Fluorescent proton sensors based on energy transfer

Article abstract of DOI:10.1021/jo2005654

Photophysical data and orbital energy levels (from electrochemistry) were compared for molecules with the same BODIPY acceptor part (red) and perpendicularly oriented xanthene or BODIPY donor fragments (green). Transfer of energy, hence the photophysical

Full text of DOI:10.1021/jo2005654

ARTICLE
pubs.acs.org/joc
Fluorescent Proton Sensors Based on Energy Transfer
Cliferson Thivierge, Junyan Han, Roxanne M. Jenkins, and Kevin Burgess*
Department of Chemistry, Texas A & M University, Box 30012, College Station, Texas 77841, United States
S Supporting Information
b
ABSTRACT: Photophysical data and orbital energy levels (from
electrochemistry) were compared for molecules with the same
BODIPY acceptor part (red) and perpendicularly oriented xanthene
or BODIPY donor fragments (green). Transfer of energy, hence the
photophysical properties of the cassettes, including the pH depen-
dent fluorescence in the xanthene-containing molecules, correlates
with the relative energies of the frontier orbitals in these systems.
Intracellular sensing of protons is often achieved via sensors that
switch off completely at certain pH values, but probes of this type are
not easy to locate inside cells in their “off-state”. A communication
from these laboratories (J. Am. Chem. Soc., 2009, 131, 1642ꢀ3)
described how the energy transfer cassette 1 could be used for
intracellular imaging of pH. This probe is fluorescent whatever the pH, but its exact photophysical properties are governed by the
protonation states of the xanthene donors. This work was undertaken to further investigate correlations between structure,
photophysical properties, and pH for energy transfer cassettes. To achieve this, three other cassettes 2ꢀ4 were prepared: another
one containing pH-sensitive xanthene donors (2) and two “control cassettes” that each have two BODIPY-based donors (3 and 4).
Both the cassettes 1 and 2 with xanthene-based donors fluoresce red under slightly acidic conditions (pH < ∼6) and green when the
medium is more basic (>∼7), whereas the corresponding cassettes with BODIPY donors give almost complete energy transfer
regardless of pH. The cassettes that have BODIPY donors, by contrast, show no significant fluorescence from the donor parts, but
the overall quantum yields of the cassettes when excited at the donor (observation of acceptor fluorescence) are high (ca. 0.6 and
0.9). Electrochemical measurements were performed to elucidate orbital energy level differences between the pHꢀfluorescence
profiles of cassettes with xanthene donors, relative to the two with BODIPY donors. These studies confirm energy transfer in the
cassettes is dramatically altered by analytes that perturb relative orbital levels. Energy transfer cassettes with distinct fluorescent
donor and acceptor units provide a new, and potentially useful, approach to sensors for biomedical applications.
’ INTRODUCTION
Application of probe 1 to monitoring intracellular pH has been
communicated.16 It fluoresces red at pH values less than about
6.5 and green at pH values above about 7 (Figure 2). Probe 1
compared favorably with commercial pH sensors such as carboxy-
SNARF-1 (Invitrogen), insofar as its pH response range is
complementary and its quantum yields are higher. Further, probe
1 exhibits a greater wavelength difference (∼80 nm) between the
two ratiometric emission peaks than carboxySNARF-1. It also
has acid functionality to conjugate to proteins, while carboxy-
SNARF-1 does not. When BSA labeled with probe 1 was
imported inside COS 7 cells, the pH values of the protein and
in the cytosol and endosomes were quantified.
Research described in this paper was undertaken to explore
how the photophysical properties of energy transfer cassettes
correlate with their structures. Nagano has published on the
photoinduced electron transfer (PeT) between meso-phenyl
groups and BODIPY cores.17 These PeT processes can nega-
tively impact the fluorescence quantum yields of BODIPYs, and
we therefore set out to prepare cassette 2, a modification of 1
Fluorescent sensors are widely used for detection of protons
and metals in several applications,1,2 especially intracellular
imaging.3 Indicators may be divided into three types: (i) ones
that are insignificantly fluorescent in the absence of analyte but are
much more emissive when it is present; (ii) the inverse, where
fluorescence of the probe is quenched by the analyte; and (iii)
sensors which have observable spectroscopicdifferenceswhenthe
analyte is present compared to when it is absent. The third type of
sensor is “always on”; this is a significant advantage because it is
clear that the probe is present even if the analyte is not (Figure 1).
Intracellular pH (pHi) is a fundamental property that corre-
lates with many events in cell biology. To measure this, research-
ers tend to rely on subtle changes in sensors that are “always
on”;4ꢀ12 for instance, they observe emission intensities as a
function of excitation wavelength.12ꢀ15 However, these sensors
do not change emission wavelength maxima as the pH is varied; if
they did, they would be far easier to use. That is why we were
excited to realize cassette 1 is always fluorescent at pH ranges
around the physiological region but with emission maxima that
vary over a very significant spectral region, ca. 530 and 600 nm.
Received: November 8, 2010
Published: May 27, 2011
r
2011 American Chemical Society
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Figure 1. Fluorescent sensors may be activated (i) or quenched (ii) by
analytes; ones that are “always on” (iii) but change wavelength of
fluorescence emissions on binding.
wherein the meso-phenyl functionality is absent. For comparison,
cassettes 3 and 4 were also targeted; these maintain the basic
design, two donors perpendicularly aligned to the same central
BODIPY18ꢀ21 acceptor. However, they are fundamentally dif-
ferent insofar as the donors are also BODIPY-based, and there
was no obvious reason to suspect transfer of energy from the
donors in these molecules to the acceptors should be pH
dependent. These were envisaged as reference compounds for
comparison of the extent of energy transfer in these systems.
To outline, the research described here investigates (i)
syntheses of the cassettes, (ii) effects of changing of the meso-
substituent on the central BODIPY {acceptor} fragment repre-
sented by the difference between cassettes 1 and 2 and between 3
and 4, and (iii) changes of donor fragment from xanthene to
BODIPY represented by the structural differences between 1 or 2
and 3 or 4. Comparison of the cassettes is made on the basis of
photophysical and electrochemical data measured at different pH
levels. The latter measurements are related to orbital levels in the
donor and acceptor fragments.
Sonogashira reactions24,25 were used to assemble the cassettes
from the acceptor components 6 and 8, and the fluorescein-based
and BODIPY-based donor components C26,27 and D28
(Scheme 2). The diacetate intermediate 9 is of some importance
because this compound has been shown to be cell permeable and
hydrolyzes in the cytosol to give green fluorescence.16 The
fluorescein-based cassettes 1 and 2 are soluble in lower alcohol
solvents giving pink solutions.
Cassette 2 (Scheme 3) was difficult to purify. Flash chroma-
tography did not give pure material, but the compound was
isolated via preparative reversed-phase HPLC in 4% yield. Both
cassettes 3 and 4 are soluble in lipophilic solvents like CH2Cl2
and give strongly colored pink or red solutions.
’ RESULTS AND DISCUSSION
Syntheses of the Cassettes 1ꢀ4. Two key diiodinated
BODIPY intermediates were prepared to make the cassettes
featured in this paper. The first, compound 6 (Scheme 1a), was
designed to have a triethylene glycol linker that would somewhat
separate the dye from the protein as well as increase solubility in
polar solvents. Conditions for the diiodination reactions shown
in Scheme 1 were based on work from Nagano et al.22 The
second, compound 8 (Scheme 1b), was formed via a route that is
analogous to one used for a homologue formed from glutaric
anhydride.23 Synthons 6 and 8 lead to cassettes with different
meso substituents: aryl and alkyl functionalities.
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Scheme 1. Syntheses of Pivotal Diiodinated Synthons: (a)
BODIPY 6 and (b) BODIPY 8
Figure 2. Three forms of the energy transfer cassette that were used as a
pH probe for intracellular imaging.
The methyl ester of cassette 4 was crystallized for single-crystal
X-ray diffraction studies (Figure 3). They show the BODIPY
donor fragments resting perpendicular to the acceptor part. In
part, it is this molecular twist that differentiates cassettes from
planar dyes consisting of a single conjugated chromophore.
Interestingly, the molecule appears to “sag” around the central
BODIPY fragment because the alkyne parts are not exactly in the
same plane; an ideal linear arrangement would give a 180° angle,
but the observed angle was 168.2 degrees. This parameter may
have some relevance because if the angle were 180° and rigid
then the transition dipoles of the BODIPY acceptor and the two
donor fragments (which are aligned with their long axes)29 would
be exactly perpendicular in any conformation about the alkyne.
In that orientation there can be no dipoleꢀdipole coupling;
hence, fluorescence resonance energy transfer (FRET) could not
occur. The fact that the molecule is not perfectly linear means
that FRET cannot be completely excluded because rotation
about the alkyne bond could place the BODIPY donors in
conformations in which weak dipoleꢀdipole coupling could
occur. However, the “sag-angle” is small, and conformations that
allow dipoleꢀdipole coupling also take the phenyl group out of
conjugation with the rest of the acceptor; consequently, the solid state
data indicates energy transfer via this mechanism30 is unfavorable.
An important set of new acceptor fragments 11ꢀ14 and known
BODIPY E31 and xanthene F32 reference compounds were also
generated for this study. Photophysical and electrochemical
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Scheme 2. Syntheses of Cassettes: (a) 1 and (b) 3
electrochemical studies were performed on these constituents, thus
avoiding the need for destructive experiments (electrochemistry)
on the valuable cassette samples. Syntheses of the new materials are
outlined in the Supporting Information.
Photophysical Properties. Salient photophysical properties
of the cassettes are shown in Table 1. The acceptor fragment
is formed by the 2,6-(alkyneꢀaryl) substituents because
these impose a dramatic red-shift on the absorbance and
properties in cassettes tend to be accurately represented by the
individual donor and acceptor fragments.33 Consequently,
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Table 1. Photophysical Properties of 1ꢀ4 in 1:1 Ethanol/CH2Cl2
absorptionb
fluorescencec
λmax donor (nm)/
λmax acceptor (nm)/
ε ꢁ 10ꢀ4
λmax donor
λmax acceptor
φacceptor excited at
φacceptor excited at
ETE
(%)f
g
compd
basea
ε ꢁ 10ꢀ4
(nm)
(nm)
acceptord
donore,j
φdonor
1
1
2
2
3
3
4
4
ꢀ
þ
ꢀ
þ
ꢀ
þ
ꢀ
þ
505/5.6
505/11
506/3.7
505/13
502/14
502/14
502/11
502/11
575/3.5
578/3.0
560/1.9
561/2.8
566/6.6
566/6.6
561/4.7
560/5.2
521
529
528
529
513
516
521
515
600
579
0.30i
0.01i
0.24j
0.01j
0.62i
0.66i
0.84j
0.93j
0.15
0.09
51
<5
38
<5
93
92
94
91
0.05
0.09
0.07
0.08
h
606
604
588
588
0.58
0.60
0.79
0.85
-
h
-
h
-
h
-
a With nBu4NOH at a concentration of 8 ꢁ 10ꢀ5 M. b At 1 ꢁ 10ꢀ5 M. c At 1 ꢁ 10ꢀ6 M. d Quantum yield of acceptor when excited at the acceptor.
e Quantum yield of acceptor when excited at the donor. f Energy-transfer efficiency calculated with the quantum yield of the acceptor with excitation at
donor divided by that with excitation at the acceptor. g Fluorescein (φ = 0.92 in 0.1 M NaOH)34 was used as a standard. h Donors in these cassettes show
no significant fluorescence emission. i Rhodamine 101 (φ = 1.00 in EtOH)35 was used as a standard. j Rhodamine B (φ = 0.97 in EtOH)34 was used as a
standard.
Scheme 3. Syntheses of Cassettes: (a) 2 and (b) 4
Figure 3. Single-crystal X-ray structure of 10.
molar absorptivities and absorb/fluoresce at wavelengths
that are characteristic of the free dye fragments (see Table 2
below).
Through-bond energy transfer cassettes are usually designed
to absorb light at the donor excitation wavelength, relay it to the
acceptor part, then emit fluorescence from there. The term
“energy transfer efficiency” (ETE, %) quantifies this; it is defined
as follows:
ETE (%) is a measure of the quantum yield of the cassette
when irradiated at the donor. It reflects the extent of energy
transfer including the negative effects of nonradiative loss in the
transfer process. The product of the molar absorptivity of the
donor in the cassette and the ETE give a measure of the
brightness of the acceptor in the system.
fluorescence properties of the BODIPY core.25 The fluores-
cein and BODIPY donor parts exhibit characteristically large
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Table 2. Photophysical Properties of Reference Compounds
in 1:1 Ethanol/CH2Cl2
a
compd basec λabs (nm)/ε ꢁ 10ꢀ4 λem (nm) fwhm (nm)
φb
11
11
12
12
13
13
14
14
E
_
560/2.6
559/2.6
565/0.9
563/1.0
574/3.2
574/3.2
573/2.3
578/2.5
506/8.7
506/8.7
508/7.4
508/11
587
587
593
592
606
605
612
613
511
511
517
517
41
41
46
43
44
53
53
52
16
17
26
25
0.72
0.71
0.46
0.61
0.52
0.54
0.42
0.44
0.98
0.90
0.76
0.95
þ
_
þ
_
þ
_
þ
_
E
þ
_
F
F
þ
a Absorption data recorded at 1 ꢁ 10ꢀ5 M. Emission data taken at 1 ꢁ
10ꢀ6 M. b Rhodamine 101 (φ = 1.0 in ethanol)35 was the standard for
11ꢀ14 and fluorescein (φ = 0.92 in 0.1 M NaOH)34 for E and F. c With
Bu4NOH at a concentration of 8 ꢁ 10ꢀ5 M.
Values of the ETE (%) for the cassettes 1ꢀ4 are shown in
Table 1. Several important observations are clear. First, the
fluorescein-based cassettes 1 and 2 have moderate ETE values
in the absence of base, but not when Bu4NOH is added. Second,
the BODIPY-based cassettes 3 and 4 have excellent ETE values,
and these are not influenced by base.
Effects of base on the cassettes are probably best visualized from
their absorbance and fluorescence spectra (Figure S1,Supporting
Information, and Figure 4). Addition of nBu4NOH to cassettes 1
and 2 increases the absorption corresponding to the fluorescein
component relative to that from the BODIPY acceptor part. This
is logical because addition of base forces the fluorescein donor into
its ring-opened phenolate carboxylate form. Absorbance spectra of
cassettes 3 and 4 are almost completely insensitive to base, as
would be predicted since the electronic spectra of BODIPY dyes
are not significantly affected by pH.
In the absence of base, excitation of the fluorescein donor of
cassettes 1 and 2 leads to significant fluorescence from the
BODIPY acceptor. However, no significant fluorescence is
observed from the BODIPY part when base is added to the same
solutions (Figure 4a and b). Conversely, addition of base has no
significant effect on the extent of energy transfer for the cassettes
3 and 4 that have BODIPY donors. Thus, in 1:1 ethanol/CH2Cl2
the fluorescein donor parts of cassettes 1 and 2 are, at least
partially, protonated, and energy transfer to the BODIPY accep-
tor occurs. Energy transfer is quenched when the fluorescein
donors are completely deprotonated. Cassettes 3 and 4 have
BODIPY, not fluorescein, donor parts, but they are otherwise
identical to 1 and 2.
Table 2 shows photophysical properties for the reference
compounds 11ꢀ14, E, and F. None of the building blocks that
were assembled to give cassettes 1 ꢀ 4 have fluorescence
characteristics that are significantly changed by added base.
The largest change in quantum yield is seen for the dicarboxylate
13 and xanthene F: for these compounds a 25% increase in the
presence of base. There are no appreciable shifts in λabs, λem, or
even the peak width values when these fragments are compared
without and with base.
Figure 4. (aꢀd) Fluorescence spectra of cassettes 1ꢀ4 (1 ꢁ 10ꢀ6 M in
1:1 ethanol/CH2Cl2); throughout, spectra recorded without added base are
shown in blue, and those with nBu4NOH (concentration of 1 ꢁ 10ꢀ4 M)
are shown in red.
None of the reference compounds can undergo changes like
that shown in equilibrium 1, but this has been comprehensively
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studied for fluorescein in aqueous media.14 These data show that
the quantum yield of fluorescein is highest in its dianion state.
However, Table 1 shows that the quantum yield of the donor part
in the fluorescein-based cassettes 1 and 2 are less than 0.1, with or
without base. Without base there is significant energy transfer to
the acceptor, so some quantum yield reduction is anticipated.
With base, however, less of the energy transferred between the
donor and acceptor is emitted as acceptor fluorescence. Further,
the xanthene quantum yields in the cassettes are much less than
for fluorescein in any of the accessible protonation states or for
the xanthene F. Thus, integration of the xanthene donors in
cassettes 1 and 2 reduces their quantum yields relative to the
parent fragments.
Table 3. Electrochemical Data for Reference Compounds E,
F, 13, 14, and Cassette 3a
compd Eonset,ox (V) HOMO (eV) Eonset,red (V) LUMO (eV) Eg (eV)
Eb
Fc
þ0.68
þ0.33
þ0.04
þ0.99
þ0.49
þ0.49
þ0.99
5.84
5.48
5.19
6.15
5.64
5.64
6.15
ꢀ1.32
ꢀ1.03
ꢀ1.67
ꢀ0.91
ꢀ0.93
ꢀ0.92
ꢀ0.92
ꢀ1.18
3.84
4.12
3.48
4.28
4.22
4.23
4.24
3.98
2.00
1.36
1.71
1.87
1.42
1.41
1.91
2.17
c,e
FNa
13b
14c
14d
3b
a Cyclic voltammograms were recorded using a glassy carbon working
electrode (A = 0.071 cm2) referenced to Fc/Fcþ and a Pt counter
electrode at a scan rate of 200 mV/s. All potentials are reported vs Fc/
Fcþ, and all HOMO and LUMO energies are derived from electro-
chemical results based on Fc/Fcþ = 5.15 eV (DMF) and 5.16 eV
(CH2Cl2) vs vacuum. All solvents were flushed with Ar(g) before use. b In
CH2Cl2. c In DMF. d In DMF and 0.1 M pyridine. e Xanthene was first
reacted with NaOH to obtain the sodium salt.
Electrochemical Studies. Oxidation and reduction potentials
were measured for the reference fragments E, F, 13ꢀ14, and for
cassette 3 relative to the ferrocene/ferrocinium couple (Table 3).
The electrochemical measurements provide an approximation of
the band gap magnitude. The HOMO and LUMO energies were
estimated on the basis of the onset of REDOX events as described
by Reynolds et al.36 BODIPY 13 shows a reversible reduction
wave, while for 14 and E the wave is quasi-reversible for the first
reduction events. For F and FNa the first reduction wave is
irreversible. The oxidation events for all compounds are irrever-
sible. In a method similar to the one used by Reynolds et al.,36 the
ferrocene/ferrocenium couple (in volts) is estimated to an
orbital level of 5.15 and 5.16 eV relative to vacuum in DMF
and CH2Cl2, respectively.37,38 Thus, energy levels of HOMO and
LUMOs can be pegged relative to this reference point. The same
data were acquired for compounds 11 and 12 (see the Support-
ing Information).
Figure 5a plots HOMO and LUMO energy levels for the
reference BODIPY E and the acceptor mimic 13 that represent
cassette 3 (BODIPY donor and acceptor). The LUMO levels of
the donor and acceptor parts are close in energy with the latter
0.44 eV lower. This corresponds to the system for which high
ETE was observed (93%) and for which no pH dependence
was observed or expected. Figure 5b,c shows data correspond-
ing to the pH-dependent cassette 1. Under conditions favoring
Figure 5. HOMO and LUMO levels of the reference compounds
representing cassettes: (a) 3; (b) 1 (under neutral conditions); and
(c) 1 (basic conditions).
protonation of the phenolic-O atoms, the two LUMO levels are
also close in energy (0.1 eV), and again, significant ETE was
observed. However, for the same cassette but under conditions
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wherein the phenol groups would be completely deprotonated,
poor energy transfer corresponds with a large energy gap
between the donor and acceptor LUMO levels.
The work described here may represent the beginning of a new
paradigm in which electronically coupled dye pairs can be used to
sense analytes in biomedical applications. There is considerable
scope for molecular modifications in this series because the donor,
acceptor, and linker fragments59 could all be modified to give sensors.
’ CONCLUSIONS
Rapid and efficient energy transfer may be possible when a
fluorescent donor is joined to an acceptor in such a way that the
two fragments would be electronically conjugated if they became
planar but are sterically prevented from doing so.39ꢀ42 The fact
that they cannot easily achieve planarity means that the absorption
spectra of the cassette resembles the sum of the donor and
acceptor parts. However, the donor part will not fluoresce when
it is excited in an efficient energy transfer cassette; instead the
energy will be rapidly transferred to the acceptor that will then
fluoresce (Figure 6).43ꢀ55
’ EXPERIMENTAL SECTION
1.16 A mixture of 6 (65 mg, 0.074 mmol), diacetylfluorescein alkyne
C26 (82 mg, 0.186 mmol), Et3N (0.11 mL, 0.74 mmol), Pd(PPh3)4
(8 mg, 0.007 mmol), and CuI (3 mg, 0.014 mmol) was dissolved in THF
(2 mL). After the solution was degassed three times via the freezeꢀthaw
method, the mixture was heated up to 45 °C for 16 h. The reaction
solvent was removed under reduced pressure, and the crude product was
purified by flash column eluting with 50% hexane/ethyl acetate to give 9
1
as a light yellow solid (80 mg, 72%): H NMR (500 MHz, CDCl3) δ
8.08 (m, 2H), 7.73 (dd, J = 8.0, 1.5 Hz, 2H), 7.65(d, J = 8.0 Hz, 2H), 7.29
(d, J = 8.5 Hz, 2H), 7.15 (d, J = 8.2 Hz, 2H), 7.10 (d, J = 2.0 Hz, 4H), 6.83
(bs, 4H), 6.83 (d, J = 2.0 Hz, 4H), 4.48 (s, 2H), 4.02 (s, 2H), 3.80ꢀ3.82
(m, 2H), 3.70ꢀ3.75 (m, 10H), 2.75 (s, 6H), 2.32 (s, 12H), 1.58 (s, 6H),
1.47(s, 9H); 13C NMR (125 MHz, CDCl3) δ 169.6, 168.8, 168.2, 159.1,
152.1, 151.8, 151.5, 144.6, 142.1, 137.9, 134.1, 132.8, 131.1, 128.9, 127.9,
127.7, 126.6, 125.8, 124.3, 124.2, 117.8, 116.0, 115.6, 110.5, 94.7, 87.2,
85.3, 84.1, 81.8, 81.5, 70.7, 70.6 (2 C), 70.5, 69.5, 69.0, 59.2, 28.1, 21.1,
Figure 6. Concepts of through-bond energy-transfer cassettes.
13.8, 13.7; MALDI MS calcd for C86H71BF2N2NaO20 (M þ Na)þ
þ
1523.46, found 1523.26; TLC (1:1 EtOAc/hexane) Rf = 0.20.
To 9 (12 mg, 0.01 mmol) in 5 mL of 2:1 methanol/THF in was added
Na2CO3 (3.5 mg, 0.03 mmol). The mixture was stirred for 3 h at 25 °C
under N2. The reaction was quenched by adding aqueous HCl (0.1 M,
10 mL), and the product was extracted out of the solution with 75%
CH2Cl2/iPrOH (5 mL ꢁ 3). The organic layers were washed with brine
solution (10 mL) and dried with magnesium sulfate. Compound 1 was
Previous studies from these laboratories provided evidence
that energy transfer mechanisms besides FRET can be dominant
in cassettes like the ones described in this paper. Specifically,
energy transfer rates between anthracene (donor) and BODIPY
(acceptor) fragments were much faster than would be expected
from a FRET mechanism.29,43,45 Further, some of those cassettes
have the donor and acceptor parts oriented in ways that should
largely exclude the possibility of FRET, yet fast transfer rates were
observed. Others in this field had documented similar effects for
energy transfer cassettes.33,56ꢀ58
In this work, we do not profess to know the mechanisms by
which energy is transferred between the donor and the acceptor.
It seems highly probable that all possible mechanisms compete;
these include dipoleꢀdipole coupling (FRET), electron transfer,
and processes in which relaxation of energy from the donor
excited state leads to excitation of the acceptor via through-bond
mechanisms that rely on orbital overlap. Moreover, the propor-
tions of each energy transfer mechanism operative will be
influenced by the protonation state of the donor, at least for
the xanthene dyes. Comparisons with the all-BODIPY donorꢀ
acceptor systems 3 and 4 reveal that the relative orbital energy
levels between the donor and acceptor fragments in these
cassettes are comparable to those in the pH-sensitive systems 1
and 2 in protonation states where efficient energy transfer is
observed. Moreover, the meso-aromatic substituent of 1 and 3 has
no significant effect on the photophysical properties of these
cassettes; this can be inferred from the data collected for 2 and 4
which have only aliphatic groups in that position.
1
isolated as a purple solid (10 mg, 99%): H NMR (500 MHz, 75%
CD3OD/CDCl3), δ 8.00 (s, 2H), 7.74 (dd, J = 8.0 Hz, 1.5 Hz, 2H), 7.65
(d, J = 7.5 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 7.15 (d, J = 8.0 Hz, 2H), 6.67
(d, J = 2.5 Hz 4H), 6.59 (d, J = 8.0 Hz, 4H), 6.51 (dd, J = 9.0 Hz, 2.5 Hz,
4H), 4.47 (s, 2H), 4.00 (s, 2H), 3.79ꢀ3.81(m, 2H), 3.71ꢀ3.73(m, 2H),
3.66ꢀ3.69 (m, 8H), 2.71 (s, 6H), 1.58 (s, 6H), 1.44(s, 9H); 13C NMR
(125 MHz, 75% CD3OD/CDCl3) δ 170.9, 170.1, 169.8, 159.5, 153.5,
145.2, 142.9, 138.3, 134.8, 133.4, 131.7, 131.2, 129.6, 128.7, 128.5, 128.0,
126.1, 125.9, 124.9, 116.3, 110.3, 108.2, 103.3, 95.6, 87.4, 86.0, 84.1, 82.7,
71.0, 70.9 (2 C), 70.8 (2 C), 69.7, 69.3, 59.5, 30.2, 28.3, 14.0; MS (MALDI)
calcd for C78H63BF2N2O16þ (M þ H)þ 1333.42, found 1333.44.
2. A mixture of 8 (30 mg, 0.05 mmol), C26 (55 mg, 0.13 mmol), Et3N
(0.30 mL, 2.1 mmol), PdCl2(PPh3)2 (5 mg, 0.01 mmol), and CuI (2 mg,
0.01 mmol) was dissolved in 1.0 mL of DMF under N2. The solution was
degassed three times via the freezeꢀthaw method and then stirred at
40 °C for 4 h and then at 25 °C for 12 h under N2. The solvent was
removed under reduced pressure and the crude product partially purified
via flash silica column eluting with 7% methanol/CH2Cl2 to give the
acetate-protected form of 2 as a purple solid (15 mg).
The product from above (15 mg) was treated with sodium carbonate
(6 mg, 0.05 mmol) in 5.0 mL of methanol. The mixture was stirred at
25 °C for 3 h. The solvent was removed under reduced pressure, and
extraction was performed using CH2Cl2 and 0.1 M HCl aqueous
solution. The aqueous layer was washed with CH2Cl2 (2 ꢁ 5 mL),
and the organic fractions were combined and dried over magnesium
sulfate. The solvent was then removed under reduced pressure and
purified via C-18 preparative HPLC eluting with a 50 ꢀ 95% MeOH and
0.1% TFA/water linear gradient over 25 min to give the desired product
with a retention time of 18 min as a purple solid (2 mg, 4%): 1H NMR
(500 MHz, CD3OD) δ 8.08 (s, 2H), 7.87 (d, J = 8.0 Hz, 2H), 7.23 (d, J =
8.0 Hz, 2H), 6.70 (s, 4H), 6.67 (d, J = 8.5 Hz, 4H), 6.58 (d, J = 7.5 Hz,
4H), 3.45 (m, 2H), 3.12 (m, 2H), 2.70 (s, 6H), 2.68 (s, 6H); MALDI
Communication between the donor and the acceptor in these
systems, by whatever mechanisms, does appear to be impacted by
the relative energies of the frontier orbitals involved. Specifically,
maximal energy transfer was observed when the electrochemical
studies showed the donor and acceptor LUMO levels were close,
but with the latter being lowest. However, ETE was least in the
series when there was an appreciable gap between the LUMO
energy levels. This may be a reflection on a larger divergence of
the LUMO wave function distributions in the latter case.
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HRMS calcd for C60H37BF2N2O12 (M ꢀ 2H/2)ꢀ2 513.6243, found
513.6244; TLC (5% MeOH/CH2Cl2) Rf = 0.20.
’ REFERENCES
3. A mixture of 6 (80 mg, 0.09 mmol), D45 (69 mg, 0.20 mmol), Et3N
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(4 mg, 0.01 mmol) was dissolved in 3.0 mL of THF. The solution was
degassed three times via the freezeꢀthaw method, and the mixture was
heated to 50 °C for 16 h under N2. The reaction solvent was removed
under reduced pressure, and the crude product was purified via flash
silica column eluting with 67% hexane/ethyl acetate to give the desired
product as a purple solid (89 mg, 74%): 1H NMR (500 MHz, CDCl3)
δ 7.64 (d, J = 8.0 Hz, 2H), 7.59 (d, J =8 .0 Hz, 4H), 7.29 (d, J = 8.5 Hz,
2H), 7.27 (d, J = 8.0 Hz, 4H), 5.99 (s, 4H), 4.48 (s, 2H), 4.02 (s, 2H),
3.80ꢀ3.82 (m, 2H), 3.70ꢀ3.75 (m, 10H), 2.76 (s, 6H), 2.55 (s, 12H),
1.58 (s, 6H), 1.47(s, 9H), 1.43 (s, 12H); 13C NMR (125 MHz, CDCl3)
δ 169.6, 158.9, 155.7, 144.1, 143.0, 141.7, 140.7, 134.8, 134.3, 132.8,
131.9, 131.2, 128.2 (2 C), 128.0, 124.1 (2 C), 121.3, 116.0, 96.0, 87.1,
85.3, 82.9, 81.6, 70.7, 70.6 (3 C), 70.5, 69.5, 69.0, 59.2, 28.1, 1þ4.6 (2 C),
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4. A mixture of 8 (30 mg, 0.05 mmol), D45 (47 mg, 0.14 mmol), Et3N
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’ ASSOCIATED CONTENT
S
Supporting Information. Preparation of compounds
b
1ꢀ14, cyclic voltammetry (CV) spectra of 11ꢀ-14 and EꢀF,
and synthesis of cassette 10. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: burgess@tamu.edu.
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’ ACKNOWLEDGMENT
Financial support for this work was provided by the National
Institutes of Health (087981) and by The Robert A. Welch
Foundation (A1121). TAMU/LBMS-Applications Laboratory
headed by Dr. Shane Tichy provided mass spectrometric sup-
port, and the X-ray Diffraction Laboratory (Dr. J. Reibenspies)
generated the crystallographic data. Dr. Yuichiro Ueno and Mr.
Juan Castro are thanked for the synthesis of fluorescein pre-
cursors. We are grateful to Professor Marcetta Y. Darensbourg
for providing equipment and expertise regarding the electro-
chemical experiments and to Dr. Cꢀesar Pꢀerez-Bolívar and
Professor Pavel Anzenbacher (Bowling Green University) for
helpful discussions regarding energy transfer in the molecules.
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