A boronate-based fluorescent probe for the selective detection of cellular peroxynitrite

Article abstract of DOI:10.1039/c4cc02943g

A boronate-based fluorescent probe 1 for the selective monitoring of intracellular peroxynitrite has been developed. The probe takes advantage of the fast reaction of an arylboronate group with peroxynitrite, yielding a corresponding phenol that undergoes spontaneous subsequent reactions to produce a strongly fluorescent product associated with a large turn-on signal.

Full text of DOI:10.1039/c4cc02943g

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A boronate-based fluorescent probe for the
selective detection of cellular peroxynitrite†
Jiyoung Kim,a Jeesook Park,a Hawon Lee,b Yongdoo Choi*b and Youngmi Kim*a
Cite this: Chem. Commun., 2014,
50, 9353
Received 21st April 2014,
Accepted 21st June 2014
DOI: 10.1039/c4cc02943g
www.rsc.org/chemcomm
A boronate-based fluorescent probe 1 for the selective monitoring utility in many biological studies, small-molecule fluorescent
of intracellular peroxynitrite has been developed. The probe takes probes offer a promising approach for detecting highly reactive
advantage of the fast reaction of an arylboronate group with ONOOÀ in vivo.5 The most popular fluorescent probes devel-
peroxynitrite, yielding a corresponding phenol that undergoes oped for this purpose thus far are based on fluorescein or
spontaneous subsequent reactions to produce a strongly fluores- rhodamine dyes, in which nonfluorescent forms are converted
cent product associated with a large turn-on signal.
to fluorescent products.6 Although enabling detection of ONOOÀ,
these probes also respond to other highly reactive oxygen species
Peroxynitrite (ONOOÀ) in living systems is receiving increasing (hROS) such as the hydroxyl radical (ꢀOH) and hypochlorite
attention owing to the diverse roles it plays as a highly reactive (OClÀ), often with higher sensitivity, and are extensively auto-
oxidant and an efficient nitrating agent in many physiological oxidized. Recent studies have identified several fluorescent probes
and pathological processes.1 ONOOÀ is formed by the diffusion- for ONOOÀ that are based on other chemical transformations
controlled reaction between nitric oxide (ꢀNO) and superoxide including aromatic nitration,7 ketone oxidation reactions8 and
anion (ꢀO2À) in inflammatory cells such as neutrophils and redox reactions of organoselenium/organotellurium compounds.9
macrophages.2 In contrast to its beneficial effects in mechan- Such probes displayed a response to ONOOÀ with variable degrees
isms employed by hosts to defend against microbial invasion, of sensitivity, however, some of them suffer from a few limitations
excessive generation of ONOOÀ could have deleterious effects on such as nonspecific reactivity with other ROS, poor photostability,
a wide array of biomolecules in cells, including lipids, proteins, or low water solubility. In order to have practical applications,
and nucleic acids, leading to cell apoptosis or necrosis,3 and has next-generation ONOOÀ probes need to be easily synthesized, and
been implicated in the genesis and progression of numerous should possess high analyte selectivity and limited susceptibility
ailments, such as cardiovascular diseases, circulatory shock, to auto-oxidation.
inflammation, cancers, ischemic stroke, reperfusion injury,
atherosclerosis, and diabetes.4
Boronate-containing fluorogenic compounds have been
used as the basis of the most effective probes for detecting
Despite the importance of ONOOÀ in human health and intracellular H2O2.10 However, the results of recent investiga-
diseases, the complex biological roles played by this species in tions, utilizing a stopped-flow kinetic technique and HPLC
cellular signaling pathways and various diseases have not yet analysis, have demonstrated that arylboronates react with
been fully elucidated. One of the main reasons for this lies in ONOOÀ to yield corresponding hydroxyl derivatives nearly six
the lack of effective chemical tools to measure dynamic orders of magnitude faster than with H2O2.11 We envisioned
changes in ONOOÀ concentrations occurring within localized that the rapid reactions with ONOOÀ would make arylboronates
regions of cells. Owing to their documented sensitivity and ideal substances for use in ONOOÀ specific fluorescent probes.
Along these lines, observations made in a recent investigation
show that a boronate-containing fluorescent probe based on the
blue emissive pyrene fluorophore is more responsive to ONOOÀ
over other ROS.12 However, positive fluorescence response of the
pyrene–boronate sensor was also seen upon treatment with
H2O2, ClOÀ, or OBrÀ for 15 min with an insufficient degree of
selectivity (ca. 3 : 1 in favor of ONOOÀ over H2O2, ClOÀ, or OBrÀ),
indicating the need for the development of new ONOOÀ selective
probes. Below, we describe the design and synthesis of the new
a Department of Chemistry, Institute of Nanosensor and Biotechnology, Dankook
University, 152 Jukjeon-ro, Suji-gu, Yongin-si, Gyeonggi-do, 448-701, Korea.
E-mail: youngmi@dankook.ac.kr; Fax: +82-31-8005-3148; Tel: +82-31-8005-3156
b Molecular Imaging & Therapy Branch, National Cancer Center, 323 Ilsan-ro,
Goyang-si, Gyeonggi-do 410-769, Korea. E-mail: ydchoi@ncc.re.kr;
Fax: +82-31-920-2529; Tel: +82-31-920-2512
† Electronic supplementary information (ESI) available: Synthetic details, addi-
tional spectral data, kinetic study, and experiments using living cells. See DOI:
10.1039/c4cc02943g
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boronate-derived probe 1, and demonstrate its application as a
highly sensitive and selective fluorescent probe for the detection
of ONOOÀ in living cells.
A benzothiazolyl iminocoumarin scaffold, serving as an analogue
of a coumarin dye, was employed in the design of the new
fluorescent probe for monitoring ONOOÀ. This selection was
guided by the excellent photophysical properties, such as high
photostabilities and high fluorescence quantum yields, of
members of this family. Moreover, iminocoumarins have excita-
tion and emission wavelengths that are longer than those of the
related coumarins, making the former more suitable than the
latter for biological applications.13 The boronate probe 1,
designed using these considerations, contains a phenol moiety
masked by a p-dihydroxyborylbenzyloxy group. This probe is
expected to display a low fluorescence quantum yield in solution,
because of fast non-radiative decay of the singlet excited state
Fig. 1 (a) Time-dependent emission spectra (lex = 430 nm) of probe 1
(10 mM) upon treatment with ONOOÀ (10 mM) in phosphate buffer (10 mM,
pH 7.4, 10% ethanol) at 37 1C. Spectra were recorded every 1 min (0–30 min).
Inset: photographs of probe 1 in the absence (left) and presence (right) of
ONOOÀ after incubation for 30 min at 25 1C under UV light (365 nm)
illumination. (b) Change in fluorescence intensity at 540 nm in the presence
of ONOOÀ (10 mM) as a function of incubation time.
that is facilitated by free rotation about the C–C double bond of the formation of aggregates in solvents like water, in which 1 has a
an alkene linker. In addition, we hypothesized that reaction of 1 low solubility. These properties along with the large fluorescence
with ONOOÀ would promote oxidative hydrolysis of the aryl- quantum yield (FF) of 3 of 0.62 in aqueous solution clearly
boronate group to generate the corresponding phenol (see demonstrate that 1 could serve as an ideal off–on fluorescent
Scheme S3 (ESI†) for the detailed mechanism), which would probe for ONOOÀ.
undergo rapid elimination of p-quinomethane to produce
The fluorescence response of 1 toward ONOOÀ in aqueous
phenolate 2b. Rapid cyclization of 2b would then generate the buffer solution (10 mM phosphate buffer, pH 7.4, 10% ethanol)
highly fluorescent benzothiazolyl iminocoumarin 3 (Scheme 1). at 37 1C is displayed in Fig. 1. Inspection of the spectra shows
Probe 1 was prepared using the two-step sequence outlined in that a time-dependent change takes place upon addition of
Scheme S1 (ESI†). Benzothiazolyl iminocoumarin 3, the expected 1 equiv. ONOOÀ to the solution of 1 (10 mM). Specifically, over a
product resulting from reaction of 1 with ONOOÀ, was also 0–30 min incubation period (plateaus at 15 min, Fig. 1b), a
prepared utilizing a previously described general method.13
large blue-shift of the emission spectrum of 1 occurs in concert
The photophysical properties of 1 and 3 were evaluated in with a distinct increase in the intensity of emission at 540 nm,
order to demonstrate that they are compatible with the use of 1 as which is the emission maximum of 3 (Fig. 1a). At the same
a turn-on fluorescent probe for ONOOÀ (Table S1, Fig. S1 and S2, time, a significant change in the emission color from pale red
ESI†). The results show that 1 in ethanol has an absorption to intense green occurs (Fig. 1a, inset). Finally, the fluorescence
maximum at 482 nm and a fluorescence maximum at 536 nm intensity of 1 does not change under aerated assay conditions
corresponding to weak green emission (FF = 0.01). The absorption when ONOOÀ is not present in the solution (Fig. 1b).
and fluorescence emission maxima of 3 in the same solvent occur
HPLC-MS analysis of the mixture generated from the reaction
at 480 nm and 530 nm (FF = 0.96), respectively. In aqueous buffer of ONOOÀ with 1 confirmed that the fluorescence increase at
solution (10 mM phosphate buffer, pH 7.4, 10% ethanol), 1 has an 540 nm is a consequence of the formation of 3 (Fig. S9, ESI†).
emission maximum at 646 nm (FF = 0.01). This significantly red- The conversion of 1 to 3 is fast (complete within 30 min), and the
shifted emission compared with that in ethanol is attributed to proposed intermediates, 2a and 2b, in the pathway for formation
of 3 are not detected by HPLC-MS. These observations show that
the new probe 1 should be useful for the detection of ONOOÀ,
which is rapidly metabolised in biological systems.
The response of 1 (10 mM) to different concentrations
of ONOOÀ in the range of 0–20 mM after incubation times of
30 min was elucidated (Fig. 2a). As expected, the fluorescence
intensity at 540 nm increases with increasing ONOOÀ concen-
tration within the measured range (Fig. 2a, inset). A good linear
relationship exists between the normalized fluorescence intensity at
540 nm (F/F0) and the concentration of ONOOÀ in the concen-
tration range of 3–10 mM (R2 = 0.998) (Fig. S5, ESI†). The detection
limit for ONOOÀ using probe 1 was determined on the basis of the
signal-to-noise ratio (S/N = 3) to be 2.5 mM, a value that is comparable
to those of previously described fluorescent probes for ONOOÀ.
Finally, the concentration dependence of the rise in fluorescence
intensity yields a rate constant (k) of 1 to 3 of ca. 15 100 MÀ1 minÀ1
for the ONOOÀ induced conversion (Fig. S8, ESI†).
Scheme 1 Chemical structure of 1 and its reaction with ONOOÀ to
release the fluorescent reporter group 3.
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Fig. 2 (a) Fluorescence titration spectra of 1 (10 mM) in the presence of
different concentrations of ONOOÀ. [ONOOÀ] = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 12, 14, 16, 18, 20 mM. Inset shows a plot of the relative fluorescence
intensity (F/F0) at 540 nm as a function of [ONOOÀ]. (b) Fluorescence
emission spectra of 1 (10 mM) in the presence of various ROS (20 mM
ONOOÀ and 100 mM for other ROS). All spectra were obtained 30 min after
addition of each analyte to 1 in phosphate buffer (10 mM, pH 7.4, 10%
ethanol) at 37 1C. lex = 430 nm.
Fig. 3 Confocal fluorescence microscope images of 1 (10 mM)-loaded
living macrophage cells (J774A.1) under different conditions. (a) Macro-
phages were incubated with 1 for 20 min at 37 1C and then imaged;
The selectivity of 1 (10 mM) toward ONOOÀ was determined
by measuring time-dependent fluorescence changes that take (b) 1-loaded macrophage cells were incubated with 50 mM H2O2 for
place in the presence of various ROS (20 mM for ONOOÀ and
30 min; (c) 1-loaded macrophage cells were incubated with 50 mM NaOCl
for 30 min; (d) macrophage cells were co-incubated with SIN-1 (1 mM) and
100 mM for hydrogen peroxide H2O2, hypochlorite OClÀ, super-
1 for 20 min; (e) macrophage cells were stimulated with LPS (1 mg mLÀ1
)
oxide ꢀO2À, hydroxyl radical ꢀOH, tert-butoxyl radical ꢀOtBu,
tert-butyl hydroperoxide t-BuOOH, nitric oxide ꢀNO) in phos-
and IFN-g (50 ng mLÀ1) for 4 h, and incubated with 1 for 20 min at 37 1C;
(f) NOS inhibitor, AG (5 mM) was co-incubated during LPS/IFN-g stimula-
phate buffer (10 mM, pH 7.4, 10% ethanol) at 37 1C. As the tion for 4 h, and incubated with 1 for 20 min at 37 1C; (g) superoxide
spectra in Fig. 2b show, only ONOOÀ promotes a dramatic
enhancement in fluorescence intensity at 540 nm (F/F0 = 47.1).
scavenger, TEMPO (300 mM) was co-incubated during LPS/IFN-g stimula-
tion for 4 h; the other procedures were the same; (h) graph showing
quantification of mean (SD) fluorescence intensity of each cell in a–g
In contrast, no changes in emission intensities occur when the
correspondingly (n 4 50). (Upper: fluorescence images, bottom: merged
probe is incubated with other ROS even at higher concentra-
tions. An exception is the case of H2O2, which induces a small
fluorescence response after 120 min (F/F0 = 1.6).14 These results
images, Ex = 405 nm, Em = 529–614 nm.)
indicate that probe 1 displays a high selectivity in vitro for by lipopolysaccharide (LPS), an inducible nitric oxide synthase
ONOOÀ over other ROS. The observations also corroborate the (iNOS), combined with interferon-g (IFN-g).17 J774A.1 cells
previous finding that boronate-containing small molecules were treated with these stimulants (IFN-g; 50 ng mLÀ1 and
react with ONOOÀ much faster than with H2O2 or OClÀ.11
LPS; 1 mg mLÀ1) for 4 h and then incubated with 1 (10 mM) for
The potential utility of 1 for the specific imaging of ONOOÀ in 20 min. In contrast to cells that are not treated with the
living cells was evaluated. For this purpose, J774A.1 macrophage stimulants, stimulated cells display a 14.4-fold increase in
cells were incubated with 10 mM 1 for 20 min. After washing with fluorescence intensity (Fig. 3e and h, P o 0.001). Moreover,
PBS to remove the remaining 1, the cells were treated with the treatment with the nitric oxide synthase inhibitor aminoguanidine
ONOOÀ donor SIN-1 (3-morpholinosydnonimine),15 which releases (AG)18 during stimulation of the cells with IFN-g/LPS prior to
equimolar amounts of nitric oxide and superoxide. Confocal incubation with probe 1 leads to greatly attenuated fluorescence
microscope images of 1-loaded macrophages that were treated with within the cells, in a manner that depends on the concentrations
SIN-1 show a marked increase in fluorescence intensity arising of AG (Fig. 3f and Fig. S13, ESI†). In addition, co-incubation with
inside the living cells with a high local concentration in the the superoxide scavenger TEMPO (2,2,6,6-tetramethylpiperidine-
cytoplasm, whereas the cells that are not treated with SIN-1 show N-oxyl)18 during stimulation with IFN-g/LPS leads to a 5-fold
negligible intracellular fluorescence (Fig. 3a and d). In the case of decrease in the fluorescence intensity of the cells (Fig. 3g and h,
cells treated with 1 mM SIN-1,16 a more-than 10-fold increase in P o 0.001), compared with that of 1-loaded activated macrophage
fluorescence intensity takes place compared to that of 1-loaded cells (Fig. 3e). The significantly reduced fluorescence signals
control cells (Fig. 3h). In addition, 1-loaded macrophages, which arising from AG- and TEMPO-treated cells clearly indicate that
are treated with either exogenous H2O2 (50 mM) or NaOCl (50 mM) strong fluorescence from the activated macrophage cells (Fig. 3e) is
for 30 min, respectively, do not show a noticeable increase in a consequence of the formation of ONOOÀ and its rapid reaction
fluorescence intensity (Fig. 3b and c).
with 1 to form 3. These results demonstrate that arylboronate
Encouraged by its specific response to ONOOÀ in living probe 1 enables effective detection and visualization of both
cells, we next determined whether 1 can be employed to image exogenous and endogenous ONOOÀ in living cells.
endogenously generated ONOOÀ in activated J774A.1 macro-
In summary, we developed an arylboronate-derived fluorescent
phage cells during phagocytosis. Macrophage cells are known probe, 1 for monitoring of ONOOÀ concentrations in cellular
to produce ROS at high levels upon physiological stimulation systems, which possesses (1) a fast response, (2) excellent selectivity
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and sensitivity toward ONOOÀ over other ROS, and (3) a large
fluorescence turn-on signal. These features should make 1 a highly
effective tool for probing the biological roles of ONOOÀ at the
cellular level and for exploring diagnostic markers for cellular
ONOOÀ formation.
This research was supported by a National Research Foundation
grant funded by the Korean government (NRF-2013M2B2A4041354
and NRF-2012R1A1A2038694).
7 T. Ueno, Y. Urano, H. Kojima and T. Nagano, J. Am. Chem. Soc.,
2006, 128, 10640.
8 D. Yang, H.-L. Wang, Z.-N. Sun, N.-W. Chung and J.-G. Shen,
J. Am. Chem. Soc., 2006, 128, 6004.
9 F. Yu, P. Li, G. Li, G. Zhao, T. Chu and K. Han, J. Am. Chem. Soc.,
2011, 133, 11030.
10 A. R. Lippert, G. C. Van de Bittner and C. J. Chang, Acc. Chem. Res.,
2011, 44, 793.
11 J. Zielonka, A. Sikora, M. Hardy, J. Joseph, B. P. Dranka and
B. Kalyanaraman, Chem. Res. Toxicol., 2012, 25, 1793.
12 F. Yu, P. Song, P. Li, B. Wang and K. Han, Analyst, 2012, 137, 3740.
13 T.-I. Kim, H. Kim, Y. Choi and Y. Kim, Chem. Commun., 2011, 47, 9825.
14 The rate constant (k) for the H2O2-induced conversion of 1 to 3 was
Notes and references
found to be ca. 2.24 MÀ1 minÀ1
.
1 C. Ducrocq, B. Blanchard, B. Pignatelli and H. Ohshima, Cell. Mol. 15 N. Ashki, K. C. Hayes and F. Bao, Neuroscience, 2008, 156, 107.
Life Sci., 1999, 55, 1068.
2 G. Ferrer-Sueta and R. Radi, ACS Chem. Biol., 2009, 4, 161.
16 N. Kuzkaya, N. Weissmann, D. G. Harrison and S. Dikalov, Biochem.
Pharmacol., 2005, 70, 343.
´
3 C. Szabo and H. Oshima, Nitric Oxide, 1997, 1, 373.
17 R. B. Lorsbach, W. J. Murphy, C. J. Lowenstein, S. H. Snyder and
S. W. Russell, J. Biol. Chem., 1993, 268, 1908.
4 P. Pacher, J. S. Beckman and L. Liaudet, Physiol. Rev., 2007, 87, 315.
5 X. Chen, X. Tian, I. Shin and J. Yoon, Chem. Soc. Rev., 2011, 40, 4783. 18 R. B. R. Muijsers, E. van den Worm, G. Folkerts, C. J. Beukelman,
6 I. Johnson and M. T. Z. Spence, The Molecular Probes Handbook:
A Guide to Fluorescent Probes and Labelling Technologies, 2010.
A. S. Koster, D. S. Postma and F. P. Nijkamp, Br. J. Pharmacol., 2000,
130, 932.
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