An improved singlet oxygen sensitizer with two-photon absorption and emission in the biological transparency window as a result of ground state symmetry-breaking

Article abstract of DOI:10.1039/c2cc15904j

A chromophore featuring a diyne bridge that connects two dibromobenzene moieties shows a much improved two-photon singlet oxygen generation ability in the biological transparency window compared to a related and relevant literature benchmark, as a result

Full text of DOI:10.1039/c2cc15904j

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COMMUNICATION
An improved singlet oxygen sensitizer with two-photon absorption and
emission in the biological transparency window as a result of ground
state symmetry-breakingw
a
a
a
ab
´
Thibault Gallavardin, Chloe Armagnat, Olivier Maury, Patrice L. Baldeck,
Mikael Lindgren,ac Cyrille Monnereau*a and Chantal Andraud*a
Received 22nd September 2011, Accepted 6th December 2011
DOI: 10.1039/c2cc15904j
A chromophore featuring a diyne bridge that connects two
dibromobenzene moieties shows a much improved two-photon
singlet oxygen generation ability in the biological transparency
window compared to a related and relevant literature benchmark,
as a result of a distorted ground state.
Dioxygen in its lowest singlet excited state (usually referred to
as singlet oxygen) could be of great interest for various
applications: photodynamic therapy (PDT),1 near infrared
optical microscopy2 and oxygen pressure (PO2) imaging.3 In
all cases, singlet oxygen is generated upon irradiation of a
photo-sensitizer that ideally possesses a strong absorption at
the incident wavelength and a long living triplet state able to
interact with ground state tissue oxygen, and promote its
excitation by energy transfer.4–6 Since pioneering works of
Fig. 1 Structure of chromophores 1 and 2.
Webb and co-workers,7 two-photon excitation (TPE), using
confocal microscopies and tuneable fs-pulsed laser sources,
became of great interest for biology related applications such
as imaging, diagnosis, or PDT.8–16 TPE presents several
advantages over its one-photon counterpart: (i) the excitation
focussing limits the irradiation to an approximately 1 mm3
volume, decreasing out-of-focus photo-damage and intrinsically
allowing a higher 3D resolution; (ii) using the tuneable Ti–sapphire
laser in the 700–1100 nm spectral range enables excitation
within the ‘‘biological transparency window’’, in which water,
biomolecules and tissues are less absorbing and scattering, and
thereby leads to a deeper penetration into biological systems.
Although numerous chromophores with optimized two
photon absorption cross-sections (sTPA) have been reported
in the literature,17,18 only a few of them allow singlet oxygen
generation.19–24 Generally speaking, these molecules present a
conjugated charge transfer (CT) structure with dipolar or
quadrupolar geometry.25 In this context, a dibromobenzene
derivative 1 (Fig. 1, top) was shown to combine a significant
two-photon absorption cross-section, owing to its quadrupolar
CT structure, along with a good singlet oxygen generation
quantum yield (fD), resulting from an intersystem crossing
process, due to the presence of the dibromobenzene ring.26,27
Recently we showed that the use of Pluronics nanoparticles
allowed the introduction of this hydrophobic molecule into
biological cells and led to cell death upon one-photon resonant
irradiation.27
However, this molecule presents a TPA maximum at 730 nm,
far from wavelengths of 800–840 nm classically used for two-
photon bio-imaging applications.27 To tackle this drawback,
chromophore 2 (Fig. 1) was designed to obtain red-shifted
TPA properties. Compared to 1, this molecule features a more
extended conjugated bridge between the two strong dialkylamino
donor groups. This bridge consists of two dibromobenzene rings
connected with a diyne moiety. We hoped that enhancing
its conjugation length would lead to a bathochromic shift of
its TPA response. Herein, we report on the spectroscopic
one-photon (absorption, emission, singlet oxygen generation)
and two-photon properties of 2, and compare them with
those of the benchmark chromophore 1. We show that the
structural modifications introduced in the former lead to much
a
´
CNRSUMR 5182, Universite Lyon 1, Ecole Normale Superieure
de Lyon, 46 allee d’Italie, 69364 Lyon Cedex 07, France.
´
´
E-mail: chantal.andraud@ens-lyon.fr, cyrille.monnereau@ens-lyon.fr
b
´
Laboratoire de Spectrometrie Physique Universite Joseph Fourier,
CNRS–UMR 5588, BP 87 F-38402 Saint Martin d’He`res, France
´
c Department of Physics, Norwegian University of Science and
Technology, 7491, Trondheim, Norway
w Electronic supplementary information (ESI) available: General
procedures, materials and methods, synthetic procedures, analytical
data, 1H and 13C NMR of compound 2, fluorescence QY plots. See
DOI: 10.1039/c2cc15904j
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1689–1691 1689

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Scheme 1 Synthesis of compound 2: reagents and conditions: (i) THF/
Et3N, Pd(PPh3)2Cl2 (0.02 eq.), CuI (0.04 eq.) 12 h, RT; (ii) TMSA,
THF/Et3N, Pd(PPh3)2Cl2 (0.02 eq.), CuI (0.04 eq.) 12 h, RT; (iii)
K2CO3 (3 eq.), THF/MeOH (1 : 1); (iv) THF/Et3N, Pd(PPh3)2Cl2
(0.01 eq.), CuI (0.01 eq.) 12 h, RT.
improved figure of merit (sTPAfD) at 800 nm compared to that
of the latter.
Chromophore 2 was obtained in a straightforward synthesis,
involving a limited number of steps: (i) a desymmetrization
step, using Sonogashira coupling between N-hexyl substituted
p-ethynylaniline and excess 1,4-diiodo-2,5-dibromobenzene;
(ii) a second Sonogashira coupling between the product
obtained in the first step and trimethylsilylacetylene, followed
by deprotection of the silyl protecting group, (iii) Glaser
homocoupling of the resulting product, to afford 2 in an overall
25% yield (Scheme 1).z
Fig. 2 One- (full) and two-photon (full with marks) absorption and
emission (dashed-dotted) spectra of 1 (top) and 2 (bottom) in dichloro-
methane at 298 K.
0.55 ꢁ 0.05 for chromophore 2 in dichloromethane (ESIw).
This value is, within experimental error, identical to that of
0.53 measured for 127 in spite of the increased number of heavy
atoms in 2.
Photophysical properties of 2 were studied in dichloro-
methane. Absorption and emission spectra are shown in
Fig. 2 along with those of molecule 1. Both chromophores
present broad, intense absorption and emission bands localised
in the visible part of the spectrum, typical of CT transitions.27
In spite of its significantly extended conjugated skeleton, 2
presents only a slightly red-shifted absorption spectrum with
respect to that of 1, with maxima at 440 and 421 nm,
respectively. In contrast, molecule 2 shows a much red-shifted
emission spectrum with respect to that of 1 (593 vs. 458 nm
respectively), and thereof a dramatically larger Stokes shift
value nea (5646 vs. 1825 cmꢀ1). By analogy with previously
reported ethynyl-linked (porphinato)metal chromophores,28,29
we tentatively assign this behaviour to a partly distorted
conformation of molecule 2 in its ground state; thus, the very
large Stokes shift observed in compound 2 could be attributed
to a reorganisation of the chromophore geometry in its excited
state towards a more planar conformation with an enhanced
conjugation. This property could be of great interest for the
target application, since red-shifted luminescence allows easier
localisation of the chromophore in biological media.
TPE spectra of 1 and 2 are also displayed in Fig. 2.
Although the positions of their one-photon absorption
(OPA) band differ only by 20 nm, a much larger difference is
seen when considering the TPA properties of both molecules
(140 nm): in the case of 2, the wavelength-halved TPE
spectrum strongly overlaps the OPA one, whereas a significant
blue shifted TPE spectrum is observed for 1. These results can
be rationalized on the basis of selection rules for TPA electronic
transitions, which are dependent on molecular symmetry:
quadrupolar centrosymmetric structures have a significantly
blue shifted TPE spectrum (i.e. OPA and TPA are mutually
exclusive, lower lying allowed two-photon induced transition
leading to promotion of the molecule to a more energetic
excited state than its one-photon counterpart), while non-
centrosymmetric structures show a strong overlap of their
OPA and TPA spectra (i.e. the same electronic transitions
are involved).30–32 Consequently, it can be deduced that 2 and
1 display electronic non-centrosymmetric and centrosymmetric
behaviour, respectively, in spite of the fact that both molecules
are supposed to feature an inversion center, if considering their
respective ‘‘flat’’ structural formula. By analogy with previous
literature studies on similar two photon absorbing chromo-
phores having flexible carbon backbones and showing spectral
properties similar to those of 2,33 it is possible to conclude that
the non-centrosymmetric behaviour of 2 is the result of a non-
planar conformation of the chromophore in its ground state,
resulting in a ‘‘symmetry breaking’’ in the molecule. Thus, the
experimental differences in the TPE spectra unambiguously
illustrate the significant contribution of a non-centrosymmetric
octupolar (D2d) geometry to the molecule’s ground state in 2
(Fig. 3).28,33 Noteworthily, this is also in good agreement
with the important Stokes shift observed for this molecule,
as discussed above in this communication. Finally, this
ground state symmetry breaking is also responsible for the
Both systems present modest, and very similar, fluorescence
quantum yields (0.25 and 0.20 for 1 and 2, respectively). This is
due to the presence of heavy bromine atoms that drive in both
systems deactivation of the singlet excited state to a lower
lying triplet state by intersystem crossing, and constitutes the
first step towards 1O2 generation. Generation of singlet oxygen
could be characterised and quantified by the observation of its
emission band at 1275 nm in dichloromethane, which corres-
ponds to the radiative transition from the dioxygen singlet
excited state towards its ground triplet state. Such a lumines-
cence is observed upon excitation in the chromophore CT
absorption band in diluted dichloromethane solution. Quantum
efficiency of singlet oxygen generation fD was estimated to be
c
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(490 vs. 570 GM, Fig. 2) in spite of its extended skeleton.
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s
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In conclusion, we introduced a molecular engineering
strategy, relying on a Glaser coupling to extend the chromophore
p-conjugated backbone. We showed that chromophore 2,
featuring diyne moieties presents a distorted ground state
geometry which results in a strong red-shift of both its emission
spectrum (because of the increased reorganization energy in the
excited state) and its TPA spectrum (because of different
selection rules associated with the octupolar contribution in
ground state’s geometry) when compared to chromophore 1.
Consequently, chromophore 2 displays two-photon absorption
and emission in the biological transparency window, which
makes it a much improved candidate as a two-photon singlet
oxygen photosensitizer, compared to benchmark chromophore 1.
The authors thank the ANR program (NanoPDT),
MACODEV and Cluster5 (PhD grant for T.G.) for financial
support. J. Bernard is also acknowledged for his technical
support in two-photon absorption measurements.
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z Abbreviations: TMSA: trimethylsilylacetylene, THF: tetrahydrofuran,
Et3N: triethylamine, RT: room temperature.
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c
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Chem. Commun., 2012, 48, 1689–1691 1691