Self-organized lipid-porphyrin bilayer membranes in vesicular form: Nanostructure, photophysical properties, and dioxygen coordination

Article abstract of DOI:10.1002/1521-3765(20021202)8:23<5469::AID-CHEM5469>3.0.CO;2-1

An amphiphilic tetraphenylporphyrin and its iron complex bearing four phospholipid substituents, in which a trimethylolethane residue connects the two acyl chains (lipid-porphyrins), have been synthesized. The free-base lipid-porphyrin 6a self-organizes i

Full text of DOI:10.1002/1521-3765(20021202)8:23<5469::AID-CHEM5469>3.0.CO;2-1

FULL PAPER
Self-Organized Lipid-Porphyrin Bilayer Membranes in Vesicular Form:
Nanostructure,Photophysical Properties,and Dioxygen Coordination
[a]
Teruyuki Komatsu,Miho Moritake,Akito Nakagawa,and Eishun Tsuchida*
Abstract: An amphiphilic tetraphenyl-
porphyrin and its iron complex bearing
four phospholipid substituents, in which
a trimethylolethane residue connects the
two acyl chains (lipid-porphyrins), have
been synthesized. The free-base lipid-
porphyrin 6a self-organizes in aqueous
media to form spherical unilamellar
vesicles with a diameter of 100 nm and
a uniform thickness of 10 nm, which
corresponds to twice the length of the
molecule. In the visible absorption spec-
trum, the porphyrin Soret band was
significantly red-shifted (12 nm) relative
to that of the monomer in benzene/
MeOH solution due to the excitonic
interaction of the porphyrin chromo-
phores. The P A isotherm of 6a gave
an area per molecule of 2.2 nm2, which
allowed the estimation of the number of
molecules in a single vesicle (2.3 Â 104).
(LB) films of 6a on a glass surface
exhibited an absorption spectrum iden-
tical to that of the 6a vesicles in bulk
aqueous solution, and this suggests that
they contain similar geometric arrange-
ments of the porphyrin moieties. Exci-
ton calculations on the basis of our
structural model reproduced the bath-
ochromic shift of the Soret band well. In
the photophysical properties of the 6a
vesicles, the characteristics of J-aggre-
gated porphyrins substantially predom-
inate: strong fluorescence and extremely
short triplet lifetime. The iron complex
6b with a small molar excess of 1-dode-
cylimidazole (DIm) also formed spher-
ical unilamellar vesicles (100 nm F).
Scanning force microscopy after evapo-
ration on a graphite surface revealed 6b/
DIm vesicles with a vertical height of
19.8 nm, which coincided with the thick-
ness of the double bilayer membranes.
The ferrous 6c formed a bis(DIm)-
coordinated low-spin FeII complex un-
der an N2 atmosphere. Upon addition of
O2 to this solution, a kinetically stable
O2 adduct was formed at 378C with a
half-life of 17 h. Distinct gel-phase (liq-
uid-crystal) transitions of the lipid-por-
phyrin membranes were clearly ob-
served; the free base 6a displayed a
higher transition temperature (568C)
than the iron complex. Magnetic circular
dichroism and infrared spectroscopic
studies proved that molecular O2 coor-
dinates to the self-organized lipid-por-
phyrinatoiron(ii) vesicles in aqueous
media.
Keywords:
nanostructures
dioxygen ligands
porphyrinoids
¥
¥
¥
self-assembly ¥ vesicles
Double-layered
Langmuir Blodgett
tron transfer,[4, 5] dioxygen (O2) transport,[6] selective oxida-
Introduction
[8]
tion reactions,[7] and ATP production have been reported.
It is of current interest to construct self-organized porphyrin
assemblies by formation of noncovalent bonds (metal ligand
coordination, hydrogen bonding, and electrostatic attraction)
to mimic the diverse biological reactivity of porphyrinoids in
nature.[1, 2] Such vectorial binding forces have built highly
complicated porphyrin architectures that are difficult to
prepare by general organic synthetic procedures. However,
most of them were prepared in organic solvents. If we are to
reproduce the various biochemical reactions that rely on the
natural porphyrins, aqueous systems are particularly impor-
tant. Hence, over the past few decades substantial efforts have
directed towards embedding synthetic model porphyrins in
phospholipid vesicles;[3] ground-state or photoinduced elec-
Unfortunately, the locations of the porphyrin co-factors in
these ensembles are not always accurate, because of the low
guest-to-host molecular ratio. To counter this problem,
porphyrin amphiphiles become attractive building blocks for
constructing well-defined three-dimensional porphyrin as-
semblies in aqueous media. Coulombic forces were first
successfully employed in the preparation of beautiful por-
phyrin fibers, but they are relatively unstable and tend to
precipitate because of their low solubility.[9] We are now
convinced that hydrophobic interaction is the most useful
driving force for preparing large-scale supramolecular sys-
tems in which thousands of metalloporphyrins are aligned
with great regularity and longevity. Indeed, some porphyrins
with amphiphilic side chains at the periphery self-organize in
water to form supramolecular aggregates (fibers, tubes,
vesicles, and sheets).[10, 11] A tetraphenylporphyrin (TPP)
derivative with four dialkylglycerophosphocholine groups on
one side of the ring plane produced spherical unilamellar
[a] Prof. E. Tsuchida, Dr. T. Komatsu, M. Moritake, A. Nakagawa
Advanced Research Institute for Science and Engineering
Waseda University, Tokyo 169-8555 (Japan)
Fax : (‡81) 3-3205-4740
E-mail: eishun@waseda.jp
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E. Tsuchida et al.
FULL PAPER
vesicles with reversible O2-binding ability, similar to those of
hemoglobin and myoglobin under physiological conditions
(pH 7.3, 378C).[12] The only drawback of this compound is
laborious introduction of the phospholipid substituents, in
which a glycerol residue connects the two acyl chains. We
recently found that the role of glycerol can be adopted by a
similar triol, namely, trimethylolethane. To the best of our
knowledge, there has been no report on a phospholipid
amphiphile involving a trimethylolethane residue as the
connecting moiety of the acyl chains, and its self-assembly
behavior. Here we report the first synthesis of and bilayer
membrane formation by novel TPP derivatives with four
phospholipid substituents on the porphyrin ring (lipid-por-
phyrins): free base 6a, FeIII complex 6b, and FeII complex 6c.
The nanostructure of the lipid-porphyrin aggregate was
elucidated by transmission electron microscopy (TEM) and
scanning force microscopy (SFM). Exciton calculations on the
basis of our bilayer model reproduces well the bathochromic
shift observed in the UV/Vis absorption of the 6a membranes.
The free-base lipid-porphyrin vesicles fluoresce strongly, and
the vesicles of the FeII complex formed a stable and reversible
O2 adduct under physiological conditions, with an O2-binding
affinity similar to that of human erythrocytes. Magnetic
circular dichroism (MCD) and infrared (IR) spectroscopic
studies on the O2 coordination mode have also been made.
a
b
HO
OH
OH
TrtO
OH
OH
1
(19%)
O
O
c
TrtO
OC(CH2)16CH3
OH
TrtO
OC(CH2)16CH3
OCCH2CH2COH
O
O
2
(63%)
3
(83%)
O
TPP-(C18OH)4
d
TrtO
OC(CH2)16CH3
OCCH2CH2CO
e
C18-TPP
O
O
4
4
(94%)
O
f
HO
OC(CH2)16CH3
OCCH2CH2CO
6a
C18-TPP
(80 %)
O
O
4
5a
g
f
(92%)
6b
(79%)
5b
(81%)
TPP-(C18OH)4: 5,10,15,20-Tetrakis{α,α,α,α-o-(2,2-dimethyl-20-
hydroxyicosanamido)phenyl}porphine
Scheme 1. Synthetic scheme for lipid-porphyrin 6a and its iron(iii)
bromide complex 6c. a) Trityl chloride/DMF, pyridine. b) Stearic acid
anhydride, 4-DMAP/THF. c) Succinic acid anhydride/THF. d) DCC,
4-DMAP/CH2Cl2. e) BF3 ¥ Et2O/CH2Cl2. f) 2-chloro-1,3,2-dioxaphospho-
lane 2-oxide/CH2Cl2, TEA. g) FeBr2/THF, 2,6-lutidine. DCC ˆ dicyclo-
hexyl carbodiimide, 4-DMAP ˆ 4-dimethylamino pyridine.
steps are required to obtain the corresponding material from
glycerol. The coupling of 3 with the parent TPP derivative
[13]
TPP-(C18OH)4
was performed by dicyclohexyl carbodi-
imide (DCC) in DMF at room temperature to give 4a in 94%
yield. BF3 ¥ MeOH selectively cleaved the trityl protecting
groups to yield 5a (92%). The phosphocholine head groups
were introduced in the final step by a general procedure with
2-chloro-1,3,2-dioxaphospholane 2-oxide. This is in contrast to
our glycerol-based lipid-porphyrin, for which only mild, acidic
phosphorylation could be used to introduce phosphocholine
residues into the 3-hydroxyl group of glycerol.[12] As a result,
the free-base lipid-porphyrin 6a was synthesized in six steps
by using the parent TPP-(C18OH)4, whereas the glycerol
analogue requires twelve steps, including zinc insertion at the
porphyrin center and complicated reactions in low yields. The
FeIII complex 6b was prepared from 5b in the same manner as
for 6a from 5a; all the compounds are available in gram
quantities. The a4 structure of the porphyrins did not
atropisomerize throughout the entire process, as was con-
1
firmed by H NMR spectroscopy: the chemical shift of the
singlet for the 2,2-dimethyl groups (d ˆ À0.4) remained
1
constant. For the FeIII complex 6b, H NMR spectra were
recorded after removing the iron center with FeCl2 in HCl/
acetic acid.[14] The Satisfactory analytical data were obtained
for all compounds described here (see Experimental Section).
Results and Discussion
Synthesis: The synthesis of lipid-porphyrins is summarized in
Scheme 1. The phospholipid precursor 3 is easily synthesized
in three steps starting from trimethylolethane, whereas five
Morphology and nanostructure of the free-base lipid-por-
phyrin assembly: The free-base lipid-porphyrin 6a was
dispersed in deionized water by ultrasonication ([6a] ˆ 1
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Lipid-Porphyrin Bilayer Membranes
5469 5480
3000mm) to give a red solution that was stable for a year
without precipitation. The TEM of the negatively stained and
evaporated sample revealed that 6a forms perfectly spherical
vesicles with a diameter of 100 nm (Figure 1a). The thickness
Figure 2. UV/Vis absorption spectra of lipid-porphyrin 6a at 258C.
chromophores. Hence, we are certain that the hydrophobic
TPP planes of 6a form two-dimensional planar sheets in the
vesicles, and stack at the middle of the bilayer membranes
(Figure 1b). The quantitative evaluation of the excitonic
interactions of these stacked porphyrin sheets are described
below.
Surface pressure molecular area (P A) isotherm and
exciton calculation of the Langmuir-Blodgett (LB) film: The
P
A isotherm of the giant porphyrin amphiphile 6a ex-
hibited essentially the same features as that of typical
amphiphilic lipids At large areas
(Figure 3).[15]
Figure 1. a) Transmission electron micrograph of a vesicle of the free-base
lipid-porphyrin 6a negatively stained with uranyl acetate. b) Schematic of
the bilayer membrane.
of the unilamellar membrane was 10 nm, which corresponds
to twice the length of the 6a molecule (4.6 nm) (Figure 1b).
Light-scattering measurements gave a sharp peak with a
narrow distribution (100 Æ 36 nm) and supported the TEM
result. In general, ultrasonic irradiation of phospholipids
provides small unilamellar vesicles. However, the spherical
membrane consisting of the giant porphyrin amphiphile 6a
(M ˆ 4564) cannot produce a large curvature; therefore, the
vesicles are much larger than the usual ones. They neither
coagulate nor photodegrade for a long time.
The visible absorption spectrum of the 6a vesicles showed a
porphyrin Soret band at 434 nm (emax ˆ 3.1  105 mÀ1 cmÀ1),
which was significantly shifted to the red region relative to
that of the monomer in benzene/MeOH (4:1; lmax ˆ 422 nm;
Figure 2). The bandwidth at half-height (Dl1/2 ˆ 16 nm) was
slightly wider than that of the monomer (Dl1/2 ˆ 14 nm). The
intensity and absorption maxima of the Q-bands demonstrat-
ed even smaller shifts of 3 nm, which may be a van der Waals
shift caused by the replacement of solvent. Thus, the larger
bathochromic shift of the Soret band (12 nm) should include
some excitonic interactions owing to a lateral arrangement
(J-aggregate) of the transition moments of the porphyrin
Figure 3. P A isotherm of lipid-porphyrin 6a. The subphase was pure
water, and the film was compressed at a rate of 10 mNminÀ1
.
(>6.0 nm2 moleculeÀ1) the film is in an expanded state. Upon
compression, the film undergoes a transition from the
expanded phase to the liquid expanded phase
(4.5 nm2 moleculeÀ1). Further compression led to the liquid-
expanded/liquid-condensed transition (3.5 nm2 moleculeÀ1),
and finally to a solid condensed phase. The collapse pressure
reached 43 mNmÀ1, and this indicates formation of a rela-
tively close packed solid membrane. Extrapolation of the
second linear portion of the isotherm to zero surface pressure
gave, as the intercept, the area per 6a molecule. The value of
2.2 nm2 coincides with the sum of the areas of four phospho-
choline head groups, and we infer that the lipid-porphyrin in
Chem. Eur. J. 2002, 8, No. 23
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E. Tsuchida et al.
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the oriented layer is likely to have a cylindrical form with a
TPP bottom of 1.5 nm2 (see the space-filling model above).
Based on this value, the number of constituent molecules of
6a in one vesicle (100 nm F) was estimated to be 2.3 Â 104
(1.4Â 104 in the outer phase and 9.0 Â 103 in the inner phase).
The 6a monolayer on the LB trough was then slowly
transferred onto a glass plate to give a porphyrin monolayer in
which the hydrophilic head groups likely face the glass
surface, while the hydrophobic TPP plane is toward the air.
Further immersion and removal of the substrate perpendic-
ular to the trough should result in formation of trilayered LB
films. Interestingly, the visible absorption spectra of these
films provided good clues to the in-plane and interlayer
arrangements of the TPP moieties in the 6a vesicles. The
monolayer showed distinct TPP absorption with sufficiently
high intensity for spectroscopic analysis (Figure 4a). The
Soret band at 430 nm is clearly red-shifted (8 nm) relative to
that of the monomer in benzene/MeOH (lmax ˆ 422 nm). The
absorption intensity of the 6a trilayers is three times as high as
that of the monolayer with a small bathochromic shift (2 nm)
in the Soret band; the lmax of the Q-bands were essentially
unaltered. This observation suggested formation of triple-
layer 6a films on the glass plate. Here, it is of interest to
subtract the spectrum of the monolayer from that of the
trilayer, presumably to give the absorption of a bilayer in
which the outer and inner TPP sheets are stacked at the center
of the membrane (Figure 4b). In fact, the difference spectrum
(lmax ˆ 433, Dl1/2 ˆ 15 nm) closely resembles that of the 6a
vesicles in bulk aqueous solution (lmax ˆ 434, Dl1/2 ˆ 1 6 nm,
Figure 2), that is, the spherical 6a vesicles and double-layered
films on a solid substrate contain similar porphyrin arrange-
ments.
calculated to be À519.9 and À515.4 cmÀ1, respectively. The
wavelength difference between the corresponding transitions
is much smaller than the half-width of the Soret band of the
monomer. Consequently, splitting of the Soret band could not
Then we employed simple point-dipole exciton coupling
theory to predict the porphyrin alignment in the LB mono-
and bilayers.[16] First, for the 6a monolayer film on the glass
surface, we assumed that the tightest packing of the TPP
moieties is realized by a rhomboidal lattice with diagonal
distances of 2.6 nm for 2a and 1.48 nm for 2b (Figure 4c), as
observed in the triclinic unit cell of a single crystal of
unsolvated free-base TPP.[17] The head-group area of 2.2 nm2
could be fitted on an individual porphyrin platform without
overlap. This assumption was also made in our excitonic
calculation on the monomolecular porphyrin platelets.[11h] The
exciton interaction DE between two porphyrins is given by
Equation (1):
À3
DE ˆ M2r (1 À 3cos2q)
(1
)
mn
where M is the transition dipole moment of 6a, rmn is the
center-to-center distance between the original porphyrin (Po)
and neighboring porphyrin (Pmn), and q is the tilt angle
between the line connecting the centers and the molecular
axes. We took into account all the interactions with the infinite
porphyrin neighbors on the 22 lines through the origin of the
coordinate axes. The excitonic interaction DE is always
Figure 4. a) UV/Vis absorption spectra of LB films of lipid-porphyrin 6a
on a glass plate at 258C. b) Schematics of the mono-, tri-, and bilayers of
lipid-porphyrin 6a on the glass surface. c) Predicted arrangement of the
porphyrin planes as a model for the bilayer membranes of lipid-porphyrin
6a. The porphyrin Po is located at the origin of the coordinate axes.
considered as Vmn and V' in two different Soret transitions
mn
Sx and Sy (see Experimental Section). With our 6a monolayer
model, the difference was very small. With M2 ˆ 81.8D2 for
the dipole strength of the monomer, total V and V' were
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Lipid-Porphyrin Bilayer Membranes
5469 5480
be expected, and the result was one exciton band, red-shifted
by 517.7 cmÀ1 with respect to the monomer. This calculated
shift is almost identical to the observed value of the 6a
monolayer (only 76.8 cmÀ1 larger). A van der Waals shift,
which occurs on going from solution to a dense-packed LB
film, was not seen. The real lattice of the LB monolayer might
be slightly different from our model, because of the direct
contact between the phosphocholine head groups and the
solid glass surface. The use of an extended transition dipole
instead of the point dipole also gave nearly the same
results.[18, 19]
fluorescence emission maxima (lem ˆ 649, 713 nm) correlates
with the Soret band absorption of the vesicles (lmax ˆ 434 nm),
which indicates that the strong fluorescence is due to the
membrane, and not to a small amount of monomer dissoci-
ated from the aggregates. Thus, the lifetime of the excited
To predict the absorption of the bilayer, we made the
additional hypothesis that the interlayer TPP arrangements
are also similar to those found in crystals of free-base TPP.
Based on the structural data reported by Strauce et al., two
postulates are made: 1) a slipped face-to-face stacking of the
porphyrin sheets with a minimum spacing d of 4.1ä (Fig-
ure 4c),[17] and 2) a porphyrin core in the upper layer is shifted
by ‡4.0, ‡2.7, and ‡4.1ä in the x, y, and z directions,
respectively, from the center of the nearest core in the layer
below. The distances and angles from the porphyrin Po to the
neighbors in the upper layer were estimated. Then we
calculated all the interactions of these porphyrin pairs (total
of 46 combinations), but stopped summation at a distance of
some 5 nm. The difference was relatively wide in this
calculation: À805.9 and 323.8 cmÀ1 for Sx and Sy, respectively.
If this is true, the Soret bands should be split or blue-shifted
with significant broadening. However, the reverse was the
case; only a 3 nm bathocromic shift from the monolayer and
still narrow bandwidth were observed. In our structural
model, the most unreliable parameter is the layer spacing d.
For a slightly larger value of d ˆ 6.0 ä the difference
decreased (À152.0 and 268.2 cmÀ1 for the x and y directions).
This means a rather small shift from lmax of the monolayer,
and is in good agreement with the fact that the absorptions of
the monolayer and the bilayer are quite similar (Figure 4a).
Since the spherical 6a vesicles in bulk aqueous solution
showed a spectrum similar to that of the double-layered LB
films of 6a on the glass plate, we can conclude that they are
both describable by equivalent exciton interactions. The small
wavelength difference between the predicted lmax in our
Figure 5. Fluorescence emission spectra of lipid-porphyrin 6a (1.7 mm) at
258C (excitation at 428 nm). The inset shows emission excitation corre-
lation image.
singlet state of the vesicles (tF ˆ 6.8 ns) was not dramatically
different from that of the monomers (tF ˆ 10 ns).
Laser flash photolysis experiments were carried out with
nanosecond laser excitations. The transient absorption spec-
trum of the 6a monomer in benzene/MeOH after laser flash
illumination displayed the typical triplet triplet (TT) ab-
sorption of TPP, in which the dark decay obeyed first-order
kinetics (triplet lifetime tT ˆ 2.0 ms) and is strongly acceler-
ated by the presence of O2 (Figure 6). Excitation of the 6a
model (431nm) and experimentally obtained values ( lmax
ˆ
433 nm on the glass surface and 434 nm in water) is likely due
to the van der Waals shift. In this case, the head group glass
contact effect, which was considered in the monolayer, can be
ignored, since the stacking of the second porphyrin layer
probably weakens it. Of course, the porphyrin lattice in the
bilayers is somewhat flexible, because of the fluidity of the
membrane, despite its being below the phase transition
temperature (vide infra); therefore, the observed shift in the
Soret band always comes from the averaged exciton inter-
action.
Figure 6. Transission absorption differential spectra of lipid-porphyrin
6a in solution 50 ns after laser-flash photolysis (excitation at 516 nm) under
an Ar atmosphere ([6a] ˆ 1.7mm). The inset shows the absorption decay at
450 nm.
Photophysical properties of free-base lipid-porphyrin vesi-
cles: The most remarkable photophysical property of the
aqueous solution of 6a vesicles is to fluoresce strongly; its
fluorescence emission intensity was 76% of that of the
monomer in benzene/MeOH (4:1; Figure 5). The three-
dimensional excitation emission spectrum revealed that the
bilayer vesicles, on the other hand, gave small-magnitude
signals with a lifetime that was too small to observe with our
nanosecond instrumentation. If internal conversion is the only
process influenced by self-assembly, the triplet state would be
quenched to the same extent as the fluorescence, but this was
not seen. These observations on the excited singlet and triplet
Chem. Eur. J. 2002, 8, No. 23
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E. Tsuchida et al.
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states of the 6a vesicles are almost the same as those on
monolayered porphyrin platelets, which consist of laterally
aligned porphyrins.[11h] Whitten et al. also reported a signifi-
cant decrease in tT for 5,10,15,20-tetrakis(a4-hexanamidophe-
nyl)porphyrin J-aggregates in the presence of a small amount
of surfactant in water.[20] There are two possible interpreta-
tions for the reduction of the triplet yield: 1) a decrease in the
rate constant of intersystem crossing kisc, and 2) an increase in
the rate constant of triplet deactivation ko. Based on our
previous calculation for a similar porphyrin assembly,[11h] we
can predict that the triplet lifetime of the 6a vesicles may
decrease by some 2 ns. The extremely short-lived triplet state
of the lipid-porphyrin vesicles could be further evaluated
using a picosecond laser flash apparatus.
Nanostructure of lipid-porphyrinatoiron assembly: The iron
(iii) complex of lipid-porphyrin 6b also self-organizes in water
to form spherical unilamellar vesicles with a diameter of
100 nm. The presence of a small excess of 1-dodecylimidazole
(DIm, DIm/6b ˆ 2.2 3.4 molmolÀ1) as an axial base did not
hinder vesicle formation. The TEM of the evaporated 6b/
DIm solution on the copper grid showed an identical
membrane with a thickness of 10 nm, which again corresponds
to twice the length of the molecule (Figure 7). Light-scatter-
ing measurements gave a main diameter peak at 97 nm with a
distribution of Æ73 nm. The visible absorption spectrum
(lmax ˆ 421, 570 nm) suggested the formation of a six-coor-
dinate low-spin iron(iii) complex with DIm ligands on both
axial sites.
The aqueous 6b/DIm was then transferred to highly
oriented pyrolytic graphite (HOPG) and subjected to SFM
under ambient conditions. The collapsed vesicles became
detectable; they are obviously images of flattened 6b/DIm
assemblies on the graphite surface (Figure 7b). The thickness
measured from the height profile of the evaporated vesicle
was 19.5 nm (vertical distance a a in Figure 7b), which
corresponds to twice the thickness of the bilayer membrane.
For evaluation of the lateral size, we should normally consider
the horizontal broadening effect due to the shape of the tip,
but this is not necessary for the vertical distance.[21] We can
therefore conclude that the iron complex with DIm produces
bilayer vesicle membranes, like free-base 6a.
Figure 7. a) Transmission electron micrograph of vesicles of the lipid-
porphyrinatoiron(iii) complex 6b/DIm negatively stained with uranyl
acetate. b) SFM images (tapping mode) of the evaporated sample of 6b/
DIm vesicles on HOPG. Image size is 600 Â 600 nm (z range: 50 nm), and
its cursor profile 19.5 nm (vertical distance a a).
O2 coordination to lipid-porphyrinatoiron(ii) vesicles: The
UV/Vis absorption spectrum of a phosphate buffer solution of
the FeII 6c/DIm vesicles revealed the presence a typical six-
coordinate low-spin FeII species (lmax ˆ 436, 538, 568 nm)
(Figure 8).[22] The TEM of the solution again exhibited
spherical unilamellar vesicles with a wall of bilayer thickness
(10 nm, not shown). Upon exposure of these vesicles to O2
gas, the absorption spectrum immediately changed to that of
an O2 adduct (lmax ˆ 435, 543 nm). This dioxygen complex was
kinetically stable in the wide range of 5 508C, depending on
the O2 partial pressure. Reversion of the O2 adduct to the
bis(DIm)-ligated species on bubbling N2 gas was observed,
that is, the dissociated DIm remains in the membrane interior
during the dioxygenation and rebinds rapidly to the iron
center after deoxygenation. The constant morphology of the
vesicle throughout the reversible O2 coordination cycle was
Figure 8. Visible absorption spectral changes for vesicles of the lipid-
porphyrinatoiron(ii) complex 6c/DIm in phosphate buffer solution (pH 7.3)
at 258C.
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Lipid-Porphyrin Bilayer Membranes
5469 5480
confirmed by TEM. Bubbling carbon monoxide into this
solution gave the more stable carbonyl complex (lmax ˆ 433,
545 nm).
porphyrin array shows a parallel arrangement of the nearest
neighbor phenyl groups and a constant repeat distance. The
stacking of all the phenyl rings is expected to provide some
lattice stabilization. By contrast, the FeIII TPP complex
aligns in a random conformation.[17] The porphyrin moieties
in the 6a bilayer are presumably oriented in a fashion similar
to that in the free-base TPP crystal, which may afford
additional stabilization to the membrane packing. Indeed,
the temperature dependence of the absorption spectrum of
the 6a vesicles showed a small hypsochromic shift with
broadening and a decrease in intensity at Tc (568C; Fig-
ure 10). This was observed only for the free-base 6a porphyrin
vesicles, and not for the iron complex.
Oxidation to the FeIII species was very slow; the half-life of
the O2 adduct was 17 h at 378C. The relationship between the
O2 binding ratio and O2 pressure, that is, the O2 equilibrium
curve, was plotted on the basis of the UV/Vis absorption
spectral changes on O2 titration (Figure 9). The apparent O2
binding affinity [P1/2 is the gas pressure at half O2 coordination
of porphyrinatoiron(ii)] of the 6c/DIm vesicles was deter-
mined to be 30 Torr at 378C, a value similar to that of human
erythrocytes.[23] Although the O2-binding profile did not show
any cooperativity like that seen in hemoglobin, their O2-
transporting efficiency between the lungs (PO ˆ 110 Torr) and
2
muscle tissue (PO ˆ 40 Torr) is 20%, which is nearly the same
2
value (22%) as that of human erythrocytes.
Figure 10. Temperature dependence of the Soret band absorption lmax and
half-width Dl1/2 of vesicles of the lipid-porphyrin 6a in water.
Figure 9. O2 equiblium curve of vesicles of the lipid-porphyrinatoiron(ii)
complex 6c/DIm in phosphate buffer solution (pH 7.3) at 378C.
Although the lipid-porphyrin vesicles have distinct phase
transitions, their low DH values mean the alkyl chains are less
dense than those of a 1,2-diparmytoyl-sn-glycero-3-phospha-
tidylcholine (DPPC) membrane. The increased amount of
Differential scanning calorimetry (DSC): The thermal behav-
ior of the lipid-porphyrin bilayer membranes was studied by
DSC. Three samples, 6a, 6b, and 6b/DIm vesicles, demon-
strated gel-phase (liquid-crystal) transitions (Table 1). Free
entropy per methylene unit (DSCH ), which is often used to
2
evaluate the molecular packing (0.90 for DPPC),[24] was also
low (0.54 and 0.56 for the 6a and 6b vesicles, respectively).
The thermotropic characteristics of the iron-complex vesicles
were further altered by addition of DIm, which resulted in a
significant decrease in Tc and a drop of 2.2 kcalmolÀ1 in the
enthalpy change of the transition.
Table 1. Phase transition temperatures of the lipid-porphyrin vesicles and
their thermodynamic parameters.
System
Tc
[oC]
DH
[kcalmolÀ1
DS
DSCH
2
[calmolÀ1degÀ1
[a]
]
[calmolÀ1degÀ1
]
]
Magnetic circular dichroism (MCD) and IR spectroscopy: For
porphyrins and hemoproteins, MCD is a sensitive probe for
oxidation state, spin state, and axial ligation of the metal
center. It has been therefore used as a fingerprint for the
coordination structure of various model hemes.[25] We applied
the MCD and IR spectroscopy to characterize the O2 adduct
of the self-organized 6c/DIm vesicles. Under an N2 atmos-
phere, the MCD showed a well-characterized spectrum of
bis(imidazole)-ligated six-coordinate low-spin FeII porphyrin,
which differs strongly to that of a five-coordinate high-spin
complex with mono-imidazole ligation (Figure 11). This
observation indicates that axial coordination is not affected
by the dense packing of the four phospholipid groups on the
porphyrin plane. Admission of O2 gas yields the six-coordi-
nate 6c/DIm(O2) complex, which shows an S-shaped A-term
MCD in the Soret region, as observed in the spectra of other
dioxygenated FeIITPP derivatives.[25] The CO adduct is also
6a
6b
56
49
40
418.6
4.173.2
3.7
1.5
0.54
75.5
29.4
0.56
6b/DIm
DPPC[b]
27.3
0.90
[a] DS ˆ DHTcÀ1
.
[b] DPPC ˆ 1,2-diparmytoyl-sn-glycero-3-phosphatidyl-
choline.[24]
base 6a showed a relatively sharp transition at 568C with an
enthalpy change DH of 4.1kcalmol À1; this suggests that the
long acyl chains on the porphyrin are responsible for
formation of close-packed layers. We are certain that the
trimethylolethane residue in the lipid group plays the same
role as a glycerol residue. It is remarkable that the ferric
complex 6b had a lower phase-transition temperature Tc of
498C. This decrease may result from the different geometric
alignments of the TPP planes in the membrane. As described
above, free-base TPP forms triclinic crystals in which each
Chem. Eur. J. 2002, 8, No. 23
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E. Tsuchida et al.
FULL PAPER
of these findings, we conclude that the self-organized 6c
bilayer vesicles coordinate molecular O2 like hemoglobin and
model hemes,[26, 27] without any hinderance by aggregation.
Conclusion
The synthesized lipid-porphyrins are giant porphyrin amphi-
philes with the ability to form perfectly round bilayer vesicles
in aqueous solution. The glycerol in the phospholipids can be
replaced by trimethylolethane. Indeed, phospholipids in
which trimethylolethane residues links diparmytoyl chains
and phosphocholine head group assemble in water to give
unilamellar vesicles similar to those formed by DPPC.[28] The
advantages of the trimethylolethane-based approach are:
1) the synthetic procedures to connect the acyl chains are
easier than with glycerol, as shown in this work; and 2) the
two acyl groups are bound in a symmetric form, which is not
realized in natural lecithin.[29] This could give parallel geo-
metric arrays of alkyl chains in membrane layers. For instance,
for radical polymerization of unsaturated lipids, the double
bonds oriented side by side will provide high polymerization
yields. In the interior of the lipid-porphyrin vesicle, two sheets
made of J-aggregated TPP planes presumably stack with a
lateral displacement. Exciton coupling theory confirmed our
predicted models, which is similar to that seen in the triclinic
unit cell of free-base TPP crystals, except for the slightly wider
interlayer distance (ca. 6.0 ä), and it reproduced the Soret
band bathochromic shifts in the visible absorption of the 6a
vesicles. The free-base lipid-porphyrin bilayers form a strong-
ly fluorescent aggregate, but show an extraordinarily short
triplet lifetime. We consider that this unique photophysical
behavior is due to the edge-to-edge alignment of the
porphyrin platforms. Furthermore, the N-coordinated high-
spin FeII complex 6c/DIm membranes can bind one equiv-
alent of O2 molecules. An aqueous solution of the 6c/DIm
vesicles with high concentration (>mm) is an entirely
synthetic O2 carrier that could act as a substitute for red
blood cells. These bilayer membranes possess a spherical
architecture consisting of 23000 porphyrins in water, and are a
new class of a supramolecular assembly that can realize
important biological reactions of porphyrinoids in nature: not
only O2 transport, but also metabolic oxidation and photo-
induced electron or energy transfer. In particular, there has
been substantial recent interest in the construction of cyclic
multiporphyrin assemblies, since a wheel-shaped bacterio-
chlorophyll circuit has been found in a light-harvesting
antenna complex.[30] More recently, we have prepared multi-
component vesicles made of differently metalated lipid-
porphyrins, in which efficient energy transfer due to the
tightest packing of the chromophores is observed. A new
investigation of the photoinduced electronic communication
in the lipid-porphyrin architecture is underway.
Figure 11. MCD spectra of vesicles of the lipid-porphyrinatoiron(ii)
complex 6c/DIm in phosphate buffer solution (pH 7.3) at 258C.
low-spin and is expected to show a similar A-term MCD band
with strong intensity; such a band was actually observed. In all
cases, the complicated pattern in the Q-band regions coin-
cided well with those previously reported (Table 2).[25]
Table 2. Absorption maxima of MCD spectra of the 6c/DIm vesicles in
phosphate buffer solution (pH 7.3) at 258C.
Atmosphere
N2
MCD lmax [nm]
416(‡), 429(À), 439(‡), 454(À), 519(‡)
531(‡), 537(À), 549(À), 568(‡), 574(À)
421(‡), 438(À), 531(‡), 541(‡)
555(À), 572(‡), 581(‡), 593(À)
422(‡), 438(À), 538(‡), 543(‡)
557(À), 578(‡), 545(À)
O2
CO
Difference IR spectra in solution of 6c/DIm vesicles for CO
versus 16O2 complexes displayed a distinct positive band at the
stretching frequency of coordinated CO at 1969 cmÀ1 (Ta-
ble 3). In the spectrum for the 16O2 versus the 18O2 adduct, we
Table 3. IR spectral data of vesicles of the lipid-porphyrinatoiron(ii)
complex 6c/DIm in phosphate buffer solution (pH 7.3) at 258C.
System
n(16O 16O)
n(18O 18O)
n(12C 16O)
6c/DIm vesicles
FeTpivPP/MIm[a]
hemoglobin[27]
O2Àor CO gas
O2
1155
1159
1107
1556
1145
1081
1075
1065
1969
1969
1951
2143
[a] FeTpivPP/MIm ˆ 5,10,15,20-Tetrakis(a,a,a,a-o-pivalamidophenyl)por-
phyrinatoiron(ii) 1-methylimidazole complex.[26]
observed the appearance of a positive band at 1155 cmÀ1 and a
negative peak at 1081 cmÀ1, both of which correspond to the
stretching frequencies of the coordinated O2 molecules. The
frequency shifts between 16O2 and 18O2 adducts (74 cmÀ1) is in
good agreement with the value calculated from the harmonic-
À
oscillator prediction of the O O stretching vibration. Their
Experimental Section
À
n(O O) bands are close to not only those of ionic superoxides,
but also to those of the bent end-on O2 bound to other
synthetic FeIITPP systems in benzene or Nujol.[26] On the basis
Materials and apparatus: All commercially available reagents of high-
purity grades were used without further purification, unless otherwise
5476
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Lipid-Porphyrin Bilayer Membranes
5469 5480
stated. Solvents were normally purified by distillation before use. Dichloro-
methane was refluxed over and distilled from P2O5. Tetrahydrofuran was
refluxed over and distilled from sodium wire. Water was deionized by using
an ADVANTEC GS-200 system. Thin-layer chromatography (TLC) was
carried out on 0.2 mm precoated plates of silica gel 60 F254 (Merck).
Purification was performed by column chromatography on silica gel
(Merck, Keiselgel 60, Art 7736) or Sephadex LH-60 gel (Amersham
Pharmacia Biotech). Infrared spectra were recorded on a JASCO FT/IR-
410 spectrometer. 1H NMR spectra were measured on a JEOL Lambda 500
spectrometer (500 MHz), and chemical shifts are expressed in parts per
million downfield from Me4Si as internal standard. FAB-MS spectra were
obtained on a JEOL JMS-SX102A spectrometer. Elemental analysis was
performed on a Yanagimoto MT3 CHN corder. UV/Vis absorption spectra
were recorded on a JASCO V-570 spectrophotometer, and steady-state
fluorescence spectra on a HITACHI F-4500 spectrofluorometer. Magnetic
circular dichroism spectra were obtained on a JASCO J-820 spectropo-
larimeter. Light-scattering measurements were performed on a COULT-
ER Model N4SD. All these measurements were normally carried out at
258C. 5,10,15,20-Tetrakis[a,a,a,a-o-(2,2-dimethyl-20-hydroxyicosanami-
do)phenyl]porphine [TPP-(C18OH)4] and 2-chloro-1,3,2-dioxaphospholane
2-oxide were prepared according to the previously reported procedures.[13]
5,10,15,20-Tetrakis{a,a,a,a-o-{20-[3-(2-octadecanoyloxymethyl-2-trityloxy-
methylpropanoxycarbonyl)propanoyloxy]-2,2-dimethylicosanamido}phe-
[13]
nyl}porphine (4): TPP-(C18OH)4 (607 mg, 0.299 mmol) was added to a
solution of 3 (2.4 g, 1.19 mol), DCC (676 mg, 3.27 mmol), and 4-DMAP
(182 mg, 0.3 mmol) in dry CH2Cl2 (25 mL). The mixture was stirred for 20 h
at room temperature in darkness. After evaporation of CH2Cl2, the residue
was redissolved in benzene, and the undissolved white DC urea was
removed by filtration. The mother liquor was brought to dryness on a
rotary evaporator. Then a CHCl3 solution of the obtained porphyrin was
added dropwise to excess MeOH to give a purple precipitate. The filtered
crude porphyrin was finally purified by column chromatography on silica
gel with CHCl3/Et2O (30:1) as eluent. The main band was collected and
dried at room temperature for several hours in vacuo to give compound 4 as
a purple solid (1.36 g, 94%). Rf ˆ 0.50 (CHCl3/Et2O, 30:1); IR (NaCl): nÄ ˆ
1156 (COC, ether), 1692 (C O, amide), 1739 (C O, ester), 3437 cmÀ1 (NH,
amide); UV/Vis (CHCl3): lmax ˆ 419, 513, 545, 587, 642 nm; 1H NMR
(500 MHz, CDCl3, 258C, TMS): d ˆ À2.6 (s, 2H; inner H), À0.2 (s, 24H;
2,2-dimethyl), 0.8 (t, 12H; (CH2)16CH3), 1.0 (s, 12H; CH3), 1.0 1.3 (m,
ˆ
ˆ
ˆ
112H; (CH2)14CH3, 128H; (CH2)16), 1.5 (m, 8H; OC( O)CH2CH2(CH2)14
8H; CH2CH2OC( O)), 2.2 (t, 8H; OC( O)CH2(CH2)14), 2.5 (s, 16H;
OC( O)(CH2)2C( O)O), 3.0 (m, 8H; CH2OTri), 4.0 (m, 16H;
C(CH2OC( O))2, 8H; CH2OC( O)(CH2)2), 7.1(s, 4H; amide), 7.2 7.3
(m, 60H; Tri), 7.4 (t, 4H; Ph H4), 7.7 (t, 8H; Ph H5), 7.8 (d, 4H; phenyl H3),
8.7 (d, 4H; Ph H6), 8.8 ppm (s, 8H; pyrrole).
,
ˆ
ˆ
ˆ
ˆ
ˆ
ˆ
2,2-Bis(hydroxymethyl)-1-trityloxypropane (1): A solution of trityl chlor-
ide (13 g, 46.6 mmol) in dry DMF (150 mL) was added dropwise over about
90 min to a solution of 2-hydroxymethyl-2-methyl-1,3-propanediol (6.0 g,
50 mmol) and pyridine (8.4 mL) in dry DMF (24 mL) at 858C under an N2
atmosphere. Then the mixture was stirred for a further 3 h at 958C. The
DMF and pyridine were removed in vacuo, and the residue was extracted
with CHCl3 and washed with water. The organic layer was dried over
anhydrous Na2SO4 and the solvent was evaporated. Excess methanol was
then poured into the mixture, and the resulting precipitate was filtered off.
The mother liquor was again evaporated. The product was separated by
column chromatography on silica gel with CHCl3/MeOH (10:1) as eluent.
The major band was collected and dried at room temperature for several
hours in vacuo to give compound 1 as a viscous solid (3.37 g, 19%). Rf ˆ
0.40 (CHCl3/MeOH, 10:1); IR (NaCl): nÄ ˆ 1154 (COC, ether), 3390 cmÀ1
(OH, alcohol); 1H NMR (500 MHz, CDCl3, 258C, TMS): d ˆ 0.9 (s, 3H;
CH3), 3.0 (s, 2H; CH2OTri), 3.5 (s, 4H; CH2OH), 7.2 7.4 ppm (m, 15H;
5,10,15,20-Tetrakis{a,a,a,a-o-{20-[3-(2-octadecanoyloxymethyl-2-hydroxy-
methylpropanoxycarbonyl)propanoyloxy]-2,2-dimethylicosanamido}phe-
nyl}porphine (5a): Boron trifluoride methanol complex (0.85 mL,
1.64 mmol) was added to a solution of 4 (1.33 g, 0.273 mmol) in dry CH2Cl2
(60 mL). After stirring for 4 h at room temperature, the green solution was
carefully poured into ice/water to stop the reaction, and the mixture
extracted with CHCl3. The organic layer was dried over anhydrous Na2SO4
and evaporated. Then a CHCl3 solution of the residue was added dropwise
to the excess MeOH to precipitate the porphyrin. The filtered crude
product was purified by column chromatography on silica gel with CHCl3/
MeOH (20:1) as eluent. The major fraction was collected and dried at room
temperature for several hours in vacuo to give compound 5a as a purple
solid (1.07 g, 92%). Rf ˆ 0.3 0.4 (CHCl3/MeOH, 20:1); IR (NaCl): nÄ ˆ
‡
1691 (C O, amide), 1738 (C O, ester), 3436 (NH, amide), 3502 cmÀ1 (OH,
alcohol); UV/Vis (CHCl3): lmax ˆ 419, 513, 545, 587, 642 nm; 1H NMR
(500 MHz, CDCl3, 258C, TMS): d ˆ À2.6 (s, 2H; inner H), À0.2 (s, 24H;
2,2-dimethyl), 0.8 (t, 12H; (CH2)16CH3), 1.0 (s, 12H; CH3), 1.0 1.3 (m,
ˆ
ˆ
ˆ
Tri). MS: m/z calcd for C24H26O3: 362.19; found: 362.26 [M ].
2-Hydroxymethyl-2-octadecanoyloxymethyl-1-trityloxypropane (2): Com-
pound 1 (3.37g, 9.30 mmol), stearic acid anhydride (5.12 g, 9.30 mmol), and
4-DMAP (568 mg, 4.65 mmol) were dissolved in dry THF (100 mL), and
the solution was refluxed for 12 h under an N2 atmosphere. After
evaporation of the solvent, excess methanol was added, and a white
precipitate was removed. The mother liquor was evaporated and the
resulting mixture was separated by column chromatography on silica gel
with CHCl3/Et2O (30:1) as eluent. The major band was collected and dried
at room temperature for several hours in vacuo to give compound 2 as a
viscous liquid (3.67 g, 63%). Rf ˆ 0.50 (CHCl3/Et2O, 30:1); IR (NaCl): nÄ ˆ
112H; (CH2)14CH3, 128H; (CH2)16), 1.7 (m, 8H; OC( O)CH2CH2(CH2)14
8H; CH2CH2OC( O)), 2.3 (t, 8H; OC( O)CH2(CH2)14), 2.6 (s, 16H;
OC( O)(CH2)2C( O)O), 3.4 (d, 8H; CH2OH), 4.1(m, 61H;
C(CH2OC( O))2, 8H; CH2OC( O)(CH2)2), 7.1(s, 4H; amide), 7.5 (t,
,
ˆ
ˆ
ˆ
ˆ
ˆ
ˆ
4H; Ph H4), 7.8 (t, 8H; Ph H5), 7.9 (d, 4H; Ph H3), 8.7 (d, 4H; Ph H6),
8.8 ppm (s, 8H; pyrrole); elemental analysis calcd (%) for C240H394N8O32
¥
C6H6 (3981.9):
N 2.71.
C 74.20, H 10.12, N 2.81; found: C 74.34, H 9.72,
1176 (COC, ether), 1737 (C O, ester), 3490 cmÀ1 (OH, alcohol); 1H NMR
ˆ
(500 MHz, CDCl3, 258C, TMS): d ˆ 0.8 (t, 3H; (CH2)16CH3), 0.9 (s, 3H;
Iron complex of 5a (5b): A solution of 5a (296 mg, 75.9 mmol) and 2,6-
lutidine (62.5 mL) in dry THF (40 mL) was added dropwise to anhydrous
FeBr2 (0.7 g) under dry N2. The mixture was refluxed for 2 h under an N2
atmosphere. The resulting solution was brought to dryness, and the residue
was extracted with CHCl3 and washed with water. After drying over
anhydrous Na2SO4, the organic layer was evaporated, and the crude
compound was separated by column chromatography on silica gel with
CHCl3/MeOH (20:1) as eluent. The major band was collected and dried at
room temperature for several hours in vacuo to give compound 5b as dark
purple solid (249 mg, 81%). Rf ˆ 0.2 0.3 (CHCl3/MeOH, 20:1); IR
ˆ
CH3), 1.3 (s, 28H; (CH2)14CH3), 1.6 (m, 2H; C( O)CH2CH2), 2.3 (t, 2H;
ˆ
C( O)CH2), 3.1(q, 2H; CH2OTri), 3.5 (q, 2H; CH2OH), 4.2 (q, 2H;
ˆ
CH2OC( O)), 7.2 7.5 ppm (m, 15H; Tri). MS: m/z calcd for C42H60O4:
‡
628.45; found: 627.5 [M À H].
2-(3-Carboxypropanoyloxy)methyl-2-octadecanoyloxymethyl-1-trityloxy-
propane (3): A solution of 2 (3.67 g, 5.85 mmol), succinic acid anhydride
(4.09 g, 40.9 mmol), and 4-DMAP (1.43 g, 11.7 mmol) in dry THF (170 mL)
was refluxed for 12 h under an N2 atmosphere. The resulting mixture was
brought to dryness on a rotary evaporator and extracted with CHCl3. The
organic layer was washed with water and dried over anhydrous Na2SO4.
The solvent was then evaporated, and the residue was separated by column
chromatography on silica gel with CHCl3/MeOH (20:1) as eluent. The
major band was collected and dried at room temperature for several hours
in vacuo to give compound 3 as a white solid (3.42 g, 83%). Rf ˆ 0.45
ˆ
ˆ
(NaCl): nÄ ˆ 1692 (C O, amide), 1737 (C O, ester), 3433 (NH, amide),
3460 cmÀ1 (OH, alcohol); UV/Vis (CHCl3): lmax ˆ 357, 417, 506, 577, 644,
683 nm.
5,10,15,20-Tetrakis{a,a,a,a-o-{20-[3-(2-octadecanoyloxymethyl-2-trimeth-
ylammonioethoxyphosphonatoxymethylpropanoxycarbonyl)propanoyl-
oxy]-2,2-dimethylicosanamido}phenyl}porphinatoiron(ii) bromide (6b):
2-Chloro-1,3,2-dioxaphospholane 2-oxide (372 mL) was added to a solution
of 5b (249 mg, 61.6 mmol) and triethylamine (0.637 mL, 4.58 mmol) in dry
CH2Cl2 (20 mL) at 08C under an argon atmosphere. After stirring the
mixture for 1h and an additional 1h at room temperature, the solvent was
removed under reduced pressure. Then, the resultant phosphate triester
was redisolved in dry DMF (30 mL), and anhydrous trimethylamine
ˆ
(CHCl3/MeOH, 20:1); IR (NaCl): nÄ ˆ 1160 (COC, ether), 1714 (C O,
carboxyl), 1741 cmÀ1 (C O, ester); 1H NMR (500 MHz, CDCl3, 258C,
ˆ
TMS): d ˆ 0.9 (t, 3H; (CH2)16CH3), 1.0 (s, 3H; CH3), 1.3 (s, 28H;
ˆ
(CH2)14CH3), 1.6 (m, 2H; C( O)CH2CH2(CH2)14), 2.2 (t, 2H;
ˆ
OC( O)CH2(CH2)15), 2.6 (m, 4H; (CH2)2COOH), 3.1(s, 2H; C H2OTri),
4.2 (d, 4H; CH2OC( O)), 7.3 7.5 ppm (m, 15H; Tri). MS: m/z calcd for
ˆ
‡
C46H64O7: 728.47; found: 751.53 [M ‡Na].
Chem. Eur. J. 2002, 8, No. 23
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5477

E. Tsuchida et al.
FULL PAPER
(4.0 mL) was immediately added. The mixture was sealed in a pressure
bottle and allowed to react for 1h at room temperature. After an additional
16 h at 608C, the solvent was removed, and the residue was washed with
MeOH. The remaining brown crude porphyrin was purified by gel column
chromatography on Sephadex LH-60 with benzene/MeOH (2:1) as eluent
to give a compound 6b as dark purple solid (202 mg, 79%). IR (NaCl): nÄ ˆ
applied perpendicular to the monolayer surface and slowly pulled up to
give porphyrin trilayers.
Exciton calculation
Monolayer: The center-to-center distance rmn of the dipole moments
between the origin porphyrin Po and the neighbor Pmn (Figure 4c) is given
by Equation (2)
ˆ
ˆ
1087 (POC, phosphate), 1216 (P O, phosphate), 1692 (C O, amide), 1735
À1
ˆ
(C O, ester), 3431cm (NH, amide); UV/Vis (CHCl3): lmax ˆ 353, 417,
501, 574, 635, 680 nm; elemental analysis calcd (%) for C260H440N12O44P4-
FeBr 2H2O (4734.07): C 65.97, H 9.45, N 3.55; found: C 66.23, H 9.46, N
3.18.
rmn ˆ [(ma)2 ‡ (nb)2]1/2
(2)
the angle qmn by Equation (3)
tanqmn ˆ nb/ma
Free-base porphyrin (6a): Phosphocholine groups were introduced into
compound 5a by using the same procedure as for 6b (Yield: 80%). IR
(3)
ˆ
ˆ
(NaCl): nÄ ˆ 1089 (POC, phosphate), 1236 (P O, phosphate), 1692 (C O,
and the exciton interaction energies Vmn for the Sx transitions and V' for
mn
À1
ˆ
amide), 1734 (C O, ester), 3430 cm (NH, amide); UV/Vis (CHCl3):
the Sy transitions by Equations (4) and (5).
lmax ˆ 422, 516, 549, 590, 646 nm; 1H NMR (500 MHz, CDCl3/CD3OD,
258C, TMS): d ˆ À2.6 (s, 2H; inner H), À0.4 (s, 24H; 2,2-dimethyl), 0.7 (t,
12H; (CH2)16CH3), 0.8 (s, 12H; CH3), 0.8 1.2 (m, 112H; (CH2)14CH3,
ˆ
À3
Vmn ˆ M2r (1 À 3cos2qmn
)
(4)
(5)
mn
À3
Vm' n ˆ M2r (1 À 3sin2qmn
)
128H; (CH2)16), 1.4 (m, 8H; OC( O)CH2CH2(CH2)14
,
8H;
mn
ˆ
ˆ
CH2CH2OC( O)), 2.1(t, 8H; OC( O)CH2(CH2)14), 2.4 (s, 16H;
‡
ˆ
ˆ
The total energy is the sum of the interactions of the infinite porphyrin
alignments on the 22 lines crossing the origin of the coordinate axes: V for
the Sx transitions and the V' for the Sy transitions.
OC( O)(CH2)2C( O)O), 3.0 (s, 36H; N (CH3)3), 3.4 (t, 8H; CH2
‡
ˆ
ˆ
N (CH3)3), 3.6 (s, 8H; P( O)OCH2), 3.8 (m, 16H; C(CH2OC( O))2,
8H; CH2OC( O)(CH2)2), 4.0 (t, 8H; CH2CH2N (CH3)3), 7.1(s, 4H;
amide), 7.3 (t, 4H; Ph H4), 7.6 (t, 8H; Ph H5), 7.7 (d, 4H; Ph H3), 8.4 (d,
4H; Ph H6), 8.6 ppm (s, 8H; pyrrole).
‡
ˆ
Bilayer: The core of the porphyrin Pkl in the upper layer is assumed to be
shifted by ‡4.02, ‡2.68, and ‡d ä in the x, y, and z directions, respectively,
from the center of the nearest porphyrin Pmn in the layer below. The center-
to-center distance rkl of the dipole moments between porphyrin Po and each
porphyrin Pkl is given by Equation (6).
Preparation of aqueous lipid-porphyrin solutions
Free-base lipid-porphyrin solution: A benzene/methanol stock solution of 6a
(50 mL, 2.0mm) was placed in a 5 mL round bottom flask and slowly
evaporated on a rotary evaporator under reduced pressure to give a thin film
of the porphyrin at the bottom. The film was then dried in vacuo for 3 h;
deionized water (5 mL) heated to 608C was then injected. The mixture was
homogenized by vortex mixing with several small glass beads (ca. 10) and
briefly ultrasonicated by a bath-type sonicator. The resulting red solution
(20 mm) was incubated for at least 12 h at room temperature before use.
rkl ˆ [(ma ‡ 4.02)2 ‡ (nb ‡ 2.68)2 ‡ d2]1/2
(6)
The angles qkl for the Sx transitions and q' for the Sy transitions are are
kl
given by Equations (7) and (8).
À3
cosqkl ˆ (ma ‡ 4.02)r
(7)
(8)
kl
Lipid-porphyrinatoiron(ii) solution. A benzene/methanol stock solution of
6b (50 mL, 2.0mm) and a solution of 1-dodecylimidazole in CHCl3 (110
165 mL, 2.0mm) was slowly evaporated by using a rotary evaporator, as
described above, to give a thin film at the bottom of the flask. The film was
dried in vacuo for 3 h, and phosphate buffer (pH 7.3, 1m m, 5 mL) heated at
608C was added. Homogenization by a tip-type ultrasonicator (60 mW,
3 min) in a water bath gave a yellow-brown solution, which was incubated
for at least 12 h at room temperature. Then the 6b solution (20 mm) was
deaerated by N2 bubbling; addition of aqueous sodium dithionate (15mm,
20 mL) gave a solution of the bis-imidazole iron(ii) complex 6c. Excess
sodium dithionate was removed by quick passage through a Sephadex G-25
(coarse) column.
À3
cosqk'l ˆ (nb ‡ 2.68)r
kl
The exciton interaction energy Vkl for the Sx transitions and V' for the Sy
kl
transitions is given by Equations (9) and (10).
À3
Vkl ˆ M2r (1 À 3cos2qkl)
(9)
kl
À3
Vk'l ˆ M2r (1 À 3cos2q' †
(10)
kl
kl
The total energy is the sum of the interactions between Po and
46 porphyrins on the upper layer within a circle of about 5 nm: VI for the
Sx transitions and V' for the Sy transitions. Then the overall excitonic
I
interaction in our bilayer model is finally defined as the sum of Vand VI for
Transmission electron microscopy (TEM): The negatively stained speci-
mens for TEM were prepared as in previously reported procedures.[11f] The
grids were observed in a JEOL JEM-100CX electron microscope at an
accelerating voltage of 100 kV.
the Sx transitions and V' and V' for the Sy transitions.
I
Excited-state lifetimes: Singlet lifetimes of 6a were measured by using a
Horiba NAES-500 nanosecond fluorometer with an N2 lamp (excitation-
side band-path filter: MC560, emission-side color filter: R62). The samples
Scanning force microscopy (SFM): A droplet of porphyrin solution (1
5mm) was pipetted onto freshly cleaved, highly oriented pyrolytic graphite
(HOPG STM-1, Advanced Ceramics Co.). After 1 min, excess fluid was
carefully blotted off by filter paper, and air-drying was carried out for a
further 1h. SFM measurements were carried out on a Nanoscope III
system (Digital Instruments Inc.) in the tapping mode under ambient
laboratory conditions. Silicon cantilevers (length 125 mm) with a spring
constant between 21and 78 Nm À1 and a resonance frequency in the range
260 410 kHz were used. The scanning rate was usually 1.0 Hz. Imaging
was simultaneously performed by displaying the amplitude signal and the
height signal.
were held in
a cuvette (optical path length 1cm). Triplet lifetime
measurements on the nanosecond timescale were performed by using a
Unisoku TSP-600 time-resolved spectrophotometer system with a Con-
tinuum Surelite I-10 Q-switched Nd:YAG laser, which generated a second-
harmonic (532 nm) pulse of 6 ns duration with an energy of 200 mJ per
pulse (10 Hz). A 150 W Xenon arc lamp was used as the monitor light
source. The triplet decay of 6a was monitored by transient absorption at
450 nm. The concentration of the lipid-porphyrin was 1.7mm, and experi-
ments were carried out at 258C. Time-resolved transient absorption spectra
after nanosecond laser flash photolysis were measured by an ANDOR
DH520-18F-WR ICCD detector with an ORIEL MS-257 imaging mono-
chromator. The excitation laser pulse with a wavelength of 516 nm was
generated from the THG (355 nm; pulse width, 5 ns) of a Spectron
SL803G-10 Nd:YAG laser by using a GWU BBO-OPO photoparametric
oscilator. A pulsed xenon flash lamp (10 W, 10 Hz, 100 ns pulse width) was
used as the monitor light source. The path length of the cuvette was 10 mm,
and all measurements were carried out by a Tokyo Instruments two-
channel simultaneous detection system at 258C.
Langmuir Blodgett layer and P A isotherm: A monolayer of lipid-
porhyrin was prepared at the air/water interface in a Nippon Laser &
Electronics LB Film Deposition System. Compound 6a was spread from a
1.17mm solution in CHCl3/MeOH (10:1) onto the pure water subphase.
The area per 6a molecule was estimated from its surface pressure versus
molecular area (P A) isotherm. Furthermore, the monolayer was pulled
onto a clean glass plate to give a lipid-porphyrin monolayer, which was
5478
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Lipid-Porphyrin Bilayer Membranes
5469 5480
O2 Binding: O2-binding to the lipid-porphyrinatoiron(ii) complex is ex-
pressed by Equation (11):
M. Lehn, Chem. Commun. 1996, 337 338; m) C. Ikeda, N. Nagahara,
E. Motegi, N. Yoshioka, H. Inoue, Chem. Commun. 1997, 1759 1760;
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7834; o) K. Sugou, K. Kitajima, T. Iwaki, Y. Kuroda, J. Am. Chem. Soc.
2002, 124, 1182 1183.
FeLP ‡ O2 > FeLP O2
(11)
where FeLP is lipid-porphyrinatoiron(ii) complex 6c. The apparent O2-
binding affinity (gaseous pressure at half O2 binding for 6c, P1/2) was
determined by spectral changes at various partial pressure of O2, as
described in the literature.[12] The half-life of the dioxygenated species was
determined by the time dependence of the absorption intensity at 540 nm,
which is due to the O2 adduct.
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Acknowledgements
We thank Prof. Naoki Yoshioka and Prof. Hidenari Inoue, Keio University
(Yokohama) for the LB measurements, and JASCO Corporation for the
use of the MCD spectrometers. This work was partially supported by
Health Science Research Grants (Artificial Blood Project) of the Ministry
of Health, Labor and Welfare and by a Grant-in-Aid for Scientific
Research (No. 13650938) from the Ministry of Education, Culture, Sports,
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Received: May 22, 2002 [F4116]
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