Two-photon fluorescent probe for peroxynitrite

Article abstract of DOI:10.1016/j.tetlet.2016.01.025

Peroxynitrite (ONOO^-), a strong oxidant found in biological systems, and an increase in its levels is related to numerous diseases, immune responses, and redox regulation of signaling pathways. Herein, we report a new two-photon probe that sensitively and selectively detects ONOO^- among other ROS/RNSs (reactive oxygen/nitrogen species) using peroxynitrite-triggered dearylation reaction.

Full text of DOI:10.1016/j.tetlet.2016.01.025

Tetrahedron Letters xxx (2016) xxx–xxx
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Two-photon fluorescent probe for peroxynitrite
JinHee Park , Cheol Ho Heo b,c, Hwan Myung Kim b,c, , Jong-In Hong a,
a
⇑
⇑
a
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea
Department of Chemistry, Ajou University, Suwon 443-749, Republic of Korea
Department of Energy Systems Research, Ajou University, Suwon 443-749, Republic of Korea
b
c
a r t i c l e i n f o
a b s t r a c t
ꢀ
Article history:
Peroxynitrite (ONOO ), a strong oxidant found in biological systems, and an increase in its levels is
related to numerous diseases, immune responses, and redox regulation of signaling pathways. Herein,
Received 7 December 2015
Revised 31 December 2015
Accepted 7 January 2016
Available online xxxx
ꢀ
we report a new two-photon probe that sensitively and selectively detects ONOO among other ROS/
RNSs (reactive oxygen/nitrogen species) using peroxynitrite-triggered dearylation reaction.
Ó 2016 Published by Elsevier Ltd.
Keywords:
Two-photon fluorescence probe
Peroxynitrite
Dearylation reaction
ꢀ
Peroxynitrite (ONOO ), a strong oxidant found in biological sys-
tems, causes oxidation of biomolecules involved in a variety of
physiological and pathological processes. Many reports suggest
However, a two-photon peroxynitrite probe with improved two-
photon absorbing property is still needed for further exploration.
Herein, we report
a new probe—JH-PN4—that selectively
ꢀ
ꢀ
that an increase in ONOO levels is related to many diseases, such
as cardiovascular, neurodegenerative, inflammatory diseases,
metabolic diseases, pain, and cancer. Moreover, ONOO has been
reported to play important roles in immune responses and redox
regulation of signaling pathways. However, the effects of ONOO
detects ONOO among other ROS/RNSs (reactive oxygen/nitrogen
species) using peroxynitrite-triggered dearylation reaction. Perox-
ynitrite was recently reported to trigger oxidative N-dearylation
reaction, which can be used to generate a fluorescence turn-on
1
ꢀ
2
ꢀ
8
ꢀ
response. JH-PN4 is highly selective and sensitive to ONOO and
are not clearly understood owing to its short half-life (<20 ms)
under typical physiological conditions and low concentration. It
has better two-photon excitation properties compared to a previ-
ously reported probe.
1
14
is therefore challenging and highly desired to develop novel chem-
ical tools for detecting peroxynitrite.
JH-PN4 is derived from 2-methylamino-6-acetylnaphthalene
(acedan derivative, compound 1), a well-known two-photon fluo-
1
5
Fluorescence probes have been developed as powerful tools for
rophore, as a reporting group and N-methyl-p-hydroxyaniline
as a targeting moiety. N-Aryl group can quench the fluorescence
ꢀ
peroxynitrite detection since they can detect subcellular ONOO
directly.
3
–9
ꢀ
However, these probes require short excitation wave-
of the acedan derivative efficiently and be eliminated by ONOO
8
lengths ranging from ultraviolet (UV) to visible light, limiting their
use in tissues or animals owing to cellular autofluorescence, artifi-
cial reactive oxygen species (ROS) generation, and shallow tissue
penetration depth.1
with good selectivity among other ROS/RNSs. The fluorescence
of compound 1 can be recovered by dearylation, which leads to a
‘push–pull’ structure of compound 1. JH-PN4 was synthesized
according to Scheme 1B. The detailed synthetic procedures, nuclear
magnetic resonance (NMR) spectra and high resolution mass spec-
trometry (HRMS) spectra are displayed in the Supporting informa-
tion (Scheme 1A). We also synthesized probes JH-PN1–3 to
perform systematic studies on the N-, O-substituent effects.
0
Over the past decade, two-photon microscopy (TPM), which
utilizes two near-infrared (NIR) photons as the excitation source,
has become a useful tool for biomedical research, offering several
advantages including localized excitation, reduced photodamage,
longer observation time, and greater tissue penetration
ꢀ
With JH-PN1–4, we tested their selectivity toward ONOO
1
1–13
depth.
two-photon excitable have been reported.
oxynitrite in live cells and animals using two-photon microscopy.
Nevertheless, only few peroxynitrite probes that are
among other ROS/RNSs in 10 mM phosphate buffer saline (PBS,
0.4% dimethyl formamide (DMF), pH 7.4). As expected, JH-PN1
and JH-PN3 having a methoxymethyl ether group did not give
8,14
They can detect per-
ꢀ
any remarkable response to ONOO and other ROS species (See
the intensity range in the y-axis of Fig. S1A and C). JH-PN2, having
a diarylamino (ArANHAAr ) group, showed only a slight increase in
⇑
Corresponding authors.
E-mail addresses: kimhm@ajou.ac.kr (H.M. Kim), jihong@snu.ac.kr (J.-I. Hong).
0
http://dx.doi.org/10.1016/j.tetlet.2016.01.025
040-4039/Ó 2016 Published by Elsevier Ltd.
0
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2
J. Park et al. / Tetrahedron Letters xxx (2016) xxx–xxx
2
Scheme 1. (A) Sensing mechanism and (B) synthesis of probes JH-PN1–4. TEA = trimethylamine, Tf O = trifluoromethanesulfonic anhydride, dba = dibenzylideneacetone,
0
0
BINAP = (±) 2,2 -bis(diphenylphosphino)-1,1 -binaphthyl, TFA = trifluoroacetic acid, DCM = dichloromethane.
ꢀ
the fluorescence intensity upon the addition of ONOO or other
ROS species (Fig. S1B). This is probably due to oxidation of diary-
ꢀ
16
300
250
lamine by ONOO . Different fluorescence responses of JH-PN1–
ꢀ
4
to ONOO can be explained by the N-substituent effects accord-
ing to a recent systematic study on the fluorescence intensities of
1
7
acedan derivatives depending on the amine donor structure.
However, the fluorescence intensity of JH-PN4 at 509 nm
increased more than 32-fold when 2 equiv of ONOO were added.
There was no significant change with other excess ROS/RNSs
Fig. 1). Although nitric oxide (NO) showed a slight increase in flu-
2
1
1
00
50
00
ꢀ
(
orescence intensity, it is negligible because we added a large
amount of NO (2 mM), which cannot exist in a biological condition
and fluorescence change is small compared to the change with
ꢀ
ONOO . Thus, further experiments were performed with JH-PN4.
50
0
To determine the sensitivity of JH-PN4, fluorescence titration
ꢀ
was performed with ONOO (Fig. 2). As expected, the fluorescence
intensity of JH-PN4 at 509 nm increased more than 57-fold when
JH-PN4
-
H2O2
-
-
.
NO
ONOO
ClO
O2
HO
ꢀ
5
equiv of ONOO were added (Fig. 2A), which resulted from the
elimination of a quencher by dearylation. The detection limit of
Figure 1. Fluorescence intensity (kem = 509 nm) of JH-PN4 (20
lM) with various
ꢀ
reactive oxygen/nitrogen species [ROS/RNSs, 2.0 equiv of ONOO , 10 equiv of other
ROSs, and 100 equiv of nitric oxide (NO)]. Data were acquired at 25 °C in 10 mM
phosphate buffer with 0.4% dimethyl formamide (DMF) at pH 7.4 with excitation at
JH-PN4 was estimated to be as low as 359 nM (Figs. 2B and S2),
ꢀ
which means that JH-PN4 has high sensitivity to ONOO . However,
the fluorescence intensity decreased when more than 5 equiv of
360 nm. Reactions were carried out for 1 h at room temperature before the
ꢀ
ONOO were added. This phenomenon is probably due to further
fluorescence intensities of the probe solutions were measured.
ꢀ
oxidation of 1 generated by ONOO triggered dearylation of JH-
PN4. Compound 1, having a secondary amine, can be oxidized with
ꢀ
8,16
itself. Since the emission spectra of the mixture of JH-PN4 and
ONOO are almost identical to that of 1 itself, we can assume that
excess ONOO , which reduces the fluorescence of 1.
The maxi-
ꢀ
mum fluorescence intensity of JH-PN4 upon addition of 5 equiv
ꢀ
1
is produced by the dearylation of JH-PN4, but the dearylation of
ONOO is weaker than the fluorescence intensity of compound 1
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J. Park et al. / Tetrahedron Letters xxx (2016) xxx–xxx
3
ꢀ
Figure 2. (A) Fluorescence spectra and (B) fluorescence intensity at 508 nm of JH-PN4 (20
l
M) with ONOO (0–8 equiv). Data were acquired at 25 °C in 10 mM phosphate
buffer (0.4% dimethyl formamide (DMF)) at pH 7.4 with excitation at 360 nm. Reactions were carried out for 30 min at room temperature before the fluorescence intensities
of the probe solutions were measured.
Figure 3. (A) Time-dependent fluorescence change. (B) Time course of fluorescence intensity (kem = 508 nm) of JH-PN4 (10
dimethyl formamide (DMF), pH 7.4) in the presence of 1.0 equiv ONOO (kex = 360 nm).
lM) in 10 mM phosphate buffer saline (PBS) (0.2%
ꢀ
ꢀ
ꢀ
Figure 4. (A) Two-photon fluorescence spectra of JH-PN4 (10
l
M) with ONOO . Inset shows two-photon fluorescence titration curve for JH-PN4 with ONOO . The excitation
ꢀ
wavelength was 740 nm. (B) Two-photon action cross section spectrum of probe JH-PN4 in the absence (j) and in the presence (h) of ONOO . These data were measured in
1
0 mM phosphate buffer saline (PBS).
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J. Park et al. / Tetrahedron Letters xxx (2016) xxx–xxx
ꢀ
sensitivity and selectivity for ONOO among other ROS/RNSs and
improved two-photon excitation properties compared with previ-
ous reported probes. These results show that JH-PN4 can be a use-
ful tool for peroxynitrite imaging in biosystems.
JH-PN4
1
70
60
50
40
30
20
10
Acknowledgments
This work was supported by the NRF grant (No.
2
013R1A1A2074468) funded by the MSIP.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.01.
0
25.
0
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4
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ꢀ
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r
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8
.8 GM at 740 nm, which is an improved value compared with a
14
previous reported probe. Moreover, the value of two photon
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ꢀ
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ꢀ
ꢀ
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1
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ꢀ
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ꢀ
change in fluorescence intensity with ONOO addition. The fluo-
rescence intensity of product 1 decreased at pH 4, but it was also
negligible. This suggests that JH-PN4 has good stability under var-
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1
1
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ꢀ
In conclusion, we developed a two-photon ONOO turn-on sen-
sor using peroxynitrite-triggered dearylation. JH-PN4 shows good
Please cite this article in press as: Park, J.; et al. Tetrahedron Lett. (2016), http://dx.doi.org/10.1016/j.tetlet.2016.01.025