3-Hydroxychromone Derivatives
FULL PAPER
tyroxychromone derivatives 11a,b and 12a,b were also pho-
tophysically characterised (Table 1). These derivatives are
not able to exhibit dual fluorescence, ESIPT-mediated for-
mation of T*, since the protected hydroxyl group in the 3-
position lacks the intermolecular proton-transfer ability
(Scheme 1B). As expected, the spectra of 11a,b and 12a,b
exhibit only one emission band, assigned to the normal ex-
cited species (N*), in this case centred around 555 nm. The
slight displacement of these emission maxima (ca. 10 nm)
compared with 7a,b and 10a,b can be explained by the dif-
ference in electronic structure when attaching the 3-isobu-
tyroxy group to the chromone system. Thus, the measure-
ments on 11a,b and 12a,b confirm the conclusion about the
emission of 7a,b and 10a,b originating from the N* state.
In addition, when comparing 11a,b and 12a,b with the un-
protected derivatives 7a,b and 10a,b, we observe higher
molecular extinction values and dramatically decreased
quantum yields (from FF of 0.4–0.5 to 0.1) (Table 1). In
spite of this, the results indicate that esterification of the 3-
hydroxyl group results in compounds with acceptable and
useful fluorescence properties.
become widely used to study cellular processes and diseases
in several biomedical research areas.[2]
To explore the utility of 3-hydroxychromone derivatives
as fluorophores for cellular imaging, HeLa cells were incu-
bated with either compound 9 or 10a in two separate ex-
periments. The fluorescence was imaged by multiphoton
laser scanning microscopy (MPLSM) using two-photon exci-
tation (2PE). 2PE was employed as these compounds are
normally excited in the UV range. 2PE will also increase
cell viability and allow the use of an optimal excitation
wavelength. Live-cell imaging at 378C showed that both
compounds penetrated rapidly into the cells. The uptake
was already observed within a minute with an accumulation
localised between the cellular membrane and the nucleus. In
Figure 1, the uptake is shown at two different time points. In
All the 3-hydroxychromone derivatives (1–10) studied
here display high extinction coefficient values (e>
12000mꢀ1 cmꢀ1) with the highest values for 7a,b (e=
27000mꢀ1 cmꢀ1; see Table 1). On the contrary, as has been
discussed above, the difference in fluorescence quantum
yields between the derivatives is more pronounced
(Table 1). The extinction coefficients for the 3-isobutyroxy-
substituted derivatives (11, 12) were even higher (e=32000–
36000mꢀ1 cmꢀ1) than those for 7a,b, but again the quantum
yields are considerably lower. Thus, the 3-hydroxy group is
important for obtaining high quantum yields, but it is evi-
dentially possible to use esterified derivatives for fluores-
cence studies.
To investigate the utility of 3-hydroxychromones as fluo-
rophores for cellular imaging, the fluorescence quantum
yield of two compounds, 10a,b, which showed a more hy-
drophilic character, was established in water, and was found
to be virtually zero. It has been shown that the fluorescence
of a solution of 4’-diethylamino-3-hydroxyflavones in ethyl
acetate changes with the addition of water,[34] and becomes
completely quenched in pure water.[35] Instead, 2-(2-furyl)-
and 2-(2-benzofuryl)-3-hydroxychromones have shown that
ESIPT in water and the increase in quantum yields was es-
pecially pronounced for derivatives containing an electron-
donating substituent in the 7-position.[35] However, we rea-
soned that the quenching in water for 10a,b could be used
as an advantage since they would not be detectable in a hy-
drophilic cellular environment but instead exhibit fluores-
cence properties when moving into more hydrophobic areas,
for example, into a hydrophobic active site, a hydrophobic
receptor binding pocket or into membrane structures. We
therefore decided to investigate how these compounds
would behave as fluorophores for live-cell imaging.
Figure 1. Two-photon excitation (2PE), live-cell imaging showing uptake
of compound 9 (A, C, E) and 10a (B, D, F). Panels A and B: Single
plane, larger field of view (215ꢂ215 mm2), after approximately 20 min.
Scale bar=50 mm. Panels C and D: Uptake after 12 min, single planes
(74ꢂ74 mm2) from the middle of 9 mm thick z stacks. Scale bar=10 mm.
Panels E and F: Cross-sections through the z stacks along the dashed
lines in C and D. Panels C–F have been modified for clarity (negative
picture mode).
Figure 1A and B, larger fields of view of the uptake after ap-
proximately 20 min can be seen, whereas the uptake after
12 min is shown for a few cells in Figure 1C–F. Both com-
pounds seem to be taken up by endosomal structures, indi-
cated by the bright punctuate structures in the images, and
by weaker fluorescent membrane-network structures that re-
semble the endoplasmic reticulum. Furthermore, experi-
ments at 48C showed no cellular uptake, thereby supporting
Cellular studies: Fluorescence microscopy, including live-cell
imaging, is an essential tool in chemical biology and has
Chem. Eur. J. 2009, 15, 9417 – 9423
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
9421
Dyrager, Christine
