Generation of catalytic amphiphiles in a self-reproducing giant vesicle

Article abstract of DOI:10.1246/cl.160107

Giant vesicles capable of generating a catalytic amphiphile that promoted vesicular membrane production were prepared. The catalyst was derived from decylaniline on the vesicles and a catalytic aldehyde present outside the vesicles. Once the catalyst was

Full text of DOI:10.1246/cl.160107

CL-160107
Received: January 31, 2016 | Accepted: February 29, 2016 | Web Released: March 11, 2016
Generation of Catalytic Amphiphiles in a Self-reproducing Giant Vesicle
Li Sheng1,2 and Kensuke Kurihara*1,2,3
1Department of Bioorganization Research, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences,
5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787
2Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science,
National Institutes of Natural Sciences, 38 Nishigo-naka, Myodaiji, Okazaki, Aichi 444-8585
3Research Center for Complex Systems Biology, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902
(E-mail: kkurihara@ims.ac.jp)
Giant vesicles capable of generating a catalytic amphiphile
that promoted vesicular membrane production were prepared.
The catalyst was derived from decylaniline on the vesicles and a
catalytic aldehyde present outside the vesicles. Once the catalyst
was generated inside the vesicular membrane, they proliferated
via a peeling-off process after membrane aldehyde addition.
Keywords: Giant vesicle
| Model protocell | Self-reproduction
The origin of life is one of the most challenging research
topics for contemporary scientists.1-3 The construction of model
protocells from simple molecules has attracted much attention
from researchers involved in the exploration of the origin of life
and the creation of artificial life.4-11 Model protocells comprise
three critical components: an informational substance, one
compartment, and a catalyst.12-14 The catalyst plays a key role
in protocell growth and division, that is, self-reproduction
(autonomous replication). Both the self-reproduction of com-
partments and the self-replication of informational substances
have been studied extensively.1,15 For example, Sugawara et al.
combined DNA amplification and self-reproduction of supra-
molecular giant vesicles (GVs) to develop a system with a
primitive cell cycle.16,17 Although a functioning protocell
requires a self-generating catalyst, current protocell model
systems depend largely on the addition of an external catalyst.
In such systems, the decrease in the amount of catalyst from
generation to generation diminishes the ability of the vesicles to
self-reproduce and eventually leads to protocell “death”.18
To address this issue, we have designed a unique system in
which a GVencapsulates a catalyst-producing system (Figure 1).
The GV produces a water-insoluble catalyst (catalytic imine, CI)
by reaction of water-soluble catalytic aldehyde (CA) located
outside the GV with decylaniline (O) located on the GV
membrane. The produced amphiphilic imine catalyst, which
has a hydroimidazolium ion as the active site, catalyzes a
reaction between membrane aldehyde (MA) and O to generate
water-insoluble vesicular membrane molecules (VM), enabling
GV self-reproduction (Figure 1b). Similar supramolecular GV
systems based on dynamic imine chemistry have been widely
studied.19
Figure 1. (a) Schematic of the reactions to form catalytic imine
(CI) and vesicular membrane molecule (VM). (b) Schematics of
the proposed catalyst production on the surface of an original
vesicle and generation of a newly formed vesicle.
Figure 2. Synthesis of the catalytic aldehyde CA, membrane
aldehyde MA, and vesicular membrane molecule VM. (i) 1,2-
Dibromoethane, cat. K2CO3/acetone, 40 °C, 72 h. (ii) Imidazole,
cat. NaH/DMF, 25 °C, 20 h. (iii) 0.5 M hydrochloric acid/THF,
H2O, 25 °C, 10 min. (iv) 30% aqueous trimethylamine solution/
acetonitrile, 25 °C, 48 h. (v) 4-Decylaniline (O), EtOH, 80 °C, 5 h.
The component molecules were synthesized by the proce-
dures shown in Figure 2. Specifically, 4-(2-bromoethoxy)benz-
aldehyde, which was prepared from 4-hydroxybenzaldehyde by
the Williamson ether synthesis, was treated with a tertiary amine
or with an imidazolium salt to afford aldehyde MA or CA,
respectively. A dehydrocondensation reaction between MA and
O in ethanol produced amphiphilic imine VM. All products were
ionization time-of-flight/mass spectroscopy (MALDI-TOF/MS)
(Supporting Information section S1).
We used thin-film hydration to prepare decylaniline-con-
taining GVs whose membranes were composed of VM and O
in a molar ratio of 1:2 (see Supporting Information section S1).
1
characterized by H NMR and matrix-assisted laser desorption/
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© 2016 The Chemical Society of Japan

Figure 3. Differential interference contrast and fluorescence
microscope images. GVs containing decylaniline molecules (a)
before, (b) 5 min after, and (c) 5 h after the addition of the catalytic
aldehyde. Bar = 20 ¯m.
For detection of the daughter vesicles via microscopy, we
stained the GVs with 1,2-dihexadecanoyl-sn-glycero-3-phos-
phoethanolamine-Texas Redμ (Texas Redμ DHPE) by using
VM + O and Texas Redμ DHPE in a molar ratio of 100:0.1. The
final concentration of lipids was 2 mM in 2 mL of the GV
dispersion (pH 6.94). Vesicles could be easily observed by
differential interference contrast (DIC) and fluorescence mi-
croscopy (Figure 3a). Catalytic aldehyde CA was added to the
GV dispersion at a VM/O/CA molar ratio of 2:4:1 (note that
this ratio indicates the amount of CA added to a GV dispersion
consisting of VM and O at a molar ratio of 2:4.). Despite a
decrease in pH from 6.94 to 5.47 upon addition of CA, no GV
disruption was observable by DIC and fluorescence microscopy
Figure 4. Differential interference contrast (DIC) images (b-g)
and fluorescence micrographs (a, h) showing the self-reproduction
of GVs. (a, b) 14 h, (c) 14 h 10 s, (d) 14 h 30 s, (e) 14 h 50 s, (f) 14 h
60 s, and (g, h) 14 h 15 min after the addition of the membrane
aldehyde MA. Arrows indicate newly formed vesicles. (i, j)
Fluorescence micrographs of GVs 15 h after the addition of MA.
VM on this outer membrane leaflet before finally giving rise to
vesicles via peeling off (Figure 1b). Moreover, similar, newly
generated vesicles emerged from other GVs 15 h after the
addition of MA (Figures 4i and 4j, white arrows).
1
at 5 min or 5 h after CA addition (Figures 3b and 3c). H NMR
spectroscopy revealed that the relative concentration of CA
gradually decreased in the system and reached an equilibrium
value of 70% after 5 h (Figure S1). The formation of a 30%
yield of catalytic imine CI by dehydrocondensation between O
and a portion of aldehyde CA was confirmed by MALDI-TOF/
In a control experiment, we added N-octadecylimidazolium
chloride catalyst CN, which does not form imine bonds with the
GV dispersion. Under these conditions, we observed frequent
GV self-reproduction via a nesting process (Figure S4). The
occurrence of this process may have been due to the location of
catalyst CN, which was homogeneously distributed in the GV
membranes instead of on the outer surface. This result supports
the proposed peeling-off mechanism (Figure 1b) and suggests
that new membrane molecules VM were generated mainly
around the catalyst (CI or CN) and that the generation of VM
promoted GV self-reproduction.
1
MS (Figure S2). Note that the H NMR signal of CI was not
detected in D2O, which suggests that the CI existed only in
the membrane phase. This result is consistent with the findings
of Sugawara et al., who demonstrated the effectiveness of a
vesicular membrane as a reaction environment for imine
coupling between benzaldehyde- and aniline-type amphiphiles.20
After 5 h, the reaction of CA to form CI reached equi-
librium, at which point membrane aldehyde MA was added to
the GV system at a VM/O/CA/MA molar ratio of 2:4:1:2. In
fluorescence microscope images of a GV obtained 5 h after MA
addition (Figure S3), the fluorescence of the GV was lower than
that prior to MA addition, which is consistent with the gradual
consumption of decylaniline (O) concomitant with GV self-
reproduction. Figures 4a-4h show a newly formed smaller GV
(arrows) generated at the outermost layer of the original vesicle
via peeling off. This self-reproduction process, which was also
recorded in real time (see Supporting Information, movie), was
triggered by the increase in the number of membrane molecules
VM on the outer membrane leaflet, resulting from the catalytic
dehydrocondensation between MA and O. CA reacted with O
on the outermost layer of the vesicle to yield CI, which tended to
localize on the outer leaflet of the original vesicular membranes.
The addition of membrane aldehyde MA, which underwent CI-
catalyzed reaction with O, produced new membrane molecules
1
GV self-reproduction was further studied by H NMR. A
vesicular dispersion was prepared in D2O, and after the addition
of CA, the dispersion was incubated for 5 h under ambient
conditions. When vesicular MA was added to the dispersion at
a VM/O/CA/MA molar ratio of 2:4:1:2, the formation of new
membrane molecules VM on the vesicular membrane was
catalyzed by CI, which was also located inside the membrane.
The yields of CI and VM (Figure 1a) were estimated from the
conversions of CA and MA, which are water soluble (Figure 5).
1
The H NMR results showed that, under catalysis by CI, the
added MA molecules were continuously converted into mem-
brane molecules VM, the formation of which induced vesicle
self-reproduction. These results are consistent with the DIC
and fluorescence microscopy results (Figure 4 and Supporting
Information, movie). Aldehyde CA, which had already reached
equilibrium with CI before MA was added (Figure S1), under-
Chem. Lett. 2016, 45, 598–600 | doi:10.1246/cl.160107
© 2016 The Chemical Society of Japan | 599

the Promotion of Science) and a Grant-in-Aid for Young
Scientists (B) (No. 15K17850, Japan Science and Technology
Agency). Additional support was provided by a grant from the
Noguchi Institute, the Kurita Water and Environment Founda-
tion (KWEF), and the Sasakawa Scientific Research Grant from
the Japan Science Society.
Supporting Information is available on http://dx.doi.org/
10.1246/cl.160107.
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reproducing system because both the catalyst and membrane
molecules were generated autonomously. The initially prepared
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This study was supported by the Okazaki ORION Project
(Okazaki Institute for Integrative Bioscience), the Astrobiology
Center Project (No. AB271006) of the National Institutes of
Natural Sciences (NINS), a Grant-in-Aid for Scientific Research
on Innovative Areas “Dynamical ordering of biomolecular
systems for creation of integrated functions” (Japan Society for
600 | Chem. Lett. 2016, 45, 598–600 | doi:10.1246/cl.160107
© 2016 The Chemical Society of Japan