Selective ratiometric detection of H2O2 in water and in living cells with boronobenzo[b]quinolizinium derivatives

Article abstract of DOI:10.1039/c4cc02283a

Boronobenzo[b]quinolizinium derivatives exhibit several favorable properties for the fluorimetric detection of hydrogen peroxide, namely quantitative transformation to a product whose emission maximum is well separated from the one of the substrate, water solubility, and the ability to operate in living cells. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc02283a

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Selective ratiometric detection of H2O2 in
water and in living cells with
boronobenzo[b]quinolizinium derivatives†
Cite this: Chem. Commun., 2014,
50, 8242
Received 27th March 2014,
Accepted 6th June 2014
Roberta Bortolozzi,a Sebastian von Gradowski,b Heiko Ihmels,*b Katy Schaferb and
¨
Giampietro Viola‡a
DOI: 10.1039/c4cc02283a
www.rsc.org/chemcomm
Boronobenzo[b]quinolizinium derivatives exhibit several favorable enabling a ratiometric analysis. Based on this principle, several
properties for the fluorimetric detection of hydrogen peroxide, ratiometric H2O2-sensitive probes were developed.8 Nevertheless,
namely quantitative transformation to a product whose emission the development of novel ratiometric fluorescent chemosensors
maximum is well separated from the one of the substrate, water for the detection of H2O2 is still an important goal, especially
solubility, and the ability to operate in living cells.
those with sufficient water solubility. The latter is an essential
requirement for studies in biological media. In this context we
Reactive oxygen species (ROS), such as e.g. hydrogen peroxide, have established the benzo[b]quinolizinium as a versatile, water-
singlet oxygen, the hydroxyl radical, the superoxide anion, soluble fluorophore for fluorimetric detections.9 In addition, we
peroxynitrite, or ozone,1 have fundamental functions in health have discovered that the hydroxy-substituted benzo[b]quinolizinium
and disease related processes.2 Specifically, increased concentra- derivative 2b has fluorosolvatochromic properties with a large red-
tions of H2O2 may cause oxidative damage of cellular proteins, shift of the emission band in water (600 nm).10 At the same time,
ageing and diseases such as cancer or diabetes.3 Therefore, the the 9-boronobenzo[b]quinolizinium (1a) exhibits the same emission
detection of H2O2 and other ROS is a challenging and important bands as the parent compound (417 nm).11 Considering this large
task in biological and chemical research. Along these lines, difference of emission color of borono- and hydroxy-substituted
fluorescent probes have been established as sensitive and selec- derivatives we proposed that the formation of the latter by the
tive tools for the detection of H2O2.4a Fluorescent light-up probes reaction of H2O2 with boronobenzo[b]quinolizinium derivatives may
offer one approach to respond to H2O2 by an emission increase.5 be monitored by a drastic shift of the emission wavelength. Mean-
These intensity-based probes may have practical advantages; while, it was shown that resembling quinolinium-type boronic acid
however, they suffer from the fact that the emission intensity esters may indeed be used for that purpose.8a Herein, we will
is also effectively influenced by other internal and external demonstrate that the boronic acids and ester derivatives 1a–d may
factors.4b A complementary approach towards fluorimetric detec- be generally employed as ratiometric fluorescence probes for the
tion of ROS is based on ratiometric probes operating at two detection of H2O2 in aqueous solution and in living cells (Scheme 1).
different detection wavelengths that enable an accurate internal
The benzo[b]quinolizinium derivatives 1a,b and 2a,b were
calibration.6 In this context aryl boronates were identified as synthesized by the cyclodehydration method,12 and the two acid
probe molecules whose selective reaction with H2O2 may be derivatives 1a,b were transformed to the corresponding pinacol-
employed for the development of ratiometric fluorescent boronates 1c,d (cf. ESI†). Known compounds (1a, 2a,b) were
probes.7 In general the transformation of the boronic acid (ester) identified by comparison with literature data.10,11,12b All new
1
group into an hydroxy functionality results in a significant change compounds (1b–d) were characterized by H-NMR and 13C-NMR
of the absorption and emission properties of the fluorophore thus spectroscopy, mass spectrometry and elemental analysis (cf. ESI†).
a Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia,
University of Padova, via Giustiniani 2, I-35128 Padova, Italy
b
¨
Department Chemie - Biologie, Universitat Siegen, Adolf-Reichwein-Str. 2,
57068 Siegen, Germany. E-mail: ihmels@chemie.uni-siegen.de
† Electronic supplementary information (ESI) available: Experimental proce-
dures, full characterization and NMR spectra, spectroscopic studies. See DOI:
10.1039/c4cc02283a
‡ Author names in alphabetical order that does not reflect the specific contribu-
tion of each author.
Scheme 1 Reaction of boronobenzo[b]quinolizinium derivatives 1a–d
with H2O2.
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The absorption spectra of the derivatives 1a–d in phosphate
buffer solution reveal the characteristic long-wavelength
absorption band of the benzo[b]quinolizinium chromophore13
with a maximum at 380 nm (1a,c) and 377 nm (1b,d) (cf. ESI†).
The hydroxy-substituted derivatives have absorption maxima at
387 nm (2a) and 379 nm (2b),10 along with a weak, very broad
redshifted charge transfer (CT) band at ca. 450 nm.10 The
emission properties of the boronic acid derivatives 1a–d resemble
the ones of the parent benzo[b]quinolizinium (lfl = 410–415 nm;
Ffl = 0.03–012). In contrast, the fluorescence maxima of the
hydroxybenzo[b]quinolizinium derivatives are significantly red-
shifted with maxima at 527 nm (2a) and 600 nm (2b).10
The reaction of the derivatives 1a–d with H2O2 in buffer solution
produced the corresponding hydroxybenzo[b]quinolizinium deriva-
tives 2a and 2b (Scheme 1). Most notably, in the case of 1a–d the
1H-NMR-spectroscopic analysis of the reaction revealed a quantita-
tive transformation with no detectable side products (cf. ESI†). The
reaction of compounds 1a–d with H2O2 in buffer solution was also
monitored by emission spectroscopy (Fig. 1). The progress of the
reaction is clearly indicated by a continuous decrease of the
intensity of the emission maximum of compounds 1a–1d (415 or
417 nm) that is accompanied by the simultaneous increase of a
new redshifted emission band at 527 nm and 600 nm, respectively.
The latter emission bands are identical to the ones obtained with
authentic samples of the hydroxy-substituted derivatives 2a,b. The
ratios of the intensities of the emission maxima increase by a factor
of 17 (1a) and 0.7 (1b) at 140 min after addition of H2O2, as
quantified by the term Ired/Iblue. Control experiments showed that
the formation of 2a and 2b does not take place in buffer solution in
the absence of H2O2. The results indicate full conversion of the
substrates 1a,b to the products 2a,b within 140 min (1a) and
160 min (1b). The limit of detection was determined to be
3.0 mM for 1a and 5.9 mM for 1c (cf. ESI†), which is comparable
to that of established probes.5b,8a,c The reaction rate was analyzed
considering pseudo first-order kinetics according to an established
protocol.8c By this method, rate constants of kobs = 4.3 Â 10À4 sÀ1
(1a) and kobs = 2.9 Â 10À4 sÀ1 (1b) were obtained (cf. ESI†). To
assess the selectivity of the oxidation reaction of compounds 1a–d
they were also treated with other oxidants, namely O2 À, 1O2, ClOÀ,
ꢀ
NOꢀ, peroxides ROOꢀ, HOꢀ, and ONO2À. The reaction was mon-
itored by fluorescence spectroscopy, and the ratiometric analysis,
Ired/Iblue, was used to determine the progress of the reaction (Fig. 2).
Notably, the compounds 1a and 1c are efficiently oxidized by H2O2,
whereas the other oxidants only induce the foÀrmation of the
Fig. 1 Fluorimetric monitoring of the reaction of H2O2 (1.0 mM) with
1a (A, 10 mM, lex = 366 nm), 1b (B, 10 mM, lex = 361 nm), 1c (C, 10 mM, lex
=
366 nm), or 1d (D, 10 mM, lex = 366 nm) in phosphate buffer (0.1 M, pH = 7.4).
Arrows indicate the development of emission bands with time. Inset: plot of
the fluorescence maxima of 1a–d (K) and 2a,b (J) versus time.
À
ꢀ
oxidation product 2a to marginal or moderate (O2 , HOꢀ, ONO2
)
extent under resembling reaction conditions. A similar trend was
observed for the derivatives 1b and 1d, however, the selectivity is
less pronounced, and the ratio of Ired/Iblue is smaller because of the The cellular uptake was rapid and reached a plateau after
lower emission intensity of the reaction product 2b. 15–20 min of incubation in Jurkat cells and after 1 h in HeLa
The ability of the derivatives 1c and 1d to detect varying cells suggesting an efficient delivery of the compound inside
levels of H2O2 in living cells was assessed. For this purpose, we the cells. After 1 h of incubation 1c and 1d emit at 450 nm
used HeLa cell lines, i.e. an adenocarcinoma that grows as (FL9 channel), while no emission was detectable at 540 nm
monolayer, and Jurkat cells, i.e. leukemia cell lines that grow in (FL10 channel) (cf. ESI†). To evaluate the ability of derivatives 1c
suspension. In preliminary experiments the cellular uptake by and 1d to respond to H2O2 in a living cell, HeLa cells were
compound 1c was evaluated by flow cytometry taking advantage treated with H2O2 (100 mM) after the incubation with 1c and 1d,
of the intrinsic fluorescence of the compound (cf. ESI†). and the relative fluorescence intensities of the cells were
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Fig. 2 Ratiometric fluorimetric analysis of the reaction of 1a (A, black),
1c (A, grey), 1b (B, black), and 1d (B, grey) (c = 10 mM) with selected ROS
(c = 1.6 Â 10À4 M, resp.) in phosphate buffer (c = 0.1 M, pH = 7.4); t = 60 min;
1a, 1c: lex = 366 nm, 1b, 1d: lex = 361 nm; ROOꢀ = C4H9N2OOꢀ.
Fig. 4 Flow cytometric analysis of HeLa (A) and Jurkat cells (B) treated for
24 h with staurosporine (1 mM) and subsequently incubated with 1c for 20 min.
analyzed by flow cytometry (Fig. 3). After 10 min the fluorescence
intensity of 1c at 450 nm decreased, and a signal at 540 nm
developed (Fig. 3A and B). This development of emission signals
may be followed also by a ratiometric analysis (cf. ESI†). In the
case of 1d, however, only the signal at 450 nm disappeared, but
no redshifted emission was observed.
Furthermore, we evaluated if 1c could be useful to detect
physiological generation of H2O2 under pharmacological induction.
Thus, both HeLa and Jurkat cells were treated with staurosporine,
that is known to induce apoptosis through mitochondrial membrane
depolarization. The latter has been associated with mitochondrial
production of reactive oxygen species (ROS), in particular H2O2.14
HeLa and Jurkat cells were treated with the drug for 12 h and
afterwards incubated with 1c for 30 min. Subsequent analysis by
flow cytometry showed a significant increase of the population of
cells that emit at 540 nm in both cell lines (Fig. 4), thus demon-
strating the capability of compound 1c to indicate pharmaco-
logically stimulated formation of intracellular H2O2.
quantitative transformation to a product whose emission maxi-
mum is well separated from the one of the substrate thus
allowing ratiometric analysis, and the general ability to operate
in living cells. With respect to a relationship between function
and structure, it appears that benzo[b]quinolizinium fluoro-
phores, in which the ROS-sensitive borono-functionality as well
as the eventually formed hydroxy functionality are linearly
conjugated with the pyridinium unit, provide the more effective
structural platform for fluorimetric detection as compared to
the cross-conjugated system. Specifically, the 9-substituted
derivatives 1a and 1c exhibit a more pronounced light-up effect
and a higher selectivity towards H2O2 than the 8-substituted
ones. It should be noted that these probes may be easily
varied by the attachment of other functionalities R1 that are
transformed to the strongly solvatochromic hydroxy-substituted
derivative 2a.
Generous support by the Deutsche Forschungsgemeinschaft is
gratefully acknowledged.
In summary we have demonstrated that boronobenzo[b]-
quinolizinium derivatives exhibit several favorable properties for
fluorimetric detection of hydrogen peroxide, namely water solubility,
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