Characterization of the reactivity of luciferin boronate - A probe for inflammatory oxidants with improved stability

Article abstract of DOI:10.1016/j.dyepig.2020.108693

Boronate derivatives of luciferin, containing oxidant-activated self-immolative moieties, recently have been developed for bioluminescent detection of hydrogen peroxide in animal models. Here, we report the synthesis and characterization of luciferin boronic acid pinacol ester (LBE) as a probe for detection of hydrogen peroxide, hypochlorous acid, and peroxynitrite, with improved stability and response time. HPLC analyses showed that LBE quickly hydrolyzes in phosphate buffer to luciferin boronic acid (LBA). Hydrogen peroxide oxidizes LBA slowly, with the formation of luciferase substrate, luciferin (Luc-OH), as the only product. Hypochlorite also oxidizes LBA to luciferin, but the subsequent reaction of Luc-OH with hypochlorite gives a chlorinated luciferin Luc–OH–Cl, which has a higher fluorescence quantum yield than luciferin at pH 7.4 and is also a substrate for luciferase (Takakura H, et. all. ChemBioChem 2012; 13:1424). Similar to other boronate probes, LBA is oxidized by peroxynitrite in two pathways. Luc-OH is the product of the major pathway, common for all the oxidants tested, whereas the non-fluorescent nitrated derivative, Luc-NO₂, is formed in the minor pathway, specific for peroxynitrite. Formation of luciferin radical intermediate in the minor pathway has been confirmed by EPR spin trapping and mass spectrometric analyses of the spin adducts. We conclude that LBE shows potential as an improved probe for the detection of inflammatory oxidants in biological settings. Complementation of the bioluminescence measurements by HPLC or LC-MS-based identification of chlorinated and nitrated luciferin(s) will help identify the oxidants detected.

Full text of DOI:10.1016/j.dyepig.2020.108693

Journal Pre-proof
Characterization of the reactivity of luciferin boronate - a probe for inflammatory
oxidants with improved stability
Marcin Szala, Aleksandra Grzelakowska, Julia Modrzejewska, Przemysław
Siarkiewicz, Daniel Słowiński, Małgorzata Świerczyńska, Jacek Zielonka, Radosław
Podsiadły
PII:
S0143-7208(20)31390-5
DOI:
https://doi.org/10.1016/j.dyepig.2020.108693
DYPI 108693
Reference:
To appear in: Dyes and Pigments
Received Date: 27 May 2020
Revised Date: 1 July 2020
Accepted Date: 3 July 2020
Please cite this article as: Szala M, Grzelakowska A, Modrzejewska J, Siarkiewicz P, Słowiński D,
Świerczyńska M, Zielonka J, Podsiadły R, Characterization of the reactivity of luciferin boronate - a
probe for inflammatory oxidants with improved stability, Dyes and Pigments, https://doi.org/10.1016/
j.dyepig.2020.108693.
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Characterization of the reactivity of luciferin boronate - a probe for inflammatory
oxidants with improved stability
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Marcin Szala1*, Aleksandra Grzelakowska1, Julia Modrzejewska1, Przemysław Siarkiewicz1, Daniel
Słowiński1, Małgorzata Świerczyńska1, Jacek Zielonka2* and Radosław Podsiadły1†
1 Institute of Polymer and Dye Technology, Faculty of Chemistry, Lodz University of Technology,
Stefanowskiego 12/16, 90-924 Lodz, Poland
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2 Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI
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53226, United States
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Abstract
Boronate derivatives of luciferin, containing oxidant-activated self-immolative moieties,
recently have been developed for bioluminescent detection of hydrogen peroxide in animal
models. Here, we report the synthesis and characterization of luciferin boronic acid pinacol
ester (LBE) as a probe for detection of hydrogen peroxide, hypochlorous acid, and
peroxynitrite, with improved stability and response time. HPLC analyses showed that LBE
quickly hydrolyzes in phosphate buffer to luciferin boronic acid (LBA). Hydrogen peroxide
oxidizes LBA slowly, with the formation of luciferase substrate, luciferin (Luc-OH), as the
only product. Hypochlorite also oxidizes LBA to luciferin, but the subsequent reaction of
Luc-OH with hypochlorite gives a chlorinated luciferin Luc-OH-Cl, which has a higher
fluorescence quantum yield than luciferin at pH 7.4 and is also a substrate for luciferase
(Takakura H, et. all. ChemBioChem 2012;13:1424). Similar to other boronate probes, LBA is
oxidized by peroxynitrite in two pathways. Luc-OH is the product of the major pathway,
common for all the oxidants tested, whereas the non-fluorescent nitrated derivative, Luc-NO2,
is formed in the minor pathway, specific for peroxynitrite. Formation of luciferin radical
intermediate in the minor pathway has been confirmed by EPR spin trapping and mass
spectrometric analyses of the spin adducts. We conclude that LBE shows potential as an
improved probe for the detection of inflammatory oxidants in biological settings.
Complementation of the bioluminescence measurements by HPLC or LC-MS-based
identification of chlorinated and nitrated luciferin(s) will help identify the oxidants detected.
Keywords: luminogenic probes, firefly luciferin-based probe, peroxynitrite-specific product
† Corresponding authors.
E-mail addresses: marcin.szala@p.lodz.pl, jzielonk@mcw.edu, radoslaw.podsiadly@p.lodz.pl,
aleksandra.grzelakowska@p.lodz.pl, , julia.modrzejewska@edu.p.lodz.pl,
przemyslaw.siarkiewicz@edu.p.lodz.pl, daniel.slowinski@edu.p.lodz.pl,
malgorzata.swierczynska@edu.p.lodz.pl
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Abbreviations: BLI, bioluminescent imaging; CAT, catalase; CBA, coumarin-7-boronic
acid; dppf, 1,1’-ferrocenediyl-bis(diphenylphosphine); DMSO, dimethyl sulfoxide; DPBS,
Dulbecco's phosphate-buffered saline; dtpa, diethylenetriaminepentaacetic acid; DMEM,
Dulbecco's Modified Eagle Medium; EPR, electron paramagnetic resonance; ESI,
electrospray ionization; HO-Bz-OH, 4-hydroxybenzyl alcohol; HPLC, high performance
liquid chromatography; Hz, hertz; IFN-γ, interferon γ; iNOS, inducible nitric oxide synthase;
isoAmONO, isoamyl nitrite; L-NAME, L-NG-nitroarginine methyl ester; LBA, luciferin 6’-
boronic acid; LBE, luciferin 6’-boronic acid pinacol ester; LC-MS, liquid chromatography-
mass spectrometry; LPS, lipopolysaccharide; Luc-Bz•, PCL-1-derived phenyl radical; Luc-
Bz-H, luciferin-6'-benzyl ether; Luc-Bz-NO2, luciferin-6'-p-nitrobenzyl ether; Luc-Bz-OH,
luciferin-6'-p-hydroxybenzyl ether; Luc-NO2, 6’-nitroluciferin; Luc-OH, D-luciferin or firefly
luciferin; Luc-OH-Cl, 7’-chloro-D-luciferin; Luc-OH-NO2, 7’-nitro-D-luciferin; MeCN,
acetonitrile; MNP, 2-methyl-2-nitrosopropane; MRM, multiple reaction monitoring; MS,
mass spectrometry; NMR, nuclear magnetic resonance; ONOO−, peroxynitrite; PCL-1,
peroxy-caged luciferin; PEG, polyethylene glycol; PMA, phorbol 12-myristate 13-acetate; 2-
PrOH, 2-propanol; QM, para-quinone methide; RNS, reactive nitrogen species; ROS, reactive
oxygen species; SIM, single ion monitoring; TFA, trifluoroacetic acid
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1. Introduction
Understanding the (patho)physiological role of reactive oxygen species/reactive
nitrogen species (ROS/RNS) requires the availability of probes for their detection and
(semi)quantitative analyses in the in vitro and in vivo settings. Bioluminescent imaging (BLI)
is commonly used for sensitive monitoring of various biomolecular processes in cells and
living animals [1-7]. The popularity of bioluminescence assays in biomedical research has
resulted in significant progress in the syntheses of new luminescent analogs of luciferin [8]
and luminogenic probes based on the firefly luciferin (Luc-OH) skeleton [9]. Among the
caged probes, oxidant-sensitive luciferin derivatives have been reported for the detection of
ROS/RNS in the in vitro and in vivo systems [10-15]. One of the first luciferin-based
bioluminogenic probes designed to image hydrogen peroxide (H2O2) in living systems was
peroxy-caged luciferin (PCL-1) (Scheme 1) [10]. The hydroxyl group of Luc-OH is alkylated
by the boronobenzyl moiety in PCL-1, which prevents recognition of the probe by luciferase.
H2O2 reacts slowly with PCL-1 (k = 1.2 M-1 s-1 ) [11], releasing Luc-OH and p-quinone
methide (QM) (Scheme 1) [12]. Release of Luc-OH from PCL-1 is followed by its oxidation
to oxyluciferin and the appearance of the bioluminescence signal in luciferase-transfected
cells.
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Scheme 1. Reaction of the PCL-1 probe with H2O2, HOCl, and ONOO–, leading to the
luminogenic substrate for luciferase (Luc-OH) after the release of QM, and to oxidant-specific
products [10-12].
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Recently, we have characterized in detail the reaction intermediates and stable
products formed in the reaction between PCL-1 probe and selected inflammatory oxidants
(H2O2, hypochlorous acid [HOCl], peroxynitrite [ONOO−]) [12]. Similar to previously studied
boronates [16,19,21,22], the reaction of PCL-1 with ONOO− proceeds via two pathways. The
major pathway is the same as for H2O2 (Scheme 1) and involves the formation of the phenolic
intermediate (Luc-Bz-OH), which after the elimination of QM yields Luc-OH as the stable
end-product. The minor pathway produces nitrated and reduced products, Luc-Bz-NO2 and
Luc-Bz-H, respectively. [12]. We have demonstrated that ONOO−-specific product, Luc-Bz-
NO2, is formed by activated macrophages incubated in the presence of the PCL-1 probe [12],
which indicates that the PCL-1 probe can be applied to specifically detect and identify
peroxynitrite in biological systems.
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Hypochlorite is another oxidant that converts PCL-1 to Luc-OH [11,12]. Previously, it
was shown that HOCl oxidizes boronate probes to nonspecific phenolic products [16, 17, 26].
However, the subsequent reaction of the phenolic product with HOCl yields chlorophenol(s),
which can serve as a footprint for HOCl [16,17]. Reaction of PCL-1 with HOCl leads to the
formation of Luc-OH, which may undergo subsequent chlorination to yield Luc-OH-Cl
(Scheme 1) [12]. Therefore, Luc-OH-Cl detection, in addition to bioluminescence in vivo,
may help identify HOCl as an oxidant responsible for the bioluminescence signal.
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During the characterization of the PCL-1 probe, we identified some potential
disadvantages of its use for the detection of oxidants in chemical, enzymatic, and/or biological
systems, as follows: (i) During the uncaging of PCL-1 and other luciferin-based redox probes
QM [10-12], quinoneimine [13], or benzoylhydrazine [15] are released. When probing the
oxidant production on the scale of seconds or minutes, the self-immolation of those moieties
may control the dynamics of the formation of the bioluminescent signal. (ii) QM, being an
electrophile, may potentially affect the cellular redox status and, thus, affect the redox
environment being probed. (iii) The PCL-1 probe decomposes significantly during prolonged
incubation in a phosphate buffer (pH 7.4). Up to 50% of PCL-1 was lost (with Luc-OH
detected as one of the products formed) over the 24-h incubation in the buffer [12]. This may
translate into a high background bioluminescence signal, limiting the probe’s sensitivity.
Here, we report the synthesis and characterization of a novel boronate-based luciferin
derivative, LBE, with the boronate moiety attached directly at the site of the hydroxyl group
(Scheme 2). Unexpectedly, this probe shows significantly higher stability, as compared with
the PCL-1 probe, under the experimental conditions used to detect cell-based oxidants. In
aqueous solutions containing a phosphate buffer (pH 7.4), we observed fast hydrolysis of LBE
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to luciferin boronic acid (LBA). Compared with the PCL-1 probe, oxidation of LBA leads to
instantaneous formation of luciferin, without any delay due to the requirement to eliminate the
self-immolative QM moiety in PCL-1. This provides an opportunity for real-time monitoring
of the dynamics of oxidant formation using the LBA probe, when coupled to fluorescence or
bioluminescence detection, which produces lower background signal, as compared with the
PCL-1 probe.
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Scheme 2. Boronate-based luciferin derivatives
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2. Experimental
2.1. General
2-Cyano-6-hydroxybenzothiazole (1), 2-cyanobenzothiazole (6),
benzothiazole (12), N-phenyl-bis(trifluoromethanesulfonimide), trietylamine, D-cysteine,
bis(pinacolato)diboron, [1,1’-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
2-amino-6-nitro-
(Pd(dppf)Cl2), 1,1’-ferrocenediyl-bis(diphenylphosphine) (dppf), zirconium(IV) oxynitrate
hydrate, copper(II) chloride, polyethylene glycol (PEG), and isoamyl nitrite (isoAmONO)
were purchased from Sigma-Aldrich (Poznan, Poland). Solvents used for syntheses were
reagent grade. The structures of the LBE probe and all synthesized standards – Luc-H, Luc-
OH, Luc-NO2, Luc-OH-Cl, Luc-OH-NO2 – were confirmed by nuclear magnetic resonance
1
(NMR) spectroscopy and mass spectrometry (MS) (see Supporting Information). H NMR
and 13C NMR spectra were recorded with a Bruker Avance DPX 250 and Bruker Avance 400
spectrometers at 250 or 400 MHz (1H) and 100 MHz (13C), respectively. Compounds were
dissolved in CDCl3 or DMSO-d6 and tetramethylsilane was added as internal reference for 1H
NMR and 13C NMR spectra, respectively. Chemical shifts (δ) are reported in ppm, and
coupling constant J values in hertz (Hz). The electrospray ionization (ESI)-MS spectra were
recorded on a Finnigan MAT 95 spectrometer (Thermo Fischer Scientific, USA).
The stock solutions of oxidants (HOCl, and H2O2) were prepared freshly before each
experiment and theirs concentrations were determined by spectrophotometry, using the
procedure described previously [12]. Peroxynitrite was synthesized in reaction of 0.6 M nitrite
with 0.7 M hydrogen peroxide at pH 13 [12]. 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 concentration of
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peroxynitrite was determined spectrally at 302 nm (ε302 = 1.7×103 M-1cm-1), after dilution in
0.1 M NaOH to ~10 mM concentration, immediately prior to each experiment.. When
studying LBE oxidation by HOCl, the LBE stock solution (1 mM) in acetonitrile (MeCN) was
added to phosphate buffer (100 mM, pH 7.4) to receive a final concentration of 100 µM of the
probe. Dimethyl sulfoxide (DMSO) solvent was avoided due to known rapid quenching of
HOCl by DMSO [12, 33]. For other experiments, stock solutions of LBE were prepared in
DMSO at 10 mM concentration, and this solution was added directly to the buffer to obtain
the desired concentration. High performance liquid chromatography (HPLC) analyses indicate
that LBE (pinacolate ester) undergoes fast hydrolysis to the boronic acid form (LBA) upon
dilution in the aqueous phosphate buffer. Therefore, although we added LBE to the reaction
mixtures, LBA is the species that reacted with the oxidants tested.
2.2. Synthesis
All synthetic pathways are shown in condensed form in Scheme 2. Detailed synthetic
procedures and 1H and 13C NMR spectra are included in Supporting Information.
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Scheme 3. Synthetic pathways used to obtain LBE probe and Luc-OH, Luc-H, Luc-OH-Cl,
Luc-OH-NO2, Luc-NO2 standards. Reagents and conditions: (i): PhN(SO2CF3)2, Et3N, CHCl3,
ꢀ, 3 h; (ii): B2pin2, Pd(dppf)Cl2, dppf, KOAc, 1,4-dioxane, 100 °C, microwave irradiation,
Ar; (iii): D-Cys, K2CO3, MeOH/H2O, 30 min; (iv): SO2Cl2, DCM, 16 h; (v): ZrO(NO3)2xH2O,
acetone, 100 °C, microwave irradiation, Ar, 15 min; (vi): CuCl2, PEG, isoAmONO, MeCN,
65 °C, Ar, 3 h; (vii): NaCN, DMSO, 110 °C, Ar, 5 h.
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2.3. HPLC analyses
HPLC analyses of all synthesized derivatives of luciferin, namely LBE, LBA, Luc-H, Luc-
OH, Luc-NO2, Luc-OH-Cl, and Luc-OH-NO2 were performed using UFLC Shimadzu
equipped with UV–Vis absorption and fluorescence detectors. Analyses were done using a
Kinetex C18 column (Phenomenex, 100 mm × 4.6 mm, 2.6 µm), which was equilibrated with
10% of MeCN in water, containing 0.1% trifluoroacetic acid (TFA). The analytes were eluted
by an increase of MeCN concentration from 10–70% over 12 min at the flow rate of 1.5
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mL/min. The LBA probe and all products formed in its reaction with H2O2, HOCl, and
ONOO─ was detected by monitoring the absorbance at 330 nm. Additionally, Luc-OH was
monitored using the fluorescence detector with the excitation set at 330 nm and emission set
at 520 nm.
2.4. Fluorescence spectra analyses
Collection of fluorescence spectra and spectroscopic characterization were performed using a
FLS-920 spectrofluorometer (Edinburgh Instruments, UK). The response of the LBA probe to
oxidants was investigated by fluorescence titration experiments in aqueous solutions,
containing a 100 mM phosphate buffer (pH 7.4), 10 µM dtpa, and 20% MeCN. The probe
(100 µM) was incubated with H2O2, HOCl, or ONOO─ for 24 h, 15 min, and 5 min,
respectively.
2.5. EPR spin-trapping experiments
The spin-trapping experiments were conducted utilizing 2-methyl-2-nitrosopropane (MNP) as
a spin trap using a Bruker EMX electron paramagnetic resonance (EPR) spectrometer, as
described previously [12, 21]. Typically, the EPR spectra were recorded immediately after a
bolus addition of peroxynitrite (200 ꢁM) to mixtures consisting of LBE (200 ꢁM) and MNP
(40 mM) in a phosphate buffer (100 mM, pH 7.4) containing dtpa (100 ꢁM) and MeCN (5%).
The instrument parameters were as follows: scan range, 150 G; time constant, 1.28 ms; scan
time, 84 s; modulation amplitude, 1 G; modulation frequency, 100 kHz; receiver gain, 1×105;
and microwave power, 20 mW. The spectra shown are the averages of 10 scans.
2.7. LC-MS analyses
Liquid chromatography-mass spectrometry (LC-MS) analyses of LBA, its oxidation products,
and spin adducts, were performed as described previously [12] using a Shimadzu LC-MS
8030 triple quadrupole mass detector coupled with a Shimadzu Nexera 2 UHPLC system
equipped with a Cortecs C18 column (Waters, 50 mm × 2 mm, 1.6 µm). The column was
equilibrated with 10% of MeCN in water containing 0.1% of formic acid. To separate the
reaction mixture, a gradient elution with increasing concentration of MeCN in the mobile
phase from 10–80% over 4 min was used. The flow rate was set at 0.5 mL/min, and the flow
was diverted to waste during the first minute and after 4 min, counting from the time of
injection. LBA and luciferin were detected in their deprotonated forms in the negative mode
using single ion monitoring (SIM), set at m/z values of 307 and 279, respectively. MNP and
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Luc-MNP spin adduct were detected in their protonated forms in positive mode using SIM,
set at m/z values of 88 and 352, respectively.
2.8. Real-time monitoring of peroxynitrite formation from activated RAW 264.7
macrophages
RAW 264.7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco)
supplemented with 10% fetal bovine serum (Omega Scientific), 2 mM L-glutamine, 100
units/mL penicillin, and 100 µg/mL streptomycin antibiotic at 37 °C in 5% carbon dioxide
and 95% air atmosphere. The cells were seeded in 96-well plates at a concentration of 2×104
cells (in 150 µl) per well and incubated at 37° C and 5% carbon dioxide overnight.
For stimulations of the cells to produce nitric oxide, cells were incubated for 8 h with
LPS (0.5 µg/mL) and INF-γ (50 units/mL). To stimulate NADPH oxidase-dependent
superoxide production, the cells were washed twice with DMEM medium and treated with
phorbol 12-myristate 13-acetate (PMA, 1 µM) (Sigma Aldrich) in Dulbecco's phosphate-
buffered saline (DPBS) buffer supplemented with sodium pyruvate (0.3 mM) and glucose (5.5
mM) (DPBS-GP) (Gibco). At the time of addition of PMA, PCL-1 or LBE probe (20 µM)
was also added. Where indicated, L-N-nitroarginine methyl ester hydrochloride (L-NAME, 1
mM) (Cayman Chemical) or catalase (CAT, 1 kU/mL) (Sigma Aldrich) was added at the time
of addition of PMA and the probe. For cell-based experiments, stock solutions of LBE and
PCL-1 probes were prepared in DMSO at 10 mM concentrations, and their final
concentrations were 20 µM. Thus, the final concentration of DMSO was kept minimal, <
0.3% (v/v), upon dilution. For the control samples, cells were treated with the probes in the
absence of stimulators and inhibitors.
Oxidation of probes was monitored in a 96-well fluorescence plate reader. The 96-well
plate with cells was placed immediately after the addition of DPBS-GP containing the probe
with or without PMA, L-NAME, and CAT in a plate reader prewarmed to 37 °C. Total
fluorescence intensities were acquired using a FLUOstar Omega plate reader (BMG
LABTECH) 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.
3. Results
3.1 Synthesis and spectroscopic properties of luciferin’s derivatives
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To test if direct luciferin boronation may improve the performance the probe for oxidant
detection, we synthesized luciferin boronic acid pinacol ester LBE and the anticipated minor
products of its reactions with ONOO– and HOCl (see Scheme 4). First, we conducted
spectroscopic characterization of these products; the results are shown in Table 1. Among all
the compounds tested, only Luc-OH and Luc-OH-Cl exhibited significant fluorescence. It is
worth noting that under physiological pH conditions, chlorinated luciferin exhibits a higher
fluorescence quantum yield than luciferin (Table 1, Supplementary Fig. 1A). The
deprotonation of luciferin results in increased fluorescence yield [29], which we attribute to
the increased acidity of luciferin upon chlorination. In fact, the reported pKa values for Luc-
OH and Luc-OH-Cl are 8.5 and 6.7, respectively [30].
Analyses of the fluorescence spectra recorded after reacting the LBE probe with H2O2,
HOCl, and ONOO– (Supplementary Fig. 1B-D) indicate that fluorescent products are formed
for all three oxidants. However, the concentration-dependence of the fluorescence intensity
suggests some difference between the oxidants. While in the presence of excess H2O2, the
fluorescence intensity does not change; in case of HOCl, the intensity further increases; and in
the case of ONOO–, the intensity decreases. Based on the determined spectroscopic properties
of Luc-OH, Luc-OH-Cl, and Luc-OH-NO2, these results suggest that luciferin formed upon
oxidation of the LBA probe undergoes chlorination by excess HOCl [12,16,17] or nitration by
excess ONOO– [16]. To better understand the chemical mechanisms of oxidation of the probe
by those three oxidants, we performed detailed identifications and quantifications of the
products formed.
Table 1. Spectroscopic properties of the LBA probe and the products of its reaction with the
oxidants tested in aqueous phosphate buffer (100 mM, pH = 7.4):MeCN (4:1)
λmax
(nm)
300
ε (λmax
(M-1·cm-1)
)
λexc
(nm)
-
λem
(nm)
-
Φem
Stokes
Compound
(%) shift (nm)
10 600
-
-
LBA
Luc-OH
Luc-H
328 (326 a) 11 900 (10 700 a) 334 (331 a) 536 (417 a) 43 (1 a) 202 (86 a)
298
310
390
434
366
14 500
19 200
14 300
7 200
-
-
-
-
-
-
-
-
Luc-NO2
Luc-OH-Cl
391
-
532
-
67
-
141
-
Luc-OH-NO2
23 000
a
260
261
In pure MeCN
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3.2. Identification of the primary product formed during the oxidation of LBA
The spectroscopic data presented in Table 1 indicate that although the nitration products are
non-fluorescent, chlorination of luciferin yields a highly fluorescent product. Therefore, the
quantitative detection of inflammatory oxidants especially of HOCl and ONOO–, based solely
on fluorescence intensity measurements, may be inadequate. Moreover, it has been reported
the presence of the chlorine atom in Luc-OH-Cl does not prevent the interaction with
luciferase, but that it affects the bioluminescence signal intensity [30]. Thus, similar to the
PCL-1 probe [12], the application the LBE probe for quantification of H2O2, HOCl, or
ONOO─ production in vitro or in vivo requires knowledge of the probe's chemistry, and
should involve the identification of the oxidant-specific product(s). First, we identified the
products formed during oxidation of LBE by three different biologically relevant,
inflammatory oxidants known to oxidize boronic compounds.
3.2.1. Oxidation of LBA by H2O2
As mentioned, H2O2 oxidizes LBA to luciferin. The HPLC chromatograms presented in Fig.
1A show that Luc-OH is the sole product formed in this reaction. The slow reaction kinetics
required a 24-h incubation period to ensure completion of the reaction between the probe and
H2O2, when present at submillimolar concentrations. Stoichiometric analysis of the reaction
between LBA and H2O2 is shown in Fig. 1B. Over the 24-h incubation period, LBA is stable
in aqueous solution containing phosphate buffer (0.1 M, pH 7.4) and MeCN (20%), as
indicated by the fact that almost 99% of the initial amount of LBA is still detected. Similar
results were obtained in the phosphate buffer solution (0.1 M, pH 7.4) containing DMSO
(1%). Stoichiometric analysis also shows that LBA is completely consumed by H2O2, and the
maximum yield of Luc-OH was achieved when the probe was reacted with a small excess of
the oxidant (less than 1.5 equivalents of H2O2), consistent with the 1:1 stoichiometry
previously reported for mono-boronated probes [16, 19]. The yield of luciferin was ca. 96%
and did not change in the presence of oxidant excess. Since probe purity was 98% and we
identified Luc-H as an impurity, one can conclude that the reaction of LBA with H2O2 led to
the formation of luciferin as the sole product.
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Figure 1. (A) HPLC chromatograms of the standards (100 µM each) and reaction mixtures of
LBA (100 µM) with H2O2 (200 µM) after 24 h of incubation. The traces were collected using
an absorption detector set at 330 nm. (B) HPLC-based titration of LBA (100 ꢁM) with H2O2
after 24 h incubation in aqueous solution containing phosphate buffer (100 mM, pH 7.4), dtpa
(10 µM), and MeCN (20%). Data are means ± standard deviation of three independent
experiments.
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3.2.2. Oxidation of LBA by HOCl.
HPLC chromatograms of the reaction mixtures of LBA with HOCl are presented in Fig. 2A.
In addition to Luc-OH, a new product with a retention time of 5.7 min was formed during the
reaction of the LBA probe with HOCl. The same product was formed when Luc-OH was
mixed with HOCl. Comparison of the retention times of the synthesized authentic standard of
7’-chloroluciferin (Luc-OH-Cl) and the product formed in the reaction of LBA with HOCl
confirms that Luc-OH-Cl is one of the products found in the reaction mixture. Formation of
this product may explain why the maximum yield of Luc-OH reaches only ca. 60%. Luc-OH-
Cl was previously found as a minor product of the reaction between of PCL-1 and HOCl [12].
The stoichiometric analysis of the oxidation reaction of LBA by HOCl is presented in Fig. 2B.
The complete consumption of the probe and formation of luciferin requires use of more than
1.5 HOCl equivalents. Unlike the reaction of LBA with H2O2, an excess of HOCl causes
disappearance of luciferin. This is consistent with our previous investigation using the PCL-1
probe, which demonstrated that released luciferin undergoes further reaction with HOCl,
leading to the formation of Luc-OH-Cl, a product specific for HOCl.
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Figure 2. Reaction between LBA and HOCl in aqueous solution containing phosphate buffer
(100 mM, pH 7.4), dtpa (10 µM), and MeCN (10%). (A) HPLC chromatograms of the
standards (100 µM each) and reaction mixtures of LBA (100 µM) with HOCl (80 µM) after
15 min of incubation. The traces were collected using an absorption detector set at 330 nm.
(B) HPLC-based titration of LBA (100 ꢁM) with HOCl. Data are means ± standard deviation
of three independent experiments
3.2.2. Oxidation of LBA by peroxynitrite
HPLC chromatograms and stoichiometric analyses of the reaction mixtures of LBA with
ONOO– are shown in Fig. 3A and 3B, respectively. The results indicate that the main product
of oxidation of the LBA probe by ONOO– is luciferin. Moreover, the yield of Luc-OH
reached maximum (ca. 80% yield) when the LBA probe was reacted with an equimolar
amount of ONOO–, confirming a 1:1 reaction stoichiometry. Based on the two established
pathways of oxidation of arylboronic acids by ONOO–, we anticipated that the major (non-
radical) pathway would yield luciferin and the minor (radical-mediated) pathway would yield
Luc-NO2 and Luc-H (see Scheme 2), the products derived from the phenyl-type radical
formed [12, 21, 22]. Moreover, we expected that nitration of Luc-OH by •NO2 formed in the
minor pathway and during decomposition of excess ONOO– would produce Luc-OH-NO2, as
shown in Scheme 2. The chromatograms of the standards and of the reaction mixture
confirmed the formation of Luc-H, Luc-NO2, and Luc-OH-NO2 when LBA was reacted with
ONOO– (Fig. 3A).
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Figure 3. Reaction between LBA and ONOO– in aqueous solution containing phosphate
buffer (100 mM, pH 7.4), dtpa (10 µM), and DMSO (1%). (A) HPLC chromatograms of the
standards (100 µM each) and reaction mixtures of LBA (100 µM) with ONOO– (80 µM) after
5 min of incubation. The traces were collected using the absorption detector set at 330 nm.
(B) HPLC-based titration of LBA (100 ꢁM) with ONOO–. Data are means ± standard
deviation of three independent experiments.
The formation of minor products in the reaction mixture, anticipated for the radical
pathway of the reaction, encouraged us to perform an EPR spin trapping experiment using an
MNP spin trap. Reacting LBA with ONOO– in the presence of the MNP spin trap resulted in
appearance of the EPR spectrum of the spin adduct, as shown in Fig. 4A (trace a). The
intensity and resolution of this EPR signal are not sufficient to determine the g value and
hyperfine coupling constants, but the signal clearly consists of three major lines (due to the
hyperfine splitting from the nitrogen atom) with an additional structure (due to the hyperfine
splitting caused by the phenyl ring hydrogen atoms). The addition of a bolus amount of
ONOO– to incubations containing LBA, MNP, and 10% of 2-PrOH, a known scavenger of
phenyl radicals, resulted in significantly reduced intensity of the EPR signal (Fig. 4A, trace
b). The EPR spectrum recorded with MNP in the reaction mixture of LBA with ONOO– was
not observed when any of those reaction components was absent (Fig. 4A, traces c and e).
Unexpectedly, when ONOO– was added to a solution of LBE in the buffer and DMSO (in the
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absence of the MNP spin trap), a single line was detected (Fig. 4A trace d). A similar EPR
spectrum was observed for a long-lived DMSO radical cluster generated in neat DMSO with a
small amount of base [31].
To further confirm the formation of Luc• and to identify the molecular structure of
spin-trapped radical, we combined the spin trapping experiment with LC-MS analysis of the
spin adduct. The LC-MS analyses (Fig. 4B) enabled detection of the MNP spin trap in the
positive mode using SIM, set at an m/z value of 88, and LBA (m/z = 308) consistent with
hydrolysis of the boronate ester in the buffer used. Luc-OH (m/z = 280) was detected in all
cases when the LBA probe was mixed with ONOO–. In the presence of LBA, ONOO–, and
MNP, the spin adduct of MNP and Luc• radical was detected (Fig. 4B, m/z = 352, peak
detected at 2.33 min). This peak can be assigned to the spin adduct present in the form of
nitroxide and/or protonated nitrone. The spin-trapping technique combined with the LC-MS-
based analysis of the molecular weight of the radical adduct confirm the formation of the
phenyl radical during the oxidation of LBA by ONOO–.
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Scheme 4. Transformation of the LBA probe, leading to luciferin and ONOO–-specific minor
products. The colored structures of the stable products correspond to the color coding of the
product profiles shown for different oxidants in Figures 1-3.
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Figure 4. Spin trapping of the phenyl radical formed during the reaction of LBA with ONOO–
. (A) EPR spectra recorded using MNP as a spin trap. (B) LC-MS analyses of the luciferin
boronic acid (LBA, M-H+, m/z = (-)307), luciferin (Luc-OH, M-H+, m/z = 279), MNP
(M+H+, m/z = (+)88), and Luc-MNP (M+H+, m/z = (+)352) spin-adduct. ONOO– was added
to the mixture of LBA (200 µM) and MNP (40 mM) in phosphate buffer (50 mM, pH 7.4)
containing dtpa (10 µM), MeCN (5%), and DMSO (2.5%). The reaction mixture was
transferred to an EPR capillary immediately after a bolus addition of ONOO−, and the spectra
were recorded at room temperature. Experiments were repeated three times independently.
Representative result displayed.
We also analyzed the reaction mixture from the spin trapping experiments to detect
ONOO–-specific minor products. Figure 5 shows the product analyses of the oxidation of
LBA by ONOO–. The major product Luc-OH (Fig. 5, m/z = (-)279, peak detected at 1.61
min) is always detected when the LBA probe is mixed with ONOO–. However, the yields of
the nitrated products Luc-NO2 (Fig. 5, m/z = (-)308, peak detected at 2.19 min), Luc-OH-NO2
(Fig. 5, m/z = (-)325, peak detected at 2.02 min) and of the Luc• reduction product, Luc-H,
(Fig. 5, m/z = (-)263, peak detected at 2.04 min) depend on the composition of reaction
mixture. When MNP is in the reaction mixture (Fig. 5, traces a), the yields of nitrated
luciferins (Luc-NO2, Luc-OH-NO2) are significantly lower compared with the yields of the
same products when oxidation was performed in the absence of MNP (Fig. 5, traces d). The
addition of 2-propanol (2-PrOH) into the reaction mixture suppressed the formation of Luc-
NO2 and Luc-OH-NO2 (Fig. 5, traces b) but increased the amount of Luc-H formed to the
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level observed when LBA was oxidized by ONOO– in solution without a spin trap (Fig. 5,
traces d). Together with the observed inhibitory effects of 2-PrOH on the formation of the
phenyl radical spin adduct (Fig. 4), these data strongly point to the role of 2-PrOH as the
quencher and reductant of the phenyl-type radical formed from the LBA probe. These
observations are consistent with the occurrence of a minor radical pathway that is specific to
the reaction between ONOO– and LBA. Overall, the EPR spin trapping experiments, the spin
trapping combined with LC-MS analyses, and the effect of the phenyl radical scavengers on
the product distribution demonstrate the involvement of the phenyl-type radical Luc• in the
minor pathway of the reaction of the LBA probe with ONOO–.
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Figure 5. LC-MS analyses of the products of LBA oxidation by ONOO–. The reactions were
carried out under conditions similar to those described in Fig. 4. LC-MS traces of the reaction
mixtures of (a) LBA (200 µM), MNP (40 mM), ONOO– (200 ꢁM); (b) LBA (200 µM), MNP
(40 mM), ONOO– (200 µM), 2-PrOH (10%); (c) LBA (200 µM), MNP (40 mM); (d) LBA
(200 µM), ONOO– (200 µM); and (e) MNP (200 µM), ONOO– (200 µM), DMSO (2.5%).
LBA, luciferin, Luc-NO2, Luc-H, and Luc-OH-NO2 were detected in negative mode using
SIM set at m/z values of 307, 279, 308, 263, and 325, respectively. Experiments were
repeated three times independently. Representative result displayed.
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3.3. Limit of detection and quantification
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Based on our HPLC analyses, we estimated the lowest concentrations of oxidant that are
needed to detect the Luc-OH, Luc-OH-Cl, and Luc-OH-NO2 (Supplementary Table 1).
Results show that the detection limit of the LBA probe for ONOO−, HOCl, and H2O2 was in
the submicromolar to low micromolar range. Limits of detection of the ONOO–-specific
product Luc-NO2 and hypochlorite-specific product Luc-OH-Cl were in the low micromolar
range. As with other fluorescent products, the actual sensitivity will strongly depend on the
detection modality and instrumentation. Furthermore, significantly higher sensitivity is
expected in the case of bioluminescence-based detection of Luc-OH and Luc-OH-Cl, as
observed for other luminescent probes.
3.4. Dynamics of the product formation
Formation of Luc-OH upon oxidation of the PCL-1 probe requires the release of the QM
moiety from the primary oxidation product (Scheme 1). Therefore, to determine if this
reaction may affect the dynamics of Luc-OH production, we compared the kinetic profiles of
Luc-OH formation during the oxidation of LBA and PCL-1 to the profile of the product
formation from a simple boronate probe, coumarin-7-boronic acid (CBA). Figure 6 shows the
buildup of absorption at 380 nm during the reaction between those boronic probes and H2O2
(25 mM). It is evident that direct derivatization of luciferin using the boronate group results in
a faster formation of the product (Luc-OH or 7-hydroxycoumarin) than when using the
boronobenzylation approach, as in the case of the PCL-1 probe. The sigmoidal shape of the
kinetic profile of the Luc-OH buildup observed in the case of the PCL-1 probe is consistent
with a multistep process, following the kinetics of the formation of a product of two
consecutive reactions.
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Figure 6. Kinetic profiles of product formation during the oxidation of probes LBA, PCL-1,
and CBA (10 µM each) by H2O2 (25 mM). Experiments were repeated three times
independently. Representative result displayed.
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3.5. Oxidation of LBA and PCL-1 by activated macrophages
To confirm that the LBE probe can report ONOO– in biologically relevant cellular systems,
we determined its performance in cultured RAW 264.7 cells activated to produce ONOO–.
RAW 264.7 macrophages were stimulated with a mixture of lipopolysaccharide (LPS),
interferon γ (IFN-γ), and phorbol myristate acetate (PMA), as described previously [18,24]. In
•–
this cellular system, PMA activates NADPH oxidase to produce superoxide (O2 ) whereas
LPS/IFN-γ pretreatment induces expression of nitric oxide synthase (iNOS), resulting in
•
•–
•
increased production of NO. Co-generation of both O2 and NO leads to the formation of
ONOO– and induces an increase in fluorescence intensity due to oxidation of LBE or PCL-1
boronate probe (Fig. 7A,B). The addition of LPS and IFN-γ or PMA alone to incubations
containing macrophages and PCL-1 or LBE caused only a minor increase in fluorescence
intensity (Fig. 7) as compared with the fully activated (LPS, IFN-γ, and PMA) cells.
Interestingly, although both probes respond similarly to the treatments used, in case of
untreated cells, a slow increase in the signal is observed for the PCL-1 probe but not for the
LBE probe (Fig. 7, control). This indicates that LBA is more stable than PCL-1 under the
experimental condition used.
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Figure 7. Real-time monitoring of the oxidation of the LBE (A) and PCL-1 (B) probes by
activated RAW 264.7 macrophages. (C, D) Rate of increase in the fluorescence signal
intensity from RAW 264.7 macrophages activated by different stimulators in the absence or in
the presence of L-NAME and CAT. Incubations contained PCL-1 (20 µM) or LBE (20 µM)
and RAW 264.7 macrophages in DBPS-GP buffer in different cellular conditions. Conditions
were as follows: control cells, PMA added at the time of the measurement, LPS and INF-γ
pre-treated cells, and LPS and INF-γ pre-treated cells with PMA added before measurements.
(Details are described in the Experimental section.) Oxidation of the probes was followed by
the measurement of the fluorescence intensity (λex 355 nm, λem 520 nm).
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To confirm the identity of the oxidants involved in the probes’ oxidation, we tested the effect
of L-NAME (iNOS inhibitor, preventing ONOO– formation) and catalase (H2O2 scavenger)
on the rate of the probes’ oxidation. As shown in Figs. 7C and 7D, L-NAME showed the
strongest inhibitory effects when cells were coexposed to PMA and LPS-/IFN-γ. Under those
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conditions, catalase had only a minor effect. These results indicate that both luciferin-based
boronic probes tested can be used for real-time detection of ONOO– formed from
•
•–
macrophages activated to co-generate NO and O2 . Interestingly, catalase completely
inhibited LBE probe oxidation in cells treated with PMA. This indicates that under these
conditions H2O2 is the major oxidant.
4. Discussion
The application of probes to detect biologically relevant oxidants in inflammatory processes
occurring in vitro and in vivo requires a detailed knowledge of probes’ chemical reactivity,
their stability in medium, and identification of the oxidant-specific product(s) together with
their spectroscopic characterization. The PCL-1 probe represents the first boronate-based
sensor to detect oxidants in vivo using the bioluminescence imaging modality [10]. Recently,
we reported the application of the PCL-1 probe to monitor oxidants in tumor tissues in vivo in
the mouse xenograft model of breast cancer, in combination with low-temperature EPR
analyzes of inactivated aconitase signals [39]. Our recent investigation of the oxidation
chemistry of the PCL-1 probe [12] showed that boronobenzylated luciferin (PCL-1 probe)
decomposes significantly in phosphate buffer during prolonged incubation. This may lead to a
relatively high background signal preventing the probe from detecting small, physiologically
relevant levels of the oxidants. Here, we show that direct boronation of luciferin results in a
more stable probe, LBE, which may represent a better choice when lower levels of the
oxidants are to be detected.
Over the last decade, several reports and reviews have emphasized that it is impossible
to categorically identify specific oxidants formed in cells without the application of HPLC- or
LC-MS-based methods to detect oxidant-specific product(s) [12, 20, 32-37]. For example, 2-
hydroxyethidium (a fingerprint of the reaction between hydroethidine and superoxide) and
ethidium (nonspecific product of two-electron oxidation of hydroethidine by various oxidants)
have overlapping fluorescence emission spectra but can be separated, identified, and
quantified with the aid of HPLC or LC-MS, using appropriate standards [38]. The most
promising probes for the detection of several nonradical ROS/RNS, produced under
inflammatory conditions, are based on the oxidation chemistry of arylboronic acids or esters.
Masking of hydroxyl or amino group(s) of a fluorophore by boronate or boronobenzyl
moieties turns off the fluorescence [28]. Originally, this class of probes was developed for
specific detection of H2O2 [28], which slowly uncaged the hydroxyl group and thus turned on
the fluorescence [16,17]. Subsequent works proved that under physiological pH, boronate
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probes are oxidized by HOCl and ONOO– at significantly higher rates than is the case for
H2O2 [16]. The additional advantage of the fluorogenic boronate probes used to detect
ONOO– is their ability to replace the boronate moiety by the -NO2 (nitro) group and thus to
form the ONOO–-specific nitro derivatives [12,17,21]. In the presence of excess ONOO–, the
phenolic product may undergo nitration to form corresponding nitrophenol. Among the two
nitration products, nitrobenzene-type (Luc-NO2) and nitrophenol-type (Luc-OH-NO2), only
the nitrophenolic product can be formed via myeloperoxidase-catalyzed nitration. Since the
nitrobenzene-type product is not generated during incubation of boronate probe with
myeloperoxidase/H2O2/nitrite systems, its detection confirms the presence of ONOO– [16,24].
Bioluminescence imaging using luciferin-based probes is currently a standard technique for in
vivo imaging. Based on our in vitro data with the use of activated macrophages, we predict
that the LBE probe can be applied as a stable probe with low background for sensitive
detection of ROS/RNS in in vivo animal models.
5. Conclusions
In this work, we synthesized the LBE probe, which undergoes hydrolysis to LBA in aqueous
solutions. We have identified the products of LBA oxidation by selected inflammatory
oxidants. The major, common product formed during the reaction of LBA with H2O2, HOCl,
and ONOO– is luciferin, a substrate for bioluminescence imaging. However, oxidation of
LBA by ONOO– proceeds via two pathways, with Luc-NO2 being an ONOO–-specific
product formed in the minor pathway involving the phenyl-type radical Luc• intermediate.
Reaction of LBA with HOCl yields luciferin that is further chlorinated to Luc-OH-Cl, a
product specific for HOCl. We propose combining LBA-based fluorescence or
bioluminescence measurements with chromatographic analyses of those specific reaction
products to identify the oxidants responsible for probe oxidation. Because the LBE probe is
more stable than PCL-1, it may be a better choice for detecting inflammatory oxidants when
present at low levels and/or when exhibiting slow reaction kinetics, such as H2O2.
Acknowledgment
This work was supported by a grant from the Polish National Science Centre within the
SONATA BIS 6 program (Grant no. 2016/22/E/ST4/00549) to RP, a grant from the Polish
National Science Centre within the MINIATURA
3
program (Grant no.
2019/03/X/ST4/00748) to MS, and an Institutional Research Grant IRG (#16-183-31) from
the American Cancer Society and the Medical College of Wisconsin Cancer Center to JZ. The
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LC-MS analyses were performed in Medical College of Wisconsin Cancer Center Redox and
Bioenergetics Shared Resource.
References
[1] McCaffrey A, Kay MA, Contag CH. Advancing molecular therapies through in vivo
bioluminescent imaging. Mol Imaging 2003;2:75-86.
https://doi.org/10.1162/15353500200303124.
[2] Luker KE, Smith MCP, Luker GD, Gammon ST, Piwnica-Worms H, Piwnica-Worms D.
Kinetics of regulated protein-protein interactions revealed with firefly luciferase
complementation imaging in cells and living animals. Proc Natl Acad Sci USA 2004;101:
12288-12293. https://doi.org/10.1073/pnas.0404041101.
[3] d’Enfert C, Vecchiarelli A, Brown AJP. Bioluminescent fungi for real-time monitoring of
fungal infections. Virulence 2010;1:174-176. https://doi.org/10.4161/viru.1.3.11119.
[4] Suff N, Waddington S. The power of bioluminescence imaging in understanding host-
pathogen interactions. Methods 2017;127:69-78. https://doi.org/10.1016/j.ymeth.2017.07.001.
[5] Roy I, McAllister DM, Gorse E, Dixon K, Piper CT, Zimmerman NP, Getschman AE,
Tsai S, Engle DD, Evans DB, Volkman BF, Kalyanaraman B, Dwinell MB. Pancreatic cancer
cell migration and metastasis is regulated by chemokine-biased agonism and bioenergetic
signaling. Cancer Res 2015;75:3529-3542. https://doi.org/10.1158/0008-5472.CAN-14-2645.
[6] Luwor RB, Stylli SS, Kaye AH. Using bioluminescence imaging in glioma research. J
Clin Neurosci 2015;22:779-784. https://doi.org/10.1016/j.jocn.2014.11.001.
[7] Mezzanotte L, van‘t Root M, Karatas H, Goun EA, Löwik CWGM. Trends in
Biotechnology 2017;35:640-652. https://doi.org/10.1016/j.tibtech.2017.03.012.
[8] Podsiadły R, Grzelakowska A, Modrzejewska J, Siarkiewicz P, Słowiński D, Szala M,
Świerczyńska M. Recent progress in the synthesis of firefly luciferin derivatives. Dyes Pigm
2019;170:107627. https://doi.org/10.1016/j.dyepig.2019.107627.
[9] Li J, Chen L, Du L, Li M. Cage the firefly luciferin! - a strategy for developing
bioluminescent probes. Chem Soc Rev 2013;42:662-676. https://doi.org/10.1039/c2cs35249d.
[10] Van de Bittner GC, Dubikovskaya EA, Bertozzi CR, Chang CJ. In vivo imaging of
hydrogen peroxide production in a murine tumor model with a chemoselective bioluminescent
reporter. Proc Natl Acad Sci USA 2010;107:21316-21321.
https://doi.org/10.1073/pnas.1012864107.
23

586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
[11] Sieracki NA, Gantner BN, Mao M, Horner JH, Ye RD, Malik AB, Newcomb ME,
Bonini MG. Bioluminescent detection of peroxynitrite with a boronic acid-caged luciferin.
Free Radic Biol Med 2013;61:40-50. https://doi.org/10.1016/j.freeradbiomed.2013.02.020.
[12] Zielonka J, Podsiadły R, Zielonka M, Hardy M, Kalyanaraman B. On the use of peroxy-
caged luciferin (PCL-1) probe for bioluminescent detection of inflammatory oxidants in vitro
and in vivo – Identification of reaction intermediates and oxidant-specific minor products.
Free Radic Biol Med 2016;99:32-42. https://doi.org/10.1016/j.freeradbiomed.2016.07.023.
[13] Wu W, Li J, Chen L, Ma Z, Zhang W, Liu Z, Cheng Y, Du L, Li M. Bioluminescent
probe for hydrogen peroxide imaging in vitro and in vivo. Anal Chem 2014;86:9800-9806.
https://doi.org/10.1021/ac502396g.
[14] Kojima R, Takakura H, Kamiya M, Kobayashi E, Komatsu T, Ueno T, Terai T, Hanaoka
K, Nagano T, Urano Y. Development of a sensitive bioluminogenic probe for imaging highly
reactive oxygen species in living rats. Angew Chem Int Ed 2015;54:14768-14771.
https://doi.org/10.1002/anie.201507530.
[15] Chen P, Zheng Z, Zhu Y, Dong Y, Wang F, Liang G. Bioluminescent turn-on probe for
sensing hypochlorite in vitro and in tumors. Anal Chem 2017;89:5693-5696.
https://doi.org/10.1021/acs.analchem.7b01103.
[16] Sikora A, Zielonka J, Lopez M, Joseph J, Kalyanaraman B. Direct oxidation of boronates
by peroxynitrite: mechanism and implications in fluorescence imaging of peroxynitrite. Free
Radic Biol Med 2009;47:1401-1407. https://doi.org/10.1016/j.freeradbiomed.2009.08.006.
[17] Zielonka J, Sikora A, Hardy M, Joseph J, Dranka BP, Kalyanaraman B. Boronate probes
as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides. Chem Res
Toxicol 2012;25:1793-1799. https://doi.org/10.1021/tx300164j.
[18] Zielonka J, Zielonka M, Sikora A, Adamus J, Joseph J, Hardy M, Ouari O, Dranka BP,
Kalyanaraman B. Global profiling of reactive oxygen and nitrogen species in biological
systems: high-throughput real-time analyses. J Biol Chem 2012;287:2984-2995.
https://doi.org/10.1074/jbc.M111.309062.
[19] Zielonka J, Sikora A, Joseph J, Kalyanaraman B. Peroxynitrite is the major species
formed from different flux ratios of co-generated nitric oxide and superoxide: direct reaction
with boronate-based fluorescent probe. J Biol Chem 2010;285:14210-14216.
https://doi.org/10.1074/jbc.M110.110080.
[20] Michalski R, Zielonka J, Gapys E, Marcinek A, Joseph J, Kalyanaraman B. Real-time
measurements of aminoacid and protein hydroperoxides using coumarin boronic acid. J Biol
Chem 2014;289:22536-22553. https://doi.org/10.1074/jbc.M114.553727.
24

620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
[21] Sikora A, Zielonka J, Lopez M, Dybala-Defratyka A, Joseph J, Marcinek A,
Kalyanaraman B. Reaction between peroxynitrite and boronates: EPR spin-trapping, HPLC
analyses, and quantum mechanical study of the free radical pathway. Chem Res Toxicol
2011;24:687-697. https://doi.org/10.1021/tx100439a.
[22] Sikora A, Zielonka J, Adamus J, Debski D, Dybala-Defratyka A, Michałowski B, Joseph
J, Hartley RC, Murphy MP, Kalyanaraman B. Reaction between peroxynitrite and
triphenylphosphonium-substituted arylboronic acid isomers: identification of diagnostic
marker products and biological implications. Chem Res Toxicol 2013;26:856-867.
https://doi.org/10.1021/tx300499c.
[23] Zielonka J, Sikora A, Adamus J, Kalyanaraman B. Detection and differentiation between
peroxynitrite and hydroperoxides using mitochondria-targeted arylboronic acid, In: Weissig
V., Edeas M. (eds) Mitochondrial Medicine. Methods in Molecular Biology, vol 1264.
Humana Press, New York, NY. http://doi.org/10.1007/978-1-4939-2257-4_16.
[24] Zielonka J, Zielonka M, VerPlank L, Cheng G, Hardy M, Ouari O, Ayhan MM,
Podsiadly R, Sikora A, Lambeth JD, Kalyanaraman B. Mitigation of NADPH oxidase 2
activity as a strategy to inhibit peroxynitrite formation. J Biol Chem 2016;291:7029-7044.
https://doi.org/10.1074/jbc.M115.702787.
[25] Smulik R, Debski D, Zielonka J, Michałowski B, Adamus J, Marcinek A, Kalyanaraman
B, Sikora A. Nitroxyl (HNO) react with molecular oxygen and forms peroxynitrite at
physiological pH: biological implication. J Biol Chem 2014;289:35570-35581.
https://doi.org/10.1074/jbc.M114.597740.
[26] Shu W, Yan L, Wang Z, Liu J, Zhang S, Liu C, Zhu B. A novel visual and far-red
fluorescent dual-channel probe for the rapid and sensitive detection of hypochlorite in
aqueous solution and living cells. Sensor Actuat B-Chem 2015;221:1130-1136.
https://doi.org/10.1016/j.snb.2015.07.066.
[27] Sedgwick AC, Han HH, Gardiner JE, Bull SD, He XP, James TD. Long-wavelength
fluorescent boronate probes for the detection and intracellular imaging of peroxynitrite, Chem
Commun 2017;53:12822-12825. https://doi.org/10.1039/c7cc07845e.
[28] Miller EW, Albers AE, Pralle A, Isacoff EY, Chang CJ. Boronate-based fluorescent
probes for imaging cellular hydrogen peroxide. J Am Chem Soc 2005;127:16652-16659.
https://doi.org/10.1021/ja054474f.
[29] Morton RA, Hopkins TA, Seliger HH. The spectroscopic properties of firefly luciferin
and related compounds. An approach to product emission. Biochemistry 1969;8:1598-1607.
https://doi.org/10.1021/bi00832a041.
25

654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
[30] Takakura H, Kojima R, Ozawa T, Nagano T, Urano Y. Development of 5′- and 7′-
substituted luciferin analogues as acid-tolerant substrates of firefly luciferase, ChemBioChem
2012;13:1424-1427. https://doi.org/10.1002/cbic.201200142.
[31] Øpstad CL, Melø T-B, Sliwka H-R, Partali V. Formation of DMSO and DMF radicals
with minute amounts of base. Tetrahedron 2009;65:7616-7619.
https://doi.org/10.1016/j.tet.2009.06.109.
[32] Prolo C, Álvarez MN, Ríos N, Peluffo G, Radi R, Romero N. Nitric oxide diffusion to
red blood cells limits extracellular, but not intraphagosomal, peroxynitrite formation by
macrophages. Free Radic Biol Med 2015;87:346-355.
https://doi.org/10.1016/j.freeradbiomed.2015.06.027.
[33] Rios N, Piacenza L, Trujillo M, Martínez A, Demicheli V, Prolo C, Álvarez MN, López
GV, Radi R. Sensitive detection and estimation of cell-derived peroxynitrite fluxes using
fluorescein-boronate. Free Radic Biol Med 2016;101:284-295.
https://doi.org/10.1016/j.freeradbiomed.2016.08.033.
[34] Prolo C, Rios N, Piacenza L, Álvarez MN, Radi R. Fluorescence and chemiluminescence
approaches for peroxynitrite detection. Free Radic Biol Med 2018;128:59-68.
https://doi.org/10.1016/j.freeradbiomed.2018.02.017.
[35] Zielonka J, Kalyanaraman B. Small-molecule luminescent probes for the detection of
cellular oxidizing and nitrating species. Free Radic Biol Med 2018;128:3-22.
https://doi.org/10.1016/j.freeradbiomed.2018.03.032.
[36] Hardy M, Zielonka J, Karoui H, Sikora A, Michalski R, Podsiadły R, Lopez M, Vasquez-
Vivar J, Kalyanaraman B, Ouari O. Detection and characterization of reactive oxygen and
nitrogen species in biological systems by monitoring species-specific products. Antioxid
Redox Sign 2018;28:1416-14132. https://doi.org/10.1089/ars.2017.7398.
[37] Kalyanaraman B, Hardy M, Podsiadły R, Cheng G, Zielonka J. Recent developments in
detection of superoxide radical anion and hydrogen peroxide: Opportunities, challenges, and
implications in redox signaling. Arch Biochem Biophys 2017;617:38-47.
http://dx.doi.org/10.1016/j.abb.2016.08.021.
[38] Zielonka J, Hardy M, Kalyanaraman B. HPLC study of oxidation products of
hydroethidine in chemical and biological systems: ramifications in superoxide measurements.
Free Radic Biol Med 2009;46:329-338. https://doi.org/10.1016/j.freeradbiomed.2008.10.031.
[39] Cheng G, Pan J, Podsiadly R, Zielonka J, Garces AM, Machado LGDD, Bennett B,
McAllister D, Dwinell MB, You M, Kalyanaraman B. Increased formation of reactive oxygen
species during tumor growth: Ex vivo low-temperature EPR and in vivo bioluminescence
26

688
689
analyses. Free Radic Biol Med 2020;147:167-174.
https://doi.org/10.1016/j.freeradbiomed.2019.12.020.
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Highlights
> Firefly luciferin-based probe for detection of peroxynitrite > Luciferin boronic acid (LBA)
is oxidized to luciferin by H2O2, HOCl, and peroxynitrite > 7’-Chloroluciferin is a product
specific for HOCl > 6’-Nitroluciferin serves as peroxynitrite-specific product > LBA enables
real-time monitoring of the ONOO– formation in activated RAW 264.7 macrophages