Rhodamine cyclic hydrazide as a fluorescent probe for the detection of hydroxyl radicals

Article abstract of DOI:10.1039/c3cc44627a

A new rhodamine fluorescent probe for monitoring OH has been developed based on the oxidative C-H abstraction reaction of rhodamine cyclic hydrazide. The probe exhibits excellent selectivity for OH with virtually no interference by other ROS/RNS species. Fluorescent imaging of A549 and RAW264.7 cells is also successfully demonstrated to detect intracellular OH in live cells.

Full text of DOI:10.1039/c3cc44627a

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Rhodamine cyclic hydrazide as a fluorescent probe for
the detection of hydroxyl radicals†
Cite this: Chem. Commun., 2013,
49, 7959
Minjeong Kim, Sung-Kyun Ko, Hyemi Kim, Injae Shin* and Jinsung Tae*
Received 20th June 2013,
Accepted 11th July 2013
DOI: 10.1039/c3cc44627a
www.rsc.org/chemcomm
A new rhodamine fluorescent probe for monitoring ꢀOH has conjugated with various fluorophores.6 And the oxidation reac-
been developed based on the oxidative C–H abstraction reaction tion of the hydrocyanine probes to the corresponding cyanine
of rhodamine cyclic hydrazide. The probe exhibits excellent selec- dyes by oxidative C–H hydrogen abstraction reaction has been
tivity for ꢀOH with virtually no interference by other ROS/RNS applied to detect OH and O2ꢁ.7 Alternatively, O–H hydrogen
ꢀ
ꢀ
species. Fluorescent imaging of A549 and RAW264.7 cells is also abstraction reaction by ꢀOH and ONO2ꢁ could trigger deprotection
successfully demonstrated to detect intracellular ꢀOH in live cells.
of 2-[6-(40-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid to
furnish the strongly fluorescent fluorescein molecule.4a Although
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) these known fluorescent probes for ꢀOH show relatively good
are associated with numerous pathological processes including selectivity, elimination of the interferences by other ROS is crucial
neurodegenerative disease, ischemic or traumatic brain injury, for precise cellular imaging.
cancer, diabetes, liver injury, and AIDS.1,2 Therefore, over the
Herein, we report a highly sensitive and selective fluorescent
ꢀ
past decade, a lot of fluorescent and luminescent detection rhodamine cyclic hydrazide probe for OH by employing C–H
methods have been developed for the detection of ROS/RNS, hydrogen abstraction reaction of hydrazine derivatives. The
such as hydroxyl radicals (ꢀOH), hydrogen peroxide (H2O2), ꢀOH-mediated C–H abstraction reaction of the pyrazolidine
superoxide radicals (ꢀO2ꢁ), hypochlorous acid (HOCl), singlet moiety of probe 1 is expected to proceed at the most electron rich
oxygen (1O2), nitric oxide (NOꢀ), and peroxynitrite (ONOOꢁ).1 3-position to generate the radical intermediate A. The spirocycle in
The hydroxyl radical (ꢀOH), one of the most deleterious intra- A will eventually open to form the strongly fluorescent compound
cellular ROS, can cause severe damage to essentially all bio- B as shown in Scheme 1.
molecules due to its high reactivity.3 Thus, the ability to detect
and quantify intra- and extracellular ꢀOH could provide key according to the literature procedure (1, NaOH, H2O–EtOH; 2,
information about the location and the extent of redox damage POCl3, CH2Cl2; 3, pyrazolidine, Et3N, CH2Cl2).9 Probe 1 forms a
Probe 1 8 was prepared from Rhodamine 6G in three steps
and cellular responses.
colorless solution in Tris–HCl buffer at pH 7.4 and shows
Recently, several fluorescent probes for the detection of ꢀOH negligible fluorescence, indicating that it exists in the spirocyclic
have been reported.4 Among them, the fluorescent probes form predominantly as expected. Upon treatment with 1.0 equiv.
based on the characteristic reactions of OH, such as aromatic of ꢀOH, probe 1 (10 mM) in Tris–HCl buffer (DMF 1% v/v)
ꢀ
addition, spin trap, and hydrogen abstraction, show relatively good solution shows strong fluorescence at 550 nm and pink-red
selectivity over other ROS. For example, the weakly fluorescent color in less than 2 minutes. This observation indicates that the
coumarin-3-carboxylic acid probes react with ꢀOH to produce ꢀOH-induced reaction of probe 1 takes place rapidly at room
the fluorescent 7-HO-coumarin-3-carboxylic acid derivatives temperature and opens the spirocyclic ring system. We
by aromatic addition reaction.5 The spin trapping method of attempted to detect the ring-opened product B, however, we
nitroxyl radical probes with ꢀOH in DMSO has also been were unable to isolate compound B. Thus, we then examined
developed, where the methyl radical (ꢀMe) generated by the the same ring-opening reaction with probe 2 which would
reaction of DMSO and ꢀOH traps the nitroxyl radicals which are yield the stable conjugated imine product 3 (Scheme 1).
Fortunately, we could fully characterize 3 to confirm the proposed
Department of Chemistry, Yonsei University, Seoul 120-749, Korea.
reaction mechanism, but the initial product 3 seemed to decom-
pose under the reaction conditions to give low isolated yield
E-mail: jstae@yonsei.ac.kr, injae@yonsei.ac.kr; Fax: +82 2-364-7050;
Tel: +82 2-2123-2603
(see ESI†).
† Electronic supplementary information (ESI) available: Experimental procedures
ꢀ
for the synthesis and copies of 1H NMR and 13C NMR of 1–3, data for UV-vis,
fluorescence titration of 1, and other data. See DOI: 10.1039/c3cc44627a
Fluorescence intensity changes of 1 upon additions of OH
(1.0 equiv.) and other ROS/RNS (5.0 equiv. of H2O2, ROOꢀ, NOꢀ,
c
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Fig. 2 Sensitivity of 1 towards ꢀOH. Fluorescence titration of 1 (10 mM) in Tris–HCl
(DMF 1% v/v) at 550 nm. (a) With increasing molar equiv. of ꢀOH. (b) With
increasing [H2O2] in Fenton’s reagent with fixed FeSO4 (10 mM).
in the presence of ꢀOH at pH 4–10 (see ESI†), therefore, we
then investigated biological imaging of intracellular hydroxyl
radicals.
Thus, we performed cell experiments to evaluate its potential
utility for fluorescent bioimaging of hydroxyl radicals. In this
study, A549 cells (human lung adenocarcinoma epithelial cells)
and RAW264.7 cells (murine macrophage-like cells) were incu-
bated with 20 mM probe 1 for 1 h. After being washed with PBS to
remove the remaining probe 1, the cells were further treated with
20 mM of Fenton’s reagent (FeSO4 : H2O2 = 1 : 10) to generate
hydroxyl radicals.11,4g Confocal microscopy analysis reveals that
the cells treated with Fenton’s reagent exhibit strong red fluores-
cence but the cells incubated with probe 1 alone display very
weak fluorescence (top panels in Fig. 3a and b). When cells
were exposed to the 20 mM probe and a membrane-permeable
radical scavenger (5 mM TEMPOL, 4-hydroxy-2,2,6,6-tetramethyl
piperidine-N-oxyl)12 prior to incubation with 20 mM of Fenton’s
reagent, remarkably attenuated fluorescence was observed in
the treated cells (bottom panels in Fig. 3a and b), revealing that
Scheme 1 Hydrogen abstraction of rhodamine probes by ꢀOH.
ꢀO2ꢁ, HOCl, ONOOꢁ, O2) in Tris–HCl buffer (DMF 1% v/v) at
1
pH 7.4 are shown in Fig. 1a and b. Only ꢀOH leads to a dramatic
enhancement in fluorescence intensity, but other ROS/RNS
species develop no significant fluorescence intensity changes.
In addition to the fluorescent changes, the colorless to pink-red
color changes associated with the reaction of probe 1 with ꢀOH
are readily detectable visually (Fig. 1c).
ꢀ
Then, we examined the ability of 1 to quantify OH in vitro.
When probe 1 was treated with ꢀOH generated by Fenton
reaction,10 the fluorescence intensity was linearly proportional
to the molar equivalents of ꢀOH (Fig. 2a). In other way, increasing
H2O2 concentrations in Fenton’s reagent with fixed amounts
of FeSO4 (10 mM) also linearly enhanced the fluorescence
intensity of 1 (Fig. 2b). Probe 1 showed strong fluorescence
Fig. 1 (a) Fluorescence intensity changes of 1 (10 mM) upon addition of ꢀOH
(1.0 equiv.) and other ROS/RNS (5.0 equiv.) in Tris–HCl buffer (DMF 1% v/v) at
pH 7.4 (25 1C, excitation at 500 nm). (b) Fluorescence intensity changes at 550 nm:
1, none; 2, ꢀOH; 3, H2O2; 4, ROOꢀ; 5, NOꢀ; 6, ꢀO2ꢁ; 7, HOCl; 8, ONOOꢁ; 9, 1O2.
(c) Color changes of 1 (20 mM) in the presence of ROS/RNS (5.0 equiv.).
Fig. 3 Fluorescence images of cells incubated with probe 1 and Fenton’s
reagent. (a) A549 and (b) RAW264.7 cells were incubated with 20 mM probe 1
for 1 h, and then incubated for 1 h with 20 mM of Fenton’s reagent in the absence
(top panels) or the presence (bottom panels) of 5 mM TEMPOL (left; fluorescence
microscope images, right; merged bright field images). Scale bar = 10 mm.
c
7960 Chem. Commun., 2013, 49, 7959--7961
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the rhodaminꢀe fluorescent probes. Probe 1 exhibits excellent
selectivity for OH with virtually no interference by other ROS/
RNS species. Fluorescent imaging of A549 and RAW264.7 cells
is also successfully demonstrated to detect intracellular ꢀOH in
live cells. We expect that this imaging technique will serve as a
ꢀ
practical tool for OH-related biological studies.
This work was supported by Basic Science Research
(No. 2011-0007015) and National Creative Research Initiative
programs (NRF).
Notes and references
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Fig. 4 Fluorescence images of cells incubated with probe 1 and PMA. (a) A549
and (b) RAW264.7 cells were incubated with 20 mM probe 1 for 1 h, and then
incubated for 1 h with 10 ng mLꢁ1 of PMA in the absence (top panels) or the
presence (bottom panels) of 5 mM TEMPOL (left; fluorescence microscope
images, right; merged bright field images). Scale bar = 10 mm.
the fluorescence signal is a consequence of the response to
intracellular hydroxyl radicals.
Encouraged by the fluorescent imaging of hydroxyl radicals
generated by Fenton’s reagent in live cells, we then examined
whether probe 1 can sense hydroxyl radicals which are produced
by the physiological stimulation with phorbol 12-myristate
13-acetate (PMA).4f,6 In this study, A549 and RAW264.7 cells
were treated with 20 mM probe 1 for 1 h. After being washed
with PBS to remove the remaining probe 1, the cells were
incubated with 10 ng mLꢁ1 PMA to generate hydroxyl radicals.
In contrast to cells untreated with PMA, a marked increase in
fluorescence was observed in both cells after stimulation with
PMA (top panels in Fig. 4a and b). To confirm that fluorescence
turn-on was due to the response of probe 1 to the generated
hydroxyl radicals, cells treated with probe 1 were incubated
with 5 mM TEMPOL prior to stimulation with PMA. It was found
that a radical scavenger remarkably reduced the red fluorescence
in both cells (bottom panels in Fig. 4a and b), indicating that the
probe monitors hydroxyl radicals generated by the physiological
8 We have tested several rhodamine cyclic hydrazides prepared from
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intracellular hydroxyl radicals in live cells.
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sensing ꢀOH is based on the oxidative C–H abstraction reaction
of rhodamine cyclic hydrazide, which is the first example in
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c
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Chem. Commun., 2013, 49, 7959--7961 7961