A boronate-based ratiometric fluorescent probe for fast selective detection of peroxynitrite

Article abstract of DOI:10.1016/j.tetlet.2017.12.004

Given that peroxynitrite (ONOO^?) is profoundly associated with health and diseases, a new fluorescent probe ABT was designed and synthesized for detection of ONOO^?. ABT manifested not only ratiometric fluorescence signals simultaneou

Full text of DOI:10.1016/j.tetlet.2017.12.004

Tetrahedron Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A boronate-based ratiometric fluorescent probe for fast selective
detection of peroxynitrite
⇑
Qi Li, Zhengyin Yang
College of Chemistry and Chemical Engineering, State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China
a r t i c l e i n f o
a b s t r a c t
Given that peroxynitrite (ONOOÀ) is profoundly associated with health and diseases, a new fluorescent
probe ABT was designed and synthesized for detection of ONOOÀ. ABT manifested not only ratiometric
fluorescence signals simultaneously in response to concentrations of ONOOÀ (within 10 s), but high
selectivity and sensitivity towards ONOOÀ over other physiological relevant species (detection limit =
26.3 nM). Moreover, ABT worked in a broad pH range with biological relevance. Thus, ABT could be used
to quantitative detection of ONOOÀ concentration and has the potential to efficiently monitor ONOOÀ in
living organisms.
Article history:
Received 22 September 2017
Revised 23 November 2017
Accepted 1 December 2017
Available online xxxx
InChIKey:
NGNQMOWXYMMDSY-UHFFFAOYSA-N
Ó 2017 Published by Elsevier Ltd.
Introduction
developed for peroxynitrite detection in biological systems.12–20
Nevertheless, most of these probes are based on a single channel
Peroxynitrite (ONOOÀ), a highly reactive oxidant in living
organisms, is mostly produced by a diffusion-controlled reaction
between nitric oxide (ÁNO) and superoxide (ÅO2À) in mitochondria.1
On the one hand, as an oxidant and efficient nitrite agent, ONOOÀ
plays a vital role in diverse pathophysiological conditions like mod-
ulating cell signal transduction.2–6 For example, ONOOÀ oxidizes
reactive cysteine residues in proteins, as a result, deactivation or
activation of the proteins.7,8 But on the other hand, abnormality level
of ONOOÀ in living cells can cause serious damage to cellular biomo-
lecules involving lipids, proteins, and nucleic acids, resulting in cell
apoptosis or necrosis. Moreover, abnormal concentrations of ONOOÀ
is associated with some ailments such as cardiovascular diseases,
circulatory shock, inflammation, cancers, and so on.9,10 Thus, it is sig-
nificant for assays of ONOOÀ detection which may be useful for the
diagnosis of relative diseases and the exploration of its various
pathophysiologies.
Although peroxynitrite in human health and diseases is of sig-
nificance, the elucidation of the biological functions of peroxyni-
trite remains a challenge. One of the main obstacles to explore
its roles in organisms is short of suitable approaches to detect it
in vivo due to its short lifetime (<10 ms) and numerous antioxidant
in cell. Fluorescent probe is of course a promising method to detect
ONOOÀ in vivo.11 Recently, a lot of fluorescent probes have been
with a turn-on or turn-off fluorescence signal, which limits their
applications in quantitative measurement of peroxynitrite concen-
tration in vitro and vivo.21 By contrast, the fluorescent probes on a
basis of independent two-channel ratiometric signal enable reduc-
ing of the errors caused by various external conditions: excitation
power, fluorescence decay and probe distribution.22,23 However, it
is still a heavy task to distinguish ONOOÀ from other reactive oxy-
gen species (ROS) such as H2O2 and ClO- with similar properties
and selectively detect ONOOÀ in the presence of reducing mole-
cules such as hydrogen sulfide and glutathione.24 Therefore, it is
indeed urgent to find more excellent fluorescent probes for detect-
ing and monitoring ONOOÀ in vivo.
As ONOOÀ can react with arylboronates forming corresponding
phenols much faster than H2O2 and HClO, arylboronate derivatives
are considerable sources for the ONOOÀ specific fluorescent
probes.25 Moreover, HMBT (2-(2-hydroxy-3-methoxyphenyl) ben-
zothiazole) is regarded as an available fluorophore used in many
fields successfully in light of large Stokes shift, good photostability
and ratiometric detection capability.26,27 Consequently, a new
ratiometric fluorescent probe named as ABT (4-[(2-(2-benzithia-
zolyl)-5-methoxy) phenoxymethyl] phenylboronic acid pinacol
ester), for detecting ONOOÀ was designed and synthesized by
modifying HMBT with an arylboronate moiety. This probe ABT
showed ultra-short response time and highly sensitive quality
towards ONOOÀ. Additionally, ABT performed prominent
selectivity for ONOOÀ over other coexisted ROS such as H2O2, HClO,
⇑
Corresponding author.
E-mail address: yangzy@lzu.edu.cn (Z. Yang).
Å
and OH.
https://doi.org/10.1016/j.tetlet.2017.12.004
0040-4039/Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Li Q., Yang Z. Tetrahedron Lett. (2017), https://doi.org/10.1016/j.tetlet.2017.12.004

2
Q. Li, Z. Yang / Tetrahedron Letters xxx (2017) xxx–xxx
Results and discussion
Synthesis of probe ABT
Probe ABT was easily synthesized through two steps outlined in
Scheme 1, and the details for synthetic data were given in Support-
ing information.
Spectral response of the probe to ONOOÀ
As the probe ABT and HMBT were in hand, their UV–vis absorp-
tion spectra and fluorescence emission spectra were investigated
in PBS buffer (10 mM, pH 7.4, 40% ethanol) at room temperature.
ABT showed maximal absorption/emission bands at 309 nm/405
nm, however, upon addition of ONOOÀ (2 equiv), the absorption
band at k = 309 nm was red-shifted to 317 nm and the emission
Scheme 1. Synthetic route of the probe ABT.
Fig. 1. Thespectral profiles were obtained in PBSbuffer (10 mM, pH 7.4, 40% ethanol).
(A) UV–vis absorption spectra of ABT (50 mM), HMBT (50 mM) and ABT (50 mM) in the
presence of 2 equiv of ONOOÀ. (B) Fluorescence spectra of ABT (5 mM), HMBT (5 mM)
and ABT (5 mM) in the presence of 2 equiv of ONOOÀ, kex. = 317 nm, slits: 5. 5.
Fig. 2. (A) Fluorescence spectra of ABT (5 mM) in the presence of various
concentrations of ONOOÀ (0–14 mM). (B) The relationship between fluorescence
emission intensity ratio (F483/F405) and ONOOÀ concentration (0–14 mM). Profiles
were obtained in PBS buffer (10 mM, pH 7.4, 40% ethanol), kex. = 317 nm, slits: 5. 5.
Please cite this article in press as: Li Q., Yang Z. Tetrahedron Lett. (2017), https://doi.org/10.1016/j.tetlet.2017.12.004

Q. Li, Z. Yang / Tetrahedron Letters xxx (2017) xxx–xxx
3
band at 405 nm was also red-shifted to 483 nm. HMBT has two
emission bands (enol and keto emission). As modified by an aly-
boronate group, HMBT exhibited enol-like emission band at 405
nm. But when ABT reacted with ONOOÀ, it would release free
HMBT exhibiting keto emission at 483 nm. Moreover, the conse-
quences showed that the UV–vis absorption spectra and emission
spectra of HMBT were likely to that of the ABT solution in the pres-
ence of ONOOÀ. As a result, it implicated that the binding of ABT
with ONOOÀ furnished HMBT (Fig. 1).
According to the assay of fluorescence titration, an obvious
change in ratiometric fluorescence signal (F483/F405) could be
observed when ONOOÀ was added to the solution of ABT and
excited at 317 nm. As shown in Fig. 2A, upon incremental addition
of ONOOÀ (0–14
lM), the emission band of ABT at 405 nm dramat-
ically decreased while another emission band at 483 nm rose up
and reached a constant value eventually. The fluorescence emis-
sion intensity ratio between 483 nm and 405 nm (F483/F405
)
enhanced to about 35 times from 0.43 to 15.18. Meanwhile, the
Fig. 3. Linear curve derived from the emission intensity ratio (F483/F405) and ONOOÀ
concentration (0–5 mM) in PBS buffer (10 mM, pH 7.4, 40% ethanol). kex. = 317 nm,
slits: 5. 5.
Fig. 5. (A) Fluorescence emission intensity ratio (F483/F405) of ABT after treatment
with ONOOÀ (10 mM) and other analytes (50 mM) including H2O2, NOÀ2 ÅOH, S2À
, ,
t-BUOOÅ, t-BUOOH, ÅO2À, HClO, FÀ, ClÀ, BrÀ, IÀ, NO3À, SO42À, and SO23À. (B) The black
column represents the ratio (F483/F405) of ABT (5 mM); the blue columns represent the
ratio of ABT (5 mM) in the presence of other analytes (50 mM) referred to above; the
red columns represent the ratio when ONOOÀ was added into ABT solution
containing other analytes. Profiles were obtained in PBS buffer (10 mM, pH 7.4, 40%
ethanol), kex. = 317 nm, slits: 5. 5. The data were gotten after addition of the analytes
in two minutes.
Fig. 4. Time-dependent fluorescence spectra of ABT upon addition of ONOOÀ
(10 mM) and other analytes (50 mM) in PBS buffer (10 mM, pH 7.4, 40% ethanol).
kex. = 317 nm, slits: 5. 5.
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Q. Li, Z. Yang / Tetrahedron Letters xxx (2017) xxx–xxx
emission intensity ratio (F483/F405) exhibited a good linear relation-
ship over ONOOÀ concentration range from 0 to 5
M or 5 to 10
M (see Fig. 2B and Supporting information). Hence, the linear
curve of the ratio (F483/F405) allowed for the convenient quantita-
tive detection of ONOOÀ over this concentration range. In addition,
detection limitation of ABT for sensing ONOOÀ was calculated to
be as low as 2.63 Â 10À8 molÁLÀ1 in accord with some previously
reported probe for ONOOÀ24,9,18 (Fig. 3).
Although, the ratio increased a little to about 2.5 times in the pres-
ence of HClO (Fig. 5A), other analytes barely interfered with the reac-
l
l
tion between ONOOÀ and ABT. The emission intensity ratio (F483
/
F
405) increased to 21–27 times when ONOOÀ added into the mixed
solution of ABT and one of the analytes mentioned above in most
cases; even though the ratio only increased to nearly 12 times, as
Å
the mixed solution in the presence of HClO, OH and t-BUOOÅ (Fig.
5B). May it result from high concentration of HClO, ÅOH and t-BUOOÅ
reacting with ABT in terms of previous reports.17 In a word, the
probe ABT displayed high selectivity towards ONOOÀ over other
physiological species in vitro.
Furthermore, temporal fluorescence responses of ABT (5 lM)
towards ONOOÀ and other analytes were examined as shown in
Fig 4. The ratio (F483/F405) rapidly and drastically went up within
10 s, and gradually reached a plateau at 50 s in the presence of
Since the pH values in typical mammalian cells usually range
from 4.5 in lysosomes to 8.0 in mitochondria, the effective pH
range of ABT for peroxynitrite detection was determined to be
ONOOÀ (10
l
M), while the ratio shows little variety even after 2
Å
mins in the case of other analytes (H2O2, NOÀ2 , OH, S2À, t-BUOOÅ,
t-BUOOH, and OÀ2 ) except HClO leading to slight enhancement of
from 5 to 9, a broad range with biological relevance (see
Å
the ratio (the concentration of other analytes is all 50
lM). In terms
Supporting information).
of kinetic studies, the rate constant of the reaction between ABT and
ONOOÀ was 1.16 Â 104 MÀ1ÁSÀ1. However, rate constants for the
reaction of ABT with H2O2, and HClO were 0.23 MÀ1ÁSÀ1 and 4.81
MÀ1ÁSÀ1 respectively. Compared to the former rate constant, the
later ones were much smaller. These results suggested that not only
ABT had great potential to efficiently capture ONOOÀ in biological
systems, in spite of its short lifetime and high activity, but selectivity
of ABT for ONOOÀ over other analytes referred to above was extre-
mely high.
Proposed sensing mechanism
The results from the spectral response of ABT to ONOOÀ
indicated that HMBT might be the product of reaction between
ABT and ONOOÀ. Furthermore, the main product of reaction
between ABT and ONOOÀ was identified by the separate reaction
of ABT and 3 equiv ONOOÀ in DMF (N,N-Dimethylformamide) at
room temperature for 1 h and the isolated major product was
HMBT which was characterized by 1H NMR spectra. Moreover,
the crude reaction mixture of ABT and ONOOÀ was analyzed using
MS showing intense peak of at m/z 258, consistent with that of
compound HMBT. Meanwhile, the intense peak of [ABT + H]+
(m/z 474) was disappeared (see Supporting information).
Obviously, it could be proved that the main product was HMBT.
So, the sensing mechanism was probably that the reaction of ABT
with ONOOÀ promoted oxidative hydrolysis of the arlyboronate
To evaluate the selectivity and competition of the probe ABT,
fluorescence spectra were obtained when ONOOÀ (10
l
M) and
M) including H2O2,
NOÀ2 , OH, S2À, t-BUOOÅ, t-BUOOH, O2À, FÀ, ClÀ, BrÀ, IÀ, NO3À, SO24À
and SO23À were added into ABT solution (5
M) respectively. Results
other physiologically relevant species (50
l
Å
Å
,
l
observed were analogous to previous time-dependent responses,
only ONOOÀ made the emission intensity ratio (F483/F405) significant
enhancement, but other species tested hardly caused any changes.
Scheme 2. Proposed mechanism for ABT sensing ONOOÀ.
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Q. Li, Z. Yang / Tetrahedron Letters xxx (2017) xxx–xxx
5
group with the rapid elimination of p-quinomethane to generate
free HMBT (Scheme 2 for mechanism in detail).28
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.tetlet.2017.12.004.
Please cite this article in press as: Li Q., Yang Z. Tetrahedron Lett. (2017), https://doi.org/10.1016/j.tetlet.2017.12.004