Diaroyl(methanato)boron Difluoride Compounds as Medium-Sensitive Two-Photon Fluorescent Probes

Article abstract of DOI:10.1002/chem.200305321

This paper evaluates the use of diaroyl(methanato)boron difluoride compounds for designing efficient fluorescent probes through two-photon absorption. Three different pathways allowing for the syntheses of symmetrical and dissymmetrical molecules are reported. The stable diaroyl(methanato)-boron difluoride derivatives can be easily obtained in good yields. They exhibit a large one-photon absorption that is easily tuned in the near-UV range. Their strong fluorescence emission covers the whole visible domain. In addition to these attractive linear properties, several diaroyl(methanato)-boron difluoride derivatives possess significant cross sections for two-photon absorption. The derived structure-property relationships are promising for designing new generations of molecules relying on the diaroyl(methanato)boron difluoride backbone.

Full text of DOI:10.1002/chem.200305321

FULL PAPER
Diaroyl(methanato)boron Difluoride Compounds as Medium-Sensitive Two-
Photon Fluorescent Probes
Emmanuelle Cognÿ-Laage,[a] Jean-FranÁois Allemand,*[a, b] Odile Ruel,[a]
Jean-Bernard Baudin,[a] Vincent Croquette,[b] Mireille Blanchard-Desce,[c] and
Ludovic Jullien*[a]
Abstract: This paper evaluates the use
of diaroyl(methanato)boron difluoride
compounds for designing efficient fluo-
rescent probes through two-photon ab-
sorption. Three different pathways al-
lowing for the syntheses of symmetrical
and dissymmetrical molecules are re-
ported. The stable diaroyl(methanato)-
boron difluoride derivatives can be
easily obtained in good yields. They ex-
hibit a large one-photon absorption
that is easily tuned in the near-UV
range. Their strong fluorescence emis-
sion covers the whole visible domain.
In addition to these attractive linear
properties, several diaroyl(methanato)-
boron difluoride derivatives possess
significant cross sections for two-
photon absorption. The derived struc-
ture property relationships are promis-
ing for designing new generations of
molecules relying on the diaroyl(me-
thanato)boron difluoride backbone.
Keywords: boron
¥
fluorescent
probes ¥ structure activity relation-
ships ¥ two-photon absorption
Introduction
evaluate the polarity or the viscosity of molecular environ-
ments. For fluorescence-depolarization experiments, fluoro-
phores with well-defined electronic-transition dipoles in a
molecular frame are preferred. Despite extensive studies,
few series of molecules satisfy these criteria, as revealed by
the catalog contents of the main purchasers of fluorescent
molecular probes; therefore, searching for new series of fluo-
rescent species is still an important issue. In fact, the lack of
appropriate fluorescent labels or probes that cover the
whole UV/Vis-wavelength range is regarded as critical for
the development of promising experiments that rely on the
observation of singly fluorescent molecules.[2] Finally, the
growing interest in fluorescence imaging based on two-
photon excitation[3,4] requires the identification of fluoro-
phores exhibiting large cross sections for multiple-photon
absorptions.[5,6]
Extrinsic fluorophores are increasingly used as reporter mol-
ecules for labeling, for probing environments, for evaluating
intramolecular distances or molecular motions, and so
forth.[1] Relatively small molecules exhibiting large fluores-
cence quantum yields are always sought after for such appli-
cations. In addition, other features have to be considered in
answer to specific questions. A high sensitivity of the photo-
physical features to the surrounding medium is favored to
[a] Dr. E. Cognÿ-Laage, Dr. J.-F. Allemand, Dr. O. Ruel,
Dr. J.-B. Baudin, Prof. Dr. L. Jullien
Dÿpartement de Chimie (C.N.R.S. U.M.R. 8640)
…cole Normale Supÿrieure, 24 rue Lhomond
75231 Paris Cedex 05 (France)
Fax : (+33)1-44-32-33-25
E-mail: Jean-Francois.Allemand@ens.fr
Ludovic.Jullien@ens.fr
In relation to the preceding paragraph, one of the most
recently developed fluorescent chromophores is 4,4-di-
fluoro-4-bora-3a-azonia-4a-aza-s-indacene
(BODIPY)
[b] Dr. J.-F. Allemand, Dr. V. Croquette
Dÿpartement de Physique (C.N.R.S. U.M.R. 8550)
…cole Normale Supÿrieure, 24 rue Lhomond
75231 Paris Cedex 05 (France)
11]
(Scheme 1).[7
BODIPY-based fluorophores exhibit spec-
tral features that make them especially attractive when the
excitation is performed in the 500 600 nm range, thus pro-
ducing a fluorescence emission at 500 650 nm. Their molar
absorption coefficient is very high (e>8î104 mꢀ1 cmꢀ1)
along with their fluorescence quantum yield (fF) that is
close to one, even in water. For imaging, such properties are
well suited for fluorescence microscopy relying on one-
photon excitation, in which they compete with most fluores-
cein and rhodamine derivatives. In contrast, BODIPY-based
Fax : (+33)1-44-32-34-92
E-mail: Jean-Francois.Allemand@ens.fr
[c] Dr. M. Blanchard-Desce
Universitÿ de Rennes 1, Institut de Chimie (C.N.R.S. U.M.R. 6510)
Campus scientifique de Beaulieu, 35042 Rennes (France)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author. It contains Figur-
es 1Sa, 1Sb, 2S, and 3S.
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DOI: 10.1002/chem.200305321
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

FULL PAPER
dibenzoyl(methanato)boron difluoride (9), whose emis-
sion[14,15] and reactivity[16] of the singlet excited state have
been investigated. Some related compounds were reported
to exhibit attractive absorption and emission features, such
as large molar absorption coefficients, or large fluorescence
20]
quantum yields.[17
In particular, the range of absorption
wavelengths (ca. 350 500 nm) is especially suitable for two-
photon excitation with femtosecond Ti sapphire pulsed
lasers that can be tuned between 700 and 1100 nm.
The present paper is organized as follows: We first de-
scribe three different synthetic pathways to access the di-
aroyl(methanato)boron difluoride derivatives 1 9. Some
photophysical properties of these compounds are then re-
ported and discussed (one- and two-photon absorption,
steady-state fluorescence emission). In view of the possible
use of Ar1Ar2BF2 compounds as labels, we also examined
both the dependence of the emission features upon the envi-
ronment and the chemical stability of the present fluores-
cent molecules.
Results and Discussion
Scheme 1. Generic structures of the BODIPY and the diaroyl(methana-
to)boron difluoride Ar1Ar2BF2 compounds prepared during this study.
Synthesis: To derive structure property relationships, we
synthesized eight compounds differing in 1) the aryl back-
bones: phenyl (Phe), biphenyl (Bi), and naphthalene (Na),
2) the aromatic substituents: H, or methoxy (MeO), and 3)
their symmetry (Ar1 =Ar2 or Ar1 ¼Ar2). The diaroyl(metha-
nato)boron difluoride compounds (Ar1Ar2BF2 1 9) were
easily obtained in good yields from the reaction of boron tri-
fluoride/diethyl ether (BF3/Et2O) with the corresponding
1,3-diketones (Ar1Ar2) in benzene or dichloromethane
(Scheme 2).[21] The Ar1Ar2 derivatives were synthesized by
three different pathways (Scheme 2).
Diketones BiBi, BiNa, MeOBiPhe, MeOPhePhe, NaNa,
and NaPhe were prepared in three steps according to path-
way A. In the first step, the ethyl aroyl acetates 1A-Ar1
(1=step 1, A=pathway A, Ar1 =Bi or Na) were obtained
from condensation of the dianion of monoethyl malonate
with aroyl chloride.[22] The monoanions from the 1A-Ar1
species were then condensed with the appropriate aroyl
chloride to yield 2A-BiBi, 2A-BiNa, 2A-MeOBiPhe, 2A-
MeOPhePhe, 2A-NaNa, and 2A-NaPhe.[23] Finally, the 2A-
Ar1Ar2 compounds were decarboxylated in the presence of
sodium chloride in DMSO to yield the desired diketones.[24]
BiPhe was synthesized in three steps (pathway B). The
condensation of 4-acetylbiphenyl with benzaldehyde first
provided the chalcone 1-biphenyl-4’-yl-3-phenylprop-2-
enone 1B-BiPhe.[25] This compound was subsequently bro-
minated[26] to yield 2B-BiPhe, which finally gave BiPhe after
elimination under basic conditions.[27] Lastly, MeOPheMeO-
Phe was directly obtained in one step from acylation of eth-
enyl acetate by 4-methoxybenzoyl chloride (pathway C).[28]
fluorophores are less satisfactory when two-photon excita-
tion is used. For instance, the two-photon fluorescence ab-
sorption (TPA) cross section (d) of the disodium salt of 4,4-
difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-inda-
cene-2,6-disulfonic acid is about two and ten times lower
than the TPE cross sections of fluorescein and rhodamine B,
respectively.[12]
Aiming at keeping the satisfactory aspects of the
BODIPY chromophores while improving their features
toward two-photon absorption, we became interested in a
series of diaroyl(methanato)boron difluoride compounds
(Ar1Ar2BF2 1 9), with structures related to BODIPY
(Scheme 1). b-Diketone boron difluoride complexes have
been known for more than 75 years;[13] nevertheless, the di-
aroyl series remains largely unexplored, excepting the inves-
tigations performed on the parent compound of this series,
Abstract in French: Cet article ÿvalue l×intÿrÜt de complexes
difluoroboroniques de diaroyl-b-dicÿtones dans l×ÿlaboration
de sondes fluorescentes aprõs excitation ‡ deux photons. Trois
diffÿrentes voies de synthõse de dÿrivÿs symÿtriques et di-
symÿtriques sont dÿcrites. Elles permettent d×obtenir de gran-
des quantitÿs de complexes avec de bons rendements. Ces
composÿs prÿsentent une absorption intense ‡ un photon, ac-
cordable dans le proche UV. Leur forte ÿmission de fluores-
cence couvre la gamme du visible. En sus de ces intÿressantes
propriÿtÿs linÿaires, certains des complexes difluoroboroni-
ques de diaroyl-b-dicÿtones synthÿtisÿs possõdent une impor-
tante section efficace d×absorption ‡ deux photons. Les rela-
tions structure-propriÿtÿs obtenues permettent d×envisager le
Chemical stability against hydrolysis: During experiments
performed in our laboratory, some BODIPY derivatives
were prone to color fading in the presence of water. For ex-
ample, during preparation of egg lecithin vesicles by deter-
gent removal, the signal arising from a BODIPY-labeled
dÿveloppement de molÿcules performantes construites
partir du squelette ÿtudiÿ.
‡
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Chem. Eur. J. 2004, 10, 1445 1455

Fluorescent Probes
1445 1455
Scheme 2. Pathways for syntheses of the target compounds Ar1Ar2BF2 1 9.
Table 1. Single-photon absorption maxima (lmax), steady-state fluorescence emission maxima (lem), molar ab-
sorption coefficients for single-photon absorption (e(lmax)Æ5% [mmꢀ1 cmꢀ1]), fluorescence quantum yields
(fF Æ5 [%]), Stokes shift values (Dn), two-photon excitation spectra maxima (l2max), two-photon excitation
cross sections (dmax Æ20% in GM; 1GM=10ꢀ50 cm4 s(photonꢀmolecule)ꢀ1) for the Ar1Ar2BF2 derivatives in
dichloromethane at 298 K.
lipid essentially disappeared in
less than 12 h, even when pro-
tected from light with alumin-
ium foil. This behavior proba-
bly originates from slow hydro-
lysis of the boron difluoride
bridge. In view of the structural
Ar1Ar2
lmax
e(lmax
)
lem
fF
[%]
Dn
[cmꢀ1
l2max
dmax
[nm]
[mmꢀ1 cmꢀ1
]
[nm]
]
[nm]
[GM]
1
2
3
4
5
6
7
8
9
411
416
394
410
410
396
420
398
364
75000
45000
61000
59000
89000
62000
47000
40000
46000
458
473
450
540
436
431
476
482
395
50
35
70
75
85
85
40
35
20
2500
2900
3160
5900
1050
2050
2800
4380
2150
795
745
775
835
785
787
775
30
60
30
200
20
25
85
30
<5
similarity
between
the
BODIPY dyes and the present
Ar1Ar2BF2 series, we investigat-
ed the stability of the series to-
wards water hydrolysis. In view
of the poor water solubility of
805
[a]
the
presently
synthesized
Ar1Ar2BF2 compounds, we con- [a] The signal remained in the 2–3 GM range between 700 nm and 750 nm.
sidered incorporating some of
them into micelles that are
known to remain quite permeable to water molecules, while
solubilizing hydrophobic molecules. Compounds 4 and 7
were dissolved in an aqueous solution of a-d-octylglucopy-
ranoside (OG 25mm), which forms micelles above 23mm at
room temperature,[29,30] to yield dye solutions (80mm). The
resistance to hydrolysis was evaluated by following the fluo-
rescence emission (vide infra) as a function of time at room
temperature. In fact, no significant drop of the signal was
noticed over a few days, so it was concluded that Ar1Ar2BF2
compounds are rather stable. The better resistance to hy-
drolysis of the present series could be related to the greater
affinity of boron to oxygen, compared with nitrogen.[31,32]
maximum one-photon absorption (lmax) is redshifted when
the conjugation path length is increased. Linear dependence
of the absorption on concentration without any spectrum al-
teration was observed in dichloromethane in the 1 20mm
range, suggesting that only monomeric species are present
in this concentration range, as reported for 9.[15] We investi-
gated the solvatochromism of some Ar1Ar2BF2 derivatives
by recording the UV/Vis absorption spectra in several sol-
vents (Table 2 and Figure 2a and b). The correlation be-
tween the transition energy associated with light absorption
and the empirical parameter ET(30) (representing solvent
polarity)[34] is displayed in Figure 2a for 3, 8, and 9. It is rea-
sonably linear except for a few deviating points originating
from the solvent dependence of the largest vibronic band
used to determine the absorption maximum. The observed
slopes are negative (positive solvatochromism). In the ab-
sence of any evidence suggesting the formation of aggre-
gates (except for 4 and 7 in cyclohexane), this result was in-
terpreted as revealing an increase of molecular-dipole
Linear absorption and emission properties: Derivatives 1 9
exhibit a strong one-photon absorption (e>50000mꢀ1 cmꢀ1)
in the 350 450 nm range (Figure 1 and Table 1). The corre-
sponding band is rather narrow (50 75 nm at half-width).
This band was interpreted as corresponding to a p!p* tran-
sition in the parent compound 9.[14,33] As anticipated, the
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L. Jullien, J.-F. Allemand et al.
FULL PAPER
Figure 1. Single-photon absorption (molar absorption coefficient e in mꢀ1 cmꢀ1, solid line), steady-state fluorescence emission (fluorescence emission in-
tensity IF in arbitrary units, dotted line), and two-photon excitation spectra (clouds of points) of a) h) 1 8, respectively, in dichloromethane at 298 K.
Spectra a) h) all have the same scale unless stated otherwise.
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Chem. Eur. J. 2004, 10, 1445 1455

Fluorescent Probes
1445 1455
Table 2. Solvatochromism of some Ar1Ar2BF2 derivatives. UV/Vis single-photon absorption and emission features under different conditions. Maxima of
absorption lmax [nm], molar absorption coefficients e(lmax)Æ5% [mmꢀ1 cmꢀ1], steady-state fluorescence emission lem [nm], and fluorescence quantum
yields fF Æ5 [%]. Also see Table 1.
Ar1Ar2
Acetone, 42.2[a]
Acetonitrile, 45.6[a]
385/60000/468/65
Cyclohexane, 30.9[a]
375/52000/408/15
DMSO, 45.1[a]
Ethanol, 51.9[a]
OG micelles
3
4
7
8
9
385/51000/451/50
406/60000/469/1
420/56000/610/15
378/45000/454/40
[b]
[c]
394/ [b]/432/
/
/
[c]/566/40[d]
[c]/510/50[d]
[b]
[c]
409/ [b]/420/
397/41000/501/50
364/46000/394/10
370/38000/523/50
364/45000/402/10
395/39000/407/35
359/43000/407/5
373/38000/502/30
371/46000/405/1
406/41000/530/1
371/46000/405/<1
[a] ET(30) values from ref. [34]. [b] The Ar1Ar2 compounds were sometimes insoluble or only partially soluble in cyclohexane. In the latter case, only
lmax and lem are indicated. [c] The light scattering induced by the presence of the OG micelles prevented extraction of reliable data from the absorption
spectrum. [d] The fluorescence quantum yields of 4 and 7 in OG micelles were crudely evaluated using Equation (1) under the assumption that the absor-
bances of the micellar solutions at the excitation wavelength were identical to the corresponding CH2Cl2 solutions at the same concentration.
Compounds 1 9 are strongly fluorescent in the 400
550 nm range in dichloromethane at 298 K (Figure 1 and
Table 1). The fluorescence quantum yields are good-to-ex-
cellent for the most conjugated molecules (0.35<fF <0.85).
As observed for one-photon absorption, fluorescence
emission becomes redshifted when the solvent polarity is in-
creased, consistent with an increase of dipole moment upon
excitation (Table 2). In addition, a more pronounced solva-
tochromism is observed for the emission than for the ab-
sorption, as revealed by an increase of the Stokes shift with
increasing polarity for the derivatives bearing the most do-
nating groups (Table 2). The large Stokes shift values can be
related to a major reorganization of the solvent shell during
the excitation lifetime. Interestingly, 4 exhibits both the
larger Stokes shift values (220 cmꢀ1 in cyclohexane,
5900 cmꢀ1 in dichloromethane, 7400 cmꢀ1 in DMSO), as well
as the most definite fluorescence solvatochromism (Fig-
ure 2b). This behavior can be attributed to a large nuclear
reorganization after excitation prior to emission, in addition
to a significant electronic redistribution occurring upon exci-
tation. Within this framework, the more pronounced fea-
tures of 4 can be tentatively interpreted as resulting from
the larger donor character of the methoxybiphenyl moiety,
leading to a larger excited-state dipole.[35]
The sensitivity of the fluorescence maxima to the solvent
polarity makes such compounds attractive as polarity
probes. From this point of view, the fluorescence characteris-
tics of 4 suggest that a-d-octylglucopyranoside (OG) acts as
a medium of intermediate polarity between dichlorome-
thane and dimethylsulfoxide (Tables 1 and 2). In fact, al-
though being structurally related to cyclohexane, the micel-
Figure 2. a) Dependence of the transition energies associated with the
lar core is strongly permeable to polar water molecules. In
processes of absorption (DEabs =hc/labs, filled markers) and emission
addition to the shift of the emission band, the solvent was
(DEem =hc/lem, empty markers) on the empirical parameter ET(30) repre-
senting solvent polarity[34] for 3 (circles), 8 (squares), and 9 (diamonds);
found to exert a significant influence on the fluorescence
quantum yield, hence supporting the view of Ar1Ar2BF2
molecules as polarity reporters.
b) single-photon absorption (molar absorption coefficient e in mꢀ1 cmꢀ1
,
filled markers) and steady-state fluorescence emission (fluorescence
emission intensity IF in arbitrary units, empty markers) of 4 in cyclohex-
ane (diamonds), dichloromethane (squares), DMSO (circles), and OG
micelles (crosses) at 298 K.
The fluorescence quantum yields were highest in dichloro-
methane and considerably decreased in polar media, such as
DMSO (Tables 1 and 2). The fluorescence quantum yields
in OG micelles are difficult to evaluate owing to light scat-
tering precluding the evaluation of sample absorbances;
nevertheless, the crude estimates derived suggest the corre-
sponding quantum yields are intermediary between apolar
and polar media. A possible explanation to account for such
sensitivity to polarity relies on the presence of two excited
moment upon light absorption. These observations are in ac-
cordance with the donor acceptor structure of the
Ar1Ar2BF2 molecules. In fact, 9 possesses a ground dipole
moment of 6.7 debye;[21] the BF2 moiety has a withdrawal
effect, whereas the aromatic rings act as donating groups.
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L. Jullien, J.-F. Allemand et al.
FULL PAPER
states whose energy would be solvent dependent: a non-
fluorescent excited state arising from a n!p* transition,
and a fluorescent excited state arising from p!p*. Such an
explanation was already proposed in closely related series,
to discuss the relationships between molecular conformation
and photophysical features.[36]
As a partial conclusion, the linear properties of the pres-
ent Ar1Ar2BF2 series satisfactorily compare with the corre-
sponding existing fluorescent series such as stilbene, couma-
rin, or pyrene derivatives, which exhibit comparable photo-
physical features.
contribution is a dominant effect, as observed in other
push pull systems.[47,54,55] We observe that the substituent
effect is much more pronounced in the case of the elongated
derivative 4, leading to an increase of nearly one order of
magnitude in the TPA response. This effect can be tentative-
ly attributed to a synergetical effect between donating sub-
stituent and conjugated linker length, as observed for optical
nonlinearities of push pull derivatives.[62]
The nature and length of the aryl moieties also influences
the TPA response. Increasing its donating strength or its size
leads to an increase of the TPA response, as indicated by
the comparison of 1) symmetrical molecules in the series 9
(<5), 1 (30), and 7 (85) and 2) dissymmetrical molecules in
the series 9 (<5), 3 (30), and 8 (30), as well as in the series
6 (25) and 4 (200). It should be stressed that the length
effect is much more pronounced in the case of a dissymmet-
rical compound with a donating substituent, leading again to
an order of magnitude increase of the TPA cross section.
We conclude from this observation that the distinctly larger
TPA characteristics of 4, among the nine related derivatives,
could be related to the major electronic redistribution occur-
ring upon excitation (vide supra), supporting the role of the
dominant dipolar contribution. In contrast, the present in-
vestigation suggests that symmetrization of the structure
does not bring any distinct advantage. In fact, dmax values
are identical for the couples (1,3) and (5,6).
Two-photon absorption properties: In contrast to single-
photon absorption, two-photon absorption is a nonlinear op-
tical process causing a molecule to simultaneously absorb
two photons to access an excited state. As a consequence,
the two-photon absorption is related quadratically to light
intensity. Provided that satisfactory chromophores will be
available, the favorable features of two-photon absorption
are anticipated to be useful for application in various
fields, such as two-photon fluorescence microscopy,[3,37,38]
two-photon-induced biological caging investigations,[39,40]
46]
and optical limiting.[41
Despite work already reported
dealing with the same issue, there is still a need to ac-
cumulate experimental data to derive relevant structure
property relationships for optimizing two-photon absorp-
58]
tion.[46
Two-photon excitation spectra of 1 9 were recorded in di-
chloromethane at 298 K with a home-built set up relying on
the design of Webb et al.[60] (see Figures 1S 3S in the Sup-
porting Information and Experimental Section). Figure 1a h
displays the spectra (for 1 8) together with the correspond-
ing single-photon absorption and fluorescence emission
spectra. The quadratic dependence of two-photon-excited
fluorescence of the compounds listed in this paper was
checked at several wavelengths (see Figure 3S in the Sup-
porting Information for a typical example); it revealed satis-
Conclusion
We have prepared a series of novel fluorophores of definite
interest for various applications in biological imaging (TPEF
microscopy, microenvironment probes). Their absorption
and emission properties have been investigated, demonstrat-
ing that these molecules can be used as sensitive fluorescent
probes of micropolarity. These new fluorochromes combine
large fluorescence quantum yields and enhanced chemical
stability against hydrolysis in aqueous environment, as com-
pared to the most popular BODIPY probes. Their TPA
cross-section action has been determined using an original
experimental protocol. Structure property relationships
have been derived providing evidence that their TPA cross
sections can be significantly enhanced in elongated dissym-
metrical derivatives bearing an electron-donating group.
These combined features of the new series of fluorophores
provide an interesting route towards optimized TPA fluoro-
phores/probes for various applications.
factory behavior over the 20 200 mW range. The position of
ð2Þ
the two-photon absorption maximum (l ) and the peak
max
two-photon absorptivity maximum (dmax) are summarized in
ð2Þ
Table 1. The observed values of l
satisfactorily compare
max
with twice the corresponding values of lmax; this suggests
that the same excited states are reached regardless of the
excitation mode. Such an observation goes against the be-
havior of symmetrical molecules, such as fluorescein, but is
in line with other reports making use of a comparable tech-
nique on unsymmetrical donor acceptor compounds. For in-
ð2Þ
stance, coumarin 307 is reported to exhibit l =800 nm in
max
methanol[12] and lmax =395 nm in ethanol.[61] In the present
series, the dmax range from 20 to 200 GM (1 GM=
10ꢀ50 cm4 s(photonꢀmolecule)ꢀ1).
Experimental Section
The effectiveness of electron-donating substituents on the
aryl group to increase the two-photon fluorescence absorp-
tivity (dmax), is supported by the trend observed in both
series of 1) dissymmetrically substituted molecules: 6 (25)
versus 9 (<5) and 4 (200) versus 3 (30) and 2) symmetrically
substituted molecules: 5 (20) versus 9 (<5). Thus, much
lower TPA values are observed in the absence of donating
substituents on the aryl moieties, suggesting that the dipolar
General procedures: Anhydrous solvents were freshly distilled before
use. Column chromatography (CC): silica gel 60 (0.040 0.063 mm) Merck
or silica gel SDS. Analytical: TLC: silica gel plates (Merck), detection by
UV (254 nm); melting point: B¸chi 510; 1H NMR spectra: AM-250
Bruker, chemical shifts in ppm related to the protonated solvent as inter-
nal reference (1H: CHCl3 in CDCl3, 7.27 ppm, CHD2SOCD3 in
CD3SOCD3, 2.49 ppm; 13C: 13CDCl3 in CDCl3, 76.9 ppm, 13CD3SOCD3 in
CD3SOCD3, 39.6 ppm); coupling constants (J) in Hz; mass spectrometry
(chemical ionization with NH3) was performed at the Service de Spectro-
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Chem. Eur. J. 2004, 10, 1445 1455

Fluorescent Probes
1445 1455
mÿtrie de masse de l×ENS; microanalyses were obtained from the Ser-
vice de Microanalyses de l×Universitÿ Pierre et Marie Curie, Paris. Acyl
chlorides were prepared from the corresponding acids by reaction with
thionyl chloride.[65] Compound 9 was synthesized according to the pres-
ently reported procedure starting from the commercially available b-di-
ketone PhePhe.
134.3, 139.6, 146.8, 165.8, 190.5 ppm; MS (CI, NH3): m/z (%): 466 (8)
[M++18], 449 (37) [M++1], 377 (100), 269 (78), 198 (28); elemental
analysis calcd (%) for C30H24O4 (448): C 80.34, H 5.54; found: C 78.06, H
5.55.
Ethyl 3-(2-naphthyl)-3-oxo-2-(biphenyl-4-carbonyl) propionate (2A-
BiNa): Same method as for 2A-NaPhe, but using ethyl naphthalene-2-
carbonyl acetate (795 mg, 3.28 mmol), and biphenyl-4-carbonyl chloride
(710 mg, 3.28 mmol). Compound 2A-BiNa was obtained as colorless
crystals after recrystallization in ethanol (478 mg, 35%). M.p. 1548C; 1H
NMR (250 MHz, CDCl3, 258C, TMS): d=1.27 (t, 3J(H,H)=7.1 Hz, 3H),
Syntheses of the diketones Ar1Ar2 alongpathway A
Ethyl 3-(biphenyl-4’-yl)-3-oxo propionate (1A-Bi): Butyllithium in hexane
(1m, 16 mL) was added dropwise to a cooled (ꢀ788C) solution of mono-
ethyl malonate (1.32 g, 10 mmol; 2 equiv) in THF (40 mL) with a crystal
of 2,2’-bipyridine as indicator.[22] The solution was allowed to warm to
ꢀ58C and stirred for 15 min. After cooling (ꢀ708C), biphenyl-4-carbonyl
chloride (1.08 g, 5 mmol) in THF (5 mL) was slowly added. The cooling
bath was removed and the mixture was stirred for 1 h. The mixture was
poured into diethyl ether (35 mL) and hydrochloric acid (15 mL, 1m).
The organic phase was separated and washed with saturated sodium hy-
drogencarbonate, then dried over magnesium sulfate. After filtration and
evaporation, the crude product was purified by CC (cyclohexane/di-
chloromethane (C6H12/CH2Cl2), gradient 40:60 to 0:100) to yield 1A-Bi
as a white powder (942 mg, 70%). M.p. 768C; 1H NMR (250 MHz,
CDCl3, 258C, TMS): d=1.27 (t, 3J(H,H)=7.2 Hz, 3H), 4.02 (s, 2H), 4.23
(q, 3J(H,H)=7.2 Hz, 2H), 7.32 7.52 (m, 3H), 7.54 7.74 (m, 4H), 7.97
8.07 ppm (m, 2H); 13C NMR (63 MHz, CDCl3, 258C, TMS) (CE: enol-
ester tautomer; CK: keto-ester tautomer): d=14.1, 14.3, 46.0 (CK), 60.3,
61.5, 87.3 (CE), 126.5, 127.1, 127.2, 127.3, 127.4, 127.9, 128.4, 128.9, 129.0,
129.1, 132.3, 134.8, 139.6, 140.1, 144.0, 146.4, 167.5, 171.1, 173.2,
192.1 ppm; MS (CI, NH3): m/z (%): 286 (24) [M++18], 269 (100) [M+
+1], 197 (40).
Ethyl naphthalene-2-carbonyl acetate (1A-Na):[66] Same procedure as for
1A-Bi, but with naphthalene-2-carbonyl chloride (952.5 mg, 5 mmol). Pu-
rified by CC (C6H12/CH2Cl2, 50:50 to 0:100). 1A-Na obtained as a white
powder (930 mg, 76%). 1H NMR (250 MHz, CDCl3, 258C, TMS) (HE:
enol-ester tautomer; HK: keto-ester tautomer): d=1.27 (t, 3J(H,H)=
7.15 Hz, 3HK), 1.36 (t, 3J(H,H)=7.15 Hz, 3HE), 4.13 (s, 2HK), 4.24 (q,
3J(H,H)=7.15 Hz, 2HK), 4.30 (q, 3J(H,H)=7.15 Hz, 2HE), 5.82 (s,
1HE), 7.48 7.70 (m, 2HK+2HE), 7.74 8.07 (m, 4HK+4HE), 8.37 (brs,
1HE), 8.47 (brs, 1HK), 12.69 ppm (s, 1HE); 13C NMR (63 MHz, CDCl3,
258C, TMS) (CE: enol-ester tautomer; CK: keto-ester tautomer): d=
14.2, 14.4, 46.1 (CK), 60.4, 61.5, 87.9 (CE), 122.6, 123.9, 126.7 (2C), 126.8,
127.0, 127.6, 127.7, 127.9, 128.3, 128.7, 128.9, 129.1, 129.7, 130.6, 132.5,
132.7, 133.5, 134.7, 135.7, 167.7, 171.3, 173.3, 192.5 ppm.
3
4.33 (q, J(H,H)=7.1 Hz, 2H), 6.38 (s, 1H), 7.37 7.74 (m, 9H), 7.84 8.04
(m, 6H), 8.47 ppm (brs, 1H); 13C NMR (63 MHz, CDCl3, 258C, TMS):
d=14.0, 62.5, 64.6, 123.9, 127.1, 127.3, 127.6, 127.8, 128.5, 129.0, 129.1,
129.3, 129.8, 130.7, 132.4, 133.0, 134.3, 135.9, 139.5, 146.7, 165.8, 190.3,
190.6 ppm; MS (CI, NH3): m/z (%): 440 (33) [M++18], 424 (34) [M+
+2], 423 (100) [M++1]; elemental analysis calcd (%) for C28H22O4 (422):
C 79.60, H 5.25; found: C 79.59, H 5.23.
Ethyl 3-(4’-methoxybiphenylyl)-3-oxo-2-benzoyl propionate (2A-MeOBi-
Phe): Same method as for 2A-NaPhe, but with ethyl benzoyl acetate
(960 mg, 5 mmol), 4’-methoxybiphenylcarbonyl chloride (1.23 g, 5 mmol)
to yield 2A-MeOBiPhe as white crystals after washing several times with
C6H12/Et2O (50:50) and after recrystallization in ethanol (1.105 g, 54%).
M.p. 1358C; 1H NMR (250 MHz, CDCl3, 258C, TMS): d=1.26 (t,
3J(H,H)=7.1 Hz, 3H), 3.87 (s, 3H), 4.25 (q, 3J(H,H)=7,1 Hz, 2H), 6.22
(s, 1H), 6.98 7.03 (m, 2H), 7.43 7.70 (m, 7H), 7.90 8.02 ppm (m, 4H);
13C NMR (63 MHz, CDCl3, 258C, TMS): d=14.0, 55.4, 62.4, 64.4, 114.5,
126.9, 128.4, 128.6, 130.0, 129.3, 131.8, 133.6, 134.0, 135.6, 146.3, 160.1,
165.8, 190.1, 190.7 ppm; MS (CI, NH3): m/z (%): 420 (25) [M++18], 403
(71) [M++1], 331 (100), 299 (41); elemental analysis calcd (%) for
C25H22O5 (402): C 74.61, H 5.51; found: C 74.08, H 5.54.
Ethyl benzoyl-4-methoxybenzoyl acetate (2A-MeOPhePhe): Same
method as for 2A-NaPhe, but with ethyl benzoyl acetate (960 mg,
5 mmol) and 4-methoxybenzoyl chloride (853 mg, 5 mmol). Compound
2A-MeOPhePhe was obtained as white crystals after recrystallization in
ethanol (1.25 g, 76%). M.p. 1048C; 1H NMR (250 MHz, CDCl3, 258C,
TMS): d=1.25 (t, 3J(H,H)=7.1 Hz, 3H), 3.87 (s, 3H), 4.29 (q, 3J(H,H)=
7.1 Hz, 2H), 6.14 (s, 1H), 6.88 6.99 (m, 2H), 7.43 7.53 (m, 2H), 7.56
7.65 (m, 1H), 7.87 7.96 ppm (m, 4H); 13C NMR (63 MHz, CDCl3, 258C,
TMS): d=14.0, 55.6, 62.4, 64.4, 114.2, 128.8, 128.9, 131.1, 133.9, 135.7,
164.2, 165.9, 189.0, 190.7 ppm; MS (CI, NH3): m/z (%): 344 (42) [M+
+18], 327 (100) [M++1], 279 (51); elemental analysis calcd (%) for
C19H18O5 (326): C 69.92, H 5.56; found: C 69.78, H 5.57.
Ethyl benzoylnaphthoyl acetate (2A-NaPhe): A solution of ethyl benzoyl
acetate (960 mg, 5 mmol) dissolved in THF (2 mL) was added dropwise
to a suspension of sodium hydride (220 mg, 5.5 mmol) in THF (3 mL),
under an atmosphere of nitrogen at 08C.[23] The mixture was stirred until
the hydrogen evolution ceased (10 min). Naphthalene carbonyl chloride
(952 mg, 5 mmol) in THF (5 mL) was then added and the stirring contin-
ued at RT for 16 h. THF was evaporated and the mixture was hydrolyzed
with sulfuric acid (2m, 10 mL). The residue was extracted three times
with CH2Cl2 and the organic phase was washed with water. After drying
(magnesium sulfate), the solvent was evaporated and the crude residue
was washed several times with cyclohexane/diethyl ether (50:50, 3 mL) to
yield 2A-NaPhe as a white powder (986 mg, 58%). M.p. 1218C; 1H
NMR (250 MHz, CDCl3, 258C, TMS): d=1.24 (t, 3J(H,H)=7.1 Hz, 3H),
4.31 (q, 3J(H,H)=7.1 Hz, 2H), 6.36 (s, 1H), 7.4 7.52 (m, 2H), 7.52 7.68
(m, 3H), 7.82 8.04 (m, 6H), 8.44 ppm (brs, 1H); 13C NMR (63 MHz,
CDCl3, 258C, TMS): d=14.0, 62.5, 64.5, 123.9, 127.2, 127.9, 128.7, 129.0,
129.2, 129.8, 130.7, 132.5, 133.1, 134.1, 135.7, 136.0, 165.9, 190.7,
190.9 ppm; MS (DEI): m/z (%): 346 (3) [M+], 300 (14), 155 (100), 127
(38), 105 (45), 77 (14); elemental analysis calcd (%) for C22H18O4 (346):
C 76.28, H 5.24; found: C 76.33, H 5.14.
Ethyl di(naphthalene-2-carbonyl) acetate (2A-NaNa): Same method as
for 2A-NaPhe, but with ethyl naphthalene-2-carbonyl acetate (930 mg,
3.84 mmol) and naphthalene-2-carbonyl chloride (731.5 mg, 3.84 mmol).
Compound 2A-NaNa was obtained as a white powder after washing sev-
eral times with C6H12/Et2O 50:50 (1.02 g, 67%). M.p. 1508C; 1H NMR
(250 MHz, CDCl3, 258C, TMS): d=1.26 (t, 3J(H,H)=7.12 Hz, 3H), 4.33
3
(q, J(H,H)=7.12 Hz, 2H), 6.50 (s, 1H), 7.49 7.69 (m, 4H), 7.81 8.07 (m,
8H), 8.48 ppm (brs, 2H); 13C NMR (63 MHz, CDCl3, 258C, TMS): d=
14.0, 62.5, 64.7, 123.9, 127.1, 127.8, 129.0, 129.1, 129.8, 130.7, 132.5, 133.1,
135.9, 165.9, 190.6 ppm; MS (CI, NH3): m/z (%): 414 (3) [M++18], 397
(11) [M++1], 260 (60), 243 (100); elemental analysis calcd (%) for
C26H20O4 (396): C 78.77, H 5.085; found: C 78.74; H 5.19.
1-(2-Naphthyl)-3-phenylpropane-1,3-dione (NaPhe): A solution of ethyl
benzoylnaphthoyl acetate (692 mg, 2 mmol), water (72 mg, 4 mmol;
2 equiv) and sodium chloride (120 mg, 2.2 mmol; 1.1 equiv) in DMSO
(2 mL) was heated at 1408C until the release of carbon dioxide ceased.[24]
After cooling to room temperature, water was added and the mixture
was extracted with ethyl acetate and cyclohexane (1:1). The organic
phase was washed three times with water, then dried over magnesium
sulfate. After filtration and evaporation of the solvent, NaPhe was ob-
tained as a white solid after recrystallization in ethanol (510 mg, 93%).
Ethyl di(biphenyl-4-carbonyl) acetate (2A-BiBi): Same procedure as for
2A-NaPhe, but with ethyl biphenyl-4-carbonyl acetate (804 mg, 3 mmol),
sodium hydride (132 mg, 3.3 mmol), biphenyl-4-carbonyl chloride
(650 mg, 3 mmol). Compound 2A-BiBi was obtained as colorless crystals
after recrystallization in ethanol (1.27 g, 94%). M.p. 1638C; 1H NMR
(250 MHz, CDCl3, 258C, TMS): d=1.28 (t, 3J(H,H)=7.1 Hz, 3H), 4.33
(q, 3J(H,H)=7.1 Hz, 2H), 6.24 (s, 1H), 7.37 7.53 (m, 6H), 7.58 7.67 (m,
4H), 7.67 7.74 (m, 4H), 7.98 8.07 ppm (m, 4H); 13C NMR (63 MHz,
CDCl3, 258C, TMS): d=14.0, 62.5, 64.6, 127.3, 127.6, 128.5, 129.0, 129.3,
1
M.p. 988C (ref. [67] 998C); H NMR (250 MHz, CDCl3, 258C, TMS): d=
4.77 (s, 2H), 7.45 7.65 (m, 5H), 7.87 8.11 (m, 6H), 8.55 ppm (brs, 1H);
13C NMR (63 MHz, CDCl3, 258C, TMS): d=93.5, 123.2, 126.8, 127.2,
127.8, 128.1, 128.3, 128.5, 128.7, 129.4, 132.5, 132.75, 132.8, 135.3, 135.6,
185.5, 185.8 ppm.
1,3-Di(biphenyl-4-yl)propane-1,3-dione (BiBi): Same method as for
NaPhe, but with ethyl di(biphenyl-4-carbonyl) acetate (248 mg,
1451
Chem. Eur. J. 2004, 10, 1445 1455
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

L. Jullien, J.-F. Allemand et al.
FULL PAPER
0.55 mmol). Compound BiBi was obtained as pale yellow plates (172 mg,
83%). M.p. 2208C (ref. [68] 218 2208C); 1H NMR (250 MHz, CDCl3,
258C, TMS):d=6.95 (s, 1H), 7.37 7.54 (m, 6H), 7.57 7.82 (m, 8H), 8.04
8.13 (m, 4H), 14.58 14.76 ppm (m, 1H); 13C NMR (63 MHz, CDCl3,
258C, TMS): d=93.2, 127.3, 127.4, 127.8, 128.2, 129.0, 134.4, 140.0, 145.3,
185.3 ppm.
under reflux for 2 h, concentrated hydrochloric acid (0.5 mL) was added
to the mixture. Reflux was continued for 5 min and water (5 mL) was
added. After evaporation of the solvent, the crude residue was purified
by CC (C6H12/CH2Cl2 75:25 to 30:70) to yield BiPhe as a white solid
(620 mg, 21%). M.p. 1118C (ref. [69] 1128C); 1H NMR (250 MHz,
CDCl3, 258C, TMS): d=6.91 (s, 1H), 7.37 7.63 (m, 8H), 7.60 7.70 (m,
2H), 7.68 7.78 (m, 4H), 7.93 8.12 (m, 4H), 14.73 ppm (brs, 1H); 13C
NMR (63 MHz, CDCl3, 258C, TMS): d=93.1, 127.0, 127.15, 127.2,
127.25, 127.3, 127.7, 128.1, 128.2, 128.7, 128.9, 128.95, 130.1, 132.4, 134.3,
135.6, 139.9, 145.2, 145.6, 185.3, 185.7 ppm.
1-(Biphenyl-4’-yl)-3-(2-naphthyl)propane-1,3-dione (BiNa): Same method
as for NaPhe, but with ethyl 3-(2-naphthyl)-3-oxo-2-(biphenyl-4-carbon-
yl) propionate (336 mg, 0.8 mmol). Compound BiNa was obtained as col-
orless crystals after recrystallization in ethanol (140 mg, 50%). 1H NMR
(250 MHz, CDCl3, 258C, TMS): d=7.06 (s, 1H), 7.37 7.72 (m, 7H), 7.75
(d, 3J(H,H)=8.4 Hz, 2H), 7.86 8.10 (m, 4H), 8.13 (d, 3J(H,H)=8.4 Hz,
2H), 8.57 (brs, 1H), 14.60 ppm (brs, 1H); 13C NMR (63 MHz, CDCl3,
258C, TMS): d=64.7, 123.9, 127.1, 127.3, 127.6, 127.9, 128.5, 129.0, 129.1,
129.3, 129.8, 130.7, 132.5, 133.1, 134.4, 136.0, 139.6, 146.7, 165.8, 190.3,
190.6 ppm.
Syntheses of the diketones Ar1Ar2 alongpathway C
1,3-Di(4-methoxyphenyl)propane-1,3-dione (MeOPheMeOPhe):[28] A sus-
pension of 4-methoxybenzoyl chloride (1.67 g, 9.8 mmol) and aluminium
chloride (1.4 g, 10.25 mmol) in tetrachloroethane (5 mL) was stirred at
558C for 2 h. Then, ethenyl acetate (430 mg, 5 mmol) was added and the
mixture was further stirred at 558C for 20 h. After cooling to room tem-
perature, hydrochloric acid (2m, 2 mL) and water (20 mL) were succes-
sively added. After extraction with ethyl acetate, the organic phase was
washed with sodium hydrogencarbonate and water. After drying (magne-
sium sulfate) and filtration, the organic solvent was evaporated to yield
MeOPheMeOPhe as pale yellow crystals after recrystallization in ethanol
(357 mg, 25%). M.p. 1148C (ref. [28] 116 1178C); 1H NMR (250 MHz,
CDCl3, 258C, TMS): d=3.89 (s, 6H), 6.74 (s, 1H), 6.93 7.02 (m, 4H),
7.92 8.01 ppm (m, 4H); 13C NMR (63 MHz, CDCl3, 258C, TMS): d=
55.5, 91.5, 114.0, 128.2, 129.1, 131.4, 163.0, 184.6 ppm.
(4’-Methoxybiphenylyl)-3-phenylpropane-1,3-dione (MeOBiPhe): Same
method as for NaPhe, but with ethyl benzoyl-4-methoxybenzoyl acetate
(1.08 g, 2.7 mmol). Compound MeOBiPhe was obtained as pale yellow
crystals after recrystallization in ethanol (515 mg, 57%). M.p. 1768C; 1H
NMR (250 MHz, CDCl3, 258C, TMS): d=3.87 (s, 3H), 6.90 (s, 1H),
6.98 7.06 (m, 2H), 7.44 7.66 (m, 5H), 7.65 7.72 (m, 2H), 7.97 8.09 ppm
(m, 4H); 13C NMR (63 MHz, CDCl3, 258C, TMS): d=55.4, 93.0, 114.4,
126.7, 127.2, 127.8, 128.3, 128.7, 132.3, 132.4, 133.6, 135.7, 144.8, 159.9,
185.4, 185.5 ppm; MS (CI, NH3): m/z (%): 348 (2) [M++18], 331 (100)
[M++1]; elemental analysis calcd (%) for C22H18O3 (330): C 79.98, H
5.49; found: C 79.82, H 5.69.
Syntheses of the Ar1Ar2BF2 compounds
Compound 1: A solution of BiBi (276 mg, 0.73 mmol) and BF3/Et2O
(0.14 mL, 1.1 mmol) in benzene (2 mL) was heated to reflux for 1 h.[21]
After evaporation of the solvent, 1 was obtained as yellow crystals after
recrystallization in benzene (230 mg, 74%). M.p. >2908C; 1H NMR
(250 MHz, CDCl3, 258C, TMS): d=7.28 (s, 1H), 7.40 7.57 (m, 6H), 7.66
7.72 (m, 4H), 7.76 7.84 (m, 4H), 8.21 8.31 ppm (m, 4H); 13C NMR
(63 MHz, CDCl3, 258C, TMS): d=93.3, 127.4, 127.8, 128.9, 129.1, 129.6,
130.8, 139.2, 148.1, 182.4 ppm; 19F NMR (235 MHz, CDCl3, 258C, CFCl3):
d=ꢀ140.24 (75%), ꢀ140.17 ppm (25%); MS (CI, NH3): m/z (%): 442
(100) [M++18], 405 (18), 361 (5); elemental analysis calcd (%) for
C27H19BF2O2 (424): C 76.44, H 4.51; found: C 76.45, H 4.51.
1-(4-Methoxyphenyl)-3-phenylpropane-1,3-dione (MeOPhePhe): Same
method as for NaPhe, but with ethyl benzoyl 4-methoxybenzoyl acetate
(654 mg, 2 mmol). Compound MeOPhePhe was obtained as colorless
crystals after recrystallization in ethanol (393 mg, 77%). M.p. 1378C; 1H
NMR (250 MHz, CDCl3, 258C, TMS): d=3.89 (s, 3H), 6.80 (s, 1H),
6.95 7.04 (m, 2H), 7.43 7.60 (m, 3H), 7.94 8.06 ppm (m, 4H); 13C NMR
(63 MHz, CDCl3, 258C, TMS): d=55.5, 92.4, 114.0, 127.0, 128.2, 128.6,
129.3, 132.1, 135.6, 163.3, 184.0, 186.2 ppm; MS (CI, NH3): m/z (%): 272
(21) [M++18], 256 (37) [M++2], 255 (100) [M++1]; elemental analysis
calcd (%) for C16H14O3 (254): C 75.57, H 5.55; found: C 75.56, H 5.65.
1,3-Di(2-naphthyl)propane-1,3-dione (NaNa): Same method as for
NaPhe, but with ethyl di(naphthalene-2-carbonyl) acetate (792 mg,
2 mmol). Compound NaNa was obtained as colorless crystals after recrys-
tallization in ethanol (501 mg, 77%). M.p. 1718C (ref. [69] 1728C); 1H
NMR (250 MHz, CDCl3, 258C, TMS): d=7.16 (s, 1H), 7.51 7.68 (m,
4H), 7.84 8.20 (m, 8H), 8.60 (brs, 2H), 14.58 ppm (brs, 1H); 13C NMR
(63 MHz, CDCl3, 258C, TMS): d=93.6, 123.3, 126.6, 127.6, 128.2, 128.4,
128.5, 129.4, 132.8, 132.9, 135.4, 185.6 ppm.
Compound 2: Same procedure as for 1, but with BiNa (140 mg,
0.4 mmol) and BF3/Et2O (0.05 mL, 1.1 mmol). 2 was obtained as yellow
1
crystals after recrystallization in benzene (113 mg, 71%). M.p. 2588C; H
NMR (250 MHz, CDCl3, 258C, TMS): d=7.39 (s, 1H), 7.43 7.57 (m,
3H), 7.57 7.64 (m, 4H), 7.82 (d, 3J(H,H)=8.5 Hz, 2H), 7.91 7.92 (m,
1H), 7.92 8.04 (m, 1H), 8.03 8.09 (m, 1H), 8.10 8.17 (m, 1H), 8.30 (d,
3J(H,H)=8.5 Hz, 2H), 8.81 ppm (brs, 1H); 13C NMR (100 MHz, CDCl3,
258C, TMS): d=93.6, 123.5, 127.3, 127.4, 127.7, 127.9, 128.9, 129.1, 129.1,
129.6, 129.8, 130.0, 130.65, 131.6, 132.5, 136.5, 139.5, 148.0, 182.4,
182.6 ppm; 19F NMR (235 MHz, CDCl3, 258C, CFCl3): d=ꢀ140.185
(75%), ꢀ140.12 ppm (25%); MS (CI, NH3): m/z (%): 416 (100) [M+
+18], 379 (23), 351 (1), 335 (1); elemental analysis calcd (%) for
C25H17BF2O2 (398): C 75.40, H 4.30; found: C 75.20, H 4.31.
Syntheses of the diketones Ar1Ar2 alongpathway B
1-Biphenyl-4’-yl-3-phenylprop-2-enone (1B-BiPhe):[25] Sodium hydroxide
(5m, 4 mL) was slowly added to a heated (508C) solution of benzalde-
hyde (1.06 g, 10 mmol) and 4-acetylbiphenyl (1.96 g, 10 mmol) in ethanol
(50 mL) and THF (10 mL). The mixture was stirred for 20 h at room tem-
perature. After evaporation of the solvents, the residue was extracted
with CH2Cl2. The organic phase was washed several times with water and
was dried (magnesium sulfate). After filtration and evaporation of the
solvent, 1B-BiPhe was obtained as a white solid (2.67 g, 94%) that was
used without further purification for the following step. 1H NMR
(250 MHz, CDCl3, 258C, TMS): d=7.35 7.53 (m, 6H), 7.60 (d,
3J(H,H)=15.7 Hz, 1H), 7.58 7.78 (m, 6H), 7.86 (d, 3J(H,H)=15.7 Hz,
1H), 8.06 8.17 ppm (m, 2H).
Compound 3: Same method as for 1, but with BiPhe (620 mg, 2.1 mmol)
and BF3/Et2O (0.27 mL, 2.1 mmol) in CH2Cl2 (20 mL). 3 was obtained as
orange crystals after recrystallization in benzene (556 mg, 76%). M.p.
2288C; 1H NMR (250 MHz, CDCl3, 258C, TMS): d=7.25 (s, 1H), 7.40
7.63 (m, 5H), 7.63 7.77 (m, 3H), 7.77 7.83 (m, 2H), 8.13 8.21 (m, 2H),
8.21 8.28 ppm (m, 2H); 13C NMR (63 MHz, CDCl3, 258C, TMS): d=
93.3, 127.3, 127.7, 128.9, 129.1, 129.2, 129.6, 130.5, 132.0, 135.2, 139.1,
148.1, 182.7, 182.9 ppm; 19F NMR (235 MHz, CDCl3, 258C, CFCl3): d=
ꢀ140.20 (75%), ꢀ140.14 ppm (25%); MS (CI, NH3): m/z (%): 366 (100)
[M++18], 329 (8), 300 (8), 285 (7); elemental analysis calcd (%) for
C21H15BF2O2 (348): C 72.45, H 4.34; found: C 72.36, H 4.40.
1-(Biphenyl-4-yl)-2,3-dibromo-3-phenylpropanone (2B-BiPhe): An excess
of bromine (0.75 mL, 20 mmol) in CH2Cl2 (7.5 mL) was slowly added at
RT to a solution of 1B-BiPhe (2.67 g, 9.4 mmol) in CH2Cl2 (30 mL).[26]
The mixture was stirred for 80 min. After evaporation of the solvent, 2B-
BiPhe was obtained as a white solid (4.17 g) that was used without fur-
ther purification for the following step. 1H NMR (250 MHz, CDCl3,
258C, TMS): d=5.67 (d, 3J(H,H)=11.3 Hz, 1H), 5.88 (d, 3J(H,H)=
11.3 Hz, 1H), 7.34 7.64 (m, 8H), 7.60 7.70 (m, 2H), 7.72 7.82 (m, 2H),
8.12 8.23 ppm (m, 2H).
Compound 4: Same method as for 1, but with MeOBiPhe (330 mg,
1 mmol) and BF3/Et2O (0.14 mL, 1.1 mmol). 4 was obtained as a yellow
1
powder after recrystallization in benzene (160 mg, 42%). M.p. 2458C; H
NMR (250 MHz, [D6]DMSO, 258C, TMS): d=3.81 (s, 3H), 7.01 7.12 (m,
2H), 7.59 7.72 (m, 2H), 7.77 7.88 (m, 3H), 7.90 8.01 (m,2H), 7.97 (s,
1H), 8.33 8.49 ppm (m, 4H); 13C NMR (63 MHz, [D6]DMSO, 258C,
TMS): d=55.7, 94.7, 115.0, 127.0, 129.0, 129.6, 129.8, 130.6, 130.7, 131.8,
136.1, 147.2, 160.6, 182.2, 182.4 ppm; 19F NMR (235 MHz, CDCl3
(CFCl3), 258C, CFCl3): d=ꢀ140.25 (67%), ꢀ140.19 ppm (33%); MS (CI,
1-(Biphenyl-4-yl)-3-phenylpropane-1,3-dione (BiPhe): Sodium methylate
(460 mg, 18.8 mmol; 2 equiv) in methanol (6 mL) was added to a suspen-
sion of 2B-BiPhe (4.17 g, 9.4 mmol) in methanol (30 mL).[27] After being
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Fluorescent Probes
1445 1455
NH3): m/z (%): 396 (100) [M++18], 359 (9), 331 (8); elemental analysis
calcd (%) for C22H17BF2O3 (378): C 68.87, H 4.53; found: C 69.73, H
4.70.
vacuum for one hour. For solvatochromism investigations, the appropri-
ate amount of desired spectroscopic grade solvent (Aldrich, except for
the CH2Cl2 which was freshly home-distilled over calcium hydride) was
added to each tube and the mixture was sonicated. For experiments in-
volving micelles, b-octylglucopyranoside was added to stock solutions of
dye before evaporation of dichloromethane. Transparent micellar solu-
tions were obtained after addition of ultra-pure water and brief sonica-
tion (final concentration in detergent 25mm).
Compound 5: Same method as for 1, but with MeOPheMeOPhe
(284 mg, 1 mmol) and BF3/Et2O (0.14 mL, 1.1 mmol). 5 was obtained as
yellow crystals after recrystallization in benzene (283 mg, 85%). M.p.
2368C; 1H NMR (250 MHz, CDCl3, 258C, TMS): d=3.93 (s, 6H), 6.97
7.06 (m, 4H), 7.02 (s, 1H), 8.10 8.19 ppm (m, 4H); 13C NMR (63 MHz,
CDCl3, 258C, TMS): d=55.8, 91.5, 114.6, 124.6, 131.3, 165.3, 180.9 ppm;
19F NMR (235 MHz, CDCl3, 258C, CFCl3): d=ꢀ141.53 (75%),
ꢀ141.47 ppm (25%); MS (CI, NH3): m/z (%): 350 (92) [M++18], 313
(100), 269 (1); elemental analysis calcd (%) for C17H15BF2O4 (332): C
61.48, H 4.55; found: C 61.62, H 4.63.
Measurements of the cross sections for two-photon absorption
Method: In the absence of photobleaching, the photon flux F(t) emitted
under two-photon excitation is proportional to the fluorophore concen-
tration c (assumed to be homogeneous and stationary) and to the square
!
of the incoming power I( r ,t) [Eq. (2)]:
Compound 6: Same method as for 1, but with MeOPhePhe (254 mg,
1 mmol) and BF3/Et2O (0.14 mL, 1.1 mmol). 6 was obtained as yellow
crystals after recrystallization in benzene (125 mg, 41%). M.p. 2168C; H
Z
1
2
!
1
FðtÞ ¼ aꢀFdc
I2ð r ; tÞdV
ð2Þ
v
NMR (250 MHz, CDCl3, 258C, TMS): d=3.94 (s, 3H), 7.00 7.09 (m,
2H), 7.11 (s, 1H), 7.51 7,61 (m, 2H), 7.65 7.73 (m, 1H), 8.09 8.21 ppm
(m, 4H); 13C NMR (63 MHz, CDCl3, 258C, TMS): d=55.8, 92.5, 114.7,
124.2, 128.6, 129.1, 131.7, 132.4, 134.7, 165.8, 181.6, 182.4 ppm; 19F NMR
(235 MHz, CDCl3, 258C, CFCl3): d=ꢀ140.84 (75%), ꢀ140.78 ppm
(25%); MS (CI, NH3): m/z (%): 320 (58) [M++18], 283 (18), 166 (100);
elemental analysis calcd (%) for C16H13BF2O3 (302): C 63.62, H 4.34;
found: C 63.71, H 4.31.
in which fF is the fluorescence quantum yield (assumed to remain identi-
cal to the value determined by the study of the linear photophysical
properties), d is the two-photon absorption cross section to be measured
(in cm4 s(photonꢀmolecule)ꢀ1) and a is the set-up detection efficiency.
During an acquisition, the amount of fluorescence detected is shown by
using Equation (3):
Compound 7: Same method as for 1, but with NaNa (324 mg, 1 mmol)
and BF3/Et2O (0.14 mL, 1.1 mmol). 7 was obtained as a yellow powder
after recrystallization in benzene (264 mg, 71%). M.p. >2768C; 1H
NMR (250 MHz, [D6]DMSO, 258C, TMS): d=7.73 7.97 (m, 4H), 8.18
8.26 (m, 2H), 8.27 8.43 (m, 4H), 8.37 (s, 1H), 8.49 8.58 (m, 2H),
9.30 ppm (brs, 2H); 13C NMR (63 MHz, [D6]DMSO, 258C, TMS): d=
95.2, 123.9, 127.6, 127.9, 128.7, 129.1, 130.1, 130.2, 132.1, 132.2, 136.2,
182.1 ppm; 19F NMR (235 MHz, CDCl3 (CFCl3), 258C, CFCl3): d=
ꢀ143.32 (75%), ꢀ143.30 ppm (25%); MS (CI, NH3): m/z (%): 390 (100)
[M++18], 353 (9), 309 (9); elemental analysis calcd (%) for C23H15BF2O2
(372): C 74.22, H 4.06; found: C 74.26, H 4.01.
Z
1
2
!
hFðtÞi ¼ aꢀFdchP2ðtÞi
Sð r ÞdV
ð3Þ
v
!
in which S( r ) is the temporal distribution of the incident light and P(t)
is the instantaneous photon flux for the sample (photonsꢀ1).
The signal detected is proportional to hP2(t)i, whereas most detectors are
2
sensitive to hP(t)i . An accurate measurement of d thus requires the de-
Compound 8: Same method as for 1, but with NaPhe (274 mg, 1 mmol)
and BF3/Et2O (0.14 mL, 1.1 mmol). 8 was obtained as a yellow powder
(261 mg, 81%). M.p. 2298C; 1H NMR (250 MHz, CDCl3, 258C, TMS):
d=7.34 (s, 1H), 7.48 7.75 (m, 5H), 7.85 8.14 (m, 4H), 8.14 8.22 (m,
2H), 8.76 ppm (brs, 1H); 13C NMR (63 MHz, CDCl3, 258C, TMS): d=
93.7, 123.4, 127.5, 127.9, 128.9, 129.1, 129.2, 129.8, 130.0, 131.6, 132.0,
132.5, 135.2, 136.5, 182.9 ppm; 19F NMR (235 MHz, CDCl3, 258C, CFCl3):
d=ꢀ140.03 (75%), ꢀ139.98 ppm (25%); MS (CI, NH3): m/z (%): 340
(100) [M++18], 303 (12), 275 (45), 259 (63); elemental analysis calcd
(%) for C19H13BF2O2 (322): C 70.85, H 4.07; found: C 70.72, H 4.10.
termination of the temporal structure of the excitation beam and its
second-order temporal coherence: g(2) =hP2(t)i/hP(t)i . This is usually
2
achieved by calibration at each wavelength by using a known standard,
such as fluorescein or rhodamine, which causes heavy and repetitive ma-
nipulations. An alternative elegant method has been described by Xu
et al.[60] By adding a Michelson interferometer to the laser path, the usual
set up is transformed into an autocorrelator, using the fluorescent sample
as the quadratic medium (Figure 1Sa and 1Sb in the Supporting Informa-
tion). The major interest of this set-up configuration is evidently to get
rid of the otherwise necessary determination of the pulse temporal shape,
for this information is now intrinsically contained in the interference pat-
tern. The only unknown quantity remaining is the a factor that may be
determined by calibration at one single wavelength. In the interference
conditions it was then shown that [Eq. (4)]:
UV/Vis spectroscopic measurements: All experiments were performed at
293 K in spectroscopic grade solvents. UV/Vis absorption spectra were
recorded on a Kontron Uvikon-930 spectrophotometer. Molar absorption
coefficients were extracted, while checking the validity of the Beer Lam-
bert law. Corrected fluorescence spectra were obtained from a Photon
Technology International LPS 220 spectrofluorimeter. The concentrations
of solutions for fluorescence measurements were adjusted so that the ab-
sorption was below 0.15 at the excitation wavelength. The overall fluores-
cence quantum yields fF were calculated from the relation shown in
Equation (1):
ꢀ
ꢁ
T
R
FðtÞdt ꢀ2 TF1
ꢀT
d ¼
ꢂ þ1
R
ꢃ
ð4Þ
2
2acAꢀF
PðtÞdt
ꢀ1
ꢀ
ꢁ
2
ArefðlexcÞ D
n
ð1Þ
ꢀF ¼ ꢀref
in which T is the integration time (2T larger than the interference pat-
Dref nref
Aðlexc
Þ
R
tern width). A= S2(!r )dVꢁ2.55/l when overfilling the back-aperture
v
of the focusing lens (in the case of a plane wave truncated by an aper-
ture, giving an Airy diffraction pattern). F¥ is the mean fluorescence
signal without any interference; it is then simply twice the fluorescence
generated by one pulse from one of the Michelson paths. It was also
shown that the ratio between the fluorescence maximum and F¥ must
be 8:1, which is also a probe for the quadratic dependence on the excita-
tion power.
in which the subscript ref stands for standard samples, A(lexc) is the ab-
sorbance at the excitation wavelength, D is the integrated emission spec-
trum, and n is the refractive index for the solvent. The uncertainty in the
experimental value of fF was estimated to be Æ10%. The standard fluo-
rophore for the quantum yield measurements was quinine sulfate in 0.1m
H2SO4 with fS =0.50.[70]
Samples for solvatochromism and stability studies were prepared in the
following manner: Fixed volumes of a stock solution of dye in dichloro-
methane were transferred into pyrex hemolysis tubes. The volatile sol-
vent was evaporated under a fume hood for one whole night or under
Set up and data treatment: The experiments were performed at room
temperature in spectroscopic grade dichloromethane at 10ꢀ4 m. Freshly
prepared solutions were sealed in square capillary tubes (1 mmî1 mm;
VitroCom, USA), kept at +58C, and away from light afterwards.
1453
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

L. Jullien, J.-F. Allemand et al.
FULL PAPER
The scheme of the home-made experimental set up was inspired from
ref. [60] (Figure 1Sa and 1Sb in the Supporting Information). The excita-
tion source was a pulsed Ti sapphire laser (mira - Coherent, USA)
pumped at 532 nm by a Nd-YVO4 laser (Verdi - Coherent, USA). This
femtosecond laser delivered pulses in the near-infrared region from 690
to 1000 nm.
[8] E. Vos de Wael, J. A. Pardoen, J. A. van Koeveringe, J. Lugtenburg,
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The Michelson interferometer consisted of a beamsplitter (BS) and two
corner-cubes (Figure 1Sb). One corner-cube (CC1) was fixed on the dia-
phragm of a loudspeaker powered by a low-frequency generator. A con-
tinuous He-Ne laser was also included in the interferometer to calibrate
the diaphragm displacement. The resulting interference pattern was re-
corded at the end of the Michelson interferometer on a photodiode
(PD1). Typical pulse widths at the sample were less than 200 fs.
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After a beam-expander 6î (BE, Melles Griot), the IR laser beam over-
filled the back aperture of the lens (Nikon, 20î, N.A. 0.35), which colli-
mates it onto the sample. A lens focused most of the excitation beam on
a second photodiode (PD2; the fraction collected remains unchanged
with wavelength). A long-pass dichroic filter (DC, Chroma, USA) sepa-
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a set of short-pass filters (from Melles Griot or OmegaOptics), the re-
maining IR background (from elastic or inelastic scattering) was totally
removed and the pure fluorescence emission was divided by a beam-split-
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PMT2, RC135 - Hamamatsu, Japan). PMT2 is a photon-counting tube,
but its minimal integration delay is limited to 10 ms, hence requiring the
auxiliary use of PMT1 with a faster rate cut energy-dependent signal. The
signals of the two photodiodes and the fluorescence in energy and in
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time on a PC, thanks to the acquisition card PCI-DAS1602/16 (Comput-
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nals were then corrected by the sensitivities of the detectors given by the
manufacturers. For PD2, the excitation wavelength was first determined
by the following method: both interference signals from the two photodi-
odes were treated by the Hilbert transform (HT) method. The phase of
the HT was integrated as a function of time, and the slope was propor-
tional to the interference spatial frequencies, that is, to the wavelength.
The ratio of the slopes for the excitation IR to the He-Ne then gave the
ratio between the wavelengths; the signal of the He-Ne on PD2 was used
as the reference wavelength (lHe-Ne =632.8 nm). With this determination
of the excitation wavelength, we then referred to the constructor sensitiv-
ity charts to extract the required correction factor. For the two PMs, the
correction was taken at the maximum emission, determined by fluorime-
try (vide supra). We also checked that the correction was modified within
the experimental uncertainty when integrating it on the whole emission
spectrum.
The value of d was computed according to Equation (4), directly from
the input signals: PD2 leads to hP(t)i, PM1 and PM2 supply F(t) and F¥
(Figure 2S in the Supporting Information). To optimize its determination,
the acquisition is triggered on the GBF signal, and the final value of d
was averaged over 15 to 20 successive acquisitions. The whole spectrum
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corresponds to the width of the excitation wavelength distribution. It is
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Received: July 10, 2003
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