A rhodamine-hydroxamic acid-based fluorescent probe for hypochlorous acid and its applications to Biological imagings

Article abstract of DOI:10.1021/ol802822t

A new rhodamine-hydroxamic acid-based fluorescent chemosensor for the rapid detection of HOCl in aqueous media was developed. The system, which utilizes an irreversible HOCl-promoted oxidation reaction, responds instantaneously at room temperature with li

Full text of DOI:10.1021/ol802822t

ORGANIC
LETTERS
2009
Vol. 11, No. 4
859-861
A Rhodamine-Hydroxamic Acid-Based
Fluorescent Probe for Hypochlorous
Acid and Its Applications to Biological
Imagings
Young-Keun Yang, Hyungseoph Jason Cho, Jihyun Lee, Injae Shin,* and
Jinsung Tae*
Department of Chemistry, Yonsei UniVersity, Seoul 120-749, Korea
jstae@yonsei.ac.kr; injae@yonsei.ac.kr
Received December 8, 2008
ABSTRACT
A new rhodamine-hydroxamic acid-based fluorescent chemosensor for the rapid detection of HOCl in aqueous media was developed. The
system, which utilizes an irreversible HOCl-promoted oxidation reaction, responds instantaneously at room temperature with linear proportionality
to the amount of HOCl. This system is highly selective for HOCl over other reactive oxygen species (ROS) and highly sensitive in aqueous
solutions. Biological imaging studies using living cells and organisms (A549 cells and zebrafish) to detect HOCl are successfully demonstrated.
Reactive oxygen species (ROS) and reactive nitrogen
species (RNS) are known to be essential to several
biological functions.1 Hypochlorous acid (HOCl), one of
the biologically important ROS, is weakly acidic and
partially dissociates into the hypochlorite ion (OCl-) in
the physiological pH solutions.2 Biologically, the hy-
pochlorite ion is synthesized from hydrogen peroxide and
chloride ions in activated neurophils catalyzed by my-
eloperoxidase (MPO).3 Hypochlorous acid reacts with
various biomolecules including DNA, RNA, fatty acids,
cholesterol, and proteins. While hypochlorous acid has
strong antibacterial properties, excess amounts of HOCl
can also cause serious damage to the biological systems.4
Therefore, a lot of effort has been given to the studies of
the various biological effects of HOCl.5
(3) (a) Kettle, A. J.; Winterbourn, C. C. Redox Rep. 1997, 3, 3–15. (b)
Hidalgo, E.; Bartolome, R.; Dominguez, C. Chem. Biol. Interact. 2002,
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8162. (c) Sugiyama, S.; Kugiyama, K.; Aikawa, M.; Nakamura, S.; Ogawa,
H.; Libby, P. Atrerioscler Thromb. Vasc. Biol. 2004, 24, 1309–1314. (d)
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D. Q.; Li, J.; Zhu, D. B. J. Am. Chem. Soc. 2004, 126, 11543–11548.
(2) In the physiological pH solutions, HOCl rather than OCl- is the
actual species. See refs 6a, c, and d for the discussion.
(5) (a) Kawai, Y.; Kiyokawa, H.; Kimura, Y.; Kato, Y.; Tsuchiya, K.;
Terao, J. Biochemistry 2006, 45, 14201–14211. (b) Pattison, D. I.; Davies,
M. J. Biochemistry 2005, 44, 7378–7387.
10.1021/ol802822t CCC: $40.75
Published on Web 01/23/2009
2009 American Chemical Society

Recently, fluorescent chemosensor-based detections of
HOCl have been given great attention.6 Known fluorescent
chemosensors for HOCl are based on the strong oxidation
property of HOCl. Oxidation reactions of p-methoxyphenol
to benzoquinone,6a dibenzoylhydrazine to dibenzoyl dii-
mide,6b thiol to sulfonate derivative,6c and p-alkoxyani-
line6d have been employed as the key reacting components
in the design of HOCl-selective fluorescent probes. Although
these reported chemosensors have demonstrated reasonable
selectivity for HOCl over other ROS, fluorescent probes of
better sensitivity and reactivity for HOCl are still required
for the biological imaging applications. Herein, we report a
highly sensitive fluorescent probe for hypochlorous acid and
its applications in biological imaging studies.
indicates that the HOCl-induced oxidation of 1 takes place
rapidly at room temperature. The reaction responsible for
the change completes within 20 s to yield the ring-opened
acyl nitroso compound 2 (Scheme 2). Elimination of HCl
Scheme 2. HOCl-Induced Oxidation of Fluorescent Probe 1
The oxidation reactions of thiols and amines by HOCl
involve chlorination reactions where -S-Cl7 and -N-Cl8
species are generated. Similarly, the HOCl-mediated oxida-
tion reaction of hydroxamic acid is expected to follow the
same chlorination pathway to give the acyl nitroso compound
after elimination of HCl (Scheme 1).
from the proposed chlorination intermediate A or B (Figure
1) is expected to be facile under the reaction conditions.
Scheme 1. Oxidation of Hydroxamic Acid by HOCl
We envisioned that this irreversible reaction could be
incorporated into the rhodamine amide system to convert
the nonfluorescent spirocyclic form to the fluorescent ring-
opened one.9,10 Therefore, the rhodamine derivative 1 was
prepared from rhodamine 6G in three steps (1, NaOH,
H2O-EtOH; 2, POCl3, CH2Cl2; 3, NH2OH, Et3N, CH2Cl2).11
Fluorescent probe 1 forms a colorless solution in PBS
buffer-DMF (0.1%) at pH 7.4, indicating that it exists in
the spirocyclic form predominantly. Addition of aqueous
NaOCl (10 equiv) to 1 developed instantaneous color and
strong fluorescence intensity changes. Although probe 1
showed no fluorescence at pH 5-13, it showed strong
fluorescence intensity changes in the presence of NaOCl at
pH 5-10 (see Supporting Information). This observation
Figure 1. Proposed chlorination intermediates.
Interestingly, the hydroxamic acid derivatives of rhodamine
B and fluorescein showed only slight fluorescence intensity
changes (see Supporting Information).12 Although we were
not able to detect the highly unstable acyl nitroso13 com-
pound 2, we could confirm the formation of rhodamine 19
1
(3) from the reaction mixture using H NMR and ESI-MS
experiments.14
(6) (a) Sun, Z. N.; Liu, F. Q.; Chen, Y.; Tam, P. K. H.; Yang, D. Org.
Lett. 2008, 10, 2171–2174. (b) Chen, X.; Wang, X.; Wang, S.; Shi, W.;
Wang, K.; Ma, H. Chem.-Eur. J. 2008, 14, 4719–4724. (c) Kenmoku, S.;
Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 7313–
7318. (d) Shepherd, J.; Hilderbrand, S. A.; Waternan, P.; Heinecke, J. W.;
Weissleder, R.; Libby, P. Chem. Biol. 2007, 14, 1221–1231. (e) Bizyukin,
A. V.; Korkina, L. G.; Velichkovskii, B. T. Bull. Exp. Biol. Med. 1995,
119, 347–351.
Fluorescence intensity changes of 1 (1 µM) upon additions
of HOCl (5.0 equiv) and other ROS15 (100 equiv of H2O2,
-
NO·, ·OH, ROO·, ·O2 ) in PBS buffer-DMF (0.1%) at pH
7.4 are shown in Figure 2. The selectivity profile proved to
(12) It seems that the hydroxamic acid derivatives of rhodamine B and
fluorescein are less likely to promote ring opening of the chlorinated
spirocyclic intermediates to give the corresponding acyl nitroso compounds.
From our previous studies, we found that the irreversible spirocyclic ring-
opening reaction is generally favored by NHEt over NEt2 or OH at the
xantene ring (see ref 10a).
(7) (a) Pereira, W. E.; Hoyano, Y.; Summons, R. E.; Bacon, V. A.;
Duffield, A. M. Biochim. Biophys. Acta 1973, 313, 170–180. (b) Davies,
M. J.; Hawkins, C. L. Free Radical Res. 2000, 33, 719–729.
(8) Slates, H. L.; Taub, D.; Kuo, C. H.; Wendler, N. L. J. Org. Chem.
1964, 29, 1424–1429.
(9) Kim, H. N.; Lee, M. H.; Kim, H. J.; Kim, J. S.; Yoon, J. Chem.
(13) Cohen, A. D.; Zeng, B.-B.; King, S. B.; Toscano, J. P. J. Am. Chem.
Soc. 2003, 125, 1444–1445.
Soc. ReV. 2008, 37, 1467–1472
.
(10) (a) Yang, Y. K.; Yook, K. J.; Tae, J. J. Am. Chem. Soc. 2005, 127,
16760–16761. (b) Ko, S. K.; Yang, Y. K.; Tae, J.; Shin, I. J. Am. Chem.
Soc. 2006, 128, 14150–14155. (c) Yang, Y. K.; Ko, S. K.; Shin, I.; Tae, J.
(14) We could detect rhodamine 19 (3) from the 1H NMR spectrum
obtained from the reaction mixture run in an NMR tube. Furthermore,
rhodamine 19 is isolated as the major product after flash column chroma-
tography of the reaction mixture (see Supporting Information).
(15) Additions of 5∼100 equiv of ROS other than NaOCl showed no
fluorescence intensity changes at all (see Supporting Information).
Nat. Protocols. 2007, 2, 1740–1745
.
(11) For the synthesis and characterization of fluorescent probe 1, see
Supporting Information.
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Org. Lett., Vol. 11, No. 4, 2009

Figure 2. (a) Fluorescence intensity changes of 1 (1 µM) upon
addition of NaOCl (5.0 equiv) and other ROS (100 equiv of H2O2,
NO·, ·OH, ROO·, ·O2-) in PBS buffer-DMF (0.1%) at pH 7.4 (25
°C, excitation at 500 nm). (b) Fluorescence intensity changes at
546 nm.
Figure 4. Images of A549 (lung cancer) cells and zebrafish. (a)
Microscopic image of A549 cells treated with 1 (5 µM) in the
absence of NaOCl (control). (b) Microscopic image of A549 cells
treated with both NaOCl (10 µM) and 1 (5 µM). (c) Fluorescence
image of A549 cells treated with 1 (5 µM) in the absence of NaOCl
(control). (d) Fluorescence image of A549 cells treated with both
NaOCl (10 µM) and 1 (5 µM). (e) Fluorescence images of zebrafish
treated with 1 (20 µM) in the absence of NaOCl (control). (f)
Microscopic image of zebrafish treated with 1 (20 µM) and NaOCl
(100 µM). (g) Fluorescence image of zebrafish treated with 1 (20
µM) and NaOCl (100 µM).
be excellent in aqueous solutions. Only HOCl enhanced the
fluorescence intensity dramatically, while other ROS are
silent to 1. In addition to the fluorescent changes, the
colorless to pink color changes associated with the reaction
of 1 with HOCl are readily detectable visually, but no
significant color changes were promoted by other ROS.
The fluorescence titration profile of 1 (0.1 µM) with HOCl
demonstrates that the detection of HOCl is possible at the
25 nM level (Figure 3). Under these conditions, the
but the cells treated with NaOCl displayed strong red
fluorescence (Figure 4d). The fluorescence intensities in-
creased as the concentration of NaOCl increased (see
Supporting Information). Similarly, the imaging of 5-day old
zebrafish treated with 100 µM NaOCl and 1 (20 µM) was
also conducted using a dissecting microscope and a fluo-
rescence microscope. The zebrafish treated with probe 1 only
exhibited no fluorescence (Figure 4e). However, strong red
fluorescence was observed from the zebrafish treated with
both NaOCl and 1 (Figure 4g).
In conclusion, we have described a highly selective and
sensitive fluorescent chemosensor for the detection of HOCl
in aqueous media. The hydroxamic acid unit of the fluores-
cent probe is oxidized by HOCl to produce a highly
fluorescent acyl nitroso compound. The chemical reaction
is fast at room temperature and irreversible. The probe can
detect ∼25 nM concentration of HOCl in aqueous media.
Fluorescent imagings of A549 cells and zebrafish are also
successfully demonstrated to detect HOCl in living cells and
organisms. We expect that this imaging technique will serve
as a practical tool for hypochlorous acid-related biological
studies.
Figure 3. (a) Fluorescence response of 1 (0.1 µM) upon addition
of NaOCl (by 25 nM) in PBS buffer-DMF (0.1%) at pH 7.4 (25
°C, excitation at 500 nm). (b) The fluorescence intensities at 547
nm.
fluorescence intensity of the solution of 1 is linearly
proportional to the amounts of HOCl added (Figure 3b).
Because the fluorescent probe 1 is highly reactive and highly
sensitive toward HOCl, it could be ideal for the biological
imaging applications.16
Next, we studied bioimaging applications of 1 for HOCl
detection in biological systems. A549 (lung cancer) cells
were incubated with and without 10-50 µM NaOCl for 20
min at 37 °C and washed with PBS buffer (pH 7.4) to remove
the remaining NaOCl, and then the treated cells were
incubated with 1 (5 µM) in culture medium for 10 min at
37 °C. After incubation under these conditions, they were
imaged using a confocal fluorescence microscope. The cells
untreated with NaOCl showed no fluorescence (Figure 4c),
Acknowledgment. This work was supported by KOSEF
(R01-2008-000-10245-0), CBMH (Yonsei University) and
NRL program.
Supporting Information Available: Experimental pro-
1
cedures for the synthesis, spectral data, copies of H NMR
and 13C NMR of 1 and 3, and data for fluorescence titrations
of 1. This material is available free of charge via the Internet
at http://pubs.acs.org.
(16) It is not easy to directly compare properties of 1 with those of the
known probes for HOCl. But we believe that the readily available rhodamine
amide derivative 1 could find broad biological applications.
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