Gemini peptide lipids with ditopic ion-recognition site. Preparation and functions as an inducer for assembling of liposomal membranes

Article abstract of DOI:10.1016/j.tet.2004.08.072

Gemini amphiphiles having two peptide lipid units and a spacer group connected at the polar heads were synthesized. A gemini peptide lipid bearing l-histidyl residues, hydrophobic double-chain segments, and a tri(oxyethylene) spacer performed as an inducer of the reversible assembling of liposomal membranes through ditopic ion recognition toward transition metal ions and alkali metal ions. The vesicular assembling behavior induced by the gemini peptide lipids was sensitive to the structural difference in the amino acid residue and the spacer group of the lipids. Graphical Abstract

Full text of DOI:10.1016/j.tet.2004.08.072

Tetrahedron 60 (2004) 9841–9847
Gemini peptide lipids with ditopic ion-recognition site.
Preparation and functions as an inducer for assembling of
liposomal membranes
*
Shintaro Iwamoto, Masashi Otsuki, Yoshihiro Sasaki, Atsushi Ikeda and Jun-ichi Kikuchi
Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma,
Nara 630-0192, Japan
Received 10 July 2004; revised 19 August 2004; accepted 23 August 2004
Available online 15 September 2004
Abstract—Gemini amphiphiles having two peptide lipid units and a spacer group connected at the polar heads were synthesized. A gemini
peptide lipid bearing L-histidyl residues, hydrophobic double-chain segments, and a tri(oxyethylene) spacer performed as an inducer of
the reversible assembling of liposomal membranes through ditopic ion recognition toward transition metal ions and alkali metal ions. The
vesicular assembling behavior induced by the gemini peptide lipids was sensitive to the structural difference in the amino acid residue and the
spacer group of the lipids.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
such as Ca2C and Mg2C to influence the aggregate
morphology of the lipid membranes. Up to the present
time, a considerable number of investigations have been
reported on synthetic gemini surfactants consist of two
surfactant molecules via a spacer group.21–23 The gemini
surfactants provide novel and interesting opportunities to
investigate various supramolecular morphologies depend-
ing on the structural characteristics following to attractive
Liposomal membranes formed with phospholipids or
bilayer-forming synthetic lipids have been widely employed
as biomembrane models, drug carriers, nano-reactors, and
scaffolds for supramolecular devices.1–9 On functionaliza-
tion of the liposomal membranes, the lipid assemblies have
been usually handled as the uni-vesicular state, but not as
the multi-vesicular assembly. In the light of the superiority
of the multi-cellular organisms in biological systems as
compared with the corresponding unicellular states, how-
ever, establishment of the methodology to construct the
multi-vesicular assemblies as artificial tissues is of great
significance. On these grounds, many kinds of approaches
for assembling of the liposomal membranes have been
proposed up to the present time.10–18 In this article, we are to
report a novel strategy to create reversible assembling
system of the liposomal membranes by using an ion-
recognizable gemini peptide lipid (1) as an inducer (Fig. 1).
Our molecular design of the lipid 1 was inspired by a
naturally occurring gemini lipid, cardiolipin, having unique
dimeric lipid structure and interesting biological func-
tions.19,20 Especially, we paid attention to the specific ion
binding behavior of the cardiolipins toward divalent ions
Keywords: Gemini peptide lipid; Ditopic ion-recognition; Vesicular
assembling; Pseudo-crown ether; Supramolecular chemistry.
* Corresponding author. Tel.: C81-743-72-6090; fax: C81-743-71-6099;
e-mail: jkikuchi@ms.naist.jp
Figure 1. Schematic representation of the assembling of liposomes
containing the gemini peptide lipid as triggered by specific ion recognition.
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.08.072

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S. Iwamoto et al. / Tetrahedron 60 (2004) 9841–9847
applications. Although much attention has been focused on
physicochemical properties of the synthetic gemini surfac-
tants, there are few reports on the synthetic gemini lipids
composed of two double-chain segments like the cardio-
lipin. Thus, we prepared the gemini lipids having two
double-chain units and two kinds of ion recognition sites, by
extending our previous studies on the bilayer-forming
synthetic peptide lipids having an amino acid moiety
interposed between the polar head moiety and a hydro-
phobic double-chain segment.24
the vesicles or the followed fusion (Fig. 2). The similar
behavior was also observed for the hybrid vesicles of 2,
but not for the hybrid vesicles containing the gemini lipid
3 or the non-gemini lipid 4 and the liposomes formed
with DMPC alone under the comparable concentration
conditions.
Figure 2. Effects of Cu2C and KC ions for the vesicular assembling as
evaluated by DLS at pH 9 and 30 8C. Concentrations in mmol dmK3
[DMPC], 0.5; [gemini lipid], 0.05; [4], 0.1; [Cu2C], 0.5; [KC], 5.0.
:
The present hybrid vesicle of 1 showed a phase transition
from gel to liquid-crystalline state with the peak maximum
(Tm) at 24.3 8C and its enthalpy change (DH) of 4.3 kJ molK1
per the matrix lipid, as evaluated by differential scanning
calorimetry (DSC). Upon addition of Cu2C ions to the
vesicular solution, the phase transition behavior was
scarcely affected, reflecting that the metal ions did not
give perturbation in the packing shape of the lipid molecules
in the membrane so as to change the aggregate morphology;
the Tm and DH values were 24.3 8C and 4.0 kJ molK1
,
2. Results and discussion
respectively, in the presence of Cu2C ions in an equimolar
amount to the phospholipid. In general, remarkable changes
in the phase transition can be detected by DSC for the
morphological transformation from small vesicles to the
corresponding larger ones.25,26 In addition, the increased
Dhy value was immediately recovered to the original value
in the metal-free system upon addition of an excess amount
of EDTA. The results clearly indicate that the assembling of
the hybrid vesicles formed with DMPC and 1 was triggered
by Cu2C ions without accompanying fusion of the vesicles
as illustrated in Figure 1.
2.1. Synthesis of the gemini peptide lipids
In order to enhance the ion-recognition ability of the
synthetic gemini lipid in the liposomal membranes, we
introduced two L-histidyl residues as the polar heads and a
tri(oxyethylene) group as a spacer between the head
moieties, expecting that the former and the latter groups
act as effective binding sites for transition metal ions and
alkali metal ions, respectively. The gemini peptide lipid (1)
was synthesized through condensation of the peptide lipids
with the corresponding bis(acyl chloride). As the reference
lipids of 1, we also prepared a gemini peptide lipid having a
hydrocarbon spacer (2), a gemini peptide lipid bearing
L-aspartate residues (3), and a non-gemini peptide lipid (4).
The syntheses of these lipids were basically referred to the
synthetic procedures of the peptide lipids.24
The ion binding to the surface of the lipid bilayer vesicle
was qualitatively examined by z-potential measurements.27
pH-Dependences of the z-potential for the DMPC vesicles
containing the gemini peptide lipid 1 in the presence
and absence of Cu2C ions were shown in Figure 3. In the
metal-free system, deprotonation of the imidazolyl
groups in 1 caused the decrease in the z-potential from
C40 to K20 mV. On the other hand, significant increase in
the z-potential was observed in a pH region over 7 in the
presence of Cu2C ions. The results strongly suggest that
Cu2C ions bind to the histidyl residues of the gemini peptide
lipids to induce the vesicular assembling.
2.2. Ion recognition behavior of the gemini peptide lipids
and its consequence in assembling of the liposomal
membranes
The hybrid bilayer vesicles were prepared by sonication of
an aqueous dispersion of dimyristoylphosphatidylcholine
(DMPC) and a gemini peptide lipid in a 10:1 molar ratio
with a cup-type sonicator at 30 W for 6 min. The
hydrodynamic diameter (Dhy) of the hybrid vesicle of 1
was evaluated to be 160 nm by means of dynamic light
scattering (DLS) measurements. Upon addition of Cu2C
ions to the hybrid vesicles at pH 9 and 30 8C, the Dhy value
was significantly increased, suggesting the assembling of
Specificity of the ion recognition by the gemini peptide lipid
to induce the vesicular assembling was evaluated from the
extent of the increased Dhy values upon addition of various
transition metal ions (Table 1). The ion selectivity for the
hybrid vesicles composed of DMPC with 1 or 2 was well
correlated to the general stability sequence of divalent

S. Iwamoto et al. / Tetrahedron 60 (2004) 9841–9847
9843
Figure 3. pH-Dependences of z-potential for the DMPC vesicle containing
the gemini peptide lipid 1 at 30 8C in the presence (square) and absence
(circle) of Cu2C ions (0.5 mmol dmK3). Concentrations in mmol dmK3
:
[DMPC], 0.5; [1], 0.05; [NaC] 0.5.
Table 1. Hydrodynamic diameters for the DMPC vesicles containing the
gemini peptide lipid at pH 9.0 and 30 8Ca
Metal ions
Hydrodynamic diameter/nmb
Lipid 1
Lipid 2
None
Mn2C
Fe2C
Co2C
Ni2C
Cu2C
Zn2C
150 (20)
140 (10)
150 (10)
270 (20)
320 (40)
540 (20)
350 (50)
190 (20)
190 (20)
210 (20)
230 (30)
240 (20)
500 (30)
360 (20)
a
Hydrodynamic diameters were determined by DLS. Concentrations in
mmol dmK3: [DMPC], 0.5; [1] or [2], 0.05, [metal ions], 0.5.
Standard deviation of three independent measurements is in
parenthesis.
Figure 4. (A) CD spectral changes for the DMPC (0.5 mmol dmK3) vesicle
containing the gemini peptide lipid 1 (0.05 mmol dmK3) upon addition
of Cu2C ions. The concentrations of Cu2C ions in mmol dmK3: a, 0; b,
0.015; c, 0025; d, 0.05; e, 0.2; f; 0.5. (B) Change in the molar CD at 214 nm
(DD3214) upon titration of the gemini peptide lipid 1 with Cu2C ions.
b
transition metal complexes, the Irving–Williams series;
Mn2C!Fe2C!Co2C!Ni2C!Cu2COZn2C 28,29
Under
.
the similar concentration conditions, alkali and alkaline-
earth metal ions did not act as the trigger for assembling of
the hybrid vesicles.
constants for the 4:1 complex of the imidazolyl groups
in the lipid and Cu2C ions. Whereas the binding constant
of Cu2C ions toward the non-gemini lipid 4 with one
imidazolyl group per one lipid molecule was 1.5!
1017 dm12 molK4 for the 1:4 complex, being much weaker
than those of the corresponding gemini lipids. Under the
conditions in Figure 2, for example, the imidazolyl groups
of the gemini lipid 1 completely form the copper complex,
whereas only 75% of the histidyl residue of 4 binds Cu2C
ions and 25% of the imidazolyl group is free from the metal
coordination.
In order to elucidate the stoichiometry and the binding
constant for the copper complexes formed in the hybrid
vesicles, circular dichroism (CD) spectra were taken in
aqueous solution by monitoring the conformational changes
in the chiral histidyl moieties of the lipid upon complexa-
tion.30,31 The CD spectral changes of the gemini peptide
lipid 1 embedded in the DMPC vesicle upon addition of
Cu2C ions are shown in Figure 4(A). The titration isotherm
shown in Figure 4(B) was obtained by monitoring the CD
intensity change at 214 nm (DD3214), which was applied to
determine the binding constant of Cu2C ions with 1. The
Job’s plot analyses in the vesicular system revealed that
Cu2C ions bind to the imidazolyl groups in a ratio of 1:4.
The binding constants for the other lipids, 2 and 4, were also
determined in the similar manner. Thus the binding
constants for the 1:2 complexes of Cu2C ions with the
gemini lipids 1 and 2 in the DMPC vesicle were 1.6!1011
and 1.3!1011 dm6 molK2, respectively. These values are
also represented to be 2.6!1022 and 1.7!1022 dm12 molK4
for the lipid 1 and 2, respectively, as apparent binding
Thus, we can image the mechanism for the Cu2C-triggered
assembling of the liposomal membranes containing the
gemini peptide lipid as follows. In order to keep two lipid
bilayer surfaces at separation less than about 2 nm in
aqueous media, some attractive force which overcomes the
repulsive hydration force observed on the surface of the
hydrated lipid membranes32,33 would be necessary. Thus,
our observation that the DMPC liposomes containing the
non-gemini lipid 4 did not form the vesicular assembly in
the presence of Cu2C ions indicates that the intra-vesicular
1:4 complex of the metal ions with the imidazolyl groups is

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S. Iwamoto et al. / Tetrahedron 60 (2004) 9841–9847
thermodynamically more stable than the corresponding
inter-vesicular complex under the present conditions,
presumably owing to the repulsive hydration force. On the
other hand, the gemini peptide lipids with two imidazolyl
groups can stabilize the inter-vesicular complex reinforced
with the linkage of the spacer unit, as shown in Figure 5.
Although the corresponding intra-vesicular complex may be
also formed in the hybrid liposome of the gemini peptide
lipid, the inter-vesicular complex would play pivotal role as
the membrane junction to induce the vesicular assembling.
We also observed that the assembling of the hybrid vesicles
was triggered by Cu2C ions in the liquid crystalline state
above the Tm of the lipid membrane, but not below the Tm at
which the inter-vesicular complex would be hardly formed
in the rigid gel matrix with highly restricted molecular
motion. This observation strongly support that the inter-
vesicular complex shown in Figure 5 is a key species for the
vesicular assembling in this study.
Figure 6. Effect of alkali metal ions (MC) on hydrodynamic diameter (Dhy
of the DMPC vesicles containing gemini peptide lipid (1) in the presence of
Cu2C ion at 30 8C and pH 9. Concentrations in mmol dmK3: [DMPC], 0.5;
[1], 0.05; [Cu2C], 0.5.
)
interesting ditopic ion-recognition systems in solutions36–38
to those in the lipid membrane systems as the biomembrane
models.
3. Conclusion
We have shown here that the transformation between the
assembling and disassembling of the liposomal membranes
can be reversibly controlled as a response of heteroditopic
ion recognition by the gemini peptide lipid embedded in
the vesicles. We believe our results revealed a new function
of the gemini surfactants and propose a guidepost to design
supramolecular assemblies exhibiting ditopic molecular
recognition and its response.
Figure 5. Plausible key species of the copper complex of the gemini peptide
lipid having L-histidyl residues which stabilizes the vesicular assembly by
forming the membrane junction.
2.3. Heteroditopic ion-recognition by the gemini peptide
lipid and its consequence in disassembling of the
liposomal membranes
The gemini peptide lipid 1 has a pseudo-crown ether moiety
at the spacer unit, and is expected to behave as a ditopic ion
receptor. As shown in Figure 2, the hydrodynamic diameter
of the assembled vesicles in the presence of Cu2C ions was
decreased from 540 to 200 nm upon further addition of KC
ions (5.0 mmol dmK3). On the other hand, there was no
significant change of the vesicular size upon addition of KC
ions in the absence of Cu2C ions. The results clearly
indicate that the assembling of the liposomal membranes is
reversibly controlled by ditopic ion recognition of 1 with
Cu2C and KC ions. Since the disassembling of the vesicles
was not observed for the hybrid vesicles of DMPC and the
gemini peptide lipid lacking the pseudo-crown ether moiety
2 upon addition of KC ions after the Cu2C-triggered
vesicular assembling. Thus the disassembling behavior of
the hybrid vesicles of DMPC and 1 was interpreted by
replacement of the Cu2C-binding to the imidazolyl groups
with the KC-binding to the pseudo-crown ether moiety to
breakdown the inter-vesicular copper complex.
4. Experimental
4.1. General
Organic solvents were purified, dried by standard pro-
cedures and kept over a drying agent before use. Unless
otherwise mentioned, reagents were commercially available
as a guaranteed grade and used without further purification.
Thin layer chromatography (TLC) was performed on a silica
gel 70 FM plate-Wako (Wako Pure Chemical Industries)
with fluorescence detection under UV light. Liquid
chromatography was performed using a silica gel column
(Wako-gel C-300) and/or high performance liquid
chromatography (HPLC) using a JAI Model instrument
(Japan Analytical Instruments) equipped with JAIGELK1H
1
and -2H columns. H NMR spectra were taken on a JEOL
JNM-LA400 spectrometer. HR-MS (FABC) was recorded
with a JEOL JMS-700 MStation mass spectrometer.
4.2. Synthesis
In addition, marked selectivity for alkali metal ions was
observed in the vesicular disassembling behavior (Fig. 6).
Tendency of the disassembling effect seems to be correlated
with the binding affinity of alkali metal ions to the pseudo-
crown ether moiety;34,35 KCONaCOLiCORbCzCsC.
Our present results would bring up possibilities to extend the
4.2.1. N,N-Dihexadecyl-N2-t-butoxycarbonyl-Nt-tosyl-L-
histidinamide [Boc-His(Tos)2C16]. N2-t-Butoxycarbonyl-
Nt-tosyl-L-histidine (3.00 g, 7.33 mmol) was added to a
solution of dihexadecylamine (2.85 g, 6.11 mmol) and

S. Iwamoto et al. / Tetrahedron 60 (2004) 9841–9847
9845
triethylamine (0.74 g, 7.33 mmol) in dry dichloromethane
(20 dmK3) at 0 8C with stirring for 15 min. Diethylcyano
phosphate (1.20 g, 7.33 mmol) was added to the solution
and stirred for 30 min at 0 8C and then for 1 h at 25 8C. The
solution was evaporated in vacuo to give a solid. The crude
product was purified by liquid chromatography on a column
of silica gel (Wako-gel C-300) with chloroform–methanol
(99.5:0.5 v/v) as eluent giving a white solid, yield 5.18 g
(99%); mp 52.6–53.4 8C. 1H NMR (400 MHz, CDCl3,
TMS): d 0.88 (6H, t, JZ6.7 Hz, (CH2)13CH3), 1.24 (52H, m,
NCH2(CH2)13CH3), 1.35 (9H, s, (CH3)3CO), 1.43 (4H, m,
NCH2CH2(CH2)13CH3), 2.42 (3H, s, C6H4CH3), 2.80 (2H, m,
CHCH2Im), 2.99–3.42 (4H, m, NCH2CH2(CH2)13CH3), 4.81
(1H, m, CHCH2Im), 5.27 (1H, d, JZ8.3 Hz, CONH), 7.09
(1H, s, Im-5H), 7.33 (2H, d, JZ8.4 Hz, o-C6H4), 7.79 (2H,
d, JZ8.4 Hz, m-C6H4), 7.89 (1H, s, Im-2H). Anal. calcd for
C50H88N4O5S: C, 70.05; H, 10.35; N 6.54%. Found: C,
70.21; H, 10.62; N, 6.55%.
CONH), 6.88 (2H, s, Im-5H), 7.51 (2H, s, Im-2H). Anal.
calcd for C84H158N8O8: C, 71.64; H, 11.31; N, 7.96%.
Found: C, 71.30; H, 11.53; N, 7.68%. HR-MS (FABC):
exact mass calcd for C84H159N8O8 [MCH]C 1408.2281,
found 1408.2275.
4.2.4. N,N-Dihexadecyl-N2-t-butoxycarbonyl-Nt-benzyl-
oxymethyl-L-histidinamide [Boc-His(Bom)2C16]. N2-t-
Butoxycarbonyl-Nt-benzyloxymethyl-L-histidine (6.76 g,
18 mmol) was added to a solution of 1-hydroxybenzotiazole
(2.43 g, 18.0 mmol) in dry dichloromethane (20 dmK3) at
0 8C with stirring for 5 min. Then N,N0-dicyclohexylcarbo-
diimide (2.97 g, 14.4 mmol) was added to the solution and
stirred at 0 8C. After 15 min, dihexadecylamine (5.74 g,
12.0 mmol) was added to the solution and stirred for 3 h at
0 8C and then for 42 h at room temperature. The precipi-
tation was removed by filtration and the solvent was
removed in vacuo. The residue dissolved in ethyl acetate
(300 dmK3) was washed sequentially with 10% aqueous
citric acid, 5% aqueous sodium hydrogen carbonate and
saturated aqueous sodium chloride. After being dried, the
solution was evaporated in vacuo to give a yellow oil. The
crude product was purified by liquid chromatography on a
column of silica gel (Wako-gel C-300) with chloroform–
methanol (99.5:0.5 v/v) as eluent giving a white solid, yield
3.02 g (31%); mp 49.8–50.2 8C. 1H NMR (400 MHz,
CDCl3, TMS): d 0.88 (6H, t, JZ6.7 Hz, (CH2)13CH3),
1.26 (52H, m, (CH2)13CH3), 1.39 (9H, s, (CH3)3CO), 1.47
(4H, m, NCH2CH2(CH2)13CH3), 2.92 (2H, m, CHCH2Im),
3.00–3.44 (4H, m, NCH2CH2(CH2)13CH3), 4.45 (1H, d,
JZ11.9 Hz, ImCH2OCHHC6H5), 4.51 (1H, d, JZ11.9 Hz,
ImCH2OCHHC6H5), 4.79 (1H, m, CHCH2Im), 5.29 (1H, d,
JZ11.0 Hz, ImCHHOCH2C6H5), 5.32 (1H, d, JZ8.5 Hz,
CONH), 5.41 (1H, JZ11.0 Hz, ImCHHOCH2C6H5), 6.89
(1H, s, Im-5H), 7.32 (5H, m, OCH2C6H5), 7.46 (1H, s, Im-
2H). Anal. calcd for C51H90N4O4: C, 74.40; H, 11.02; N
6.81%. Found: C, 74.27; H, 10.91; N, 6.87%.
0
4.2.2. N2,N2 -2,5,8,11-Tetraoxadodecanedioyl-bis(N,N-
dihexadecyl-Nt-tosyl-L-histidinamide) [Teo-Bis(His(Tos)
2C16)]. Trifluoroacetic acid (8.90 g, 78.0 mmol) was added
to a solution of Boc-His(Tos)2C16 (3.34 g, 3.90 mmol) in
dry dichloromethane (10 dmK3) at 25 8C with stirring for
2 h. The solvent was evaporated in vacuo, and the residue
and triethylamine (3.95 g, 39.0 mmol) was dissolved in dry
dichloromethane at 0 8C. 2,5,8,11-Tetraoxadodecanedioyl
dichloride (0.536 g, 1.95 mmol) dissolved in dichloro-
methane (10 dmK3) was added to the solution for 20 min.
The mixture was stirred for 1 h at 25 8C and washed with
brine. The solvent was evaporated in vacuo, and the crude
product was purified by liquid chromatography on a column
of silica gel (Wako-gel C-300) with chloroform–methanol
(99:1 v/v) as eluent and then HPLC with chloroform as
1
eluent to give a colorless oil, yield 0.75 g (22%). H NMR
(400 MHz, CDCl3, TMS): d 0.88 (12H, t, JZ6.7 Hz,
(CH2)13CH3), 1.26 (104H, m, (CH2)13CH3), 1.41 (8H, m,
NCH2CH2(CH2)13CH3), 2.42 (6H, s, C6H4CH3), 2.84 (4H,
m, CHCH2Im), 2.93–3.42 (8H, m, NCH2CH2(CH2)13CH3),
3.62 (4H, s, CH2CH2O(CH2)2OCH2CH2), 3.65 (4H, m,
CH2CH2OCH2CH2OCH2CH2), 4.12–4.32 (4H, m, CH2CH2
OCH2CH2OCH2CH2), 4.81 (2H, m, CHCH2Im), 5.73 (2H,
d, JZ8.3 Hz, CONH), 7.09 (2H, s, Im-5H), 7.33 (4H, d,
JZ8.4 Hz, o-C6H4), 7.79 (4H, d, JZ8.4 Hz, m-C6H4), 7.89
(2H, s, Im-2H). Anal. calcd for C98H170N8O12S2$5H2O: C,
65.15; H, 10.04; N, 6.20%. Found: C, 65.15; H, 9.67; N,
6.12%.
0
4.2.5. N2,N2 -Octanedioyl-bis(N,N-dihexadecyl-Nt-
benzyloxymethyl-L-histidinamide) [C6-Bis(His(Bom)2C16)].
Trifluoroacetic acid (8.16 g, 71.6 mmol) was added to a
solution of Boc-His(Bom)2C16 (2.95 g, 3.58 mmol) in dry
dichloromethane (40 dmK3) at room temperature with
stirring for 2 h. The solvent was evaporated in vacuo, and
the residue and triethylamine (2.95 g, 28.6 mmol) was
dissolved in dry dichloromethane (40 dmK3) at 0 8C.
Octanedioyl dichloride (0.61 g, 2.86 mmol) dissolved in
dichloromethane (7 dmK3) was added to the solution for
1 h. The mixture was stirred for 4 h at 25 8C and the solvent
was removed in vacuo. The residue dissolved in dichloro-
methane (200 dmK3) was washed sequentially with 10%
aqueous citric acid, 5% aqueous sodium hydrogen carbonate
and saturated aqueous sodium chloride. After being dried,
the solution was evaporated in vacuo to give a yellow oil.
The crude product was purified by liquid chromatography
on a column of silica gel (Wako-gel C-300) with chloro-
form–methanol (99:1 v/v) as eluent giving a colorless oil,
0
4.2.3. N2,N2 -2,5,8,11-Tetraoxadodecanedioyl-bis(N,N-
dihexadecyl-L-histidinamide) (1). Aqueous NaOH
(1.0 mol dmK3, 10 dmK3) was added to Teo-Bis(His(Tos)
2C16) (0.43 g, 0.28 mmol) in methanol (100 dmK3) with
stirring for 1 h at 0 8C. The solvent was removed in vacuo,
and the residue was purified by liquid chromatography on a
column of silica gel (Wako-gel C-300) with chloroform–
methanol (97:3 v/v) to give a colorless oil, yield 0.145 g
1
(41%). H NMR (400 MHz, CDCl3, TMS): d 0.88 (12H, t,
1
JZ6.7 Hz, (CH2)13CH3), 1.26 (104H, m, (CH2)13CH3), 1.47
(8H, m, NCH2CH2(CH2)13CH3), 3.02 (4H, m, CHCH2Im),
3.22–3.39 (8H, m, NCH2CH2(CH2)13CH3), 3.64 (4H, s,
CH2CH2OCH2CH2OCH2CH2), 3.67 (4H, m, CH2CH2
OCH2CH2OCH2CH2), 4.12–4.32 (4H, m, CH2CH2OCH2
CH2OCH2CH2), 4.81 (2H, m, CHCH2Im), 6.41 (2H, br,
yield 2.42 g (85%). H NMR (400 MHz, CDCl3, TMS): d
0.88 (12H, t, JZ6.7 Hz, (CH2)13CH3), 1.26 (108H, m,
(CH2)13CH3, CO(CH2)2(CH2)2(CH2)2CO), 1.47 (8H, m,
NCH2CH2(CH2)13CH3), 1.58 (4H, m, COCH2CH2(CH2)2-
CH2CH2CO), 2.12 (4H, m, COCH2(CH2)4CH2CO), 3.02 (4H,
m, CHCH2Im), 3.11–3.43 (8H, m, NCH2CH2(CH2)13CH3),

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S. Iwamoto et al. / Tetrahedron 60 (2004) 9841–9847
4.42 (2H, d, JZ11.9 Hz, ImCH2OCHHC6H5), 4.54 (2H, d,
JZ11.9 Hz, ImCH2OCHHC6H5), 5.17 (2H, m, CHCH2Im),
5.31 (2H, d, JZ11.0 Hz, ImCHHOCH2C6H5), 5.41 (2H,
JZ11.0 Hz, ImCHHOCH2C6H5), 6.89 (2H, s, Im-5H), 7.22
(2H, d, JZ8.5 Hz, CONH), 7.32 (10H, m, OCH2C6H5), 7.51
(2H, s, Im-2H). Anal. calcd for C100H174N8O7$1H2O: C,
74.95; H, 11.07; N, 6.99%. Found: C, 74.87; H, 10.90; N,
6.92%.
4.2.8. N2-Acetyl-N,N-dihexadecyl-L-histidinamide (4).
Pd-C 10% (0.223 g) was added to Ac-His(Bom)2C16
(0.893 g, 1.16 mmol) dissolved in 80% aqueous acetic
acid (20 dmK3). The mixture was placed under hydrogen at
atomospheric pressure and room temperature for 24 h with
stirring. The mixture was filtered on celite and the filtrate
was concentrated under reduced pressure. The crude
product was purified by liquid chromatography on a column
of silica gel (Wako-gel C-300) with chloroform–methanol
(97:3 v/v) as eluent giving a white solid, yield 0.254 g
(33%); mp 60.7–61.5 8C. 1H NMR (400 MHz, CDCl3,
TMS): d 0.88 (6H, t, JZ6.7 Hz, (CH2)13CH3), 1.26 (52H,
m, (CH2)13CH3), 1.47 (4H, m, NCH2CH2(CH2)13CH3), 2.00
(3H, s, CH3CO), 2.98 (2H, m, CHCH2Im), 3.01–3.50 (4H,
m, NCH2CH2(CH2)13CH3), 5.03 (1H, m, CHCH2Im), 6.57
(1H, d, JZ8.5 Hz, CONH), 6.83 (1H, s, Im-5H), 7.57 (1H, s,
Im-2H). Anal. calcd for C40H76N4O2$0.5H2O: C, 73.45; H,
11.87; N, 8.57%. Found: C, 73.35; H, 11.83; N, 8.39%. HR-
MS (FABC): exact mass calcd for C40H77N4O2 [MCH]C
645.6047, found 645.6041.
0
4.2.6. N2,N2 -Octanedioyl-bis(N,N-dihexadecyl-L-histidin-
amide) (2). Pd-C 10% (0.36 g) was added to C6-
Bis(His(Bom)2C16) (1.13 g, 0.69 mmol) dissolved in 80%
aqueous acetic acid (20 dmK3). The mixture was placed
under hydrogen at atomospheric pressure and room
temperature for 15 h with stirring. The mixture was filtered
on celite and the filtrate was concentrated under reduced
pressure. The crude product was purified by liquid
chromatography on a column of silica gel (Wako-gel
C-300) with chloroform–methanol (97:3 v/v) as eluent
giving a white solid, yield 72.1 mg (17%); mp 95.4–
96.1 8C. 1H NMR (400 MHz, CDCl3, TMS): d 0.88 (12H, t,
JZ6.7 Hz, (CH2)13CH3), 1.26 (108H, m, (CH2)13CH3,
CO(CH2)2(CH2)2(CH2)2CO), 1.58 (4H, m, COCH2CH2
(CH2)2CH2CH2CO), 2.12 (4H, m, COCH2(CH2)4CH2CO),
3.02 (4H, m, CHCH2Im), 3.13–3.39 (8H, m, NCH2CH2
(CH2)13CH3), 5.17 (2H, m, CHCH2Im), 6.83 (2H, s, Im-
5H), 7.22 (2H, d, JZ8.5 Hz, CONH), 7.63 (s, 2H, Im-2H).
Anal. calcd for C84H158N8O4$2CH3CO2H: C, 72.18; H,
11.43; N, 7.65%. Found: C, 72.45; H, 11.47; N, 7.72%. HR-
MS (FABC): exact mass calcd for C84H159N8O4 [MCH]C
1344.2484, found 1344.2483.
0
4.2.9. N2,N2 -2,5,8,11-Tetraoxadodecanedioyl-bis(N,N-
dihexadecyl-L-aspartic 1-amide) (3). The gemini peptide
lipid having aspartate residues (3) was prepared in a manner
analogous to that for the synthesis of 1 by using N2-t-
butoxycarbonyl-O4-benzyl-L-aspartic acid instead of N2-t-
butoxycarbonyl-Nt-tosyl-L-histidine. In the final step, the
benzyl groups were removed by the Pd-C catalyzed
hydrogenation. A colorless oil. 1H NMR (400 MHz,
CDCl3, TMS): d 0.88 (12H, t, JZ6.7 Hz, (CH2)13CH3),
1.26 (104H, m, (CH2)13CH3), 1.58 (8H, m, NCH2CH2
(CH2)13CH3), 2.65 (2H, m, CHCH2COOH), 3.21–3.43 (8H,
m, NCH2CH2(CH2)13CH3), 3.67 (4H, s, CH2CH2OCH2
CH2OCH2CH2), 3.69(4H, m, CH2CH2OCH2CH2OCH2CH2),
4.14–4.33 (4H, m, CH2CH2OCH2CH2OCH2CH2), 4.97 (2H,
m, CHCH2COOH), 6.89 (2H, br, CONH). Anal. calcd for
C80H154N4O12$0.5H2O: C, C, 69.98; H, 11.38; N, 4.08%.
Found: C, 69.98; H, 11.50; N, 4.16%. HR-MS (FABC):
exact mass calcd for C80H155N4O12 [MCH]C 1364.1642,
found 1364.1649.
4.2.7. N2-Acetyl-N,N-dihexadecyl-Nt-benzyloxymethyl-
L-histidinamide [Ac-His(Bom)2C16]. Trifluoroacetic acid
(7.04 g, 61.8 mmol) was added to a solution of Boc-
His(Bom)2C16 (2.54 g, 3.08 mmol) in dry dichloromethane
(10 dmK3) at room temperature with stirring for 2 h. The
solvent was evaporated in vacuo, and the residue and
triethylamine (3.13 g, 30.1 mmol) was dissolved in dry
dichloromethane (10 dmK3) at 0 8C. Acetic anhydride
(0.921 g, 9.02 mmol) was added to the solution for 1 h.
The mixture was stirred for 8 h at room temperature and the
solvent was removed in vacuo. The residue dissolved in
dichloromethane (100 dmK3) was washed sequentially with
10% aqueous citric acid, 5% aqueous sodium hydrogen
carbonate and saturated aqueous sodium chloride. After
being dried, the solution was evaporated in vacuo to give a
yellow oil. The crude product was purified by liquid
chromatography on a column of silica gel (Wako-gel C-300)
with chloroform–methanol (99:1 v/v) as eluent giving a
colorless oil, yield 1.13 g (49%). 1H NMR (400 MHz,
CDCl3, TMS): d 0.88 (6H, t, JZ6.7 Hz, (CH2)13CH3), 1.26
(52H, m, (CH2)13CH3), 1.47 (4H, m, NCH2CH2(CH2)13-
CH3), 2.00 (3H, s, CH3CO), 2.98 (2H, m, CHCH2Im), 3.01–
3.50 (4H, m, NCH2CH2(CH2)13CH3), 4.46 (1H, d, JZ11.7 Hz,
ImCH2OCHHC6H5), 4.58 (1H, d, JZ11.7 Hz, ImCH2-
OCHHC6H5), 5.11 (1H, m, CHCH2Im), 5.31 (1H, d,
JZ11.0 Hz, ImCHHOCH2C6H5), 5.41 (1H, JZ11.0 Hz,
ImCHHOCH2C6H5), 6.36 (1H, d, JZ8.3 Hz, CONH), 6.83
(1H, s, Im-5H), 7.32 (5H, m, OCH2C6H5), 7.46 (1H, s, Im-
2H). Anal. calcd for C48H84N4O3: C, 75.34; H, 11.06; N,
7.32%. Found: C, 74.97; H, 11.18; N, 7.27%.
4.3. Preparation of hybrid vesicles
Hybrid liposomes employed in this study were generally
prepared according to established protocol1 as follows.
Appropriate amounts of synthetic lipid and dimyristoyl-
phosphatidylcholine (DMPC) were dissolved in chloroform.
The solvent was evaporated under nitrogen gas flow and the
residual trace solvent was completely removed in vacuo.
Hydration of the thin film thus obtained on the wall of a vial
was performed at 40 8C with an appropriate amount of
water. Multi-walled bilayer vesicles were formed upon
vortex mixing of the aqueous dispersion. The corresponding
single-walled vesicles were prepared by sonication of the
dispersion sample with a cup-type sonicator above the phase
transition temperature for 5 min.
4.4. Measurements
4.4.1. Differential scanning calorimetry (DSC). The phase
transition behavior of the lipid bilayer vesicles was
measured with a differential scanning calorimeter (VP-
DSC, MicroCal). The measurements were performed

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Acknowledgements
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