Banana-shaped biphotonic quadrupolar chromophores: From fluorophores to biphotonic photosensitizers

Article abstract of DOI:10.1039/c1nj20073a

This study aims at designing dual-role biphotonic chromophores that could be used for photodynamic therapy (PDT) while maintaining some fluorescence in order to locate them, thus allowing selective irradiation of cancer cells when combined with targeting.

Full text of DOI:10.1039/c1nj20073a

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New Journal of Chemistry
A journal for new directions in chemistry
www.rsc.org/njc
Volume 35 | Number 9 | September 2011 | Pages 1761–1908
ISSN 1144-0546

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PAPER
Banana-shaped biphotonic quadrupolar chromophores: from fluorophores
to biphotonic photosensitizersw
ab
c
Cedric Rouxel, Marina Charlot,ab Youssef Mir,ab Celine Frochot,
´
´
Olivier Mongin*ab and Mireille Blanchard-Desce*ab
Received (in Montpellier, France) 27th January 2011, Accepted 25th March 2011
DOI: 10.1039/c1nj20073a
This study aims at designing dual-role biphotonic chromophores that could be used
for photodynamic therapy (PDT) while maintaining some fluorescence in order to locate them,
thus allowing selective irradiation of cancer cells when combined with targeting. Quadrupolar
two-photon absorbing fluorophores were synthesized from the symmetrical functionalization
of a fluorene core bearing elongated conjugated rods made from arylene–vinylene or
arylene–ethynylene building blocks in order to test modifications which could increase singlet
oxygen production ability while retaining some fluorescence and high two-photon absorption
(TPA) cross-sections in the biological spectral range of interest. All chromophores show a polar
emissive excited state whose dipole moment is highly dependent on the nature of the conjugated
linker. Interestingly, the largest TPA responses in the NIR region as well as singlet oxygen
quantum yield are correlated with the smallest dipole moment of the emissive excited-state.
The molecular optimization study led to a multifunctional biphotonic chromophore combining
high TPA cross-sections in the whole spectral range of interest (700–900 nm), reasonable singlet
oxygen production efficiency, significant remaining fluorescence, and alcohol end-groups
for further covalent grafting. This compound offers thus interesting potentialities for highly
selective spatially-resolved two-photon PDT by incorporation in nanostructures.
advantages for biological or biomedical applications, including
Introduction
the ability for highly selective excitation in biological media,
intrinsic three-dimensional resolution, and reduction of photo-
damage by using lower excitation intensities. Depending on the
targeted applications, TPA chromophores have to fulfil different
requirements: molecular engineering depends on the desired
application, in terms of spectral range and specific additional
features. For instance, in the case of biological imaging, high
fluorescence quantum yields, photostability and absence of
toxicity as well as very large TPA cross-sections in the bio-
logical spectral window (700–1100 nm) are required.18–20 Such
large TPA cross-sections are also required for photodynamic
therapy in addition to high singlet oxygen production quantum
yield or reactive oxygen species sensitization (harmful to cancer
cells).3,21 Hence biphotonic chromophores (i.e. chromophores
having very large TPA cross-sections) suitable for PDT or
imaging would have different excited state outcomes: fluores-
cence (for imaging applications) or redox behavior or sensiti-
zation of oxygen for PDT. The present work aims at designing
dual-role biphotonic chromophores that could be used for PDT
while maintaining some fluorescence in order to locate them,
thus allowing selective irradiation of cancer cells when combined
with targeting. Such chromophores should thus combine signifi-
cant fluorescence and singlet oxygen quantum yields while having
very large TPA cross-sections in the biological spectral window.
Molecular two-photon absorption (TPA) has gained increasing
attention over recent years,1–4 in relation with the wide-ranging
applications it offers, such as microfabrication,5–7 3D optical
data storage,8,9 optical power limiting,10,11 two-photon laser
scanning microscopy12,13 or photodynamic therapy14–17 (PDT).
Most of these applications require both pulsed sources (to increase
the probability of this nonlinear process) i.e. femtosecond,
picosecond, or possibly nanosecond lasers and chromophores
exhibiting very high TPA cross-sections (intense but also
broad TPA bands are highly desirable due to the versatility
these offer in terms of laser sources), allowing either more
efficient excitation or decrease of the excitation intensity (use of
less expensive laser sources). This also offers a number of
a
´
CNRS, Chimie et Photonique Moleculaires (UMR 6510),
´
´
263 avenue du General Leclerc, 35042 Rennes, France.
E-mail: mireille.blanchard-desce@univ-rennes1.fr,
olivier.mongin@univ-rennes1.fr
b Universite´ de Rennes 1, UMR 6510, Campus de Beaulieu,
Baˆtiment 10A, Case 1003, 35042 Rennes, France
´
LRGP UPR 3349, Nancy-Universite, CNRS, 1 rue Grandville,
BP 20451, F-54001 Nancy, France
c
w Electronic supplementary information (ESI) available: Photophysical
properties, emission spectra and Perrin plots of compounds Q1, Q2 and
Q02 in different solvents. See DOI: 10.1039/c1nj20073a
c
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Most of the one-photon photosensitizers are porphyrin
derivatives; however, single porphyrins have low TPA cross-
sections in the spectral range of interest (15 GM for tetra-
phenylporphine).22 Giant TPA cross-sections can be obtained
with expanded porphyrins,23,24 diyne porphyrin dimers,25
fused porphyrin dimers,26 porphyrin arrays27 or supramolecular
assemblies,25,28 however, at the expense of a definite decrease
of the fluorescence quantum yield (if not suppression) and
residual one-photon absorption overlapping with the TPA
band. Indeed the one-photon resonance enhancement at the
origin of the giant TPA values results in a loss of some of the
advantages resulting from selective TPA, in particular the 3D
resolution. In this context, our aim was to develop alternative
two-photon fluorescent photosensitizers based on banana-
shaped quadrupolar systems. We have previously shown that
such derivatives can combine very large TPA cross-sections
and high fluorescence quantum yields.29–35 Among others, this
approach led to membrane markers for two-photon cellular
imaging,30,36 but also to nanodots37–39 with outstanding two-
photon brightness as ‘‘soft’’ biocompatible substitutes to toxic
quantum dots for in vivo imaging in living animals.38 Based on
these results, our purpose was to investigate structural variation
of the quadrupolar systems, in order to increase intersystem
crossing while maintaining significant fluorescence quantum
yields in order to obtain biphotonic sensitizers that retain
fluorescence to allow in vivo monitoring and subsequent
localized irradiation.
and by adding terminal grafting moieties. Such moieties
could be of interest for conjugation of photosensitizers with
hydrophilic targeting moieties, such as peptides43–45 or folic
acid.46,47 This would provide an expeditious way to provide
water-solubility. Alternatively, in lieu of direct conjugation,
terminal grafting moieties are also intended for covalent
anchoring of the photosensitizers within the matrix of
silica nanoparticles48 or the addition of shielding dendritic
branches.38,49 Such routes would allow isolating the lipophilic
chromophores from the water environment, in particular from
the deleterious effects of water on TPA50 but also on the
singlet oxygen and fluorescence quantum yields.38 In addition,
nanostructures such as nanoparticles or dendrimers offer high
flexibility for further surface-functionalization with hydrophilic
targeting moieties.48,51
Results and discussion
Design
Four biphotonic quadrupolar chromophores have been studied
in the present work (Chart 1). Model compounds Q1 and Q2
were chosen as lead compounds, since they combine
photostability, in relation with triple bonds in the conjugated
system, broad and intense TPA bands in the 700–900 nm range,
as well as high fluorescence quantum yield (Ff).35 Molecule Q2
exhibits a larger TPA cross-section (s2) than Q1, but a
lower fluorescence quantum yield, which leads to a similar
two-photon brightness (s2Ff) for both molecules. However
for the targeted application (fluorescence + PDT), Q2 could
be more efficient than Q1 (s2FD being the figure of merit
for the singlet oxygen production, where FD is the singlet
oxygen quantum yield) if increase of intersystem crossing
is responsible for the decrease of the fluorescence quantum
yield. Compound Q02, a functionalized analogue of Q2
Ogilby and coworkers40–42 have implemented a successful
strategy based on the adjunction of heavy atoms such as
bromine on a quadrupolar backbone. This leads to efficient
intersystem crossing and singlet oxygen generation quantum
yields but concomitantly to vanishing fluorescence. We herein
explore an alternative approach by modifying aromatic building
blocks in the conjugated system (replacing phenylene units by
thienylene units, in order to increase intersystem crossing),
Chart 1 Chemical structures of Q1, Q2, Q02 and Q03 (Non = n-nonyl, Bu = n-butyl).
c
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1772 New J. Chem., 2011, 35, 1771–1780

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possessing terminal grafting moieties, has also been synthesized,
in order to evaluate the potential influence of these non-
conjugated auxiliary groups on the photophysical properties,
via environmental effect. Finally Q03, in which phenylene
units have been replaced by thienylene units in the
conjugated system, has also been prepared. According to the
essential-state model52,53 such a replacement is expected to
lead to TPA enhancement, but it could also increase inter-
system crossing,54 which could in turn facilitate singlet oxygen
production.
Photophysical properties
One-photon absorption (OPA) and photoluminescence
properties. Table 1 and Fig. 1 show, respectively, the compared
photophysical properties and the absorption/emission spectra
of compounds Q1, Q2, Q02 and Q03 in toluene. All chromo-
phores present an intense absorption band from the near
UV to the blue visible range. The maximum molar extinction
coefficients of the four chromophores gathered in Table 1 are
very large and all the oscillator strengths are much higher than
1, concomitantly with large full widths at half maximum. On
one hand fluorescence quantum yields of Q1, Q2 and Q02 are
relatively high, ranging between 0.6 and 0.8, which is of interest
for imaging applications. On the other hand, compound Q03
shows a lower fluorescence quantum yield indicative of the
onset of non-radiative deactivation processes competing with
fluorescence emission.
Synthesis
Quadrupolar chromophores Q1 and Q2 were synthesized
according to the procedures reported in ref. 35. The synthesis
of chromophores Q02 and Q03 is described in Scheme 1. The
fluorene core 1c was obtained from 9,9-dibutyl-2,7-diiodo-
9H-fluorene (1a)55 by means of a twofold Pd(II)-catalyzed
cross-coupling with 2-methyl-3-butyn-2-ol, followed by base-
promoted deprotection of the resulting intermediate 1b. Double
Sonogashira coupling of 1c with 4-bromobenzaldehyde (2)
afforded bis-aldehyde 3. In contrast, analogous reaction with
5-bromothiophene-2-carboxaldehyde failed in our hands and
this compound had to be replaced by the corresponding iodo
derivative 5,56 to afford 6 very efficiently. Wittig condensations
of 3 and 6 with phosphonium salt 457 led to chromophores Q02
and Q03, respectively, as single (E,E)-stereoisomers. It should
be noted that protection of the alcohol function of 4 proved
unnecessary.
As expected, substitution of a triple bond (compound Q1)
by a double bond (compound Q2) in the conjugated path
induces a bathochromic shift of both absorption and emission
bands (Fig. 1), in agreement with improved electronic delocali-
zation. As well as being more transparent, compound Q1 is
also more fluorescent than its analogue Q2, this effect being
related mostly to a smaller non-radiative decay rate due to reduced
conformational flexibility. Replacing the long lipophilic chains
on the amino donating end groups (compound Q2) by grafting
moieties (compound Q02) induces a slight blue shift of the
absorption band and reductions of both the fluorescence
quantum yield (due to a larger non-radiative decay rate) and
anisotropy (in relation with the smaller size). Finally, replacement
Scheme 1 (a) 2-Methyl-3-butyn-2-ol, Pd(PPh3)2Cl2, CuI, toluene/Et3N, 40 1C, 16 h (87%); (b) KOH, toluene/i-PrOH, reflux, 0.5 h (71%);
(c) Pd(PPh3)2Cl2, CuI, toluene/Et3N, 40 1C, 16 h (75% for 3 and 97% for 6); (d) 4, tBuOK, anhyd CH2Cl2, 20 1C, 16 h (53% for Q02 and 35%
for Q03).
c
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Table 1 Photophysical properties of compounds Q1, Q2, Q02 and Q03 in toluene
Cpd labs/nm emaxa/Mꢀ1 cmꢀ1 FWHM/cmꢀ1 fb
l
em/nm Stokes shift/cmꢀ1 Ff td /ns kre (109 sꢀ1) knr (109 sꢀ1) t0 /ns rg
FD FD
c
e
f
h
i
Q1 387
Q2 411
Q02 406.5
Q03 436.5
1.3 ꢁ 105
4390
4730
4740
4780
2.3 433
2.5 464
— 463.5
2750
2780
3030
2690
0.82 0.75 1.09
0.71 0.82 0.87
0.67 0.79 0.85
0.48 0.93 0.52
0.24
0.35
0.42
0.56
0.91
1.15
1.18
1.94
0.24 0.05 0.02
0.23 0.03 0.01
0.19 0.02 0.01
0.16 0.22 0.12
1.3 ꢁ 105
j
—
1.0 ꢁ 105
1.9 494.5
a
b
Molar extinction coefficient. Oscillator strength. Fluorescence quantum yield determined relative to quinine in 0.5 M H2SO4. Fluorescence
c
d
e
f
lifetime determined by using time-correlated single-photon counting (TCSPC). Radiative (kr) and non-radiative (knr) decay rates. Radiative
g
h
lifetime. Fluorescence anisotropy. Singlet oxygen formation quantum yield determined relative to tetraphenylporphyrin in toluene (FD[TPP] = 0.68
in toluene). i Singlet oxygen formation quantum yield determined relative to tetraphenylporphyrin in chloroform (FD[TPP] = 0.55 in chloroform).
j
The molar extinction coefficient of Q02 has not been measured due to its low solubility in toluene.
Fig. 1 Normalized absorption and emission spectra of compounds Q1, Q2, Q02 and Q03 in toluene.
of the two phenylene units by two thienylene units (Q02 - Q03)
leads to a marked red-shift of both absorption and emission
and induces a significant reduction of the fluorescence quantum
yield as a result of combined decrease of the radiative decay
rate (in relation with red-shifted emission) and increase of the
non-radiative decay rate. This is consistent with a facilitated
inter-system crossing (S1 - T1).58
Solvatochromism. Table 2 illustrates the dependence of the
optical properties of chromophore Q03 on solvent polarity
and viscosity. All compounds display similar solvatochromic
behaviour (see ESIw). Increasing the solvent polarity induces a
slight red shift of the absorption band and a marked batho-
chromic shift of the emission band as clearly illustrated in
Fig. 2 for chromophore Q03. This marked positive solvato-
chromism provides evidence of a highly polar emissive excited-
state, in agreement with a localization of the excitation on a
dipolar chromophoric subunit in low polarity to high polarity
environments.59
Whereas compounds bearing linear alkyl chains Q1 and Q2
present a similar fluorescence anisotropy in agreement with a
similar size, compound Q02 lacking terminal long alkyl
chains shows a smaller anisotropy ratio. Q03 having similar
peripheral (and central) alkyl groups to chromophore Q02
reveals a lower anisotropy ratio in agreement with the smaller
size of its conjugated system.
In low to medium polarity solvents Q03 displays significant
fluorescence (Ff values ranging from 0.5 to 0.6) whereas its
fluorescence significantly decreases in high polarity solvents
Table 2 Photophysical properties of compound Q03 in different solvents
Solvent
labs/nm
lem/nm
Stokes shift/cmꢀ1
Ffa
t/ns
kr (109 sꢀ1
)
knr (109 sꢀ1
)
t0b/ns
rc
Toluene
Bu2O
CHCl3
AcOEt
DCM
436
493
491
522.5
544
549.5
555
591.5
604
617
2650
2700
4035
4605
4870
4865
5950
6355
6090
0.48
0.55
0.48
0.51
0.61
0.51
0.22
0.052
0.051
0.93
0.99
1.22
1.42
1.43
1.51
0.88
1.03
0.49
0.52
0.56
0.39
0.36
0.42
0.34
0.25
0.05
0.10
0.56
0.45
0.43
0.34
0.27
0.32
0.89
0.92
1.94
1.94
1.79
2.54
2.77
2.35
2.93
4.02
19.99
9.7
0.16
0.19
0.15
0.12
0.10
0.12
0.13
0.21
0.28
433.5
431.5
435
433.5
437
437.5
436.5
448.5
THF
Acetone
CH3CN
DMSO
a
b
Fluorescence quantum yield determined relative to quinine in 0.5 M H2SO4. Radiative lifetime. Fluorescence anisotropy.
c
c
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Fig. 2 Normalized emission spectra of fluorophore Q03 in solvents of different polarities.
like DMSO, acetone and CH3CN due to the combination of
reduced radiative decay rates (in relation with red-shifted emission)
and significant enhancement of the non-radiative decay rates,
indicating efficient competing deactivation processes.
Table 3 Molecular sizes (long axis R) and excited-state dipole moments
of chromophores Q1, Q2, Q02 and Q03 derived from fluorescence
solvatochromism and anisotropy studies
Lippert–Mataga
slopec (103 cmꢀ1
Compound
Ra/A
ab/A
)
Dm/D
As shown in Fig. 3, the solvatochromic behaviour of the
quadrupolar chromophores in medium to high polarity solvents
can be fitted with a Lippert–Mataga relationship, given in
eqn (1).60,61 The Stokes shift values show a linear dependency
on the polarity–polarizability function Df:
Q1
13.1
12.9
11.6
11.1
6.55
6.45
5.8
24.6
19.6
19.1
17.1
27
23
20
17
Q2
Q02
Q03
5.55
a
b
Molecular radius derived from fluorescence anisotropy. Dipolar
nabs ꢀ nem = 2(Dm2/hca3)Df + const
(1)
c
Onsager cavity radius. Slope derived from the linear dependence of
the Stokes shift on the solvent polarity–polarizability function Df.
where nabs (nem) is the wavenumber of the absorption (fluores-
cence) maximum, h is the Planck constant, c is the light
velocity, a is the radius of the Onsager cavity, and Df = (e – 1)/
(2e + 1) – (n2 – 1)/(2n2 + 1), where e is the dielectric constant
and n the refractive index of the solvent while Dm is the change of
dipole moment of the solute between ground and excited states.
This behaviour is consistent with (medium to high polarity)
solvent-induced symmetry breaking occurring in the excited
state of quadrupolar chromophores, leading to excitation
localization on a dipolar chromophoric subunit (i.e. DA
portion of the DAD quadrupolar structure).59 Hence the
emission solvatochromism is similar to that of corresponding
push–pull chromophores showing strong intramolecular
charge transfer transition. The slopes of the corresponding
linear regressions (i.e. 2Dm2/hca3) are collected in Table 3. To
further derive Dm values, accurate estimation of the Onsager
cavity radius (a) is required. To do so, anisotropy (r) and fluores-
cence lifetime (t) measurements have been conducted in solvents
of varying viscosity (Z) in order to determine the molecular size of
the quadrupolar chromophores.
Using the Perrin equation (eqn (2)),62 one can derive—from the
linear variation of (0.4/r ꢀ 1) as a function of t/Z (see Fig. 4)—
the effective molecular volume (v) and subsequently estimate the
Onsager cavity radius a (estimated as one-half of the quadrupolar
molecule radius in relation with excitation localization on a
dipolar chromophoric subunit).
0:4
rmax
¼
ð2Þ
tkT
Zv
1 þ
Fig. 3 Lippert–Mataga correlations for chromophores Q1, Q2, Q02
and Q03.
Fig. 4 Perrin plot from the variation of the fluorescence anisotropy of
Q03 in a series of solvents of different viscosities.
c
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The data are collected in Table 3. As expected quadrupolar
chromophores Q1/Q2 (bearing long alkyl chains) exhibit
larger molecular radius than chromophores that only have
short alkyl chains on the end-groups (Q02/Q03). In contrast
quadrupolar chromophore Q03 has a smaller radius due its
shorter and less rigid conjugated system. We observe that
chromophore Q1 having the more rigid extended system leads
to the largest Dm value (27 D) whereas the shortest chromo-
phore Q03 shows the smallest one (17 D). We also note that the
presence of the hydroxyethyl chain on Q02 leads to a slightly
smaller excited-state dipole than for Q2, indicating that the
presence of the alcohol moiety influences the intramolecular
charge transfer transition.
by investigating their two-photon excited fluorescence (TPEF)
in solution. The measurements were performed under excita-
tion with 150 fs pulses from a Ti:sapphire laser by using the
experimental protocol of Xu and Webb.65 Table 4 and Fig. 5
show, respectively, TPA cross-section values and spectra of the
compounds Q1, Q2, Q02 and Q03 in THF (which is a relevant
model of the polarity that such lipophilic chromophores could
experiment when wrapped within dendrimeric sheaths).39
For all compounds, we observe intense and broad TPA
bands in the NIR region. For strictly centrosymmetrical
quadrupoles, the one-photon excited state is expected to
have no or little TPA activity and the lower energy two-
photon-allowed excited state to lie at higher energy. However,
due to the banana-shape of the investigated series of compounds,
we observe in all cases a TPA band (or shoulder in the case of
chromophore Q03) which lies at about twice the one-photon
(OPA) maximum absorption wavelength. The higher lying
maximum (as well as an additional higher lying strongly
allowed maximum) is observed only in the case of chromo-
phore Q03. We notice that all chromophores present broad
absorption bands with a shoulder extending to an even lower
energy than the one photon-allowed excited state. This is
indicative of strong electron–phonon coupling.
1
Photosensitizing ability: singlet oxygen generation. The O2
luminescence at 1272 nm of the four samples (Q1, Q2, Q02 and
Q03) was measured both in toluene and in chloroform by
comparison with a tetraphenylporphyrin reference solution
measured in the same solvents. The singlet oxygen quantum
yield (FD) values are gathered in Table 1. For each compound,
we observe an increase of the singlet oxygen quantum yield
from chloroform to toluene and for both solvents the highest
values of FD obtained are for the quadrupole Q03. Further-
more the same trend is observed whatever the solvent used.
The first three chromophores present an important fluorescence
emission which induces very low singlet oxygen quantum
yields (FD). In contrast, the less aromatic thienyl fluorescent
compound Q03 favours a non-negligible generation of singlet
oxygen as well as a reasonable fluorescence emission as
was initially required for allowing both targeting monitoring
(by fluorescence imaging) and therapy (by photosensitized
production of singlet oxygen). It should be noticed that the
largest singlet oxygen quantum yield is correlated with the smallest
dipole moment of the emissive excited state, in agreement
with the fact that an increase of intramolecular charge transfer
usually competes with processes leading to singlet oxygen
production (i.e. intersystem crossing and energy transfer
to oxygen).63,64
As illustrated in Fig. 5 and already reported for analogous
rod-like chromophores,35 the substitution of a triple bond
(Q1) by a double bond (Q2) leads to a large increase of the
TPA cross-section values in the NIR region (with a significant
broadening and red-shift of the TPA spectrum). Indeed, the
fluorophores built from vinylene linkers (Q2, Q02 and Q03)
instead of ethynylene linkers (Q1) reveal much larger TPA
cross-section values in the whole NIR region, this effect being
Based on the experimental results, to further increase the
singlet oxygen quantum yield (but at the expense of a decrease
of the residual fluorescence), a possible way for the optimiza-
tion of these systems could be the introduction of additional
thienylene units in the conjugated system. This would most
probably lead to an improved singlet oxygen/fluorescence
ratio for application in PDT.
Fig. 5 TPA spectra of Q1, Q2, Q02 and Q03 in THF (with solid
Two-photon absorption (TPA). The TPA spectra of the
fluorophores were determined in the NIR range (700–980 nm)
curves as guides for the eyes only).
Table 4 Two-photon absorption data of compounds Q1, Q2, Q02 and Q03 in THF
s2a/GM
At lmax1
TPA
TPA
TPA
2lmaxOPA/nm
lmax1TPA/nm
lmax2TPA/nm
lmax3TPA/nm
At lmax2
At lmax3
Cpd
Q1
774
824
822
874
750
820
820
870
o700
r700
r700
820
—
—
—
730
1130
1090
980
—
—
—
—
1340
Q2
Z 1430
Z 1190
940
Q02
Q03
600
a
1 GM = 10ꢀ50 cm4 s photonꢀ1; TPA measurements were performed using a mode-locked Ti:sapphire laser delivering 150 fs pulses at a repetition
rate of 76 MHz, using fluorescein as a reference.
c
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more pronounced in the longer wavelength range. More
strikingly, chromophore Q02 seems to show slightly lower
TPA responses than chromophore Q2 (however close to the
limit of the experimental errors), which could indicate that the
slightly different environment provided by the presence of only
two alcohol moieties—although non-conjugated—in close
proximity to the chromophoric unit affects the charge redistri-
bution (as was suggested by the slightly lower excited-state
dipole) occurring upon excitation and also the TPA responses.
Finally chromophore Q03 owns both the largest TPA
responses and the broader spectrum in the spectral range of
interest for biomedical applications (three different maxima
are observed in the 750–950 nm range). This result can be
related to the introduction of low-aromaticity thiophene hetero-
cycles in the conjugated system, in agreement with the essential-
state model52,53 and previously reported experimental results.35
Hence this chromophore meets all the prerequisites for its
use as a biphotonic sensitizer for PDT by two-photon excita-
tion in the spectral range of interest for biomedical applica-
tions while allowing 3D mapping by two-photon excited
fluorescence imaging. In addition, the presence of two alcohol
end-groups provides an easy way for further functionalization
by covalent grafting on various platforms and/or covalent
attachment of targeting moieties.
sensitizers15,70,71 within nanoparticles has been shown to be of
high interest for preserving photophysical properties in
aqueous media (including singlet oxygen production) and to yield
nano-object with very high TPA. In addition the nanoparticle
approach offers high flexibility for combination of chromophores
and targeting units within single nano-objects.48,51 We are
currently implementing such a modular strategy.
Experimental section
Synthetic procedures
General methods. All air- or water-sensitive reactions were
carried out under dry argon. Solvents were generally dried and
distilled prior to use. Reactions were monitored by thin-
layer chromatography on Merck silica gel 60 F254 precoated
aluminium sheets. Column chromatography: Merck silica gel
Si 60 (40–63 mm, 230–400 mesh or 63–200 mm, 70–230 mesh).
Melting points were determined on an Electrothermal IA9300
digital melting point instrument. NMR: Bruker ARX 200
(1H: 200.13 MHz, 13C: 50.32 MHz) or Avance AV 300
(1H: 300.13 MHz, 13C: 75.48 MHz), in CDCl3 solutions;
1H chemical shifts (d) are given in ppm relative to TMS as
internal standard, J values in Hz and 13C chemical shifts
relative to the central peak of CDCl3 at 77.0 ppm. High and
low resolution mass spectra measurements were performed
at the Centre Regional de Mesures Physiques de l’Ouest
(C.R.M.P.O., Rennes), using a Micromass MS/MS ZABSpec
TOF instrument with EBE TOF geometry; LSIMS (Liquid
Secondary Ion Mass Spectrometry) at 8 kV with Cs+ in
m-nitrobenzyl alcohol (mNBA); ES+ (electrospray ionization,
positive mode) at 4 kV; EI (electron ionization) at 70 eV.
Elemental analyses were performed at C.R.M.P.O. Compounds
Q1,35 Q2,35 1a,55 457 and 556 were synthesized according to the
respective literature procedures.
Conclusion
Symmetrical banana-shaped bis-donor fluorophores were
prepared from the symmetrical functionalization of a fluorenyl
core bearing elongated conjugated rods made from arylene–
vinylene or arylene–ethynylene building blocks in order to test
modifications which could increase singlet oxygen production
ability while maintaining some fluorescence and large TPA
responses. As was anticipated, the replacement of phenylene
moieties by less aromatic thienylene moieties in the conjugated
rods gave rise to both enhancement and broadening of the
TPA responses in the spectral region of interest for biomedical
applications. Moreover, this replacement also led to a major
increase of the singlet oxygen production ability (by a factor of
ten compared to a structural analogue) while keeping residual
fluorescence, in relation with the increase of the intersystem
crossing. The experimental study also gives some trends for
further tentative improvement of the biphotonic sensitizer.
This could possibly be achieved by increasing the number of
thienyl units in the conjugated linker (also leading to red-shift
of the TPA spectrum), keeping in mind that combination with
phenyl units seems also needed not to decrease the TPA
responses35 and maintain a suitable triplet excited state energy
level. Hence a subtle compromise has to be achieved to
improve both singlet oxygen production and TPA.
4,40-(9,9-Dibutyl-9H-fluorene-2,7-diyl)bis(2-methyl-3-butyn-
2-ol) (1b). Air was removed from a solution of 1a55 (6.00 g,
11.3 mmol) in 37.5 mL of toluene/Et3N (5/1) by blowing argon
for 20 min. Then CuI (86 mg, 0.45 mmol), Pd(PPh3)2Cl2
(316 mg, 0.45 mmol) and 2-methyl-3-butyn-2-ol (2.84 g,
33.8 mmol) were added, and the mixture was stirred at
40 1C for 16 h. After evaporation of the solvent, the residue
was purified by column chromatography (heptane/CH2Cl2
30 : 70 then CH2Cl2) to yield 4.37 g (87%) of 4,40-(9,9-dibutyl-
1
9H-fluorene-2,7-diyl)bis(2-methyl-3-butyn-2-ol) (1b): H NMR
(200.13 MHz, CDCl3) d 7.60 (d, J = 8.6 Hz, 2H), 7.40
(d, J = 8.6 Hz, 2H), 7.38 (s, 2H), 2.16 (s, 2H), 1.94
(m, 4H), 1.66 (s, 12H), 1.07 (m, 4H), 0.66 (t, J = 7.3, 6H),
0.52 (m, 4H); 13C NMR (50.32 MHz, CDCl3) d 150.8, 140.5,
130.7, 126.0, 121.3, 119.8, 93.9, 82.9, 65.7, 55.0, 40.1, 31.5,
25.7, 23.0, 13.8; HRMS (EI) calcd for C31H38O2 (M+ꢂ) m/z
442.2872, found 442.2859. Anal. calcd for C31H38O2 (442.64):
C, 84.12; H, 8.65. Found: C, 84.01; H, 8.71%.
The molecular optimization study shows that chromophore
Q03—which combines large TPA responses in the whole
spectral range of interest, reasonable singlet oxygen production
ability and residual fluorescence—offers promises for highly
selective spatially-resolved two-photon PDT, based on the
combination of such a graftable sensitizer with targeting units
that could allow sensitive detection (3D mapping by two
photon-excited fluorescence) of tumour cells. In particular
encapsulation of biphotonic fluorophores66–69 or biphotonic
2,7-Diethynyl-9,9-dibutyl-9H-fluorene (1c). To a solution of
1b (1.04 g, 2.35 mmol) in 20 mL of toluene/i-PrOH (3/1) was
added solid KOH (0.39 g). The mixture was heated under
reflux for 0.5 h. After cooling, KOH was filtered off and
the solvents were evaporated and the residue was purified
c
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by column chromatography (heptane/CH2Cl2 70 : 30 then
1
20 : 80) to yield 0.54 g (71%) of 1c: H NMR (200.13 MHz,
(t, J = 5.5 Hz, 4H), 3.47 (q, J = 7.0 Hz, 4H), 1.99 (m, 4H),
1.19 (t, J = 7.0 Hz, 6H), 1.07 (m, 4H), 0.69 (t, J = 7.3 Hz,
6H), 0.60 (m, 4H); HRMS (ESI) calcd for C61H65N2O2
[(M + H)+] m/z 857.5046, found 857.5046. Anal. calcd for
C61H64N2O2 (857.19): C, 85.47; H, 7.53; N, 3.27. Found: C,
85.01; H, 7.71; N, 3.32%.
CDCl3) d 7.63 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H),
7.46 (s, 2H), 3.15 (s, 2H), 1.94 (m, 4H), 1.07 (m, 4H), 0.67
(t, J = 7.2 Hz, 6H), 0.54 (m, 4H); 13C NMR (50.32 MHz,
CDCl3) d 151.0, 140.9, 131.2, 126.5, 120.8, 119.9, 84.5, 77.4,
55.1, 40.0, 25.8, 22.9, 13.7; HRMS (EI) calcd for C25H26
(M+ꢂ) m/z 326.2035, found 326.2036. Anal. calcd for C25H26
(326.48): C, 91.97; H, 8.03. Found: C, 92.17; H, 8.07%.
Fluorophore Q03. To a solution of 6 (0.241 g, 0.441 mmol)
and 457 (0.576 g, 1.016 mmol) in anhydrous CH2Cl2 (10 mL)
was added tBuOK (0.149 g, 1.325 mmol). The mixture was
stirred at 20 1C for 16 h, then filtered through a short pad of
silica gel. The solvent was removed under reduced pressure,
and the crude product was purified by column chromato-
graphy (CH2Cl2/AcOEt 98 : 2 then 95 : 5) to yield 134 mg
(35%) of Q03; 1H NMR (300.13 MHz, CDCl3) d 7.66
(d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.48 (m, 2H),
7.35 and 6.73 (AA0XX0, JAX = 8.9 Hz, 8H), 7.17 and 6.89
(AX, JAX = 3.6 Hz, 4H), 6.98 (d, J = 16.0 Hz, 2H), 6.85
(d, J = 16.0 Hz, 2H), 3.81 (t, J = 5.7 Hz, 4H), 3.50 (t, J = 5.7 Hz,
4H), 3.50 (q, J = 7.1 Hz, 4H), 1.99 (m, 4H), 1.66 (s, 2H), 1.18
(t, J = 7.0 Hz, 6H), 1.07 (m, 4H), 0.69 (t, J = 7.3 Hz, 6H),
0.59 (m, 4H); 13C NMR (75.46 MHz, CDCl3) d 151.1, 148.1,
145.8, 140.7, 132.7, 130.5, 129.6, 127.9, 125.6, 125.0, 124.8,
121.8, 120.4, 120.0, 117.2, 112.5, 94.9, 84.0, 60.3, 55.2, 52.4,
45.6, 40.2, 25.9, 23.1, 13.9, 12.0; HRMS (ESI) calcd for
C57H61N2O2S2 [(M + H)+] m/z 869.41745, found 869.4173.
4,40-(9,9-Dibutyl-9H-fluorene-2,7-diyldi-2,1-ethynediyl)bis-
benzaldehyde (3). Air was removed from a solution of 1c
(0.251 g, 0.766 mmol) and 2 (0.354 g, 1.914 mmol) in 10 mL of
toluene/Et3N (4/1) by blowing argon for 20 min. Then CuI
(6.0 mg, 0.031 mmol) and Pd(PPh3)2Cl2 (22 mg, 0.031 mmol)
were added, and deaeration was continued for 10 min. There-
after the mixture was stirred at 40 1C for 16 h. The solvent was
removed under reduced pressure, and the crude product was
purified by column chromatography (heptane/CH2Cl2 50 : 50
1
then 40 : 60) to yield 307 mg (75%) of 3; H NMR (300.13
MHz, CDCl3) d 9.94 (s, 2H), 7.80 (d, J = 8.4 Hz, 4H), 7.63
(d, J = 8.4 Hz, 4H), 7.62 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.7 Hz,
2H), 7.46 (s, 2H), 1.94 (m, 4H), 1.05 (m, 4H), 0.60 (t, J =
7.2 Hz, 6H), 0.53 (m, 4H); 13C NMR (75.46 MHz, CDCl3)
d 191.3, 151.2, 141.1, 135.4, 132.0, 131.1, 129.6, 126.2, 121.4,
120.3, 94.5, 89.2, 55.3, 40.2, 25.9, 22.9, 13.8; HRMS (ESI)
calcd for C39H35O2 [(M + H)+] m/z 535.26371, found
535.2638.
Photophysical methods
All photophysical properties measurements have been performed
with freshly-prepared air-equilibrated solutions at room tempera-
ture (298 K). UV/Vis absorption spectra were recorded on a
Jasco V-570 spectrophotometer. Steady-state and time-resolved
fluorescence measurements were performed on dilute solutions
(ca. 10ꢀ6 M, optical density o 0.1) contained in standard 1 cm
quartz cuvettes using an Edinburgh Instruments (FLS920)
spectrometer in photon-counting mode. Fully corrected emission
5,50-(9,9-Dibutyl-9H-fluorene-2,7-diyldi-2,1-ethynediyl)bis-2-
thiophenecarboxaldehyde (6). Air was removed from a solution
of 1c (0.150 g, 0.460 mmol) and 556 (0.251 g, 1.06 mmol) in
6.5 mL of toluene/Et3N (4/1) by blowing argon for 20 min.
Then CuI (7.0 mg, 0.037 mmol) and Pd(PPh3)2Cl2 (27 mg,
0.039 mmol) were added, and deaeration was continued for
10 min. Thereafter the mixture was stirred at 40 1C for 16 h.
The solvent was removed under reduced pressure, and the
crude product was purified by column chromatography (heptane/
CH2Cl2 50 : 50 then 40 : 60) to yield 245 mg (97%) of 6;
1H NMR (300.13 MHz, CDCl3) d 9.80 (s, 2H), 7.61 and 7.26
(AX, JAX = 3.9 Hz, 4H), 7.62 (d, J = 8.5 Hz, 2H), 7.47
(d, J = 8.5 Hz, 2H), 7.45 (s, 2H), 1.93 (m, 4H), 1.02 (m, 4H),
0.60 (t, J = 7.3 Hz, 6H), 0.51 (m, 4H); 13C NMR (75.46 MHz,
CDCl3) d 182.4, 151.4, 143.9, 141.4, 136.1, 133.0, 132.5, 131.0,
126.0, 120.9, 120.4, 98.9, 82.6, 55.3, 40.1, 25.9, 23.0, 13.8;
HRMS (ESI) calcd for C35H30O2S2 (M+ꢂ) m/z 546.16872,
found 546.1687.
abs
spectra were obtained, for each compound, at lex = lmax
with an optical density at lex r 0.1 to minimize internal
absorption. Fluorescence quantum yields were measured according
to literature procedures.72,73 Fluorescence lifetimes were
measured by time correlated single-photon counting (TCSPC)
by using the same FLS 920 fluorimeter. Excitation was
achieved by a hydrogen-filled nanosecond flashlamp (repetition
rate 40 kHz). The instrument response (FWHM ca. 1 ns)
was determined by measuring the light scattered by a Ludox
suspension. The TCSPC traces were analyzed by standard
iterative reconvolution methods implemented in the software
of the fluorimeter. All compounds displayed strictly mono-
exponential fluorescence decays (w2 o 1.1).
Fluorophore Q02. To a solution of 3 (0.100 g, 0.187 mmol)
and 457 (0.244 g, 0.430 mmol) in anhydrous CH2Cl2 (4 mL)
was added tBuOK (0.063 g, 0.561 mmol). The mixture was
stirred at 20 1C for 16 h, then filtered through a short pad of
silica gel. The solvent was removed under reduced pressure,
and the crude product was purified by column chromato-
graphy (CH2Cl2/AcOEt 98 : 2 then 95 : 5) to yield 85 mg
(53%) of Q02; 1H NMR (200.13 MHz, CDCl3) d 7.67
(d, J = 7.7 Hz, 2H), 7.53 (d, J = 7.7 Hz, 2H), 7.52
(m, 2H), 7.53 and 7.47 (AA0BB0, JAB = 8.5 Hz, 8H), 7.41
and 6.76 (AA0XX0, JAX = 8.7 Hz, 8H), 7.08 (d, J = 16.1 Hz,
2H), 6.90 (d, J = 16.1 Hz, 2H), 3.83 (t, J = 5.5 Hz, 4H), 3.51
Two-photon absorption
Two-photon absorption (TPA) measurements were conducted
by investigating the two-photon excited fluorescence (TPEF)
of the fluorophores in THF at room temperature on air-
equilibrated solutions (10ꢀ4 M), according to the experimental
protocol established by Xu and Webb.65 This protocol avoids
contributions from excited-state absorption that are known to
result in largely overestimated TPA cross-sections. To span the
700–980 nm range, a Nd:YLF-pumped Ti:sapphire oscillator
c
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was used generating 150 fs pulses at a 76 MHz rate. The
excitation was focused into the cuvette through a microscope
objective (10ꢁ, NA 0.25). The fluorescence was detected in
epifluorescence mode via a dichroic mirror (Chroma 675dcxru)
and a barrier filter (Chroma e650sp-2p) by a compact CCD
spectrometer module BWTek BTC112E. Total fluorescence
intensities were obtained by integrating the corrected emission
spectra measured by this spectrometer. TPA cross-sections
(s2) were determined from the two-photon excited fluorescence
(TPEF) cross-sections (s2Ff) and the fluorescence emission
quantum yield (Ff). TPEF cross-sections were measured relative
to fluorescein in 0.01 M aqueous NaOH for 715–980 nm,65,74
and the appropriate solvent-related refractive index corrections.75
Data points between 700 and 715 nm were corrected according
to ref. 76. The quadratic dependence of the fluorescence
intensity on the excitation power was checked for each sample
and all wavelengths, indicating that the measurements were
carried out in intensity regimes where saturation or photo-
degradation did not occur.
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The excitation source consisted of a Xe-arc, the light was
separated in a SPEX 1680, 0.22 mm double monochromator.
The detection at 1270 nm was done through a PTI S/N 1565
monochromator, and the emission was monitored by a
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The optical density of the reference and the sample solution
(at 405 nm) were set equal to 0.2.
Acknowledgements
Financial support by ANR PNANO 07-102 is gratefully
acknowledged. We thank Region Bretagne for a fellowship
to CR. Part of the work was supported by GDR Photomed.
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