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

Article abstract of DOI:10.1016/j.freeradbiomed.2016.07.023

Peroxy-caged luciferin (PCL-1) probe was first used to image hydrogen peroxide in living systems (Van de Bittner et al., 2010 [9]). Recently this probe was shown to react with peroxynitrite more potently than with hydrogen peroxide (Sieracki et al., 2013 [11]) and was suggested to be a more suitable probe for detecting peroxynitrite under in vivo conditions. In this work, we investigated in detail the products formed from the reaction between PCL-1 and hydrogen peroxide, hypochlorite, and peroxynitrite. HPLC analysis showed that hydrogen peroxide reacts slowly with PCL-1, forming luciferin as the only product. Hypochlorite reaction with PCL-1 yielded significantly less luciferin, as hypochlorite oxidized luciferin to form a chlorinated luciferin. Reaction between PCL-1 and peroxynitrite consists of a major and minor pathway. The major pathway results in luciferin and the minor pathway produces a radical-mediated nitrated luciferin. Radical intermediate was characterized by spin trapping. We conclude that monitoring of chlorinated and nitrated products in addition to bioluminescence in vivo will help identify the nature of oxidant responsible for bioluminescence derived from PCL-1.

Full text of DOI:10.1016/j.freeradbiomed.2016.07.023

Free Radical Biology and Medicine 99 (2016) 32–42
Contents lists available at ScienceDirect
Free Radical Biology and Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Original article
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
b
a
c
Jacek Zielonka a, , Radosław Podsiadły , Monika Zielonka , Micael Hardy ,
n
Balaraman Kalyanaraman a,
n
a Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, United States
b Institute of Polymer and Dye Technology, Faculty of Chemistry, Lodz University of Technology, Stefanowskiego 12/16, 90-924 Lodz, Poland
c Aix-Marseille Université, CNRS, ICR UMR 7273, 13397 Marseille, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Peroxy-caged luciferin (PCL-1) probe was first used to image hydrogen peroxide in living systems (Van de
Bittner et al., 2010 [9]). Recently this probe was shown to react with peroxynitrite more potently than
with hydrogen peroxide (Sieracki et al., 2013 [11]) and was suggested to be a more suitable probe for
detecting peroxynitrite under in vivo conditions. In this work, we investigated in detail the products
formed from the reaction between PCL-1 and hydrogen peroxide, hypochlorite, and peroxynitrite. HPLC
analysis showed that hydrogen peroxide reacts slowly with PCL-1, forming luciferin as the only product.
Hypochlorite reaction with PCL-1 yielded significantly less luciferin, as hypochlorite oxidized luciferin to
form a chlorinated luciferin. Reaction between PCL-1 and peroxynitrite consists of a major and minor
pathway. The major pathway results in luciferin and the minor pathway produces a radical-mediated
nitrated luciferin. Radical intermediate was characterized by spin trapping. We conclude that monitoring
of chlorinated and nitrated products in addition to bioluminescence in vivo will help identify the nature
of oxidant responsible for bioluminescence derived from PCL-1.
Received 25 April 2016
Received in revised form
24 June 2016
Accepted 21 July 2016
Available online 22 July 2016
Keywords:
Boronate probes
Luciferin
Bioluminescence
Spin traps
& 2016 Elsevier Inc. All rights reserved.
ꢀ
1. Introduction
to peroxynitrite (ONOOꢁ) and nitrogen dioxide radical ( NO2).
Rigorous identification of those species is crucial for under-
standing their role in cellular signaling and pathology.
Reactive oxygen species (ROS) and reactive nitrogen species
(RNS) have emerged as important mediators of cellular signaling
and damage [1–3]. ROS and RNS comprise of different species of
very diverse chemical reactivity, lifetime and target specificity in
extracellular and intracellular milieu [4,5]. The term ‘ROS’ typically
Molecular imaging of ROS/RNS is an emerging area of research
in redox and free radical biology [6–8]. Bioluminescence or fluor-
escence modalities are typically used. Peroxy-caged luciferin (PCL-
1) (Fig. 1) is one of the first cell-permeable small molecular weight
probes used to image ROS in living systems [9,10]. H2O2 slowly
reacts with PCL-1 probe (k¼1.2 M ꢁ1 sꢁ1 [11]) to form luciferin
in situ that is oxidized by the luciferase enzyme (using ATP as a co-
factor) emitting a green bioluminescent signal [9,10] (Fig. 1). Upon
oxidation, PCL-1 probe eliminates the para-quinone methide (QM),
with the formation of luciferin which gets oxidized to oxyluciferin
in luciferase-transfected cells generating bioluminescence (Fig. 1).
It was shown that administration of bolus H2O2 to mice over-
expressing luciferase increased the bioluminescent signal from
PCL-1 [9,10]. Subsequently, PCL-1 was shown to be oxidized to
luciferin in the presence of hypochlorite (HOCl) and ONOOꢁ [11].
Importantly, it was shown that in the presence of plasma, the
probability of oxidation of PCL-1 probe by H2O2 is very low;
however, under this condition, the probe was still oxidized by
ꢀ
refers to superoxide radical anion (O2 ꢁ), hydrogen peroxide
ꢀ
ꢀ
(H2O2), hydroxyl radical ( OH), lipid peroxy radicals (LOO ), lipid
hydroperoxides (LOOH), and singlet oxygen (1O2) and ‘RNS’ refers
Abbreviations: AA-OOH, amino acid hydroperoxide; DIPPMPO, diisopropox-
yphosphoryl-5-methyl-1-pyrroline N-oxide; DPI, diphenyleneiodonium; HO-Bz-
ꢀ
OH, 4-hydroxybenzyl alcohol; L-NAME, L-NG-Nitroarginine methyl ester; LOO , li-
pid peroxy radicals; LOOH, lipid hydroperoxide; Luc-Cl, chloroluciferin; MNP, 2-
methyl-2-nitroso propane; MRM, multiple reaction monitoring; PCL-1, peroxy-
caged luciferin; Pr-OOH, protein hydroperoxide; QM, para-quinone methide; RNS,
reactive nitrogen species; ROS, reactive oxygen species; SIM, single ion monitoring
n
Corresponding authors.
E-mail addresses: jzielonk@mcw.edu (J. Zielonka),
radoslaw.podsiadly@p.lodz.pl (R. Podsiadły), mzielonka@mcw.edu (M. Zielonka),
micael.hardy@univ-provence.fr (M. Hardy), balarama@mcw.edu (B. Kalyanaraman).
http://dx.doi.org/10.1016/j.freeradbiomed.2016.07.023
0891-5849/& 2016 Elsevier Inc. All rights reserved.

J. Zielonka et al. / Free Radical Biology and Medicine 99 (2016) 32–42
33
described elsewhere [12] and stored at ꢁ80 °C.
L
-NAME and DPI
were from Cayman. All other chemicals were from Sigma-Aldrich
and were of highest purity available. The stock solutions of
ONOOꢁ, HOCl and H2O2 were prepared freshly each day and the
concentration was determined by spectrophotometry, using the
extinction coefficients values of 1.7 ꢃ 103 Mꢁ1 cmꢁ1 (at 302 nm, in
0.1 M NaOH), 350 Mꢁ1 cmꢁ1 (at 292 nm, in 0.1 M NaOH) and
39.4 Mꢁ1 cmꢁ1 (at 240 nm, in water), respectively. PCL-1 stock
solution (1 mM) was typically prepared using ethanol (EtOH) as a
solvent to minimize scavenging of HOCl by DMSO, a solvent ty-
pically used for boronate probes. Of the four organic solvent (EtOH,
acetonitrile, DMSO and DMF) tested for interference with HOCl-
induced oxidation of coumarin boronic acid (CBA) to hydro-
xycoumarin (COH), EtOH exhibited the smallest inhibitory effect
(Suppl. Fig. 1). For the spin trapping of phenyl radical, DMSO was
used to prepare the stock solution of PCL-1 to avoid scavenging of
phenyl radical by EtOH. 5-Diisopropoxyphosphoryl-5-methyl-1-
pyrroline N-oxide (DIPPMPO) was synthesized according to the
published procedure [20].
Fig. 1. A hypothetical scheme showing the application of PCL-1 probe for biolu-
minescent detection of H2O2, HOCl or ONOOꢁ in luciferase-transfected mice tumor
xenografts.
ONOOꢁ. This is consistent with our previous reports demon-
strating that ONOOꢁ reacts directly and rapidly with boronate
probes forming corresponding phenols as the major products. This
brings into question the identity of the oxidant(s) responsible for
in vivo bioluminescence measurements using PCL-1 probe.
2.2. HPLC analyses
HPLC analyses of PCL-1 and its oxidation products were per-
formed using Agilent 1100 HPLC equipped with UV–vis absorption
and fluorescence detectors. The compounds were loaded onto
Kinetex C18 column (Phenomenex, 100 mm ꢃ 4.6 mm, 2.6 mm)
equilibrated with 10% of acetonitrile in water, containing 0.1%
trifluoroacetic acid. The products were eluted by an increase of the
acetonitrile concentration from 10-100% over 7 min. The flow rate
was kept at 1.5 mL/min. PCL-1 and the products containing luci-
ferin moiety were detected by monitoring absorbance at 330 nm,
and the product of water added to QM was detected at 220 nm.
Additionally, luciferin was also monitored using the fluorescence
detector with the excitation set at 330 nm and emission set at
520 nm.
We have demonstrated that in addition to H2O2, other biolo-
gically-relevant oxidants, including HOCl and ONOOꢁ, are able to
oxidize aromatic boronates to the corresponding phenolic pro-
ducts [12–15]. More recently, we have shown that selected amino
acid hydroperoxides (AA-OOH) and protein hydroperoxides (Pr-
OOH) also oxidize boronic compounds [16]. With all of the oxi-
dants tested so far, the mechanism appears to be similar; a 1:1
stoichiometry between oxidant and boronate probes was ob-
served, resulting in the same major product. However, the main
difference is the rate constant of the reaction between different
oxidants and the boronate probe. The rate constants varied from
100, 101, 104 to 106 Mꢁ1 sꢁ1 for H2O2, AA-OOH, HOCl and ONOOꢁ
,
respectively [13,16]. Therefore, depending on the experimental
settings, boronates may be used to detect different oxidants by
monitoring the reaction products. Unlike other listed oxidants,
ONOOꢁ oxidizes boronic compounds in two pathways: major
(ꢂ90%), non-radical pathway, leading to the corresponding phe-
nol; and minor (ꢂ10%), radical pathway, forming a phenyl-type
2.3. LC-MS analyses
LC-MS analyses of PCL-1, its oxidation products and spin ad-
ducts were performed using Shimadzu LC-MS 8030 triple quad-
rupole mass detector coupled to Shimadzu Nexera 2 UHPLC sys-
tem. The reaction mixture was injected on Cortecs C18 column
(Waters, 50 mm ꢃ 2 mm, 1.6 mm) equilibrated with 10% of acet-
onitrile in water containing 0.1% of formic acid. The compound was
eluted by increasing the acetonitrile concentration in the mobile
phase from 10-80% over 4 min. 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. PCL-1, luci-
ferin, Luc-Bz-NO2, Luc-Bz-H and Luc-Cl were detected as positive
ions using multiple reaction monitoring (MRM) mode, using the
primary/fragment ion pairs of 4154135, 2814235, 4164234,
371491 and 3154269, respectively. Luc-Bz-OH was detected in
positive mode using single ion monitoring (SIM), set at the m/z
value of 387.
ꢀ
radical, nitrogen dioxide ( NO2) and stable products formed from
them [12,13,17]. We propose that these ONOOꢁ-specific products
may serve as specific markers for ONOOꢁ. By determining the
ONOOꢁ-specific products, we recently confirmed the formation of
ONOOꢁ from nitroxyl (HNO) reaction with O2 [18] and tested the
effect of inhibition of NADPH oxidase on the production of ONOO–
by activated macrophages [19].
Here we investigate in detail the products formed from the
oxidative and nitrative chemistry of PCL-1 that will help to better
interpret in vivo bioluminescence results. We compared the pro-
ducts formed during the oxidation of PCL-1 by H2O2, HOCl and
ONOOꢁ, the likely in vivo inflammatory oxidants. As several oxi-
dant species react with PCL-1 to generate bioluminescence, the
oxidant-specific minor product(s) may be used to confirm the
identity of ROS/RNS species.
2.4. EPR spin-trapping
2. Materials and methods
EPR spin trapping experiments were performed using Bruker
EMX EPR spectrometer, as reported previously [17]. The instru-
ment parameters were as follows: scan range, 150 G; time con-
stant, 1.28 ms; scan time, 84 s; modulation amplitude, 1 G; mod-
ulation frequency, 100 kHz; receiver gain, 1 ꢃ 105; and microwave
power, 20 mW. The spectra shown are the averages of 5 scans.
2.1. Chemicals, preparation of solutions
PCL-1 probe was synthesized as described below. D-Luciferin
(potassium salt) was purchased from Gold Biotechnology. H2O2
and HOCl were from Sigma-Aldrich. ONOOꢁ was synthesized as

34
J. Zielonka et al. / Free Radical Biology and Medicine 99 (2016) 32–42
Fig. 2. Synthetic pathways used to obtain (A) PCL-1 probe, Luc-Bz-H and Luc-Bz-NO2 and (B) Luc-Cl standards.
2.5. NMR analyses
chlorine atom is located in the position C-2. The NMR spectra of
chloroluciferin, including two-dimensional sequences (hetero-
nuclear multiple bond coherence, HMBC and heteronuclear single
quantum coherence, HSQC) are shown in Supplementary
Figs. 2 and 3.
NMR analyses for determination of the structure of synthesized
standards of Luc-Bz-NO2 and Luc-Cl were performed at the Aix-
Marseille Université (Spectropole). 1H NMR and 13C NMR spectra
were recorded with a Bruker DPX 600 spectrometer at 400.13 or
600.13 MHz and 75.54 MHz, respectively. Solutions were prepared
in CDCl3 as a solvent, using TMS or CDCl3 as internal reference for
2.7. Cell culture and extraction of PCL-1 oxidation products
1H NMR and 13C NMR spectra respectively. Chemical shifts (
δ
) are
RAW 264.7 cells were cultured in DMEM medium (Invitrogen)
reported in ppm, and coupling constant J values in hertz (Hz). NMR
peak multiplicities are described as follows: s, singlet; d, doublet;
dd, doublet of doublets; brdd, broad doublet of doublets; and m,
multiplets.
supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL
penicillin and 100 mg/mL streptomycin. For stimulation of the cells
to produce nitric oxide, cells were incubated overnight (12–16 h)
with LPS (0.5 mg/mL) and IFN (50 units/mL). To stimulate NADPH
γ
oxidase-dependent superoxide production, the cells were washed
and treated with phorbol 12-myristate 13-acetate (PMA, 1 mM) in
DPBS supplemented with pyruvic acid and glucose (DPBS-GP). At
the time of addition of PMA, PCL-1 probe (100 mM) was also added
and the cells were incubated for 1 h at 37 °C in a CO2-free in-
2.6. Synthesis of PCL-1 and standards of the products
PCL-1, Luc-Bz-H and Luc-Bz-NO2 (Fig. 2A) were synthesized by
modifying the published protocol [9] using 4-(bromomethyl)ben-
zeneboronic acid pinacol ester, benzyl bromide, and 4-nitrobenzyl
bromide, respectively, as the starting materials. The synthetic
cubator. Where indicated, L-NAME (1 mM) or diphenyleneiodo-
nium (DPI, 0.1–10 mM) was added 30 min before PMA. After 1 h, an
aliquot of the medium was collected and frozen in liquid nitrogen.
Cells were washed twice with ice-cold PBS, harvested and cen-
trifuged (1 min, 1000g). The supernatant was discarded and the
cell pellet was frozen in liquid nitrogen.
For LC-MS analyses, cells were lysed in ice-cold DPBS contain-
ing 0.1% Triton X100. The cell lysates and cell media were mixed
(1:1) with ice-cold acetonitrile and left for 30 min on ice, followed
by centrifugation (30 min, 20,000g, 4 °C). Clear supernatants were
subsequently mixed (1:1) with ice-cold 0.1% formic acid in water
and centrifugated (15 min, 20,000g, 4 °C). The supernatants were
analyzed by LC-MS, as described above.
protocol was modified to include the use of free amino acid, D-
cysteine, rather than hydrochloride form. The crude products were
purified on a silica column (hexane:ethyl acetate, 9:1). This syn-
thetic method allowed us to obtain the compound of sufficient
purity, without the need for HPLC-based purification. Therefore,
relatively large amounts of the probe can be conveniently pre-
pared using this protocol. Nitrobenzylated luciferin (Luc-Bz-NO2)
structure was confirmed by NMR analysis: 1H NMR, (400.13 MHz):
δ
8.29 (2H, d, J¼8.4), 8.10 (1H, dd, J¼8.7, 18.6), 7.87 (1H, d, J¼7.9),
7.77 (2H, d, J¼7.5), 7.38–7.29 (1H, m), 5.40 (2H, s), 5.2 (1H, m),
3.81–3.61 (2H, m).
Chloroluciferin (Luc-Cl) was synthesized by reacting
D-luciferin
with HOCl, as shown in Fig. 2B. -Luciferin potassium salt (100 mg,
D
0.314 mmol) was dissolved in deionized water (10 mL). The solu-
tion was stirred in the dark, and HOCl (208 mL, 10 mM) was added.
The reaction's progress was monitored by HPLC. After depletion of
luciferin, hydrochloric acid (2 M, 20 mL) was added to the mixture.
The precipitate was filtered off, washed with water until washings
were neutral, and dried under reduced pressure. Crude product
was purified using preparative HPLC. The position of chlorination
3. Results
3.1. Identification of the primary product formed during the oxida-
tion of PCL-1
To understand the mechanism of oxidation of PCL-1 probe to
luciferin, we detected and quantitated the products formed upon
PCL-1 reaction with H2O2, HOCl and ONOOꢁ. In agreement with
previous reports [9–11], luciferin was detected as the major stable
product in all cases (Fig. 3). However, HPLC-based monitoring of
the product formation over time indicates that before luciferin is
formed, elution of a peak (marked by an asterisk next to the PCL-1
probe peak) corresponding to a different species, was observed
(Fig. 4A-C, top). As this species is formed from PCL-1 reaction with
all three oxidants and its decomposition is accompanied by luci-
ferin formation, we attributed this species to the primary phenolic
product (Luc-Bz-OH in Fig. 5) formed prior to the elimination of
the QM (Fig. 5). Oxidation of PCL-1 by ONOOꢁ or HOCl resulted in
the formation of the primary product immediately after mixing
was determined based on NMR analyses: 1H NMR, (600.13 MHz):
δ
7.94 (1H, d, J¼9.0), 7.30 (1H, d, J¼9.0), 5.35 (1H, br.dd, J¼10.4,
8.4), 3.75 (1H, dd, J¼10.9, 10.4), 3.71 (1H, dd, J¼10.9, 8.4); 13C
NMR (75.47 MHz):
δ 171.3 (1C, s), 163.3, (1C, s), 157.4 (1C, s), 153.4
(1C), 145.7 (1C), 137.1 (1C), 123.5 (1C), 117.6 (1C), 110.5 (1C), 78.9
(1C), 35.2 (1C). The lack of a signal of proton at the carbon atom
C-2, in the 1H NMR spectrum of chlorinated luciferin (Suppl.
Fig. 2A), points to its replacement by the chlorine atom. The as-
signment has been confirmed by 13C NMR (Suppl. Fig. 2B) and 2D-
NMR analyses (Suppl. Fig. 3). The 13C chemical shift of the carbon
atom C-2 at 110.5 ppm combined with the 3J correlation between
proton atom Hb (7.3) and the carbon atom C-2 prove that the

J. Zielonka et al. / Free Radical Biology and Medicine 99 (2016) 32–42
35
of the QM (Fig. 5) moiety follows the first-order kinetics, with the
rate constant of ꢂ2 ꢃ 10ꢁ3 sꢁ1 determined at pH 7.4 from the
time-dependent decay of Luc-Bz-OH (Fig. 4B-C, bottom).
Next we confirmed the identity of the primary product de-
tected under acidic conditions, using HPLC and mass spectrometry.
The mass of the detected species (m/z¼385) is consistent with the
deprotonated phenol Luc-Bz-Oꢁ (Fig. 5). The decomposition of
Luc-Bz-OH into luciferin is accompanied by elimination of QM
through a self-immolative reaction mechanism [21]. We reasoned
that in aqueous solution, QM would quickly react with water to
form 4-hydroxybenzyl alcohol (HO-Bz-OH, Fig. 5 [22]). The de-
composition of Luc-Bz-OH intermediate produced two HPLC
peaks: one corresponding to luciferin, and another one at a shorter
retention time. This peak detected at a shorter retention time co-
eluted with the authentic standard of HO-Bz-OH, but not of
4-methylresorcinol. Thus we conclude that in the absence of other
nucleophiles, QM exclusively produces HO-Bz-OH in aqueous
solutions.
Fig. 3. HPLC chromatograms obtained after a 30-min incubation of PCL-1 probe
(100 μM) alone or in the presence of H2O2 (10 mM), HOCl (80 μM) or ONOOꢁ
(80 μM) in aqueous solutions containing phosphate buffer (0.1 M, pH 7.4) and dtpa
(10 μM). The traces were collected using the absorption detector set at 330 nm.
3.2. Oxidation of PCL-1 by H2O2
(o1 min), as compared to the time resolution of the HPLC ana-
lyses (Fig. 4B and C, bottom). This is consistent with relatively high
rate constants of the reaction of boronates with HOCl and ONOOꢁ
(104 and 106 Mꢁ1 sꢁ1, respectively) [12,13]. Under the conditions
used, the reaction of PCL-1 with ONOOꢁ was completed within
1 s, and with HOCl within 1 min, after mixing. In contrast, the
reported rate constant of the reaction of boronates with H2O2 is
ꢂ1 Mꢁ1 sꢁ1 [12]. Thus, the reaction half-life when using 10 mM
H2O2 can be calculated to be 70 s. This is consistent with the ob-
served time course of PCL-1 consumption (Fig. 4A, bottom) and
slower formation of the intermediate Luc-Bz-OH (Fig. 4, bottom),
as compared to the other oxidants tested. When Luc-Bz-OH was
generated rapidly from HOCl and ONOOꢁ reaction with PCL-1, its
decomposition kinetics could be easily monitored. The elimination
As shown in Fig. 6, oxidation of PCL-1 probe by H2O2 (10 mM)
yields the highest level of luciferin as compared to the other two
oxidants tested. This is consistent with our previous studies using
other boronates, demonstrating a 1:1 stoichiometry for the bor-
onate:H2O2 reaction, with the corresponding phenol as the sole
product. Because of the slow reaction kinetics, a 24-h incubation
was needed to ensure completion of the reaction between PCL-1
and H2O2. As shown in Fig. 6A, the PCL-1 probe was completely
consumed, and the yield of luciferin reached maximum when the
probe was reacted with equimolar amount of H2O2, confirming a
1:1 reaction stoichiometry. Of note, the yield of the product was
only ca. 75%. This could be explained by the low stability of PCL-1
and/or luciferin over
a 24-h incubation period in aqueous
Fig. 4. Time course of the conversion of PCL-1 probe (0.1 mM) into luciferin product in the presence of (A) H2O2 (10 mM), (B) HOCl (80 μM) and (C) ONOOꢁ (80 μM) in
aqueous solutions containing phosphate buffer (0.1 M, pH 7.4) and dtpa (10 μM). The traces were collected using the absorption detector set at 330 nm. The asterisk indicates
the position of a Luc-Bz-OH peak.

36
J. Zielonka et al. / Free Radical Biology and Medicine 99 (2016) 32–42
Fig. 5. Scheme showing the oxidative conversion of PCL-1 probe into luciferin, with the elimination of para-quinone methide (QM).
solutions. Indeed, the PCL-1 concentration determined after 24-h
incubation was only 50 mM, indicating a significant decomposition
of the probe during the prolonged incubation period. Under those
conditions luciferin was one of the products formed in the absence
of H2O2, but only accounted for 20% of the amount of PCL-1
decomposed.
the formation and trapping of the PCL-1-derived phenyl radical is
shown in Fig. 8A. With both spin traps (MNP and DIPPMPO) in the
reaction mixtures of PCL-1 with ONOOꢁ, we recorded the EPR
spectra that were qualitatively different than observed in reaction
mixtures of spin traps with ONOOꢁ, but without the PCL-1 probe
(Fig. 8B,C). Although the signal intensity was significantly lower
than detected for simple 4-acetylphenylboronic acid, we were able
to attribute the spectra to the spin adducts of the PCL-1-derived
phenyl radical. The spectrum obtained with MNP spin trap con-
sisted 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) (Fig. 8B).
With DIPPMPO cyclic nitrone spin trap, the spectrum (Fig. 8C)
corresponds to a mixture of spin adducts. To confirm the forma-
tion of the phenyl radical adduct, we performed LC-MS analysis of
the reaction mixture containing both MNP (Fig. 8D) and DIPPMPO
(Fig. 8E) spin traps. When PCL-1 was reacted with ONOOꢁ the
peaks of luciferin were detected with both spin traps, regardless of
the presence of the spin trap. In the presence of DIPPMPO, we
observed a decrease in peak intensity due to luciferin, which we
attribute to ion suppression by excess of DIPPMPO eluting over the
whole LC-MS analysis time. In the presence of PCL-1, ONOOꢁ and
3.3. Oxidation of PCL-1 by HOCl
The stoichiometric analysis of the reaction between PCL-1
probe and HOCl is shown in Fig. 6B. Unlike reaction with H2O2, a
slight excess of HOCl was required for complete consumption of
PCL-1. Again, the yield of luciferin was significantly less than 100%,
despite the fact that the probe was stable enough over the in-
cubation period (30 min). Based on the effect of ethanol on the
extent of oxidation of CBA to COH (Suppl. Fig. 1), interference by
EtOH (10%) used as a solvent for PCL-1 cannot account for more
than 10% decrease in the yield of luciferin. We tentatively attribute
the lower yield of luciferin to formation of additional, minor
product(s) of the reaction of PCL-1 with HOCl (not detected). With
excess HOCl, the product luciferin undergoes further reaction with
HOCl, leading to decreased yield of luciferin. This is supported by
the detection of a new HPLC peak assigned to chlorinated luciferin
under the conditions of excess of HOCl (Fig. 7). The assignment of
this HPLC peak to chlorinated luciferin was supported by the ob-
servation that this product was also formed when authentic luci-
ferin was reacted with HOCl (Fig. 7). The structure was further
confirmed by mass spectrometry (m/z¼315), and the position of
chlorination was established using NMR analyses (see Section 2).
ꢀ
MNP, the spin adduct of MNP and Luc-Bz radical was detected
(Fig. 8D, m/z¼458, peak detected at 3.03 min). Similarly, only in
the presence of PCL-1, ONOOꢁ and DIPPMPO did we detect the
ꢀ
spin adduct of Luc-Bz to DIPPMPO (Fig. 8E, m/z¼632, peak de-
tected at 3.07 min). This peak can be assigned to the spin adduct
present in the form of nitroxide and/or protonated nitrone. The
spin trapping results confirm the formation of the phenyl radical
during the oxidation of PCL-1 by ONOOꢁ. Notably, under the
conditions used, no elimination of the QM occurred from PCL-1-
derived phenyl radical. To detect the stable end-products derived
from the phenyl radical, we synthesized the authentic standards of
the expected compounds, including Luc-Bz-NO2 (the product of
3.4. Oxidation of PCL-1 by ONOOꢁ
Similar to H2O2 and HOCl, ONOOꢁ also oxidized PCL-1 probe,
forming a maximal yield of luciferin at an equimolar ratio of the
probe and ONOOꢁ (Fig. 6C). Although the yield of luciferin was
significantly lower than 100%, this was expected for ONOOꢁ re-
action with PCL-1 based on two pathways of oxidation: major
(non-radical) pathway yielding luciferin, and minor (radical-
mediated) pathway yielding products derived from the phenyl
radical formed. To demonstrate the occurrence of the free radical
pathway and formation of the corresponding phenyl radical, we
performed EPR spin trapping experiments, using 2-methyl-2-ni-
troso propane (MNP) and DIPPMPO spin traps (Fig. 8). The basis of
ꢀ
ꢀ
recombination of Luc-Bz phenyl and NO2 radicals) and Luc-Bz-H
ꢀ
(Luc-Bz reduction product) and performed LC-MS analyses of the
reaction mixtures.
3.5. LC-MS analyses
Fig. 9 shows the product analyses of the reaction of oxidation of
PCL-1 by three oxidants: H2O2, HOCl, and ONOOꢁ. The proposed
reaction mechanism and the structures of compounds detected are

J. Zielonka et al. / Free Radical Biology and Medicine 99 (2016) 32–42
37
Fig. 7. HPLC chromatograms of the reaction mixtures of PCL-1 and luciferin with
HOCl. PCL-1 (0.1 mM) was mixed with HOCl (175 μM), and luciferin (90 μM) was
reacted with 80 μM HOCl. The arrow indicates the position of a new peak attributed
to chloroluciferin (Luc-Cl). Reactions were carried out in aqueous solutions con-
taining phosphate buffer (0.1 M, pH 7.4) in the absence of dtpa, with 10% EtOH in
case of PCL-1 reaction.
dimethyl sulfoxide (DMSO), a known HOCl scavenger (Suppl.
Fig. 1), inhibited oxidation of PCL-1 and formation of the Luc-Cl
product.
3.6. Oxidation of PCL-1 by activated RAW 264.7 cells
To test the feasibility of formation and detection of the perox-
ynitrite-specific product, Luc-Bz-NO2 in biological systems, we
utilized RAW 264.7 cells activated to produce ONOO– (Fig. 10).
Stimulation of RAW 264.7 cells with lipopolysaccharide (LPS), in-
terefon
γ
(IFN ) and phorbol myristate acetate (PMA) leads to the
γ
formation of ONOO– [14,19] and induces oxidation of PCL-1 to
luciferin and nitration to Luc-Bz-NO2. These products were de-
tected both intracellularily (Fig. 10A) and in the cell media
(Fig. 10B) and were inhibitable by preincubation of the cells with L-
NAME or DPI in a concentration-dependent manner. We have re-
cently demonstrated that DPI blocks the formation of ONOO– in
activated macrophages [19].
4. Discussion
Fig. 6. HPLC-based titration of PCL-1 (100 mM) with (A) H2O2 (24 h), (B) HOCl
(30 min) and (C) ONOOꢁ (30 min) in aqueous solutions containing phosphate
buffer (0.1 M, pH 7.4) and dtpa (10 μM).
4.1. Rigorous identification of ROS and RNS
The use of molecular probes for detection and quantification of
reactive cellular oxidizing and nitrating species requires detailed
knowledge of the probes’ chemistry, reaction kinetics, and possibly
the identification of the oxidant-specific product(s), as well. In
many cases, ROS/RNS-specific marker products have been char-
acterized [12–14,18–20,22–24]. Because of overlapping fluores-
cence characteristics of products formed from the reaction be-
tween fluorophores and oxidants, it is nearly impossible to cate-
gorically identify specific oxidants formed in cells using confocal
fluorescence technique [23,25]. For example, both 2-hydro-
xyethidium (specific marker product of hydroethidine and super-
oxide) and ethidium (non-specific, two electron oxidation product
of hydroethidine and various oxidants) have overlapping fluores-
cence spectral characteristics. HPLC-based methods are clearly
more suitable for separating, identifying and quantifying them by
comparing with appropriate standards [26]. The most significant
progress in ROS detection over the last decade was the develop-
ment of a new class of boronate-based probes, initially proposed
for specific detection of H2O2. H2O2 slowly oxidizes boronates to
the corresponding hydroxyl derivatives (phenolic products in case
of aromatic boronates) [12,13]. The design of boronate probes is
shown in Fig. 9A and the mass spectra of every analyte are pre-
sented in Fig. 9B. With all three oxidants, the peak detected im-
mediately (injected in less than 2 min) after mixing corresponds to
Luc-Bz-OH, which upon further incubation (1 h) decomposes to
form luciferin (Fig. 9C). No other products were detected in the
presence of H2O2. The LC-MS peaks observed in the Luc-Bz-NO2
channel matched the retention time of PCL-1 and were attributed
to relatively low resolution of the mass detector and only one unit
difference (415 vs. 416) of the m/z values for PCL-1 and Luc-Bz-NO2
(Fig. 9B). The nitrated product (Luc-Bz-NO2) was observed with
ONOOꢁ immediately after mixing, and its yield did not change
upon further incubation. This is consistent with a rapid re-
ꢀ
ꢀ
combination of Luc-Bz and NO2 radicals. Small amounts of the
ꢀ
Luc-Bz reduction product (Luc-Bz-H, Fig. 9) was also detected.
The yield of Luc-Bz-H was significantly increased when the reac-
tion between PCL-1 and ONOOꢁ was performed in the presence of
2-propanol (2-PrOH), a known scavenger of phenyl radicals. Dur-
ing the reaction between PCL-1 and HOCl, an additional product,
Luc-Cl (Fig. 9) detected under the conditions of excess HOCl was
attributed to the chlorinated product from luciferin. Addition of

38
J. Zielonka et al. / Free Radical Biology and Medicine 99 (2016) 32–42
ꢀ
Fig. 8. Spin trapping of the phenyl radical formed during the reaction of PCL-1 with ONOOꢁ. (A) Scheme of the formation and trapping of Luc-Bz radical; (B) EPR spectra
registered using MNP spin trap; (C) EPR spectra registered with the use of DIPPMPO spin trap; (D) LC-MS analyses of luciferin and Luc-Bz-MNP spin adduct; (E) LC-MS
analyses of luciferin and Luc-Bz-DIPPMPO spin adduct. Incubation mixture contained the following compounds: PCL-1 (250 mM), DIPPMPO (10 mM) or MNP (40 mM), in
Tris–HCl buffer (100 mM, pH 9.5) containing dtpa (100 mM), catalase (100 U/ml), and DMSO (0.25%). The reaction mixture was transferred to an EPR capillary immediately
after bolus addition of ONOOꢁ (resulting in the 200 mM ONOOꢁ concentration in the sample), and the spectra were recorded at room temperature.

J. Zielonka et al. / Free Radical Biology and Medicine 99 (2016) 32–42
39
Fig. 9. LC-MS analyses of the products of PCL-1 oxidation. (A) Scheme of the transformation of PCL-1, leading to luciferin and oxidant-specific minor products, (B) online
mass spectra recorded for each product and (C) LC-MS traces of the reaction mixtures of PCL-1 (100 mM) alone or after addition of H2O2 (10 mM), ONOOꢁ (80 μM) or HOCl
(90 μM or 200 μM).
typically based on the substitution of the hydroxyl group of the
fluorescent compound with the boronate moiety. When direct
substitution of the hydroxyl group leads to undesired in-
tramolecular interactions or is synthetically challenging, a simpler
boronobenzylation is used (e.g., PCL-1 probe), leading to the cor-
responding boronobenzyl derivatives.
In a series of papers we have characterized the mechanism of
the reaction of boronates with peroxynitrite and identified minor,
ONOO–-specific products, in which the boronate moiety is replaced
by the nitro group [12,13,17,19,27,28]. While the nitration of the
phenolic products is a common feature of the chemical reactivity
of peroxynitrite and peroxidase/H2O2/nitrite systems, replacement
of the boronate moiety by the nitro group is specific for ONOO–
and results in nitrobenzene-like product [13,17]. Incubation of
boronates with myeloperoxidase(MPO)/H2O2/nitrite systems does
not produce the nitrobenzene-like product, but leads to nitration
of the phenolic product (formed during the reaction of boronates
with H2O2), leading to nitrophenols (in contrast to nitrobenzenes)

40
J. Zielonka et al. / Free Radical Biology and Medicine 99 (2016) 32–42
Fig. 10. Profiles of PCL-1 oxidation products formed in RAW 264.7 macrophages activated to produce ONOOꢁ. RAW 264.7 cells were activated to produce ONOOꢁ by
overnight incubation of LPS (0.5 μg/ml) and IFNγ (50 units/mL) followed by addition of 1 mM PMA (I/LþP). At the time of addition of PMA, PCL-1 (100 μM) was also added and
the cells were incubated for 1 h before harvesting. L-NAME (1 mM) or DPI (0.1–10 μM) were added 30 min before addition of PMA. (A) LC-MS/MS traces of PCL-1, luciferin and
# ##
Luc-Bz-NO2; (B) Results of quantitative analyses of the LC-MS/MS data. ** - po0.01 vs. control; and
- po0.05 and po0.01, respectively vs. I/LþP.
[27,28].
One of the advantages of boronate probes is that the reaction
standard-of-care drugs. Typically tumor growth is assessed by
measuring the intensity of bioluminescence signal (light intensity)
in luciferase-transfected cancer cell mice xenografts [30]. The
substrate, luciferin, is injected as needed and the green biolumi-
nescent signal intensity from the luciferase-transfected tumor cells
is measured [30]. Using the PCL-1 probe, one can monitor in vivo
bioluminescence imaging of tumor-derived ROS/RNS. Mice bearing
luciferase-transfected cancer cells are administered with PCL-1
probe on different days after tumor implantation. This approach
enables selective monitoring of ROS/RNS in tumor cells due to
selective localization of luciferase in tumor cells. Upon reaction
with H2O2 or ONOOꢁ or HOCl, luciferin is formed in situ (from
PCL-1) which is oxidized by the luciferase enzyme (using ATP as a
co-factor) to generate green bioluminescence (Fig. 1). As biolumi-
nescence will depend not only on ROS/RNS but also on tumor size,
the number of tumor cells, and intracellular ATP, parallel analysis
should be performed with luciferin as the substrate. To distinguish
between H2O2 and ONOOꢁ, appropriate antioxidant enzymes (e.g.,
PEG catalase), nitric oxide synthase inhibitors or superoxide dis-
mutase mimetics inhibitors may be used in addition to measuring
the specific nitrated product derived from PCL-1 in tumor tissues.
chemistry and kinetics remain unchanged, independent of the
actual scaffold used in the probe design, with a few exceptions.
The advantage of in vivo probes based on boronates’ chemistry is
the relatively low probability for interference of the heme pro-
teins, and biological reductants, which limit the in vivo application
of other probes, including hydroethidine and cyclic nitrone spin
traps. Recently, a boronate-caged positron emission tomography
(PET) tracer was used to image H2O2 in renal carcinoma cells [29].
As PET imaging can be readily translated to the clinical setting,
boronate-caged PET tracers may find wider applications in oxi-
dative stress/nitrative stress imaging in vivo.
4.2. Potential applications
The application of PCL-1 probe to monitor production of oxi-
dants in tumor and/or tumor environment by monitoring biolu-
minescence in rodent models may enable better understanding of
the role of ROS/RNS in tumor growth and immunosuppressive
effects of tumor microenvironment. This in turn may provide a
better approach to increase the efficiency of cancer im-
munotherapy and/or combination of redox modulators with the

J. Zielonka et al. / Free Radical Biology and Medicine 99 (2016) 32–42
41
5. Conclusions
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K. Sakai, K. Morimoto, K. Todoroki, O. Inoue, In vivo imaging of reactive oxygen
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1. In this work we have identified the primary product of oxida-
tion of the PCL-1 probe, Luc-Bz-OH (Fig. 5). Decomposition of
Luc-Bz-OH (via a self-immolative reaction) leads to the forma-
tion of luciferin with the elimination of QM. In the absence of
other nucleophiles, QM reacts with water to form HO-Bz-OH
(Fig. 5). However, in a cellular environment, other nucleophiles,
including thiols, will likely react with QM.
2. The major product identified by HPLC with all three oxidants
tested was luciferin. However, in the case of ONOOꢁ, the reac-
tion proceeds via two pathways, with the minor pathway
leading to the formation of ONOOꢁ-specific minor product, Luc-
ꢀ
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imaging: in vivo bioluminescence detection of hydrogen peroxide and caspase
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1783–1795, http://dx.doi.org/10.1021/ja309078t.
Bz-NO2, via intermediate phenyl radical Luc-Bz (Fig. 9A). This
radical has been detected using the spin trapping technique,
and the nitrone adduct identified by LC-MS.
3. The minor, ONOO–-specific product, Luc-Bz-NO2 is formed by
activated macrophages incubated in the presence of the PCL-1
probe, and can be detected and quantified by LC-MS analyses.
4. Although, reaction with HOCl seems to proceed via a single,
non-radical pathway, the product formed, luciferin, undergoes
further reaction with HOCl, leading to the formation of Luc-Cl, a
product specific for HOCl.
5. Here we propose the combination of non-invasive biolumines-
cence monitoring of oxidant production in vivo in luciferase-
expressing cells, with HPLC or LC-MS analyses of tissues to
detect oxidant-specific minor products. This will provide more
detailed information on the identity(ies) of the species detected.
Identification of the oxidants produced under pathophysiolo-
gical conditions will allow for more precise interventions to
inhibit their formation.
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E. Newcomb, M.G. Bonini, Bioluminescent detection of peroxynitrite with a
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[12] A. Sikora, J. Zielonka, M. Lopez, J. Joseph, B. Kalyanaraman, Direct oxidation of
boronates by peroxynitrite: mechanism and implications in fluorescence
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Real-time measurements of amino acid and protein hydroperoxides using
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org/10.1074/jbc.M114.553727.
Acknowledgment
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B. Kalyanaraman, Reaction between peroxynitrite and boronates: EPR spin-
trapping, hplc analyses, and quantum mechanical study of the free radical
pathway, Chem. Res. Toxicol. 24 (2011) 687–697, http://dx.doi.org/10.1021/
tx300499c.
This work was supported by a grant from NIH (R01 HL073056)
to B.K. The LC-MS analyses were performed in Medical College of
Wisconsin Cancer Center Redox and Bioenergetics Shared
Resource.
[18] R. Smulik, D. Dębski, J. Zielonka, B. Michałowski, J. Adamus, A. Marcinek,
B. Kalyanaraman, A. Sikora, Nitroxyl (HNO) reacts with molecular oxygen and
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DIPPMPO, a crystalline analog of the nitrone DEPMPO: synthesis and spin
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Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.freeradbiomed.
2016.07.023.
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