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ChemComm
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DOI: 10.1039/C7CC04635A
Journal Name
COMMUNICATION
performing Griess assay (Figure S7). The percentage of NO Further, to find out the suitability of ONB-COU-DEA-NONOate
generated was found to be 84 % (1 PE) and 25 % (2 PE). for photoresponsive anticancer therapy, dose dependent
Further the gradual rise in the fluorescence during photolysis cytotoxicity study has been performed on MDA-MB-231 cell
(Figure S8), due to COU generation has been monitored by lines by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium
recording the emission at constant intervals. From figure 3 it bromide assay (MTT assay), both before and after exposure to
could be stated that, the rise in emission intensity is in direct UV irradiation.18 For this study two 96 well plates with MDA-
correlation with the amount of NO generated.
MB-231 cells were incubated with different concentrations of
ONB-COU-DEA-NONOate and ONB-COU-Ac (4,5-dimethoxy-2-
nitrobenzyl-7-hydroxy-(coumarin-3-yl)methyl-O2-conjugated
acetate-cascade photocage of inert metabolite acetate) under
same conditions. One set of cells were exposed to UV light
(≥365 nm, 30 min) where as the other was left in normal
condition (30 min) and eventually both the plates were
incubated for 24 h. From the cell viability results obtained after
irradiation, the effective concentration of ONB-COU-DEA-
NONOate was found to be 10 μM (Figure 5). After photolysis
the percentage of cell viability for ONB-COU-DEA-NONOAte
and ONB-COU-Ac obtained (Figure 5) implies that the cell
death is solely induced by the released DEA-NONOate and not
by other photoproducts.
Figure 3. (a) % NO released and (b) % emission intensity upon photolysis of ONB-COU-
DEA-NONOate (10-4 M in 3:7 ACN:PBS; pH 7.4) by 1 and 2 PE.
The evolution of two new peaks (PP, COU) in HPLC pattern and
high percentage of NO generated, indicate that cascade
photocaging of DEA-NONOate has assisted in surpassing the
limitations associated with direct photocaging (path B in
Scheme 1) as anticipated. From analyzing above observations
the following mechanism has been proposed for photo-
uncaging of ONB-COU-DEA-NONOate (Scheme S2): Firstly,
upon irradiation ONB-COU-DEA-NONOate gets excited and
activates the photorelease of PP and phenolate of COU-DEA-
NONOate via the aci-nitro intermediate.11 Subsequently,
phenolate of COU-DEA-NONOate thus obtained undergoes
spontaneous 1,8 elimination resulting in the formation of
coumarinyl quinone methide intermediate and release of DEA-
NONOate anion. In aqueous medium (pH 7.4) coumarinyl
quinone methide intermediate spontaneously rearranges itself
to the fluorophore COU, where as DEA-NONOate anion rapidly
releases 2 molecules of NO and DEA.
Figure 5. Dose-dependent photoinduced cytotoxicity study of ONB-COU-Ac and ONB-
COU-DEA-NONOate, obtained before irradiation (a) and after irradiation (b), with UV
light (30 min). Values are presented as means ± standard deviations of three different
observations.
In summary, we have developed a simple and novel strategy
i.e. Cascade photocaging, for the protection of DEA-NONOate,
that helps in evading undesirable photochemical pathways and
carcinogenic byproduct formation. Further successful photo-
uncaging of ONB-COU-DEA-NONOate upon 1 PE and 2 PE has
been demonstrated. The release of fluorescent COU molecule
was utilized in self monitoring the NO release. ONB-COU-DEA-
NONOate has shown potential anticancer activity on MDA-MB-
231 cell line and its real time reporting facility also has been
studied using confocal imaging. Further, this proof of concept
can aid in successful design and development of various
cascade photocages of diazeniumdiolates comprising
combinations from a wide variety of phototriggers and a
suitable light activable linker based on quinone-methide
reactivity.
In addition, to comprehend the in vitro photoinduced real time
reporting facility of ONB-COU-DEA-NONOate, time dependent:
cell imaging was carried out. For this, MDA-MB-231 cells were
treated with 10 μM (effective concentration) of ONB-COU-
DEA-NONOate and exposed to UV light (≥365 nm) for different
time intervals, under similar conditions. Later on, confocal
images of the cells were obtained at regular intervals over a
period of 30 min irradiation. From the images it could be
observed that there is a gradual increase in the cytoplasmic
fluorescence of cells with irradiation exposure time (Figure 4).
This signifies the internalisation of ONB-COU-DEA-NONOate
and light triggered diazeniumdiolate and COU release.
We thank DST-FIST for 400MHz NMR. K. K. Behara (IF110732)
and Y.R (IF130658) are thankful to DST (AORC Programme)
for Inspire fellowship.
F
igure 4. Confocal images of MDA-MB-231 cells incubated with ONB-COU-NONOate
(10 µM), obtained after exposing to UV light (≥ 365 nm) at irradiation time intervals (a)
control (t = 0 min), (b) 10 min, (c) 20 min, (d) 30 min. (Scale bars: 20 μm)
Notes and references
1
L. K. Keefer, ACS Chem. Biol., 2011, 6, 1147–1155.
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