Anthracene - BODIPY cassettes: Syntheses and energy transfer

Article abstract of DOI:10.1002/chem.200304754

Compounds based on the 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) framework are excellent fluorescent markers. When BODIPY dyes of this type are conjugated to functionalities that absorb at relatively short wave-lengths, those functionalities can, in some molecules, transmit the absorbed energy to the BODIPY which then fluoresces. In such cases the BODIPY fragment acts as an acceptor while the other group serves as a donor. Energy transfer efficiencies in such donor-acceptor cassette systems must vary with the relative orientation of the two components, and with the structure of the linkers that attach them. This study was designed to probe these issues for the special case in which the linkers between the donor and acceptor fragments are conjugated. To do this, the cassettes 3-10 were prepared. Electrochemical studies were performed to provide insight into the degree of donor -acceptor conjugation in these systems. X-ray Crystallographic studies on single crystals of compounds 7 and 9 revealed the favored conformations of the donor and acceptor fragments in the solid state. Absorption, fluorescence, and time-resolved fluorescence spectra of the compounds were recorded, and quantum yields for the cassettes excited at the donor λ_max were measured. Fluorescence steady-state anisotropy data were determined for cassettes 3 and 9 to provide information about the mutual direction of the transition dipole moments.

Full text of DOI:10.1002/chem.200304754

FULL PAPER
Anthracene BODIPY Cassettes: Syntheses and Energy Transfer
Chi-Wai Wan,[a] Armin Burghart,[a] Jiong Chen,[a] Fredrik Bergstrˆm,[b]
Lennart B.-ä. Johansson,[b] Matthew F. Wolford,[c] Taeg GyumKim, [c] Michael R. Topp,[c]
Robin M. Hochstrasser,[c] and Kevin Burgess*[a]
Abstract: Compounds based on the 4,4-
difluoro-1,3,5,7-tetramethyl-4-bora-
the relative orientation of the two com-
ponents, and with the structure of the
linkers that attach them. This study was
designed to probe these issues for the
special case in which the linkers between
the donor and acceptor fragments are
conjugated. To do this, the cassettes 3
10 were prepared. Electrochemical stud-
ies were performed to provide insight
into the degree of donor acceptor con-
jugation in these systems. X-ray Crystal-
lographic studies on single crystals of
compounds 7 and 9 revealed the favored
conformations of the donor and accept-
or fragments in the solid state. Absorp-
tion, fluorescence, and time-resolved
fluorescence spectra of the compounds
were recorded, and quantum yields for
the cassettes excited at the donor lmax
were measured. Fluorescence steady-
state anisotropy data were determined
for cassettes 3 and 9 to provide informa-
tion about the mutual direction of the
transition dipole moments.
3a,4a-diaza-s-indacene
(BODIPY)
framework are excellent fluorescent
markers. When BODIPY dyes of this
type are conjugated to functionalities
that absorb at relatively short wave-
lengths, those functionalities can, in
some molecules, transmit the absorbed
energy to the BODIPY which then
fluoresces. In such cases the BODIPY
fragment acts as an acceptor while the
other group serves as a donor. Energy
transfer efficiencies in such donor ac-
ceptor cassette systems must vary with
Keywords: dyes/pigments ¥ energy
transfer
¥
fluorescent probes
¥
UV/Vis spectroscopy
are usually connected via non-conjugated linker systems,
hence the predominant energy transfer mechanism is
through-space (usually Fˆrster mechanism; Figure 1a).[4]
A
Introduction
Fluorescent labels that emit light at wavelengths distant from
that of the source used to excite them have many applications
in biotechnology. For single dye systems the difference
between the S0 absorption and the S1 emission wavelengths
is the Stokes× shift.[1] When the Stokes× shift of a single dye is
insufficient for a particular application, strategies that exploit
through-space energy transfer between two dyes are fre-
quently used.[2, 3] An absorbing molecular fragment (a donor)
is arranged to be proximal to an emitting fragment (an
acceptor); the donor is then excited at wavelengths close to its
lmax, and the fluorescence of the acceptor is observed. For
biotechnological applications, the donor and acceptor units
[a] Prof. K. Burgess, Dr. C.-W. Wan, Dr. A. Burghart, Dr. J. Chen
Texas A & M University, Chemistry Department
P.O. Box 30012, College Station, Texas 77842 (USA)
E-mail: burgess@tamu.edu
Figure 1. Through-space and through-bond energy transfer.
[b] F. Bergstrˆm, Prof. L. B.-ä. Johansson
Department of Chemistry: Biophysical Chemistry
Umea University, 90187 Umea (Sweden)
requirement for Fˆrster energy transfer is that the emission
spectrum of the donor must overlap with the absorption
spectrum of the acceptor. That requirement places an upper
limit on the range of fluorescence wavelengths from two-dye
cassettes irradiated using a single excitation wavelength.
[c] Dr. M. F. Wolford, Dr. T. G. Kim, Prof. M. R. Topp,
Prof. R. M. Hochstrasser
Department of Chemistry, University of Pennsylvania
Philadelphia, PA 19104 (USA)
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In contrast to through-space energy transfer cassettes,
donor and acceptor units connected by conjugated linker
fragments may transfer energy via several pathways. These
include through-space energy transfer, but also other path-
ways that may be collectively referred to as through-bond
energy transfer mechanisms (Figure 1b), which include Dex-
ter energy transfer and others.[5] The literature on molecules
that exhibit through-bond energy transfer may be divided into
that which deals with oligomeric conjugated materials,[6] and
other contributions featuring models for biological systems
11]
(e.g. porphyrin-containing systems).[7
Energy transfer
through bonds does not require the emission spectrum of
the donor to overlap with the lowest energy excited states of
the acceptor. Of course, excitation of any molecule above the
S1 state will lead to S1 to S0 fluorescence, but the higher energy
absorption cross sections are often low and they vary
considerably between molecules. In conjugated donor ac-
ceptor cassettes, however, a given donor part can be
incorporated to absorb strongly at a given wavelength, and
the acceptors can be varied to emit at different wavelengths
that are distant from that of the source used to excite them.
We are interested in designing fluorescent dyes for biotech-
nology that exploit this phenomenon.[12, 13]
For conjugated cassettes of the type shown in Figure 1b, it
may not be possible to ascertain how much energy transfer
proceeds via through-bond mechanisms relative to the
through-space pathways. This is very likely to be the case
when the donor and acceptor fragments are relatively close
together. Nevertheless, the overall rates of energy transfer can
be measured; they in turn will be influenced by the
structure of the donor, acceptor, and linker fragments,
and the orientation of the donor linker acceptor connec-
tivity.
The work described herein focuses on the last of the issues
outlined above: overall rates of energy transfer in donor
acceptor cassettes as a function of the linker structure and
orientation. To do this, compounds 1 and 2 were used as
mimics of the donor and acceptor fragments in cassettes 3
10; all of which feature dyes of the BODIPY type as
acceptors.[7, 14, 15] The structures of these cassettes were
determined in part by synthetic accessibility. For instance, it
is much easier to prepare molecules with aryl substituents at
C8 of the BODIPY framework, than with alkyne groups there,
but it is relatively straightforward to put either type of
substituent at C4. Another consideration in the design of
cassettes 3 10 was future applications of these materials.
Thus, in molecules 4 6 a bromine is included to allow these
cassettes to be attached to other molecules via organometallic
coupling reactions. Spectroscopically, the bromine substituent
is unlikely to have any appreciable effect, and comparisons
between cassettes 3 and 4 allow this assumption to be
explored in one representative case.
these orientation/connection issues. Specifically, compounds
3
6, each having an anthracene donor connected to the side
of a BODIPY fragment via different linkers, and the ™axially-
linked∫ systems 7 10 were compared. Absorption and
fluorescence emission data collected for these compounds
were interfaced with results from X-ray crystallographic,
electrochemical, and time resolved fluorescence experiments
to begin to understand how the structure and electronic
properties of these molecules correlate with their spectro-
scopic characteristics.
Efficiencies of energy transfer in a conjugated donor ac-
ceptor cassettes must depend on the orientation of the donor
and acceptor fragments (a key factor for Fˆrster transfer),[16]
and the nature of the p-system that connects them (partic-
ularly relevant to through-bond transfer mechanisms). The
work that is described here features several derivatives of
BODIPY 2 which were investigated to explore some of
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BODIPY 13. Suzuki coupling[18] of 13 to the 9-anthracenyl
boronic acid gave the desired cassette 7.
Results and Discussion
Syntheses of the cassettes: Anthracene derivative 1 was
prepared via the Sonogashira coupling shown in Scheme 1;
this was the key reaction used for most of the syntheses of the
Cassette 8 was also prepared via a route that involved the
iodo-BODIPY 13 (Scheme 2b). Successive Sonogashira cou-
plings[19, 20] of this with trimethylsilylacteylene and with
Scheme 1. Preparation of compounds 1 and 3.
Scheme 2b. Preparation of cassette 8.
cassettes described here. Thus cassette 3 was prepared via
similar coupling of the alkyne-functionalized BODIPY.[13]
Cassettes 5, 6, and 7, were prepared in the same way, but
beginning with starting materials for which syntheses have
been reported by this group before.[12, 13, 17]
Cassette 7 was prepared by the sequence in Scheme 2a
beginning with commercially available 3,5-dimethylpyrrole-2-
carbaldehyde. Iodination, under basic conditions, gave the
4-iodo derivative 12, and this was then condensed with 2,4-
dimethylpyrrole and treated with boron trifluoride to give the
9-bromoanthracene gave the desired product. Possibly the
most difficult step to develop in this synthesis was the removal
of the trimethylsilyl group, because the BODIPY frameworks
are not stable to prolonged treatment with fluoride ion. This
transformation was accomplished by careful control of the
reaction temperature and time.
Scheme 2c outlines the synthesis devised for dye 9. This
features a Suzuki, silyl deprotection, Sonogashira sequence.
Similarly, cassette 10 was prepared by a Sonogashira, silyl-
deprotection, Sonogashira sequence as shown in Scheme 2d
involving compound 19.[13]
Cyclic voltammetry: Redox potential data can be related the
electronic conjugation of the chromophores within the
cassettes, hence the cassettes 3 and 7 10 were studied by
cyclic voltammetry. The single chromophore models 1 and 2
were also studied to provide references for the various
molecular components. Table 1 summarizes the data obtained
(throughout these experiments, chloroform solvent was used
and the scan rate was 100 mVsÀ1).
All compounds exhibited irreversible redox waves, with the
exception of the reduction waves of 2 and 7 for which the
waves are partially reversible. For cassettes 3, 7, 9, 10 it was
shown that two electrons could be removed from (and added
to) the system. Those electron transfer steps are most likely
related to redox events on the chromophore parts of the
molecules. The oxidation potentials of the model dyes 1 and 2
were almost the same (0.85 and 0.88 V, respectively), so that in
the cassettes the oxidation steps corresponding to the
anthracene and to the BODIPY, respectively, coalesced.
Scheme 2a. Preparation of cassette 7.
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Table 1. Redox potentials of compounds 1 3 and 7 10 in CH2Cl2.
(Ferrocene was used as internal standard. Scan rate 100 mVsÀ1.)
Compound
Oxidation
potentials
Epa [V]
Reduction
potentials
Epc [V]
1
2
‡ 0.88
‡ 0.85
--[a]
À 1.75
(E1/2 ˆ À1.66 V, ipa/ipc ˆ 0.20)
À 1.69
3
7
‡ 0.85 (2 e)
‡ 0.80 (2 e)
2nd wave: À 2.21
À 1.71
(E1/2 ˆ À1.65 V, ipa/ipc ˆ 0.22)
À 1.54
8
9
‡ 0.58
‡ 0.85 (2 e)
À 1.66
2nd wave: À 2.26
À 1.57
10
‡ 0.83 (2 e)
2nd wave: À 2.20
[a] Redox wave not accessible under conditions employed.
1, the redox wave is beyond the accessible range (< À2.50 V).
This anthracene reduction is also not visible for 7, only the
BODIPY reduction at À1.71 V was observed. This potential
is the most negative of those measured for 3, 7 10 hence this
cassette is the one that showed the least conjugation within
the series. Compounds 3 and 9, which possess the same linker,
display some small degree of conjugation because their
reduction potentials corresponding to the BODIPY fragment
are less negative (À1.66 and À1.69 V, respectively) than for 2
and 7. Moreover, the reduction corresponding to the anthra-
cene part of 3 and 9 occurred in the observable range
(ꢀ À 2.2 V); this also indicates that it was more conjugated in
these molecules than in compound 2. These trends were
observed to continue for compound 10, indicating a higher
degree of conjugation. Interestingly, the compound 8 was
shown to have a relatively low oxidation potential, a modestly
negative reduction potential, and no second oxidation or
reduction. These electrochemical characteristics are indica-
tive of a well-conjugated system.
Scheme 2c. Preparation of cassette 9.
The data described above indicate increasing conjugation in
the series 7 < 9 and 3 < 10 < 8. This observation is chemically
reasonable. Cassette 7, having a direct connection between the
BODIPY and 9-anthracenyl components is likely to adopt a
twisted conformation in which two chromophores are elec-
tronically independent and therefore less conjugated. The
BODIPY-to-benzene connection in cassettes 3 and 9 is also
likely to impede conjugation for similar reasons. However, the
1,4-bis(ethynyl)phenyl linker in 10 allows good electronic
communication between the dyes, and when the phenyl linker
is removed, as in 8, there is almost no barrier to p-conjugation.
Single crystal X-ray studies: Figure 2 shows representations of
compounds 7 and 9 generated from X-ray crystallographic
coordinates. The bond lengths and angles are unexceptional
for these systems, except that the structures do show the
degree of twist between the BODIPY fragment and the
peripheral aryl substituent (9-anthracenyl for 9 and a sub-
stituted benzene ring for 3). Inspections of these models
shows that the anthracenyl fragment of 7 clearly encounters
Scheme 2d. Preparation of cassette 10.
Conversely, reduction gave two distinct waves, at least for
compounds 3, 9, and 10 and the reduction potential data was
useful for probing the p-conjugation in the systems. For model
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K. Burgess et al.
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Figure 2. Chem3D diagrams from single crystal X-ray diffraction studies
of a) cassette 7, and b) cassette 9.
more steric interactions with the BODPY framework than the
1,4-disubstituted phenyl substituent of 9. Thus the twist
between the donor and acceptor planes is more rigidly
enforced in 7 than in 9.
Figure 3. Normalized absorption spectra of a) cassettes 3 6 and b) 7 10
in CHCl3.
Spectroscopic properties: The absorption spectra of the
cassettes in ethanol and chloroform display peaks due to
strong and weak S0 ! S1 transitions with maxima at 500
645 nm and 365 445 nm, for the BODIPY group and
anthracene groups, respectively. A strong higher-energy
transition of anthracene was observed at about 265 nm. For
the model compound 20, absorptions are observed at about
300 nm that can be ascribed to the S0 ! S1 transition of the
conjugated phenylethynyl system; similar absorptions for the
cassettes are therefore likely to be due to the phenylethynyl
linker system (Figure 3). For most of these samples, the
absorption spectrum approximates that which would be
expected from adding the components together.
The following experiment was performed to test whether
attaching the chromophores causes a weak perturbation of the
absorbance for the component parts. The molar absorption at
the BODIPY component of the cassette studied most in this
paper (see below), cassette 5, was measured as 40000mÀ1 cmÀ1
in CHCl3. The value measured from the absorption spectrum
of 5 in Figure 3 is 20000mÀ1 cmÀ1 for the absorption in the
anthracene region. These values are perfectly consistent with
that for anthracene derivatives including, for instance, com-
pound 20 (30000mÀ1 cmÀ1), which is more highly conjugated
than the residue used in the cassettes. These data show that
attaching the anthracene compenent to the acceptor system
causes minimal perturbation of the absorptivity of that
component.
Compound 8 does not have a sterically enforced twist to
prevent the donor and acceptor coming into a planar
conformation. Consequently, the absorption spectrum for this
compound differs slightly from the others, but perhaps not as
much as might be expected. The absorption maximum for
cassette 8 is red-shifted by about 40 nm compared with 9, 10, 3
where the linker is a phenylethynyl group. These shifts agree
qualitatively with that between anthracene and 9,10-bis(phe-
nylethynyl)anthracene (BPEA) 20.[21]
Comparisons of the absorption spectra of the acceptor
components may use cassette 3 as a reference point since its
lmaxabs closely resembles that of the free BODIPY 2 (500 and
504 nm when dissolved in ethanol and chloroform, respec-
tively). For cassettes 7 and 9, in which the short edge of the
acceptor (i.e., C2) is directly bound to an aromatic group, the
lmaxabs values are red-shifted by about 20 nm compared with 3.
Cassettes 8 and 10, where the immediate linkage is to an
ethynyl group, have lmaxabs values red-shifted by about 40 nm
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compared with 3. Furthermore, the spectral band shape of the
cassettes 7 10 are broader than 3. All cassettes show a
modest solvent dependence with a few nanometers blue-shift
upon replacing the solvent ethanol with chloroform.
The fluorescence emission spectra of the cassettes show
maxima varying between 515 and 650 nm (Figure 4). The shift
between fluorescence and absorption maxima is between
tested). Based on lifetime measurements described below, we
estimate the donor fluorescence quantum yields in the
cassettes to be <5 Â 10À5.
The acceptor fluorescence time profiles of the cassettes
appear to be mono-exponential. For example, the acceptor
lifetimes of the cassettes in chloroform vary between 0.5 to
4.0 ns (Table 2), and in most cases are significantly longer than
when the compounds are dissolved in ethanol (0.13 to 3.2 ns).
The shortest lifetimes were measured found for cassette 8.
Table 2. Absorption and fluorescence spectroscopic properties of compounds 3, 4,
7, 8, 9, and 10 in ethanol and chloroform.
Cassette Solvent lfl (SA0 † D(S1) À A(S1) D(S2) À A(S1) F[c]
t[d] ET
[ns] Effic.
[%]
nm[a]
103 cmÀ1[b]
103 cmÀ1[b]
3
3
4
4
7
7
8
8
9
EtOH 514
5.6
5.5
6.0
5.9
11.1
10.3
8.8
8.8
7.5
7.4
7.7
7.6
19.0
18.4
19.3
18.8
23.3
22.1
22.5
22.3
20.9
20.2
21.5
20.8
0.16 Æ 0.04 1.1 > 95
0.39 Æ 0.05 2.1 > 95
0.57 Æ 0.03 4.0 > 95
0.67 Æ 0.03 4.0 > 95
0.05 Æ 0.01 1.0 > 95
0.25 Æ 0.02 2.7 > 95
0.01 Æ 0.01 0.3 > 95
0.02 Æ 0.01 0.5 > 95
0.28 Æ 0.03 1.7 > 95
0.75 Æ 0.06 3.7 > 95
0.22 Æ 0.02 1.3 > 95
0.42 Æ 0.03 2.2 > 95
CHCl3 517
EtOH 541
CHCl3 545
EtOH 615
CHCl3 594
EtOH 640
CHCl3 647
EtOH 568
CHCl3 569
EtOH 588
CHCl3 590
9
10
10
[a] The peak wavelength of fluorescence of the acceptor is denoted lfl (SA0 †.
[b] D(S1) À A(S1) denotes the wavenumber difference between the peak wave-
length of the first vibronic absorption band of the S0 ! S1 transition in the donor and
the emission maximum of the acceptor. D(S2) À A(S1) denotes the corresponding
quantity for the higher-energy transition of the donor, near 260 nm. [c] F is the
quantum yield of fluorescence. The wavelengths [nm] used to excite of the donor
(D) and acceptor (A) groups were as follows: 3, 420 (D) and 490 (A); 4, 420 (D) and
490 (A); 7 385 (D) and 490 (A); 8, 420 (D) and 490 nm (A); 9, 420 (D) and 490 (A);
and, 10, 420 (D) and 490 (A).
The fluorescence quantum yields (F) of compounds 3, 4,
and 7 10 in chloroform and ethanol were determined
(Table 2). In ethanol, F takes values between 0.01 and 0.57
and in chloroform between 0.02 and 0.75. The F values on
were the same whether the donor or the acceptor groups were
excited. The absence of a F(lex) dependence suggests nearly
complete energy transfer from the donor to the acceptor. The
quantum yields measured were always higher when chloro-
form was used as the solvent than ethanol. Cassette 9 exhibits
the highest quantum yield in CHCl3, and 8 exhibits the lowest
fluorescence quantum yield in EtOH.
The fluorescence excitation anisotropies of cassettes 9 and 3
provide information about the alignments of excitation and
emission transition dipole moments. The excitation anisotro-
py experiments with 9 in propane-1,2-diol at 262 K (Figure 5),
show the anisotropy is about 0.35 for the S0 ! S1 transition
(450 560 nm). The steady-state fluorescence anisotropy of
the compound as a cold glass corresponding to the anthracene
S0 ! S1 transition (400 nm) is about 0.3. The corresponding
limiting fluorescence anisotropies of BODIPY 2 and free
anthracene are 0.37[22] and 0.34,[23] respectively. This implies
that the overall transition dipoles of cassette 9 are very similar
to the polarization of its donor and acceptor constituents. For
Figure 4. Corrected normalized fluorescence emission spectra of a) com-
pounds 3 6 and b) 7 10 in CHCl3.
5500 11000 cmÀ1, when the cassettes are excited at the S0 ! S1
transition of the donor. The corresponding difference is
18400 23300 cmÀ1 when the donor is excited at the S0 ! S2
transition. The spectral band shapes of the cassettes are
similar to those of parent BODIPY 2, although somewhat
broader. Cassettes 9 and 3, which only differ in linker
attachment site on the BODIPY group, have very different
spectra. The spectrum of 3 much resembles that of BODIPY
2, while the spectrum of 9 is considerably shifted to the red
with a fluorescence maximum at 569 nm. This difference may
be due to the fact that substitution at the BODIPY C2 position
lowers the symmetry, and this perturbs the S0 and S1 states. In
unpublished studies, we have also found a similar red-shift for
N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-in-
dacene-2-yl)iodoacetamide, which is also substituted at C2.[22]
The fluorescence from the donors in all the cassettes was very
weak, indicating rapid energy transfer from donor to acceptor
(the solvents chloroform, ethanol and propane-1,2-diol were
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K. Burgess et al.
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is invariant to rotation about the linker axis because of the
parallel dipoles, so that the anisotropy is expected to be close
to 0.4.[24] The observed value (0.37) is consistent with this
reasoning. However, the chemical structure of 3 allows
internal rotational freedom of the donor and acceptor about
the linker -C6H4-CC- component. A random distribution of
orientations about the linker axis would give an expected
anisotropy value close to 0.1. The lower value observed
suggests that the molecular planes of anthracene and BOD-
IPY moieties preferentially adopt non-coplanar orientations
possibly in the range 50 608C.
The interpretation of the ultrafast spectroscopic measure-
ments required formulation of a generic energy level Scheme
for the cassettes. To do this, compounds 4 and 6 were selected
because these have very different BODIPY acceptor frag-
ments in the series 3 10, and within that set of compounds it
is the acceptor rather than the anthracene-based donor
fragments that show the most variation. In the UV spectra
of the cassettes (Figure 3) the anthracene component is
almost constant and the main differences are with respect to
substitution pattern and orientation. The lifetime of the
cassettes× lower-lying anthracene level was of primary interest
in this study. Figure 6 represents the energy level scheme,
Figure 5. Fluorescence excitation anisotropies and absorption spectra of
a) compound 3 and b) compound 9 in propane-1,2-diol at 262 K. The
fluorescence emissions were monitored at 570 and 550 nm, respectively.
cassette 3, the limiting fluorescence anisotropy is about 0.35 in
the spectral region arising from the BODIPY S0 ! S1 tran-
sition (450 560 nm), while it is about À0.15 for the region
originating from the anthracene S0 ! S1 transition (ꢀ400 nm).
The angle (d) between the absorption and emission transition
dipoles is related to the fluorescence steady-state anisotropy
(r) through the relationship:
Figure 6. Energy level diagram depicting the anthracene and BODIPY
energy levels and showing the origins of different UV emissions in ultrafast
two-photon experiments.
showing that synchronization of pulses at 415 and 830 nm
leads to high-energy emission, in principle reaching the high-
energy limit imposed by the sum of the photon energies
(ꢀ275 nm). Delaying the arrival of the 830 nm pulses allows
vibrational dephasing and other relaxation processes to occur,
so that the accessible region of UV fluorescence is signifi-
cantly reduced.
Cassette 5 was one of the first prepared, and more sample
was available than for some of the other cassettes, so this
compound was studied in more depth. The following dis-
cussion illustrates the approach used. Figure 7 shows how the
ultraviolet emission spectrum depended on the pulse interval
for 5 in chloroform. At t ˆ 0, when the pulses were synchron-
ized in the sample, emission was observed close to the short-
wavelength limit of 275 nm, as expected, in addition to a
stronger emission band having a Franck Condon maximum
near 320 nm. This emission band is consistent with observa-
tions made by excitation with two photons at 532 nm, and with
r0
rˆ (3cos2 d À 1)
2
where the limiting anisotropy, and r0 is theoretically 2³5. The
blue edge of the S0 ! S1 transition band of anthracene is short-
axis polarized.[24] It therefore follows from the anisotropy
values obtained for the S0 ! S1 transition of the BODIPY
group in compounds 9 and 3 that the S0 ! S1 transition dipole
of BODIPY is long-axis polarized. Consequently, energy
transfer in cassette 3 cannot be ascribed to point-dipole
interactions. The limiting anisotropy of 3 is about zero for the
260 nm absorption of anthracene, but it is significantly smaller
(more negative) for 9. For cassette 9 the transition dipole for
the 260 nm absorption of anthracene should be perpendicular
to the S1 ! S0 emission dipole of the BODIPY fragment, but
parallel for system 3. In cassette 9, the fluorescence anisotropy
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Cassettes for Fluorescence Energy Transfer
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depend on the state of relaxation of this complex molecule
was real; however, the same decay time against different
amounts of background signal was measured over the entire
range from 300 390 nm. This experiment detected no
significant variation in the apparent dynamics with the
sampling wavelength, and we are confident that the signal
represents a single relaxation time of the anthracene moiety.
Figure 8 also shows, for comparison, a similar decay trace
obtained for cassette 6 under the same conditions. That trace,
after deconvolution of the instrument response function,
showed a relaxation time of only 310 fs.
Table 3 shows the collected data from lifetime measure-
ments of the anthracene moiety in a series of BODIPY
compounds.[25] The data reveal some significant differences in
Figure 7. UV emission spectra of cassette 5 resulting from synchronized
(t ˆ 0) and separated (t ˆ 20 ps).
the existence of the abovementioned absorption maximum
near 300 nm. In fact, the entire emission spectrum from 275
380 nm is stronger at t ˆ 0 than when the pulses are separated.
When the 830 nm probe pulses were delayed by 20 ps, the
system is seen to have relaxed since the ultraviolet emission is
less intense and has shifted to longer wavelengths. This is
consistent with relaxation to the BODIPY, from which
excitation at 830 nm can generate fluorescence at >310 nm.
Therefore, an experiment involving 830 nm probing, with
monitoring at <300 nm, should reveal a decay consistent with
the energy transfer rate from the anthracene species followed
by a more constant signal attributable to vibrationally ™hot∫
lower levels. Time-correlated single-photon counting experi-
ments as well as optically gated fluorescence measurements
confirmed that the preliminary relaxation was complete in
50 ps, the only remaining dynamics being associated with
nanosecond-scale fluorescence decay.
Table 3. Ultrafast relaxation times of cassettes 3 10 in CHCl3 solution as
measured by pump-probe spectroscopy.
Compound
S1 Absorption
maximum [nm]
Energy relaxation
t [fs]
3
4
5
6
7
8
9
10
500
500
520
635
520
540
525
540
320
490
550
310
< 200
< 200
ꢀ 200
ꢀ 200
behavior of the cassettes. Thus, 3 6 tend to give stronger UV
emission signals, with measurable lifetimes attributable to the
anthracene component in the range 300 800 fs (including a
solvent effect measured for 5). The other cassettes tend to give
weaker, shorter-lived signals, some of which were apparently
decaying within the instrument response profile, which is
estimated to be ꢀ150 fs (fwhm).
Figure 8 shows time profile for 5, which was obtained by
scanning the pulse separation, while monitoring the UV
fluorescence signal at 300 nm. The data fit a decay time of
550 fs, which we associate with relaxation of the anthracene
moiety. The possibility that the UV emission spectrum could
Conclusion
In the series of compounds 3 10, two, 8 and 10, are not truly
cassettes, as defined here, since they are fully conjugated
systems without an internal twist to break that conjugation.
This is reflected in the slightly red-shifted absorption spectra,
which also show broader bands than the other compounds in
this study. Similarly, the electrochemical studies (Table 1)
showed that the degree of donor acceptor conjugation was
greatest for 8, then 10.
Compounds 3 7 and 9 are true cassettes. X-ray crystallo-
graphic studies on single crystals of compounds 7 and 9
revealed the favored conformations of the donor and acceptor
fragments, at least in the solid state (Figure 2). In 7, the
anthracene donor and the BODIPY acceptor are directly
attached and significant steric interactions prevent the donor
and acceptor fragments becoming planar. The corresponding
steric effects for compound 9 are evident, but not so severe.
Consistent with this, electrochemical studies of 3, 7, and 9
revealed that donor acceptor conjugation decreases in the
order 3 > 9 > 7. In studies of absorption and time-resolved
Figure 8. Time profiles resulting from detecting UV emission at 300 nm
(see Figure 7) and scanning the time separation of the 415 and 830 nm
ultrashort pulses. The relaxation of the anthracene component is caused by
energy transfer within the cassettes.
Chem. Eur. J. 2003, 9, 4430 4441
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4437

K. Burgess et al.
FULL PAPER
*
*
all cases where the donor was attached to the long axis of
the BODIPY acceptor, as in 7 and 9, exhibit significantly
faster energy transfer than if the donors are coupled to the
short axis as in 3; but
although the sequence of molecules 7 10 was chosen to
focus on the possible influence of molecular separation on
the transfer rate, the experiments so far employed had
insufficient time resolution (ꢀ150 fs) to make a useful
comparison.
fluorescence spectra, cassette 8 was shown to have different
spectroscopic properties from the other compounds in the
series; unlike the other cassettes studied, it did not fluoresce
significantly when excited at the donor lmax. Quantum yields
for the cassettes excited at the donor lmax increased in the
order 8 < 7 < 3 < 10 < 4 < 9. Near complete energy transfer
from the donor to the acceptor is suggested by the fact the
quantum yields do not vary significantly if the compounds are
excited at the donor or acceptor absorbance. The quantum
yields measured were always higher when chloroform was
used as the solvent than ethanol. Cassette 9 gave the highest
fluorescence quantum yield, and 8 the lowest. The average
fluorescence lifetimes change in reasonable agreement with
changes of the measured quantum yields. Fluorescence
emission spectra of the cassettes show maxima varying
between 514 and 647 nm. For compounds 3 and 9 it is shown
that the S0 ! S1 transition dipole of BODIPY group is long-
axis polarized. Fluorescence steady-state anisotropy data of
cassette 3 suggest that the molecular planes of anthracene and
BODIPY are preferentially oriented in a non-coplanar
conformation.
The molecules in this study can be grouped into two
structural classes. The first, compounds 3 6, have the
BODIPY group attached so that its short axis is parallel to
the phenylethynyl linker and to the short axis of the
anthracene moiety. The substituents on the BODIPY accept-
or were varied within this compound set. These cassettes show
measurably different relaxation times in the range 300 550 fs
(in chloroform). However, despite the wide range of anthra-
cene BODIPYenergy gaps, there was no obvious correlation
between the energy gap and the relaxation time. Thus, the
lowest absorption bands of 6 are red-shifted relative to the
other cassettes in this set, but the relaxation times for all the
members fall within the same range. Comparative measure-
ments for 5 in cyclohexane, ethanol and chloroform solutions
revealed a variation in lifetime from 500 800 fs, for which
ethanol showed the shortest, and cyclohexane solution
showed longest decay time. These results reveal the need for
caution when interpreting rates in terms of the physical
properties of the solute molecule alone.
Experimental Section
General synthetic procedures: Melting points are uncorrected. High field
NMR spectra were recorded on Varian Unity Plus (1H at 300 MHz, 13C at
75 MHz, 31P at 121 MHz) or Inova (1H at 500 MHz, 13C at 125 MHz) NMR
spectrometers. Chemical shifts are reported in units of ppm relative to the
solvent peak (CDCl3: 7.27 ppm for 1H and 77.0 ppm for 13C; D2O: 4.63 ppm
for 1H; [D6]acetone: 2.04 ppm for 1H and 29.9 ppm for 13C; [D6]DMSO:
2.50 ppm for 1H and 39.5 ppm for 13C; CD3OD: 3.30 ppm for 1H and
49.0 ppm for 13C; CD3CN: 1.93 ppm for 1H and 1.3 ppm for 13C). Mass
spectra were obtained from the Mass Spectrometry Applications Labo-
ratory at Texas A&M University using electrospray unless otherwise
indicated; the matrix used for the MALDIanalyses was 2,4,6-trihydrox-
yacetophenone. Thin-layer chromatography was performed using silica gel
60 F254 plates. Flash chromatography was performed using silica gel (230
600 mesh). CH2Cl2, THF, DMF, triethylamine, and toluene were distilled
from appropriate drying agents. Other chemicals were purchased from
commercial suppliers and used as received. The product cassettes were
made in relatively small quantities, so molar absorptivity and micro-
analytical data were not obtained.
(2'-Phenylethynyl)-9-anthracene 1:
A mixture of 9-bromoanthracene
(0.5 g, 1.94 mmol), phenylacetylene (0.42 mL, 3.89 mmol) and [Pd(PPh3)4]
(0.23 g, 0.20 mmol) in triethylamine (2 mL) and THF (5 mL) was stirred at
608C under nitrogen for 12 h. The mixture was diluted using EtOAc
(20 mL), washed with water, dried (MgSO4) and evaporated to yield crude
product. It was purified via silica gel chromatography (hexane/EtOAc 50:1)
giving compound 21 (0.45 g, 83%). M.p. 88 908C; Rf 0.54 (hexane/EtOAc
8:1); 1H NMR (CDCl3): d ˆ 7.51 7.61 (m, 5H), 7.69 7.74 (m, 2H), 7.93 (d,
J ˆ 7.5 Hz, 2H), 8.04 (d, J ˆ 8.4 Hz, 2H), 8.41 (s, 1H), 8.82 (d, J ˆ 8.4 Hz,
2H); 13C NMR (CDCl3): d ˆ 86.4, 100.7, 117.1, 123.6, 125.5, 126.5, 126.6,
127.6, 128.3, 128.4, 128.6, 131.0, 131.6, 132.5; HRMS: m/z: calcd for C22H14
278.1096; found: 278.1093 [M] .
:
‡
Cassette 7: nBuLi (1.6m in hexane, 1.5 mL, 2.10 mmol) at À788C under
nitrogen was added to a solution of 9-bromoanthracene (0.5 g, 1.95 mmol)
in THF (10 mL). After stirring for 15 min at À788C, trimethylborate
(0.25 mL, 2.10 mmol) was added in one portion. The mixture was allowed
to warmed to 258C over 30 min, then quenched with dilute HCl. The
mixture was extracted with EtOAc (20 mL), washed with water, dried
(MgSO4) and evaporated to obtain the crude 9-anthracenylboronic acid
that was used without further purification.
The second set of molecules 7 10 have different separation
of the anthracene and BODIPY moieties, and, unlike the first
set, have the BODIPY unit attached with its long axis parallel
to the linker. All the relaxation times in this group were
substantially shorter than for 3 6, in the general range
ꢀ200 fs. The above observations suggest that the orientation
of the BODIPY moiety has the greater effect on the
relaxation rates of the anthracene subunit than its substitu-
tion, or the separation of the two chromophores within this
group. The conjugated systems, 8 and 10, were not exceptional
within this group, that is, 7 10 all exhibited much faster
relaxation dynamics than the first set.
9-Anthracenylboronic acid, compound 13 (0.20 g, 0.55 mmol) and
[Pd(PPh3)4] (60 mg, 0.05 mmol) were heated reflux in toluene (10 mL),
ethanol (5 mL) and 2m Na2CO3 (2 mL) for 6 h. The mixture was cooled to
258C and diluted with EtOAc (20 mL). The mixture was washed with
water, dried (Mg2SO4) and evaporated to yield crude product that was then
purified via silica gel chromatography (hexane/CH2Cl2 5:1) to give
compound 7 (0.16 g, 71%). M.p. 255 2568C; Rf 0.35 (hexane/CH2Cl2
1:1); 1H NMR (CDCl3): d ˆ 1.95 (s, 3H), 2.24 (s, 3H), 2.32 (s, 3H), 2.64
(s, 3H), 6.14 (s, 1H), 7.26 (s, 1H), 7.41 7.52 (m, 4H), 7.76 (d, J ˆ 8.4 Hz,
2H), 8.08 (d, J ˆ 8.1 Hz, 2H), 8.53 (s, 1H); 13C NMR (CDCl3): d ˆ 10.3,
11.3, 13.3, 14.8, 119.3, 120.3, 125.2, 125.9, 126.2, 127.3, 127.7, 128.0, 128.7,
130.9, 131.4, 132.9, 133.8, 140.3, 141.7, 156.4, 157.5; HRMS: m/z: calcd for
In summary:
‡
C27H23BF2N2: 424.1922; found: 424.1927 [M] .
*
The energy transfer rates measured for cassettes 3 6 vary
Cassette 8: A mixture of compound 4-(trimethylsilylalkynyl)phenylboronic
acid (60 mg, 0.22 mmol), 9-bromoanthracene (85 mg, 0.33 mmol) and
[Pd(PPh3)4] (25 mg, 0.022 mmol) in triethylamine (2 mL) and THF
(5 mL) was stirred at 608C under nitrogen for 12 h. The mixture was
by about a factor of two in chloroform solvent, but no
correlation has been established with the structure of the
acceptor chromophore;
4438
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 4430 4441

Cassettes for Fluorescence Energy Transfer
4430 4441
diluted using EtOAc (20 mL), washed with water, dried (MgSO4) and
evaporated to the yield crude product. This was purified via silica gel
chromatography (hexane/CH2Cl2 3:1) giving compound 8 (60 mg, 61%).
M.p. 3008C (decomp); Rf (hexane/CH2Cl2 1:1); 1H NMR (CDCl3): d ˆ 2.34
(s, 3H), 2.59 (s, 3H), 2.63 (s, 3H), 2.90 (s, 3H), 6.17 (s, 1H), 7.19 (s, 1H),
7.53 7.58 (m, 2H), 7.61 7.66 (m, 2H), 8.06 (d, J ˆ 8.7 Hz, 2H), 8.46 (s, 1H),
8.66 (d, J ˆ 8.7 Hz, 2H); 13C NMR (CDCl3): d ˆ 11.1, 11.4, 13.9, 14.9, 92.5,
93.4, 117.7, 120.1, 120.5, 125.7, 126.6, 127.3, 128.8, 131.2, 132.2, 140.5, 143.0,
149.5, 159.4; MALDI-MS: m/z: calcd for C29H22BF2N2: 448.19; found:
(2 mL, 15.3 mmol) was added, and the reaction mixture was stirred at 258C
for 10 min. BF3 ¥ OEt2 (3.2 mL, 25.5 mmol) was added and the mixture was
heated to 708C for 2 h. The mixture was diluted with EtOAc (100 mL),
washed with water, dried (MgSO4) and evaporated to obtain crude product.
Purification via flash chromatography on silica gel using hexane/CH2Cl2 5:1
gave compound 13 as a orange solid (1.50 g, 36%). M.p. 212 2158C; Rf ˆ
0.25 (1:1 hexane/CH2Cl2); 1H NMR (CDCl3): d ˆ 2.20 (s, 3H), 2.24 (s, 3H),
2.54 (s, 3H), 2.57 (s, 1H), 6.10 (s, 1H), 7.05 (s, 1H); 13C NMR (CDCl3): d ˆ
11.3, 13.6, 14.8, 15.4, 80.5, 120.1, 132.2, 134.1, 142.2, 143.2, 155.3, 159.2;
‡
‡
447.99 [M] .
HRMS: m/z: calcd for C13H14BF2IN2: 374.0263; found: 374.0266 [M] .
Cassette 9: A mixture of compound 17 (20 mg, 0.057 mmol), 9-bromoan-
thracene (26 mg, 0.10 mmol) and [Pd(PPh3)4] (6 mg, 0.05 mmol) in triethyl-
amine (2 mL) and THF (5 mL) was stirred at 608C under nitrogen for 12 h.
The mixture was diluted using EtOAc (20 mL), washed with water, dried
(MgSO4) and evaporated to yield crude product. This was purified via silica
gel chromatography (hexane/CH2Cl2 5:1) giving compound 9 (12 mg,
BODIPY 14: A mixture of compound 12 (0.2 g, 0.53 mmol), [Pd2(dba)3] ¥
CHCl3 (56 mg, 0.062 mmol), CuI(35 mg, 0.18 mmol), phenol (46 mg,
0.49 mmol) and nBu4NI(0.68 g, 1.85 mmol) was flushed with nitrogen,
then, DMF (5 mL) and iPr2Et (0.25 mL) were added, and the mixture was
stirred at 258C for 5 min. It was then allowed to cool to 08C, trimethylsi-
lylacetylene (0.13 mL, 0.93 mmol) was added, and the mixture was stirred
at room temperature for 2 h. The mixture was diluted with EtOAc (20 mL),
washed with water, dried (MgSO4) and evaporated to yield crude product.
This was purified via silica gel chromatography (hexane/EtOAc 15:1)
giving compound 14 (0.15 g, 82%). M.p. 212 2138C; Rf 0.51 (hexane/
EtOAc 4:1); 1H NMR (CDCl3): d ˆ 0.28 (s, 9H), 2.23 (s, 3H), 2.27 (s, 3H),
2.54 (s, 3H), 2.61 (s, 3H), 6.08 (s, 1H), 7.03 (s, 1H); 13C NMR (CDCl3): d ˆ
0.1, 10.5, 11.2, 13.3, 14.7, 97.4, 100.5, 113.5, 119.9, 120.6, 131.3, 134.5, 141.5,
142.9, 157.7, 158.9; HRMS: m/z: calcd for C18H23BF2N2Si: 344.1692; found:
1
40%). M.p. 221 2238C; Rf 0.25 (hexane/CH2Cl2 1:1); H NMR (CDCl3):
d ˆ 2.31 (s, 3H), 2.32 (s, 3H), 2.63 (s, 3H), 2.64 (s, 3H), 6.13 (s, 1H), 7.17 (s,
1H), 7.38 (d, J ˆ 8.1 Hz, 2H), 7.54 7.59 (m, 2H), 7.63 7.69 (m, 2H), 7.88 (d,
J ˆ 8.1 Hz, 2H), 8.07 (d, J ˆ 8.1 Hz, 2H), 8.49 (s, 1H), 8.72 (d, J ˆ 8.1 Hz,
2H); 13C NMR (CDCl3): d ˆ 10.3, 11.3, 13.4, 14.8, 86.8, 100.6, 117.2, 119.4,
120.3, 122.2, 125.7, 126.6, 126.7, 127.8, 128.7, 129.8, 131.1, 131.2, 131.7, 132.6,
133.9, 134.0, 137.0, 141.8, 154.3, 157.7; HRMS: m/z: calcd for C35H27BF2N2:
‡
524.2235; found: 524.2238 [M] .
‡
344.1698 [M] .
Cassette 10: The mixture of compound 13 (200 mg, 0.53 mmol), compound
19 (200 mg, 0.66 mmol) and [Pd(PPh3)4] (6 mg, 0.05 mmol) in triethylamine
(2 mL) and THF (10 mL) was stirred at 608C under nitrogen for 12 h. The
mixture was diluted using EtOAc (20 mL), washed with water, dried
(MgSO4) and evaporated to yield crude product. This was purified via silica
gel chromatography (hexane/CH2Cl2 5:1) giving compound 10 (210 mg,
BODIPY 15: A solution of trimethylsilylethynyl-BODIPY 14 (0.15 g,
0.44 mmol) in THF (5 mL) was stirred at À788C under nitrogen while
TBAF (1.0m in THF, 0.65 mL, 0.65 mmol) was added dropwise over a few
minutes. The solution was warmed to 258C and stirred at this temperature
until TLC analysis indicated completion of reaction (ꢀ30 min). The
solution was poured into water (50 mL) and extracted with EtOAc
(20 mL). The organic layer was washed with water, dried (MgSO4) and
evaporated to yield crude product. This was purified via silica gel
chromatography (hexane/EtOAc 15:1) giving compound 15 (60 mg,
1
71%). M.p. 265 2668C; Rf 0.16 (hexane/CH2Cl2 1:1); H NMR (CDCl3):
d ˆ 2.28 (s, 3H), 2.39 (s, 3H), 2.57 (s, 3H), 2.70 (s, 3H), 6.11 (s, 1H), 7.10 (s,
1H), 7.50 7.65 (m, 6H), 7.74 (d, J ˆ 8.7 Hz, 2H), 8.03 (d, J ˆ 8.2 Hz, 2H),
8.45 (s, 1H), 8.65 (d, J ˆ 8.4 Hz, 2H); 13C NMR (CDCl3): d ˆ 10.7, 11.3, 13.6,
14.9, 84.1, 88.2, 95.5, 100.6, 117.0, 120.1, 120.5, 123.1, 123.6, 125.7, 126.7,
128.0, 128.7, 131.2, 131.3, 131.6, 132.2, 132.6, 134.7, 135.2, 140.8, 143.0, 157.6,
159.4; HRMS: m/z: calcd for C37H27BF2N2: 548.2235; found: 548.2248
1
51%). M.p. 173 1758C; Rf 0.40 (hexane/EtOAc 4:1); H NMR (CDCl3):
d ˆ 2.25 (s, 3H), 2.29 (s, 3H), 2.56 (s, 3H), 2.62 (s, 3H), 3.35 (s, 1H), 6.10 (s,
1H), 7.06 (s, 1H); 13C NMR (CDCl3): d ˆ 10.4, 11.3, 13.2, 14.8, 76.2, 83.0,
112.1, 120.1, 120.7, 131.1, 134.7, 141.8, 143.2, 157.4, 159.4; HRMS: m/z: calcd
‡
[M] .
‡
for C15H15BF2N2: 272.1296; found: 272.1296 [M] .
Cassette 3: A mixture of compound 19 (0.1 g, 0.29 mmol), 9-bromoan-
thracene (0.14 g, 0.57 mmol) and [Pd(PPh3)4] (33 mg, 0.029 mmol) in
triethylamine (2 mL) and THF (5 mL) was stirred at 608C under nitrogen
for 12 h. The mixture was diluted using EtOAc (20 mL), washed with water,
dried (MgSO4) and evaporated to yield crude product. This was purified via
silica gel chromatography (hexane/CH2Cl2 2:1) giving compound 3 (0.1 g,
66%). M.p. 2608C (decomp); Rf 0.28 (hexane/CH2Cl2 1:1); 1H NMR
(CDCl3): d ˆ 1.55 (s, 6H), 2.62 (s, 6H), 6.06 (s, 2H), 7.42 (d, J ˆ 8.1 Hz, 2H),
7.55 7.60 (m, 2H), 7.64 7.70 (m, 2H), 7.94 (d, J ˆ 8.4 Hz, 2H), 8.08 (d, J ˆ
8.4 Hz, 2H), 8.51 (s, 1H), 8.71 (d, J ˆ 8.7 Hz, 2H); 13C NMR (CDCl3): d ˆ
14.6, 14.7, 87.7, 99.9, 116.7, 121.4, 124.4, 125.8, 126.6, 126.8, 128.2, 128.4,
128.8, 131.2, 132.3, 132.7, 135.1, 140.8, 143.0, 155.8; HRMS: m/z: calcd for
BODIPY 16: nBuLi (1.6m in hexane, 1 mL, 1.6 mmol) at À788C under
nitrogen was added to a solution of 4-(trimethylsilylethynyl) iodobenzene
(0.3 g, 1.13 mmol) in THF (5 mL). After stirring for 15 min at À788C,
trimethylborate (0.2 mL, 1.6 mmol) was added in one portion. The mixture
was warmed to 258C, stirred for 30 min, and quenched with dilute HCl
solution. The mixture was extracted with EtOAc (20 mL), washed with
water, dried (MgSO4) and evaporated to obtain the crude 4-(trimethylsi-
lylethynyl)phenylboronic acid that was used without further purification.
The boronic acid isolated from above procedure, compound 9 (0.10 g,
0.27 mmol) and [Pd(PPh3)4] (30 mg, 0.027 mmol) in toluene (10 mL),
ethanol (5 mL) and 2m Na2CO3 (2 mL) were heated under reflux for 5 h.
The mixture was cooled to 258C and diluted with EtOAc (20 mL). The
mixture was washed with water, dried (MgSO4) and evaporated to yield the
crude product. This was purified via silica gel chromatography (hexane/
CH2Cl2 3:1) giving compound 16 (90 mg, 80%). M.p. 209 2108C; Rf 0.39
(hexane/CH2Cl2 1:1); 1H NMR (CDCl3): d ˆ 0.27 (s, 9H), 2.23 (s, 3H), 2.30
(s, 3H), 2.53 (s, 3H), 2.59 (s, 3H), 6.11 (s, 1H), 7.14 (s, 1H), 7.21 (d, J ˆ
8.4 Hz, 2H), 7.55 (d, J ˆ 8.1 Hz, 2H); 13C NMR (CDCl3): d ˆ 0, 10.2, 11.3,
13.3, 14.7, 94.8, 104.9, 119.4, 120.3, 121.7, 129.5, 129.6, 131.1, 132.0, 132.2,
133.9, 137.0, 141.7, 154.3, 157.7; HRMS: m/z: calcd for C24H27BF2N2Si:
‡
C35H27BF2N2: 524.2235; found: 524.2242 [M] .
4-Iodo-3,5-dimethylpyrrole-2-carbaldehyde 12: A mixture of 3,5-dimethyl-
pyrrole-2-carbaldehyde (2.37 g, 19.27 mmol), iodine (5.30 g, 20.88 mmol)
and K2CO3 (3.93 g, 28.48 mmol) in methanol (50 mL) was stirred under
nitrogen at 08C for 12 h. The reaction mixture was extracted with Et2O
(100 mL), washed with saturated sodium thiosulfate (50 mL, saturated),
dried (MgSO4) and evaporated to yield a crude product. This residue was
recrystallized from hexanes/dichloromethane (5:1) to give compound 12
(4.10 g, 85%). M.p. 161 1638C; Rf 0.17 (hexanes/EtOAc 4:1); 1H NMR
(CDCl3): d ˆ 2.31 (s, 3H), 2.39 (3H), 9.52 (s, 1H), 10.66 (s, 1H); 13C NMR
(CDCl3): D ˆ 12.6, 14.3, 72.6, 129.1, 136.8, 140.1, 176.2; HRMS: m/z: calcd
‡
420.2005; found: 420.2008 [M] .
BODIPY 17: A solution of compound 16 (90 mg, 0.21 mmol) in THF
(5 mL) was stirred at À788C under nitrogen while TBAF (1.0m in THF,
0.26 mL, 0.26 mmol) was added dropwise over a few minutes. The mixture
solution was warmed to 258C and stirred until TLC analysis indicated
completion of the reaction (ꢀ30 min). The solution was poured into water
(50 mL) and extracted with EtOAc (20 mL). The organic layer was washed
with water, dried (MgSO4) and evaporated to yield crude product. This was
purified via silica gel chromatography (hexane/CH2Cl2 4:1) giving com-
pound 17 (50 mg, 68%). M.p. 204 2058C; Rf 0.16 (hexane/CH2Cl2 1:1);
1H NMR (CDCl3): d ˆ 2.23 (s, 3H), 2.29 (s, 3H), 2.55 (s, 3H), 2.59 (s, 3H),
‡
for C7H8INO: 249.9729; found: 249.9727 [M] .
BODIPY 13: Phosphorus oxytrichloride (1.00 mL, 11 mmol) was added to
a solution of 3,5-dimethylpyrrole (1.02 g, 11 mmol) and iodopyrrole 12
(2.66 g, 11 mmol) in n-pentane (5 mL) and CH2Cl2 (5 mL) at 08C with
stirring over 5 min. A red precipitate formed; this was removed by filtration
and washed with cold pentane. The filtrates were neutralized with aqueous
Ca(OH)2 at 08C. The organic layer was extracted by CH2Cl2 (100 mL), and
washed with water, dried (MgSO4) and evaporated to obtain orange solid
(1.52 g). The solid was dissolved into benzene (20 mL) then triethylamine
Chem. Eur. J. 2003, 9, 4430 4441
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4439

K. Burgess et al.
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3.16 (s, 1H), 6.11 (s, 1H), 7.15 (s, 1H), 7.24 (d, J ˆ 8.1 Hz, 2H), 7.58 (d, J ˆ
8.1 Hz, 2H); 13C NMR (CDCl3): d ˆ 10.2, 11.3, 13.3, 14.7, 77.6, 83.5, 119.5,
120.3, 120.6, 129.6, 130.9, 132.2, 132.5, 133.9, 134.3, 136.9, 141.8, 154.1, 157.8;
impurities, decomposition products and scattered laser radiation. However,
this ultrashort-lived emission can be detected in real time through the
application of ultrafast spectroscopy. One approach uses an optical gate, in
which some of the emitted fluorescence is mixed with a laser pulse in a
nonlinear crystal to generate optical sum-frequency radiation. This signal
can then be detected against a much reduced background signal via a
monochromator/photomultiplier combination. Such an approach was used
by in this laboratory to time-resolve the emission from 5, and provides a
reference point for the data presented here.[25] However, intrinsically low
signals and sample quantities limited the application of this approach.
‡
HRMS: m/z: calcd for C21H19BF2N2: 348.1609; found: 348.1601 [M] .
Silyldiyne 18: A mixture of 9-ethynylanthracene (200 mg, 0.78 mmol),
4-(trimethylsilylethynyl)iodobenzene (250 mg, 0.86 mmol), [Pd(PPh3)4]
(8 mg, 0.07 mmol) in triethylamine (2 mL) and THF (10 mL) was stirred
at 608C under nitrogen for 12 h. The mixture was diluted using EtOAc
(20 mL), washed with water, dried (MgSO4) and evaporated to yield crude
product. The residue was purified via silica gel chromatography (hexane/
CH2Cl2 10:1) giving compound 18 (220 mg, 75%). M.p. 45 468C; Rf 0.32
(hexane/CH2Cl2 6:1); 1H NMR (CDCl3): d ˆ 0.32 (s, 9H), 7.49 7.64 (m,
6H), 7.71 (d, J ˆ 8.4 Hz, 2H), 8.01 (d, J ˆ 8.4 Hz, 2H), 8.42 (s, 1H), 8.63 (d,
J ˆ 8.4 Hz, 2H); 13C NMR (CDCl3): d ˆ 0, 88.3, 96.4, 100.3, 104.7, 116.9,
123.0, 123.6, 125.7, 126.6, 126.7, 128.0, 128.7, 131.1, 131.4, 132.0, 132.6;
Pump-probe techniques provide an alternative way to monitor ultrafast
transient events in electronically excited molecules, providing potentially
larger signals in the limit of short time resolution. It is well established that,
when ultrashort-lived higher-energy electronic states of a molecule are
populated by the consecutive absorption of photons from two laser pulses,
weak fluorescence can be observed against nearly zero background. This
technique was used by Topp and co-workers at 10 30 ps time resolution to
time-resolve the singlet lifetimes of weakly fluorescent heterocyclic
aromatic molecules and some other species.[30] The present experiments
on the anthracene BODIPY cassettes have excited the anthracene moiety
by 415 nm pulses, and subsequently populated an even higher-energy state
via 830 nm pulses, leading to fluorescence at l > 275 nm. The dependence
of the resulting ultraviolet fluorescence intensity on pulse separation
provides a record of the dynamics of the intermediate state.
‡
HRMS: m/z: calcd for C27H22Si: 374.1491; found: 374.1493 [M] .
Diyne 19: A solution of compound 7 (220 mg, 0.80 mmol) in THF (10 mL)
was stirred at À788C under nitrogen while TBAF (1.0m in THF, 1.2 mL,
1.2 mmol) was added dropwise over several minutes. The mixture solution
was warmed to room temp until TLC analysis indicated completion of
reaction (ꢀ30 min). The solution was poured into water (50 mL) and
extracted with EtOAc (20 mL). The organic layer was washed with water,
dried (MgSO4) and evaporated to yield crude product. This was purified via
silica gel chromatography (hexane/CH2Cl2 10:1) giving compound 19
(200 mg, 75%). M.p. 45 468C; Rf 0.32 (hexane/CH2Cl2 6:1); 1H NMR
(CDCl3): d ˆ 7.49 7.64 (m, 6H), 7.73 (d, J ˆ 8.4 Hz, 2H), 8.01 (d, J ˆ 8.4 Hz,
2H), 8.42 (s, 1H), 8.63 (d, J ˆ 8.4 Hz, 2H); 13C NMR (CDCl3): d ˆ 79.1,
83.3, 88.4, 100.1, 116.8, 122.0, 124.0, 125.7, 126.6, 126.7, 128.1, 128.7, 131.1,
131.4, 132.2, 132.6; HRMS: m/z: calcd for C24H14: 302.1096; found:
Apparatus: The experiments used a Ti/sapphire laser oscillator/amplifier
combination, delivering 200 mW of average power near 830 nm at a
repetition rate of 1 kHz. The pulse autocorrelation profile had a width of
<60 fs. Second-harmonic radiation near 415 nm (ꢀ10 mW) was generated
in a 1 mm thick BBO crystal, after which the beams were split and then
recombined after passing the 830 nm pulses through a variable optical
delay line, which was scanned in steps of 1.5 mm (/10 fs increment), and a
620 Hz beam chopper. The experiment used a flowing sample cell ꢀ1 mm
thick, of which fluorescence from the front portion was imaged onto the
entrance slit of a double monochromator. The cross-correlation profile of
the two pulses, as measured by third-harmonic generation at the surface of
a silica slide was ꢀ90 fs, and the overall cross-correlation profile of the
experiment, which was limited by group velocity dispersion in the sample
cell, was measured to be ꢀ150 fsfwhm. The output from a photomultiplier
mounted on the back of the monochromator was sampled by a lock-in
amplifier synchronized to the beam chopper, and the output was fed to a
computer. The experiment involved either a) scanning the fluorescence
signal as a function of wavelength at a fixed delay time to generate a
transient emission spectrum, or b) scanning the delay line for a fixed
detection wavelength to record a time profile.
‡
302.1095 [M] .
Spectroscopic studies: Spectroscopic grade ethanol was purchased from
Kemetyl, chloroform for UV spectroscopy was obtained from Fluka, and
synthetic grade propane-1,2-diol from Merck-Schuchardt. Absorption
spectra were recorded on a GBC 912 (GBC Scientific Equipment Pty,
Ltd, Australia). A SPEX fluorolog 112 instrument (SPEX Industries,
Metuchen, NJ, USA) equipped with Glan-Thompson polarizers was used
for depolarization experiments. The excitation and emission bandwidths
were 5.6 and 2.7 nm, respectively. All fluorescence spectra were corrected.
To avoid re-absorption, the maximum absorbance was kept below 0.08.
Time-resolved fluorescence experiments were performed on a modified
PRA 3000 instrument (PRA Inc., London, Ontario, Canada) using light
emitting diodes, NanoLED-01 and NanoLED-05 (IBH, Glasgow, Scotland,
UK), for excitation. Excitation wavelengths were isolated through inter-
ference filters centered at 440 nm (HBWˆ 11 nm), 500 nm (HBWˆ
12 nm) and 520 nm (HBWˆ 28 nm) (Omega/Saven, Sweden). Fluores-
cence was observed either through an interference filter at 550 nm
(HBWˆ 40 nm) or a long-pass filter above 610 nm (Schott, Germany).
The results for the above approach were compared with preliminary
measurements that had been made earlier for cassettes 4 6 by fluores-
cence upconversion.[25] Since the results agreed closely, we are satisfied that
the two-photon approach is indeed measuring the lifetime of the
anthracene excited state. This approach was then used for all cassettes
because the signal-to-noise ratio was more favorable than for fluorescence
upconversion.
The fluorescence quantum yield was calculated using the following
relationship:
F ˆ F{1 À exp(ÀAref ln10)}n2 ¥ F(l)dl[{1 À exp(ÀAln10)}nr2ef ¥ Fref(l)dl]À1
where A, n and F denote the absorbance at the excitation wavelength, the
refractive index of the medium and the corrected fluorescence spectrum.
Acknowledgement
28]
The reference systems used were perylene in ethanol (F ˆ 0.92)[26 and
3,3',4,4'-difluoro-1,3,5,7-tetramethyl-4-borata-3a-azonia-4a-aza-s-indacene
in methanol (F ˆ 0.94).[29]
The concentrations used in the determination of quantum yields were
decided upon by the following procedure. To avoid re-absorption, the
absorption maximum of BODIPY was chosen to be less than 0.08. Using an
estimated molar absorptivitity of 60000mÀ1 cmÀ1, the concentrations were
therefore always in the micromolar range.
Use of the TAMU/LBMS-Applications Laboratory directed by Dr. Shane
Tichy are acknowledged. Support for this work was provided by The
National Institutes of Health (HG 01745) and by The Robert A. Welch
Foundation.
[1] J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum, New
York, 1983.
Ultrafast spectroscopic measurements
General considerations: The time constant for energy transfer from the
substituted anthracene moiety to the BODIPY component was measured
by ultrafast spectroscopy, by directly probing absorption and fluorescence
signals attributable to the anthracene species. Low-quantum-yield emis-
sions from the anthracene chromophores (F < 5 Â 10À5) were not readily
detected by the usual fluorescence methods, in competition with intrinsic
[2] L. Stryer, Annu. Rev. Biochem. 1978, 47, 820.
[3] R. M. Clegg, Biotechnology 1995, 6, 103.
[4] T. Fˆrster, Ann. Phys. 1948, 2, 55.
[5] S. Speiser, Chem. Rev. 1996, 96, 1953.
[6] J. M. Tour, Chem. Rev. 1996, 96, 537.
[7] R. W. Wagner, J. S. Lindsey, Pure Appl. Chem. 1996, 68, 1373.
4440
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Chem. Eur. J. 2003, 9, 4430 4441

Cassettes for Fluorescence Energy Transfer
4430 4441
[8] M. S. Vollmer, F. W¸rthner, F. Effenberger, P. Emele, D. U. Meyer, T.
St¸mpfig, H. Port, H. C. Wolf, Chem. Eur. J. 1998, 4, 260.
[9] M. S. Vollmer, F. Effenberger, T. St¸mpfig, A. Hartschuh, H. Port,
H. C. Wolf, J. Org. Chem. 1998, 63, 5080.
[19] K. Sonogashira, Y. Tohda, N. Hagihara, Tetrahedron Lett. 1975, 16,
4467.
[20] I. B. Campbell, in Organocopper Reagents: a PracticalApproach ,
1994, pp. 217.
[10] F. Li, S. I. Yang, Y. Ciringh, J. Seth, C. H. Martin, D. L. Singh, D. Kim,
R. R. Birge, D. F. Bocian, D. Holten, J. S. Lindsey, J. Am. Chem. Soc.
1998, 120, 10001.
[11] R. W. Wagner, J. Seth, S. I. Yang, D. Kim, D. F. Bocian, D. Holten, J. S.
Lindsey, J. Org. Chem. 1998, 63, 5042.
[12] K. Burgess, A. Burghart, J. Chen, C.-W. Wan, in SPIE BiOS 2000 The
InternationalSymposium on BiomedicalOptics , San Jose, CA, 2000.
[13] A. Burghart, L. H. Thoresen, J. Chen, K. Burgess, F. Bergstrˆm, L. B.-
ä. Johansson, Chem. Commun. 2000, 2203.
[21] I. B. Berlman, Handbook of Fluorescence Spectra of Aromatic
Molecules, 2nd ed., Academic Press, London, New York, 1971.
[22] J. Karolin, L. B.-ä. Johansson, L. Strandberg, T. Ny, J. Am. Chem. Soc.
1994, 116, 7801.
[23] L. B.-ä. Johansson, J. G. Molotkovsky, L. D. Bergelson, Chem. Phys.
Lipids 1990, 53, 185.
[24] J. Michl, E. W. Thulstrup, Ber. Bunsenges. Phys. Chem. 1974, 78, 575.
[25] M. F. Wolford, Ph. D. Dissertation thesis, University of Pennsylvania,
2001.
[14] A. Treibs, F.-H. Kreuzer, Liebigs Ann. Chem. 1968, 718, 208.
[15] E. V. d. Wael, J. A. Pardoen, J. A. v. Koeveringe, J. Lugtenburg, Recl.
Trav. Chim. Pays-Bas 1977, 96, 306.
[16] J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed.,
Kluwer Academic/Plenum Publishers, New York, 1999.
[17] J. Chen, A. Burghart, A. Derecskei-Kovacs, K. Burgess, J. Org. Chem.
2000, 65, 2900.
[26] W. H. Melhuish, J. Phys. Chem. 1960, 64, 762.
[27] J. N. Demas, G. A. Crosby, J. Phys. Chem. 1961, 65, 229.
[28] W. Dawson, M. W. Winsor, J. Phys. Chem. 1968, 72, 3251.
[29] I. A. Johnson, H. C. Kang, R. P. Haugland, Anal. Biochem. 1991, 198,
228.
[30] H.-B. Lin, M. R. Topp, Chem. Phys. 1979, 36, 365.
[18] N. Miyaura, A. Suzuki, Chem. Rev. 1995, 95, 2457.
Received: January 22, 2003 [F4754]
Chem. Eur. J. 2003, 9, 4430 4441
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