Product-boosted fluorescence signal: A new approach for designing small-molecule probes for detection of peroxynitrite

Article abstract of DOI:10.1039/d0cc02963g

In situ self-assembled boronate ester comprising commercially available 2-formylphenylboronic acid and 2-(2′,3′-bihydroxyphenyl)benzothiazole (BHBT) is explored for the detection of ONOO- with product-boosted fluorescence. The self-assembly can detect ONO

Full text of DOI:10.1039/d0cc02963g

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DOI: 10.1039/D0CC02963G
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Product-boosted fluorescence signal: a new approach for
designing small-molecule probes for detection of peroxynitrite
Received 00th January 20xx,
Accepted 00th January 20xx
Dejia Wang, Yuchen Huyan, Xiaojing, Nan, Hongjuan Li, Shiguo Sun and Yongqian Xu*
DOI: 10.1039/x0xx00000x
In situ self-assembled boronate ester comprising of commercially mode saccharide sine it has a high binding affinity with
available 2-formylphenylboronic acid and 2-(2',3'- phenylboronic acid) were often added to enable the boronic acid to
1
6-18
bihydroxyphenyl)benzothiazole (BHBT) is explored for the form stable boronic ester.
In addition, indicators with two
-
detection of ONOO with product-boosted fluorescence. The self- neighboring hydyoxyl groups, e.g., Alizarin Red (ARS) were
assembly can detect ONOO in endoplasmic reticulum of living introduced into boronate-based probes to produce indicator
-
cells.
displacement assay (IDA) to enhance the selectivity of peroxynitrite
1
7
2
over H O
2
.
Although these approaches that are based on self-
Accumulating evidence showed that the homeostasis of reactive
oxygen/nitrogen species in cells or tissues is associated with a
variety of human diseases, such as Alzheimer's disease, arthritis,
assemblies can reduce the procedure of synthesis and improve
selectivity, they still exist some drawbacks. First, turn-off but not
turn-on fluorescence response to peroxynitrite was observed,
16-19
1
cancer, autoimmune and inflammatory diseases. Peroxynitrite
which is inferior for detection because the former was easily
interfered with autofluorescence. Second, the synthetic procedure
for these probes of which boronate groups was integrated is
laborious. Moreover, these IDA-based probes can only release
original indicator or fluorophore, but no amplified signal which is
stronger than indicator or fluorophore itself was obtained to detect
peroxynitrite, resulting in low sensitivity of probes. Recently, Tian
group reported the first example of protein-probe hybrid-based
method in which protein was utilized to “stabilized” peroxynitrite-
released fluorophore to boost fluorescence signal, to improve the
-
(
ONOO ), a strong oxidant that is formed via the reaction between
-
superoxide ( O
2
) and nitric oxide (NO), has been recognized as a
2
crucial biomarker of pathological process. Peroxynitrite can cause
irreversible damage to a range of biological targets including
phospholipids, proteins, and nucleic acids. The detection of ONOO
-
would be helpful for understanding the pathological functions of
-
3
ONOO and diagnosis of related diseases. But in biological systems,
-
4
ONOO is difficult to be monitor due to its short half-life (10-20 ms).
Therefore, it is of significant interest to develop powerful tools to
-
detect ONOO .
20
sensitivity.
However, this host-guest-based-method is only
Fluorescent probes have the potential to detect biological species
due to the advantages of high sensitivity, good selectivity,
spatiotemporal resolution, noninvasiveness, as well as potential
applicable to hydrophobic molecules. Therefore, new strategy is
substantially required.
5
applications in living cells. A number of small-molecule fluorescent
-
probes with simple synthesis have been reported to detect ONOO
5
-19
over the last two decades. . Among of them, small-molecular
fluorophores conjugated with boronic acid/boronate ester receptor
have been proven useful, which undergo peroxynitrite-triggered
oxidation to form the corresponding phenol to produce distinct
1
5-19
fluorescence change.
Boronate ester receptors showed shorter
response time and higher selectivity to peroxynitrite than boronic
acid receptors probably due to the optimized redox potential. 1,2-
and 1,3-diol such as monosaccharide (D-fructose was chosen as a
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of
Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi, 712100, P.R.
China. xuyq@nwsuaf.edu.cn
Scheme 1. The strategy of designing boronate-based fluorescent probe for the
detection of peroxynitrite.
Electronic Supplementary Information (ESI) available: [The detailed experimental
process, synthesis of compounds, additional fluorescence spectra and SEM, DLS
spectra]. See DOI: 10.1039/x0xx00000x
Herein, we present a novel platform of a boronic ester-based
self-assembly
comprising
commercially
available
2-
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Journal Name
formylphenylboronic
acid
and
2-(2',3'- aldehyde, producing more thermodynamically stable boronic esters
View Article Online
DOI: 10.1039/D0C C2 30- 22 69 63G
-
bihydroxyphenyl)benzothiazole (BHBT) for the detection of ONOO which are benefitial to recognize peroxynitrite (Scheme 1).
The
with turn-on fluorescence and high sensitivity. This self- aldehyde group probably feature the similar function of neighboring
1
6, 27
assemble has following advantages: 1) different from amine reported by James and coworkers.
convential methods,
To further prove this
1
6-22
1,2-diol but not boronate acid hypothesis, we studied the fluorescence responses of BHBT to
segment was functioned into fluorephore, minimizing the meta- and para-aldehyde phenylboronic acid (m-B and p-B).
synthese of probes, 2) in situ formed boronic ester improve the Compared to the obvious turn-on fluorescence change after
-
selectivity of ONOO over H
2
O
2
, 3) the self-assembly experiences treatment with o-B, no change or even turn-off responses were
integrated, released and recombined processes. The released observed as m-B or p-B was added (Fig. S5-6). The result implied
product, salicylaldehyde can immobilize BHBT through hydrogen- that no stable boronic esters were formed in the presence of m-B or
bond-triggered cross-linking to boost detection signal, improving p-B. Correspondingly, the solution of BHBT with m-B or p-B showed
-
the sensitivity. Moreover, the self-assembly can detect ONOO in no fluorescence enhancement towards peroxynitrite, which is
endoplasmic reticulum of living cells.
similar to those with boronic acid derivatives without aldehyde. The
2
-(2'-hydroxyphenyl)benzothiazole (HBT), a typical excited state specific fluorescence change confirmed the unique function of
intramolecular proton transfer (ESIPT) molecule, have been widely ortho-aldehyde group in the formation of boronic ester and process
used as fluorophore of probes because it has high fluorescence of sensing of peroxynitrite.
quantum yield, excellent photo-stability, facile synthesis, as
2
1-22

well as large Stokes shift
.
1,2-diol featured BHBT, besides with
1000
0  
1
2
3
4
5
6
7
8
9
0 
0 
0 
0 
0 
0 
0 
0 
0 
the photochemical properties of BHT, could also provide an
effective motif which is similar to monosaccharide to react with
boronic acid to form fluorescent boronic ester (Scheme 1). As far as
we know, this is first sample that 1,2-diol featured fluorogen
coupled with simple phenylboronic acid derivative was used to
structure peroxynitrite fluorescence probe with amplified signal
originated from “indicator”.
800
600
400
200
0
BHBT was achieved through facile synthesis with two steps.
Condensation reaction between o-vanillin and 2-aminothiophenol is
followed by demethylation with BBr
3
. The detailed synthetic
450
500
550
600
Concentration (M)
procedure was described in the Supporting Information. The
1
13
compounds were fully characterized by H NMR, C NMR and MS.
The fluorescence response of BHBT alone to peroxynitrite was Fig. 1 Fluorescence spectra change of BHBT (10 μM) in the presence of 20 equivalents
-
of o-B upon addition of different concentration of ONOO in PBS buffer (10 mM, pH
first studied. fluorescence quenching of BHBT upon addition of the
8
.0).
peroxynitrite was observed probably due to the oxidation of
hydroxyl in BHBT to ketone by peroxynitrite (Fig. S1). Next, different
kinds of boronic acid derivatives including phenylboronic acid (PBA),
We further tested the response of the self-assembled BHBT-o-B
towards peroxynitrite. In buffer solution, the fluorescence intensity
gradually increased with addition of peroxynitrite (Fig. 1). The
quantum yields before and after addition of peroxynitrite are about
2
-methylphenylboronic
acid
(2-MPBA),
4-
(bromomethyl)phenylboronic acid (4-BPBA) and its ester (4-BPBE),
and 2-formylphenylboronic acid (o-B) were separately added to the
solution of BHBT, turn-on fluorescence change was obtained,
suggesting these phenylboronic acid derivatives can interact with
0
.53% and 1.34%, respectively. There is a good linear relationship
between fluorescence intensity at 494 nm and the concentration of
peroxynitrite in the region of 0-30 μM (Fig. S7). The detection limit
was calculated as low as 3.64 nM. We also investigated the
selectivity of self-assembled BHBT-o-B to other relative reactive
1
6
BHBT to form corresponding boronic ester (Fig. S2-3). To check
the response of these self-assembled boronic esters to
peroxynitrite, we added peroxynitrite to these solutions to monitor
the fluorescence change. Except for that with o-B, all self-
assembled boronic esters showed fluorescence quenching change
1
-
-
oxygen and nitrogen species, such as ·OH, O
2
, ClO , H
2
O
2
, NO·, NO
2
-
-
-
,
NO
3
and O
2
· . Among them, only ClO caused a relative
fluorescence enhancement (Fig. 2 and S8). As reported previously,
(Fig. S3a-d). In contrast, the boronic ester derived from o-B (with
- 16
boronic ester can be oxidized into phenol by ClO . But in
ortho-aldehyde group) showed turn-on fluorescence response after
addition of peroxynitrite (Fig. S3e). Boronic esters derivatives with
or without ortho-aldehyde group demonstrated opposite
fluorescence change to peroxynitrite, suggesting that the ortho-
aldehyde group plays important role in the sensing process of
peroxynitrite.
It is presumed that the aldehyde group with electron-
withdrawing property would increase the acidity of boronic acid
through intramolecular hydrogen bond to enhance its affinity to
-
physiological and pathological conditions, ClO is not present or is at
very low concentration. In addition, the activity of peroxynitrite is
- 28
much strong than that of ClO . Therefore, the complex of BHBT-o-
B can selectively detect peroxynitrite with high sensitivity. Besides
facile synthesis and assemble, BHBT-o-B demonstrates comparable
-
performance of some other small-molecular ONOO probes
reported previously in the aspects of detection limit, response time
and selectivity (Table S1).
To shed insight into the sensing mechanism, we utilize HPLC
technique to check whether 2-formylphenol (salicylaldehyde), the
1
,2-diol featured BHBT. Furthermore, in the presence of BHBT,
…
there is B O interaction via adjacent oxygen positioned on
2
| J. Name., 2012, 00, 1-3
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oxidized product of 2-formylbenzeboronic acid, occurs after leads to the formation of large aggregation through intermolecular
View Article Online
2
9-30
DOI: 10.1039/D0CC02963G
The increasing size of particles can also be observed from
addition of peroxynitrite. Under identical condition, we monitored HBs.
the retention time of solutions including BHBT, BHBT-o-B and dynamic light scatting (DLS) spectra (Fig. S14-15). The OH groups on
BHBT-o-B with addition of peroxynitrite, respectively (Fig. S9-10). BHBT and 2-formylphenol act as intra- and intermolecular donors of
The solution of BHBT-o-B treated with peroxynitrite has two peaks HB to formyl groups. Complementary to other interactions such as
with retention time of 6.2 and 3.9 min, which were assigned to the C(Ar)−H···O and C(π)···C(π) interactions, two-dimensional
3
0
BHBT and 2-formylphenol, respectively, confirming that 2- arrangement formed (Fig. S16). The fluorescence change of BHBT
formylbenzeboronic acid was oxidized into 2-formylphenol.
induced by 2-formylphenol is like to that of BHBT in
cetyltrimethylammonium bromide (CTAB) micelles (Fig. S17).
Rigidity of BHBT existing in netlike assembly would increase and
nonradiative decay reduce, leading to the enhancement of
fluorescence.
2
1
1
0
0
.0
.5
.0
.5
.0
a)
b)
c)
d)
-
-
-
-




O
H
1
O 2
l
O
H 2O 2 NO
O 2
O 3
O 2
O
O
C
N
N
N
O
Fig. 2 The relative fluorescence intensity change of BHBT (10 μM) at 490 nm upon
addition of various ROS/RNS. Inset: Photographs of BHBT (a), BHBT in the presence of
-
-
o-B (b), and sequential addition of ClO (c) and ONOO (d), respectively.
In reported assay based on IDA, original indicator was released
after addition of targeted analytes, producing spectral recovery of
1
6-19
indicator for the detection of targets.
In the system of self-
assembled BHBT-o-B, BHBT and 2-formylphenol would be released
after addition of peroxynitrite. Interestingly, not the fluorescence
recovery but increased fluorescence of BHBT was observed. To seek
the underlying reason, we investigate the effect of corresponding
product, 2-formylphenol, on the fluorescence spectra of BHBT. The
fluorescence intensity of BHBT increases with continuous addition
of 2-formylphenol (Fig. S11). HPLC spectra revealed that there is no
reaction between BHBT and 2-formylphenol (Fig. S9b). It is assumed
that there is intermolecular hydrogen bonds (HBs) that induces the
fluorescence enhancement of BHBT. But for 3-, 4-formylphenol,
catechol resorcinol or hydroquinone, they did not induce any
fluorescence change of BHBT (Fig. S12). Therefore, the fluorescence
increasing from HBs should include the synergistic participation of
2
9
Fig. 3 Confocal fluorescence images of liver cancer cells (HepG2 cells). (a) blank of BHBT
ortho-aldehyde group. The distinctive resonance effect of π-
(
2
10 µM); (b) BHBT with 200 µM 2-aldehydephenylboronic acid; (c) BHBT with 200 µM
-aldehyde phenylboronic acid and subsequent treatment of 200 µM ClO ; (d) BHBT
electrons in 2-formylphenol strengths HBs for additional
-
3
0
stabilization of BHBT.
Product (2-formylphenol)-boosted
with 200 µM 2-aldehydephenylboronic acid and subsequent treatment of 200 µM
-
fluorescence signal significantly improves the sensitivity for ONOO .
-
ONOO . Images were acquired by using excitation wavelength of λex= 405 nm. Scare
2
-formylphenol (salicylaldehyde) is not only a kind of perfume, but bar: 20 µm.
also an important organic synthesis intermediate with wide
application. BHBT alone can selectively recognize salicylaldehyde
with turn-on fluorescence response.
In order to study the self-assembled process of BHBT and 2-
formylphenylboric acid, and after treatment with peroxynitrite, the
morphological changes of the assemblies were studied by field
emission scanning electron microscopy (FESEM). As shown in Fig.
3
1
In order to further study the biological applications of self-
assemblies, we performed fluorescence microscopic imaging
experiments on hepatoma cells (HepG2). As shown in Fig. 3, HepG2
0
cells were incubated with BHBT at 37 C for 30 minutes, no obvious
fluorescence was found. The cells were further incubated with 200
μM 2-formylphenylboric acid for 20 minutes, no fluorescence
enhancement was observed. When the cells were incubated for 30
S13, BHBT has
a dispersed needle-like structure. As 2-
-
minutes with 200 ONOO , strong blue fluorescence was observed.
formylphenylboric acid was added, cross-linking structure was
observed, indicating that BHBT and 2-formylphenylboric acid were
assembled to form boranic ester. The addition of peroxynitrite
The results showed that BHBT had good membrane permeability,
-
and BHBT-o-B self-assembly could be used to detect ONOO in living
cells. The ability of BHBT-o-B to image endogenous and exogenous
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J. Name., 2013, 00, 1-3 | 3
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Journal Name
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7 M. Weber, A. B. Mackenzie, S. D. Bull and T. D.ViJeawmAretiscl,e AOnnlainle.
ONOO in live cells were evaluated in a number of different cell lines
Chem., 2018, 90, 10621-10627.
DOI: 10.1039/D0CC02963G
(
HepG2, HL7702 and HeLa). These cells were pre-treated with
lipopolysaccharide (LPS), in which ONOO was stimulated
intracellularly. As shown in Fig. S18-20, a turn-on fluorescence
response of BHBT-o-B in these cells was found. In contrast, no ‘‘turn
on’’ response was observed when the cells were pre-treated with
the ONOO scavenger TEMPO (superoxide scavenger) and amino
guanidine (nitric oxide synthase inhibitor) (Fig. S21). These results
suggested that BHBT-o-B self-assembly has the capability of imaging
endogenous and exogenous ONOO in live cells. To identify the
8
9
1
1
1
1
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1
34, 18479-18482.
-
ONOO with different organelle trackers were performed (Fig.
1
4 T. Peng, N. K. Wong, X. M. Chen, Y. K. Chan, D. H. H. Ho, Z. N.
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S22). The commercial Mitochondrial tracker Rhodamine 123, ER
tracker ER-Tracker™ Red and Lysosomal tracker LysoTracker® Green
DND-26 were used in A549 cells. The Peason’s correction
coefficient between BHBT-o-B and these organelle trackers are
1
1
1
5 L. Wu, Y. Wang, M. Weber, L. Liu, A. C. Sedgwick, S. D. Bull, C.
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Qian, E. V. Anslyn, S. D. Bull and T. D. James, Chem. Sci., 2015, 6,
0
.21, 0.94 and 0.56, respectively. These results indicated that
-
the response of BHBT-o-B to ONOO was in the endoplasmic
reticulum organelle of living cells. The cytotoxicity of BHBT-o-B
self-assembly or BHBT with salicylaldehyde is tested by MTT assay.
As shown in Fig. S23, the survival rate of cells cultured with the
concentration for imaging is over 80%, showing very low
cytotoxicity for biological application.
In conclusion, we have reported new approach for detection
of ONOO with product-boosted fluorescence. In the process of
sensing, the self-assembly experiences “integrated”,
released” and “recombined” processes
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This work was supported by the National Natural Science
Foundation of China (Grant No. 21676218, 21978241 and
2
2
1
1878249) and Shaanxi Province Science and Technology (2019JM-
73 and 2018JM2008).
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Conflicts of interest
There are no conflicts to declare.
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Graphic abstract