Size and branching effects on the fluorescence of benzylic dendrimers possessing one apigenin fluorophore at the core

Article abstract of DOI:10.1016/j.tet.2013.10.002

Different generations of dendrimers incorporating one fluorescent core of apigenin and three Fréchet benzylic dendrons have been prepared. The chief geometric features of these dendrimers have been obtained by Molecular Dynamics simulations. These computa

Full text of DOI:10.1016/j.tet.2013.10.002

Tetrahedron 69 (2013) 10361e10368
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Size and branching effects on the fluorescence of benzylic
dendrimers possessing one apigenin fluorophore at the core
Petr Vin ꢀs a , Martina Vermachov aꢁ , Pavel Dra ꢀs ar , Melisa del Barrio , Carmen Jarne ,
,
b
a
a
c
c
c
,
b
,
d
,
e
d,
e
b,
d
, ,
y
*
ꢁ
*
Vicente L. Cebolla , Abel de Cozar
Institute of Chemical Technology, Prague, Technick aꢁ 5, 16628 Praha, Dejvice, Czech Republic
Donostia International Physics Center (DIPC), P.O. Box 1072, 20018 San Sebasti aꢁ n e Donostia, Spain
Instituto de Carboquímica, ICB-CSIC, P.O. Box 549, 50080 Zaragoza, Spain
, Ronen Zangi , Fernando P. Cossío
a
b
c
d
ꢀ
Department of Organic Chemistry I, Universidad del País Vasco e Euskal Herriko Unibertsitatea (UPV/EHU), P Manuel de Lardizabal 3, 20018 San
Sebasti aꢁ n e Donostia, Spain
e
Ikerbasque, Basque Foundation for Science, E-48011 Bilbao, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 June 2013
Received in revised form 27 September 2013
Accepted 3 October 2013
Available online 10 October 2013
Different generations of dendrimers incorporating one fluorescent core of apigenin and three Fr eꢁ chet
benzylic dendrons have been prepared. The chief geometric features of these dendrimers have been
obtained by Molecular Dynamics simulations. These computational data suggest that the asphericities of
dendrimers belonging to the third and fourth generations are considerably larger than those associated
with lower radii of gyration. Fluorescence spectra of high generation dendrimers evolve along time and
quantum yields show an appreciable lowering for the fourth generation dendrimer. All these data sug-
gest aggregation phenomena and lower quantum yields for nonspheric dendrimers in solution.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Dendrimers
Apigenin
Fluorescence
Photophysical properties
Quantum yield
1
1
1
. Introduction
increase towards the periphery. However, this general model was
12
refined and now it is generally admitted that this density profile is
more complex, thus resulting in dense core regions and relatively
less dense peripheries.
Fluorescent dendrimers are particularly interesting because
of their potential as analytical tools or in light-harvesting de-
Since the discovery in 1978 of ‘cascade molecules’ by V o€ gtle
1
2
et al., dendrimers (as termed by Tomalia ) have emerged as a very
important class of macromolecules with well-defined architec-
tures. The nature of the cores, the different spacers, and end groups,
as well as the degrees of branching can be chosen among many
13
14
15
16
6a
vices, organic light emitting diodes (OLEDs), solar cells or
displays. The chosen fluorophores can be installed at different
3
17
different building blocks, thus giving rise to a plethora of different
4
families of dendrimers with many applications in, among other
parts of the macromolecule. In most cases, diverse fluorophores
and/or donor/acceptor systems can be incorporated within the
different branches or at the periphery. In these cases, however, the
structure optical response relationship is difficult to rationalize. In
previous work on dendrimers carried out in our group, we have
analyzed the catalytic activity of dendrimers with one active site
at the core. In this case a rationale of the catalytic activity of dif-
fields, catalysis,5 materials science, supramolecular chemistry,
6
7
and biomedicine.8
The structure and shape of dendrimers are difficult to determine
9
and actually the usual analytical techniques are pushed to their
limits to characterize the identity, purity, and structural elucidation
of these macromolecules. In this respect, computational methods
are very useful to describe adequately both the geometrical and
dynamic behavior of dendritic molecules. Pioneering studies
proposed that dendrimers in general exhibit density profiles in
which a global minimum is located at the center, with a monotonic
18
ferent dendrimers possessing Fr eꢁ chet dendrons was developed.
10
Following this approach, in this paper we would like to present
a simple model describing the structure activity quantitative re-
lationship of fluorescent dendrimers incorporating only one fluo-
19
rophore at the core. As a case study, we have chosen apigenin,
a weak blue light emitting fluorophore that possesses three phe-
nol groups amenable to Williamson coupling reactions with dif-
ferent Fr eꢁ chet dendron bromides. The interplay between the 4H-
*
y
Corresponding authors. E-mail address: fp.cossio@ehu.es (F.P. Cossío).
Fax: þ34 943 015270.
0
040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.10.002

10362
P. Vin ꢀs et al. / Tetrahedron 69 (2013) 10361e10368
chromen-4-one fluorophore and the phenyl chromophores of the
branches and at the periphery should result in different photo-
physical responses for the successive generations of dendrimers.
As we will see, this design has permitted to find a simple model
that connects the quantum yield of the fluorescent emission with
an easy to compute and intuitive combined geometrical
parameter.
The reaction conditions reflect the increasing difficulty to ac-
complish the formation of the three CeO bonds along the different
dendritic generations. Thus, formation of small compounds 3aec
(Apigenin derivatives 3b, c being the zeroth and first generation G0
and G1 in the dendrimer series) required NaH as a base and 20 h at
room temperature. In the case of dendrimers 3def, Williamson
couplings involving Fr eꢁ chet dendrons 2def required potassium car-
ꢀ
bonate, 18-crown-6 as a cryptand of potassium and heating at 80 C.
2
2
. Results and discussion
2
.2. Molecular simulations and structural parameters of
.1. Preparation of apigenin derivatives
dendrimers 3bef
We prepared compounds 3aef by Williamson reaction between
and halides 2aef in the presence of different bases (Scheme 1).
In order to gain a better understanding of the structural properties
of compounds 3bef and their relationship with the observed prop-
1
OH
O
MeI or RBr
OR
O
2a
2b-f
i
HO
O
RO
O
1
3a-f (G0-G4)
OH
OR
a: R=Me
BnO
O
b: R=Bn (G0) OBn
c: R=
OBn
(
G1)
BnO
BnO
OBn
BnO
O
OBn
BnO
OBn
O
O
OBn
O
O
d: R=
O
(
G2)
f: R=
O
O
OBn
(
G4)
O
O
OBn
BnO
OBn
O
BnO
OBn
O OBn
O
OBn
O
BnO
BnO
O
BnO
O
O
OBn
OBn
BnO
O
e: R=
OBn
OBn
(
G3)
O
O
OBn
O
BnO
OBn
ꢀ
2 3
CO (for G2eG4), 18-crown-6 (for G2eG4), rt (for G0, G1) or 80 C
Scheme 1. Synthesis of ethers 3aef (G0eG4) from apigenin 1. Reagents and conditions: i: NaH (for G0, G1) or K
for G2eG4). Bn¼Benzyl.
(
The chemical yields of purified products were relatively low
15e40%, see Experimental section) but satisfactory enough for our
purposes.
erties (vide infra), we performed atomistic molecular dynamics (MD)
simulations on these compounds using the MM3 force field. Evo-
lution of dendrimers 3bef (G0eG4) was simulated along 1000 ps.
2
0
(

P. Vin ꢀs et al. / Tetrahedron 69 (2013) 10361e10368
10363
0
1
During the production time of the MD simulations, the fluctuating
energy values were observed to be stable within the thermal limits
the relative root mean squared fluctuation in the energy ranged from
.01 for the smallest system to 0.001 for the largest system), as it can
2
1
L
0
0
2
B
2
C
trðSÞ ¼ Rg ¼ tr@ 0
L
0 A
(4)
2
(
0
2
0
0
L
3
be seen by inspection of the energy profiles gathered in Fig. 1.
In Eq. 3 M stands for the total mass of the dendrimer. In the last
term of Eq. 4 the tensor S is diagonalized such that the components
L
i
(i¼1,2,3) of R
g
with respect to the corresponding main axes are
assigned according to the following criterion:
2
2
2
3
L > L > L
(5)
1
2
i
These L values can be used to quantify the shape of the molecule
being considered. Thus, the asphericity a
geometry is defined as
s
of one given molecular
21
ꢀ
ꢁ
1
2
2
¼ L ꢁ
1
2
2
3
a
s
L þ L
(6)
2
According to Eq. 6, a
s
reflects the anisotropy of the molecule
with respect to the principal axes. For a perfect sphere, the mag-
nitudes of the three main radii are identical (L ¼L ) and
¼L
therefore a >0 reflect the increasing
¼0. In our case, values of a
anisotropy of dendrimers 3bef by departure from perfect sphe-
ricity. The average values of L and a are gathered in Table 1.
1
2
3
s
s
Fig. 1. MM3 potential energy profiles for dendrimers 3bef (G0eG4) along the MD
simulations.
i
s
Our computed values for asphericity show closely related values
for G0 and G2. First generation dendrimer G1 is more spheric than
apigenin derivatives G0 and G2, G3 showing a somewhat larger
The general shape of compounds 3b-G0 and dendrimers 3cef
G1eG4) can be appreciated in Fig. 2, in which we have super-
ꢂ
2
(
value of about 64 A . However, the largest asphericity value was
imposed the 10 most stable geometries obtained during the pro-
duction time of the respective MD simulations. From these data it
can be readily seen that lower dendrimers 3b-G0, 3c-G1 and 3d-G2
permit contacts between the apigenin and the external medium,
whereas in higher dendrimers 3e-G3 and 3f-G4 these contacts are
much more difficult.
computed for G4, with a value of a almost twice than that obtained
for G3 (See Fig. 4 and Table 1). This suggests that the hydrodynamic
behavior of G4 is determined by a larger departure from sphericity
s
and aggregation phenomena to form more isotropic structures with
2
2
lower a values should be expected.
s
In order to quantify these geometric differences, we computed the
radii of gyration for all the points generated along the MD simula-
2.3. Photophysics of compounds 3aef
tions. The radius of gyration R
g
is given by the following expression:
Absorption spectra of compounds 3aef show two zones, which
provide different structural information (Fig. 5). One of them, as-
sociated with a maximum near 315 nm, is related to the fluo-
rophore core. The other zone, with a maximum absorption near
280 nm, may provide some information on the fluorescence gen-
erated by the benzylic dendrons. In all cases, we observed that
absorbances at both 280 and 315 nm increase linearly with con-
centration. Inspection of Fig. 5 shows quite similar UV spectra for
G0eG2, with a distinctive profile for G4, G3 being between both
subsets. This can be explained in terms of an effect of the medium.
2
3
1=2
P
6
n
i ¼ 1
!
! ! ! 7
6
¼ 6
4
m
i
ð r
i
ꢁ g Þð r
i
ꢁ g Þ7
R
g
P
7
5
(1)
n
m
i ¼ 1
i
!
where m
i
is the mass of atom i (i¼1,2,.,n), r is its position vector,
i
!
and g is the position vector of the center of masses:
P
n
!
mi
23
!
i ¼ 1
m
i
r
i
According to Toptygin the extinction coefficient as well as the
g ¼
P
(2)
n
radiative decay rate depend on the refractive index. In spite of the
fact that the increase with the refractive index is higher for the
radiative decay rate, it also occurs for the extinction coefficient and
it was observed for the first generations of dendrimers.
i ¼ 1
The fluctuation of the R
g
values along the MD simulations for the
different generations G0eG4 are gathered in Fig. 3 and the average
values are reported in Table 1. Our results indicate that the R values
g
Quantum yields were measured at the previously described two
excitation wavelengths. Apigenin exhibits double fluorescent
of 3b-G0 and 3c-G1 are similar during most of the production time.
The values obtained for 3d-G2 are slightly higher. Dendrimers 3e-
G3 and 3f-G4 exhibit consistently larger radii of gyration and lower
fluctuations, which indicate more rigid structures and larger atom
densities at the respective cores and peripheries.
24
emission in methanol, with two maxima at 430 nm and 534 nm.
When excitation is done at 300 nm, emission shows a maximum at
430, and a shoulder at 534 nm. By exciting at 357 nm, only emission
at 534 nm is obtained. The emission spectra of the dendrimers
when exciting at 280 nm are similar than those when exciting at
g
The square of R is equal to the trace of the gyration tensor S of
a given molecule and it is defined by Eq. 3:
315 nm (see Supplementary data for further information). This has
P
P
P
0
1
m ðx ꢁ xcmÞðx ꢁ xcm
Þ
Þ
Þ
m ðx ꢁ xcmÞðy ꢁ ycm
Þ
Þ
Þ
m ðx ꢁ xcmÞðz ꢁ zcm
Þ
Þ
Þ
i
i
i
i
i
i
i
i
i
i
i
i
B P
P
P
C
C
1
B
m ðy ꢁ ycmÞðx ꢁ xcm
m ðy ꢁ ycmÞðy ꢁ ycm
m ðy ꢁ ycmÞðz ꢁ zcm
i
i
i
i
i
i
i
i
i
S ¼
B
C
A
(3)
M @ P
i
P
i
P
i
m ðz ꢁ zcmÞðx ꢁ xcm
m ðz ꢁ zcmÞðy ꢁ ycm
m ðz ꢁ zcmÞðz ꢁ zcm
i
i
i
i
i
i
i
i
i
i
i
i

10364
P. Vin ꢀs et al. / Tetrahedron 69 (2013) 10361e10368
Fig. 2. Snapshots from the MD simulations of dendrimers 3b-G0, 3c-G1, 3d-G2, 3e-G3 and 3f-G4. The apigenin core in each dendrimer is highlighted in blue. The most stable
structures are gathered in ball & stick representation. The remaining structures correspond to the 10 most stable structures within the 1000 ps simulation time. The hollow arrow in
3f-G4 indicates the position of the apigenin core.
Table 1
Average values of radius of gyration (hR i, in A), its components along the main axes
ꢂ
2
a
ꢂ
g
ꢂ
L
1
eL
3
(in A) and asphericities (ha
s
i, in A ) of dendrimers 3bef (G0eG4)
Dendrimer hR
g
i
hL
1
i
hL hL ha i
2
i
3
i
s
3
3
3
3
3
b-G0
c-G1
d-G2
e-G3
f-G4
6.07ꢃ0.07
6.53ꢃ0.02
8.36ꢃ0.01
11.21ꢃ0.12
5.59ꢃ0.26 2.07ꢃ0.05 1.20ꢃ0.15
5.05ꢃ0.79 3.52ꢃ0.06 2.23ꢃ0.10
6.75ꢃ0.10 4.20ꢃ0.10 2.74ꢃ0.78
9.15ꢃ0.17 5.16ꢃ0.10 3.66ꢃ0.05
28.30ꢃ1.42
16.84ꢃ1.07
32.94ꢃ0.34
63.73ꢃ0.50
14.82ꢃ0.16 12.29ꢃ0.46 6.85ꢃ0.40 4.83ꢃ0.08 116.05ꢃ1.23
a
All values correspond to the average values obtained along the MD simulations
using the MM3 force field and Eqs. 1e5.
possibility for an intramolecular proton transfer. As a consequence,
ꢁ
3
the quantum yield of 3a (1.7ꢂ10 , see Table 2) is higher than that
ꢁ
4
of apigenin (4ꢂ10 ).
r
Quantum yields and radiative constants k of dendrimers were
found to be higher than those of 3a, and increase on going from 3b-
G0 to 3d-G2 at
exc¼315 nm. However, from this latter dendrimer
these values decrease to achieve a quantum yield of 3f-G4 at
80 nm similar to that of 3a. In the case of
Fig. 3. Evolution of the radius of gyration of dendrimers 3bef (G0eG4) along the
production time of MD simulations.
l
2
l
exc¼280 nm, 3c-G1
been reported25 to be due to an intramolecular proton transfer
between a phenolic OH (in C-5) and the oxygen of carbonyl (in C-4).
In the case of 3a, the latter emission is not present since the phe-
nolic OH has been methoxylated and, therefore, there is no
showed the highest quantum yield and a monotonous decrease was
observed on going from G2 to G4. The observed enhancements of
fluorescence can be related to the contribution of the refractive
2
3
index, according to the Toptygin equation:

P. Vin ꢀs et al. / Tetrahedron 69 (2013) 10361e10368
10365
case of 3a, n is that of solvent. In the case of dendrimers G0eG4,
the n value associated with the local medium that would cor-
respond to that of the aromatic dendrons in the microenvi-
ronment of the fluorophore. It is reasonable to think that, at
least for dendrimers G0eG2, it will be higher than that of the
solvent. Likewise, it should become higher with the degree of
branching. However, our results also suggest that from a certain
degree of branching on, aggregations of dendrimers occur in
THF. This should lead to a decrease of the fluorescent emission,
as it can be seen by inspection of the quantum yields reported
in Table 2.
Emission spectra of higher dendrimers were found to evolve
over time, which also suggests aggregation phenomena. Fig. 6
shows the spectra of solutions of different concentrations of
3
e-G3 48 h after their preparation, showing that these spectra
and the corresponding aggregates become stable after sufficient
time. These data are consistent with the structural results
obtained from the MD simulations: the lower asphericity
shown by G3 is, at least in part, responsible for these aggrega-
tion phenomena and, into a higher extension, those associated
with G4.
Fig. 4. Evolution of the asphericity of dendrimers 3bef (G0eG4) along the production
time of MD simulations.
Fig. 6. Emission spectra of 3e-G3 at five different concentrations and after 48 h of their
preparation.
From the data collected in Tables 1 and 2, the evolution of the
quantum yields along the G1eG4 series can be quantified. We have
Fig. 5. Absorption spectra of compounds 3aef in THF.
found a linear correlation between the hR
g
i=ha
s
i ratio and the
quantum yield associated with the fluorescent emission with
ꢄ
a wavelength of 280 nm, namely that associated with
p
/
p
Table 2
transitions involving the Fr eꢁ chet dendrons of G1eG4. This corre-
lation is given by the following expression:
Photophysical data of apigenin derivatives 3aef
Dendrimer Va (315 nm) Va (280 nm)
s
(ns)b
k
ꢂ10ꢁ9 (s )c
ꢁ1
k
nrꢂ10ꢁ9 (s )d
ꢁ1
r
ꢂ
ꢃ
3
3
3
3
3
3
a
0.0017
0.0053
0.0095
0.0112
0.0033
0.0077
0.0018
0.0045
0.0055
0.0039
0.0021
0.0017
0.88
0.82
0.86
0.86
1.03
0.75
0.001924
0.006498
0.011017
0.012984
0.003219
0.010216
1.129810
1.219443
1.148672
1.146302
0.972390
1.316572
R
ha
g
s
F
¼ KG
(8)
b-G0
c-G1
d-G2
e-G3
f-G4
i
ꢂ
with K
G
¼0.014 A (Fig. 7). Our attempts to find a similar correlation
associated with the excitation of the apigenin fluorophore met
with no success. These results suggest that the combined hR i=ha
g
s
i
a
Quantum yields measured in THF at different excitation wavelengths using
parameter can be useful to design dendrimers with better pho-
tophysical properties by increasing the generation number (with
larger radii of gyration) while keeping asphericities as low as
possible. Note however that the relation in Eq. 8 is found to hold
for dendrimers with shapes studied in this work, which are
characterized by relatively large value of asphericity. Further
studies are needed in order to elucidate the behavior of den-
quinine sulfate as standard (See Experimental section).
b
Time-domain excited-state lifetime.
Radiative constants.
Non-radiative constants, measured in THF.
c
d
2
k
r
¼ nf k
(7)
r0
where n is the refractive index of the host medium; f is a func-
tion of n, and depends on the geometry of the local environ-
ment, and kr0 is the radiative decay constant in vacuum. In the
drimers with smaller value of ha
s
i and, in particular, for perfectly
spherical molecules ðha
s
iÞ because then the right hand side of Eq.
8 diverges.

10366
P. Vin ꢀs et al. / Tetrahedron 69 (2013) 10361e10368
4.2. General experimental methods
Reagents and solvents were purchased from commercial sup-
pliers and used without further purification. Fr eꢁ chet-type dendrons
of generation number one to four 2cef were prepared according to
3
2
procedure reported by Fr eꢁ chet. NaH was used in form of 60% (w/
w) dispersion in mineral oil. Column chromatographies were car-
ried out with silica gel 60 (0.040e0.063 mm). All melting points are
3
uncorrected. NMR spectra were recorded in CDCl and using fre-
quencies as indicated bellow. Mass spectra were measured using
electrospray ionization at positive mode. MALDI-TOF MS spectra
were measured using trihydroxyacetophenone in 60% acetonitrile
with 0.1% of TFA as matrix. MALDI-TOF spectrum of dendrimer 3f
was recorded in linear mode using sinapic acid in 60% acetonitrile
with 0.1% of TFA as matrix.
0
33
4
.2.1. 5,7,4 -Trimethoxyflavone,
3a. To a solution of apigenin
(50 mg, 0.19 mmol) in dry dimethylformamide (2 ml) was added
NaH (20 mg, 0.5 mmol), under cooling by ice bath. After stirring for
10 min under argon, methyl iodide (0.15 ml, 2.56 mmol) was added
dropwise. The reaction mixture was stirred for 20 h at room tem-
perature and then, it was diluted by dichloromethane, washed by
Fig. 7. Correlation between structural data of dendrimers G1eG4 and quantum yields
measured with an excitation wavelength of 280 nm.
3
. Conclusions
4
water, organic phases collected and dried (MgSO ), and evaporated
under reduced pressure. The residue was purified by flash chro-
In this paper we report the preparation and the main geo-
metrical and photophysical properties of different dendrimers
possessing an apigenin fluorophore at the core and different
Fr eꢁ chet dendrons. The coupling between the core and the dendrons
has been carried out via Williamson reactions between the three
phenolic groups of apigenin and the corresponding Fr eꢁ chet bro-
matography on silica gel (CH Cl /MeOH) and by precipitation by
2
2
hexanes from ethyl acetate to yield title product as yellow solid
ꢁ
1 1
(40%, 30 mg): IR 1640, 1601, 1573, 1511 cm ; H NMR (300 MHz,
ꢀ
CDCl , 25 C)
3
d
¼7.81 (d, J¼8.5 Hz, 2H), 6.98 (d, J¼8.5 Hz, 2H), 6.59 (s,
1H), 6.54 (d, J¼1.8 Hz, 1H), 6.35 (d, J¼2.3 Hz, 1H), 3.94 (s, 3H), 3.90
1
3
ꢀ
mides. A transition from nearly spherical to highly aspherical
dendrimers has been detected on going from the first three gen-
erations to G3 and, specially, G4. It is found that for lower genera-
tion dendrimers the quantum yields of the fluorescent emissions
increase with the generation number or the radius of gyration as it
could be expected for more isolated fluorophores. This maximum
quantum yield is achieved for G2. However, there is a significant
decay in the quantum yields of G3 and G4, which parallels an in-
crease in asphericity for these latter dendrimers. These data, to-
gether with the evolution of the emission spectra, indicate that
aggregation phenomena are relevant in G3 and G4, thus resulting in
more efficient nonradiative decays and therefore in lower quantum
yields. These results suggest that, in order to improve the quantum
yields of fluorescent emission of dendrimers highly spheric low
associative dendrimers should be designed.
(s, 3H), 3.87 (s, 3H) ppm; C NMR (126 MHz, CDCl , 25 C)
3
d
¼177.60, 163.88, 162.02, 160.89, 160.65, 159.82, 127.58 (2C), 123.85,
114.33 (2C), 109.23, 107.69, 96.06, 92.81, 56.40, 55.71, 55.45 ppm.
MS (ESI) m/z: found 312.9 [MþH] ; calcd 313.1.
þ
0
34
4.2.2. 4 ,5,7-Trisbenzyloxyflavone, 3b-G0. To a solution of apige-
nin (40 mg, 0.15 mmol) in dry dimethylformamide (2 ml) was
added NaH (20 mg, 0.5 mmol), under cooling by ice bath. After
stirring for 10 min under argon, benzyl bromide (0.2 ml, 1.69 mmol)
was added dropwise. The reaction mixture was stirred for 20 h at
room temperature and then, it was diluted by ethyl acetate, washed
by water, organic phases collected and dried (MgSO ), and evapo-
4
rated under reduced pressure. The residue was purified by flash
chromatography on silica gel (gradient of ethyl acetate in toluene/
hexane (60/40 v/v)) and precipitated by hexanes from ethyl acetate
to yield title product (25%, 21 mg) as slightly yellow solid: IR 1638,
ꢁ
1
1
ꢀ
4
4
. Experimental section
1601, 1509, 1163 cm ; H NMR (300 MHz, CDCl
3
, 25 C)
d
¼7.82 (d,
J¼8.5 Hz, 2H), 7.62 (d, J¼7.6 Hz, 2H), 7.28e7.49 (m, 13H), 7.07 (d,
.1. Computational methods
J¼8.5 Hz, 2H), 6.64 (d, J¼2.1 Hz, 1H), 6.58 (s, 1H), 6.49 (d, J¼1.8 Hz,
1
3
1
CDCl
H), 5.23 (s, 2H), 5.14 (s, 2H), 5.11 (s, 2H) ppm; C NMR (126 MHz,
ꢀ
All the computational studies reported in this paper were based
3
, 25 C)
d
¼177.32,162.81,161.17, 160.65, 160.54, 159.70,136.44,
2
6
upon molecular mechanics
(
(MM) and molecular dynamics
136.29, 135.73, 128.75, 128.74, 128.69, 128.55, 128.41, 128.21, 127.64,
127.59, 127.45, 126.58, 124.13, 115.23, 109.87, 107.82, 98.37, 94.28,
27
MD). In both, the MM3 method developed by Allinger et al. as
implement in the MacroModel package was used. All MD simu-
2
8
þ
70.77, 70.47, 70.17 ppm. MS (ESI) m/z: found 541.5 [MþH ];
2
9
lations were performed with SHAKE to constrain the CeH bonds.
The temperature was set up to 298 K. The system was equilibrated
for 1 ns with time steps of 1 fs. This equilibration time is 10 times
longer than the expected value for the relaxation time of den-
drimers30 of this size. The production run was started from this
point and lasted another nanosecond with time steps of 1 fs. In all
cases, we observed that during the production period, the energy
and temperature of the whole system were equilibrated. During the
production run, the coordinates were saved each picosecond,
which implies a total of 1000 structures. These structures were
used to calculate the averages of the properties specified below. To
calculate these properties, programs based on the DYNAMO li-
calcd 541.6.
4.2.3. Dendrimer 3c-G1. To
a solution of apigenin (19 mg,
0.070 mmol) in dry dimethylformamide (2 ml) was added NaH
(15 mg, 0.38 mmol), under cooling by ice bath. The mixture was
stirred for 10 min under argon and then, 2c (150 mg, 0.39 mmol)
was added. The reaction mixture was stirred for 20 h at room
temperature and then it was diluted by dichloromethane and
4
washed by water. Organic phases were collected, dried (MgSO ),
and evaporated under reduced pressure. The residue was purified
by flash chromatography on silica gel (gradient of ethyl acetate in
toluene/hexane (60/40 v/v)) and precipitated two times by pentane
from ethyl acetate to yield corresponding product (25%, 21 mg) as
31
brary were written.

P. Vin ꢀs et al. / Tetrahedron 69 (2013) 10361e10368
10367
a colorless solid: mp 113e114 C, IR 1640, 1592, 1145, 1049 cmꢁ1; 1H
ꢀ
flash chromatography on silica gel (gradient of diethyl ether in
toluene/hexane (75/25 v/v)) and precipitated by pentane from THF
to yield title product (15%, 9 mg) as a colorless solid glass: IR: 1641
ꢀ
NMR (500 MHz, CDCl
3
, 25 C)
d
¼7.81 (d, J¼8.8 Hz, 2H), 7.28e7.47
(
(
(
m, 30H), 7.04 (d, J¼9.1 Hz, 2H), 6.93 (d, J¼2.3 Hz, 2H), 6.65e6.71
ꢁ
1 1
m, 4H), 6.62 (s, 1H), 6.52e6.62 (m, 4H), 6.45 (d, J¼2.1 Hz, 1H), 5.18
(small intensity), 1590, 1142, 1040 cm ; H NMR (600 MHz, CDCl
3
,
1
3
ꢀ
s, 2H), 5.08 (s, 6H), 5.04 (s, 10H) ppm; C NMR (126 MHz, CDCl
3
,
25 C)
d
¼7.63 (d, J¼8.8 Hz, 2H), 7.19e7.37 (m, 240H), 6.88 (d,
ꢀ
2
5
C)
d
¼177.24, 162.63, 161.04, 160.56, 160.28, 160.25, 160.21,
J¼8.8 Hz, 2H), 6.80 (m, 2H), 6.70 (br s, 2H), 6.54e6.66 (m, 78H),
1
1
1
59.64, 159.53, 139.01, 138.75, 138.13, 136.98, 136.69, 136.63, 128.59,
28.47, 128.05, 128.03, 127.84, 127.67, 127.63, 127.51, 127.50, 124.19,
15.25, 109.90, 107.84, 106.41, 106.32, 105.31, 101.81, 101.68, 101.61,
6.43e6.54 (m, 53H), 6.33 (d, J¼8.8 Hz, 1H), 4.77e4.98 (m, 186H)
13
ꢀ
ppm; C NMR (200 MHz, CDCl
3
, 25 C)
d¼160.13, 160.11, 160.09,
160.07, 160.05, 160.02, 160.00, 159.94, 139.24, 139.19, 139.17, 136.76,
136.74, 136.73, 128.53, 128.51, 128.50, 127.94, 127.91, 127.89, 127.51,
106.39, 106.35, 106.33, 101.56, 101.54, 101.53, 101.49, 101.47, 70.02,
70.00, 69.96, 69.91, 69.89, 69.88, 69.84 ppm. MS (MALDI-TOF) m/z
found 10.121 [MþH]þ calcd 10.092.
9
8.32, 94.37, 70.60, 70.26, 70.17, 70.15, 70.10, 70.02 ppm. MS (ESI)
þ
m/z found 1176.8 [MþH ], calcd 1176.44; Anal. Calcd for C78
H
64
O
11
:
C, 79.57, H, 5.48. Found: C, 79.78, H, 5.64.
4
.2.4. Dendrimer 3d-G2. Apigenin (7.4 mg, 0.027 mmol) was dis-
solved in mixture of DMF and toluene (2 ml, 1/1 v/v). Then, K
2
CO
3
4
.3. Spectroscopic measurements
(
75 mg, 0.54 mmol),18-crown-6 (5 mg, 0.02 mmol) and 2d (154 mg,
ꢀ
0
.19 mmol) were added. The reaction mixture was stirred at 80 C
UV/vis absorption and photoluminescence measurements were
for 20 h. Then, it was diluted by dichloromethane and washed by
water. Organic phases were collected, dried (MgSO ), and evapo-
carried out on an absorption spectrometer (Shimadzu UV-2401-PC)
and fluorescence spectrophotometer (FLUOROMAX-P, Jobin
Yvon), respectively, at ambient conditions, using standard 10 mm
cells and THF as solvent.
4
a
rated under reduced pressure. The residue was purified by flash
chromatography on silica gel (gradient of diethyl ether in toluene/
hexane (75/25 v/v)) and precipitated by pentane from THF to yield
title product (40%, 28 mg) as a colorless solid glass: IR 1641, 1592,
x
Fluorescence quantum yields of each compound (F ) were de-
3
5
ꢁ
1
1
ꢀ
termined by the method described by Williams et al., using qui-
nine sulfate (in 0.1 M H SO ) as standard, which has a known
ST¼0.54), according to
1
145, 1044 cm
;
H NMR (500 MHz, CDCl
3
, 25 C)
d
¼7.73 (d,
2
4
J¼9.1 Hz, 2H), 7.27e7.47 (m, 60H), 7.00 (d, J¼9.1 Hz, 2H), 6.93 (d,
fluorescence quantum yield (
F
J¼2.1 Hz, 2H), 6.62e6.74 (m, 15H), 6.43e6.61 (m, 12H), 6.47 (d,
13
J¼2 Hz, 1H), 5.15 (s, 2H), 4.90e5.07 (m, 40H) ppm; C NMR
2
GradX
h
ꢀ
X
(
1
1
1
1
7
[
2
126 MHz, CDCl
3
, 25 C)
d
¼177.18, 162.65, 161.01, 160.54, 160.18,
f
¼
f
X
STGrad
2
ST
h
ST
60.14, 160.08, 159.63, 159.52, 143.15, 139.47, 139.15, 139.07, 139.00,
38.74, 138.11, 136.85, 136.73, 136.72, 128.57, 128.51, 128.00, 127.99,
27.90, 127.61, 127.54, 127.51, 127.29, 124.14, 115.18, 109.86, 107.82,
06.47, 106.41, 106.39, 105.28, 101.83, 101.70, 101.59, 101.56, 70.12,
0.07, 70.01, 70.00 ppm. MS (MALDI-TOF) m/z found 2448.5
where Grad is the slope from the plot of integrated fluorescence
intensity versus absorbance at the same excitation wavelength, and
h
stands for the refractive index of the solvent (1.3332 for sulfuric
acid solution of quinine, and 1.407 for THF).
þ
þ
MþH] , calcd 2449.9; found 2472.4 [MþNa] , calcd 2471.9; found
Absorbances of five concentrations of the standard (in
þ
488.5 [MþK] , calcd 2487.9; Anal. Calcd for C162
136
H O23: C, 79.39,
H
2
SO
tions have been chosen, which provide absorbances lower that
.1 at and above the excitation wavelength, to avoid inner filter
effects.
Wavelength at which the standard and samples absorb (either
15 nm or 280 nm) was chosen as excitation wavelength. Emission
4
) and each sample (in THF) were measured. Concentra-
H, 5.59. Found: C, 79.22, H, 5.47.
0
4
.2.5. Dendrimer 3e-G3. Apigenin (6.1 mg, 0.023 mmol) was dis-
solved in mixture of DMF and toluene (2 ml, 1/1 v/v). Then, K CO
30 mg, 0.22 mmol), 18-crown-6 (5 mg, 0.019 mmol) and 2e
2
3
(
(
3
150 mg, 0.091 mmol) were added. The reaction mixture was
spectra were recorded on the same solutions under constant con-
ditions, 48 h after stabilization (slit width: 10 nm).
ORIGIN 8.0 was used for integrating the area of emission spectra.
Residual fluorescence of solvents has been corrected. Five-point
Area-Absorbance regressions were plotted for the standard and
each compound.
Time-domain excited-state lifetimes were measured using an
IBH 5000F coaxial nanosecond flashlamp, in a FL3-11 Fluorolog
equipment.
ꢀ
stirred at 80 C for 20 h. Then, it was diluted by dichloromethane
and washed by water. Organic phases were collected, dried
(
4
MgSO ), and evaporated under reduced pressure. The residue
was purified by flash chromatography on silica gel (gradient of
diethyl ether in toluene/hexane (75/25 v/v)) and precipitated two
times by pentane from THF to yield title product (25%, 34 mg) as
ꢁ
1
1
colorless solid glass: IR: 1641, 1592, 1147, 1044 cm
; H NMR
ꢀ
(
500 MHz, CDCl
3
, 25 C)
d
¼7.67 (d, J¼8.8 Hz, 2H), 7.18e7.47 (m,
1
(
2
20H), 6.93 (m, 4H), 6.45e6.72 (m, 63H), 6.41 (br s., 1H), 5.09
13
br s., 2H), 4.78e5.04 (m, 88H) ppm; C NMR (126 MHz, CDCl
3
,
ꢀ
5
C)
d
¼160.14, 160.12, 160.08, 160.06, 160.04, 159.96, 139.48,
Acknowledgements
1
1
39.28, 139.17, 136.79, 136.74, 128.55, 128.54, 128.51, 127.96,
27.93, 127.91, 127.52, 127.51, 106.42, 106.36, 101.70, 101.63, 101.57,
01.54, 70.10, 70.06, 70.03, 69.96, 69.93 ppm. MS (MALDI-TOF) m/z
Financial support by the Spanish Ministry of Economy and
Competitiveness, with the participation of European Union
(MINECO, projects CTQ2010-16959/BQU, CTQ2012-35535 and
Consolider-Ingenio CSD2007-00006), from the University of the
Basque Country (UPV/EHU, UFI11/22 QOSYC), from the Basque
Government (GV/EJ, grant IT-324-07), from the Donostia In-
ternational Physics Center (DIPC), from the Ministry of Education,
Youth and Sports of the Czech Republic (grant MSM6046137305),
and Czech Science Foundation (projects 304/10/1951, P503/11/
0616) is acknowledged. M.d.B. thanks the CSIC for the JAE-Pre
contract funding for her PhD. The authors also thank the SGI/IZO-
SGIker UPV/EHU and the DIPC for generous allocation of compu-
tational resources.
1
þ
þ
found 4996.4 [MþH] , calcd 4995.9; found 5019.5 [MþNa] ,
calcd 5018.0. Anal. Calcd for C330
C, 79.46, H, 5.78.
280
H O47: C, 79.31, H, 5.65. Found:
4
.2.6. Dendrimer 3f-G4. Apigenin (1.6 mg, 0.0059 mmol) was dis-
solved in mixture of DMF and toluene (1 ml, 1/1 v/v). Then, K CO
15 mg, 0.11 mmol), 18-crown-6 (3 mg, 0.011 mmol) and 2f (70 mg,
2
3
(
0
8
.0208 mmol) were added. The reaction mixture was stirred at
0 C for 20 h. Then, it was diluted by dichloromethane and washed
ꢀ
by water. Organic phases were collected, dried (MgSO
4
), and
evaporated under reduced pressure. The residue was purified by

10368
P. Vin ꢀs et al. / Tetrahedron 69 (2013) 10361e10368
Supplementary data
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Jian, W.; Ju, M.; AnJun, A.; JingZhi, S.; Zhong, T. B. Sci. China Chem. 2010, 53,
409.
4. (a) Grabchev, I.; Staneva, D.; Betcheva, R. Curr. Med. Chem. 2012, 19, 4976; (b)
2
Scanned photocopies of H and 13C NMR data and MD input files
1
1
(
Maestro-Macro Model format) were supported. Supplementary
Albertazzi, L.; Brondi, M.; Pavan, G. M.; Sato, S. S.; Signore, G.; Storti, B.; Ratto,
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5. Adronov, A.; Fr eꢁ chet, J. M. J. Chem. Commun. 2000, 1701.
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1