Rational design of a fluorescent hydrogen peroxide probe based on the umbelliferone fluorophore

Article abstract of DOI:10.1016/j.tetlet.2008.03.063

In this study, we report a novel water-soluble umbelliferone-based fluorescent probe for hydrogen peroxide. This probe shows very large increases (up to 100-fold) in fluorescent intensity upon reaction with hydrogen peroxide, and good selectivity over other reactive oxygen species (ROS).

Full text of DOI:10.1016/j.tetlet.2008.03.063

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3045–3048
Rational design of a fluorescent hydrogen peroxide
probe based on the umbelliferone fluorophore
Lupei Du, Minyong Li, Shilong Zheng, Binghe Wang *
Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30302-4098, USA
Received 18 February 2008; revised 12 March 2008; accepted 12 March 2008
Available online 16 March 2008
Abstract
In this study, we report a novel water-soluble umbelliferone-based fluorescent probe for hydrogen peroxide. This probe shows very
large increases (up to 100-fold) in fluorescent intensity upon reaction with hydrogen peroxide, and good selectivity over other reactive
oxygen species (ROS).
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Hydrogen peroxide; Reactive oxygen species (ROS); Hydroxycoumarin; Umbelliferone; Fluorescence
Produced during a number of physiological processes,
such as Alzheimer’s disease, apoptosis, and phagocyto-
sis,1–3 reactive oxygen species (ROS), including hydroper-
oxide (ROOH), superoxide ðO2ꢀÞ, hydroxyl radical
(ÅOH), and various peroxides (ROOR0) react with a large
variety of easily oxidizable cellular components, such as
NADH, NADPH, catecholamines, ascorbic acid, histidine,
tryptophan, tyrosine, cysteine, glutathione, proteins, and
nucleic acids.4 ROS can also oxidize cholesterol and unsat-
urated fatty acids, causing membrane lipid peroxidation.
Among all the ROS in biological systems, hydrogen per-
oxide (H2O2) is vasoactive and has been detected under
various pathophysiological conditions such as inflammation,
hypoxia-reoxygenation, and deficiency of a co-factor for
nitric oxide (NO) synthesis. Hydrogen peroxide is gener-
ated in response to various stimuli, including cytokines
and growth factors, and is also involved in regulating bio-
logical processes as diverse as immune cell activation and
vascular remodeling in mammals,5 and stomatal closure
and root growth in plants.6 Moreover, recent evidence
demonstrated that hydrogen peroxide generated by the
mitochondrial respiration is a potent inducer of oxidative
damage and mediator of aging.7 Although a number of
reports have been published, the significance of hydrogen
peroxide in biological system and the mechanism of its
action are still poorly understood because of the limited
availability of detection methods.
There have been several types of probes reported for the
detection of hydrogen peroxide and other ROS, including
fluorescein analogues,8,9 rhodamine analogues, Amplex
Red analogues,10,11 phosphine-based fluorophores,12,13
and lanthanide coordination complexes.14,15 A recent
review has summarized the current state of this field very
well.16 Each of the reported probes has its own advantages
and disadvantages. An ideal probe should (1) be specific to
the ROS of interest; (2) have the appropriate physicochem-
ical properties to allow for permeation across the mem-
brane barriers and to allow certain solubility in water; (3)
give off a strong and easily detectable reporting signal upon
encountering the appropriate ROS, (4) have the property
O
B
HO
O
O
O
O
H2O2
O
1 (weak fluorescence)
2 Umbelliferone (strong fluorescence)
*
Corresponding author. Tel.: +1 404 413 5544; fax: +1 404 413 5543.
Scheme 1. Reaction of compound 1 with hydrogen peroxide.
E-mail address: wang@gsu.edu (B. Wang).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.03.063

3046
L. Du et al. / Tetrahedron Letters 49 (2008) 3045–3048
O
O
O
O
B
B
O
B
HO
O
O
TfO
O
O
a
O
O
O
b
3
2
1
91% yield
87% yield
Scheme 2. Synthesis of coumarin boronate. Reagents and conditions: (a) (TfO)2O, DMAP, DCM, rt; (b) PdCl2(dppf)/dppf, KOAc, dioxane, 80 °C,
microwave.
of turning on the signal upon encountering the appropriate
ROS instead of turning off; and (5) easily synthesized from
readily available starting materials. Among the detectable
signals used, fluorescence is considered one of the more
sensitive ones. Some important desirable features for a
fluorescent probe include high quantum yield and emission
in the visible region. Herein, we report the design and syn-
thesis of a fluorescent hydrogen peroxide probe that has
most of the desirable features prescribed for such a probe.
This probe complements what is already available and
should be very useful for the in vitro and in vivo detection
of hydrogen peroxide formation.
Our design strategy depends on the selective hydrogen
peroxide-mediated conversion of arylboronates into
phenols.17,18 In this design, the coumarin moiety was
selected as the fluorescent chromophore. The end product
after hydrogen peroxide oxidation is 7-hydroxycoumarin
(umbelliferone) (Scheme 1), which is a well-known fluoro-
phore with high quantum yield.19–21 Umbelliferone is also
used in sunscreen lotion as an antioxidant and has a
minimal toxicity.22 In addition, the designed probe can be
synthesized easily from readily available starting material
40
120
100
80
60
40
20
0
100 μM
30
20
0
10
0
350
400
450
500
550
600
Wavelength (nm)
350
400
450
500
550
600
50
40
30
20
10
0
Wavelength (nm)
-10
0
20
40
60
80
100
[H2O2]/μM
Fig. 1. Fluorescence response of 5 lM compound 1 to 100 lM H2O2 after
30 min. The black and red lines were recorded before and after H2O2
addition, respectively (top). The spectra were acquired in 0.1 M phosphate
buffer, pH 7.4 (kex = 332 nm). The bottom panel shows the color change
before (left) and after hydrogen peroxide addition (right).
Fig. 2. Emission spectra (top) and concentration-dependent fluorescence
intensity changes (bottom) of compound 1 at 454 nm at room tempera-
ture: experiments were conducted in 0.1 M phosphate buffer, pH 7.4 with
excitation at 332 nm. The emission spectra were obtained 30 min after the
addition of hydrogen peroxide to a 5 lM solution of compound 1.

L. Du et al. / Tetrahedron Letters 49 (2008) 3045–3048
3047
(umbelliferone) through a two-step conversion in high
overall yield (about 80%).
oxide over hydroxyl radical (ÅOH) and over twofold more
reactive over superoxide ðO2ꢀÞ. It is particularly note-
worthy that the fluorescence response of compound 1
was not influenced significantly by hypochlorite (OClꢀ),
hydroxyl radical (ÅOH), tert-butyl hydroperoxide (TBHP),
and tert-butoxy radical (t-BuOÅ). Thus, we believe that
compound 1 could be a very useful novel fluorescent probe
for detecting hydrogen peroxide.
The synthesis of the designed probe (1) started from
commercially available 7-hydroxycoumarin (Scheme 2).
After conversion to the corresponding triflate (3) in over
90% yield,23 palladium-mediated borylation using
pinacol-protected diborate gave the final probe (1) in about
87% yield.24,25
Compound 1 was evaluated for its ability to detect
hydrogen peroxide under near physiological conditions
(0.1 M phosphate buffer, pH 7.4). The probe itself (1) dis-
plays no fluorescence. The addition of hydrogen peroxide
triggers a very significant fluorescence increase (about
100-fold) at 454 nm (Fig. 1). The NMR and MS experi-
ments also confirmed that umbelliferone (2) was the prod-
uct from the reaction of coumarin-7-boronate (1) with
hydrogen peroxide.
Next, we investigated the concentration-dependent
fluorescence response of compound 1 to the addition of
hydrogen peroxide (Fig. 2). The fluorescence intensity of
compound 1 increased as a function of hydrogen peroxide
concentration at below 40 lM and then it leveled off.
We also investigated whether the fluorescence response
of compound 1 was hydrogen peroxide-specific. Figure 3
compares the relative reactivity of compound 1 toward var-
ious ROS.26 Selectivity data are displayed at several time
points over 120 min. Compound 1 exhibits a 100-fold
higher response to hydrogen peroxide over similar ROS
such as hypochlorite (OClꢀ), tert-butyl hydroperoxide
(TBHP), and tert-butoxy radical (t-BuOÅ). This probe is
also more than sixfold more responsive to hydrogen per-
In conclusion, we have designed and synthesized a novel
water-soluble fluorescent probe (1) for hydrogen peroxide.
This probe shows very large increases in fluorescent inten-
sity upon reaction with hydrogen peroxide (up to 100-fold)
at 5 lM. It also shows very good selectivity over other
ROS. In addition, the probe also has the advantage of easy
synthesis from readily available inexpensive starting mate-
rials. We hope that all these properties will make this
coumarin-based fluorescent probe very useful for the
in vitro and in vivo detection of hydrogen peroxide.
Acknowledgments
Financial support from the NIH (CA113917 and
CA123329), Georgia Cancer Coalition, Georgia Research
Alliance and the Molecular Basis of Diseases program at
GSU is gratefully acknowledged.
Supplementary data
Supplementary data (spectroscopic data for 1 and 3)
associated with this article can be found, in the online
version, at doi:10.1016/j.tetlet.2008.03.063.
References and notes
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dropwise trifluoromethanesulfonic anhydride (1 mL, 6 mmol) at 0 °C.
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Mill-Q filtration system. Quartz cuvettes were used in all studies. A
Shimadzu UV-1700 UV–visible spectrometer was used for all the
absorbance studies. A Shimadzu RF-5301PC fluorometer was used
for all the fluorescent studies. The solutions of compound
1
(5 ꢁ 10ꢀ6 M) were prepared in 0.1 M phosphate buffer at pH 7.4.
Then the solutions were transferred into a 1 cm quartz cell and UV
absorbance data were recorded immediately. The UV kmax was used
as the fluorescence excitation wavelength.