Long-wavelength fluorescent boronate probes for the detection and intracellular imaging of peroxynitrite

Article abstract of DOI:10.1039/c7cc07845e

Two boronate fluorescent probes have been developed for the detection of peroxynitrite (TCFB1 and TCFB2). TCFB1 was shown to have a low sensitvity towards peroxynitrite and have a poor solubility in aqueous solution whereas TCFB2 demonstrated high sensitivity towards peroxynitrite and mitochondria localisation with the ability to detect exogenous and endogenous peroxynitrite in live cells (Hep-G2, RAW 264.7, HeLa and A459).

Full text of DOI:10.1039/c7cc07845e

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: A. C. Sedgwick, H.
Han, J. E. Gardiner, S. Bull, X. He and T. D. James, Chem. Commun., 2017, DOI: 10.1039/C7CC07845E.
Volume 52 Number
1
4
January 2016 Pages 1–216
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
ChemComm
Chemical Communications
www.rsc.org/chemcomm
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc
first methanofullerene derivatives of Sc
3
N@C2n (2n
=
68 and 80). Synthesis of the
3
N@D5h-C80
rsc.li/chemcomm

Please do not adjust margins
ChemComm
Page 1 of 4
View Article Online
DOI: 10.1039/C7CC07845E
Journal Name
COMMUNICATION
Long-wavelength fluorescent boronate probes for the detection
and intracellular imaging of peroxynitrite
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
a
a
b
Adam C. Sedgwick,* Hai-Hao Han, Jordan E. Gardiner, Steven D. Bull,* Xiao-Peng He,* and
a
Tony D. James*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Two boronate fluorescent probes have been developed for the near infrared (NIR) probes is of particular interest because
detection of peroxynitrite (TCFB1 and TCFB2). TCFB1 was shown to longer excitation/ emission wavelengths allows deeper tissue
have a low sensitvity towards peroxynitrite and have a poor penetration and minimalises background auto-fluorescence
1
4-15
solubility in aqueous solution whereas TCFB2 demonstrated high from proteins and photodamage to the biological samples.
sensitivity towards peroxynitrite and mitochondria localisation In the literature, Sikora et al. reported that the reaction rates
-
with the ability to detect exogenous and endogenous of ONOO with aromatic boronates are 200 times faster than
-
hypochlorous acid (HOCl/ClO ) and a million times faster than
peroxynitrite in live cells (Hep-G2, RAW 264.7, HeLa and A459).
1
6
hydrogen peroxide (H
2 2
O ). Therefore, a number of boronate
-
Peroxynitrite (ONOO ), is a highly reactive nitrogen species that
fluorescent probes have been recently developed for the
-
8, 17-18
is formed via the diffusion controlled reaction between
detection of ONOO .
2-Dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran
TCF)-based fluorophores have an internal charge transfer (ICT)
-
superoxide ( O ) and nitric oxide (NO). ONOO acts as a
1-2
-
2
1, 3
signalling molecule in vivo for a number of pathways
-
.
(
However, ONOO is more commonly known for its deleterious
properties, causing irreversible damage to a range of biological
donor-π-acceptor (D-π-A) structure with long emission
wavelengths. As a result, TCF fluorophores have been used in
many applications such as non-linear optic chromophores and
4
targets such as lipids, proteins and DNA. Therefore, ONOO
-
1
9-25
has been implicated as a key pathogenic factor for a number of
diseases, which include inflammation, cancer, ischemia-
molecular probes.
With this research, we developed two
boronate TCF-based fluorescent probes for the detection of
-
ONOO (TCFB1 and TCFB2). The TCF fluorophore unit was
5
reperfusion and neurodegenerative diseases. In biological
-7
-
systems ONOO is difficult to measure due to it being short-
synthesised in one step using the reaction of 3-hydroxy-3-
methyl-2-butanone, malonitrile and NaOEt in EtOH. With the
TCF unit in hand, the (D-π-A) systems TCFB1 and TCFB2 were
1
lived with a half-life ~10-20 ms. Therefore, making the
-
development of powerful tools for the detection of ONOO is of
2
6
significant interest.
isolated in high yield using microwave reaction conditions.
The microwave irradiation of a mixture of piperidine (Cat.), TCF
and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzaldehyde in EtOH followed by filtration led to the
isolation of the desired TCFB2. For the synthesis of TCFB1
With our research, we are particularly interested in the
development of small molecule fluorescent probes for the
detection of biologically relevant analytes in vivo owing to
their high sensitivity, selectivity and high spatial and temporal
,
-
resolution. In the past few years, a number of ONOO
microwave irradiation of a mixture of piperidine (Cat.), TCF
and 4-hydroxybenzaldhyde in EtOH followed by filtration led to
the isolation of the intermediate TCF-OH. This was
subsequently alkylated with 2-(4-(bromomethyl)phenyl)-
fluorescent probes have been developed for imaging in live
-13
8
cells and mice.
However, despite significant progress in this
-
area of research, there is a lack of long-wavelength ONOO
fluorescent probes. The development of long wavelength/
4,4,5,5-tetramethyl-1,3,2-dioxaborolane using K CO and NaI in
2 3
MeCN to afford TCFB1 in a reasonable yield (47 %).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 4
View Article Online
DOI: 10.1039/C7CC07845E
COMMUNICATION
Journal Name
140
120
100
-
ONOO
80
60
40
20
0
580
590
600
610
620
630
640
650
660
1
1
1
40
20
00
Wavelength/ nm
-
Fig. 2 - Fluorescence spectra of TCFB2 (10 µM) with addition of ONOO (0 – 10 µM) in
0
PBS buffer solution, 20 % DMSO, pH 8.00 at 25 C. λex = 560 nm. Slit widths ex = 10 nm
-
8
6
4
2
0
0
0
0
0
ONOO
and em = 15 nm
Having determined the selectivity of TCFB2, we evaluated its
-
ability to image endogenous and exogenous ONOO in live
cells. TCFB2 was evaluated in a number of different cell lines
5
90
600
610
620
630
640
650
660
(Hep-G2: human hepatoma, HeLa: human cervical cancer,
Wavelength/ nm
RAW264.7: mouse macrophage and A549 cells: human lung
cancer), which were incubated with TCFB2 (10 µM) for 30
minutes and washed with PBS buffer solution three times. As
shown in Fig. 3, TCFB2 demonstrated a clear “turn on”
-
Fig. 1 - Fluorescence spectra of TCFB1 (10 µM) with addition of ONOO (0 – 100 µM) in
o
PBS buffer solution, 20 % DMSO, pH 8.00 at 25 C. λex = 560 nm. Slit widths ex = 10 nm
and em = 15 nm
-
response with the addition of SIN-1 (ONOO donor). No “turn
We initially evaluated the UV-Vis (Fig. S2) and fluorescence
on” response was observed when the cells were pre-treated
-
with the ONOO scavenger uric acid. TCFB2 also provided a
behaviour (Fig. 1 and S3) of TCFB1, in pH 8.0 buffer solution
(
20 % DMSO). DMSO was required to improve the aqueous
clear “turn on” response for the detection of stimulated
solubility of TCFB1. Under these conditions, TCFB1 produced
-
ONOO . RAW 264.7 cells were used in which ONOO was
-
an up to 6.5-fold fluorescence “turn on” in the presence of
-
ONOO (0-100 µM). (Scheme S1, S2 and Fig. S1) However, in
29
stimulated using lipopolysaccharide (LPS). This led to the
activation of the TCFB2 fluorescence intracellularly (Fig. 4). In
contrast, no “turn on” response was observed in the presence
-
of uric acid indicating the selectivity for ONOO in cells. A cell
comparison to our previously reported ESIPT probe, TCFB1 was
-
less sensitive towards ONOO despite a larger “turn on”
8
response.
proliferation assay showed that the compound was not toxic
towards all the cell lines used with concentrations well above
that used for imaging (Fig. S13).
Subsequently, we evaluated the selectivity of TCFB1 towards
other ROS (Fig. S4, S5 and S11). TCFB1 demonstrated an
-
excellent selectivity for ONOO , which permitted the
The production of superoxide occurs mainly through the
30
mitochondrial electron transport pathway; therefore the
-
mitochondria are the main source of ONOO in macrophages.
evaluation of its ability to detect exogenous and endogenous
-
ONOO in live cells. Unfortunately, due to its poor aqueous
solubility, large amounts of precipitate with TCFB1 was
observed (data not shown).
Commercial Mito-tracker Green was used to localise in the
mitochondrial compartments of RAW264.7. We then used
-
TCFB2 to investigate the subcellular distribution of ONOO . The
Therefore, we turned our attention towards the evaluation of
the UV-Vis and fluorescence properties of TCFB2, which has
results indicated that the fluorescence of the probe co-
localised with that of the tracker resulting in a Pearson
coefficient of 0.84 (Fig. 5). We have also carried out an
additional lysosome co-localisation assay, and the result
showed that the probe did not co-localise well with lysosome
-
20
previously been reported for the detection of ClO . As
previously reported for other aryl boronate fluorescent
27-28
probes,
TCFB2 was found to be initially non-fluorescent
with no UV absorption beyond ~525 nm (Fig. S6). The addition
-
of ONOO to TCFB2 resulted in the appearance of a large
(Pearson's correlation = 0.38) (Fig. S14). This suggests that
emission peak at 606 nm (Fig. 2 and S7). This was accompanied
by a colorimetric response (yellow to pink) and an appearance
peroxynitrite was produced at the mitochondria.
of
a large UV absorption peak at ~590 nm. TCFB2
demonstrated high sensitivity and rapid reaction (Fig. S8) with
-
ONOO and was able to detect very low concentrations (0-10
-
µM). As predicted, both ClO and H
2
O
2
also resulted in a
fluorescence response (Fig. S9, S10, and S12), however, larger
concentrations and reaction times were required. These
observations clearly, demonstrated the greater reactivity of
-
the boronate towards ONOO .
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 4
Please dCo h ne omt Ca do j um s mt margins
View Article Online
DOI: 10.1039/C7CC07845E
Journal Name
COMMUNICATION
Fig. 5. (a) Fluorescence co-localisation of TCFB2 (10 μM) with Mito-Tracker Green (1
μM) in RAW264.7 cells (scale bar = 20 μm). (b) Fluorescence quantification of TCFB2
Fig. 3. (a) Fluorescence imaging (scale bar = 100 μm) (b) quantification of different cells
incubated with TCFB2 (10 µM) without (-/-) or with a subsequent addition of Sin-1 (500
µM, a peroxynitrite promoter) (+/-) or a subsequent addition of and uric acid (100 µM,
a peroxynitrite quencher) and then Sin-1 (+/+). Excitation and emission wavelengths for
TCFB2 are 560-580 nm and 580-650 nm, respectively. The cell nuclei were stained by
Hoechst 33342.
and Mito-Tracker of
a selected section (the black line in “Merged” panel) of a
RAW264.7 cell. Excitation wavelength for Mito-Tracker Green and TCFB2 is 489 and 579
nm, respectively. Emission wavelength for Mito-Tracker Green and TCFB2 is 506 and
603 nm, respectively. The cell nuclei were stained by Hoechst 33342.
In conclusion, we have developed two long-wavelength
-
reaction based fluorescent probes for the detection of ONOO .
Unfortunately, TCFB1 had a low solubility in aqueous solution,
which led to the observation of precipitates in cell imaging
experiments. However, TCFB2 displayed selective and sensitive
-
turn on” with the addition of ONOO . The large fluorescence
“
response observed for TCFB2 facilitated its use in cell imaging
experiments. Therefore, TCFB2 was able to detect exogenous
-
and endogenous ONOO with a large fluorescence “turn on”
over a range of cell lines (Hep-G2, RAW 264.7, HeLa and A459)
.
Mitochondrial localisation of TCFB2 was observed by co-
localisation with Mito-Tracker Green. Overall, these results
demonstrate that TCFB2 is a useful tool to understand the role
-
of ONOO in biological systems and could lead to systems
3
1,32
capable of disease diagnosis. A glycosylation strategy
to
improve the water solubility of the insoluble TCFB1 is currently
Fig. 4. (a) Fluorescence imaging (scale bar = 100 μm) (b) quantification of RAW264.7 underway in our laboratories.
incubated with TCFB2 (10 µM) without (-/-) or with subsequent addition of
a
-
1
lipopolysaccharide (LPS, 1 μg mL ) (+/-) or a subsequent addition of both LPS and uric
acid (100 µM, a peroxynitrite quencher) (+/+). Excitation and emission wavelength for
TCFB2 are 560-580 nm and 580-650 nm, respectively. The cell nuclei were stained by
Hoechst 33342.
Acknowledgements
We would like to thank the EPSRC and the University of Bath for
funding. ACS and JEG thank the EPSRC for studentships. TDJ wishes
to thank the Royal Society for a Wolfson Research Merit Award.
NMR characterisation facilities were provided through the Chemical
Characterisation and Analysis Facility (CCAF) at the University of
Bath (www.bath.ac.uk/ccaf). The EPSRC UK National Mass
Spectrometry Facility at Swansea University is thanked for analyses.
X.-P. He thanks the National Natural Science Foundation of China
(
21722801 and 21572058), the Science and Technology Commission
of Shanghai Municipality (15540723800), the Fundamental
Research Funds for the Central Universities (222201717003) and
the Shanghai Rising-Star Program (16QA1401400) (to X.-P. He) for
financial support. The Catalysis And Sensing for our Environment
(
CASE) network is thanked for research exchange opportunities. TDJ
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 4
View Article Online
DOI: 10.1039/C7CC07845E
COMMUNICATION
Journal Name
thanks ECUST for a guest professorship. Professors Jia Li and Yi Zang 16 A. Sikora, J. Zielonka, M. Lopez, J. Joseph and B.
are thanked for their helpful advice on the cellular experiments.
Kalyanaraman, Free Radic. Biol. Med., 2009, 47, 1401.
7 X. Sun, Q. Xu, G. Kim, S. E. Flower, J. P. Lowe, J. Yoon, J. S.
Fossey, X. Qian, S. D. Bull and T. D. James, Chem. Sci., 2014,
1
1
5, 3368.
8 S. Palanisamy, P. Y. Wu, S. C. Wu, Y. J. Chen, S. C. Tzou, C.
H. Wang, C. Y. Chen and Y. M. Wang, Biosens. Bioelectron.,
Conflicts of interest
There are no conflicts to declare.
2
017, 91, 849.
1
2
2
9 Y. H. Yang, J. L. Liu, H. Y. Xiao, Z. Zhen and S. H. Bo, Dyes
Pigments, 2017, 139, 239.
0 W. Shu, L. G. Yan, Z. K. Wang, J. Liu, S. Zhang, C. Y. Liu and
B. C. Zhu, Sens Actuators B Chem., 2015, 221, 1130.
1 Y. J. Wang, Y. Shi, Z. Y. Wang, Z. F. Zhu, X. Y. Zhao, H. Nie, J.
Qian, A. J. Qin, J. Z. Sun and B. Z. Tang, Chem. Eur. J., 2016,
Notes and references
1
P. Pacher, J. S. Beckman and L. Liaudet, Physiol. Rev., 2007,
, 315.
87
2
J. S. Beckman and W. H. Koppenol, Am. J. Physiol. Cell
Physiol., 1996, 271, C1424.
22, 9784.
2 Y. R. Wang, L. Feng, L. Xu, Y. Li, D. D. Wang, J. Hou, K. Zhou,
2
3
4
A. Weidinger and A. V. Kozlov, Biomolecules, 2015,
P. Ascenzi, A. di Masi, C. Sciorati and E. Clementi,
Biofactors, 2010, 36, 264.
5, 472.
Q. Jin, G. B. Ge, J. N. Cui and L. Yang, Chem. Commun.,
2
016, 52, 6064.
2
2
3 B. C. Zhu, H. Kan, J. K. Liu, H. G. Liu, Q. Wei and B. Du,
Biosens. Bioelectron., 2014, 52, 298.
4 C. Y. Li, M. Li, Y. Li, Z. S. Shi, Z. J. Li, X. B. Wang, J. Sun, J. W.
5
6
7
8
9
1
H. Ischiropoulos and J. S. Beckman, J. Clin. Invest., 2003,
11, 163.
P. Sarchielli, F. Galli, A. Floridi and V. Gallai, Amino Acids,
003, 25, 427.
D. A. Wink, Y. Vodovotz, J. Laval, F. Laval, M. W. Dewhirst
and J. B. Mitchell, Carcinogenesis, 1998, 19, 711.
A. C. Sedgwick, X. L. Sun, G. Kim, J. Yoon, S. D. Bull and T. D.
James, Chem. Commun., 2016, 52, 12350.
Z. N. Sun, H. L. Wang, F. Q. Liu, Y. Chen, P. K. H. Tam and D.
Yang, Org. Let., 2009, 11, 1887.
0 X. Li, R.-R. Tao, L.-J. Hong, J. Cheng, Q. Jiang, Y.-M. Lu, M.-H.
Liao, W.-F. Ye, N.-N. Lu, F. Han, Y.-Z. Hu and Y.-H. Hu, J. Am.
Chem. Soc., 2015, 137, 12296.
1
Sun, D. M. Zhang and Z. C. Cui, J. Mater. Chem. C, 2016,
392.
5 T. Yu, G. X. Yin, P. Yin, Y. Zeng, H. T. Li, Y. Y. Zhang and S. Z.
Yao, RSC Adv., 2017, , 24822.
4,
2
8
2
2
2
2
2
3
7
6 M. Ipuy, C. Billon, G. Micouin, J. Samarut, C. Andraud and Y.
Bretonniere, Org. Biomol. Chem., 2014, 12, 3641.
7 E. W. Miller, A. E. Albers, A. Pralle, E. Y. Isacoff and C. J.
Chang, J. Am. Chem. Soc., 2005, 127, 16652.
8 B. C. Dickinson, C. Huynh and C. J. Chang, J. Am. Chem. Soc.,
2
010, 132, 5906.
9 A. Vazquez-Torres, J. Jones-Carson and E. Balish, Infect.
Immun., 1996, 64, 3127.
0 M. D. Brand, C. Affourtit, T. C. Esteves, K. Green, A. J.
Lambert, S. Miwa, J. L. Pakay and N. Parker, Free Radic.
Biol. Med., 2004, 37, 755.
1 X.-P. He, Y. Zang, T. D. James, J. Li, G.-R. Chen and J. Xie,
Chem. Commun., 2017, 53, 82.
2 J. Zhang, Y. Fu, H.-H. Han, Y. Zang, J. Li, X.-P. He, B. L.
1
1
1
1
1
1 F. B. A. Yu, P. Li, G. Y. Li, G. J. Zhao, T. S. Chu and K. L. Han,
J. Am. Chem. Soc., 2011, 133, 11030.
2 F. B. Yu, P. Li, B. S. Wang and K. L. Han, J. Am. Chem. Soc.,
2013, 135, 7674.
3 D. Cheng, Y. Pan, L. Wang, Z. B. Zeng, L. Yuan, X. B. Zhang
and Y. T. Chang, J. Am. Chem. Soc., 2017, 139, 285.
4 L. Yuan, W. Y. Lin, K. B. Zheng, L. W. He and W. M. Huang,
Chem. Soc. Rev., 2013, 42, 622.
3
3
Feringa and H. Tian, Nat. Commun., 2017, 8, 987.
5 R. Weissleder, Nat. Biotechnol., 2001, 19, 316.
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins