10.1002/chem.201705546
Chemistry - A European Journal
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we had confirmed sufficient emission of most compounds in
aqueous solution (Table S3). Firstly, the non-toxicity of the
compounds was confirmed by incubating A549 cells with the
dyes at 100 µM concentrations for 90 mins, followed by
treatment with Alamar Blue cell viability reagent. No dye was
found to be toxic under these conditions (Figure S22), which far
exceeded those subsequently used in imaging studies.
Therefore, A549 cells were incubated with the synthesised
naphthalimides (10 μM, 20 min) and cellular fluorescence was
visualised by confocal microscopy following excitation with 405
or 488 nm lasers. All dyes could be observed in cells, and
despite only small structural changes, there are significant
differences in their cellular localisation (Figure S23-S35). A
majority of the dyes, such as N2 (Figure 4A) and N10 (Figure
4B), exhibit cytoplasmic localisation. However, some of the more
lipophilic derivatives, such as N8 (Figure 4C) and N11 (Figure
4D), show localisation in lipid droplets, which was confirmed by
colocalisation studies with Nile red (Figure S36-S40).
Interestingly, despite the fact that N8 emission is completely
quenched in HEPES buffer (Table S3), this compound could be
clearly seen in lipophilic areas of the cell at wavelengths
corresponding to its spectrum in acetonitrile or acetone.
emission wavelengths. The structure-photophysical property
relationships that we have been able to determine will enable
the rational design of further improved fluorophores in the future.
Importantly, all new naphthalimides showed good intracellular
fluorescence and low cellular toxicity, with localisation in the
cytoplasm or lipid droplets. These fluorophores are therefore
appropriate for incorporation into a wide range of fluorescent
sensors. The breadth of photophysical and biological behaviour
will enable selection of the most appropriate fluorophore for
each specific application. With this expansion in the scope of 4-
amino-1,8-naphthalimides, the future looks bright for this class of
fluorophore.
Acknowledgements
We thank the University of Sydney for funding.
Keywords: aromatic substitution • fluorescence • naphthalimide
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a
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