Highly sensitive and selective bioluminescence based ozone probes and their applications to detect ambient ozone

Article abstract of DOI:10.1039/c5cc08622a

Highly selective and sensitive bioluminescence based probes, which respond to ozone but not to other ROS, have been developed. These probes were used to determine ozone concentrations in environmental samples.

Full text of DOI:10.1039/c5cc08622a

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Highly sensitive and selective bioluminescence
based ozone probes and their applications to
detect ambient ozone†
Cite this:DOI: 10.1039/c5cc08622a
Received 17th October 2015,
Accepted 5th November 2015
Younseok Nam, Beom Seok Kim and Injae Shin*
DOI: 10.1039/c5cc08622a
www.rsc.org/chemcomm
8
Highly selective and sensitive bioluminescence based probes, which light emission quantum yield. Bioluminescence is produced
respond to ozone but not to other ROS, have been developed. as a consequence of the luciferase catalyzed transformation of
These probes were used to determine ozone concentrations in D-luciferin or aminoluciferin into their corresponding oxyluciferin
2
+
environmental samples.
in the presence of O
emission process, unlike fluorescence, does not require the use
Reactive oxygen species (ROS), including the superoxide anion of an external light source. The advantageous features of bio-
2
, ATP and Mg (Scheme 1). Thus, this
À
ꢀ ꢀ
radical (O
), singlet oxygen ( O
known to be implicated in various physiological and patho- enzyme activities, detect biologically important species and test
2
), hydroxyl radical (HO ), hydrogen peroxide luminescence have prompted us to perform investigations targeted
1
À
(H
2
O
2
2
) and hypochlorite ion (ClO ), are at the development of bioluminescent probes to determine
1
6
logical processes. Another ROS, the strong oxidant ozone (O ), cellular uptake events. In a recent study described below, we
3
has become a harmful environmental pollutant. It is known designed and prepared the first bioluminescence based probe
that exposure to ozone causes damage to mucous and respiratory to selectively and sensitively detect ozone.
2
tissues of animals. However, ozone present in the stratosphere
(the ozone layer) is beneficial to living organisms because it
prevents harmful UV light from reaching the earth’s surface. On
the other hand, it has been suggested that ozone-like species are
3
possibly generated in cells. However, this is a controversial
proposal owing to the lack of selective and sensitive probes for
4
detecting ozone in living systems. Because of the environmental
issues and the biological significance of ozone, selective, sensitive
and facile methods to detect this ROS are in high demand.
Recently fluorescein, resorufin and near-infrared (NIR) based
fluorescent probes have been devised to detect ozone in bio-
5
logical and/or atmospheric samples. These probes respond to
ozone but not to other ROS. Although fluorescent probes have
been developed to detect in vitro and/or in vivo levels of various
6
biologically and environmentally important species, they are
often plagued by the presence of high background fluorescence.
As an alternative to using a fluorescence protocol, luciferase
based bioluminescent systems have received great attention
owing to their low backgrounds, high sensitivities and quanti-
7
tative capabilities. Firefly bioluminescence is the most well-known
Scheme 1 Bioluminescence detection of O
to generate 1,2,3-trioxolane. Retro
3+2] cycloaddition of 1,2,3-trioxolane gives aldehyde containing luciferin.
3
using probes 1 and 2. (A) The
photoemitting biological process that is associated with a high terminal alkene of 1 reacts with O
3
[
The resulting luciferin derivative subsequently undergoes b-elimination to
afford D-luciferin. (B) Probe 2 undergoes a similar process to 1 to produce
aminoluciferin. (C) The generated D-luciferin and aminoluciferin from 1
and 2, respectively, are oxidized to the corresponding oxyluciferin by
Center for Biofunctional Molecules, Department of Chemistry, Yonsei University,
Seoul 03722, Korea. E-mail: injae@yonsei.ac.kr
†
Electronic supplementary information (ESI) available: Experimental methods,
2+
supplementary figures and NMR data. See DOI: 10.1039/c5cc08622a
luciferase, in the presence of ATP, Mg and oxygen, with light emission.
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The strategy employed to design bioluminescence based
ozone probes is founded on the observation that 6-O-alkylated
and 6-N-acylated luciferin does not undergo light emitting
processes by firefly luciferase. This finding suggested that
linking 3-butenyl and 3-butenyloxycarbonyl groups to the
2
6-OH and 6-NH groups of luciferin might lead to the respective
derivatives 1 and 2 that will serve as probes for ozone (Scheme 1).
Thus, unlike the intact probes, products generated by reaction of
1
and 2 with ozone should undergo luciferase promoted light
emission. Specifically, the terminal double bonds in 1 and 2
are expected to undergo ozonolytic cleavage to generate aldehyde
containing compounds. Because aldehydes of these types are
normally not stable, it is expected that they would rapidly undergo
b-elimination to produce D-luciferin or aminoluciferin. Accord-
ingly, probes 1 and 2 would detect ozone as a consequence of the
fact that their ozonolysis leads to the generation of products that
should emit bioluminescence in the presence of luciferase, ATP,
2+
Mg and oxygen.
To test this proposal, probes 1 and 2 were synthesized accord-
ing to the procedure shown in Scheme S1 (ESI†) and characterized
employing NMR spectroscopy and MS. The abilities of 1 and 2 to
be converted by ozonolysis to respective D-luciferin and amino-
luciferin were initially explored. The results of HPLC and MS
Fig. 1 Bioluminescence spectra after treatment of (A) 1 and (B) 2 with O
3
.
Each probe was treated with various concentrations of O for 30 min. The
3
mixture was treated with 2 mM pyrrolidine for 5 min and then with
luciferase in assay buffer. Bioluminescence spectra were recorded on a
fluorimeter in bioluminescence measurement mode. The color ruler of
3
analyses show that the reaction of each probe with O for 1 h
mainly produces an aldehyde and its hydrate form, which are bioluminescence intensity is shown at the bottom.
in equilibrium in aqueous solutions (Fig. S1 and S2, ESI†).
D-Luciferin and aminoluciferin are formed as minor products in
these reactions. These findings indicate that b-elimination of O (10 mM) in the absence and presence of pyrrolidine. By using
3
initially formed aldehydes is much slower than the ozonolytic a bioluminescence microplate reader, we found that probes 1
cleavage of olefin precursors. Because it is known that pyrrolidine and 2 in the presence of pyrrolidine respond quickly to O
3
and
greatly accelerates b-elimination processes, the same reactions that the emission intensities reach maximal values within 5 min
were performed in the presence of pyrrolidine. HPLC analysis following the addition of O (Fig. S5, ESI†).
of the products reveals that D-luciferin and aminoluciferin are The ability of 1 and 2 to respond to ozone was also evaluated
generated exclusively from 1 and 2, respectively, after 1 h treat- by examining the changes in fluorescence intensities. Intact 1
ment with O in the presence of pyrrolidine (Fig. S1, ESI†). fluoresces with a wavelength maximum at 445 nm. After addition
9
3
3
The ability of probes 1 and 2 to sense O was evaluated by of ozone, the fluorescence emission from the probe red-shifts to
3
examining changes in the bioluminescence intensities of 1 and 530 nm, which is identical to fluorescence from D-luciferin
2
after treatment with O
the effect of O and pyrrolidine on luciferase activity. D-Luciferin a red shift of the fluorescence maximum from 455 nm to 500 nm,
and aminoluciferin were separately incubated with purified firefly which is characteristic of emission from aminoluciferin (Fig. S6B,
luciferase in the presence of various concentrations of O or ESI†). The results of this study also support the responses of the
pyrrolidine. The results show that neither substance influences probes to O . In addition, the findings show that 1 undergoes a
luciferase activity (Fig. S3, ESI†). Next, a solution of each probe larger ozone promoted Stokes shift (ca. 80 nm) than 2 (ca. 45 nm).
3
. This study was initiated by probing (Fig. S6A, ESI†). In the case of probe 2, ozone treatment promotes
3
3
3
(
2 mM) was first treated with O (10 mM) for 30 min and then with Moreover, the results of time-dependent studies demonstrate
3
2
mM pyrrolidine for 5 min and finally with luciferase. Ozone conclusively that 1 and 2 respond to ozone rapidly in the presence
treatment was found to promote a great increase in the bio- of pyrrolidine (Fig. S7, ESI†).
luminescence intensities of both probes (Fig. S4, ESI†). This
Because the selectivity toward ozone is a highly important
finding, along with the fact that each probe displays a very high requirement for probes, the response of the biolumines-
signal-to-background ratio, validates the strategy used for the design cence properties of 1 and 2 to various ROS was evaluated.
of the new bioluminescent probes for the detection of O
of spectra also shows that bioluminescence intensities of 1 and 2 O , 1 mM O , 1 mM H O , 1 mM OH, 1 mM O , 1 mM
increase in an ozone-concentration dependent manner and that ClO , and the myeloperoxide–H O –Cl system which gen-
under identical conditions the O3 promoted bioluminescence erates HOCl in equilibrium with Cl
3
. Analysis For this purpose, each probe (2 mM) was treated with 20 mM
1
ꢀ
À
ꢀ
3
2
2
2
2
À
À
0
2
2
1
2
.
Luciferase in assay
intensity of 1 is ca. 10 times larger than that of 2 (Fig. 1). buffer was then added to each mixture and the biolumines-
To evaluate time-dependent changes in bioluminescence cence intensity was measured by using a bioluminescent
intensities induced by ozone, 1 and 2 (2 mM) were treated with microplate reader. The results show that 1 and 2 respond to
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Table 1 Measurements of ozone concentrations by bioluminescence,
iodometric titration and UV spectroscopy methods (mean Æ s.d., n = 3)
Ozone
concentration (mM)
Sample
solution (nM)
Bioluminescence method 10.0 Æ 0.69 20.0 Æ 1.1 53.5 Æ 0.62
Iodometric titration
UV spectroscopy
9.73 Æ 0.50 19.6 Æ 1.3 N/A
10.4 Æ 0.66 21.2 Æ 1.7 N/A
N/A: not applicable. A sample solution was obtained at the main gate of
Yonsei University.
higher in areas with heavy automobile traffic. Moreover, ozone in
each of the samples could not be detected by using iodometric
titration and UV spectroscopy methods, which are typically
Fig. 2 Responses of (A) 1 and (B) 2 to various ROS. A solution of each probe
(
2 mM) in potassium phosphate buffer (5 mM, pH 7.0) was treated with 20 mM
1
ꢀ
À À
ꢀ
3
O , 1 mM
O
2
, 1 mM H
2
O
2
, 1 mM OH, 1 mM O
2
, 1 mM ClO or
myeloperoxidase (40 nM)–H
2
O
2
(200 mM)–NaCl (150 mM). After incubation employed to measure micromolar concentrations of this ROS.
for 1 h, luciferase in assay buffer was added to the mixture, and then bio- This observation demonstrates that the newly developed probe is
luminescence intensity was measured by using a bioluminescence microplate
reader (error bar: mean Æ s.d., n = 3). ‘Un’ in each graph means ‘untreated’.
highly sensitive to ozone.
In conclusion, in this investigation we designed and synthe-
sized the first bioluminescent probe that selectively and sensi-
ozone but not to other ROS, indicating their high levels of tively responds to ozone. We demonstrated the application of one
selectivity (Fig. 2).
of the probes for measuring ozone concentrations in environ-
The sensitivities of 1 and 2 for O
3
detection were determined mental samples. It is anticipated that the bioluminescence-based
next. Bioluminescence intensities of 1 and 2 linearly correlate probes may show great potential in detecting ozone in environ-
with ozone concentrations in the 0–400 nM (0–15 ppb) range mental and biological samples.
and the detection limits of 1 and 2 were determined to be 1.0 Â
This work was supported financially by a grant from the National
M, respectively (Fig. S8, ESI†). Because 1 Creative Research Initiative (2010-0018272) Program in Korea.
À11
À10
10
and 1.1 Â 10
exhibits a greater luminescence increase induced by ozone than
does 2, the former probe was used in further studies.
Recently, it was reported that amino acids, including tryptophan
Notes and references
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2
3
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11
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2
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(
11
reported procedure. As in the previous study, Indigo carmine was
used as the chemical probe. As the data given in Fig. S9A (ESI†)
show, the UV absorbance of indigo carmine at 610 nm is attenu-
ated in a concentration-dependent manner in irradiated solutions
containing Trp, Met, Cys and His, but not in those containing other
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4
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7
8
In the final phase of this effort, we demonstrated that probe 1
can be employed to detect O
convenient manner.
3
in environmental samples in a
For this purpose, open, wide mouth glass
bottles containing buffer solutions were placed at four different
5a,b
1
locations for 8 h. The ozone concentration of each sample was 11 K. Yamashita, T. Miyoshi, T. Arai, N. Endo, H. Itoh, K. Makino,
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1
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Chem. Commun.