Energy transfer in aminonaphthalimide-boron-dipyrromethene (BODIPY) dyads upon one-and two-photon excitation: Applications for cellular imaging

Article abstract of DOI:10.1002/asia.201301334

Aminonaphthalimide-BODIPY energy transfer cassettes were found to show very fast (k_EET≈10¹⁰-10¹¹ s^-1) and efficient BODIPY fluorescence sensitization. This was observed upon one-and two-photon excitation, which extends the application range of the investigated bichromophoric dyads in terms of accessible excitation wavelengths. In comparison with the direct excitation of the BODIPY chromophore, the two-photon absorption cross-section δ of the dyads is significantly incremented by the presence of the aminonaphthalimide donor [δ≈10 GM for the BODIPY versus 19-26 GM in the dyad at λ_exc=840 nm; 1 GM (Goeppert-Mayer unit)=10^-50 cm⁴ s molecule^-1 photon^-1]. The electronic decoupling of the donor and acceptor, which is a precondition for the energy transfer cassette concept, was demonstrated by time-dependent density functional theory calculations. The applicability of the new probes in the one-and two-photon excitation mode was demonstrated in a proof-of-principle approach in the fluorescence imaging of HeLa cells. To the best of our knowledge, this is the first demonstration of the merging of multiphoton excitation with the energy transfer cassette concept for a BODIPY-containing dyad. Take two: Aminonaphthalimide-BODIPY energy transfer cassettes were designed in order to take advantage for bioimaging from one-and two-photon excitation coupled with energy transfer. The bichromophoric dyads showed ultrafast and highly efficient energy transfer and were used for confocal fluorescence microscopy imaging of HeLa cells as bio-relevant models. Copyright

Full text of DOI:10.1002/asia.201301334

FULL PAPER
DOI: 10.1002/asia.201301334
Energy Transfer in Aminonaphthalimide-Boron-Dipyrromethene (BODIPY)
Dyads upon One- and Two-Photon Excitation: Applications for Cellular
Imaging
Daniel Collado,[a] Patricia Remꢀn,[b] Yolanda Vida,[a] Francisco Najera,[a] Pratik Sen,[c]
Uwe Pischel,*[b] and Ezequiel Perez-Inestrosa*[a]
Abstract:
Aminonaphthalimide–
the dyads is significantly incremented
by the presence of the aminonaphthali-
mide donor [dꢀ10 GM for the
BODIPY versus 19–26 GM in the dyad
at lexc =840 nm; 1 GM (Goeppert–
a precondition for the energy transfer
cassette concept, was demonstrated by
time-dependent density functional
theory calculations. The applicability of
the new probes in the one- and two-
photon excitation mode was demon-
strated in a proof-of-principle approach
in the fluorescence imaging of HeLa
cells. To the best of our knowledge, this
is the first demonstration of the merg-
ing of multiphoton excitation with the
energy transfer cassette concept for
a BODIPY-containing dyad.
BODIPY energy transfer cassettes
were found to show very fast (kEET
ꢀ1010–1011 sꢁ1) and efficient BODIPY
fluorescence sensitization. This was ob-
served upon one- and two-photon exci-
tation, which extends the application
range of the investigated bichromo-
phoric dyads in terms of accessible ex-
citation wavelengths. In comparison
with the direct excitation of the
BODIPY chromophore, the two-
photon absorption cross-section d of
Mayer
unit)=10ꢁ50 cm4 smoleculeꢁ1
photonꢁ1]. The electronic decoupling
of the donor and acceptor, which is
Keywords: bioimaging
· boron ·
energy transfer · fluorescence · two-
photon absorption
Introduction
dynamic therapy.[10–14] Furthermore, they have been used in
chemosensing,[8,15–17] molecular switching,[18,19] and for the re-
alization of logic operations.[12,13,15] These dyes are character-
ized by large molar absorption coefficients (e) and high fluo-
rescence quantum yields (Ffl), resulting in extraordinary
bright emitting (eꢀFfl) fluorophores.[1–3] However, the gen-
erally very small Stokes shifts of BODIPY dyes may cause
re-absorption or effects from excitation-light scattering,
which is a potential drawback for their application, especial-
ly in bioimaging. One strategy to bypass this problem is the
integration of BODIPY dyes as acceptors in bi- or multi-
chromophoric arrays, some of them qualifying as energy
transfer cassettes where donor and acceptor units are elec-
tronically non-conjugated.[8,17,20–26] The electronic energy
transfer (EET) from a donor with adequate photophysical
characteristics to the BODIPY moiety may proceed through
space (often Fçrster resonance energy transfer)[21,22,26] or
through a bond mechanism;[7,8,23,24] actually, both mecha-
nisms may act in parallel.[21] This yields an artificial increase
in the Stokes shift, also known as virtual or pseudo-Stokes
shift. A complementary approach that evolved from techni-
cal advances in multiphoton techniques[27,28] builds on the
two-photon excitation of BODIPY dyes, which facilitates
the use of near-infrared laser sources and separates the exci-
tation spectrally from the emission through upconver-
sion.[5,6,29,30] In this context, two-photon excitation (TPE)
fluorescence laser-scanning microscopy has opened a steadily
expanding field of in vivo imaging of tissues, being a tool for
the diagnosis and therapy monitoring of pathological condi-
Boron dipyrromethene (BODIPY) dyes[1–3] are nowadays
widely commercialized chromophores that have attracted
immense interest as versatile tools in biological chemistry,
for example, as pH sensors in intracellular environments,[4]
as probes in fluorescence imaging,[5–9] or as agents in photo-
[a] Dr. D. Collado, Dr. Y. Vida, Dr. F. Najera, Prof. E. Perez-Inestrosa
Department of Organic Chemistry
University of Malaga
29071 Malaga (Spain)
and
Andalusian Center for Nanomedicine and Biotechnology - BION-
AND
29590 Malaga (Spain)
Fax : (+34)952137565
E-mail: inestrosa@uma.es
[b] P. Remꢁn, Dr. U. Pischel
CIQSO - Center for Research in Sustainable Chemistry
and Department of Chemical Engineering, Physical Chemistry, and
Organic Chemistry
University of Huelva
21071 Huelva (Spain)
Fax : (+34)959219983
E-mail: uwe.pischel@diq.uhu.es
[c] Dr. P. Sen
Department of Chemistry
Indian Institute of Technology Kanpur
Kanpur 208 016, U.P. (India)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201301334.
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tions.[31–33] In the present work, we aimed at the design of
BODIPY-containing dyads that combine the possibilities of
one- and two-photon excitation (OPE and TPE, respective-
ly) with efficient EET, thereby opening a way to integrate
multiple excitation channels that lead to observation of
BODIPY fluorescence. The approach of two-photon EET
has been validated for a handful of case studies[34–38] and has
found applications in bioimaging recently.[39,40] However, to
the best of our knowledge, this is the first attempt of merg-
ing this concept with BODIPY energy transfer cassettes. In
detail, herein we report the design, synthesis, and photo-
physical properties of the new fluorescent dyads 1 and 2
that integrate 4-aminonaphthalimide and BODIPY chromo-
phores (Scheme 1).
Scheme 2. Synthesis of the dyads 1 and 2 and structures of model com-
pounds 3 and 4.
Synthesis
The dyads were synthesized (see Scheme 2) in a three-step
sequence starting by condensation of 4-bromo-1,8-naphthal-
ic anhydride with the corresponding 4-aminoalkylbenzoic
acid. The resulting products (5 or 6) were then transformed
into the aminonaphthalimides 7 or 8 by nucleophilic substi-
tution with n-butylamine at the aromatic 4-position of the
dicarboximide. Afterwards, the carboxylic acids were acti-
vated in form of their acyl chlorides (treatment with SOCl2)
and then reacted with kryptopyrrole in the presence of NEt3
as base and BF3-OEt2 to yield the BODIPY derivatives
1 and 2 (Scheme 2). The first two steps yielded the respec-
tive products in good to excellent yields (70–92%). The
final step, being an unoptimized reaction, gave the
BODIPY dyads 1 and 2 in moderate but sufficient yields
(ca. 35–40%). As a side note, the molecular design could be
potentially expanded by taking advantage of the relatively
straightforward functionalization of 4-bromonaphthalimides
by nucleophilic substitution with amines (e.g., propargyla-
mine) that provide bioconjugatable units, such as terminal
alkynes, which may be implied in click chemistry. The model
chromophore 3 is a known compound and was synthesized
according to a published procedure.[43] Details about the
preparation of the model 4 can be found in the Supporting
Information.
Scheme 1. Structures of the dyads 1 and 2.
Results and Discussion
Molecular Design
The molecular design criteria for the dyads can be resumed
as follows. The BODIPY part is expected to be the energy
acceptor. The energy donor moiety should absorb in a wave-
length region where the BODIPY has an optical window
(ca. 420–450 nm). This maximizes the antenna effect of the
donor and ensures its selective excitation. The energy trans-
fer should be efficient and thus, following the Fçrster theory
for through-space EET, the spectral overlap integral J be-
tween the donor emission and the BODIPY absorption
should be maximized.[41] Furthermore, the unquenched
donor emission quantum yield should be sufficiently high in
order to obtain large values for the critical transfer radius
(R0). The two-photon absorption properties of the energy
donor should reflect a significant absorption cross section.
Finally, for the sake of an accurate treatment of the photo-
physical processes, the geometry of the dyad (determining
the real distance and relative orientation of donor and ac-
ceptor) should be known. This requires a spacer with limited
conformational freedom.[42] The outlined design criteria are
fulfilled for the dyads 1 and 2 as will be discussed in detail
below.
Spectroscopic Properties upon One-Photon Excitation
(OPE)
The photophysical properties of the dyads 1 and 2 in aceto-
nitrile solution are summarized in Table 1. The UV/Vis ab-
sorption spectra show the typical bands of the separated
chromophores as can be easily seen by comparison with the
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Table 1. Photophysical properties of the dyads 1 and 2, and the models 3
and 4 in acetonitrile.
[a]
[b]
[d]
[e]
labs
lfl
D[c]
[cmꢁ1
Ffl
tfl
[nm]
[nm]
G
]
[ns]
1
2
522 (50100)
442 (13700)
522 (56400)
434 (15400)
521 (69600)
431 (13700)
534
534
535
535
534
523
430
3898
465
4350
467
4081
0.65[f]
0.34[g]
0.64[f]
0.39[g]
0.72[f]
0.43[g]
5.43 (96%), 2.41 (4%)
5.10 (76%), 7.58 (24%)
5.80 (97%), 2.45 (3%)
5.49 (65%), 7.65 (35%)
5.16
3
4
9.70
[a] UV/Vis absorption maximum (the corresponding molar absorption
coefficient e in Mꢁ1 cmꢁ1 is given in parenthesis). [b] Fluorescence emis-
sion maximum. [c] Stokes shift, calculated as difference between emission
and absorption maxima. [d] Fluorescence quantum yield; 15% error.
[e] Nanosecond fluorescence lifetime measured by time-correlated single-
photon-counting; 5% error, for the dyads, biexponential fitting was ap-
plied. A rise component is observed in the picosecond resolved fluores-
cence kinetic traces (see text and the Supporting Information). [f] Excita-
tion of BODIPY; rhodamine 6G as standard (Ffl =0.95 in ethanol).
[g] Excitation of aminonaphthalimide; quinine sulfate (Ffl =0.55 in 0.05m
H2SO4) as standard for 4, for the dyads 1 and 2, the value was measured
against 4.
Figure 2. UV/Vis absorption (dashed lines) and fluorescence spectra
(solid lines) of the models 3 (black) and 4 (gray) in acetonitrile (ꢀ10 mm
each). The absorption spectra are normalized to the long-wavelength
band of 3 and reflect the relative molar absorption coefficients. The fluo-
rescence spectra reproduce the relative emission quantum yields of the
models.
(see Figure 1 for dyad 1 and the Supporting Information for
dyad 2), and no sign of aminonaphthalimide emission (onset
below 500 nm) was observed. As will be discussed below,
this supports an efficient EET process. The quantum yields
of the dyads upon BODIPY excitation are rather high (Ffl
~0.65) as commonly observed for BODIPY derivatives.[1–3]
However, the quantum yields are somewhat reduced for the
excitation of the aminonaphthalimide (Ffl =0.34–0.39); see
the discussion below. The lifetime decays of the dyads are of
bi-exponential nature, but the major component of each
dyad is very similar to the lifetime of the BODIPY model 3
(see Table 1).
Figure 1. UV/Vis absorption spectrum (solid line) and fluorescence spec-
tra of dyad 1 upon excitation of the aminonaphthalimide (442 nm; dotted
line) and the BODIPY (500 nm; dashed line) in acetonitrile; [1]ꢀ10 mm.
The emission spectra are not corrected for the different amount of ab-
sorbed excitation light (see arrows that assign the excitation wave-
lengths).
Excited-State Communication between the Chromophores
As mentioned above, in the dyads the aminonaphthalimide
plays the role of the energy donor, while BODIPY is the ac-
ceptor and emitter. In accordance with the “cassette ap-
proach”, both chromophores in dyad 1 are linked by a non-
conjugated methylene bridge, thereby promoting through-
space EET. For dyad 2, geometry optimizations (Gaussi-
model compounds 3 and 4 (see Figures 1 and 2 as well as
the Supporting Information for dyad 2).[44] The band with
a maximum at about 522 nm corresponds to the S0!S1 tran-
sition of the BODIPY fragment, while the broad band with
a maximum at about 430–440 nm is typical for the charge-
transfer transition of the aminonaphthalimide chromo-
phore.[45] It can be further seen that the aminonaphthalimide
band is situated in the absorption window of the BODIPY
part. This enables the selective excitation of either chromo-
phore. The electronic independence of the two chromo-
phores in the dyads is also confirmed by time-dependent
density functional theory calculations that show chromo-
phore-specific transitions with no sign of mixing:
HOMOꢁ1!LUMO for the aminonaphthalimide and
HOMO!LUMO+1 for the BODIPY moiety (see contour
plots in the Supporting Information).
an 09)[46] at the B3LYP\6-311G
ACTHNUTRGNE(UNG d,p) level of theory have
shown that both chromophores are decoupled due to the
perpendicular orientation of the central phenylene unit (see
Figure 3 bottom). This is most likely the result of steric hin-
drance. Hence, it can be assumed that dyad 2 is also opera-
tive mainly by through-space EET.[20] Hence, the Fçrster
theory can be applied for both dyads, invoking exclusively
experimental parameters.[41]
By maximizing the donor fluorescence quantum yield and
the spectral overlap integral of donor emission and acceptor
absorption, it is relatively straightforward to arrive at
a donor–BODIPY pair with predicted unit EET efficiency.
These requirements are fulfilled in our dyads, yielding
a spectral overlap integral J of 1.6ꢀ10ꢁ10 cm6 molꢁ1 and criti-
cal EET radii R0 of 28.7 ꢃ and 40.9 ꢃ for dyad 1 and 2, re-
spectively. The different critical radius for the dyads results
from the variations in the donor–acceptor orientation factor
Regardless of the excitation wavelength, the fluorescence
of the dyads corresponds exclusively to the BODIPY part
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values obtained for the direct excitation of BODIPY (Ffl
~0.35 versus 0.65; see Table 1). This may be explained by in-
voking a photoinduced electron transfer from the excited
singlet aminonaphthalimide to the BODIPY as pathway that
competes with the EET. The application of the Rehm–
Weller equation[47] allowed us to estimate the thermodynam-
ic feasibility of such a process (DG ca. ꢁ0.1 eV).[48] These ef-
fects were observed for both dyads and they are therefore
not dependent on the bridge that connects both chromo-
phores. Hence, it can be concluded that the postulated elec-
tron transfer follows a through-space mechanism. Notewor-
thy, the opposite electron transfer from ground-state
BODIPY to excited-state aminonaphthalimide is unlikely.
This conclusion is based on observations for other amino-
napthalimides that carry the electron donor at the “imide
side” and that was reasoned with a molecular electric-field
effect and the help of theoretical calculations.[49,50] Finally,
electron transfer under involvement of the excited BODIPY
is practically excluded because the fluorescence quantum
yield upon direct excitation (lexc =500 nm) of this chromo-
phore is basically not altered in comparison to the model
compound 3 (see Table 1). In any case, the EET obtained
fluorescence quantum yields are sufficiently high to enable
the use of the dyads in fluorescence imaging as will be
shown below.
Figure 3. B3LYP\6-311GACHTUNGTRENNUNG(d,p)-optimized ground-state geometries and
data for calculation of the orientation factor k2 corresponding to dyad
1 (a) and 2 (b).
Two-Photon Absorption (TPA) Properties
k2 (0.0295 for dyad 1 and 0.2451 for dyad 2; see required
angles for the calculation in Figure 3 and more details in the
Supporting Information). The real donor–acceptor distances
of the dyads, which were calculated from the energy-mini-
mized geometries, are well below the critical radius (R=
10.3 ꢃ and 10.9 ꢃ for dyad 1 and 2, respectively). This indi-
cates an efficient and competitive energy transfer. Further-
more, with the lifetime of the unquenched donor (model
compound 4; Table 1), rate constants kEET of 4.8ꢀ1010 sꢁ1
and 2.9ꢀ1011 sꢁ1 are predicted for dyad 1 and 2, respective-
ly; see, for example, ref. [41] for the standard calculation of
EET parameters according to the Fçrster theory. The very
fast EET was experimentally confirmed by fitting the rise
component of the BODIPY emission kinetics of the dyads,
yielding kEET =1.0ꢀ1011 sꢁ1 (for 1) and 9.6ꢀ1010 sꢁ1 (for 2);
see the Supporting Information. The agreement of the order
of magnitude between the values calculated by application
of the Fçrster theory and the picosecond lifetime measure-
ments is quite satisfying when taking into account that the
structural parameters (k2 and R) were determined for gas-
phase optimized structures. In accordance with the concep-
tual approach of EET cassette designs, the Stokes shift of
the BODIPY part is effectively increased from about 450 to
approximately 4000 cmꢁ1, comparing the direct and the sen-
sitized pathways (see Table 1). The latter value is in the
order of the Stokes shift observed for typical charge-transfer
chromophores such as the aminonaphthalimide 4.
After establishing the dyads as EET cassettes, we were in-
terested in combining this feature with nonlinear optical ex-
citation of the energy donor. The TPA cross-sections d of
the dyads 1 and 2 and their model chromophores 3 and 4
were measured (see Figure 4), using rhodamine B as a refer-
ence.[51] The BODIPY fluorophore alone (model 3) shows
TPA cross-sections d of about 10 GM [1 GM (Goeppert–
Mayer unit)=10ꢁ50 cm4 smoleculeꢁ1 photonꢁ1] in the range
from 740 to 840 nm and an additional weaker band with
a maximum at about 1020 nm (d<5 GM). On the other
hand, the aminonaphthalimide model 4 shows two maxima
at 840 and 870 nm with cross-sections of 17 and 10 GM, re-
spectively. Finally, the dyads 1 and 2 combine the TPA fea-
tures of both chromophores, thus leading to increased TPA
cross-sections at 840 nm (d=19–26 GM). Hence, in compari-
son with the BODIPY model 3 the absorption cross-section
at 840 nm is effectively enhanced by 160 and 90% in the
dyads 1 and 2, respectively. The two-photonic nature of the
observed phenomena was confirmed by the quadratic de-
pendence of the fluorescence emission on the laser power
(see Figure 4 for dyad 1).[6] It was gratifying to observe that
the excitation of the aminonaphthalimide moiety in 1 under
OPE (440 nm) and TPE (880 nm) conditions yields virtually
identical BODIPY emission spectra with a maximum at
around 530–540 nm (see Figure 1 and inset in Figure 4). Sim-
ilar results were obtained for dyad 2. The wavelength of
880 nm was chosen for the TPE experiments in order to
comply with the general idea of lTPE ꢀ2lOPE (see ref. [27]).
This wavelength is slighly different from the somewhat blue-
Interestingly, the measured fluorescence quantum yields
of BODIPY emission upon excitation of the aminonaphtha-
limide donor in the dyads are somewhat lower than the
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Figure 5. Upper row: Confocal microscopic images of HeLa cells incubat-
ed for 1 h with dyad 1 (lexc =514 nm): fluorescence (left), overlaid fluo-
rescence and transmission images (middle), and a magnified image of
one cell (right). Lower row: Two-photon confocal microscopic images of
HeLa cells incubated with dyad 1. Laser excitation at 720 nm (left),
880 nm (middle), and 1050 nm (right).
Figure 4. Two-photon absorption spectra for 1 (closed circles), 2 (closed
squares), 3 (open squares), and 4 (open circles) in N,N-dimethylforma-
mide. The insets show the fluorescence spectrum of dyad 1 (top) and the
corresponding dependence of the fluorescence signal on the relative laser
power (bottom) at lexc =880 nm under TPE conditions.
with dyad 1 (dyad 2 resulted in similar images; see the Sup-
porting Information). As can be seen, dyad 1 is easily inter-
nalized by the cells and no signs of morphological damage
are observed. A closer inspection of the images revealed
that the probe is preferentially localized in the cytoplasm
and does not enter the cell nucleus. The samples were also
irradiated at wavelengths where the aminonaphthalimide
absorbs dominantly (405 and 458 nm) and, in agreement
with the efficient EET, the obtained fluorescence corre-
sponded to BODIPY (see Supporting Information).
The feasibility of TPE under conditions of cell imaging
was finally demonstrated with the HeLa-cell-incubated
dyads (Figure 5 and the Supporting Information), yielding
similar results as for OPE conditions. In Figure 6, the emis-
sion spectra of selected points of the confocal microscopic
image upon TPE are shown, corresponding to the BODIPY
emission.
shifted maximum of the TPA spectrum (840 nm), a discrep-
ancy that is often observed for TPA (see, for example,
ref. [28]). Noteworthy, it comes as a surplus that under our
conditions the enhancement of the TPA cross-section, com-
paring the dyads 1 and 2 with the BODIPY model 3, is
maximized at around 880 nm (enhancement by 330% and
130% for the dyads 1 and 2, respectively).
The involvement of the aminonaphthalimide in the two-
photon-excitation EET process in the dyads is further corro-
borated by the shape of the TPA spectra (Figure 3) that
present unambiguously the characteristics of the corre-
sponding spectrum of the energy donor model 4. As the
TPA spectra of the dyads are obtained by observing the
BODIPY emission in two-photon-excitation fluorescence
measurements,[51] this is interpreted as the population of the
S1 state of the energy donor under TPE conditions and the
subsequent EET[35] to the BODIPY acceptor. Hence, TPE
fluorescence imaging can be further improved by implying
EET. In this way multiple wavelengths are accessible for the
excitation of the energy donor and acceptor in the OPE as
well as the TPE regime.
Cell Imaging
For testing the practical applicability of these highly fluores-
cent probes, HeLa cells were treated with dyad 1 or 2 in
water/dimethylsulfoxide (0.1 vol%) for one hour at 378C.
Figure 5 shows confocal fluorescence microscopy images
Figure 6. TPE confocal microscopic image and emission spectra of select-
ed points of HeLa cells incubated with dyad 1 upon excitation at 880 nm
(spectrum c shows the background).
under direct excitation of the BODIPY chromophore (lexc
=
514 nm, OPE conditions) of HeLa cells that were incubated
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other nonlinear crystal (0.5 mm BBO, q=388, f=908) by using the funda-
mental beam as a gate pulse. The up-converted light was dispersed in
a monochromator and detected by photon-counting electronics. A cross-
correlation function obtained with use of the Raman scattering from
water displayed a full width at half-maximum (fwhm) of 250 fs. The fem-
tosecond fluorescence decays were deconvoluted using a Gaussian shape
for the exciting pulse using commercial software (IGOR Pro, WaveMet-
rics).
Conclusions
In conclusion, we demonstrated the merging of the EET
cassette concept with TPE fluorescence imaging for two
new aminonaphthalimide–BODIPY dyads. The involvement
of photoinduced electron transfer somewhat lowers the fluo-
rescence quantum yield of the BODIPY upon excitation of
the aminonaphthalimide. However, the obtained photophys-
ical data provide a reliable ground for the application of the
dyads in confocal fluorescence microscopy. Beside the favor-
able linear spectroscopic properties for EET, the significant
two-photon absorption cross-section of the aminonaphthali-
mide energy donor also allows its excited state population
and the subsequent energy transfer to the BODIPY moiety.
This provides possibilities of using OPE and TPE of the
energy donor or the direct excitation of the BODIPY chro-
mophore in fluorescence imaging of HeLa cells that were
chosen as bio-relevant model systems.
Multiphoton Microsocpy of the Fluorophores
The fluorescence properties of the dyads 1 and 2 and the model com-
pounds 3 and 4 [in N,N-dimethylformamide loaded on a glass-bottomed
dish (35 mm diameter, Mattek)] were analyzed using an inverted Leica
SP5 AOBS MP confocal microscope equipped with a MaiTai Ti:Sapphire
HP laser (Spectra-Physics, Inc.) tunable between 690 and 1040 nm. The
imaging was performed by using a 63ꢀPLAN APO oil immersion objec-
tive (NA 1.4) focused on the edge of the droplet. This allowed the simul-
taneous detection of the sample and the background fluorescence. The
emission and excitation spectra data for droplet and background regions
of interest (ROIs) were registered with the Leica LAS AF software.
Emission spectra (multi-photon excitation at 880 nm) were measured by
using a dynamic 30 nm wide emission detection window moving in 30
steps between 400 and 700 nm.
Cell Culture and Incubation with Fluorophores
Experimental Section
Glass-bottomed cell culture dishes (35 mm diameter, Mattek) were
seeded with HeLa cells. The cells were cultivated at 378C with 5% CO2
in RPMI 1640 medium (Gibco) supplemented with 2 mm l-glutamine
(Life Technologies, Inc.) and 5% fetal bovine serum (Life Technologies,
Inc until they reached 50–70% confluence. This happened typically
within 1–2 days. The dishes containing the HeLa cells were then incubat-
ed for 1 h with 1 mL of fresh, preheated RPMI 1640 culture medium con-
taining different concentrations of fluorophore (including a negative con-
trol). After incubation, each dish was washed two times with preheated
phosphate-buffered saline (PBS, pH 7.4) to remove traces of unbound
fluorophore and then fixed using 4% paraformaldehyde for 10 min at
room temperature. Finally, the cells were washed three times with PBS
and stored at 48C until analysis.
General
Commercial grade reagents and solvents were used without further pu-
rification unless otherwise stated. Acetonitrile and N,N-dimethylforma-
mide, which were used in the photophysical measurements, were of spec-
troscopic grade. 1H and 13C NMR spectra were recorded either on
a Bruker Advance III 400 MHz or a Bruker Advance III 600 MHz instru-
ment. The spectra are referenced to the residual solvent signal. The prod-
ucts were purified by silica gel chromatography (SiO2 40–63 mm). Ele-
mental analysis was carried out on a Leco TruSpec Micro CHNS ana-
lyzer.
Photophysical Measurements
Confocal and Multiphoton Microscopy of Fluorophores in Biological
Conditions
All photophysical measurements were performed with 10 mm solutions in
quartz cuvettes (1 cm optical pathlength) at room temperature (238C).
UV/Vis absorption spectra were measured with a UVPC-1603 instrument
from Shimadzu. Steady-state fluorescence measurements were done on
a Varian Eclipse fluorimeter. Fluorescence quantum yields were deter-
mined using rhodamine 6G in ethanol (Ffl =0.95)[52] or quinine sulfate in
0.05m H2SO4 as reference (Ffl =0.55).[53,54] The quantum yields were cal-
culated by taking the different refractive indices of the solvent of the
sample and the reference into account.[55] The two-photon absorption
cross-sections, d, were determined by two-photon-excitation fluorescence
measurements on a Leica SP5 AOBS MP instrument according to a pub-
lished procedure.[51] Rhodamine B (10 mm in methanol) was used as a ref-
erence, assuming that the fluorescence quantum yield is the same regard-
less whether two-photon or one-photon excitation is used.[51] The TPA
cross-sections were calculated according to standard procedures and
within a laser power regime where the fluorescence was proportional to
the square of the laser excitation power. In this way it was ensured that
only two-photon absorption occurred.[38]
The fluorescence properties upon multi-photon excitation of the cell-in-
cubated fluorophores (cells fixed in PBS) were analyzed with the same
equipment as used for the fluorophores alone (see above). Furthermore,
under OPE conditions, confocal excitation lasers (405, 458, and 514 nm)
were used. Multi-photon emission spectra were obtained for 880 nm exci-
tation.
General Synthesis of 4-Bromo-1,8-naphthalimides
4-Bromo-1,8-naphthalic anhydride (2 mmol) and the corresponding ben-
zoic acid derivative (2 mmol) were suspended in N,N-dimethylformamide
(5 mL) and stirred at 908C for 24 h. After cooling to room temperature,
the mixture was poured into 50 mL of water and the precipitate was col-
lected by filtration, washed with water, and dried to yield the product.
4-((6-Bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2
ACHTUNGTNER(NUNG 3H)-
yl)methyl)benzoic acid (5)
Time-correlated single-photon-counting measurements were performed
on a FS920 fluorescence spectrometer (Edinburgh Instruments). As exci-
tation sources, picosecond pulsed diode lasers (Edinburgh Instruments)
Pale yellow solid (730 mg, 89%). 1H NMR (400 MHz, [D6]DMSO): d=
8.60 (d, J=7.3 Hz, 1H), 8.58 (d, J=8.5 Hz, 1H), 8.36 (d, J=7.8 Hz, 1H),
8.24 (d, J=7.8 Hz, 1H), 8.00 (t, J=8.0 Hz, 1H), 7.87 (d, J=8.3 Hz, 2H),
7.47 (d, J=8.3 Hz, 2H), 5.30 ppm (s, 2H); 13C NMR (100 MHz,
[D6]DMSO): d=166.9, 162.9, 162.9, 142.0, 132.8, 131.8, 131.3, 131.2,
129.8, 129.3, 129.3, 129.5, 128.8, 128.4, 127.3, 122.6, 121.8, 42.9 ppm; ele-
mental anal. calcd (%) for C20H12BrNO4: C 58.56, H 2.95, N 3.41; found:
C 58.60, H 2.80, N 3.52.
were used (EPL-445: lexc =442.2 nm, pulse width 78.3 ps; EPL-485: lexc
=
482.0 nm, pulse width 100.6 ps). The nanosecond fluorescence decays
were deconvoluted using the instrument response function that was re-
corded with a light-scattering Ludox solution. The ultrafast fluorescence
transients were measured by a femtosecond fluorescence up-conversion
setup (FOG-100, CDP Corp.). The sample was excited at 405 nm using
the second harmonic of a mode-locked Ti-sapphire laser. To generate the
second harmonic, a nonlinear crystal (1 mm BBO, q=258, f=908) was
used. The fluorescence emitted from the sample was up-converted in an-
Chem. Asian J. 2014, 9, 797 – 804
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ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Ezequiel Perez-Inestrosa et al.
4-(6-Bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2
(6)
(3H)-yl)benzoic acid
134.8, 134.4, 131.3, 130.0, 129.2, 128.2, 126.1, 124.7, 123.0, 120.2, 109.9,
104.4, 43.4, 43.1, 31.0, 20.3, 17.0, 14.5, 13.8, 12.4, 11.8 ppm; HRMS (ESI)
m/z calcd. for C40H43BN4O2F2: 660.3447 [M]+; found: 660.3434.
Pale yellow solid (554 mg, 70%). 1H NMR (600 MHz, [D6]DMSO): d=
8.66–8.61 (m, 2H), 8.38 (d, J=7.8 Hz, 1H), 8.29 (d, J=7.8 Hz, 1H), 8.10
(d, J=8.3 Hz, 2H), 8.07 (t, J=8.0 Hz, 1H), 7.55 ppm (d, J=8.3 Hz, 2H);
13C NMR (150 MHz, [D6]DMSO): d=166.8, 163.0, 162.9, 141.5, 140.4,
139.8, 132.8, 132.3, 131.8, 131.6, 131.4, 130.8, 130.6, 130.5, 129.8, 129.7,
129.5, 129.4, 129.3, 128.8, 128.8, 124.9, 123.2, 112.9 ppm; elemental anal.
calcd (%) for C19H10BrNO4: C 57.60, H 2.54, N 3.54; found: C 58.00, H
2.81, N 3.62.
Dyad 2
Red solid (65 mg, 40%). RF =0.15 (CH2Cl2/n-hexane, 1:1); 1H NMR
(400 MHz, CDCl3): d=8.63 (d, J=7.2 Hz, 1H), 8.51 (d, J=8.4 Hz, 1H),
8.15 (d, J=8.4 Hz, 1H), 7.66 (t, J=7.9 Hz, 1H), 7.50–7.40 (m, 4H), 6.78
(d, J=8.5 Hz, 1H), 5.34 (m, 1H), 3.48–3.37 (m, 2H), 2.51 (s, 6H), 2.31
(q, J=7.5 Hz, 4H), 1.82 (t, J=7.5 Hz, 2H), 1.55 (q, J=7.4 Hz, 2H), 1.48
(s, 6H), 1.05–0.93 ppm (m, 9H); 13C NMR (100 MHz, CDCl3): d=165.6,
165.0, 154.7, 150.8, 140.3, 139.6, 137.5, 136.6, 135.8, 133.8, 132.4, 131.7,
131.2, 130.7, 129.9, 127.1, 125.7, 121.2, 110.9, 105.4, 44.4, 31.9, 21.3, 18.0,
15.5, 14.7, 13.4, 12.8 ppm; HRMS (ESI) m/z calcd. for C39H41BN4O2F2:
646.3291 [M]+; found: 646.3300.
General Synthesis of 4-Butylamino-1,8-naphthalimides
4-Bromonaphthalimide derivative (0.36 mmol) and n-butylamine
(5.1 mmol) were dissolved in dimethylsulfoxide (4 mL) and stirred at
808C overnight. The resulting solution was diluted with dichloromethane
(100 mL) and washed with a 1m aqueous HCl solution (10 mL) and
water (10 mL). The organic layer was dried over anhydrous MgSO4 and
concentrated under reduced pressure to give a yellow solid. Purification
by column chromatography (SiO2, EtOAc) afforded the corresponding 4-
butylaminonaphthalimide.
Acknowledgements
4-((6-(Butylamino)-1,3-dioxo-1H-benzo[de]isoquinolin-2ACTHNUTRGNE(NUG 3H)-
yl)methyl)benzoic acid (7)
The support by the Spanish MINECO (CTQ2010-20303 for E.P.-I. and
CTQ2011-28390 for U.P.), the Junta de Andalucꢄa (FQM-3685 for U.P.),
the European Union (FEDER), and the Science and Engineering Re-
search Board, Government of India (SR/S1/PC-08/2011 to P.S.) is grate-
fully acknowledged.
Yellow solid (135 mg, 92%). 1H NMR (400 MHz, [D6]DMSO): d=8.74
(d, J=8.5 Hz, 1H), 8.45 (d, J=7.3 Hz, 1H), 8.29 (d, J=8.6 Hz, 1H),
7.87–7.82 (m, 3H), 7.68 (t, J=7.3 Hz, 1H), 7.40 (d, J=8.4 Hz, 2H), 6.79
(d, J=8.5 Hz, 1H), 5.27 (s, 2H), 3.39 (q, J=7.2 Hz, 2H), 1.70 (q, J=
7.2 Hz, 2H), 1.43 (q, J=7.2 Hz, 2H), 0.95 ppm (t, J=7.2 Hz, 3H);
13C NMR (100 MHz, [D6]DMSO): d=164.4, 163.4, 159.3, 151.5, 135.2,
143.4, 135.2, 131.5, 130.1, 129.8, 129.4, 127.7, 124.8, 122.1, 120.7, 108.6,
107.6, 104.4, 65.5, 61.2, 43.0, 30.4, 20.2, 14.2 ppm; elemental anal. calcd
(%) for C24H22N2O4: C 71.63, H 5.51, N 6.96; found: C 71.23, H 5.32, N
7.05.
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4-(6-(Butylamino)-1,3-dioxo-1H-benzo[de]isoquinolin-2
acid (8)
ACHTUNGTREN(NGNU 3H)-yl)benzoic
Yellow solid (126 mg, 90%). 1H NMR (400 MHz, [D6]DMSO): d=8.80
(d, J=8.5 Hz, 1H), 8.44 (d, J=7.3 Hz, 1H), 8.27 (d, J=8.6 Hz, 1H), 8.05
(d, J=8.4 Hz, 2H), 7.87 (m, 1H), 7.71 (t, J=7.3 Hz, 1H), 7.40 (d, J=
8.4 Hz, 2H), 6.82 (d, J=8.5 Hz, 1H), 3.40 (m, 2H), 1.71 (m, 2H), 1.44
(m, 2H), 0.95 ppm (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, [D6]DMSO):
d=163.9, 163.0, 156.7, 150.9, 139.6, 134.4, 132.3, 131.8, 130.8, 130.6,
130.163, 130.0, 129.6, 129.1, 125,0, 124.2, 122.7,120.2,107.5, 103.7, 42.5,
29.9, 19.8 ppm; elemental anal. calcd (%) for C23H20N2O4: C 71.12, H
5.19, N 7.21; found: C 71.33, H 5.37, N 7.56.
General Synthetic Procedure for the Dyads
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benzene (10 mL) and thionyl chloride (1 mL) was added. The mixture
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Dyad 1
Red solid (56 mg, 34%). RF =0.35 (CH2Cl2/n-hexane, 1:1); 1H NMR
(400 MHz, CDCl3): d=8.61 (dd, J=7.3, 0.9 Hz, 1H), 8.50 (d, J=8.4 Hz,
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[44] The aminonaphthalimides 7 and 8, bearing the linker moieties, have
similar absorption spectra like the model chromophore 4 (see spec-
Chem. Asian J. 2014, 9, 797 – 804
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ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim