Development of highly selective and sensitive probes for hydrogen peroxide

Article abstract of DOI:10.1039/b309393j

Probes that react specifically with hydrogen peroxide to release chromophoric or fluorescent reporter groups were designed and synthesized.

Full text of DOI:10.1039/b309393j

Development of highly selective and sensitive probes for hydrogen
peroxide†
Lee-Chiang Lo* and Chi-Yuan Chu
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan. E-mail: lclo@ntu.edu.tw;
Fax: 886-2-2362-1979; Tel: 886-2-2362-1979
Received (in Cambridge, UK) 6th August 2003, Accepted 12th September 2003
First published as an Advance Article on the web 6th October 2003
Probes that react specifically with hydrogen peroxide to
release chromophoric or fluorescent reporter groups were
designed and synthesized.
(chromophore/fluorophore) is masked with a 2,3-butanediol
ester of a Dobz derivative. Probe 1a carries a chromophoric p-
nitroaniline group (lmax 383 nm), while probe 1b adopts a
fluorescent coumarin reporter (lex 348 nm, lem 440 nm). Upon
reaction with hydrogen peroxide, the reporters could be released
and serve as the basis for the determination of hydrogen
peroxide (Scheme 1).
The synthesis of the probes 1a and 1b begins with
arylboronic acid 27 in a straightforward manner (Scheme 2).
The boronic moiety was first quantitatively converted to
boronate ester 3 by reaction with 2,3-butanediol. Removal of
the TBDMS group of ester 3 was achieved by treatment with
Bu4NF to give borylbenzyl alcohol 4 in 88% yield. Borylbenzyl
alcohol 4 is a key intermediate in this synthetic scheme. It
reacted with triphosgene to generate the borylbenzylox-
ycarbonyl chloride 5 in situ, which in turn could couple with the
corresponding reporter group, namely p-nitroaniline and
7-amino-4-methylcoumarin, to offer probes 1a and 1b in 84%
and 64% yields, respectively.
Hydrogen peroxide is one of the major reactive oxygen species
(ROS) in living systems.1 Its production is linked to the
activities of certain enzymes that have been implicated in
important cellular processes, including apoptosis, cytotoxicity,
cell growth, and proliferation.2 Therefore, many efforts have
been devoted to the determination of hydrogen peroxide in
bioanalytical chemistry.3 Currently, the most widely used
method is an enzymatic approach that involves the use of
horseradish peroxidase (HRP), which catalyzes the oxidation of
suitable substrates to generate read-out signals.4 However, it
would be of great interest to develop a chemical approach that
not only is sensitive to hydrogen peroxide, but also avoids
possible interference resulting from the complex biological
samples.
It is well known that hydrogen peroxide reacts with
arylboronic acids under mild alkaline conditions to generate
phenols.5 p-Dihydroxyborylbenzyloxycarbonyl (Dobz) group
was later introduced as a special amine protecting group, as it
could be selectively removed by hydrogen peroxide.6 Based on
these results, we envision that probes highly specific for
hydrogen peroxide could be developed. Prototype probe 1 (1a
and 1b) were designed and synthesized to test the idea, as shown
in Scheme 1. In the design of probe 1, a suitable reporter group
Scheme 2 Synthesis of the probes 1a and 1b.
In the preliminary study, 200 mM of probe 1a was incubated
with various amounts of hydrogen peroxide (10 ~ 50 mM) in the
presence of 140 mM NaHCO3 (pH 8.3) at 25 °C. The UV/vis
spectra of probe 1a and p-nitroaniline in DMF are shown in Fig.
1. Although there is some overlapping in their UV/vis spectra,
the incubation mixtures were monitored and recorded with a
microplate reader at 405 nm to reduce the interference from the
unreacted probe 1a. The amount of released p-nitroaniline
(A405) quickly increased with time and reached plateau ( > 90%
yield) after 80 ~ 90 min. A good linear correlation was observed
between the released p-nitroaniline and the hydrogen peroxide
in the mixture (Fig. 2), indicating that the design of the probe
could function as depicted in Scheme 1 and that probe 1a could
Scheme 1 Structure of the probe 1 (1a and 1b) and its reaction with
hydrogen peroxide to release the reporter signals.
† Electronic supplementary information (ESI) available: general methods.
See http://www.rsc.org/suppdata/cc/b3/b309393j/
Fig. 1 UV/vis spectra of probe 1a (Ã) and p-nitroaniline (—) in DMF.
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This journal is © The Royal Society of Chemistry 2003

Fig. 2 The linear correlation of the released p-nitroaniline (A405) upon
reactions of probe 1a (200 mM) with 10, 20, 30, 40 and 50 mM of hydrogen
peroxide in the presence of 140 mM NaHCO3 (pH 8.3) for 80–90 min.
Fig. 4 The linear correlation of the fluorescence response (lex 355 nm, lem
460 nm) upon reactions of probe 1b (200 mM) with 0.1, 0.2, 0.5, 1.0, 2.5 and
5.0 mM of hydrogen peroxide. The other conditions are the same as
described in Fig. 2.
be successfully used for the determination of hydrogen
peroxide. Although lower probe concentrations could be used
when working with low concentrations of hydrogen peroxide,
we suggest keeping the probe concentration at 200 mM in order
to obtain a good response range within a reasonable time.
We have also investigated the potential interfering effect of
hydroperoxide and thiol compounds on the reaction of probe 1a
with hydrogen peroxide. The results indicated that tert-butyl
hydroperoxide reacted with probe 1a at a much slower rate
( ~ 2%). As for cysteine, it only produced a negligible increase
in A405 when incubated alone with probe 1a. However, when
added in the incubation mixture of probe 1a and hydrogen
peroxide, it lowered the amount of the released p-nitroaniline,
indicating that the thiol group might consume hydrogen
peroxide8 and should be avoided in the assay mixture.
Since hydrogen peroxide is also the co-product of many
oxidases for some important metabolites, measurements of the
concentration of these substrates or the activity of the
responsible enzymes could be indirectly achieved by monitor-
ing the hydrogen peroxide resulting from the coupled reactions.
Therefore, the probes developed in this study could be linked to
these studies and find wide applications. As a demonstration,
experiments for the determination of glucose concentrations
were carried out. In this model study, various amounts of
glucose (10 ~ 50 mM) were first treated with glucose oxidase to
generate hydrogen peroxide, of which the concentrations were
determined by using probe 1a as described above. A good linear
correlation again was successfully established between glucose
and hydrogen peroxide as shown in Fig. 3.
determination of hydrogen peroxide (Fig. 4), except a fluores-
cence microplate reader (lex 355 nm, lem 460 nm) was utilized.
It offers a more sensitive detection range (0.1 ~ 5 mM) than that
of probe 1a.
In summary, probes 1a and 1b were conveniently prepared
and were demonstrated to be probes for hydrogen peroxide on
a reaction mechanism basis. The skeleton of these probes
consists of a butanediol ester of the Dobz derivative, masking
the amino group of suitable reporter groups. These two probes
are stable compounds that could be activated and release the
reporter groups upon reaction with hydrogen peroxide. The
probe design offers great flexibility for signal output. Probes 1a
and 1b serve well in the micromolar and sub-micromolar range,
respectively. In addition, in combination with a microplate
reader they are well suited for high throughput screening
purposes. Our ongoing study is to improve the sensitivity of the
probes by exploiting new reporter groups. In the meantime, one
of the major goals is to apply these probes in studying the events
inside the cells. Preliminary data of the pH effect on the reaction
of probe 1a and H2O2 favorably indicated that the probe could
still function at pH 7.0–7.5, although at a slower rate (18–32%).
We are currently evaluating the feasibility of this potential
application.
This work was supported by the National Science Council
(NSC 92-3112-B-002-005 to LCL).
Notes and references
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One of the major advantages in the design of the probes in
this study is the flexibility in the choice of the reporter groups.
Different reporter groups, which carry special properties yet do
not affect the detecting mechanism, could be used to meet the
demands. In the meantime, the preparation of different probes
basically follows the same synthetic scheme. For example,
probe 1b, which carries a fluorescent coumarin derivative, was
also conveniently constructed. Probe 1b behaves similarly to
that of probe 1a when employed in excess (200 mM) for the
4 (a) G. G. Guilbault, P. J. Brignac Jr. and M. Juneau, Anal. Chem., 1968,
40, 1256–1263; (b) M. Zhou, Z. Diwu, N. Panchuk-Voloshina and R. P.
Haugland, Anal. Biochem., 1997, 253, 162–168; (c) M. Zhou and N.
Panchuk-Voloshina, Anal. Biochem., 1997, 253, 169–174.
5 H. G. Kuivila and A. G. Armour, J. Am. Chem. Soc., 1957, 79,
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6 (a) D. S. Kemp and D. C. Roberts, Tetrahedron Lett., 1975, 4629–4632;
(b) M. Wakselman, Nouv. J. Chim., 1983, 7, 439–447.
7 P. R. Ashton, R. Ballardini, V. Balzani, A. Credi, K. R. Dress, E. Ishow,
C. J. Kleverlaan, O. Kocian, J. A. Preece, N. Spencer, J. F. Stoddart, M.
Venturi and S. Wenger, Chem. Eur. J., 2000, 6, 3558–3574.
8 B. Haliwell, M. V. Clement and L. H. Long, FEBS Lett., 2000, 10–13.
Fig. 3 Determination of glucose using probe 1a after pretreatment with
glucose oxidase (0.9 U, 175 mM NaHCO3, pH 8.3) for 30 min. The other
conditions are the same as described in Fig. 2.
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