Boronate based fluorescence (ESIPT) probe for peroxynitrite

Article abstract of DOI:10.1039/c6cc06829d

A simple probe for the detection of peroxynitrite was developed incorporating a benzyl boronic ester "protecting" unit. The "protecting" unit of the probe is removed by peroxynitrite to "turn-on" ESIPT fluorescence (4.5 fold enhancement). Furthermore, the probe was cell permeable and was used in cell imaging experiments showing an off-on response towards peroxynitrite, in HeLa and RAW 264.7 cells.

Full text of DOI:10.1039/c6cc06829d

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Boronate based fluorescence (ESIPT) probe for peroxynitrite
Adam C. Sedgwick,a Xiaolong Sun,a Gyoungmi Kim,b Juyoung Yoon,*b Steven D. Bull*a and Tony D.
James*a
Received 00th January 20xx,
Accepted 00th January 20xx
A simple probe for the detection of peroxynitrite was developed incorporating a benzyl boronic ester “protecting” unit.
The “protecting” unit of the probe is removed by peroxynitrite to “turn-on” ESIPT fluorescence (4.5 fold enhancement).
Furthermore, the probe was cell permeable and was used in cell imaging experiments showing an off-on response towards
peroxynitrite, in HeLa and RAW 264.7 cells.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Peroxynitrite (ONOO-) is a short lived reactive nitrogen species 1).
(RNS)1,2 with a half-life of ~10-20 ms known to be a key
S
N
pathological intermediate in
Including inflammatory,
a
wide range of diseases.
S
N
S
N
ONOO-
ischemia-reperfusion and
N
neurodegenerative diseases.3-5 Peroxynitrite behaves as a
strong oxidant and nitrating agent towards a wide range of
biological targets such as lipids, proteins, and DNA.6 Therefore
the development of peroxynitrite selective fluorescent
molecular probes is highly desirable.7-15 However, current
commercial peroxynitrite probes including aminophenyl
fluorescein (APF) and hydroxyphenyl fluorescein (HPF) lack
selectivity and react with other ROS.16
1
H
N
NH
O
2
ESIPT
OH
Non-fluorescent
Fluorescent
Scheme 1.
We realised that the fluorescent N-methyl-benzothiazole fragment
2 provides a new core on which to develop a range of selective
probes. Our group is particularly interested in incorporating benzyl
boronic ester units, which have been elegantly employed by Chang
and Shabat,17-19 this “protecting group” is oxidatively cleavage by
hydrogen peroxide to generate highly fluorescent products.
Recently Li et al. developed a “turn-on” fluorescent probe 1 for
tracking the in situ generation of peroxynitrite in cells and mice
using ischemia-induced neurovascular damage.6 Benzothiazole was
shown to be a good fluorescent probe with high “turn-on” (600
fold), high selectivity towards peroxynitrite and rapid transport
across the blood brain barrier. As shown in Scheme 1 in its normal
form 2 is weakly fluorescent but when excited it can isomerise via
excited state intramolecular proton transfer (ESIPT), resulting in a
large increase in fluorescence.
Similarly, it has been shown that boronates/ boronic acids react
more rapidly (milliseconds) with peroxynitrite when compared to
hydrogen peroxide where the oxidation process can take several
hours.20 Therefore, decided to incorporate the Shabat “protecting
group” with the fluorescent N-methyl-benzothiazole core in order
to develop peroxynitrite sensor 3.
Probe 1 was designed to contain a saturated C-N bond thus blocking
the ESIPT process, however, on reaction with peroxynitrite the p-
hydroxyaniline group undergoes oxidative cleavage to form
benzoquinone when exposed to peroxynitrite to generate the
highly fluorescent N-methyl-benzothiazole proton donor (Scheme
O
N
S
B
O
N
3
Probe 3 was synthesised over three steps through the reaction of 2-
aminothiophenol and isatoic anhydride to afford 2-
(benzo[d]thiazol-2-yl) aniline in 72 % yield. A one pot reductive
amination was then carried out using sodium triacetoxyborohydride
and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde in
78 % yield. The intermediate formed was found to contain a
particularly unreactive aniline towards
a number of reagents
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including NaH, consequently an excess of reagents was required, Having determined the selectivity of probe 3, we then evaluated its
therefore, 15 equivalents of AcOH and 10 equivalents of ability to visualise endogenous and exogenous peroxynitrite using
formaldehyde were used to afford probe 3 in 14 % yield. (Scheme cell imaging experiments. The HeLa cells were incubated with probe
S1)
3 (20 µM) for 30 minutes and washed with Dulbecco’s phosphate-
buffered saline (DPBS). The cells were then observed using a
confocal laser microscope excitation λ 405 nm, emission λ 430 –
455 nm. From Figure 3 probe 3 can penetrate live cell membranes
and provide a clear “turn on” response in the presence of various
concentrations of peroxynitrite added exogenously. No “turn on”
was observed when the cells were pretreated with the peroxynitrite
scavenger ebselen.
We subsequently evaluated the fluorescence behaviour of probe 3,
in in pH 8.2 buffer solution [52 wt% methanol].21 Probe 3 produced
an up to 4.5 fold fluorescence “turn on” in the presence of low
concentrations of peroxynitrite (10 µM).
300
200
100
0
To detect peroxynitrite endogenously, RAW 264.7 cells were used
and the immune reaction was induced using 1µg/mL and 50 ng/mL
LPS, IFN-γ. As shown above in figure, probe 3 was shown to detect
peroxynitrite endogenously. Probe 3 was also evaluated at longer
wavelengths in cells λex 473nm and λem 490 – 590 nm (Fig S7).
a
c
b
d
420
440
460
480
500
520
540
Wavelength/ nm
Fig 1. Fluorescence spectra of probe 3 (0.25 µM) with the addition of
peroxynitrite (0-10 µM), λex 400 nm in pH 8.2 buffer solution [52 wt%
methanol].
Fig 3. Fluorescence property in the live cell. HeLa cells were incubated with
20 μM probe 3 for 30 min and washed with DPBS and added (a) 0, (b) 30, (c)
100 μM ONOO- and (d) after pretreatment with 100 μM ebselen, add 100
μM ONOO- for 30 min. Fluorescence images were acquired by confocal
Subsequently, we evaluated the selectivity of probe 3 towards
other ROS/RNS. As expected, ClO- (100 µM) led to a decrease in
fluorescence intensity (0.71) due to its strong oxidising ability slowly
destroying probe 3. While, as reported by Sikora et al., H2O2 led to a
small fluorescence enhancement at 100 µM. Addition of 10 mM
H2O2 is required to produce a significant fluorescence response (Fig
S3 and S4), clearly demonstrating the greater reactivity of boronic
acids/ esters towards peroxynitrite.
microscopy.
λ
ex 405 nm/
λ
ex. 430 – 455 nm. Scale bar: 10 μm.
c
a
d
b
5
4
3
2
1
0
Fig 4. Detection of peroxynitrite made by immune reaction in the
macrophage. RAW 264.7 cells were treated with (a) no, (b) 1 μg/ml LPS
16hr, 50 ng/ml interferon-γ 4hr, (c), LPS, IFN-γ + 100 μM ebselen and (d) LPS,
IFN-γ + 100 μM uric acid. The cells were stained with 20 μM probe 3 for 30
min and washed with DPBS and imaged by confocal microscopy.
em 430 – 455 nm. Scale bar: 10 μm.22-24
λex 405 nm/
λ
Conclusions
-
-
.
-
.
ONOO H2O2 HO
ClO ROO 1O2 NO
O2
Blank
In conclusion we have a developed a reaction based fluorescent
probe that can be used to detect peroxynitrite at low
concentrations allowing the detection in cells. Before exposure to
ROS, probe 3 has a low fluorescence intensity becuase the ESIPT
process is not possible. However, the fluorescence is enhanced
once the boronic ester “protecting group” is removed selectively by
Fig 2. Selectivity of Probe 3 (0.25 µM), 10 µM peroxynitrite, All other ROS
100
M. H2O2, ClO- and ROO; incubated for 30 mins, λex 400 nm/ λem 461 nm
µ
in pH 8.2 buffer solution [52 wt% methanol].
2 | J. Name., 2012, 00, 1-3
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exposure to peroxynitrite. We have demonstrated that N-methyl- 9. Z. Lou, P. Li and K. Han, Acc. Chem. Res., 2015, 48, 1358-1368.
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benzothiazole core unit 2 can be used to develop probes with
applications in exploring the pathological effect of peroxynitrite.
Furthermore, probe 3 as a very important advantage over probe 1
since could be easily developed into a theranostic agent towards
tumour cells. This approach was recently demonstrated by Kim et
al. who have used the boronic “protecting group” to weaponize a
probe by linking with the anticancer prodrug 5’-deoxy-5-
fluorouridine to the boronic acid group.25
11. X. Chen, F. Wang, J. Y. Hyun, T. Wei, J. Qiang, X. Ren, I. Shin and
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