and CI-Bz-NO2 in
macrophages activated to produce ONOO− . We propose the use of a combination of real-time monitoring of
probe oxidation using fluorimetry and fluorescence microscopy with liquid chromatography/mass spectrometry-
based product identification for rigorous detection and quantitative analyses of ONOO− in biological systems.

1. Introduction reaction between nitric oxide (•NO) and superoxide radical anion
(O2•‒) [1–3], has been considered as a key oxidizing and nitrating
Among various reactive oxygen and nitrogen species in biological species formed in various pathophysiological states, especially involving
systems, peroxynitrite (ONOO− ), the product of a diffusion-controlled inflammatory components [4–7]. Recent literature points to the

* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: aleksandra.grzelakowska@p.lodz.pl (A. Grzelakowska), julia.modrzejewska@dokt.p.lodz.pl (J. Modrzejewska), jolanta.kolinska@p.lodz.pl
(J. Kolińska), marcin.szala@p.lodz.pl (M. Szala), mzielonka@mcw.edu (M. Zielonka), karolina.debowska@p.lodz.pl (K. Dębowska), malgorzata.zaklos-szyda@p.
lodz.pl (M. Zakłos-Szyda), adam.sikora@p.lodz.pl (A. Sikora), jzielonk@mcw.edu (J. Zielonka), radoslaw.podsiadly@p.lodz.pl (R. Podsiadły).

https://doi.org/10.1016/j.freeradbiomed.2021.12.260
Received 23 October 2021; Received in revised form 7 December 2021; Accepted 14 December 2021
Available online 17 December 2021
0891-5849/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
A. Grzelakowska et al.
involvement of ONOO− in pulmonary hypertension [8], liver and kidney
injury [9–12], and the mechanism of immunosuppressive effects of
myeloid-derived suppressor cells (MDSCs) in cancer [13]. ONOO− can
react directly with thiols, carbon dioxide, heme proteins, iron–sulfur
centers, and zinc thiolates [14,15]. In addition, the products of the
decomposition of ONOO− under physiological conditions include highly
oxidizing and/or nitrating hydroxyl radical (•OH), carbonate radical
anion (CO3•–), and nitrogen dioxide (•NO2), which may directly modify
and damage cell components [10]. The accurate detection of ONOO− in
different cellular and extracellular compartments, and the estimation of
its formation rates, is required to understand how nitric oxide-derived
oxidants affect biological processes, including mitochondrial dysfunc­
tion and cell death. The detection of cellular ONOO− is challenging due
to its short lifetime in biological systems [16] and low steady-state
concentration [17,18]. Initially, indirect methods, such as analysis of
nitrotyrosine residues of modified proteins [19,20] or low-throughput
electrochemical approaches [21], were used for its detection. Nitrative
modifications of proteins and lipids have been identified as biomarkers
for reactive nitrogen species in numerous diseases [22,23]. With the
constant development and improvement of LC-MS-based methods, the
detection of nitrated products on endogenous molecules have gained
prominence and helped identify targeted for oxidative/nitrative modi­
fication of cellular components. Tyrosyl moieties in proteins may be
nitrated, however, via different biochemical pathways including not
only peroxynitrite, but also via heme peroxidase (e.g. myeloperoxidase,
eosinophil peroxidase)-catalyzed nitration in the presence of nitrite and
hydrogen peroxide [24,25].
A more recent approach to determine ONOO− in biological systems is
based on employing low-molecular-weight probes that, by a fast reac­
tion with ONOO− , form stable and characteristic products [26,27]. It is
preferable to use fluorescent probes that react with cellular oxidants to
form the products, which can be detected using fluorescence-based
techniques and allow for sensitive detection of reactive species in bio­
logical systems.
A fluorescent probe must fulfill many requirements, such as lack of
toxicity, analyte specificity, and solubility in aqueous solutions, to be
used in biological systems [28,29]. Also, the amount of the detected
product should be proportional to the amount of the oxidant produced.
Furthermore, the probe should not affect the system being investigated
(i.e., it cannot induce reactive oxygen species formation or change the
redox status of the cell). Preferably, the probe should be selective toward
a single oxidant. In case of a lack of selectivity, different oxidants should
form different, oxidant-specific products, easily distinguishable by the
detection technique used. It is, therefore, important to establish what
species may react with the probe under various conditions, and the
relative rates of those reactions. The product(s) of the reaction between
the probe and the oxidant should be sufficiently stable to accumulate
over the time of the experiment to allow quantitative analyses [26,29].
Most reported redox probes constructed via masking of hydroxyl or
amino groups in fluorophore require the use of an organic solvent to
prepare the stock solutions and to improve their solubility in water.
However, organic cosolvent may result in cytotoxicity or interfere with
the assays, for example, by scavenging the analyte of interest. It was
shown that DMSO may compete with boronate probes for hypochlorous
acid (HOCl) [30,31]. Therefore, the development of water-soluble yet
cell-permeable probes would be highly beneficial.
Over the last decade, numerous molecular probes have been pro­
posed to measure ONOO− , with different selectivity and sensitivity [20,
32,33]. Among them, boronate-based molecular probes emerge as su­
perior to other chemical sensors reported to date for this oxidant
detection due to their rapid and direct reaction with ONOO− [26,32,34].
Boronate probes react stoichiometrically with ONOO− to yield the cor­
responding hydroxylated compound as the major reaction product.
Boronates can also be converted to the same major phenolic product by
HOCl, hydrogen peroxide (H2O2), and some other biologically relevant
oxidants, but the reaction with ONOO− is kinetically most favorable. In
35
Free Radical Biology and Medicine 179 (2022) 34–46
Fig. 1. Boronate probe based on 1-ethyl-3-(4-methylene-7-hydroxy-2H-chro­
men-2-one) imidazolium chloride (CI-Bz-BE).
the case of ONOO− , the oxidation takes place nearly a million times
faster than with H2O2 (k(ONOO⁻) ~106 M-1s-1; k(H₂O₂) ~100 M-1s-1) [34].
Reaction of the boronate probes with ONOO− involves nucleophilic
addition of the oxidant to the boron atom, followed by nitrite elimina­
tion and deboronation to form a corresponding phenolic product. With a
few exceptions reported, ONOO− -induced oxidation of aryl boronates
(boronic acids, ArB(OH)2, or boronic esters, ArBPin) typically proceeds
via two pathways: the major, nonradical pathway leads to the formation
of corresponding phenol (ArOH), and the minor, radical pathway leads
to formation of phenyl radicals and radical-derived minor products
(ArH, ArNO2) [3,30,35–39]. Depending on the compound structure, the
contribution of the radical pathway in the oxidation of boronate may
range from <1% to 20%. The minor pathway is highly specific for the
reaction of boronates with ONOO− . Replacement of the boronate moiety
in boronic compounds by the nitro group strongly supports an inter­
mediary role of ONOO− and may serve as a unique marker for ONOO−
formation in cell-free and cellular systems [34]. In case of bor­
onobenzylated probes, the formation of the fluorescent product is a
two-step process [30,31,40]. The reaction of oxidant with the boronate
group in probe leads to the corresponding primary phenol, which is not
stable at physiological pH and upon spontaneous elimination of quinone
methide (QM) leads to the formation of a final, stable phenolic product
(Scheme S1).
Lipophilic cationic agents have been shown to accumulate in cell
mitochondria in response to the mitochondrial membrane potential [41,
42]. This property was utilized in developing positively charged probes
for mitochondrial membrane potential, but also to target various mo­
lecular probes and bioactive agents to mitochondria [43–45]. The most
common strategy to target the chemical agents to mitochondria is their
conjugation to a triphenylphosphonium cation (TPP+). Other organic
cations, where the positive charge is delocalized over or shielded by
aromatic rings, have also been shown to accumulate in mitochondria
[46,47]. Recently, the coumarin imidazolium derivative containing the
2,4-dinitrobenzenesulfonyl group as a thiol-sensing moiety was pro­
posed as a mitochondria-targeted two-photon fluorescent probe for the
detection of mitochondrial thiols [48]. Upon reaction with thiols, the
probe was converted into the hydroxycoumarin imidazolium (CI–OH)
product. The fluorescence colocalization measurements confirmed a
high degree of mitochondrial localization of the CI–OH product, with a
Pearson’s correlation coefficient of 0.9, similar to a TPP+-linked
hydroxycoumarin standard [48]. This provides an opportunity to target
coumarin-based sensors to mitochondria by conjugation of the coumarin
fluorophore with the alkylimidazolium cation via a methylene bridge.
Due to favorable photophysical and photochemical properties,
including high fluorescence yield and good photostability, the coumarin
skeleton often is chosen as the basis for the construction of various types
of chemosensors, by combing the 2H-chromen-2-one moiety with an
analyte-sensing moiety [49]. One of the best-studied commercially
A. Grzelakowska et al.
available pro-coumarin sensors used for the detection of ONOO− is
coumarin-7-boronic acid (CBA) [3,50–53].
Here, we designed and synthesized a new water-soluble florescent
boronate probe based on a coumarin scaffold, bearing a mitochondria-
targeting imidazolium cationic moiety (CI-Bz-BA, Fig. 1). The chemi­
cal reactivity of this probe toward selected biological oxidants is re­
ported. The minor, yet specific products of the reaction between the
probe and ONOO− have been identified, and the authentic standards
were synthesized (Scheme S2-S3). The results are complemented by a
detailed spectroscopic characterization of the probe and its oxidation/
nitration products, followed by application of the probe to detect
ONOO− formed in situ in cell-free and cellular systems.
2. Materials and methods
2.1. Materials
Myeloperoxidase (MPO) and 3-morpholinosydnonimine hydrochlo­
ride (SIN-1) were from Calbiochem (San Diego, CA). Catalase (CAT,
from bovine liver) and superoxide dismutase (SOD, from bovine eryth­
rocytes) were obtained from Sigma Aldrich (St. Louis, MO). ONOO− was
prepared as reported previously [54] and stored at − 80 ◦ C. H2O2 and
HOCl were from Merck (Kenilworth, NJ). Other reagents and starting
materials for chemical syntheses were purchased from commercial
vendors and used without further purification.
2.2. Synthesis
Detailed synthetic procedures for all compounds shown are pre­
sented in the Supplementary Information.
2.3. Spectroscopic study
The absorption spectra were measured using a Jasco V–670
UV–vis–NIR spectrophotometer (Jasco, Japan). The steady-state emis­
sion spectra, fluorescence excitation/emission matrix spectra, and
fluorescence lifetimes were recorded by a FLS-920 spectrofluorometer
(Edinburgh Instruments, Livingston, UK). Fluorescence spectra were
taken with a slit width of 0.5 nm. A picosecond-pulsed light-emitting
diode (EPLED 340, Edinburgh Instruments) was used as an excitation
light source in the time-resolved measurements. The diode had a peak
wavelength at 341.2 nm and a pulse width of 835.7 ps. The excitation
and emission bandpasses were 2.0 nm. The instrument profile was ob­
tained by replacing the sample with Ludox as a scatter. The data were
analyzed by a least-squares reconvolution procedure using the software
package provided by the Edinburgh Instruments. Fluorescence lifetimes
were calculated from intensity decay analyses [39]. Fluorescence
quantum yields were determined by the relative method using the
procedure described previously [40]. Quinine sulfate was used as a
reference standard [41,42].
2.4. Preparation of solutions
For all experiments, stock solutions of CI-Bz-BE, CI–OH, CI-Bz-H,
and CI-Bz-NO2 were prepared by dissolving the solid compounds in
DMSO (10 mM) except for the measurements involving HOCl, for which
the CI-Bz-BE probe was dissolved in water (1 mM). Although all pre­
pared compounds are soluble in water, in accordance with the recom­
mendations [55], the concentrated stock solutions were prepared in
DMSO to avoid hydrolysis of the probe in aqueous media when stored
for extended periods of time.
The working solutions of ONOO− , HOCl, and H2O2 were freshly
prepared before each experiment and kept on ice. The concentration
ONOO− was determined by spectrophotometry, using the extinction
coefficient value 1.7 × 103 M-1cm-1 (at 302 nm, in 0.1 M NaOH). The
concentrations of HOCl and H2O2 were also determined by
36
Free Radical Biology and Medicine 179 (2022) 34–46
spectrophotometry, using the extinction coefficient values of 350 M-
1
cm1 (at 292 nm, in 0.1 M NaOH) and 39.4 M-1cm-1 (at 240 nm, in
water), respectively. Solutions of oxidants were added directly to the
buffered reaction mixtures. All measurements were performed in
aqueous solutions of phosphate buffer (100 mM, pH 7.4) containing
diethylenetriaminepentaacetic acid (dtpa, 10 μM).
In reactions involving HOCl, dtpa was omitted to avoid scavenging of
HOCl by dtpa. To investigate the effect of carbon dioxide (CO2) on the
yield of the products of the reaction of CI-Bz-BA with ONOO− , we car­
ried out the reaction in the presence of sodium bicarbonate (NaHCO3).
ONOO− , HOCl, or H2O2 was rapidly mixed with CI-Bz-BA using a vortex
mixer (10 s) and incubated for 5 min, 15 min, and 24 h, respectively,
before HPLC and spectroscopic analyses.
2.5. HPLC analyses of the products of the CI-Bz-BA reaction with
oxidants in cell-free system
HPLC analyses of CI-Bz-BA and its oxidation products (CI–OH, CI-
Bz-H, CI-Bz-NO2) were performed using a Shimadzu instrument (Kyoto,
Japan) equipped with absorption and fluorescence detectors. The sam­
ples (10 μl) were separated using a reverse-phase column (Phenomenex,
Kinetex Biphenyl, 50 mm × 4.6 mm, 2.6 μm) equilibrated 0.1% (v/v)
trifluoroacetic acid aqueous solution containing 5% (v/v) MeCN.
Gradient elution was performed at a flow rate of 1.5 mL/min with
ultraviolet–visible absorption detection (λ = 254 nm and 330 nm). The
compounds were eluted by raising MeCN concentration (v/v) from 5%
to 55% over 5.5 min followed by an increase to 100% from 5.5 to 6 min.
Under these conditions, CI–OH shows a retention time of 2.50 min, CI-
Bz-BA of 3.65 min, CI-Bz-H of 4.75 min, and CI-Bz-NO2 of 4.80 min.
2.6. Oxidation of CI-Bz-BA induced by ONOO− produced from SIN-1
SIN-1 slowly decomposed in aqueous solution at pH 7.4 producing
both •NO and O2•–, which react in a diffusion-limited reaction, yielding
ONOO− [56–58]. SIN-1 stock solution (25 mM) was prepared in 50 mM
HCl.
SIN-1 (50 μM) was added to a solution of CI-Bz-BA (10 μM) in a
phosphate buffer (pH 7.4, 100 mM) containing dtpa (10 μM), and the
changes of fluorescence intensity during the generation of •NO and O2•–
obtained from the decomposition of SIN-1 in the presence or absence of
CAT (100 U/mL) and/or SOD (0.1 mg/mL) were monitored for 1 h in the
kinetic mode.
The HPLC analysis was used to monitor the decay of SIN-1 and CI-Bz-
BA, and to identify the products formed. To initiate the reaction, a SIN-1
solution (100 μM) was combined with a phosphate buffer solution (100
mM, pH 7.4) containing dtpa (10 μM) and CI-Bz-BA (200 μM). The
analysis was performed using a Shimadzu instrument equipped with an
absorption detector, as described above. Changes in the peak area were
used to determine the progress of the reaction over the time.
2.7. Oxidation of CI-Bz-BA induced by H2O2 in the presence of MPO and
NaNO2
MPO stock solution (5 μM) was prepared in a phosphate buffer (100
mM, pH 7.4), aliquoted, and stored at − 80 ◦ C. The stock solutions of
H2O2 (100 mM) and nitrite (NaNO2, 1 M) were prepared in distilled
water. CI-Bz-BA (100 μM) was incubated with MPO (0–15 nM), H2O2
(500 μM), and NaNO2 (5 mM) in a phosphate buffer (100 mM, pH 7.4)
containing dtpa (10 μM) at room temperature for 60 min. Reactions
were terminated by adding CAT (100 U/mL), and the products were
immediately analyzed by HPLC, as described above.
2.8. Kinetic studies
The determination of the rate constants of CI-Bz-BA with the
different oxidants was carried out under pseudo-first-order conditions,
A. Grzelakowska et al.
using an excess of either the probe (in the case of ONOO− and HOCl) or
the oxidant (in the case of H2O2) as described in the Supplementary
Information.
The rate constant for reaction of CI-Bz-BA with ONOO− was also
determined via competition kinetics assays utilizing boronate probes
CBA and peroxy crimson-1 (PC1) as the competitors, using the reported
rate constants of 1.1 × 106 M-1s-1 [3] and 9.9 × 105 M-1s-1 [40],
respectively. The details of the competition kinetics analyses are
described in the Supplementary Information.
2.9. Cell culture experiments
RAW 264.7 cells (ATCC, USA) were maintained in Dulbecco’s
Modified Eagle’s Medium (Gibco, USA) containing 10% (v/v) fetal
bovine serum, L-glutamine (2 mM), penicillin (100 U/ml), and strepto­
mycin (0.1 mg/ml), and grown at 37 ◦ C in a 5% carbon dioxide
atmosphere.
To determine a cytotoxic potential of CI-Bz-BA and CI–OH, cells
were incubated with the compounds for 4 h or 24 h before measure­
ments of cell viability. Two separate assays, the MTT and PrestoBlue,
were used to determine cell metabolic activity.
A stock solution of the CI-Bz-BE probe (10 mM in DMSO) was added
directly to the cell media to obtain a 20 μM concentration, resulting in
0.2% (v/v) final concentration of DMSO.
Oxidation of CI-Bz-BA was monitored in a 96-well fluorescence plate
reader. The cells were seeded in black 96-well plates at a concentration
of 3 × 104 cells per well (in 150 μL total volume) and incubated over­
night at 37 ◦ C in a 5% carbon dioxide atmosphere. To induce iNOS
expression and •NO production, cells were incubated for 8 h with
interferon γ (IFN-γ, 50 U/ml) and lipopolysaccharide (LPS, 0.5 μg/ml) in
Dulbecco’s Modified Eagle’s Medium. Subsequently, the medium was
replaced by the DPBS buffer supplemented with sodium pyruvate (0.33
mM) and glucose (5.56 mM, DPBS-GP, Gibco, USA), and O2•– production
was stimulated by the addition of phorbol 12-myristate 13-acetate
(PMA, 1 μM). Concomitant stimulation of •NO and O2•– production re­
sults in ONOO− generation by RAW 264.7 cells, as described previously
[35,53]. At the time of the addition of PMA, the CI-Bz-BA probe (20 μM)
also was added. Where indicated, L-N-nitroarginine methyl ester hy­
drochloride (L-NAME, 1 mM, Cayman Chemical, USA) or CAT (1000
U/mL) were added before stimulation with PMA. Immediately after the
addition of DPBS-GP containing the probe with or without PMA,
L-NAME, and CAT, the plate was placed in a plate reader prewarmed to
37 ◦ C. Total fluorescence intensities were acquired using a FLUOstar
Omega plate reader (BMG LABTECH, Germany) equipped with the
appropriate excitation and emission filters (λex = 355 nm, λem = 520
nm). The instrument was kept at 37 ◦ C during the measurements, and
fluorescence intensity was read from the bottom of each well. Changes in
the fluorescence intensity were monitored for 2 h in the kinetic mode.
2.10. LC-MS analyses of extraction of CI-Bz-BA oxidation products
To identify the oxidation products of CI-Bz-BA formed in cells by
endogenously generated ONOO− , RAW 264.7 macrophages were seeded
in 10-cm culture dishes (1.5 × 106 cells/dish). Cells were stimulated to
produce ONOO− using same treatments as in the plate reader experi­
ments. After the addition of CI-Bz-BA (20 μM) in DPBS-GP, dishes with
cells were incubated for 1 h at 37 ◦ C in a carbon dioxide-free incubator.
The oxidation products of CI-Bz-BA by endogenously generated ONOO−
were extracted according to a slightly modified version of a published
procedures [35,55,59].
Coumarin-based products were analyzed by liquid chromatogra­
phy–mass spectrometry (LC-MS) performed using a Shimadzu LC-MS
8030 triple quadrupole mass detector coupled to a Shimadzu Nexera 2
ultra-high-performance liquid chromatography system. The samples
were injected on a Raptor Biphenyl column (Restek, 100 mm × 2.1 mm,
2.7 μm) equilibrated with 10% of MeCN in water containing 0.1% of
37
Free Radical Biology and Medicine 179 (2022) 34–46
formic acid. The compounds were eluted by increasing the MeCN con­
centration in the mobile phase from 5% to 100% over 6 min. The flow
rate was set at 0.5 mL/min, and the flow was diverted to waste during
the first 1.5 min and after 6.5 min, counting from the time of injection.
CI–OH (retention time 2.1 min), CI-Bz-BE (retention time 5.3 min), CI-
Bz-BA (retention time 3.4 min), CI-Bz-H (retention time 4.5 min), and
CI-Bz-NO2 (retention time 4.55 min) were detected as positive ions
using the multiple reaction monitoring mode, with the primary/frag­
ment ion pairs 487.2 > 270.05, 405.1 > 270.1, 271.0 > 147.05, 406.0 >
270.0, and 361.00 > 270.0.
2.11. Two-photon fluorescence imaging
For two-photon fluorescence microscopy, cells were seeded over­
night into 35 mm glass bottom dishes (MatTek Corporation, Ashland,
MA), followed by activation to produce ONOO− , as described above.
Cells were imaged using a Carl Zeiss LSM510 META detection system
(Jena, Germany). Images were collected with a 40 × objective and
image acquisition software. Two-photon excitation properties of the
CI–OH fluorophore were characterized previously [48]. However,
before imaging ONOO− production with the CI-Bz-BE probe, the
two-photon illumination parameters were optimized using cells loaded
with the fluorescent product, CI–OH, and the excitation wavelength of
735 nm; emission light in the range 465–550 nm was selected.
3. Results and discussion
3.1. Synthesis of coumarins
The CI-Bz-BE probe and the anticipated major and minor products of
its reaction with ONOO− were obtained via two simple steps presented
in Scheme S2. Detailed synthetic protocols are included in the Supple­
mentary Information. The compounds were obtained with good yields,
ranging from 73% to 91%. NMR spectroscopy and mass spectrometry
studies confirmed the chemical structures of the compounds (See
Experimental section in Supplementary Information and Figs. S1–S8.)
3.2. Spectroscopic characterization of the probe and its oxidation/
nitration products
The spectroscopic properties of coumarins are presented in Table S1.
UV–vis absorption and fluorescence spectra at pH of 7.4 were charac­
terized for all products (Figs. S12-S13). In aqueous solutions, CI-Bz-BE
(pinacolate ester) undergoes hydrolysis to the boronic acid form (CI-Bz-
BA). Thus, the spectroscopic properties in an aqueous solution pertain to
CI-Bz-BA.
Both the probe and the anticipated products carrying the coumarin
imidazolium scaffold show absorption bands in the ultraviolet region
located at 323–330 nm. The presence of benzyl groups does not cause
any significant change in the absorption spectra as compared with
CI–OH. The fluorescence spectra of compounds CI–OH, CI-Bz-BA, and
CI-Bz-H show one fluorescence maximum, while compound CI-Bz-NO2
possess two maxima. For all compounds, the fluorescence emission
maximum is located in the visible spectral range. The incorporation of
benzyl groups causes a blue shift of the emission wavelength (λem) and a
decrease in fluorescence lifetime when compared with CI–OH. The
strong electron-withdrawing substituent –NO2 (compound CI-Bz-NO2)
largely decreases the fluorescence intensity, compared with the unsub­
stituted system CI-Bz-H and the CI-Bz-BA probe. As shown in Fig. S12,
both the probe and its main oxidation product exhibit fluorescence. A
comparison between the fluorescence emission spectra (Fig. 12B) or the
fluorescence excitation-emission matrix spectra (Fig. S14) of CI-Bz-BA
and CI–OH indicates clear distinction of the emission bands, suggesting
that the fluorescence of the probe should not interfere with the fluo­
rescence of its oxidation product. The introduction of the imidazolium
moiety to the coumarin skeleton results in a red shift of the emission
A. Grzelakowska et al. Free Radical Biology and Medicine 179 (2022) 34–46

comparison with 7-hydroxycoumarin (COH) (Table S1, Fig. S15).

3.3. Chemical reactivity of CI-Bz-BA probe toward selected biological

oxidants

The reaction of CI-Bz-BA with ONOO− leads to the batochromic shift

Fig. 2. The fluorescence spectra changes of CI-Bz-BA (10 μM) after incubation
in the presence of ONOO− (0–30 μM) in a phosphate buffer (100 mM, pH 7.4 in
the presence of 10 μM dtpa). Inset: Fluorescence intensity measured at 405 nm
and 481 nm for a solution of CI-Bz-BA (10 μM) in a phosphate buffer (100 mM,
pH 7.4 in the presence of 10 μM dtpa) after the addition of ONOO− (0–30 μM).
Data are means ± standard deviation of three independent experiments. The
emission spectra (excitation at 330 nm; ex/em slit: 0.5 nm) were collected after
5 min incubation of CI-Bz-BA with the oxidant.
band with a slight decrease in the fluorescence quantum yield in
of the fluorescence of the solution, with the spectrum of the product
resembling that of authentic CI–OH (Fig. 2). A single isosbestic point
was observed at 450 nm for this oxidative conversion. There is a distinct
linear increase in fluorescence intensity at 481 nm and depletion of the
intensity at 405 nm with successive addition of ONOO− at doses up to
10 μM, followed by complete disappearance of the band due to the probe
and saturation of the intensity due to the product. This suggests a 1:1
reaction stoichiometry between the probe and the oxidant.
Next, the HPLC analyses were performed to for the identification and
quantitative analyses of the reaction products formed during the reac­
tion between the CI-Bz-BA probe and ONOO− . Both product identifi­
cation and quantitative analyzes were done using independently
synthesized standards. HPLC analyses confirmed that CI–OH is the
major product of the reaction of CI-Bz-BA with ONOO− (Fig. 3A), while
additional, minor products (e.g., CI-Bz-NO2) are also formed, even when
the probe was present in excess of the oxidant (Fig. 3C). Titration of the
probe with ONOO− results in the product profiles shown in Fig. 3B and
D, indicating the formation of both phenolic (major) and radical-
mediated products (minor) in a dose-dependent manner. The maximal
yield of the CI–OH product was close to 92%, and a slight excess of the
oxidant was required for complete consumption of the CI-Bz-BA probe.
These data agree with the fluorescence measurements conducted under
the same experimental conditions (Fig. 2). The HPLC analyses revealed

Fig. 3. Oxidation of the CI-Bz-BA probe by ONOO− . (A, C) The HPLC chromatograms of the incubation mixtures containing CI-Bz-BA (100 μM) and ONOO− (0, 80,
and 200 μM) in a phosphate buffer (100 mM, pH 7.4 in the presence of 10 μM dtpa). (B, D) HPLC-based titration of CI-Bz-BA (100 μM) with ONOO− (0–300 μM) in a
phosphate buffer (100 mM, pH 7.4 in the presence of 10 μM dtpa). Data are means ± standard deviation of three independent experiments. The HPLC traces were
collected after 5 min incubation of CI-Bz-BA with oxidant using the absorption detector set at 330 nm.

38
A. Grzelakowska et al. Free Radical Biology and Medicine 179 (2022) 34–46

Scheme 1. The major and minor products of the reaction of CI-Bz-BA and ONOO− .

Fig. 4. Kinetics of the reaction of CI-Bz-BA with ONOO− . (A) Kinetic traces of CI-Bz-BA fluorescence decay observed at 405 nm. (B) The dependence of pseudo-first-
order rate constants of the reaction of Cl-Bz-BA probe with ONOO− on the probe concentration. Solutions consisted of the CI-Bz-BA probe (25–50 μM), phosphate
buffer (0.1 M, pH 7.4 in the presence of 200 μM dtpa), and ONOO− (5 μM). Data are means ± standard deviation of three independent experiments.

the build-up of two partially overlapping peaks with retention times of
4.75 and 4.80 min, corresponding to CI-Bz-H and CI-Bz-NO2, respec­
tively, the two products anticipated for the radical pathway of the re­
action. We expected that the •NO2 radical formed in the minor pathway
and/or during decomposition of excess ONOO− would lead to nitration
of both the primary (CI-Bz-OH) and possibly the secondary (CI–OH)
phenolic products, depending on the time required for CI-Bz-OH to
eliminate QM and form CI–OH. The nitrated CI–OH standard was not
prepared; however, after the reaction of CI-Bz-BA with ONOO− , we
detected in the reaction mixture the same product as obtained when
CI–OH was mixed with ONOO− (Fig. S16). This confirms that the
elimination of the QM moiety is fast enough to produce CI–OH, when
the •NO2 radical is still present in the solution.
We hypothesized that nitration of the primary phenolic product CI-
Bz-OH by •NO2 would produce CI-Bz-OH-NO2, which after the elimi­
nation of QM-NO2 through a self-immolative reaction mechanism would
produce CI–OH as the stable end product (Scheme 1). QM-NO2 is ex­
pected to react with water to form 4-hydroxy-3-nitrobenzyl alcohol
(OH-Bz-3NO2–OH). However, OH-Bz-3NO2–OH may be also formed as
a product of the nitration of 4-hydroxybenzyl alcohol (OH-Bz-OH),
which is formed upon elimination of QM by CI-Bz-OH. As shown in
Fig. 3C and D, with an increase in ONOO− concentration, especially at or
above the concentration of the probe, a new product eluting at 2.81 min
appeared in the HPLC traces. The retention time of this product was
identical to that of the independently synthesized standard (Scheme S3),
OH-Bz-3NO2–OH. The same product was also formed when OH-Bz-OH
was mixed with ONOO− (Fig. S17).
The rate constant of the reaction between CI-Bz-BA and ONOO− was
determined using the stopped-flow technique due to its expected high
value and the instability of ONOO− at pH 7.4. Because the fluorescent
product CI–OH is formed via a multi-step process involving the self-
immolation of QM, the pseudo-first-order rate constants were

39
A. Grzelakowska et al.
Fig. 5. Changes of fluorescence intensity of an aqueous solution of CI-Bz-BA
(10 μM) due to co-generation of •NO and O2•– from the decomposition of SIN-1
(50 μM) in the presence or absence of CAT (100 U/mL) and/or SOD (0.1 mg/
mL) in a phosphate buffer (100 mM, pH 7.4) containing dtpa (10 μM). Inset:
The rates of increase in the fluorescence signal intensity from incubations
contained CI-Bz-BA and SIN-1 in the presence or absence of CAT and/or SOD.
The solutions containing CI-Bz-BA were excited at 330 nm, and the emitted
light intensity was measured at 481 nm (excitation/emission slit = 0.5 nm).
Data are means ± standard deviation of three independent experiments.
obtained by fitting the decay of the fluorescence signal recorded at 405
nm, following the consumption of the probe (Fig. 4). From the slopes of
the plots of the observed first-order rate constants versus the concen­
tration of the reactant, the second-order rate constant was determined to
be equal (1.3 ± 0.1) × 106 M-1s-1. This value is close to the values re­
ported for other boronate-based probes [30,31,34–36,40,60]. The lim­
itation of the stopped-flow-based monitoring the product consumption
was the relatively small difference in the fluorescence intensity before
and after the reaction. This is because the probe was used in excess of
ONOO− and possibly due to smaller differences in fluorescence intensity
between the probe and the primary phenol, as compared with CI–OH.
The rate constant of the reaction of CI-Bz-BA with ONOO− was also
determined using the competition kinetic approach (Fig. S22). The
coumarin boronate (CBA) and resorufin boronate (PC1) derivatives
were used as competitors in kinetic experiments. The values, 5.4 × 105
Fig. 6. The fluorescence spectra changes of CI-Bz-BA (10 μM) after incubation in the presence of (A) H2O2 and (B) HOCl (0–30 μM) in a phosphate buffer (100 mM,
pH 7.4). The emission spectra (excitation at 330 nm; ex/em slit: 0.5 nm) were collected after 24 h and 15 min incubation of CI-Bz-BA with H2O2 and HOCl,
respectively. Insets: Data are means ± standard deviation of three independent experiments.
40
Free Radical Biology and Medicine 179 (2022) 34–46
M-1s-1 and 5.2 × 105 M-1s-1, obtained from the competition assays using
CBA and PC1 competitors, respectively, are ca. two-fold lower than the
value from the stopped-flow experiment, which may be due to the
above-mentioned limitations of the stopped-flow experiments and/or
other factors. Nevertheless, the determined values of the rate constant
are close to the values reported for other boronate probes and indicate
the ability of CI-Bz-BA to rapidly intercept and detect ONOO− .
The oxidative conversion of the CI-Bz-BA probe by ONOO− was also
investigated in the system co-generating •NO and O2•‒. To this end, the
CI-Bz-BA probe was incubated with SIN-1 in a phosphate buffer (pH 7.4)
at room temperature. Fig. 5 depicts the changes of fluorescence intensity
of CI-Bz-BA at 481 nm over time in the presence and absence of SIN-1
and/or CAT and SOD. The addition of SIN-1 to the solution of CI-Bz-
BA at pH 7.4 resulted in a strong and time-dependent increase in fluo­
rescent intensity, which was partially inhibitable by SOD, but not by
CAT. This suggests that ONOO− formed in situ from •NO and O2•‒ is
responsible for oxidation of CI-Bz-BA to the corresponding fluorescent
product.
To identify and quantify the products formed during the incubation
of CI-Bz-BA and SIN-1, the reaction mixtures were analyzed by HPLC. As
shown in Fig. S18, both the major product, CI–OH, and the minor
products, CI-Bz-H and CI-Bz-NO2, were detected, and both CI–OH and
CI-Bz-NO2 increased almost linearly with the time of incubation within
the 4-h period. These results further confirm that ONOO− , whether
added as a bolus or generated in situ, can be detected using the CI-Bz-BA
probe. Furthermore, in addition to the major and fluorescent product,
CI–OH, the reaction results in the formation of ONOO− -specific nitrated
product, CI-Bz-NO2.
As ONOO− − dependent reactions are often altered in the presence of
CO2 [61–63], we investigated the effects of varying the levels of HCO3−
on the extent of oxidation of the CI-Bz-BA probe. With increasing HCO3−
concentration, the yield of the CI–OH and CI-Bz-NO2 products
decreased (Fig. S19), suggesting that the probe competes with CO2 for
ONOO− . This is in contract with many other probes reported for
ONOO− , which detect ONOO− -derived radicals rather than ONOO− it­
self [64]. Moreover, the results obtained indicate that, even in the
presence of physiological concentrations of HCO3− , the CI-Bz-BA probe
can still intercept ONOO− - to produce the corresponding phenol,
CI–OH, as well as the ONOO− -specific nitrated product, CI-Bz-NO2.
Boronate probes have been originally proposed and utilized for the
detection of H2O2 and later shown to be also able to report HOCl [3,30,
31,34–37,60,65–67]. Therefore, the reactivity of the CI-Bz-BA probe
toward these oxidants was investigated using fluorescence spectroscopy
and HPLC analyses.
A. Grzelakowska et al. Free Radical Biology and Medicine 179 (2022) 34–46

Fig. 7. Oxidation of CI-Bz-BA by H2O2. (A–D) HPLC analyses of the products formed from the reaction of the CI-Bz-BA probe and H2O2. Incubation mixtures
consisting of CI-Bz-BA (100 μM) and H2O2 [(A,B) 10 mM and (C) 0, 80, and 200 μM] in a phosphate buffer (100 mM, pH 7.4) containing dtpa (10 μM). The probe and
its oxidation products were detected using an absorption detector at (A, C) 330 nm and (B) 254 nm. (D) HPLC-based titration of the CI-Bz-BA probe (100 μM) with
H2O2 (0–300 μM) in a phosphate buffer (100 mM, pH 7.4) containing dtpa (10 μM). Data are means ± standard deviation of three independent experiments. The
HPLC traces were collected after 24-h incubation of CI-Bz-BA with oxidant using an absorption detector set at 330 nm.

Fig. 8. Oxidation of CI-Bz-BA by HOCl. (A,B) HPLC analyses of the products formed from the reaction of the CI-Bz-BA probe and HOCl. Incubation mixtures
consisting of CI-Bz-BA (100 μM) and HOCl (0, 80, and 200 μM) in a phosphate buffer (100 mM, pH 7.4) without dtpa. The probe and its oxidation products were
detected using an absorption detector at 330 nm. (D) HPLC-based titration of the CI-Bz-BA probe (100 μM) with HOCl (0–300 μM) in a phosphate buffer (100 mM, pH
7.4) without dtpa. Data are means ± standard deviation of three independent experiments. The HPLC traces were collected after 15-min incubation of CI-Bz-BA with
oxidant using an absorption detector set at 330 nm.

41
A. Grzelakowska et al.
Fig. 9. Comparison of the products formed from the oxidation of CI-Bz-BA by
ONOO− and MPO. (A) HPLC traces of the products detected during the reaction
between the CI-Bz-BA probe (100 μM) and ONOO− (200 μM), H2O2 (500 μM),
H2O2 (500 μM) + MPO (10 nM), H2O2 (500 μM) + NaNO2 (5 mM), and H2O2
(500 μM) + NaNO2 (5 mM) + MPO (10 nM) in a phosphate buffer (100 mM, pH
7.4) with dtpa (10 μM). The products were determined 1 h after adding H2O2 at
330 nm. (B) Results of titration of CI-Bz-BA (100 μM) with MPO. CI-Bz-BA was
incubated with MPO (0–15 nM) in the presence of H2O2 (500 μM) and NaNO2
(5 mM) in a phosphate buffer (100 mM, pH 7.4) with dtpa (10 μM) for 1 h. Data
are means ± standard deviation of three independent experiments.
Similar to the oxidation by ONOO− , the reactions with H2O2 and
HOCl lead to a shift in the fluorescence spectrum, with the product
exhibiting similar spectrum to that of CI–OH (Fig. 6). Analyses of the
fluorescence spectra of the reaction mixtures of CI-Bz-BA with HOCl
point to a lower yield of the fluorescent product(s) compared with those
for ONOO− and H2O2, and the consumption of the fluorescent product in
the presence of excess HOCl.
The reaction kinetics for CI-Bz-BA oxidation by HOCl and H2O2 was
investigated under pseudo-first-order conditions, using an excess of
either the probe in the case of HOCl or of the oxidant in the case of H2O2,
and monitoring the decay of the fluorescence signal due to the probe
(Figs. S23 and 24). The rate constants for the reactions between CI-Bz-
BA and H2O2 or HOCl were determined to be 1.9 ± 0.1 and (1.4 ± 0.1) ×
104 M-1s-1, respectively, significantly lower than observed for ONOO− .
These values are, however, in the same range as those reported for other
42
Free Radical Biology and Medicine 179 (2022) 34–46
boronate-based redox probes [3,31,34–36,40,60].
Time-dependent HPLC-based analyses of the reaction mixtures of CI-
Bz-BA and H2O2 indicate that before CI–OH is formed, elution of a peak
at 3.85 min is detected. Based on our previous report [30], we assigned
this peak to the primary phenolic product (CI-Bz-OH) formed during the
oxidation of CI-Bz-BA. Decomposition of CI-Bz-OH into CI–OH is
accompanied by elimination of QM, which reacts with water to form
OH-Bz-OH. This product was detected at 1.70 min, coeluting with the
authentic standard of OH-Bz-OH (Fig. 7). Because no other peaks were
detected, it can be concluded that upon oxidation by H2O2, CI-Bz-BA
undergoes conversion to CI–OH as the sole product of the reaction. To
determine the reaction stoichiometry, CI-Bz-BA was incubated with
different concentrations of H2O2 and the HPLC analyses were performed
after 24-h incubation. The results indicate that the reaction between
CI-Bz-BA and H2O2 is stoichiometric, as the yield of the probe con­
sumption and product formation is close to 100% with respect to the
amount of H2O2. Moreover, we did not observe degradation of the
CI-Bz-BA-derived product, CI–OH, over the course of experimentation,
even after 24-h incubation with an excess of the oxidant.
HPLC analyses of the reaction mixtures of the CI-Bz-BA probe with
HOCl indicate that the maximum yield of CI–OH reached ca. 70% under
the experimental conditions used (Fig. 8). The amount of CI–OH
decreased when an excess of the oxidant was added. The disappearance
of CI–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 of the same retention
time as the product formed when CI–OH was mixed with HOCl
(Fig. S20). 3-Chloro-4-hydroxybenzyl alcohol (OH-Bz-3Cl–OH), the
product of hypothetical chlorination of primary phenolic product, CI-
Bz-OH, which is formed from of the oxidation of CI-Bz-BA and/or
chlorination of OH-Bz-OH, which is formed by the decomposition of CI-
Bz-OH, was not detected under the experimental conditions used
(Fig. S21).
3.4. Oxidation of CI-Bz-BA by the MPO/H2O2 system
One of the limitations of the common assays to detect ONOO− is the
inability to distinguish between ONOO− and MPO/H2O2/NO2− -medi­
ated oxidation/nitration. To investigate whether CI-Bz-BA can differ­
entiate between ONOO− and MPO/H2O2/NO2− , the reaction mixtures
were analyzed by HPLC (Fig. 9). Although the major product, CI–OH,
was formed in all the systems tested, due to the presence of ONOO− or
H2O2, this experiment clearly identified CI-Bz-H and CI-Bz-NO2 as
specific products for ONOO− . The formation of ONOO− − specific minor
products occurred in the presence of ONOO− but not in the presence of
MPO/H2O2/NO2− , which is consistent with the data reported previously
for the ortho-mito-phenylboronic acid probe [68]. On the other hand,
MPO in the presence of nitrite leads to nitration of the primary phenolic
product CI-Bz-OH, formed from of oxidation of CI-Bz-BA by H2O2
and/or to nitration of OH-Bz-OH, which is formed by decomposition of
CI-Bz-OH, as evidenced by the formation of OH–3NO2-Bz-OH. Incu­
bation of OH-Bz-OH with the MPO/H2O2/NO2− system leads to the
formation of the same nitration product (Fig. S17).
3.5. Application of the probe to detect cellular ONOO−
Before application for ONOO− detection, the effect of CI-Bz-BA
probe and the CI–OH oxidation product on cell viability was tested with
the use of the MTT and PrestoBlue assays. RAW 264.7 cells were treated
with various concentrations (0–200 μM) of the studied compounds for 4
or 24 h before the viability assessment (Fig. S25). CI-Bz-BA and CI–OH
did not affect cell viability in the concentration range of 0–100 μM
whether measured after 4 or 24 h of incubation.
Next, the probe was tested in RAW 264.7 cells activated to produce
•
NO and/or O2•‒. Stimulation of RAW 264.7 cells with LPS/IFN-γ and
PMA leads to the formation of ONOO− [53]. As shown in Fig. 10A,
A. Grzelakowska et al.
Fig. 10. Real-time monitoring of CI-Bz-BA oxidation by activated RAW 264.7 macrophages. (A) The increase in fluorescence signal intensity from incubations
containing RAW 264.7 macrophages activated by different stimulators as shown. (B) Rate of increase in the fluorescence signal intensity from RAW 264.7 mac­
rophages activated by different stimulators in the absence or in the presence of L-NAME and CAT. Incubations contained CI-Bz-BA (20 μM) and RAW 264.7 mac­
rophages in DBPS-GP buffer in the presence of different stimulators (λex = 355 nm, λem = 520 nm). Data are means ± standard deviation of three independent
experiments.
Fig. 11. Representative two-photon microscopy images of RAW 264.7 macro­
phages incubated with CI-Bz-BA (20 μM) (control, left panel); stimulated with
LPS (1 μg/mL), IFN-γ (50 U/mL), and PMA (1 μM); and then incubated with CI-
Bz-BA (20 μM) (right panel). The incubation time was 90 min. The images were
obtained using 735 nm as the excitation wavelength and 465–550 nm emission
windows, at 40 × magnification. (DIC, differential interference contrast).
stimulation of RAW 264.7 macrophages with LPS/IFN-γ and PMA in the
presence of the CI-Bz-BA probe led to a strong and time-dependent in­
crease in fluorescence intensity. The addition of LPS and IFN-γ or PMA
alone to incubations containing macrophages and CI-Bz-BA caused no
increase in fluorescence as compared with the control cells. This sug­
gests that ONOO− formed from co-generated •NO and O2•‒ was
responsible for probe oxidation. To confirm the identity of the species
responsible for CI-Bz-BA oxidation, the effects of L-NAME, as a nitric
oxide synthase inhibitor, and CAT, an enzymatic H2O2 scavenger, on the
rate of probe oxidation were tested. While L-NAME caused a significant
decrease of fluorescence derived from the probe oxidation, CAT had no
effect on the rate of probe oxidation, indicating that ONOO− rather than
H2O2 was the oxidant detected. The results indicate that the CI-Bz-BA
43
Free Radical Biology and Medicine 179 (2022) 34–46
probe can be used to selectively monitor ONOO− production by mac­
rophages activated to co-generate •NO and O2•‒.
Since the two-photon excitation properties of CI–OH fluorophore
have been characterized previously [48], two-photon fluorescence mi­
croscopy was applied to image probe oxidation in activated macro­
phages. As shown in Fig. 11, upon excitation at 735 nm,
LPS/IFN-γ/PMA-stimulated cells have shown significant two-photon
fluorescence response (465–550 nm) in comparison with the control,
non-stimulated cells. Based on the published report confirming a high
degree of mitochondrial localization of the CI–OH product [48], we
propose that the punctate fluorescence observed in Fig. 12 may be due to
the presence of the CI–OH product in cell mitochondria. Further studies
are needed, however, to determine whether the probe detected mito­
chondrial ONOO− or the product formed in different site(s) translocated
to mitochondria, in the investigated system.
Next, the products of CI-Bz-BA oxidation were analyzed by LC-MS in
cell lysates and cell culture media. As shown in Fig. 12, incubation of
macrophages activated to produce •NO and O2•‒ in the presence of CI-
Bz-BA resulted in the formation of CI–OH and ONOO− -specific product
CI-Bz-NO2. These products were detected both intracellularly and in the
cell media, and were inhibitable by preincubation of the cells with L-
NAME.
These results confirm that CI-Bz-BA is a suitable probe for detecting
ONOO− generated in cellular systems and that CI-Bz-NO2 may be used
as an ONOO− -specific marker to confirm the identity of the oxidant.
4. Conclusions
We report the development of a novel fluorescent probe, CI-Bz-BA,
which shows a kinetic preference for ONOO− in cell-free and cell-based
systems. The probe was synthesized with a simple two-step methodology
under microwave conditions, and provided a good overall yield and high
purity. The unequivocal advantage of using the CI-Bz-BA probe is its
solubility in water, and for this reason it is not necessary to use an
organic cosolvent that can interfere with the oxidants. The chemical
reactivity of the CI-Bz-BA probe was studied in detail, including its re­
action kinetics and reactivity toward various oxidants (ONOO− , H2O2,
HOCl). The three oxidants evaluated are able to oxidize CI-Bz-BA to
corresponding phenolic product CI–OH, resulting in a red shift of its
fluorescence emission band. However, the rate constant of the reaction
with ONOO− is more than 5 and 2 orders of magnitude higher than that
of H2O2 and HOCl, respectively. The profile of the products formed from
A. Grzelakowska et al. Free Radical Biology and Medicine 179 (2022) 34–46

Fig. 12. LC-MS analyses of products formed from the oxidation of CI-Bz-BA activated to produce ONOO− RAW 264.7 macrophages. RAW 264.7 macrophages were
activated using LPS (0.5 μg/mL), IFN-γ (50 U/mL), and PMA (1 μM), and incubated for 1 h with CI-Bz-BA (20 μM) in DPBS supplemented with glucose (5.56 mM) and
sodium pyruvate (0.33 mM), as described in the Experimental section. LC-MS traces of CI-Bz-BA, CI–OH, and CI-Bz-NO2 in the (A) cell lysates and (B) media.

the reaction between ONOO− and the CI-Bz-BA probe is highly specific ONOO− formation in a variety of biologically relevant systems. Based on
for this oxidant. The major pathway leads to the formation of CI–OH, the presence of the positive charge and published report [48], we
whereas under the conditions used the minor pathway yields two anticipate that the applications of the probe may be also extended for the
products: CI-Bz-H and CI-Bz-NO2. Reaction of the probe and ONOO− , detection of mitochondrial oxidants, including ONOO− .
added as bolus or generated in situ from superoxide and nitric oxide
fluxes, led to the formation of a characteristic nitrated product CI-Bz- Author contributions
NO2. CI-Bz-BA competes with CO2 for ONOO− and probe oxidation
products were observed under biologically-relevant levels of CO2. These A.G. wrote the draft of the manuscript. All authors contributed to and
data underscore the usefulness of the probe for ONOO− detection in gave approval to the final version of the manuscript.
biological systems. Since the nitrobenzene-type product is not generated
during incubation of boronate probe with MPO/H2O2/nitrite systems,
Declaration of competing interest
its detection demonstrates the presence of ONOO− . On the other hand,
nitration of the primary phenolic product, CI-Bz-OH, which is formed
The authors declare no conflict of interest.
from of oxidation of CI-Bz-BA and/or nitration of OH-Bz-OH, which is
formed by the decomposition of CI-Bz-OH and results in the production
Acknowledgments
of OH-Bz-3NO2–OH, cannot distinguish between ONOO− and •NO2
formed from the MPO/H2O2/NO2− system.
This work was supported by a grant from the Polish National Science
Incubation of CI-Bz-BA with RAW 264.7 macrophages activated to
Centre (NCN) within the SONATA BIS 6 program (Grant No. 2016/22/
produce ONOO− yielded the corresponding fluorescent phenolic com­
E/ST4/00549).
pound, CI–OH, as well as the ONOO− -specific nitrated product, CI-Bz-
The LC-MS analyses were performed at the Medical College of Wis­
NO2. These studies demonstrate that CI-Bz-BA fulfills the requirements
consin Cancer Center Redox and Bioenergetics Shared Resource. The
to be classified as an efficient ONOO− probe applicable to the study of
authors thank Dr. Suresh Kumar (Medical College of Wisconsin) for his

44
A. Grzelakowska et al.
help with the two-photon fluorescence imaging experiments.
A.G. was supported by a grant from the Polish National Science
Centre (NCN) within the MINIATURA 4 program (Grant No. 2020/04/
X/ST4/01002).
A.G. thanks Klaudia Salamon for initial contributions.
Appendix A. Supplementary data
Supplementary data related to this article can be found at htt
ps://doi.org/10.1016/j.freeradbiomed.2021.12.260.
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