Novel Dimethylhydrazine-Derived Spirolactam Fluorescent Chemodosimeter for Tracing Basal Peroxynitrite in Live Cells and Zebrafish

Article abstract of DOI:10.1021/acs.jafc.9b01298

The precise cellular function of peroxynitrite (ONOO^-) in biosystems remains elusive, primarily owing to being short of ultrasensitive techniques for monitoring its intracellular distribution. In this work, a novel rhodamine B cyclic 1,2-dimeth

Full text of DOI:10.1021/acs.jafc.9b01298

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New Analytical Methods
A novel dimethylhydrazine-derived spirolactam fluorescent
chemodosimeter for tracing basal ONOO- in live cells and zebrafish
Caiyun Liu, Xue Zhang, Zilu Li, Yanan Chen, Zihan Zhuang, Pan
Jia, Hanchuang Zhu, Yamin Yu, Baocun Zhu, and Wenlong Sheng
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01298 • Publication Date (Web): 14 May 2019
Downloaded from http://pubs.acs.org on May 15, 2019
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A novel dimethylhydrazine-derived spirolactam fluorescent
−
chemodosimeter for tracing basal ONOO in live cells and zebrafish
†
,
†
†
†
†
†
Caiyun Liu, * Xue Zhang, Zilu Li, Yanan Chen, Zihan Zhuang, Pan Jia,
†
†
†,
‡,
Hanchuang Zhu, Yamin Yu, Baocun Zhu, * and Wenlong Sheng *
†
School of Water Conservancy and Environment, University of Jinan, Jinan 250022,
China.
‡
Biology Institute, Qilu University of Technology (Shandong Academy of Sciences),
Jinan 250103, China.
*
Corresponding author. Fax: +86-531-82767617; Tel.: +86-531-82767617
E-mail address: liucaiyun1982072@163.com (C. Liu), lcyzbc@163.com (B. Zhu) and
5618694162@163.com (W. Sheng)
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ABSTRACT
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The precise cellular function of peroxynitrite (ONOO ) in biosystems remains
elusive, primarily owing to be short of ultrasensitive techniques for monitoring its
intracellular distribution. In this work,
a
novel rhodamine
B
cyclic
1,2-dimethylhydrazine fluorescent chemodosimeter RDMH-PN for highly specific
−
and ultrasensitive monitoring of basal ONOO in biosystems was rationally designed.
The fluorescence titration experiments demonstrated that RDMH-PN was capable of
quantitatively detecting 0-100 nM ONOO− (LOD = 0.68 nM). In addition,
RDMH-PN has the outstanding performances of ultrafast measurement, naked-eye
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detection, and preeminent selectivity toward ONOO to accurately detect intracellular
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basal ONOO . Finally, it has been confirmed that RDMH-PN not only could map the
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intracellular basal ONOO level by inhibit tests, but also could trace the fluctuations
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of endogenous and exogenous ONOO levels with diverse stimulations in live cells
and zebrafish.
KEYWORDS:
fluorescent
chemodosimeter;
peroxynitrite
(ONOO−);
1,2-dimethylhydrazine; rhodamine; fluorescence bioimaging
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INTRODUCTION
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Peroxynitrite (ONOO ), an important reactive oxygen species (ROS), is formed
•
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by a diffusion-limited coupling of NO and O
2
at the rate of 50-100 μM per min in
biological systems, and can be stable and last for hours in the nanomolar
concentration range.1-3 Peroxynitrite plays crucial roles in signal transduction and
antibacterial activities.4,5 However, growing evidence has suggested that excessively
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produced ONOO can damage various biomacromolecules because of its powerful
oxidative and nitrative capbility.6,7 An increasing number of diseases have been
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proved to be closely associated with ONOO , such as neurodegenerative disease,
chronic inflammation, and cardiovascular disorders.8-11 Unfortunately, the exact
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cellular mechanisms of ONOO are still not completely disclosed because of being
short of ultrasensitive techniques for monitoring its intracellular distribution.
Therefore, developing accurate and ultrasensitive methods for visualizing the ultralow
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concentration ONOO in the complex biosystems is urgently needed.
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The expanding emergence of ONOO fluorescent chemodosimeters shows their
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extraordinary superiority in measuring exogenous and endogenous ONOO in
biosystems.12-36 It is because that fluorescent chemodosimeters not only hold unique
imaging features of high spatiotemporal resolution, in situ detection, and
non-invasiveness, but also possess excellent selectivity because they are mainly
designed based on their specific reaction with target analytes.37-42 Up to now, available
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ONOO fluorescent chemodosimeters are mainly constructed based on different
reactive recognition moieties, including N-phenylrhodol,26-27 boronic acid pinacol
ester,30-34 electron-poor C=C double bond,43-49 ketoamide,50-52 hydrazine,53-58
organoselenium,59 and organotellurium.60 However, almost all fluorescent
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chemodosimeters for tracing intracellular ONOO suffer from the interferences of
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other ROS (e.g. H
2
O
2
and OCl ) due to their similar properties.
On the other hand,
most of available fluorescent chemodosimeters only detect enhanced concentration
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intracellular ONOO because of their low sensitivity and high reactivity, short
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half-life, ultralow basal concentration of ONOO . Therefore, developing specific
fluorescent chemodosimeters for accurately tracing intracellular basal ONOO−
without the interferences of other ROS remains a great challenge.
In this work,
a
simple fluorescent chemodosimeter employing
1,2-dimethylhydrazine as novel reactive recognition receptor was prepared for highly
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specific and sensitive detection of ONOO . Experimental results demonstrated that
the as-synthesized chemodosimeter not only hold prominent specificity for ONOO−
than other relevant species including ROS, but also can accurately detect nanomolar
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concentration ONOO with ultrafast response speed (< 3 s). The satisfactory
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biocompatibility enables it to trace intracellular basal ONOO and the fluctuations of
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exogenous/endogenous ONOO levels with various stimulations in live cells and
zebrafish. More importantly, the dimethylhydrazine-derived spirolactam has been
proven to be a unique model for constructing highly specific and ultrasensitive
chemodosimeters for ONOO−.
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EXPERIMENTAL SECTION
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General information. The chemicals and instrumentations are displayed in
Supporting Information. The fluorescence and absorption spectra were determined 1
minute after various species addition at 25 °C except for kinetics experiments. The
stock solution of chemodosimeter RDMH-PN was prepared in DMSO and the
adopted analytical condition is the PBS solution containing 5% DMSO (20 mM, pH =
7.4).
Preparation of chemodosimeter RDMH-PN. Phosphorus oxychloride (459 mg,
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3 mmol) were dissolved in absolute 1,2-dichloroethane (10 mL). Then, rhodamine B
(479 mg, 1 mmol) was introduced into the above mixture and refluxed for 4 hours.
After removing the solvent, the solution of 1,2-dimethylhydrazine dihydrochloride
(133 mg, 1 mmol) and DIPEA (1.349g, 10 mmol) in absolute CH
added slowly to the above residue. The resulted mixture was further reacted at 25 ˚C
for another 12 hours. After removing CH Cl , the crude products were refined through
Cl
2 2
(15 mL) was
2
2
silica gel chromatography to afford chemodosimter RDMH-PN (296 mg, 61%).
1H-NMR (400 MHz, CDCl
3
) δ (ppm): 1.16(t, J = 7.2 Hz, 12H), 2.31(s, 3H), 2.79(s,
3H), 3.28-3.42(m, 8H), 6.29(dd, J = 2.8, 8.8 Hz, 2H), 6.51(d, J = 2.4 Hz, 2H), 6.71(d,
J = 8.4 Hz, 2H), 7.30(d, J = 6.4 Hz, 1H), 7.48-7.56(m, 2H), 8.25(dd, J = 1.6, 7.2 Hz,
1
3
1H); C-NMR (100 MHz, CDCl ) δ (ppm): 12.58, 34.60, 38.86, 44.42, 63.33, 99.10,
3
106.27, 110.03, 127.37, 127.69, 129.03, 130.09, 130.29, 132.20, 137.95, 148.55,
+
154.21, 164.10. HRMS (ESI): Calcd for C30
485.2913.
H
37
N
4
O
2
[M+H] 485.2911; Found,
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RESULTS AND DISCUSSION
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Design of chemodosimeter RDMH-PN. An excellent fluorescent
chemodosimeter is the perfect combination of eminent fluorophore and unique
reactive recognition receptor. Xanthenes have been extensively applied in exploring
fluorescent chemodosimeters for various target analytes owing to the outstanding
photophysical properties including high fluorescence quantum yield, negligible
cytotoxicity, and the unique easy-regulation of ring-opening of the corresponding
non-fluorescent spirolactam.67-69 Based on the above-mentioned description, screening
appropriate reactive recognition unit is essential for constructing a specific and
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ultrasensitive ONOO fluorescent chemodosimeters. Recently, a large number of
hydrazide-based fluorescent chemodosimeters have been successfully constructed for
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identifying ONOO−.53-58 But the selectivity and reaction activity of those
chemodosimeters toward ONOO− are closely associated with the adopted
fluorophores,56,66 and especially the hydrazide-containing spirolactams are prone to be
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+ 67-69
hydrolyzed with the catalysis of Cu .
Therefore, to further optimize the response
properties of such chemodosimeters, a novel fluorescent chemodosimeter for ONOO−
detection was proposed by replacing hydrazide moiety with 1,2-dimethylhydrazine
recognition group. Namely, the newly designed chemodosimeter was composed of
rhodamine B dye as fluorophore and 1,2-dimethylhydrazine as the reactive unit
(RDMH-PN, Scheme 1 and 2). The 1,2-dimethylhydrazine group was integrated into
fluorophore, which formed spirocyclic ring system to reduce the fluorescence. Upon
−
the addition of ONOO , chemodosimeter RDMH-PN causes the ring-opening
reaction, and leads to the recovery of fluorescence.61-65 The design and reaction
mechanism has been also verified by HPLC and HRMS (Fig. S1-S2).
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Scheme 1. Recognition mechanism of chemodosimter RDMH-PN for ONOO−
1
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detection.
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Scheme 2. Preparation of chemodosimter RDMH-PN.
−
Determination of ONOO . Firstly, fluorescence spectra of RDMH-PN with
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19
ONOO were obtained in the PBS solution (20 mM, pH 7.4, 5% DMSO). As depicted
in Figure 1a, chemodosimeter RDMH-PN solution emitted negligible fluorescence.
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As designed, the emission peak was enhanced gradually with ONOO (0-10 μM).
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Satisfactorily, increasing ONOO concentrations (1-10 μM) manifest a linear
enhancement of the RDMH-PN fluorescence intensity (linear equation: y = 286942 ×
−
2
[ONOO ] (μM) + 235185, R = 0.9907) (Figure 1b). Furthermore, to estimate the
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capability of RDMH-PN to detect extremely low concentration ONOO , the
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additional titration of ONOO in the range of ultralow concentration was implemented.
Excitedly, the favorable linear dependence between fluorescence intensities and
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ONOO concentrations ranging from 0-100 nM was also obtained, demonstrating
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chemodosimeter RDMH-PN could accurately determine ONOO at the nanomolar
level (Figure 1c). Subsequently, the limit of detection was determined to be 0.68 nM
(3σ/k). Furthermore, the ultrafast reaction (< 3 s) between RDMH-PN and 1 equiv.
−
ONOO has been confirmed in Figure 2. These spectral studies revealed that
chemodosimeter RDMH-PN has powerful capability to implement the real-time
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determination of highly reactive ONOO with excellent sensitivity (Table S1).
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Figure 1. (a) Concentration-dependent fluorescent spectra of RDMH-PN (5 μM) with
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different concentration ONOO (0-10 μM) under the PBS solution (20 mM, pH = 7.4,
5% DMSO). (b) The linear dependence between fluorescence intensities and ONOO−
concentrations (1-10 μM). (c) The linear dependence between fluorescence intensities
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and ONOO concentrations (0-100 nM). λex = 520 nm. Slit widths: Wex = Wem = 3 nm.
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The spectra were recorded in 1 min after ONOO injection at 25 °C .
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Figure 2. Time-course of RDMH-PN (5 μM) with ONOO (5 μM) under the PBS
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solution (20 mM, pH = 7.4, 5% DMSO). λex = 520 nm, λem = 585 nm. Slit widths: Wex
= Wem = 3 nm. This experiment was performed at 25 °C .
The absorption spectrum of chemodosimter RDMH-PN for determining ONOO−
was also estimated in the PBS solution (20 mM, pH 7.4, 5% DMSO). Chemodosimter
RDMH-PN (5 μM) displayed negligible absorption intensity before reacting with
−
−
ONOO . As predicted, the reaction between chemodosimter RDMH-PN and ONOO
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led to clear enhancement of absorption intensity at 570 nm. Color change of
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RDMH-PN could be distinctly observed with and without ONOO , which was
beneficial to naked-eye observation.
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Figure 3. Absorption spectra of RDMH-PN (5 μM) with/without ONOO (5 μM)
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under the PBS solution (20 mM, pH = 7.4, 5% DMSO). Inset: photos of the
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RDMH-PN (20 μM) solution with/without ONOO (20 μM). The spectra were
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recorded in 1 min after ONOO injection at 25 °C .
These results manifested the usability of chemodosimter RDMH-PN as an
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ultrafast quantitative tool for monitoring ONOO at ultralow concentration level
under physiological conditions. The above-mentioned recognition properties of
RDMH-PN make it convenient to trace intracellular basal ONOO−.
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Specificity of chemodosimeter RDMH-PN toward ONOO . The specificity of
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RDMH-PN has been verified in the presence of ONOO and other relevant analytes
+
+
2+
2+
2+
+
2+
3+
2+
−
2−, CO
2−, NO −
,
including K , Na , Ca , Mg , Cu , Cu , Fe , Fe , Zn , Br , SO
3
3
3
•
t
•
−, 1O
−
−
NO
2
−, NO, TBHP, H
2
O
2
, O Bu, OH, O
2
2
, Cys, Hcy, GSH, OCl , and ONOO .
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As shown in Figure 4, only ONOO induced a significant increment of fluorescence
intensity at 585 nm, and other analytes could not cause the obvious changes of
fluorescence intensity. Subsequently, the possible interferences of pH on
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chemodosimeter RDMH-PN for monitoring ONOO were also studied. These results
manifested that chemodosimeter RDMH-PN possessed a satisfactory stability and
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response capability for ONOO in the physiological pH ranges (Fig. S3). Moreover,
the eminent photo-stability of chemodosimeter RDMH-PN was further proven (Fig.
S4). The above consequences implied chemodosimeter RDMH-PN could accurately
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trace intracellular ONOO in the bioimaging applications.
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Figure 4. Fluorescence changes of RDMH-PN (5 μM) with various species (100 μM
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except for annotations) under the PBS solution (20 mM, pH = 7.4, 5% DMSO). 1.
+
+
2+
2+
2+
Blank, 2. K (1 mM), 3. Na (1 mM), 4. Ca (1 mM), 5. Mg (1 mM), 6. Cu , 7.
+
2+
3+
2+
−
2−, 13. CO
2−, 14. NO −
, 15. NO −
Cu , 8. Fe , 9. Fe , 10. Zn , 11. Br , 12. SO
3
3
3
2
, 16.
•
t
•
−
1
NO, 17. TBHP, 18. H
2
O
2
(500 μM), 19. O Bu, 20. OH, 21. O
2
, 22. O
2
, 23. Cys (500
−
−
μM), 24. Hcy (500 μM), 25. GSH (1 mM), 26. OCl , and 27. ONOO (5 μM). λex
=
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520 nm, λem = 585 nm. Slit widths: Wex = Wem = 3 nm. The spectra were recorded in 1
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min after analytes injection at 25 °C .
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Bioimaging of ONOO in live cells. To study the recognition properties of
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chemodosimeter RDMH-PN for ONOO in complex living systems, we conducted
biological imaging experiments with RAW 264.7 macrophages. Initially, we checked
the cytotoxicity of chemodosimeter RDMH-PN by a cell counting kit-8 (CCK-8)
method. These results displayed chemodosimeter RDMH-PN had negligible effect on
cell survival (Figure 5). Then, we followed to test whether chemodosimeter
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RDMH-PN has capability to trace intracellular basal ONOO . Firstly, the bioimaging
of control cells was carried out, and almost no intracellular fluorescence was observed
(Figure 6a-c). As predicted, the macrophages preincubated with chemodosimeter
RDMH-PN for 30 minutes exhibited the obvious red intracellular fluorescence under
the same bioimaging conditions (Figure 6d-f). Additionally, the macrophages
pretreated with 4-amino-tempo (a superoxide scavenger, it causes the reduction of
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intracellular ONOO levels) was further treated with chemodosimeter RDMH-PN for
30 minutes, and the weaker red intracellular fluorescence than the control
macrophages incubated with only RDMH-PN was observed (Fig. S5). These data
−
demonstrated chemodosimeter RDMH-PN was quite sensitive to basal ONOO in
normal macrophages without extrinsic stimuli. Subsequently, the macrophages
preincubated with chemodosimeter RDMH-PN for 30 minutes were further treated
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with ONOO (20 µM) for another 20 minutes, and the stronger intracellular
fluorescence was successfully obtained (Figure 6g-i), demonstrating chemodosimeter
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RDMH-PN could determine the changes of exogenous ONOO concentrations. To
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estimate the capability of RDMH-PN in detecting endogenous ONOO , the
macrophages were stimulated by PMA (1.0 µg/mL) and LPS (1.0 µg/mL) for 30
minutes. With extending incubation with RDMH-PN for another 20 minutes, a
significant increase of intracellular fluorescence has been observed (Figure 6j-l).
Above results demonstrated that chemodosimeter RDMH-PN could map the basal
−
−
ONOO and the changes of endogenous/exogenous ONOO concentrations in live
macrophages effectively (Figure 6m).
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Figure 5. Macrophage viability with different concentration chemodosimeter
RDMH-PN.
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Figure 6. Fluorescence images of live macrophages: (a-c) control cells; (d-f) cells
treated with RDMH-PN; (g-i) cells preincubated with RDMH-PN, and then treated
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with ONOO ; (j-l) cells preincubated with LPS and PMA, and then incubated
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RDMH-PN; (m) the fluorescence intensities of the corresponding cells.
Bioimaging of ONOO− in zebrafish. With the favorable bioimaging
performance of RDMH-PN in live cells, we then examined whether it has the
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possibility to bioimage ONOO in vivo. Based on this consideration, the 4-day-old
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zebrafish was chosen as a suitable organism model to visualize basal ONOO and the
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changes of endogenous/exogenous ONOO concentrations in our experiments (Figure
7). Firstly, the bioimaging of control zebrafish was carried out, and almost no
fluorescence was observed (Figure 7a-c). As designed, the zebrafish preincubated
with chemodosimeter RDMH-PN for 30 minutes exhibited the obvious red
fluorescence under the same bioimaging conditions (Figure 7d-f). These results
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indicated chemodosimeter RDMH-PN was very sensitive to basal ONOO in normal
zebrafish without extrinsic stimuli. Subsequently, the zebrafish preincubated with
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chemodosimeter RDMH-PN for 30 minutes was further treated with ONOO (50 µM)
for another 20 minutes, and the stronger red fluorescence was also successfully
obtained (Figure 7g-i), demonstrating chemodosimeter RDMH-PN could monitor the
−
changes of exogenous ONOO levels in vivo. Moreover, to estimate the capability of
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RDMH-PN in detecting endogenous ONOO in vivo, the zebrafish were stimulated
by PMA (1.0 µg/mL) and LPS (1.0 µg/mL) for 30 minutes. With extending incubation
with RDMH-PN for another 20 minutes, the significant increase of fluorescence also
confirmed the bioimaging applicability in vivo (Figure 7j-l). Taking all these results in
consideration, it can be concluded that chemodosimeter RDMH-PN was able to
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accurately monitor intracellular basal ONOO− and the changes of
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endogenous/exogenous ONOO concentrations in zebrafish.
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Figure 7. Fluorescence images of zebrafish: (a-c) control zebrafish; (d-f) zebrafish
treated with RDMH-PN; (g-i) zebrafish preincubated with RDMH-PN, and then
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treated with ONOO ; (j-l) zebrafish preincubated with LPS and PMA, and then
incubated RDMH-PN.
In conclusion, we have constructed a novel fluorescent chemodosimeter for
tracing intracellular basal ONOO− in biosystems. The high specificity and
ultrasensitivity of chemodosimeter RDMH-PN was ascribed to the adoption of
reactive recognition group of 1,2-dimethylhydrazine. Bioimaging applications of
−
chemodosimeter RDMH-PN in tracing intracellular basal ONOO and the changes of
−
ONOO concentrations with different stimulations in live cells and zebrafish were
confirmed. Therefore, we anticipate that chemodosimeter RDMH-PN may be used as
−
a powerful tool to explore the generation and transport of ONOO in biosystems.
Moreover, the 1,2-dimethylhydrazine modified spirocyclic structure has been proven
to be a unique model for constructing highly specific and ultrasensitive
−
chemodosimeters for ONOO detection.
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*Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI: 10.1021/acs.jafc.×××××××.
Experimental methods and additional data.
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The authors declare no competing financial interest.
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65 ACKNOWLEDGEMENTS
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This study was financially supported by NSFC (21777053 and 21607053) and
Shandong Provincial Natural Science Foundation (ZR2017MB014).
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