Virtues and limitations of Pittsburgh green for ozone detection

Article abstract of DOI:10.1039/c4cc08244c

A recently proposed 2′,7′-dichlorofluorescein (DCF)-derived fluorescent probe for the detection of ozone shows good selectivity against a number of reactive oxygen species and good pH stability for biological and environmental applications. It is found, however, that over oxidation of the fluorescent product (Pittsburgh green) can occur. This could render quantitative measurements inaccurate due to a reduction in fluorescence and overlapping fluorescence signals from over oxidation by-products and it requires careful experimental design. Although difficult to assess by fluorescence measurements, the over oxidation can be conveniently monitored by ¹H NMR spectroscopy.

Full text of DOI:10.1039/c4cc08244c

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DOI: 10.1039/C4CC08244C
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Virtues and limitations of Pittsburgh green for
ozone detection
Cite this: DOI: 10.1039/x0xx00000x
Carlos Castelló Beltrán,a,b Elliott A. Palmer,a Benjamin R. Buckley*a and
Felipe Iza*b
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
A recently proposed 2’,7’-diclorofluorescein(DCF)-derived
fluorescent probe for the detection of ozone shows good
selectivity against a number of reactive oxygen species and
good pH stability for biological and environmental
applications. It is found, however, that over oxidation of the
fluorescent product (Pittsburgh green) can occur. This
could render quantitative measurements inaccurate due to a
reduction in fluorescence and overlapping fluorescence
signals from over oxidation by-products and it requires
careful experimental design. Although difficult to assess by
fluorescence measurements, the over oxidation can be
conveniently monitored by 1H NMR spectroscopy.
the same product
2, albeit through an alternative reaction
pathway.5 With this in mind many research groups active in this
area have set out to produce extremely selective probes specific
to each type of ROS.
Several ozone probes have been proposed in recent years. For
example, a nearꢀinfrared fluorescent probe for monitoring ozone
in cells has been recently proposed by Tang and coꢀworkers
(TrpꢀCy, Figure 1).6 They designed the probe using
tricarbocyanine (Cy), a nearꢀinfrared fluorescent dye, and Lꢀ
tryptophan (Trp), as an O3 indicator. TrpꢀCy and Cy have very
different fluorescent excitations at about 630 and 770 nm
respectively and the λmax of the fluorescent emission for TrpꢀCy is
770 nm. The difference between the excitation and emission
wavelengths avoids interferences improving the detection
sensitivity.
The detection and quantification of reactive oxygen species
(ROS) has been an area of intense interest due in part to ROS
ubiquitous involvement in a range of biological processes.1,2 The
development of sensitive mechanisms by which to monitor and
detect ROS has found wide interest across a range of scientific
disciplines. From the range of possible methods of detection,
small molecule fluorescent probes have become increasingly
applied in the determination and quantification of ROS. This is
due to their high levels of sensitivity, simplicity in terms of data
collection, and high spatial resolution.3 However, one of the
major issues surrounding the development of chemical probes
for ROS detection revolves around their selectivity. For example,
in 1989 Takeuchi and Ibusuki identified ozone in biological
samples by chemiluminescence using indigoꢀ5,5’ꢀdisulfonate
(IDS, 1; Scheme 1).4 But it was later found by Wentwoth and
coworkers that IDS also reacts with singlet oxygen (1O2) to afford
The nearꢀinfrared fluorescent probe was tested with different
concentrations of ozone and good linearity between the ozone
concentration and the fluorescence was observed in the range
0.05ꢀ7.0 ꢁM of O3.6 TrpꢀCy was also exposed to chemical
species typically encountered in intracellular environments: ROS
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DOI: 10.1039/C4CC08244C
6000
3)
Probe (
6)
Product (
4)
DCF (
4000
2000
0
0
2
4
6
8
10
12
pH
does indeed liberate fluorescent material. The fluorescence,
however, changes over time and depends on when it is
measured after KI addition (Figure 4).10 This prompted us to
analyze the crude isolated reaction mixture and we found that
although it had been reported that treatment of the probe under
ozonolysis conditions affords the fluorescent compound 6a in
quantitative yield,7 1H NMR spectroscopy questioned its
generation (Figure 5).10 Crucially, reference 7 does not report
analytical data of the product and instead it points the reader to
an earlier publication in which the authors isolate the compound
such as singlet oxygen, hydrogen peroxide and hydroxyl radical;
RNS such as nitric oxide; biological antioxidants such as
glutathione and ascorbic acid; and metals ions such as K+, Ca2+
and Mg2+. The results of these tests showed high selectivity of
TrpꢀCy towards ozone but the synthesis of the probe is not
straight forward and of low yield.
Of simpler synthesis and higher yield is the probe developed by
Koide and coworkers for the selective and quantitative detection
1
6a and report H and 13C NMR data.11 This earlier publication,
of ozone (Scheme 2). The probe
diclorofluorescein (DCF,
, Scheme 3)7,8 and works by having a
homoallylic group appended to DCF analogue that is
essentially nonꢀflorescent. On addition of ozone, rapid ozonolysis
of the double bond proceeds to form , followed by loss of
propenal and liberation of the florescent product (Pittsburgh
3 is derived from 2’,7’ꢀ
however, deals with the sensing of palladium, hence formation of
the fluorescent product is carried out under different conditions
than those present in ozonolysis reactions. Further analysis of
4
a
the crude reaction mixture indicates that both
5 and 6 exist as a
5
mixture of ringꢀopened (5a/6a) and ringꢀclosed (5b/6b) materials,
and that following the ozonolysis step (and contrary to the
assumption made in reference 7), the side chain is not readily
liberated (NMR spectra show that 5a,b remain 24h after
exposure and fluorescence is seen to increase over time, Figure
4). Addition of Al2O3 or triethylamine, however, promotes
cleavage of the side chain, as shown also in reference 12. It is
noted that the addition needs to be subsequent to the ozone
exposure as the presence of the base quenches ozone and
prevents the ozonolysis of the probe. Therefore, fluorescent
measurements cannot be performed directly in real time.
6
Green9). The probe has been used in a variety of biological and
nonꢀbiological environments.7,8
As shown in figure 2, the probe has good selectivity against a
number of ROS including singlet oxygen, hydrogen peroxide,
hydroxyl radical, superoxide, hypochlorite, and peroxynitrite; and
the fluorescence is stable over a broad biologically relevant pH
range (6ꢀ9) (See figure 3).
Encouraged by the selectivity and quantitative yield reported in
reference 7 for the probe
3, we chose to prepare 3 in order to
use it in future ozone quantification experiments (Scheme 3).
Treatment of the probe
3 using a commercial ozone generator
200
400
2h after 30s O3 exposure
3 days after 30s O3 exposure
350
300
250
200
150
100
50
160
120
80
Fluorescence increases
over time as 5 slowly
converts to 6
40
0
0
1
.
.
-
-
1
2
3
4
5
6
7
8
Blank O3(0.6µM) O2(25µM) O2 (25µM) HO (25µM) H2O2(2mM) OCl (25µM) ONOO (35µM)
500
520
540
560
580
600
620
Wavelength (nm)
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DOI: 10.1039/C4CC08244C
Scheme 3. Synthesis of probe
3
and subsequent reaction products when treated with ozone
Fig 3. 1H NMR spectra of the probe
3 4 for comparison.
before and after varying treatments of ozone. 1H NMR spectra of authentic DCF
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DOI: 10.1039/C4CC08244C
7. A. L. Garner, C. M. St Croix, B. R. Pitt, G. D. Leikauf, S. Ando and
K. Koide, Nat. Chem., 2009, , 316-321.
8. A. L. Garner, C. M. St Croix, B. R. Pitt, G. D. Leikauf, S. Ando and
K. Koide, Nat. Chem., 2010, , 422–422.
9. K. Koide, F. Song, D. D. E. Groh, A. L. Garner, V. D. Mitchell, L. A.
Davidson and N. A. Hukriede, Chem. Bio. Chem. 2007, 214–218.
1
400
380
360
340
320
300
280
260
240
220
Fluorescence emission at 520nm
2
9
10. In order to check our findings were not due to a problem with the
source of ozone, two ozone generators from different suppliers were
employed and similar results were obtained with both devices. See
supplementary information for details.
After 30 seconds probe 3 has been
consumed and fluorescence decreases
due to degradation of 6 in ozone
11. F. Song, A. L. Garner and K. Koide, J. Am. Chem. Soc. 2007, 129
,
12354-12355.
12. K. L. Leslie, D. Li and K. Koide, J. Org. Chem. 2011, 76, 6860-6865.
0
50
100
150
200
250
300
O3 exposure time (s)
Fig 6. Observed Fluorescence Over Increased O3 Exposure Time
Finally, it is noted that the fluorescent product and the
intermediates (compounds 5a,b/6a,b) undergo oxidation in the
presence of ozone, leading to the undesired loss of product 5/6
and the formation of DCF
4 (Scheme 3). Loss of product 6 and
subsequent oxidation of DCF accounts for a decrease in
fluorescence over increasing O3 exposure time (Figure 6).4
In summary, probe
3 provides a useful means for selective
ozone detection across a broad pH range. If one wished to
employ it as a quantitative ozone probe, however, attention
needs to be paid to the incomplete cleavage of the side chain
and the formation of DCF
4, especially as DCF 4 and the
fluorescent product 6a,b have very similar fluorescence spectra.9
These limitations can partially be overcome by performing the
experiments in an excess of probe
3, under wellꢀmixed
conditions and recording fluorescence at fixed intervals after the
addition (post ozone exposure) of a base to promote the sideꢀ
chain cleavage.
We thank Loughborough University for a PhD studentship (to
C.C.ꢀB.), Research Councils UK for a RCUK Fellowship (to
B.R.B.) and the UK Engineering and Physical Science Research
Council (F.I.)
Notes and references
1
X. Chen, X. Tian, I. Shin and J. Yoon, Chem. Soc. Rev., 2011, 40,
4783-4804.
2. M. E. Jun, B. Roy and K. H. Ahn, Chem. Commun., 2011, 47, 7583-
7601.
3. P. Wardman, Free Radic. Biol. Med. 2007, 43 995–1022
4. K. Takeuchi and T. Ibusuki, Anal. Chem., 1989, 61, 619-623.
5. P. Wentworth, J. E. McDunn, A. D. Wentworth, C. Takeuchi, J.
Nieva, T. Jones, C. Bautista, J. M. Ruedi, A. Gutierrez, K. D. Janda,
B. M. Babior, A. Eschenmoser and R. A. Lerner, Science
2002, 298, 2195-2199.
,
6. K. Xu, S, Sun, J. Li, L. Li, M. Qiang and B. Tang, Chem. Commun.
2012, 48, 684-686.
,
4 | J. Name., 2012, 00, 1-3
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