A fullerene-distyrylbenzene photosensitizer for two-photon promoted singlet oxygen production

Article abstract of DOI:10.1039/b922740g

Novel compounds endowed with a high two-photon absorption (TPA) cross-section and a high singlet oxygen quantum yield, are sought after for their possible application in anti-cancer therapies. In this paper we present a prototype macromolecule bearing a d

Full text of DOI:10.1039/b922740g

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PAPER
www.rsc.org/pccp | Physical Chemistry Chemical Physics
A fullerene–distyrylbenzene photosensitizer for two-photon promoted
singlet oxygen productionw
Elisabetta Collini, Ilaria Fortunati, Sara Scolaro, Raffaella Signorini,
Camilla Ferrante,* Renato Bozio, Graziano Fabbrini, Michele Maggini,*
Emiliano Rossi and Simone Silvestrini
Received 30th October 2009, Accepted 12th February 2010
First published as an Advance Article on the web 17th March 2010
DOI: 10.1039/b922740g
Novel compounds endowed with a high two-photon absorption (TPA) cross-section and a high
singlet oxygen quantum yield, are sought after for their possible application in anti-cancer
therapies. In this paper we present a prototype macromolecule bearing a distyrylbenzene dimer as
TPA unit and a [60]fullerene moiety for singlet oxygen generation. Linear absorption and
emission spectra are measured, to help understanding the interactions between the single
molecular units. The TPA absorption properties of the distyrylbenzene alone as well as bound
to the methanofullerene unit are recorded with the TPA-induced fluorescence technique. An
appreciable enhancement of the TPA cross-section was observed in the molecular conjugate.
Singlet oxygen generation has been detected exciting the sample both in the Vis and NIR through
one- and two-photon absorption processes, respectively. Although functionalization decreases
the overall singlet oxygen quantum yield of fullerene, the presence of the distyrylbenzene antenna
allows two-photon generation of singlet oxygen through an energy transfer process.
and dermatology.43 Most photosensitizers, in medically-approved
Introduction
PDT applications, are tetrapyrrole-based compounds (porphyrins,
Functionalized fullerenes are extensively studied as active
chlorines, phthalocyanines). Similar to these traditional photo-
components in photonic devices, such as, for instance, poly-
sensitizers, excitation of functionalized fullerenes in oxygenated
mer solar cells1–5 or optical limiters6,7 for their strong electron
solutions leads to extremely efficient 1O2 generation with an almost
unitary quantum yield,44 through an energy transfer process from
accepting properties and weak ground state absorption which,
the excited triplet state of the fullerene.45,46 Recent reports have
in turn, is coupled to a strong absorption of excited singlet and
triplet states in the whole visible range.8,9 For the mentioned
shown that in polar solvents, especially those containing reducing
agents, illumination can also generate O2 .
ꢁ 47 Singlet oxygen and
ꢀ
applications, a large number of derivatives have been con-
sidered, in particular fulleropyrrolidines and methanofullerenes,
for their ease of synthesis and further functionalization.10–12
superoxide pathways are analogous to the Type II and Type I
photochemical mechanisms identified in PDT with conventional
tetrapyrrolic compounds.48,49
A
wide variety of donor-linked fullerenes with porphyrins,13–19
tetrathiafulvalenes20–23 or ferrocenes24,25 have been prepared and
studied in the past. More recently, several dyad systems,
where [60]fullerene is combined with carbazole, benzothiazole,
benzothiazole-triphenylammine26,27 and diphenylammino-
fluorene28–30 moieties as well as distyrylbenzene31 and oligo-
(p-phenylene vinylene),32–34 have been synthesised to serve as
building blocks for photonic materials.
The biological activity of fullerenes in PDT has been known
for more than a decade.36 Several studies reported in the litera-
ture have demonstrated that fullerene-based photosensitizers
could lead to DNA cleavage,50 photoinactivation of viruses,51
production of oxidative damage in membranes,52 and even
PDT-induced regression of tumors in mice.53
Recently, the two-photon absorption (TPA) process has
been explored as an intriguing route to excite the photo-
sensitizer for singlet oxygen generation. This strategy could
improve many aspects of PDT: (i) the sample is excited by
simultaneous absorption of two lower energy photons in the
NIR region (750–900 nm) rather than with one higher energy
photon in the Vis region (375–450 nm); excitation in the NIR
region where the ‘‘transparency window’’ of skin falls, allows
for deeper penetration in the tissues (hundreds of microns); (ii)
absorption occurs only in the focal region of the beam, and
therefore singlet oxygen can be produced only in a well defined
volume of the sample, (iii) although not yet fully demon-
strated, photobleaching of the sensitizing chromophore should
be minimized.54,55
Water-soluble fullerenes and fullerene–polymer hybrids
have, instead, been prepared specifically as sensitizers for
singlet oxygen generation35–40 in photodynamic therapy
(PDT). PDT is a medical treatment that makes use of reactive
ꢁ
oxygen species like singlet oxygen (1O2) and superoxide (O2
)
ꢀ
as powerful oxidizing agents in cancer therapy,41 ophthalmology42
Department of Chemical Sciences and UdR INSTM,
University of Padova, Via Marzolo 1, I-35131 Padova, Italy.
E-mail: camilla.ferrante@unipd.it, michele.maggini@unipd.it;
Fax: 00390498275239; Tel: 00390498275148
w Electronic supplementary information (ESI) available: Selected
analytical and photophysical data for the investigated compounds.
See DOI: 10.1039/b922740g
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Currently, most of the molecular systems investigated as
potential TPA photosensitizers are molecules showing a high
TPA cross section, such as distyrylbenzenes (DSB)56,57 or
porphyrin dimers.58 Porphyrin derivatives were also recently
tested as photosensitizers for two-photon excited photo-
dynamic therapy (TPE-PDT) in vivo. In particular TPE-PDT
was successfully employed in the treatment of lung cancer,
leading to tumor regression at a significant tissue depth,59
and in the targeted closure of blood vessels associated with
neoplastic diseases.60
Linear absorption and emission
All the photophysical parameters obtained for dyad 1 and
relative precursor model compounds are summarized in
Table 1.
Steady-state UV-Vis absorption spectra of dyad 1 and
model compounds 3–5 were recorded in air-saturated toluene
solutions and reported in Fig. 1. The spectra of compounds 3
(triangles) and 4 (circles) show the characteristic features of
DSB chromophores, with two principal maxima at 395 and
330 nm. The absorption spectrum of the dimeric form 4 is,
within the experimental error, equivalent to twice the absorp-
tion spectrum of 3, proving that no interaction takes place
between the DSB moieties in the ground electronic state. The
fullerene derivative 5 (squares) shows the absorption spectrum
typical of functionalized methanofullerenes with the main
band at 339 nm, which is broader with respect to pristine
[60]fullerene, and an additional band at 433 nm.66 The spec-
trum also displays a very weak absorption in the whole visible
range extending up to 700 nm. (see ESI for a direct comparison
of [60]fullerene and compound 5 spectra).w The absorption
spectrum of dyad 1 (solid line, no symbols) discloses super-
imposed features of fullerene and DSB. From 450 to 380 nm
the spectrum is dominated by the absorption of the DSB dimer
4, while at shorter wavelength the contribution of the fullerene
unit becomes noticeable. Within experimental error, the
absorption spectrum of 1 matches the profile obtained by
summation of the absorption spectra of the two individual
moieties that constitute the dyad (dashed line) in the whole
investigated spectral range, except for a slight deviation in the
blue side. This deviation can originate from weak interactions
between the two units or, more likely, is the result of errors
associated with the absolute measure of the extinction coefficient,
becoming more relevant in the blue side due to increasing
scattering contributions. These results are in agreement with
previous studies on donor–acceptor dyads in solution and
suggest that there is no ground-state interaction between the
fullerene and the DSB moieties. This is also confirmed by the
presence of the weak but characteristic long wavelength
absorption signal between 500 and 700 nm, as shown in the
inset of Fig. 1. These bands are characteristic of the [60]fullerene
derivatives and their presence in the absorption spectrum of 1
means that the p-conjugated structure of [60]fullerene and thus
its photophysical properties, are not perturbed by functionali-
zation with the DSB moiety.
In the search of molecules characterized both by high TPA
cross sections and high singlet oxygen quantum yields, we
propose the fullerene–distyrylbenzene conjugate (1). The idea
is to functionalize the fullerene unit, characterized by a unitary
singlet oxygen photosensitization quantum yield, with suitable
chromophores capable of efficient TPA, acting as antennas.
After TPA, the antenna (donor) transfers its excitation via
energy transfer to the fullerene, where inter-system crossing
and singlet oxygen generation occurs. This approach is parti-
cularly versatile because it demonstrates that by employing
different chromophores or functional groups, with specialized
functions, it is possible to generate new molecular conjugates
endowed with targeted properties. Compound 1 is the first
prototype that, although not yet optimized, still shows the
basic properties, i.e. mildly efficient TPA that gives rise to
appreciable singlet oxygen production, necessary for our
purpose. Indeed amelioration of the present design should
encompass an increase of the TPA cross-section as well as an
improvement of the water solubility for application in the bio-
medical field.
Results and discussion
Synthesis
Methanofullerene 1 was synthesized in 30% yield through a
nucleophilic cyclopropanation reaction to [60]fullerene of the
iodomalonate anion that was formed in situ from malonate 4
and iodine in the presence of the base 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU, Scheme 1).64 HPLC analysis was
employed to assess the purity of 1, whereas NMR spectroscopy
(Fig. S9–S10)w and mass determination validate its proposed
molecular structure. The trans–trans configuration of the
double bonds in the distyrylbenzene moieties was established
3
The possible presence of energy or charge transfer processes
occurring after excitation of 1 between the DSB and fullerene
moieties can be ascertained through emission spectroscopy.
Fig. 2 shows the emission spectra of 1 and 4 in toluene after
excitation of the samples at 400 nm in the same experimental
conditions. The emission spectrum of 3 (not shown) presents
the same features as 4, but is characterized by an intensity
approximately halved with respect to an equimolar solution of
4, an evidence of negligible interactions between the two branches.
The emission of dyad 1 can be qualitatively described as the
sum of emissions from the DSB and fullerene components. In
the 400–600 nm region, the emission spectrum is dominated by
the appearance of two bands at 440 and 470 nm, with a
shoulder at about 500 nm, typical of DSB units, whereas in
via NMR, through measurement of the JHH of the olefin
protons which was found to be around 16 Hz, as reported in
the literature.65 Malonate 4 was, in turn, prepared in 45%
overall yield as illustrated in Scheme 2. The Horner-Emmons
Wittig (HEW) coupling reaction of p-methoxybenzaldehyde
with
2,5-Bis(diethylphosphonate)-1,4-dimethoxybenzene61
gave the trans-stylbene derivative 2 in 62% yield. A HEW
coupling of 2, 4-(2-hydroxyethoxy)-benzaldehyde,62 gave dis-
tyrylbenzene 3 in 85% yield. Malonate 4 was obtained in 86%
yield by esterification of malonyl dichloride from its distyryl
alcohol 3.
Methanofullerene 5, whose structure and synthesis are
reported in the ESI,w was used as a reference methanofullerene
derivative lacking the distyrylbenzene moiety.
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Scheme 1 Fullerene–distyrylbenzene conjugate 1 considered in this work.
Scheme 2 Reagents and conditions: (a) tBuOK, DMF, 25 1C, 4 h, 62%; (b) tBuOK, DMF, 25 1C, 3 h, 85%; NaHCO3, CH2Cl2, 25 1C, 15 h, 86%,
Table 1 Linear and nonlinear optical properties of compounds 1, 3–5
sTPA (GM) @
lexc = 740 nm
Compound
lmax abs/nm
ea/Mꢁ1 cmꢁ1
lmax fluo/nm
FQYb
336
392
570
96 700
89 000
1000
440, 468,
500(shoulder), 705
1
0.008 ꢃ 0.001
111 ꢃ 10
333
397
18 300
40 700
3
4
0.96 ꢃ 0.07
0.70 ꢃ 0.07
42 ꢃ 6
74 ꢃ 6
442, 470,
500(shoulder)
333
395
48 300
85 600
339
429
570 (broad)
40 600
2400
1000
5
705
/
/
a
b
Estimated error: 15%. Calculated in the 400–600 nm interval.
the red side of the spectrum, between 600 and 800 nm, it shows
the characteristic emission of fullerene cage at 705 nm (inset in
Fig. 2). A much higher intensity of the bands at 440 and
470 nm with respect to that at 705 nm is indicative of the
predominant contribution of DSB absorption at the excitation
wavelength. The more important feature emerging from the
comparison between the dyad and precursor compounds spec-
tra is however the nearly complete quenching of DSB fluores-
cence in the dyad: there is more than two orders of magnitude
decrease in fluorescence intensity relative to the free DSB 4. In
Fig. 2 the absolute fluorescence intensity of 1 was multiplied by
100 in order to be comparable with the fluorescence from 4 on
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excited at 330 nm, in the spectral region between 400 and
600 nm.67
Coumarin 153 (FQY = 0.56) and Coumarin 102 (FQY = 0.93)
in ethanol, are used as standards.68 The results are summarized
in Table 1. The high FQY of 3 and 4 confirms that these
compounds are extremely strong fluorophores, as already
proven for differently functionalized DSB compounds.69,70 On
the other hand, the FQY of dyad 1 is less than 1%, supporting
the presence of highly efficient non-radiative decay channels
involving the fullerene unit.
Two photon absorption
The TPA properties of 1, 3 and 4, dissolved in toluene were
investigated with the two photon induced fluorescence (TPIF)
technique in the femtosecond regime. The TPA spectrum of 5
could not be determined since its fluorescence emission in the
near-IR (680–800 nm) cannot be detected by our experimental-
setup (detection window: 350–600 nm).
Fig. 1 Steady-state UV-Vis absorption spectra of 1 (no symbol) and
model compounds 3 (triangles), 4 (circles), and 5 (squares) in air-
saturated toluene. The dashed line is the profile obtained by summation
of the absorption spectra of 4 and 5.
Absolute values of TPA cross section (sTPA) for a sample
can be obtained comparing the TPIF emission of the sample
with the emission of a standard molecule, whose sTPA and
FQY are known:
the same intensity scale. The fluorescence spectra of compound
3 in toluene and of an equimolar solution of [60]fullerene and
compound 3 are almost indistinguishable, whereas the fluores-
cence of an equimolar solution of 1 in toluene shows a dramatic
change with respect to the previous two (see ESI).w These
measurements confirm that at the concentrations investigated
in this study, interactions between [60]fullerene and DSB occur
only when they are covalently linked.
ꢀ
ꢁ
qsCr ns 2Fr
qrCs nr Fs
sTPA;s
¼
sTPA;r
ð1Þ
where sTPA,i are the TPA cross sections, qi are the coefficients
of the quadratic fit of the TPIF intensity as a function of
incident laser pulse energy, ni the refractive indices, Fi the
FQYs and Ci the concentrations of the sample (i = s) and the
reference standard (i = r).
The quenching of the DSB emission in dyad 1 can be
explained if either an energy or electron transfer process occurs
from the excited DSB unit towards the fullerene, giving rise to
an efficient non-radiative decay channel for the relaxation
dynamics of 1. In principle, the presence of energy transfer in
fullerene dyads can be verified recording the fluorescence excita-
tion spectrum for the fullerene emission, collected at 705 nm.
However, the excitation profile obtained for 1 monitoring the
705 nm emission was too weak to give any useful information.
The amount of quenching was quantified by measuring the
fluorescence quantum yields (FQY) of compounds 1, 3 and 4,
At room temperature, it is reasonable to assume that the
fluorescence efficiency, from the lowest-lying singlet electronic
state, does not change if this state is populated by one- or two-
photon excitation, making it therefore possible to insert in
eqn (1) the values of FQYs which were estimated with linear
fluorescence experiments. The reference standard used is a
solution of fluorescein in water (pH 4 11), whose sTPA in the
same spectral range is given in references 71 and 72. The FQY
of the fluorescein standard is set equal to 0.90.67
In Fig. 3 the TPA spectra of 1 (squares), 3 (circles), and 4
(triangles) in toluene are compared with the respective normalized
linear absorption spectra, also in toluene. The TPA data
points are plotted against half the laser excitation wavelength,
so that direct comparison with the one-photon absorption
spectrum is easily accomplished. The error bars are estimated
through an error propagation procedure. In particular, the
error bar affecting the TPA data of 1 is larger than the one of
3 and 4, since the relative error estimated for the FQY is 20
and 10%, respectively.
The TPA spectrum of 3 shows a steady increase of the TPA
cross section as the wavelength decreases and no maximum is
observed in the spectral range investigated. Molecule 3 is
almost a symmetrical D-p-D structure, where D indicates the
terminal electron-donor group, and p is a conjugated bridge.
The general structure D-p-D is one of the most recurring
designs for chromophores endowed with large sTPA. Experi-
mental and theoretical evidence showed indeed that bis-donor
substituted compounds could have sTPA values more than one
Fig. 2 Emission spectra of compounds 1 (solid line) and 4 (dashed
line) recorded at lex = 400 nm; the absolute fluorescence intensity for
compound 1 has been scaled by 100 in order to compare the data on
the same scale. The inset shows an enlargement of the red side of the
spectra where the fullerene moiety emits.
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quadratic dependence typical of a TPA process in the whole
range of excitation wavelengths investigated, therefore it was
possible to record its TPA spectrum. The comparison between
the absolute values of sTPA measured for 1 and 4, shows a
slight enhancement of the TPA efficiency of the fullerene dyad
with respect to the DSB model precursor 4. A similar enhance-
ment was observed in the sTPA values of different conjugated
chromophores upon attaching a terminal fullerene, both in the
ns and fs regime.28,74,75 Considering that pristine fullerene
displays negligible nonlinearities,76,77 this effect cannot be
explained invoking an additive contribution, but is more likely
due to the electron withdrawing property of fullerene. The
presence of an electron-accepting moiety like the fullerene
gives to the dyad a partial D-p-A-p-D character, which
guarantees a better TPA efficiency.
Fig. 3 Two-photon absorption spectra of compounds 1 (squares), 3
(circles), and 4 (triangles) in toluene. The corresponding normalized
linear absorption spectra are also reported. The TPA data are plotted
against half the excitation wavelength employed, in order to allow a
direct comparison with the linear spectra.
Singlet oxygen production
One-photon sensitization experiments. The quantum yield of
1
one-photon sensitized O2 formation (FD) of compound 1 is
order of magnitude larger than the corresponding pristine
unsubstituted molecules. Theoretical studies allowed correla-
ting this enhancement with intramolecular charge transfer
from the terminal D groups to the p-bridge. Each modification
of the molecular structure increasing the amount of intra-
molecular charge transfer can thus be exploited to synthesize
molecules with larger sTPA absolute values. More specifically,
it was found that sTPA increases when (i) the electron-donating
character of the D group is strengthened, (ii) the conjugation
length of the p-system is increased (since the charge is trans-
ferred over a longer distance) and (iii) when electron acceptors
(A) are attached to the p bridge, to form the general structure
D-p-A-p-D (wherein the amount of charge transferred is
increased).70,73
estimated through measurements of the 1270 nm emission
intensity of 1O2 upon excitation at 403 nm. At this wavelength
the absorption is mainly due to the DSB moiety. Toluene is
used as solvent for sample 1 as well as for the reference
compounds: [60]fullerene and meso-TPP. CS2 solutions were
also investigated for comparison. The concentration of all
solutions was adjusted to obtain a linear absorbance of about
0.3 at 403 nm; this guarantees comparable excitation con-
ditions for both the sample and the standards solutions. The
standard molecules [60]fullerene and meso-TPP in toluene
have FD equal to 1.0 and 0.88 respectively.44
A number of control experiments were preliminarily per-
1
formed in order to assign the recorded signal to O2 lumines-
cence emission. Specifically, singlet oxygen signals were not
observed from sensitizer-free solvents (toluene or CS2) and nor
from the degassed sample. The lifetime of 1O2 in the two
different solvents was also checked coupling the InGaAs
photodiodes with a preamplifier and an oscilloscope. The
signal decay was always single exponential, and the values of
the 1O2 lifetime, roughly estimated by a monoexponential fit of
the recorded decays, were consistent with those expected in the
analyzed solvents. More specifically, a lifetime of about 30 ms
was obtained upon irradiation of toluene solutions of the
standards and compound 1, whereas a longer lifetime of about
5 ms was estimated for CS2 solutions, in good agreement with
literature data.78–80 The singlet oxygen lifetimes observed
show no dependence on the laser pulse energy in the whole
range investigated.
Among D-p-D structures, functionalized DSB have received
a lot of attention, in particular, when D at the terminus phenyl
rings are N,N-dialkylamino groups.69,70 For these structures,
the maxima of the one- and two-photon absorption spectra do
not coincide, and the TPA peak is shifted towards shorter
wavelengths with respect to the one-photon maximum, as it
also happens for 3. The absolute value of the sTPA in these
compounds (up to 1000 GM) is, however, one order of magni-
tude larger than that measured for DSB 3 (up to 50 GM)
probably because of the weaker electron-donating capability
of the alkyloxy groups in 3 in comparison with the afore-
mentioned N,N-dialkyls. In our case the use of DSB 3 has
been dictated mainly by the fact that the corresponding DSB
with a N,N-dialkyl terminus is not stable under the conditions
(DBU/I2) employed for the cyclopropanation reaction to
[60]fullerene.
The detection apparatus (InGaAs photodiode and Lock In)
used to determine FD allows recording only an average signal
and is not sensitive to the temporal decay of the 1O2 emission.
In order to check if steady state conditions are reached in
all the photophysical steps involved in 1O2 production, the
measurements have been performed with 0.2, 0.5 and 1 kHz
repetition rate. The ratio between the signals of the sample and
the standard does not show any dependence on the repetition
rate, confirming that steady state conditions are reached.
For both standards and for 1 the 1O2 emission signal
displays a linear dependence on both concentration and
The TPA spectrum of 4 follows the same wavelength
dependence as 3 and its absolute value is about twice the
TPA cross section of 3 in the whole range investigated. This
experimental finding confirms what has already been observed
in the linear absorption spectra: i.e. the two DSB units in 4 do
not interact noticeably and therefore give two independent
and additive contributions to the whole TPA cross section.
Although the FQY of compound 1 in toluene in the
400–650 nm range is less than 1%, its TPIF signal shows the
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incident power, as expected for a one-photon promoted
process. The singlet oxygen quantum yield FD is determined
from the relative intensity of singlet oxygen luminescence,
measured under identical conditions, by the relation:
brSs
bsSr
FDs ¼ FDr ꢄ
ð2Þ
where subscripts s and r refer to the sample and the standard,
respectively. Si (i = s, r) is the 1O2 luminescence signal for
the ith species and bi is a correction factor accounting for the
number of absorbed photon, that, in turn, depends on the
absorbance of the solution at the wavelength of the incident
laser beam (Ai) and on its intensity (I0):
Fig. 4 Two possible mechanisms for singlet oxygen generation by
dyad 1 in toluene. A represents the C60 core and D the DSB moiety.
EnT = energy transfer; ET = electron transfer; BET = back electron
transfer; ISC = intersystem crossing; RP-ISC = radical pair inter-
1
+
3
+
ꢁ
system crossing. The energy levels (Dꢀ ꢁAꢀꢁ) and (Dꢀ ꢁAꢀ
)
were drawn with energy intermediate between Dꢁ *A and 3*DꢁA,
in analogy with previous studies on porphyrin–fullerene dyads also in
toluene (see ref. 82 and 87).
1
bi = I0(1 ꢁ 10ꢁAi
)
(3)
The estimates for FD gathered by comparison with [60]-
fullerene and meso-TPP are 0.33 ꢃ 0.08 and 0.44 ꢃ 0.06,
respectively. These values are considerably lower than the
FD of pristine fullerene.
Having ruled out the topologic and the shielding effect as
the primary cause of FD reduction, the most plausible expla-
nation is assuming the presence of competitive decay path-
ways. The mechanism most frequently invoked to describe the
generation of singlet oxygen by photoexcited fullerene adducts
(Fig. 4) involves preliminary photoexcitation of the chromo-
phoric groups attached to the fullerene core (1*DꢁC60) followed
The 1O2 generation efficiency of [60]fullerene mixed with
compound 3 in toluene does not show this reduction, and is
similar to the one of [60]fullerene, within the experimental
error (see ESI).w These measurements confirm that at the
concentrations investigated in this study, the reduction in FD
occurs only when the two units are covalently linked.
The decrease in the efficiency of 1O2 production by func-
tionalized fullerenes with respect to pristine [60]fullerene is a
well documented phenomenon, and has already been observed
in different classes of adducts.46,81–83
1
by energy transfer to the [60]fullerene moiety (Dꢁ *C60). The
resulting singlet excited state of [60]fullerene is then readily
3
converted to the long-lived triplet excited state (Dꢁ *C60) via
intersystem crossing. The final step is the energy transfer to
molecular oxygen.
It was demonstrated that for pristine fullerene and simple
fullerene derivatives, both intersystem crossing and energy
transfer to molecular oxygen occur with nearly unitary quantum
efficiency.85,86 This means that the FD reduction is most probably
due to a less operative singlet energy transfer between the two
moieties forming the dyad.
A first explanation can be found in the correlation between
photophysical properties and topology of the fullerene core.
Studies of a series of methanofullerene46,81 and epoxyfullerene46
adducts with an increasing number of addends demonstrated
that multiple functionalization results in the perturbation of
the conjugation of the fullerene core and in an ensuing rise of
the triplet states energy and abatement of triplet and singlet
oxygen quantum yields. However, in the mono-adducts the
topology effect can indeed account only for 10% decrease in
the singlet oxygen generation efficiency.81
A second factor contributing to the FD reduction could be
the shielding effect exerted by the DSB branches toward the
photoactive fullerene core. This effect was observed in dendritic
tris(bipyridine) ruthenium chelates84 and, more recently, in
dendritic-fullerene derivatives.83 In the case of fullerene adducts,
this effect led to a decrease of oxygen diffusion rate regardless
of the solvent nature, but only in polar solvents was the
delayed singlet oxygen sensitization accompanied by an effec-
tive decrease of FD. This result was attributed to the more
compact structure assumed by the dendrimer in polar solvents,
resulting in a less effective orbital overlap between the active
[60]fullerene core and the oxygen.83 Our steady-state measure-
ments are not sensitive to the dynamic aspects of the 1O2
generation, so it was not possible to verify if the presence of
the two DSB branches cause a relevant decrement of the
oxygen diffusion rate. On the other hand, the use of a non-polar
solvent like toluene should guarantee a relatively open configu-
ration and a minimization of the shielding effect.
Another possible mechanism, recently proposed for porphyrin–
fullerene dyads in toluene, is based on the results obtained
with time-resolved electron paramagnetic resonance (TR-EPR)
measurements.82,87 This alternative mechanism (Fig. 4) involves
electron transfer, intersystem crossing between radical pair
species and back electron transfer to generate the fullerene
3
triplet state (Dꢁ *C60). The activation of such a mechanism
could explain the loss of efficiency in the 1O2 generation, as well
as the small amount of energy transfer from DSB to fullerene
detected by the fluorescence excitation measurements.
The involvement of a radical species in the sensitization
mechanism can be indirectly verified checking the efficiency of
1O2 generation in more polar solvents, capable of stabilizing
radicalic intermediates. Comparison between the efficiency of
1O2 sensitization in different solvents of pristine [60]fullerene
with the one of dyad 1 shows a net decrease for the latter with
respect to [60]fullerene, when the dielectric constant of the
solvent increases. The solvents employed are toluene, chloro-
benzene and benzonitrile, characterized by a sizable change of
their dielectric constant (from 2.38 of toluene to 25.20 of
benzonitrile). These experimental findings are an indirect
confirmation of the involvement of charge separated species
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1
in the proposed decay mechanism (details on the measure-
ments are given in the ESI).w
Although corroborated by this indirect evidence, the presence
The figure indicates that the O2 data recorded upon 710 and
750 nm irradiation are consistent with a two-photon absorp-
tion scheme. The linear slopes generated by a least-squares
analysis are 1.9 ꢃ 0.1 and 1.8 ꢃ 0.15 for 710 and 750 nm
excitation, respectively, as expected for a TPA process. The
data obtained with 403 nm excitation (slope 1.01 ꢃ 0.05) are
also reported for comparison.
3
of this pathway leading to formation of Dꢁ *C60 from radical
pair species could be confirmed eventually through TR-EPR
measurements.
As a final remark it is important to point out that although
the FD of 1 is smaller than the FD of [60]fullerene, it still reaches
a 30% value, necessary for a molecule to be considered as a
suitable candidate for photodynamic therapy. Furthermore 1
absorbs strongly in the visible, where most lasers actually
employed in photodynamic therapy emit, whereas [60]fullerene
shows just a vanishingly small absorption coefficient in this
spectral range.
As a control experiment, we also irradiated in the same
conditions sensitizer-free toluene and solutions of the two
standards ([60]fullerene and meso-TPP) in toluene, both air-
equilibrated and degassed. In all cases, no signal with quadratic
dependence was recorded and after correction for scattered
light, the signal resulted in zero.
Experimental
Two-photon sensitization experiments. It is reasonable to
assume that irrespective of whether excitation is promoted
by a one- or a two-photon process, fast relaxation to the
lowest singlet excited S1 state will take place. Since the process
leading to singlet oxygen sensitization originates from S1, the
photophysics of 1O2 will be independent of the excitation
scheme. The main difference between the one-photon and
the two-photon excitation schemes is the yield of S1 popu-
lation, being about 7-8 orders of magnitude lower for the two-
photon process than for the one-photon process.57 Thus, to be
Materials
[60]fullerene was purchased from Bucky USA (99.5%). 2,5-
bis(diethylphosphonate)-1,4-dimethoxybenzene,61 4-(2-hydroxy-
ethoxy)-benzaldehyde62 and methanofullerene 5 (Fig. S13, ESI)w
were prepared as previously reported. All other reagents were
used as purchased from Sigma-Aldrich. The toluene for photo-
physical characterizations of derivatives 1–5 was a commercial
spectrophotometric-grade solvent used without further purifi-
cation. Thin layer (TLC) and column chromatography were
performed using a Polygram SilG/UV254 (TLC plates) and
silica gel MN 60 (70–230 Mesh) by Macherey-Nagel.
1
able to detect O2 signal promoted by TPA, the molecule not
only must efficiently produce singlet oxygen, but also have an
appreciable TPA cross section.
The first attempt to detect 1O2 luminescence was made
irradiating a toluene solution of 4 at 806 nm. No signal could
be detected within the noise level. This was partly expected
since the TPA cross section of 1 at 806 nm is less than 50 GM
(Fig. 3), too low to effectively populate the S1 state with the
available laser intensity.
Instrumentation
1H (250.1 MHz) and 13C (62.9 MHz) NMR spectra were
recorded on a Bruker AC-F 250 spectrometer. IR spectra were
recorded on a Perkin-Elmer FT-IR model 1720X. Oily com-
pounds were measured between KBr windows; powders were
measured making KBr pellets. HPLC analysis of 1 was carried
out on a Shimadzu LC-8A instrument, equipped with a
Shimadzu UV6000LP photodiode array detector by Thermo
Scientific, using a SiO2 Phenomenex Luna column (150 ꢅ 4.6 mm,
4 mm, eluent: toluene, flow rate: 2 ml minꢁ1). ESI-MS spectra
were recorded on a LC-MSD-Trap-SL (Agilent Technologies,
Palo Alto – USA). Samples were dissolved in methanol
(B10ꢁ5 M) and injected in a stream of the same solvent at
50 ml minꢁ1 (nitrogen nebulizing gas pressure = 20 psi; flow =
5 L minꢁ1; T = 325 1C). APPI-MS spectrum of derivative 1
was recorded on the same instrument (nitrogen nebulizing gas
pressure = 30 psi; flow = 4 L minꢁ1; T = 325 1C; vaporizer
T = 350 1C). The sample, dissolved in cyclohexane (B10ꢁ5 M),
was directly infused in the APPI source through a syringe pump
(100 ml minꢁ1).
Absorption and fluorescence spectra of all compounds
were carried out with a Cary 5 (Varian) and a Fluoromax
(Spex Jobin-Yvon), respectively. Typical concentrations were
B1 ꢅ 10ꢁ5 M for one-photon absorption and 1 ꢅ 10ꢁ6 M for
emission measurements. The TPA experiments (on samples
B1 ꢅ 10ꢁ4 M) were performed using the TPA induced
fluorescence technique (TPIF). The technique and the experi-
mental set-up were described in detail elsewhere.63 Briefly, a
tunable Ti:Sapphire femtosecond laser system (Coherent Mira
Optima 900-F), delivering pulses with approximately 130 fs
In the light of the TPA measurements presented in Fig. 3,
the experiment was repeated exciting at shorter wavelengths
(710 and 750 nm), where the TPA cross section is large enough
to generate a non negligible S1 population. At these wave-
lengths the signal was dominated by a large contribution of
scattered IR light, generated by the OPA800C, and not
adequately removed with long-pass filters. It was however
possible to discriminate between the scattered light and the
1O2 luminescence emission comparing the signal recorded for
the air-equilibrated and the degassed solution (Fig. 5a). The
signal recorded from the degassed tube arises only from
scattering processes, and shows indeed the typical linear
1
dependence on the pulse energy. Generation of O2 by one-
photon processes can be ruled out because the absorbance of
the sample solution at these wavelengths is o0.002. On the
other hand, the signal from the air-equilibrated solution shows
a super-linear dependence on the pulse energy, as expected for
multi-photon processes. After correction of the air-equilibrated
solution signal for the scattered light, a clear quadratic depen-
1
dence is found (Fig. 5b, 6). Since for TPA, the O2 intensity is
known to be proportional to the square of laser intensity, we
can ascribe these signals to sensitization of singlet oxygen
promoted by two-photon excitation.
Fig. 6 shows the double logarithmic plot of 1O2 inten-
sity against laser pulse energy at 710 and 750 nm excitation.
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Fig. 5 (a) Comparison between the signals recorded for an air-equilibrated (squares) and a degassed (circles) solution of 1 in toluene. The signal
recorded in the degassed solution is only due to scattering, not completely removed, as indeed proven by the typical linear dependence on the pulse
energy. In the inset the actual signal due to 1O2 luminescence emission obtain after subtraction of the scatter. (b) TPA promoted 1O2 generation at
different wavelengths. The signals were already corrected for the scatter contribution. The inset shows an enlargement of the data at 750 nm.
instead of fluorescence cuvettes, because they allow degassing
and sealing the sample under vacuum after several freeze-
pump-thaw cycles. This procedure was applied to all the
solutions employed, to check that the signal recorded by the
experimental set-up is generated exclusively by 1O2 emission at
1270 nm. The one-photon excited emission was collected by a
combination of lenses at 901 with respect to the excitation
pulses, and sent to a room temperature InGaAs photodiode
(Hamamatsu). The signal from the photodiode was then
averaged by a Lock-In amplifier and recorded by a computer.
The two-photon promoted 1O2 generation was measured at
three different wavelengths: 710, 750 and 806 nm. The funda-
mental emission of the laser at 806 nm was employed directly
or used to pump an optical parametric amplifier (OPA800C-
1
Fig. 6 Double logarithmic plot of O2 intensity against pulse energy
Spectra Physics) set to deliver pulses at 710 and 750 nm. A
on air-equilibrated toluene solutions of 1 at 403 nm (triangles), 710 nm
slightly different acquisition geometry was employed at these
(circles) and 750 nm (squares) excitation. Dots are experimental data
wavelengths: luminescence was detected directly from the
and the solid lines are linear fits. The signals were corrected for the
front face of the sample by a liquid-nitrogen cooled InGaAs
scatter contribution using a degassed solution of the sample.
photodiode/preamplifier (EG&G Judson) system. An inter-
ference filter centred at 1270 ꢃ 20 nm (CVI) was placed
duration at the repetition rate of 76 MHz is used to excite the
directly in front of the photodiodes to discriminate the singlet
sample from 730 to 830 nm. The laser pulses are focalized by a
oxygen emission from the laser scattered light and all other
40 cm lens inside a 1 cm path cuvette, so that the Rayleigh
sample emissions.
range extends almost over the whole depth of the cuvette. The
fluorescence signal, collected at 901 by a 4 cm lens, is measured
Syntheses and selected analytical data: methanofullerene 1
with a photomultiplier tube and recorded by a computer-
controlled digital oscilloscope as a function of input beam
intensity. The quadratic dependence of the fluorescence signal
with respect to the incident laser intensity was verified at each
excitation wavelength.
To a solution of [60]fullerene (77 mg, 0.11 mmol, 1.2 eq.),
malonate 4 (83 mg, 0.09 mmol, 1 eq.) and iodine (27 mg,
0.11 mmol, 1.2 eq) in toluene (60 ml), a solution of DBU (20 mg,
0.13 mmol, 1.2 eq) in 10 ml toluene was added dropwise under
a nitrogen atmosphere over a period of 10 min. When the
addition was complete, the brownish mixture was stirred for
2 h at rt. The solvent was removed under reduced pressure and
the crude product purified by flash chromatography (SiO2,
eluent: toluene, then toluene/ethyl acetate 95:5). The fractions
containing the product were concentrated to a small volume
under reduced pressure and were transferred into a centrifuge
tube. The product was precipitated from cyclohexane and
washed with methanol. 43.5 mg (30%) of 1 were isolated as
a brownish powder. 1H-NMR (250 MHz, CDCl3, 25 1C,
TMS) d 7.49–7.42 (t, 8H), 7.37–6.98 (m, 8H), 7.08 (s, 4H),
The one-photon and two-photon 1O2 generation perfor-
mances were investigated through direct measurement of the
dye-sensitized singlet oxygen luminescence at 1270 nm. In the
one-photon experiment, the sample was excited at 403 nm by
the frequency-doubled emission of a Ti-sapphire amplified
laser system (Spectra Physics) characterized by 150 fs long
pulses at several repetition rates in the range 0.2–1 kHz. The
laser pulses were suitably attenuated by neutral density filters
as well as a combination of a half-waveplate and a polarizer.
The sample solution was held in a 4 mm diameter quartz tube
to open air, and no oxygen was added. Quartz tubes are used,
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6.90–6.89 (d, 4H), 6.87–6.85 (d, 4H), 4.82–4.80 (t, 4H),
4.34–4.32 (t, 4H), 3.89 (s, 12H), 3.83 (s, 6H). 13C-NMR
(250 MHz, CS2CDCl3 4:1, 25 1C, TMS) d 163.05, 159.18,
157.78, 151.32, 151.27, 145.30, 145.19, 145.13, 145.04, 144.91,
144.70, 144.65, 143.90, 143.06, 143.03, 142.26, 141.85, 140.98,
139.15, 131.59, 130.78, 129.11, 128.30, 128.00, 127.86, 126.72,
126.33, 121.86, 121.25, 114.83, 114.19, 108.75, 108.63, 71.32,
65.60, 65.38, 56.02, 55.12, 51.76. HPLC: Rf (1) = 3.38 min;
purity 498%. APPI-MS m/z 1651 [M+H]+.
0.12 mmol, 1 eq) in 5 ml of dry CH2Cl2 was slowly added
dropwise over a period of 2 h. The mixture was left stirring for
additional 12 h at room temperature, then the solvent was
removed under reduced pressure. The crude product was
purified by flash chromatography (SiO2, eluent: ethyl acetate/
petroleum ether 7:3). 92.6 mg (86%) of derivative 4 were
isolated after crystallization from a THF/ethanol mixture.
1H-NMR (250 MHz, CDCl3, 25 1C, TMS) d 7.50–7.45 (q, 8H),
7.37–7.02 (q, 8H), 7.09 (s, 4H), 6.91–6.88 (d, 4H), 6.87–6.85
(d, 4H), 4.52–4.48 (t, 4H), 4.19–4.16 (t, 4H), 3.90 (s, 12H), 3.83
(s, 6H), 3.50 (s, 2H). IR(KBr) nB 3035, 2998, 2932, 2832, 1754,
1733, 1603, 1510, 1406, 1247, 1208, 1174, 1039, 965, 848, 817
cmꢁ1. ESI-MS: m/z 932 [M]+.
Phosphonate 2
To a solution of 2,5-bis(diethylphosphonate)-1,4-dimethoxy-
benzene (10.0 g, 22 mmol) and 4-methoxybenzaldehyde (3.10 g,
22 mmol) in 200 ml of DMF, a solution of tBuOK (1.72 g,
15 mmol) in 15 ml of DMF was added dropwise, under a
nitrogen atmosphere, over a period of 1 h. When the addition
was complete, the mixture was stirred at room temperature for
3 h and then concentrated at reduced pressure. The crude
product was purified by unreacted aldehyde and symmetrical
distyrylbenzene by flash chromatography (SiO2, eluent: ethyl
acetate/petroleum ether 8:2, then ethyl acetate/ethanol 9:1).
5.74 g (62%) of phosphonate 2 were isolated as a light yellow
oil that solidifies on standing. 1H-NMR (250 MHz, CDCl3,
25 1C, TMS) d 7.49–7.46 (d, 2H), 7.35–7.00 (q, 2H), 7.08 (s, 1H),
6.94–6.92 (d, 1H), 6.91–6.87 (d, 2H), 4.11–3.99 (m, 4H), 3.87
(s, 3H), 3.85 (s, 3H), 3.82 (s, 3H), 3.30–3.21 (d, 2H), 1.29–1.23
(t, 6H). 13C-NMR (250 MHz, CDCl3, 25 1C, TMS) d 159.20,
151.15, 130.51, 128.24, 127.62, 125.87, 121.05, 119.85, 114.49,
113.94, 108.29, 61.89, 56.12, 55.18, 27.67, 25.45, 16.27.
IR(KBr) nB 2983, 2905, 2838, 1603, 1514, 1413, 1265, 1222,
1036, 971, 818 817 cmꢁ1. ESI-MS: m/z 421 [M+H]+.
Conclusion
In the search for novel materials acting as photosensitizers for
two-photon excited photodynamic therapy, we synthesized
and characterized a prototype fullerene dyad in which pristine
[60]fullerene was functionalized with distyrylbenzene moieties.
The use of a two-photon absorption excitation process as
an alternative to one-photon processes in PDT treatment is
advantageous because of the possibility to focus on a confined
and small treatment area of diseased tissues with a greater
depth and at the same time avoid damage to healthy surrounding
tissues. The success of this approach relies on the availability
of water–soluble molecules with significantly large TPA cross-
section and singlet oxygen generation. The dyad proposed in
this work represents a promising prototype system in which
the high efficiency of [60]fullerene as singlet oxygen sensitizer is
exploited in combination with the high TPA cross section of
distyrylbenzene moieties.
The photophysical properties of the dyad were preliminarily
characterized by linear and nonlinear optical experiments and
compared with the properties of model precursor compounds.
Linear fluorescence experiments highlighted the presence of
strong quenching of the DSB emission and confirmed the
presence of energy or electron transfer processes from the excited
DSB unit towards the fullerene. Moreover, TPA measure-
ments showed a two-fold enhancement of the TPA cross-section
in the dyad with respect to the DSB model compound.
Characterization of singlet oxygen sensitization showed that,
although the quantum yield of the dyad is smaller than the one
of the pristine [60]fullerene, it still reaches a 30% value,
necessary for a molecule to be considered a suitable candidate
for photodynamic therapy. More interestingly, it was possible
to record sensitization of singlet oxygen promoted also by two-
photon excitation.
Distyrylbenzene 3
To a solution of 4-(2-hydroxyethoxy)-benzaldehyde (0.59 g,
3.56 mmol) and phosphonate 2 (1.50 g, 3.56 mmol) in 20 ml
DMF, a solution of tBuOK (1.72 g, 8.82 mmol) in 10 ml of
DMF was added dropwise under a nitrogen atmosphere over a
period of 1 h. When the addition was complete, the mixture
was stirred at room temperature for 2 h and then concentrated
at reduced pressure. The crude product was dissolved in
CH2Cl2 and washed with dil. HCl. The organic phase, dried
over Na2SO4, was concentrated and the residue crystallized
from CH2Cl2/hexane. 1.30 g (85%) of 3 were isolated as
yellow crystals. 1H-NMR (250 MHz, CDCl3, 25 1C, TMS)
d 7.50–7.47 (d, 4H), 7.39–7.00 (q, 4H), 7.11 (s, 2H), 6.93–6.91
(d, 2H), 6.89–6.88 (d, 2H), 4.11–4.09 (t, 2H), 3.99–3.95 (t, 2H),
3.91 (s, 6H), 3.83 (s, 3H). 13C-NMR (250 MHz, CDCl3, 25 1C,
TMS) d 159.18, 158.17, 151.33, 131.16, 130.68, 128.34, 128.14,
127.78, 126.58, 126.39, 121.39, 121.10, 114.70, 114.06, 108.90,
69.20, 61.47, 56.35, 55.30. IR(KBr) n B 3033, 2996, 2936,
2832, 1601, 1510, 1406, 1249, 1209, 1175, 1041, 963, 847,
819 cmꢁ1. ESI-MS: m/z 432 [M]+.
The approach utilized in this study provided a reliable
starting point for the design of efficient [60]fullerene-based
structures exploitable in two-photon excited PDT.
Acknowledgements
Distyryl malonate 4
Financial support was provided by the grants: FIRB
RBNE033KMA and PRIN 2007LN873M (MIUR), PROMO
(CNR-INSTM), CPDA074184 (Univ. of Padova) and MISCHA
(Fondazione CARIPARO). We wish to thank Dr E. Marotta,
Univ. of Padova for APPI-MS measurements.
To a solution of distyrylbenzene 3 (0.1 g, 0.23 mmol, 2 eq.) in
10 ml of anhydrous CH2Cl2, NaHCO3 (77.4 mg, 0.92 mmol,
8 eq) was added under a nitrogen atmosphere. After 30 min at
room temperature, a solution of malonyl dichloride (16.3 mg,
ꢂc
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36 E. Nakamura and H. Isobe, Acc. Chem. Res., 2003, 36,
807–815.
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