A fluorescent hydrogen peroxide probe based on a 'click' modified coumarin fluorophore

Article abstract of DOI:10.1016/j.tetlet.2009.12.049

Herein a water-soluble 'click' modified coumarin-based fluorescent probe for hydrogen peroxide is reported. This probe shows significant intensity increases (up to fivefold) in near-green fluorescence upon reaction with hydrogen peroxide, and good selectivity over other reactive oxygen species.

Full text of DOI:10.1016/j.tetlet.2009.12.049

Tetrahedron Letters 51 (2010) 1152–1154
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A fluorescent hydrogen peroxide probe based on a ‘click’ modified
coumarin fluorophore
a,b,
a,
a,
, Nanting Ni Y , Minyong Li a,b, , Binghe Wang
*
*
´
´
Lupei Du Y
a Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30302-4098, USA
b School of Pharmacy, Shandong University, Jinan, Shandong 250012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein a water-soluble ‘click’ modified coumarin-based fluorescent probe for hydrogen peroxide is
reported. This probe shows significant intensity increases (up to fivefold) in near-green fluorescence
upon reaction with hydrogen peroxide, and good selectivity over other reactive oxygen species.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 6 August 2009
Revised 6 December 2009
Accepted 9 December 2009
Available online 21 December 2009
Keywords:
Hydrogen peroxide
Reactive oxygen species (ROS)
Coumarin
Click chemistry
Fluorescence
As a relatively stable reactive oxygen species (ROS), hydrogen
peroxide (H2O2) generation in cellular environments has been cor-
related with various pathophysiological conditions.1,2 Hydrogen
peroxide is generated in response to various stimuli, including
cytokines and growth factors, and is involved in cellular ‘redox’ sig-
naling in regulating many severe diseases, such as cancer and Par-
kinson’s and Alzheimer’s syndromes.3–5 Ample evidence also
demonstrates that hydrogen peroxide generated by mitochondrial
respiration is a potent inducer of oxidative damage and a mediator
of aging.6 Moreover, recent studies also indicated that hydrogen
peroxide could mediate rapid wound detection in zebrafish.7
Although a number of reports have been published on the subject,
the dual characters of adverse and beneficial effects of hydrogen
peroxide generation in biological system are still poorly under-
stood because of the limited availability of detection methods.8
So far several types of chemical probes based on fluorescein,9
coumarin,10 Amplex Red,11 and luciferin,12 have been reported
for the selective detection of hydrogen peroxide and some have
been used for the detection of intracellular hydrogen peroxide lev-
els.13 A recent review has summarized the current state of this field
very well.14 Each of the reported probes has its own advantages
and limitations in terms of issues such as membrane permeability,
photostability, wavelength, ease of synthesis, synthetic modularity
or lack of, and solubility. An ideal probe should remain non-fluo-
rescent until interactions (chemical reaction or physical binding)
with its target, have the appropriate physicochemical properties
to allow for ready permeation across membrane barriers, are sta-
ble, and have certain solubility in water. In addition, ease of syn-
thesis and synthetic modularity are also important factors as the
latter would allow for easy preparation of analogs with different
chemical and spectroscopic properties and for incorporation of
additional recognition moieties. Based on the ability for hydrogen
peroxide to oxidatively convert arylboronates into phenols,15,16
we had previously reported a umbelliferone-based fluorescent
probe for the detection of hydrogen peroxide.10 In this case, oxida-
tive cleavage of the boronate moiety converts a non-fluorescent
coumarin derivative to fluorescent umbelliferone, which signals
hydrogen peroxide detection. The small size, high permeability,
and easy synthetic manipulation of coumarin compounds make
them very attractive as probes for ROS detection.17 However, this
umbelliferone probe has
a short excitation wavelength at
332 nm, which could result in photo-damage to labeled biomole-
cules and/or cells. Therefore, we were interested in the design
and synthesis of new coumarin-based probes with a longer excita-
tion wavelength. In addition, we were also interested in designing
a system that allows for synthetic modularity and ligation of addi-
tional functional groups that could be used to manipulate the
physicochemical properties, permeability, and spectroscopic prop-
erties of the system, and for tethering of a recognition moiety for
site-specific delivery of the hydrogen peroxide probe. Recently,
the laboratories of Wang and Fahrni reported that ‘click’ modifica-
tions of coumarin could be used as a way to manipulate the spec-
troscopic properties of this fluorophore.18–20 In the work from the
* Corresponding authors. Tel./fax: +86 531 8838 2076 (M.L.); tel.: +1 404 413
5544; fax: +1 404 413 5543 (B.W.).
E-mail addresses: mli@sdu.edu.cn (M. Li), wang@gsu.edu (B. Wang).
These two authors made equal contributions.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.12.049

´
L. Du Y et al. / Tetrahedron Letters 51 (2010) 1152–1154
1153
HO
O
O
O
B
H2O2
O
O
O
N
N
Ar
N
N
N
Ar
2, Ar = Phenyl
1, Ar = Phenyl
N
λem = 475 nm
λ
ex = 400 nm
Scheme 1. Reaction of compound 1 with hydrogen peroxide.
Wang laboratory, it was reported that 3-azido-7-hydroxy-couma-
120
100
80
60
40
20
0
rin was not fluorescent. However, ‘click’ modification by reaction
with an alkyne converts this compound to a very fluorescent prod-
uct. By taking advantage of the electronic and spectroscopic
changes associated with triazole ring formation after Cu(I)-cata-
lyzed azide-alkyne click reaction,21–23 we designed compound 1
(Scheme 1), which showed improved spectroscopic properties,
after hydrogen peroxide oxidation, with an increase of excitation
wavelength by about 70 nm compared with the umbelliferone-
based probe and afforded the possibility of tethering additional
functional groups for targeted delivery and/or improved physico-
chemical properties.
Synthesis of the designed probe (1) started from 3-azido-7-
hydroxycoumarin18 (3) as shown in Scheme 2. By reacting with
phenylacetylene in the presence of Cu(I), triazole-conjugated cou-
marin (2) was obtained in over 70% yield.23 After conversion to the
corresponding triflate (4) in over 80% yield palladium-mediated
borylation using pinacol-protected diborate gave the final probe
(1) in about 5% yield.24
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) displays almost no background
fluorescence. Addition of hydrogen peroxide triggered a very sig-
nificant fluorescence intensity increase (around fivefold) at
475 nm (Fig. 1). The excitation wavelength (400 nm) was about
70 nm higher than that of the umbelliferone-based hydrogen per-
oxide probe reported earlier from our laboratory.10 NMR and MS
experiments confirmed that hydroxylcoumarin (2) was the prod-
uct after reaction of 1 with hydrogen peroxide.
Concentration-dependent fluorescence response of compound 1
was also examined at 30 min after the addition of hydrogen perox-
ide (Fig. 2). The fluorescence intensity of compound 1 increased
significantly as a function of hydrogen peroxide concentration.
Compound 1’s specificity in fluorescence response for hydrogen
peroxide was examined.9,25–27 Figure 3 compares the relative reac-
tivity of compound 1 toward various kinds of ROS at several time
points over 120 min. Compound 1 exhibited a 2–4-fold higher
responses to hydrogen peroxide over other ROS species (Fig. 3).
0min
120min
400
450
500
550
600
650
700
Wavelength (nm)
Figure 1. Fluorescence response of compound 1 to 100 lM H2O2 after
5
l
M
120 min. The bottom and top spectra were recorded before and after H2O2 addition,
respectively. Spectra were acquired in 0.1 M phosphate buffer, pH 7.4
(kex = 400 nm).
Specifically, the selectivity of this probe for hydrogen peroxide is
more than threefold over hydroxyl radical (ÅOH), twofold over
superoxide ðO2ꢀ), twofold over hypochlorite (OClꢀ), and threefold
over tert-butyl hydroperoxide (TBHP) and tert-butoxy radical (t-
BuOÅ). It is important to note that hydroxyl radical (ÅOH), tert-butyl
hydroperoxide (TBHP) and tert-butoxy radical (t-BuOÅ) did not in-
duce concentration-dependent fluorescence intensity changes,
suggesting that it might be impurities in these reagents that in-
duced the low level response observed.
In conclusion, we have designed and synthesized a new water-
soluble ‘click’ modified coumarin-based fluorescent probe, which
shows large increases in fluorescent intensity upon reaction with
hydrogen peroxide (around fivefold) at 5 lM. The introduction of
the triazole ring increased the excitation wavelength by about
70 nm. The probe (1) also shows good selectivity for hydrogen per-
oxide over other ROS. In addition, the probe has the advantage of
HO
O
O
HO
O
O
(TfO)2O, DMAP
DCM
N
N
N3
sodium ascorbate
CuSO4 H2O:EtOH=1:1
3
4
N
2
O
B
O
O
O
O
N
TfO
O
O
B
B
O
O
O
N
N
PdCl2(dppf)/DPPF
KOAc dioxane
N
N
N
1
Scheme 2. Synthesis of compound 1.

´
L. Du Y et al. / Tetrahedron Letters 51 (2010) 1152–1154
1154
90
80
70
60
50
40
30
20
10
0
Acknowledgments
250 μM
100 μM
50 μM
25 μM
10 μM
5 μM
Financial support from the NIH
(CA123329, CA113917,
GM086925, and GM084933), Georgia Cancer Coalition, Georgia Re-
search Alliance and the Molecular Basis of Diseases program at
GSU is gratefully acknowledged.
0
Supplementary data
Supplementary data (experimental detail for 1) associated with
this article can be found, in the online version, at doi:10.1016/
j.tetlet.2009.12.049.
420
470
520
570
620
670
Wavelength (nm)
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Figure 2. Concentration-dependent emission intensity changes of compound 1 at
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