A novel system of self-reproducing giant vesicles

Article abstract of DOI:10.1021/ja029379a

Novel self-reproducing giant vesicles, consisting of a vesicular amphiphile with an imine group in its hydrophobic chain, were constructed. This vesicular amphiphile, the product of a dehydrocondensation reaction between amphiphilic aldehyde and a lipophi

Full text of DOI:10.1021/ja029379a

Published on Web 06/13/2003
A Novel System of Self-Reproducing Giant Vesicles
Katsuto Takakura, Taro Toyota, and Tadashi Sugawara*
Contribution from the Department of Basic Science, Graduate School of Arts and Sciences,
The UniVersity of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan
Received November 18, 2002; E-mail: suga@pentacle.c.u-tokyo.ac.jp
Abstract: Novel self-reproducing giant vesicles, consisting of a vesicular amphiphile with an imine group
in its hydrophobic chain, were constructed. This vesicular amphiphile, the product of a dehydrocondensation
reaction between amphiphilic aldehyde and a lipophilic aniline derivative, could be prepared within the
giant vesicles. When a protected form of the aldehyde precursor was added to a suspension of giant vesicles
containing the lipophilic aniline precursor and a catalyst, dehydrocondensation between the two precursors
took place inside the vesicles and produced the same amphiphile as the one which constitutes the original
vesicle. The newly formed amphiphiles self-assembled in the inner water pool to form small vesicles, which
were eventually extruded through the outer layer of the original vesicle to the bulk water. Accordingly, this
kinetic system can be designated as a self-reproducing system of giant vesicles.
Scheme 1. Formation of Bolaamphiphile Occurring in Vesicular
Membrane
Introduction
Giant vesicles (GVs) are composed of amphiphiles and have
spherical lamellar structures with diameters larger than 1 µm.
Since the structure and the dynamic behavior of GVs are similar
to those of biological cell membranes, such vesicles have drawn
much attention as plausible models for artificial cells.1 GVs
undergo characteristic morphological changes induced by varia-
tions in osmotic pressure or temperature or by the presence of
additives.2 It is particularly interesting that morphological
changes can also be induced by chemical transformation of the
amphiphiles within the vesicular membrane.3
a self-reproducing system because the product of the coupling
reaction is not a component of the original vesicle.
Recently, we discovered that a vesicular membrane serves
as an efficient reaction environment for an imine-coupling
between a benzaldehyde-type amphiphile and an aniline-type
amphiphile to produce a bolaamphiphile,4 which forms a
monolayer membrane due to the presence of polar heads at both
ends of the hydrophobic chain (Scheme 1).5 Although this
kinetic system undergoes morphological changes that result in
an increase in the number of vesicles, it cannot be regarded as
Herein we report a novel self-reproducing system of giant
vesicles as represented in Figure 1. An amphiphile, V, bearing
an imine group in its hydrophobic chain, was found to form
GVs when 5 mol % of cholesterol was added as a stabilizer.
Since amphiphile V is a coupling product of amphiphilic
aldehyde A and 4-octylaniline (B), if amphiphilic aldehyde A
is added to the suspension of GVs containing lipophilic aniline
B, amphiphile V can be prepared by the dehydrocondensation
between above two precursors as in the case of the precedent
example. In this sense, these two precursors could be regarded
as nutrients for amphiphile V. To prevent the dehydroconden-
sation between A and B from occurring in the bulk water, we
protected the formyl group of A with a 1,3-dioxolane group to
produce “locked” precursor, A′. In addition, we incorporated
C, the catalyst for the removal of the protective group into the
vesicular membrane (Figure 1). In fact, 1H NMR spectroscopy
revealed that additional V molecules were produced under the
above reaction condition.
(1) (a) Luisi, P. L.; Walde, P.; Oberholzer, T. Curr. Opin. Colloid Interface
Sci. 1999, 4, 33-39. (b) Szostak, J. W.; Bartel, D. P.; Luisi, P. L. Nature
2001, 409, 387-390.
(2) (a) Sackmann, E.; Duwe, H.-P.; Engelhardt, H. Faraday Discuss. Chem.
Soc. 1986, 81, 281-290. (b) Ka¨s, J.; Sackmann, E. Biophys. J. 1991, 60,
825-844. (c) Menger, F. M.; Balachander, N. J. J. Am. Chem. Soc. 1992,
114, 5862-5863. (d) Menger, F. M.; Gabrielson, K. J. Am. Chem. Soc.
1994, 116, 1567-1568. (e) Menger, F. M.; Lee, S. J.; Keiper, J. S. Chem.
Commun. 1998, 957-958. (f) Menger, F. M.; Angelova, M. I. Acc. Chem.
Res. 1998, 31, 789-797. (g) Nomura, F.; Nagata, M.; Inaba, T.; Hiramatsu,
H.; Hotani, H.; Takiguchi, K. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2340-
2345.
(3) (a) Wick, R.; Walde, P.; Luisi, P. L. J. Am. Chem. Soc. 1995, 117, 1435-
1436. (b) Jaeger, D. A.; Schilling, C. L., III; Zelenin, A. K.; Li, B.; Kubicz-
Loring, E. Langmuir 1999, 15, 7180-7185. (c) Kahya, N.; Pe´cheur, E.-I.;
de Boeji, W. P.; Wiersma, D. A.; Hoekstra, D. Biophys. J. 2001, 81, 1464-
1474. (d) Jaeger, D. A.; Clark, T., Jr. Langmuir 2002, 18, 3495-3499.
(4) Takakura, K.; Toyota, T.; Yamada, K.; Ishimaru, M.; Yasuda, K.; Sugawara,
T. Chem. Lett. 2002, 404-405.
(5) Fuhrhop, J.-H.; Ko¨nig, J. Membranes and Molecular Assemblies: The
Synkinetic Approach; The Royal Society of Chemistry: Cambridge, 1994;
pp 50-55.
This finding prompted us to use differential interference
contrast optical microscopy to monitor such morphological
changes. When the reaction partner A′ was added to a suspen-
sion of giant vesicles containing B, a drastic morphological
9
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J. AM. CHEM. SOC. 2003, 125, 8134-8140
10.1021/ja029379a CCC: $25.00 © 2003 American Chemical Society

Novel System of Self-Reproducing Giant Vesicles
A R T I C L E S
Figure 1. Schematic illustration of the self-reproducing giant vesicles: (i) locked precursor A′ is incorporated into a vesicle composed of V and catalyst
C and is unlocked to produce reactive precursor A; (ii) A reacts with lipophilic precursor B inside the vesicle to form vesicular molecule V; (iii) new vesicles
are generated as V is produced; (iv) generated vesicles are extruded through the membrane to the bulk water.
change, such as birthing2d or separation,2e took place inside
the original vesicle. Such a reproducing system can be regarded
as a more progressed system compared to the previously
reported ones3a because the reproducing process takes place
within the vesicle, accompanying the transfer of chemical
substances from the bulk water to the inner water pool of the
vesicle through the vesicular membranes.
synthetic amphiphile V turned out to form giant vesicles when
5 mol % of cholesterol was mixed as an additive.
Catalyst C, in which a catalytic moiety (imidazolium
hydrochloride) and a green fluorescent BODIPY7 moiety are
connected by a long alkyl chain, was prepared as shown in
Scheme 3. The presence of a fluorescent probe in catalyst C is
advantageous because the probe enables us to visualize the site
where the catalytic reaction takes place. Condensation of the
starting benzaldehyde derivative6 with 2,4-dimethylpyrrole gave
a dipyrromethane intermediate, which was treated with BF3‚
Et2O to form a BODIPY derivative. A nucleophilic substitution
reaction between imidazole and the fluorophore with a bromo-
decyloxy tail and subsequent acidification afforded catalyst C.
The absorption maximum in the fluorescent spectrum of catalyst
C was observed at 500 nm, and the emission maximum appeared
at 519 nm. The fluorescence quantum yield for catalyst C was
determined to be 0.58 upon excitation at 500 nm by using N,N′-
bis(1-hexylheptyl)-3,4:9,10-perylenebis(carboximide) as a refer-
ence compound.8
Results and Discussion
1. Synthesis and Properties of Amphiphiles. Amphiphilic
precursor A was synthesized according to the procedure reported
by Kunitake.6 Precursor A and vesicular amphiphile V were
synthesized according to the procedures outlined in Scheme 2.
The 1,3-dioxolane derivative of 4-(10′-bromo-n-decyloxy)benz-
aldehyde was treated with trimethylamine to afford locked
precursor A′. A dynamic light scattering measurement revealed
that locked precursor A′ self-assembled to form micelles
(diameter, 4 nm; standard deviation ) 1 nm). In contrast,
lipophilic precursor B was dispersed in water as an emulsion
of oil droplets. The acid-catalyzed dehydrocondensation between
A and B in EtOH produced azomethine derivative V. The
(7) Treibs, A.; Kreuzer, F.-H. Liebigs Ann. Chem. 1968, 718, 208-223.
(8) Langhals, H.; Karolin, J.; Johansson, B.-A° . J. Chem. Soc., Faraday Trans.
1998, 94, 2919-2922.
(6) Okahata, Y.; Kunitake, T. J. Am. Chem. Soc. 1979, 101, 5231-5234.
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Takakura et al.
Scheme 2. Synthesis of Vesicular Molecule V and Its Precursors,
A, A′, and B
Scheme 3. Synthesis of Catalytic Amphiphile C
Figure 2. Formation of V from various combination of starting materials:
(a) A and B (]); (b) A′ and B (4); (c) A′, B, and 5 mol % of C (O).
Figure 3. Differential interference contrast optical micrograph of a NIGV
composed of V, B, C, and cholesterol (20:20:1:2). An oil droplet with a
diameter of ca. 5 µm is included in the inner water pool of the vesicle,
which has a diameter of 20 µm. The scale bar corresponds to 10 µm.
2. Formation of Vesicular Amphiphile in Water. The
formation of vesicular amphiphile V by dehydrocondensation
between A and B in water was monitored by 1H NMR
spectroscopy. The progress of the conversion under the various
reaction conditions is plotted in Figure 2. When unlocked
precursor A and lipophilic B were mixed, the amount of product
V increased smoothly for the first 2 h, and the equilibrium was
reached after 5 h (plot a). On the other hand, V formed much
more slowly in the reaction between locked precursor A′ and
B (plot b). In contrast, the reaction was greatly accelerated by
the addition of 5 mol % of catalyst C and equilibrium was
reached after 15 min (plot c). These results show that catalyst
C catalyzed not only the cleavage of the protective group but
also the imine-coupling reaction.
3. Preparation of Nutrient-Including Giant Vesicles
(NIGVs). It is interesting to study the dynamic behavior of the
giant vesicle composed of V and a catalytic amount of C when
precursors A and B are added. Since lipophilic B formed
relatively large oil droplets, it dissolved only slightly into the
vesicular membrane composed of V when added to the bulk
water. To overcome this difficulty, we prepared a GV consisting
of V, C, and B. In other words, we set up a situation in which
one of the precursors of the vesicular molecule had been
incorporated into the vesicle in advance. We prepared GVs
composed of a mixture of V, B, C, and cholesterol (20:20:1:2)
according to the film-swelling method, and after the suspension
of GVs stood for 2 days, we found that most of them included
oil droplets in their inner water pool (Figure 3). 9 It was revealed
by fluorescence microscopy that the catalyst C was distributed
not only in the original vesicular membrane but also in oil
droplets included in the vesicle.
(9) Oil droplets were not present in the inner water pool of the giant vesicles
immediately after they were produced by the film-swelling method. We
assume that the oil droplets separated from the vesicular membranes over
time because the membranes were supersaturated with B. We observed oil
droplets not only in the inner water pool but also in the bulk water, owing
to the separation of oil droplets into the outer water phase.
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Novel System of Self-Reproducing Giant Vesicles
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Figure 4. Differential interference contrast optical micrographs of morphological changes in a NIGV: (a)-(c) images obtained 10, 20, and 23 min, respectively,
after addition of a micellar solution of A′; (d), (e) images showing the release of inner vesicles at 26 min after the addition of A′. The scale bars correspond
to 10 µm. The vesicular membranes formed at site P, birthing occurred at site Q, and separation occurred at site R.
vesicles came out through the outer membrane (birthing2d) at
the site Q. Accompanied by the birthing process, the peeling
of the outermost layer of the original vesicle (separation2e) also
took place at the site R. When the same experiment was carried
out on the NIGVs including several oil droplets, they also
exhibited the same dynamics, that is, the membrane formation
from the included oil droplets and the subsequent morphological
change. The result supports that the dynamics depicted in Figure
4 is a reproducible phenomenon.
Table 1. Types of Aggregates Formed from Aqueous Mixtures of
V, B, C, and Cholesterol
ratio of components
rujln
(V:B:C:cholesterol)
shape of aggregate
1
2
3
4
5
40:0:1:2
30:10:1:2
20:20:1:2
10:30:1:2
0:40:1:2
GV
GV
GV including oil droplets (NIGV)
oil droplet
oil droplet
To clarify that the composition of the extruded vesicles are
the same as that of the original ones, we set up a simplified
model reaction system shown in Figure 5a. In the current
vesicular system, catalyst C was dissolved in the oil droplets
in the inner water pool as well as in the membrane of the vesicle
(Figure 1). In the model system, however, C was dissolved only
in the oil droplets of lipophilic precursor B. The dynamics of
the oil droplets after the addition of a solution of locked
precursor A′ was monitored by mean of both fluorescence
microscopy and differential interference contrast optical mi-
croscopy.
To examine the ratio of the compositions for preparing GVs
that contained oil droplets, we systematically varied the ratio
and monitored the types of aggregates that were produced (Table
1). V/B ratios larger than 1 (runs 1 and 2) produced GVs that
contained no oil droplets, while ratios smaller than 1 produced
only oil droplets (runs 4 and 5). However, whenever the ratio
was close to 1 (run 3), most of GVs include oil droplets. On
the basis of these findings, we concluded that the oil droplets
that separated from the vesicular membrane were composed
mainly of lipophilic aniline B. Since B is one of the components,
or “nutrients,” of vesicular amphiphile V, we designate these
GVs as nutrient-including giant vesicles (NIGVs).
4. Morphological Change in NIGVs. The morphological
changes in NIGVs (V:B:C:cholesterol ) 20:20:1:2) were
monitored by means of differential interference contrast optical
microscopy at 23 °C after the addition of a micellar solution of
A′. Although all the NIGVs underwent some kinds of morpho-
logical change, the mode of the morphological change depended
on the volume ratio of the outer vesicle to the included oil
droplets.
When a solution of A′ was added to the suspension of B
containing fluorescent catalyst C (site L), vesicular membranes
were generated from the hydrophobic surface of the oil droplets
and they grew as the oil droplets decreased in size (Figure 5b-
d) with high reproducibility. In a control experiment carried
out on the right side of the same slide (site R), no morphological
changes occurred when A′ was added to a suspension of oil
droplets of B without containing C (Figure 5e). The above
results unequivocally demonstrated that the vesicles were
generated only after locked A′ had been transformed to reactive
A in the presence of catalyst C,10 giving rise to the vesicular
amphiphile V through the dehydrocondensation.
Among NIGVs, GVs containing several oil droplets exhibited
dramatic dynamics (Figure 4). About 10 min after A′ was added,
the oil droplets inside NIGV started to decrease in size, and
after 20 min, even the largest oil droplet was about to disappear.
In compensation for the disappearance of the oil droplets, new
vesicles were generated inside the original NIGV and the inner
The dynamics discovered in the above experiments rational-
izes the vesicular reaction system schematically depicted in
Figure 1. Namely, when amphiphilic precursor A′ is added to
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Takakura et al.
Figure 5. (a) Schematic illustration of the mixing chamber used to observe the two kinds of oil droplets: B plus C (green circle), B (gray circle). (b)-(d)
Representative microscopic images of the morphological changes in the oil droplet composed of B and 5 mol % of C at site L at 0, 60, and 90 min,
respectively, after the addition of a solution of A′ (red dots). (e) Representative microscopic image of the oil droplet composed of only B. The scale bars
correspond to 10 µm. The upper images were recorded by fluorescence microscopy and the lower by differential interference contrast optical microscopy.
the bulk water, it dissolves into the vesicular membrane
containing catalyst C and is converted to deprotected precursor
A. Then vesicular amphiphile V is produced in the inner water
pool of NIGV through the dehydrocondensation between A and
lipophilic precursor B which exists as the encapsuled oil droplet,
as proved by the model experiment (Figure 5). It should be noted
that the addition of the amphiphilic precursor A′ triggers the
increase of the number of GVs through birthing and separation
phenomena. Although all the extruded vesicles are not neces-
sarily the newly formed vesicles but may contain some
preexisting ones in the original vesicles of a multi-lamella type,
it can be said that the composition of the extruded vesicles are
the same as the original ones. Accordingly, the present vesicular
system can be regarded as a self-reproducing one because the
GV of the first generation separated into GVs of the second
generation composed of the same amphiphile as the first
generation.
giant multilamellar vesicles, Menger et al. have reported that
the birthing of a GV composed of didodecyldimethylammonium
bromide (DDAB) takes place immediately after the addition of
octyl glucoside, which does not react with DDAB.2d In contrast,
the morphological changes in our system occurred only after
an induction period of ca. 20 min, as in case of our previously
reported system.4 The induction period corresponds to the time
period necessary for the production of a sufficient amount of V
that is enough to induce the morphological changes. Jaeger et
al. have reported that vesicles are destroyed by the reaction with
2-chloroethyl phenyl sulfide11 or with O-methyl S-benzyl
phenylphosphonothiolate3d,11 because the vesicular molecule is
transformed into a nonamphiphilic molecule. In light of these
experimental results, the morphological changes we observed
in our reaction system may have resulted from the amphiphilic
nature of V.
Our reaction system can be described as a novel self-
reproducing system because the new vesicles are composed of
the same component as the original vesicle. Although the self-
reproduction of an oleic acid/oleate giant vesicle has been
reported,3a one can argue that the alkaline hydrolysis of oleic
anhydride really occurs within the vesicular membrane. The
advantage of our system is that we visually confirm the
generation of the new GVs within the original vesicle.
5. Significance of the Self-Reproducing System. The most
characteristic feature of morphological changes in our system
is that they were induced by a chemical transformation occurring
in the inner water pool of the GV. In their pioneering work on
(10) To confirm that catalyst C firmly adhered to the oil droplets, we carried
out the following experiment. We used a semipermeable membrane
(SPECTRUM Por6RC) to separate a suspension of fluorescent oil droplets
composed of B and 5 mol % of C from a suspension of nonfluorescent oil
droplets of B alone. Even after standing for 5 days, the oil droplets without
C did not fluoresce at all. This result shows that C has sufficient affinity
for the hydrophobic emulsion.
(11) Jaeger, D. A.; Li, B. Langmuir 2000, 16, 5-10.
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under reduced pressure, and the resulting solid was washed with acetone
to afford A′ (224 mg, 65%).
1H NMR (270 MHz, DMSO-d6): 7.32 (2H, d, J ) 8.6 Hz), 6.89
(2H, d, J ) 8.6 Hz), 5.62 (1H, s), 4.05-3.87 (4H, m), 3.01 (9H, s),
1.58-1.74 (4H, m), 1.20-1.46 (12H, m). HRMS-FAB (m/z): [M -
Br]+ calcd for C22H38NO3, 364.2852; found 364.2863.
[10-[4′-(4′′-Octylphenylimino)phenoxy]decyl]trimethylammoni-
um Bromide (V). Unlocked precursor A (400 mg, 1.0 mmol), which
was prepared by the literature procedure,6 and 4-octylaniline (B) were
dissolved in absolute ethanol (2 mL), and a catalytic amount of acetic
acid was added. The mixture was refluxed for 12 h and then cooled.
After the evaporation of ethanol, the residue was washed with acetone
to afford amphiphilic imine derivative V (512 mg, 87%).
1H NMR (270 MHz, CDCl3): 8.39 (1H, s), 7.82 (2H, d, J ) 8.9
Hz), 7.18 (2H, d, J ) 8.1 Hz), 7.12 (2H, d, J ) 8.1 Hz), 6.96 (2H, d,
J ) 8.9 Hz), 4.02 (2H, t, J ) 6.3 Hz), 3.55 (2H, m), 3.44 (9H, s), 2.61
(2H, t, J ) 7.8 Hz), 1.88-1.20 (28H, m), 0.88 (3H, t, J ) 6.6 Hz).
HRMS-FAB (m/z): [M - Br]+ calcd for C34H55N2O, 507.4314; found
507.4329.
1-[10′-{4′′-(1′′′,3′′′,5′′′,7′′′-Tetramethyl-4′′′,4′′′-difluoro-4′′′-bora-
3a′′′,4a′′′-diaza-s-indacen-8′′′-yl)phenoxy}decyl]imidazole Hydro-
chloride (C). 4-(10-Bromo-n-decyloxy)benzaldehyde (342 mg, 1.0
mmol) and 2,4-dimethylpyrrole (190 mg, 2.0 mmol) were dissolved in
dichloromethane (25 mL), and the solution was degassed by bubbling
nitrogen (30 min). One drop of trifluoroacetic acid was added, and the
solution was stirred for 12 h at room temperature under a nitrogen
atmosphere. Then, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (0.25 g,
1.1 mmol) was added, and the mixture was stirred for 3 h. The reaction
mixture was washed with saturated aqueous NaHCO3, and brine, dried
over Na2SO4, filtered, and concentrated. The crude product was purified
by alumina column chromatography using CHCl3 as an eluent to afford
a dipyrromethane derivative as a colored oil. The product was dissolved
in chloroform (10 mL), together with BF3‚Et2O (0.72 mL, 3.8 mmol)
and triethylamine (0.50 mL, 6.8 mmol), and the mixture was refluxed
for 1 h. The reaction mixture was washed with saturated aqueous
NaHCO3 and brine, dried over Na2SO4, filtered, and concentrated. The
fluorescent oil was purified by silica gel column chromatography using
hexane/ethyl acetate to afford a fluorescent alkyl bromide (73 mg, 13%)
as a vermilion powder.
Wick et al. have reported a vesicle-producing system in which
two kinds of precursors, a lisophospholipid and a fatty acid,
and an enzyme catalyst are artificially incorporated into a giant
vesicle by means of microinjection.12 Although new GVs are
generated by the formation of a double-chain phospholipid, they
are not released in this case. In contrast, the characteristic of
our system is that the inner vesicles were released, especially
when amphiphilic precursor A′ was added to the bulk water.
The dynamics is extremely interesting from the viewpoint that
they bear a topological resemblance to some cellular processes,
such as the budding of yeast.13 Moreover, it is advantageous to
supply GVs continuously with amphiphilic precursor as nutrients
for developing a robust and recursive self-reproducing system.
It is also to be noted that the current system is interesting
from a viewpoint of the chemical complex system.14
A
microscopic-level chemical transformation, occurring within a
vesicle, induces a macroscopic-level morphological change in
the vesicle. This morphological change, in turn, affects the
microscopic process because the change modifies the reaction
environment itself. If these two dynamics, molecular transfor-
mations and morphological changes, are suitably balanced, the
self-reproducing vesicular system renders an essence of the
cellular dynamics which draws a recursive trajectory.
Summary
We constructed a novel self-reproducing vesicular system in
which a giant vesicle produced daughter vesicles in its inner
water pool by means of a coupling reaction between the two
precursors of the amphiphile that constituted the original vesicle.
The generation of the new vesicles in the inner water pool and
their release to the bulk water were visually confirmed. Such
dynamics are extremely interesting in that they bear a topological
resemblance to some cellular processes.
Experimental Section
1H NMR (270 MHz, CDCl3): 7.14 (2H, d, J ) 8.6 Hz), 6.98 (2H,
d, J ) 8.6 Hz), 5.97 (2H, s), 4.00 (2H, t, J ) 6.6 Hz), 3.41 (2H, t, J
) 6.7 Hz), 2.55 (6H, s), 1.90-1.73 (4H, m), 1.58-1.25 (18H, m).
HRMS-FAB (m/z): [M]+ calcd for C29H38BBrF2N2O, 558.2229; found
558.2232.
The alkyl bromide (50 mg, 0.09 mmol) was mixed with imidazole
(68 mg, 1 mmol) in EtOH (0.5 mL), and the mixture was heated at
80 °C for 12 h. After the mixture was cooled, diethyl ether was added,
and the organic layer was washed with 0.5 N aqueous NaOH, dried
over Na2SO4, filtered, and concentrated. After the evaporation of diethyl
ether, the fluorescent N-substituted imidazole (37 mg, 76%) was
obtained as a vermilion powder.
1H NMR (270 MHz, CDCl3): 7.47 (1H, s), 7.14 (2H, d, J ) 8.9
Hz), 7.05 (1H, s), 6.98 (2H, d, J ) 8.9 Hz), 6.90 (1H, s), 5.97 (2H, s),
4.00 (2H, t, J ) 6.6 Hz), 3.93 (2H, t, J ) 7.0 Hz), 2.55 (6H, s), 1.88-
1.56 (4H, m), 1.53-1.18 (18H, m). HRMS-FAB (m/z): [M + H]+
calcd for C32H42BF2N4O, 547.3420; found 547.3436.
Finally, the N-substituted imidazole (35 mg, 0.06 mmol) was
dissolved in 1 N hydrochloric acid, and the solvent was removed under
reduced pressure to afford fluorescent imidazolium hydrochloride C
(37 mg, quantitative) as a vermilion powder.
General. All commercially available reagents were purchased from
Tokyo Kasei Co. or Aldrich Co. and were used without further
1
purification. Reaction solvents were distilled. H NMR spectra were
recorded on a JEOL GSX-270 spectrometer. UV-vis spectra were
recorded on a JASCO V-570 spectrometer. Fluorescence spectra were
recorded on a Shimadzu RF-503A spectrometer. High-resolution fast
atom bombardment mass spectra (HRMS-FAB) were recorded on a
JEOL JMS-700 spectrometer with m-nitrobenzyl alcohol as matrix.
[10-[4′-(1′′,3′′-Dioxolan-2′′-yl)phenoxy]decyl]trimethylammoni-
um Bromide (A′). 4-(10-Bromo-n-decyloxy)benzaldehyde (683 mg,
2.0 mmol), which was prepared according to a literature procedure,6
and ethylene glycol (140 mg, 2.3 mmol) were dissolved in benzene
(7.5 mL), and p-toluenesulfonic acid monohydrate (38 mg, 0.2 mmol)
was added. The mixture was refluxed for 15 h and then cooled. The
resulting solution was diluted with benzene, washed with 10% aqueous
sodium hydroxide and brine, dried over Na2SO4, filtered, and concen-
trated. The crude mixture was purified by recrystallization from THF/
n-hexane to afford the 1,3-dioxolane derivative (601 mg, 78%) as
colorless crystals. The obtained 1,3-dioxolane derivative (385 mg, 1.0
mmol) was added to a 30% aqueous solution of trimethylamine (10
mL), and the suspension was heated at 80 °C for 30 h. After the
suspension was cooled, water and excess trimethylamine were removed
1H NMR (270 MHz, CDCl3): 9.66 (1H, br, s), 7.38 (1H, s), 7.14-
7.11 (3H, m), 6.98 (2H, d, J ) 8.6 Hz), 5.97 (2H, s), 4.34 (2H, br, s),
4.00 (2H, t, J ) 6.4 Hz), 2.54 (6H, s), 2.00-1.72 (4H, m), 1.55-1.18
(18H, m). UV-vis (CH2Cl2): λmax (log ꢀ) 500 (4.7), 336 nm (4.0).
Fluorescence (CH2Cl2): λem 519 nm (λex 475 nm). HRMS-FAB (m/z):
[M - Cl]+ calcd for C32H42BF2N4O, 547.3420; found 547.3409.
(12) Wick, R.; Luisi, P. L. Chem. Biol. 1996, 3, 277-285.
(13) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P.
MOLECULAR BIOLOGY OF THE CELL, 4th ed.; Garland Science: New
York, 2002; pp 969-970.
(14) Kaneko, K.; Furusawa, C. Phys. A 2000, 280, 22-33.
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A R T I C L E S
Takakura et al.
Dynamic Light Scattering (DLS) Measurement. An aqueous
solution of A′ (10 mM) was used to perform DLS experiments on a
NIKKISO Microtrac 150 at room temperature after the solution had
been sonicated for 5 min.
dispersion was injected from an open site of the mixing chamber. Then,
a solution of A′ was placed on the opposite open site of the chamber.
These two solutions were gently mixed by the surface tension and
thereby a concentration-gradient of A′ was spontaneously generated in
the chamber. The dynamic behavior of the NIGVs was monitored with
an Olympus BX51 (obj. lens × 40) equipped with an image-recording
and -processing system.
Optical Microscopic Observation of Catalyst-Dependent Genera-
tion of Vesicular Membranes. An aqueous dispersion of fluorescent
oil droplets composed of B and 5 mol % of C (2.5 mM, 10 µL) was
placed on one side of a rectangular glass plate (24 × 60 mm), and an
aqueous dispersion of the nonfluorescent oil droplets composed of B
(2.5 mM, 10 µL) only was placed on the other side. Then, an aqueous
solution of A′ (10 mM, 10 µL) was placed on the middle of the plate,
and the plate was covered with a cover glass. The morphological
changes in the oil droplets on the two sides after contact with the
solution of A′ were monitored with an Olympus BX51 (obj. lens ×
40) microscope equipped with a halogen-lamp and a color CCD camera
connected to an image-recording and -processing system. An Olympus
WIB filter set (ex ) 460-490 nm; em > 515 nm) was used for
recording the fluorescent images.
1H NMR Spectroscopic Monitoring of the Formation of Vesicular
Molecule V. A 40 mM D2O solution of amphiphilic precursor A′ or A
was prepared. Lipophilic precursor B containing 5 mol % of catalyst
C in the case of b in Figure 2 was dissolved in ethanol, and then the
solvent was removed under reduced pressure. To the residue was added
D2O, and the mixture was sonicated to prepare a 40 mM aqueous
dispersion of oil droplets. Then, 5 mL of the solution of A or A′ and
an equal volume of the dispersion of oil droplets of B (with C in the
case of b) were combined and then stirred at room temperature. Aliquots
(50 mL) of the reaction mixture were withdrawn by micropipet every
15 min, and the reaction solvent was rapidly removed at room
temperature under reduced pressure. The residual mixture was dissolved
1
in 500 µL of DMSO-d6 for measurement of the H NMR spectra of
the reaction mixture on a JEOL GSX-270 spectrometer. The conversion
from A′ and B to V or from A and B to V was monitored by means of
the ratio of the signal area of the imine V to that of the starting material
at the initial stage.
Preparation and Observation of Nutrient-Including Giant Vesicle
(NIGV). A 20:20:1:2 mixture of V, B, C, and cholesterol (a membrane
stabilizer) was dissolved in ethanol. Removal of the solvent under
reduced pressure resulted in the formation of a myelin-like film.
Addition of deionized water to the resulting film at 45 °C produced a
suspension of giant vesicles. After the suspension was left standing
for 48 h, NIGVs were observed with an Olympus BX51 (obj. lens ×
40) differential interference contrast microscope equipped with an
image-recording and -processing system.
Mixture of a Solution of NIGVs with a Solution of Locked
Precursor A′ in a Mixing Chamber. An aqueous dispersion of NIGV
(2.5 mM, 15 µL) was mixed with an aqueous solution of A′ (10 mM,
15 µL) at 23 °C, in a mixing chamber made of a glass slide and a
cover slide, which were firmly fixed together with spacers.2g A NIGV
Acknowledgment. The authors thank Mr. J. Onodera (JEOL)
for his skillful measurement in terms of high-resolution mass
spectroscopy. This work was supported by grants-in-aid from
the Center of Excellence (Study of Life Science as Complex
Systems) of the Ministry of Education, Culture, Sports, Science,
and Technology, Japan.
Supporting Information Available: Representative images
of morphological changes in the NIGVs releasing no vesicles,
as well as the relevant discussion in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
JA029379A
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8140 J. AM. CHEM. SOC. VOL. 125, NO. 27, 2003