4.2. 9-[2-(3-Carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-ones (DPAXs)
The most widely used 1O2 trap is 9,10-diphenylanthracene (DPA), which reacts rapidly and
specifically with 1O2 to form a thermostable endoperoxide at a rate of k =1.3106 M1 s1.
The decrease in absorbance at 355 nm is used as a measure of the formation of the endoperoxide.
However, DPA derivatives are not very sensitive probes because the detection is based on the
measurement of absorbance [79].
Umezaka et al. [79] fused DPA with a fluorophore (fluorescein) aiming to associate the first’s
reactive characteristics with the second’s fluorescent characteristics. Fluorescein was chosen as
fluorophore since it has a high fluorescence quantum yield in aqueous solution and is able to be
excited at long wavelength. From this fusion resulted 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-
hydroxy-3H-xanthen-3-ones (DPAXs) (Fig. 11) [79]. Thus, DPAXs were the first chemical traps
for 1O2 that permitted fluorescence detection. They react with 1O2 to produce DPAX
endoperoxides (DPAX-EPs) (Fig. 11). DPAXs themselves scarcely fluoresce, while DPAXEPs
are strongly fluorescent. The mechanism accounting for the diminution of fluorescence in
DPAXs and its enhancement in DPAX-EPs remain unclear [79].
The fluorescence intensity of fluorescein derivatives is known to be decreased under acidic
conditions as a consequence of the protonation of the phenoxide oxygen atom. In order to
stabilize the fluorescence intensity at physiological pH, electron-withdrawing groups wereincorporated at the 2- and 7-positions of the xanthene chromophore, leading to Cl (DPAX-2) and
F (DPAX-3) (Fig. 11). This modification lowered the pKa value of the phenolic oxygen atom
[79].
DPAX-2 was used to detect the production of 1O2 from two different generation systems: the
MoO4
2/H2O2 system and the 3-(4-methyl-1-naphthy)propionic acid endoperoxide (EP-1)
system, which act at different pH values (10.5 and 7.4, respectively). In both cases an increase
of the probe’s fluorescence was verified when in contact with the generating system. These
results confirmed DPAXs’ advantage when detecting 1O2 in neutral or basic aqueous solutions
[79]. The behaviour of this probe towards H2O2, !NO and O2 ! was also studied, but no change in
the intensity of the fluorescence was observed for any of these reactive species. These facts
corroborate the specificity of this probe for 1O2 [79].
The detection of 1O2 in biological samples was also investigated. With this purpose, DPAX-2
diacetate (DPAX-2-DA) was prepared, since it was considered to be more permeable to cells.
DPAX-2-DA is hydrolysed by intracellular esterases to generate DPAX-2. Both DPAX-2 and
DPAX-2DA were tested and compared in the same assay systems. However, cells were stained
similarly in both cases. This observation probably means that DPAX-2 itself is also membranepermeable
[79].