19F magnetic resonance probes for live-cell detection of peroxynitrite using an oxidative decarbonylation reaction

Article abstract of DOI:10.1039/c4cc04292a

We report a newly discovered oxidative decarbonylation reaction of isatins that is selectively mediated by peroxynitrite (ONOO^-) to provide anthranilic acid derivatives. We have harnessed this rapid and selective transformation to develop two reaction-based probes, 5-fluoroisatin and 6-fluoroisatin, for the low-background readout of ONOO^- using ¹⁹F magnetic resonance spectroscopy. 5-fluoroisatin was used to non-invasively detect ONOO^- formation in living lung epithelial cells stimulated with interferon-γ (IFN-γ). This journal is

Full text of DOI:10.1039/c4cc04292a

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19F magnetic resonance probes for live-cell
detection of peroxynitrite using an oxidative
decarbonylation reaction†
Cite this: Chem. Commun., 2014,
50, 12311
Received 5th June 2014,
Accepted 12th August 2014
Kevin J. Bruemmer,a Sara Merrikhihaghi,a Christina T. Lollar,a Siti Nur Sarah Morris,b
Johannes H. Bauerbc and Alexander R. Lippert*ac
DOI: 10.1039/c4cc04292a
www.rsc.org/chemcomm
We report a newly discovered oxidative decarbonylation reaction of from a lack of selectivity and a relatively slow rate of reaction
isatins that is selectively mediated by peroxynitrite (ONOOÀ) to provide with ONOOÀ.
anthranilic acid derivatives. We have harnessed this rapid and selective
19F magnetic resonance has emerged as a powerful technique
transformation to develop two reaction-based probes, 5-fluoroisatin for the detection and imaging of biological analytes due to low
and 6-fluoroisatin, for the low-background readout of ONOOÀ using background in human cells and tissues.9 The 19F isotope is present
19F magnetic resonance spectroscopy. 5-fluoroisatin was used to at nearly 100% abundance in terrestrial fluorine and has a
non-invasively detect ONOOÀ formation in living lung epithelial cells gyromagnetic ratio that endows it with sensitivity comparable with
1
stimulated with interferon-c (IFN-c).
the H nucleus. Indeed, 19F NMR and MRI have been used for
in vitro and in vivo detection of pH,10 metal ions,11 hypochlorite,12
Peroxynitrite (ONOOÀ) is a highly reactive nitrogen oxide species thÀat NO,13 tissue oxygenation,14 hypoxia,15 enzymatic activity,16 and
is formed in vivo from the rapid recombination of superoxide (O2
)
other proteins of interest.17 These advantages prompted us to develop
and nitric oxide (NO) radicals.1 Whereas it is recognized that NO and two reaction-based 19F magnetic resonance probes, 5-fluoroisatin and
O2 are endogenously generated to mediate cellular signaling,2 6-fluoroisatin, for the detection of ONOOÀ using newly discovered
À
ONOOÀ formation is generally believed to be deleterious and oxidative decarbonylation chemistry (Scheme 1).
is increased in a number of diseases such as cancer,3 cardiac
The oxidative decarboxylation of a-ketoacids has been employed
dysfunction,4 asthma,5 and aging-related pathologies. Despite its as a chemoselective and biologically compatible reaction for
conspicuous role in many human ailments, there is currently a detecting H2O2 by hyperpolarized 13C MRI.18 This reaction is
lack of specific methods for detecting ONOOÀ in biological entropically favoured and is thought to proceed by a two-electron
samples due to its short lifetime, competition from endogenous mechanism.19 ONOOÀ, on the other hand, can access one-electron
scavengers, and background signal from other reactive species.6 oxidation pathways after forming adducts with carbonyl com-
The current method of choice for studying ONOOÀ employs pounds,20 offering an opportunity to program selectivity versus other
antibodies that stain proteins containing nitrated tyrosine residues, reactive oxygen species. We reasoned that the a-ketocarbonyl of
often considered a ‘‘footprint’’ of ONOOÀ production.7 This method isatins would be stable towards two-electron cleavage of the
has proved valuable for increasing our comprehension of ONOOÀ carbonyl–carbonyl bond, but might be vulnerable to one-electron
biology, but is an indirect technique and lacks compatibility decarbonylation mechanisms. In support of this reasoning,
with living biological specimens. Other methods include using
redox-sensitive probes dichlorodihydrofluorescein (DCHF),
dihydrorhodamine 123 (DHR 123), and other recently developed
responsive dyes.8 Although promising, these probes often suffer
a Department of Chemistry, Southern Methodist University, Dallas, TX 75275-0314,
USA. E-mail: alippert@smu.edu; Fax: +1-214-768-4089; Tel: +1-214-768-2482
b Department of Biological Sciences, Southern Methodist University, Dallas,
TX 75275-0235, USA
c Center for Drug Discovery, Design, and Delivery (CD4),
Southern Methodist University, Dallas, TX 75275-0314, USA
† Electronic supplementary information (ESI) available: Experimental procedure,
Scheme 1 ONOOÀ-mediated oxidative decarbonylation of 5-fluoroisatin
cell culture conditions, and supplementary spectral data. See DOI: 10.1039/
and 6-fluoroisatin.
c4cc04292a
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Fig. 1 Oxidative decarbonylation of isatin with ONOOÀ. (a) Fluorescence
spectra of 200 mM isatin with 0 mM ONOOÀ (red trace) and 20 mM ONOOÀ
(black trace). (b) 200 mM isatin and 0, 5, 10, 15, or 20 mM GSH was reacted
with 20 mM ONOOÀ in 100 mM HEPES at pH 7.4 and 21 1C. Emission spectra
were acquired with lex = 312 nm and the intensity was measured at 405 nm.
Fig. 2 Responses of (a) 5-fluoroisatin and (b) 6-fluoroisatin to ONOOÀ. 1. 1 mM
fluoroanthranilic acid. (2–7) Reaction of 1 mM fluoroisatin with 2. 1.5 mM
ONOOÀ 3. 1.25 mM ONOOÀ 4. 1.0 mM ONOOÀ 5. 0.75 mM ONOOÀ
6. 0.5 mM ONOOÀ 7. 0.25 mM ONOOÀ 8. 1 mM fluoroisatin. All reactions were
performed in 100 mM HEPES at pH 7.4. Spectra were acquired with 128 scans at
11.7 T using an internal reference peak of trifluoroacetic acid at À35.6 ppm.
isatins are inert to biological levels of H2O2, but react instantly with
ONOOÀ to form a fluorescent anthranilic acid product with an
emission maximum centred at 405 nm (Fig. 1). We measured an
The 19F NMR responses of these probes were then evaluated for
observed second order reaction rate constant of isatin with ONOOÀ their selectivity for ONOOÀ against other reactive sulfur, oxygen,
of 3.5 Æ 0.1 Â 104 MÀ1 sÀ1 at 21 1C using glutathione (GSH) as a and nitrogen (RSON) species (Fig. 3). Both 5-fluoroisatin and
reaction competitor (Fig. 1).21 The combination of the rate constant 6-fluoroisatin are highly selective for ONOOÀ as minimal 19F
(k) and the substrate concentration [T], k[T], can be used to compare NMR response was observed with any other RSON species at
the reactivity versus other molecules. For instance, for 1 mM isatin this concentration. Even at 120 mM H2O2, complete conversion
at 21 1C, k[T] = 35 sÀ1, comparing favourably to CO2, a key scavenger to the anthranilic acid was not observed (Fig. S4 and S5, ESI†).
of ONOOÀ in biological systems, which has k[T] = 60–100 sÀ1 under We measured a rate constant for the reaction of isatin with
physiological conditions.22 Encouraged by these properties, we H2O2 of 3.3 Æ 0.4 Â 10À3 MÀ1 sÀ1 at 21 1C, indicating that H2O2
synthesized fluorinated isatin compounds using a previously reacts with isatin B10 000 000 times slower than ONOOÀ (Fig. S6,
disclosed sulfur ylide methodology.23 We confirmed the produc- ESI†). Higher concentrations of the hydroxyl radical did not result in
tion of 5-fluoroanthranilic acid in 86% yield from the reaction of signal for the fluorinated anthranilic acid, although the isatin peak
5-fluoroisatin with 1.5 equivalents of ONOOÀ by 19F NMR, did disappear (Fig. S4 and S5, ESI†). The selectivity of 5-fluoroisatin
1H NMR, GC/MS, and HPLC analysis (Fig. S1, ESI†).
for ONOOÀ was further investigated using HPLC analysis (Fig. S8,
We tested our first generation probes, 5-fluoroisatin and ESI†). Quantification of these experiments revealed that the
6-fluoroisatin, for their 19F NMR responses to synthetic ONOOÀ hydroxyl radical and Angeli’s salt (commonly used as a nitroxyl
(Fig. 2).24 An insert tube containing a 0.1% solution of trifluoroacetic donor) displayed slight reactivity towards 5-fluoroisatin, producing
acid (TFA) in D2O provided a reference signal at À35.6 ppm. With 5-fluoroanthranilic acid in 14% and 22% yields, respectively
increasing 250 mM increments of ONOOÀ, the chemical shift (Fig. S9, ESI†). However, it should be noted that hydroxyl radical
changes from À56 to À60 ppm for 5-fluoroisatin (Dd = 4 ppm) rarely diffuses far from its site of production1 and Angeli’s salt may
and from À44 to À52 ppm for 6-fluoroisatin (Dd = 8 ppm). Complete generate ONOOÀ under certain conditions.25 Taken together, these
conversion of isatin to anthranilic acid was observed at data demonstrate that the 19F NMR probes 5-fluoroisatin and
1.5 equivalents of ONOOÀ. Integration of the 19F NMR peaks 6-fluoroisatin display good selectivity for ONOOÀ over other
of the fluoroanthranilic acids and fluoroisatins provide a quantifi- RSON species.
able evaluation of the reaction with increasing ONOOÀ concen-
tration (Fig. S2 and S3, ESI†).
Excited by these results, we applied 5-fluoroisatin towards
detecting biological ONOOÀ. When treated with the cytokine
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Fig. 4 Detection of cellular ONOOÀ. (a) A549 cells were treated with
50 mM 5-fluoroisatin and 50 ng mLÀ1 IFN-g for 12 hours. The media was
transferred to an NMR tube and spectra were acquired with 7300 scans at
11.7 T using an internal reference peak of trifluoroacetic acid at À35.6 ppm.
(b) Quantification of the anthranilic acid concentration determined from
19F NMR peak integrations in A549 cells treated for 6–18 hours with 50 mM
5-fluoroisatin and 0.1% BSA as a vehicle control, n = 5 (Control) or 50 ng mLÀ1
IFN-g, n = 5 (IFN-g). Statistical analysis was performed using a two-tailed
Student’s t-test to give p = 0.015. Error bars are Æ S.D.
Fig. 3 Selectivity of (a) 5-fluoroisatin and (b) 6-fluoroisatin versus 750 mM
RSON species, except for GSH, which was reacted at 5 mM. 500 mM
fluoroisatin was reacted with 1. ONOOÀ 2. ꢀOH 3. GSH 4. ꢀNO 5. Na2N2O3 6.
KO2 7. tBuOOH 8. GSNO 9. NO2À 10. ClOÀ 11. KHSO5 12. H2O2. All reactions,
except 2, were performed in 100 mM HEPES at pH 7.4. Reaction 2 was
performed in 100 mM PBS at pH 7.4 as described previously.12a Spectra were
acquired with 128 scans at 11.7 T using an internal reference peak of
trifluoroacetic acid at À35.6 ppm.
is observed, supporting the ability of 5-fluoroisatin to detect cellular
ONOOÀ. This 19F NMR detection of ONOOÀ provides direct
evidence for the generation of ONOOÀ by A549 epithelial cells
in response to IFN-g signalling, confirming and complementing
the results of prior investigations.26
To conclude, we have communicated a newly discovered
isatin decarbonylation reaction that is mediated by ONOOÀ
under physiological conditions. This rapid and biologically
compatible transformation was utilized to develop two new
19F NMR probes, 5-fluoroisatin and 6-fluorisatin, for the chemo-
selective detection of ONOOÀ over other RSON species. Using
5-fluoroisatin to measure ONOOÀ produced by living A549 lung
epithelial cells in response to cytokine stimulation, we detected
an increase in ONOOÀ over vehicle controls that matched the
trend observed with the fluorescent dye DHR 123. This method
non-invasively samples ONOOÀ in the cellular media, and we
anticipate that this study will give rise to a new class of reaction-
based ONOOÀ probes.
interferon-g (IFN-g), A549 lung epithelial cells generate NO and
display increased staining for nitrotyrosine residues.26 While
these observations are consistent with ONOOÀ generation, it
has never been directly detected in this system. We therefore
utilized 5-fluoroisatin to monitor ONOOÀ in this cellular model
of lung cancer inflammation. We co-incubated B106 A549 cells
with 50 mM 5-fluoroisatin and 50 ng mLÀ1 IFN-g or a vehicle
control for 6–18 hours to capture live-cell ONOOÀ production
over this interval. After incubation, a sample of the cellular
media was removed and analyzed by acquiring overnight 19F
NMR spectra using 7300 scans (Fig. 4). With this protocol,
anthranilic acid concentrations as low as 1.1 mM can be
detected. In the cells treated with IFN-g, a reproducible increase
in the 5-fluoroanthranilic acid peak at À60 ppm is observed,
indicating increased ONOOÀ production (Fig. 4a). Quantifying
the peak intensities with reference to the TFA internal standard
showed a statistically significant ( p o 0.05) increase in the
production of 5-fluoroanthranilic acid from 1.1 Æ 0.3 mM (n = 5)
in vehicle treated cells to 2.0 Æ 0.6 mM (n = 5) for IFN-g treated
cells (Fig. 4b). In order to confirm these results, we used DHR
123, a dye that responds to reactive oxygen species with
moderate selectivity for ONOOÀ,8f to provide an independent
measure of ONOOÀ production in cytokine-stimulated epithelial
cells (Fig. S10, ESI†). A clear increase in the intracellular fluorescence
Funding was provided by Southern Methodist University
(start-up funds to A.R.L.; University Research Council grant to
J.H.B.) and the Engaged Learning program (research funds to
K.J.B.). K.J.B. and C.T.L. were supported by Hamilton Under-
graduate Research Scholarships.
Notes and references
1 P. Pacher, J. S. Beckman and L. Liaudet, Phys. Rev., 2007, 87, 315.
2 (a) J. Stone and S. Yang, Antioxid. Redox Signaling, 2006, 8, 243;
(b) G. Groeger, C. Quiney and T. Cotter, Antioxid. Redox Signaling,
2009, 11, 2655.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 12311--12314 | 12313

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´
3 E. Szaleczky, L. Pronai, H. Nakazawa and Z. Tulassay, J. Clin. 14 R. P. Mason, R. L. Nunnally and P. P. Antich, Magn. Reson. Med.,
Gastroenterol., 2000, 30, 47. 1991, 18, 71.
4 P. B. Massion, O. Feron, C. Dessy and J. L. Balligand, Circ. Res., 2003, 15 K. A. Krohn, J. M. Link and R. P. Mason, J. Nucl. Med., 2008, 49, 129S.
93, 388.
16 (a) K. Yamaguchi, R. Ueki, H. Nonaka, F. Sugihara, T. Matsuda and
S. Sando, J. Am. Chem. Soc., 2011, 133, 14208; (b) K. Matsuo,
R. Kamada, K. Mizusawa, H. Imai, Y. Takayama, M. Narazaki,
T. Matsuda, Y. Takaoka and I. Hamachi, Chem. – Eur. J., 2013,
19, 12875.
5 T. Hanazawa, S. A. Kharitonov and P. J. Barnes, Am. J. Respir. Crit.
Care Med., 2000, 162, 1273.
6 R. Radi, G. Peluffo, M. N. Alvarez, M. Naviliat and A. Cayota, Free
Radical Biol. Med., 2001, 30, 463.
7 Y. Z. Ye, M. Strong, Z. W. Huang and J. S. Beckman, Methods 17 Y. Takaoka, T. Sakamoto, S. Tsukiji, M. Narazaki, T. Matsuda,
Enzymol., 1996, 269, 201. H. Tochio, M. Shirakawa and I. Hamachi, Nat. Chem., 2009, 1, 557.
8 (a) D. Yang, H. Wang, Z. Sun, N. Chung and J. Shen, J. Am. Chem. 18 A. R. Lippert, K. R. Keshari, J. Kurhanewicz and C. J. Chang, J. Am.
Soc., 2006, 128, 6004; (b) F. Yu, P. Li, B. Wang and K. Han, J. Am. Chem. Soc., 2011, 133, 3776.
Chem. Soc., 2013, 135, 7674; (c) C. Song, Z. Ye, G. Wang, J. Yuan and 19 B. Siegel and J. Lanphear, J. Org. Chem., 1979, 44, 942.
Y. Guan, Chem. – Eur. J., 2010, 16, 6464; (d) J. Tuan, H. Chen, 20 J. Massari, R. Tokikawa, D. B. Medinas, J. P. F. Angeli, P. D. Mascio,
˜
L. Zhuo, Y. Xie, N. Li and B. Tang, Chem. – Eur. J., 2011, 17, 6626;
(e) Q. Zhang, Z. Zhu, J. Cheng, N. Zhang, Y. T. Long, J. Zheng,
N. A. Assunçao and E. J. H. Bechara, J. Am. Chem. Soc., 2011,
133, 20761.
X. Qian and Y. Yang, J. Am. Chem. Soc., 2012, 134, 18479; 21 (a) D. A. Wink, J. F. Darbyshire, R. W. Nims, J. E. Saavedra and
( f ) J. P. Crow, Nitric Oxide, 1997, 1, 145.
9 J. X. Yu, R. R. Hallac, S. Chiguru and R. P. Mason, Prog. Nucl. Magn.
Reson. Spectrosc., 2013, 70, 25.
P. C. Ford, Chem. Res. Toxicol., 1993, 6, 23; (b) J. A. Reisz, C. N. Zink
and S. B. King, J. Am. Chem. Soc., 2011, 133, 11675; (c) W. H.
Koppenol, J. J. Moreno, W. A. Pryor, H. Ischiropoulos and J. S.
Beckman, Chem. Res. Toxicol., 1992, 5, 834.
10 (a) R. P. Mason, Curr. Med. Chem., 1999, 6, 481; (b) A. M. Kenwright,
I. Kuprov, E. De Luca, D. Parker, S. U. Pandya, P. K. Senanayake and 22 G. Ferrer-Sueta and R. Radi, ACS Chem. Biol., 2009, 4, 161.
D. G. Smith, Chem. Commun., 2008, 2514.
11 (a) G. A. Smith, R. T. Hesketh, J. C. Metcalfe, J. Feeney and P. G.
23 C. T. Lollar, K. M. Krenek, K. J. Bruemmer and A. R. Lippert, Org.
Biomol. Chem., 2014, 12, 406.
Morris, Proc. Natl. Acad. Sci. U. S. A., 1983, 80, 7178; (b) P. Harvey, 24 R. M. Uppu, Anal. Biochem., 2006, 354, 165.
K. H. Chalmers, E. De Luca, A. Mishra and D. Parker, Chem. – Eur. J., 25 K. M. Miranda, A. S. Dutton, L. A. Ridnour, C. A. Foreman, E. Ford,
2012, 18, 8748.
N. Paolocci, T. Katori, C. G. Tocchetti, D. Mancardi, D. D. Thomas,
M. G. Epsey, K. N. Houk, J. M. Fukuto and D. A. Wink, J. Am. Chem.
Soc., 2005, 127, 722.
12 (a) T. Doura, A. Qi, F. Sugihara, T. Matsuda and S. Sando, Chem.
Lett., 2011, 12, 1357; (b) T. Doura, R. Hata, H. Nonaka, F. Sugihara,
Y. Yoshioka and S. Sando, Chem. Commun., 2013, 49, 11421.
13 A. A. Bobko, S. V. Sergeeva, E. G. Bagryanskaya, A. L. Markel,
V. V. Khramtsov, V. A. Reznikov and N. G. Kolosova, Biochem.
Biophys. Res. Commun., 2005, 330, 367.
26 (a) K. S. Aulak, M. Miyagi, L. Yan, K. A. West, D. Massillon,
J. W. Crabb and D. J. Stuehr, Proc. Natl. Acad. Sci. U. S. A., 2001,
98, 12056; (b) S. Roy, S. Sharma, M. Sharma, R. Aggarwal and
M. Bose, Immunology, 2004, 112, 471.
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