Energy transfer among light-harvesting macrorings incorporated into a bilayer membrane

Full text of DOI:10.1021/ja8045712

Published on Web 12/16/2008
Energy Transfer among Light-Harvesting Macrorings Incorporated into a
Bilayer Membrane
Naoto Nagata, Yusuke Kuramochi, and Yoshiaki Kobuke*
Graduate School of Materials Science, Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma, Nara 630-0192, Japan
Received June 16, 2008; E-mail: kobuke@iae.kyoto-u.ac.jp
Scheme 1. Structures of the Cyclic Assembly Formed from the
Amphiphilic Trisporphyrins, the Monoporphyrin, and an Internal
Energy/Electron Acceptor
The photosynthetic antenna of purple bacteria is one of the best-
characterized membrane proteins. These proteins have two types
of light-harvesting complexes, designated as LH1 and LH2. LH1
accommodates the reaction center1 in its central cavity. LH2
constitutes large peripheral antennas that serve to increase the cross
section for light absorption and transfer the excitation energy to
the reaction center via LH1.2 In both complexes, dimeric subunits
of bacteriochlorophylls are arranged into macrorings with slipped
cofacial structures. Highly efficient excitation energy transfer is
performed by the remarkable organization of these pigments in light-
harvesting complexes.3
In recent years, great efforts in molecular design to mimic
antenna structures using covalently linked4 or self-assembled5
multiporphyrin arrays have been undertaken. At present, however,
only a few studies incorporating porphyrins into a bilayer membrane
have been reported.6 Furthermore, no examples simulating the
energy transfer systems between LH1 and LH2 exist in the literature.
In a previous paper, we reported the preparation of macroring
architectures mimicking the structure of B850 in LH2 by comple-
mentary coordination of m-phenylene gable porphyrins appended
with imidazolyl groups,7 These cyclic assemblies are regarded as
one of the most ideal elements for constructing artificial antenna
systems because their fluorescence is not quenched by the assembly
into ring structures, even though the chromophores are strongly
coupled because of their close proximity. The self-assembled cyclic
trisporphyrin trimer provides a cavity to accommodate functional
tridentate ligands with large binding constants (K > 107 dm3
mol-1).8
Small unilamellar vesicles (SUVs) containing (2Zn)3 or 3Zn in
their interior were prepared through a sonication procedure.9 The
molar ratios of (2Zn)3 and 3Zn to phospholipids were varied in
the ranges 1/300 to 1/7500 and 9/300 to 9/1500, respectively. In
all cases, no porphyrin remained at the top of the gel filtration
column after elution with buffer, indicating that the porphyrins were
incorporated quantitatively into the SUVs. The absorption spectrum
of (2Zn)3 incorporated into the vesicles [(2Zn)3/lipid molar ratio
< 1/300] showed a Soret band split into two peaks at 408 and 440
nm due to exciton coupling between the neighboring porphyrin
units, and its maxima and fwhm remained unaltered relative to that
in 9/1 CHCl3/MeOH (Figure S4 and Table S1 in the Supporting
Information). The absorption spectrum suggests that (2Zn)3 is
incorporated in the membrane while retaining the trimeric macroring
structure. In the case of 3Zn, the Soret band in the bilayer membrane
(3Zn/lipid ) 9/1500) showed a red shift and was broadened (39
nm) compared with that in the homogeneous solution. These
differences in the spectra of (2Zn)3 and 3Zn may be explained by
the fact that the monomeric porphyrin aggregates in the relatively
high concentrations in the membrane, while the cyclic trimers are
prevented from dense aggregation by their void structures with the
reported compound.6d Incorporation of (2Zn)3 into the membrane
at molar ratios less than 1/300 reduced the fluorescence due to
concentration quenching but kept the intensity higher than 25% of
the initial value (Table S1).
Here we report on the incorporation of a cyclic assembly made
of amphiphilic trisporphyrins into a liposomal membrane to mimic
the bacterial antenna system. Four amphiphilic ω-carboxyalkyl-
benzoyl groups were introduced at two positions of the terminal
porphyrins of the trisporphyrin in order to place the dissociated
carboxylates near the membrane surface. These chromospheres can
be fixed at high concentrations in the bilayer membrane with their
planes perpendicular to the membrane surface, similar to LH1 and
B850 in LH2. The energy transfer among these cyclic assemblies
has been demonstrated by the use of an energy/electron acceptor
in the antenna ring.
A trisporphyrin precursor 1Zn (Scheme 1) was prepared by
condensation of 5-(1-methylimidazol-2-yl)-10,15-bis[11-(4-ethoxy-
benzoyloxy)undecyl]-20-(3-formylphenyl)porphyrin with dipyr-
romethane, followed by Zn(II) insertion. Carboxyl-terminated
trisporphyrin 2Zn was prepared by hydrolysis of 1Zn. In order to
obtain the cyclic trimers (1Zn)3 and (2Zn)3, 1Zn and 2Zn were
reorganized according to a published procedure.8a,b The cyclic
structure was confirmed by 1H NMR and analytical gel permeation
chromatography (GPC)-HPLC. The monomeric porphyrin 3Zn
was synthesized as the reference compound.
The cyclic trimer has three coordination sites of monomeric zinc
porphyrins that seem to be the most suitable for cooperative ligation
by tridentate ligands. The coordination behavior of the trimer was
investigated by using tripodal ligand 4 appended with a C60 energy/
electron acceptor. The binding constants in CHCl3 were obtained
from spectroscopic titrations of (1Zn)3 at various concentrations
9
10 J. AM. CHEM. SOC. 2009, 131, 10–11
10.1021/ja8045712 CCC: $40.75
2009 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Schematic diagram of excitation energy transfer among mac-
rocyclic antenna complexes in a bilayer membrane.
Figure 1. Fluorescence quenching curves at 623 nm upon complexation
of macroring (2Zn)3 with tripodal ligand 4 in SUVs at (2Zn)3/lipid molar
ratios of 1/1500 (O) and 1/7500 ( ×) and that of (1Zn)3 (9.1 × 10-8 M) in
CHCl3 solution (9, observed points; solid line, theoretical curve calculated
with K ) 3.5 × 108 M-1).
(2Zn)3 macrorings maintain their binding capacity at the high initial
concentrations in the membrane and permit energy transfer among
their assemblies, as shown in Figure 2. The cyclic structure is
necessary to realize the high affinity and selectivity for binding
the tripod ligand 4.
of 4 by using the characteristic red shift of Soret bands and the
fluorescence quenching profile on ligation. The binding constant
of the (1Zn)3-4 complex was estimated to be greater than 108 M-1
(Figure S7 in the Supporting Information), indicating that 4 was
selectively and cooperatively incorporated into (1Zn)3, as we
reported previously for a cyclic trimer with 3-allyloxypropyl
groups.8b Next, we investigated the spectroscopic properties of
(2Zn)3 toward incorporation of the tripodal guest in the liposomal
membrane. (2Zn)3 and 4 were cosonicated with phospholipids, and
the resulting dispersion was isolated by gel filtration as described
above. The molar ratios of the host in the phospholipids were
examined at 1/1500 and 1/7500, where the host fluorescence was
retained. Addition of 4 to (2Zn)3 resulted in a sharp red shift of
the Soret bands at low 4/(2Zn)3 ratios, exhibiting behaviors similar
to those in homogeneous solution (Figures S6 and S9 in the
Supporting Information). Furthermore, a shoulder at 540 nm due
to an uncoordinated porphyrin moiety appeared and decreased in
intensity with increasing 4 concentration. The 540 nm peak
disappeared after the addition of 1 equiv of 4 in a homogeneous
solution. These changes can be useful in monitoring coordination
on the free porphyrinatozinc (Figure S10 in the Supporting
Information). They suggest that the cyclic structure is maintained
in SUVs and binds 4 with a large association constant for 1:1
complexation.
The efficiency of excited energy transfer among cyclic trimers
was estimated by fluorescence quenching. The relative fluorescence
intensities of (2Zn)3 determined by the addition of various amounts
of the guest molecule are illustrated in Figure 1 in comparison with
the plot obtained in a homogeneous solution of (1Zn)3 in CHCl3.
When the mixture of 4 and (2Zn)3 at a molar ratio of 1/5 was put
into phospholipid vesicles at a (2Zn)3/lipid molar ratio of 1/1500,
the quenching efficiency was 54%. This efficiency was about 3
times larger than that of (1Zn)3-4 in a homogeneous solution
(19%). The fact that the binding behavior of (2Zn)3-4 remained
similar in the bilayer membrane and in a homogeneous solution
implies that the three cyclic assemblies in the membrane were
quenched by the introduction of one acceptor molecule. Under more
dilute conditions at a (2Zn)3/lipid molar ratio of 1/7500, the
quenching efficiency was slightly decreased. In the case of
monomeric porphyrin 3Zn, the fluorescence quenching remained
as low as 28% at a 1/5 molar ratio of 4 to 3Zn and 30-40% even
at a 1/3 molar ratio, corresponding to the equimolar mixture of the
pyridyl ligand and the coordination site. Fluorescence quenching
data combined with UV-vis spectral changes demonstrate that the
In this paper, we have examined the incorporation of a cyclic
assembly consisting of amphiphilic trisporphyrins into a liposomal
membrane. The cyclic assembly (2Zn)3 was fixed in an orientation
similar to bacterial light-harvesting complexes in the phospholipid
vesicle. Under the conditions concentrated in the membrane, the
fluorescence quenching by incorporation of the energy/electron
acceptor to the inner sphere of (2Zn)3 was enhanced by energy
transfer from neighboring (2Zn)3. The amphiphilic porphyrin
macroring was demonstrated to be a suitable candidate for
constructing an artificial photosynthetic antenna or reaction center
complex in the membrane.
Supporting Information Available: Synthetic details; GPC, MS,
UV-vis, and NMR spectra; UV-vis titration data; and preparation of
small unilamellar vesicles. This material is available free of charge
via the Internet at http://pubs.acs.org.
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