Trigger lipids inducing pH-dependent liposome fusion

Article abstract of DOI:10.1016/S0009-3084(02)00053-1

Aspartic acid-derived artificial lipids (ADLs; s indicates the number of the methylene groups, s=2, 4, 6, 8, 10, 12) (Scheme 1) with various carboxyl alkyl chains as head groups are designed and synthesized, which are incorporated into liposome membranes by sonication. Fluorescence resonance energy transfer (FRET) measurements indicate that ADL6, ADL8 and ADL10 have high lipid-mixing ability in the acidic solution. The other ADLs, however, do not induce remarkable liposome fusion at acidic nor neutral pH. The hydrophobicity of the head groups of ADL6, ADL8 and ADL10 is suitable as triggers of membrane fusion.

Full text of DOI:10.1016/S0009-3084(02)00053-1

Chemistry and Physics of Lipids 119 (2002) 51Á68
/
www.elsevier.com/locate/chemphyslip
Trigger lipids inducing pH-dependent liposome fusion
Yoshikatsu Ogawa, Masato Kodaka *, Hiroaki Okuno
Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Center,
AIST Tsukuba Center 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Received 28 February 2002; received in revised form 16 May 2002; accepted 18 May 2002
Abstract
Aspartic acid-derived artificial lipids (ADLs; s indicates the number of the methylene groups, sꢀ2, 4, 6, 8, 10, 12)
/
(Scheme 1) with various carboxyl alkyl chains as head groups are designed and synthesized, which are incorporated into
liposome membranes by sonication. Fluorescence resonance energy transfer (FRET) measurements indicate that ADL6,
ADL8 and ADL10 have high lipid-mixing ability in the acidic solution. The other ADLs, however, do not induce
remarkable liposome fusion at acidic nor neutral pH. The hydrophobicity of the head groups of ADL6, ADL8 and
ADL10 is suitable as triggers of membrane fusion. # 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Trigger lipids; Liposome; Membrane
1. Introduction
sulfatide (Cestaro et al., 1983), Ca2ꢁ (Sundler and
Papahadjopoulos, 1981), polyethylene glycol
(PEG) (Yang et al., 1997) and fusion peptides
(Horth et al., 1991) are known to induce mem-
brane fusion. Since endocytosis is regarded as the
principal pathway by which liposomes enter cells
and the pH of the inside of endocytic vesicles is
mildly acidic, pH-sensitive liposomes have been
extensively studied as vehicles for cytoplasmic
delivery of drugs and nucleotides (Shangguan et
al., 1998; Connor and Huang, 1986). It is believed
that these liposomes would be destabilized in the
endocytic lumen and fuse eventually with the
endosome membranes, thereby releasing the en-
trapped contents into the cytoplasm before they
are destroyed in lysosomes (Ropert et al., 1993).
Liposomes are specially noted as potent gene
carriers in cell biology. A promising method is to
prepare a trigger-dependent fusion inducer as a
The fusion of biological membranes is of
fundamental importance in diverse cellular pro-
cesses such as fertilization (Gwatkin et al., 1976),
viral infection (Murayama and Okada, 1965), and
neuro-transmitter release from presynaptic vesicles
(Kelly et al., 1979). Although many of the
physiological functions mediated by membrane
fusion events are largely understood, the molecular
mechanism by which the fusion process itself
occurs is not clear. To gain insight into this
mechanism, various model systems are used, in
particular those involving phospholipid vesicles.
On the other hand, some chemical species like
* Corresponding author. Tel.: ꢁ
298-61-6123
E-mail address: m.kodaka@aist.go.jp (M. Kodaka).
/
81-298-61-6683; fax: ꢁ81-
/
0009-3084/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 0 9 - 3 0 8 4 ( 0 2 ) 0 0 0 5 3 - 1

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Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á68
/
lipid component, and efficient synthesis of this
type of lipid analogues have been demanded. A
carrier liposome is stable (fusion does not occur) in
an ordinary condition and cannot be used as a
carrier unless a fusion is induced by a stimulus.
Actually, we briefly showed in the previous com-
munication that liposomes including our novel
In the present work, we show a new route to
synthesize ADL2ÁADL12, in which 9-fluorenyl-
methoxycarbonyl (Fmoc) aspartic acid tert-butyl
ester is used instead of tert-butyloxy (Boc) aspartic
acid benzyl ester (Ogawa et al., 1999a). We there-
fore describe in detail the design and synthesis of
/
ADL2ÁADL12 and their functions as pH-response
/
triggers for liposome membrane fusion. In addi-
tion to the design and synthesis of the novel trigger
lipids, we have also succeeded in a theoretical
explanation of the observed relationship between
the structures and the fusion-inducing activity of
the trigger lipids.
artificial lipids (Scheme 1; ADL2ÁADL12), which
/
were designed to mimic the fundamental charac-
teristics of phosphatidylethanolamine, can induce
pH-dependent membrane fusion (Ogawa et al.,
1999a). Our new lipids are quite unique in that
they induce membrane fusion as lipid components.
Based on this idea, we also studied artificial lipids
with peptide ligands (Ogawa et al., 1999b; Yagi et
al., 1999, 2000) and 2-nitrophenol trigger (Shah et
al., 2000). Though fusion-inducing compounds
reported so far such as proteins and PEG have
strong activity to induce membrane fusion, their
sizes are too heavy to prepare liposomes in a good
reproducibility as the components and thus these
compounds are not adequate as pH-dependent
fusion inducers.
2. Results
The key step of the present study is the design
and synthesis of the trigger lipids possessing
alkylcarboxyl head groups with suitable hydro-
phobicity. For this purpose, we designed the six
aspartic acid-derived lipids (Scheme 1; ADL2Á
/
ADL12) with different alkyl length (viz. different
hydrophobicity) in the head groups. We synthe-
sized these lipids via two methods (route-I and -II)
as shown in Schemes 2 and 3. Though the both
methods gave ADL2ÁADL12, route-II is superior
/
to route-I, since the number of the steps in route-II
is less than that in route-I. Actually, the Boc and t-
Bu groups can be removed simultaneously with
trifluoroacetic acid (TFA) in the final step of
route-II (Scheme 3).
These lipids are composed of three fundamental
parts: (A) two long alkyl chains in the tail part,
which can be anchored to the hydrophobic regions
of lipid bilayer; (B) a polar moiety composed of
two aspartic acid residues in the central part; (C) a
terminal alkylcarboxylic acid group as a pH-
dependent trigger for liposome fusion. Part C
composed of the carboxyl group and the alkyl
chain was designed so that its hydrophobicity
could be controlled by pH, depending upon
whether the carboxyl group is protonated or
deprotonated. Since the pKa of the carboxyl head
groups is thought to be 4Á5 in view of the pKa of
/
an ordinary carboxyl group, part C should behave
as a hydrophobic protrusion on the lipid mem-
brane like the fusion peptide of hemagglutinin
Scheme 1. Structures of aspartic acid derived lipids and
phosphatidylethanolamine.

Scheme 2. Synthetic route-I of ADLs: (i) WSCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)/DMAP (4-dimethylaminopyridine); (ii) HOBt (1-
hydroxybenzotriazole hydrate)/TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate)/DMAP; (iii) TFA (trifluoroacetic acid); (iv) HOBt/WSCI/
DMAP; (v) TFA; (vi) HOBt/TBTU/DMAP; (vii) TFA; (viii) H2/PdÃC.
/

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Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á68
/
Scheme 3. Synthetic route-II of ADLs; (i) WSCI/DMAP, (ii) HOBt/TBTU/DMAP, (iii) TFA, (iv) HOBt/TBTU/DMAP, (v)
piperidine, (vi) HOBt/WSCI/DMAP, (vii) TFA.
(Tatulian et al., 1995) at acidic pH below 5. As
revealed in Scheme 1, ADLs (sꢀ2, 4, 6, 8, 10, 12)
in Fig. 1, which gives an exponential-type relaxa-
tion process.
/
Since the liposome fusion was actually observed
(Fig. 1), we further studied the dependency of the
fusion activity on the acyl-chains length. Fig. 2
shows the fusion % of liposomes including various
has some fundamental structural characteristics of
phosphatidylethanolamine. A typical time course
of fluorescence intensity at 530 nm for liposomes
containing ADL8 (10 mol%) at pH 4.0 is depicted
ADLs in 5 min after the acidification ([ADLs]ꢀ5,
/
10, 15, 20 mol%). A noteworthy finding is that
there are optimal lengths of the alkyl chain in the
head group of ADLs (sꢀ2, 4, 6, 8, 10, 12), among
/
which ADL6 and ADL8 (20 mol%) especially show
high fusion % (ca. 40%). The lipids with shorter or
longer chains (ADL2, ADL4 and ADL12), how-
ever, did not induce remarkable fusion, and the
membrane fusion at neutral pH was scarcely
observed in any liposome including the synthetic
lipids (up to 20 mol%) at least within 1 h (data not
shown).
The fusion activities of ADL6 and ADL8 in
various pH solutions (pH 3.6Á7.2) were examined
/
to confirm that the carboxyl groups actually
function as pH-controllable triggers. Fig. 3 shows
Fig. 1. A typical time course of fluorescence intensity (530 nm)
expressing liposome fusion at pH 4.0 using ADL8 (10 mol%).

Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á
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68
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Fig. 2. Relation between fusion % and alkyl chain lengths of head groups in ADLs; pH 4.0, 37 8C; composition (mol%) of ADLs in
liposomes is displayed inside the figure.
low fusion % between pH 3.6 and 7.2. In contrast
to ADL6, ADL8 and ADL10, the lipids with
shorter or longer chains, ADL2, ADL4 and
ADL12, did not induce remarkable fusion even
at acidic pH (data not shown).
To estimate the stability (tightness) of the
bilayers, the degree of calcein leakage through
liposome membranes was utilized as an index of
the stability of liposomes including ADLs. Fig. 4
shows the time course of calcein leakage in three
different types of ADLs. The liposomes including
ADL12 is as stable as the control liposomes
Fig. 3. Dependence of fusion % on pH of liposome solution; 20
mol% ADL6 (k), 20 mol% ADL8 (m), control (^); reaction
time 5 min, 37 8C. Curves for ADL6 and ADL8 are theore-
tically obtained.
the pH-dependency of the liposome fusion induced
by ADLs, where the curves for ADL6 and ADL8
are theoretically obtained as explained in detail in
Section 3. It is obvious that the carboxyl groups in
the head parts act as triggers in this fusion process.
The fusion of the liposomes containing ADL6 or
ADL8 was significantly accelerated below ca. pH 5
by the protonation of the carboxylic group, while
that of the control liposome (ADLs free) gave very
Fig. 4. Time course of calcein leakage from liposomes contain-
ing ADLs (20 mol%); (1) control (without ADLs); (2) ADL2; (3)
ADL8; (4) ADL12.

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Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á68
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without any ADLs. The liposomes including
ADL2 is, on the other hand, the least stable among
the liposomes used in the calcein leakage assay.
For example, the leakage in 10 min is about 1.5
times higher than that of the control liposome. The
stability of the liposome membranes including
ADL8 is situated between ADL2-liposome and
the control one.
were found to have appropriate alkyl lengths for
the pH-dependent fusogen in the liposome mem-
brane. As shown by the pH profile (Fig. 3), the
fusion proceeded up to 40Á50% in the pH region
/
lower than around 5. This clearly shows that the
protonation of the terminal carboxylate of ADLs
is the trigger of liposome fusion. Fig. 2 indicates
that there is an optimal region of hydrophobicity
between ADL6 and ADL10. The reason for the
very low ability of ADL12 to induce liposome
fusion may be that its alkyl chain in the head part
is too long and too hydrophobic to remain in an
outer aqueous phase and ADL12 is consequently
embedded in the membrane even at neutral pH. In
contrast to this, the head alkyl chains of ADL2
and ADL4 are considered to be too short and too
hydrophilic to migrate to another membrane,
which also results in low fusion %.
3. Discussion
For the last decade, liposomes have been
exploited predominantly as potent carriers of
various polar materials into cells (Yang et al.,
1997; Horth et al., 1991; Bailey and Cullis, 1997).
We have shown in the present study that artificial
lipids with simple structures can induce pH-
dependent membrane fusion in a manner similar
to authentic fusion inducers such as Spike proteins
of virus (Murayama and Okada, 1965; Tatulian et
al., 1995).
Theoretical interpretation is possible to the
above eperimental results. The hydrophobicity of
the trigger parts in ADLs can be quantitatively
estimated with log P value, which is semi-empiri-
cally given by Eq. (1) used in the CAChe system,
We have reported here the preparation of
biomimetic membrane-fusion inducers with quite
simple structures in comparison with fusion pep-
tides and proteins. In most fusion inducers such as
Spike protein of influenza virus, hydrophobic
fusion peptide is hidden at neutral pH. At acidic
pH, however, it protrudes from the membrane
surface and is incorporated into target membrane.
This makes these two membranes recognize each
other and start membrane fusion. In this point, the
carboxylic acid with long alkyl chain of ADLs,
which is protonated at low pH, is an appropriate
hydrophobic part to mimic fusion proteins as a
simple fusion inducer. Aspartic acid residues were
selected in the central part of ADLs skeleton, on
the basis of MM3 molecular mechanics calculation
(CAChe system ver 3.6, Sony Tektronix Co.).
Hydrophilic carboxylate groups of aspartic acid
residues attached to the central parts of ADLs are
thought to prevent the head group from sinking
into lipid bilayer membrane. To estimate the
appropriate balance of hydrophobicity and hydro-
philicity of part C required for pH-controlled
fusion, we synthesized six homologous lipids
having various methylene chain lengths in the
carboxylic acid part. ADL6, ADL8 and ADL10
log Pꢀꢂ0:33263ꢁ0:048959Sꢂ0:027225H
pffiffiffiffiffiffiffi
vac pffiffiffiffiffiffi
ꢁ0:03116Hwaterꢂ1:4003 NNꢂ1:0241 N0;
(1)
where Hvac and Hwater are, respectively, the heat of
formation in vacuo and in water, S is the van der
Waals area, NN and NO are, respectively, the
number of nitrogen and oxygen atoms. The values
Hvac, Hwater and S can be calculated by AM1
method in the CAChe system, where COSMO
(Conductor-like Screening Model) method is used
in an aqueous solution. In the actual AM1
calculation, model compounds, CH3NHCO-
(CH2)nCOOH and CH3NHCO(CH2)nCOOꢂ,
were used instead of the trigger part of ADLs.
Very good linear relationships have been found
between log P and n as given by Eqs. (2) and (3).
log Pꢀ0:332nꢂ1:327 (for RCOOH form); (2)
log Pꢀ0:318nꢂ3:768 (for RCOOꢂ form): (3)
Since the definition of P is the partition coefficient
of a compound between n-octanol and water, Eq.
(4) holds between the fraction distributed to n-

Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á
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68
57
octanol phase (X) and log P.
mental relation between the fusion activity and the
hydrophobicity of the trigger parts. It should be
emphasized, furthermore, that the present theore-
tical method is applicable to design any type of
trigger groups.
The pH-dependence curves of fusion % (Fig. 3)
can be also obtained theoretically as follows. The
decreasing rate of the concentration [L] of the
fluorescence-labeled liposome (Label-Lip) is ap-
proximately given by a pseudo-first order equation
(Eq. (5)), since the concentration of the fusion
liposome (Fusion Lip) is 10 times higher than
Label-Lip,
X ꢀP=(1ꢁP)ꢀ10log P=(1ꢁ10log P):
(4)
From Eqs. (2)Á(4), the relation between X and n
/
can be obtained (Fig. 5). It should be noted that
the X values of the carboxyl form (RCOOH) are
large for n ]
form (RCOOꢂ) are small for n 5
that the trigger parts with nꢀ6Á
/
6 and the X values of the carboxylate
10. This suggests
10 are apt to be
/
/
/
transferred to a hydrophobic phase, viz. lipid
membrane layer. These findings support the results
in Fig. 2 showing that ADL6, ADL8 and ADL10
induce liposome fusion efficiently. To show this
relation more clearly, Fig. 5 also illustrates the
dependence of the fusion % (20 mol% ADLs) on n.
It is obvious from Fig. 5 that the high fusion % of
ADL6, ADL8 and ADL10 is attributable to the
high affinity of the protonated trigger part toward
an organic phase (that is, large X values of the
RCOOH form) and the high affinity of the
deprotonated trigger part toward an aqueous
phase (that is, small X values of the RCOOꢂ
form). In ADL2 and ADL4 the X values of
RCOOH form are not large enough to efficiently
migrate into another liposome membrane, and in
ADL12 the X value of the RCOOHꢂ form is so
large that the trigger becomes inactive by being
embedded into its own liposome membrane. In
spite of its simple principle, the present theoretical
analysis has succeeded in describing the funda-
ꢂd[L]=dtꢀkm[L];
(5)
where k is the rate constant of liposome fusion
when all the carboxyl groups of the trigger parts
are protonated, and m is the fraction of the
protonated carboxyl group (05
/
m 51). Eq. (6) is
/
derived from the integration of Eq. (5),
fusion %ꢀ100(1ꢂ[L]=[L]0)
ꢀ100f1ꢂexp(ꢂkmt)g;
where m can be represented by Eq. (7).
(6)
(7)
mꢀ10pK ꢂpH=(10pK ꢂpH ꢁ1):
a
a
Eqs. (6) and (7) lead to,
fusion %ꢀ100[1ꢂexpfꢂ10pK ꢂpHkt=
a
(10pK ꢂpH ꢁ1)g]:
(8)
a
The experimental data can be well fitted by Eq. (8)
(tꢀ5 min) as shown in Fig. 3. The best-fit values
of k and pKa for ADL6 are 0.17 and 4.2 minꢂ1
/
,
and those for ADL8 are 0.11 and 4.6 minꢂ1. These
pKa values are reasonable in view of those values
of ordinary carboxylic acids, while the k values are
difficult to evaluate in the present study. The fact
that the experimental results can be well repro-
duced by the above theoretical methods supports
the conclusion that the carboxyl groups function
as pH-controllable triggers.
One of the structural features in ADLs is that
they have four amide bonds in one lipid molecule.
As amide bonds can interact with each other
through hydrogen bonds, the lipids can possibly
form clusters in the hydrophobic environment of
liposome membrane (Kremer et al., 2000). This
Fig. 5. Dependence of fraction (X) on number of methylene
group (n) of model trigger compounds; RCOOH form (m),
RCOOꢂ form (k), and dependence of fusion % (ADLs 20
mol%) on n (^) (see the text for detailed explanation).

58
Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á68
/
cluster formation can be easier at acidic pH than
neutral pH, because the anionic charges of carbox-
ylate attached to the head of ADLs may disturb
the cluster formation by electrostatic repulsion,
while there is no such interaction in the protonated
state. It is possible that this cluster formation has
some influence on the fusion induced by ADL6
and ADL8, as is normally observed in other
natural fusion proteins which are known to work
in a cooperative manner.
ADL8 and ADL10 have enough requirements for
pH-dependent membrane fusion in comparison
with fusogenic proteins or virus itself. Our new
trigger lipids may open a way to the development
of biomimetic liposomes as carriers of cytoplasmic
delivery of biofunctional macromolecules such as
DNA and proteins as well as ordinary drugs.
4. Experimental
It was shown that the incorporation of N-
palmitoyl phosphatidylethanolamine (NPPE) into
liposome membranes decreased the leakage of
calboxyfluorescein entrapped in the liposomes
(Domingo et al., 1993). Domingo et al. suggested
that the third acyl chain of NPPE was inserted in
the hydrophobic region of lipid bilayer and this
increased the hydrophobicity of core region of the
membranes, which can account for the suppression
of calboxyfluorescein leakage. It was demon-
strated, on the other hand, that the incorporation
of N-acetylphosphatidylethanolamine (NAcPE)
into liposome membranes increased the leakage
of calboxyfluorescein entrapped in the liposomes
(Ogawa and Epand, 1997). Here, the N-acetyl
group was supposed not to be inserted in the
hydrophobic region of bilayer. In the present
study, we have shown that the incorporation of
ADL2 to liposomes destabilizes the liposome
membrane as compared to the control one but
the incorporation of ADL12 to liposomes does
not. These results are well compatible with the
previous studies mentioned above. However, since
the calcein leakage is 5% at most, it is concluded
that the incorporation of ADLs as a whole does
not seriously disturb the membrane stability at
neutral pH.
We have designed and established efficient
routes of the syntheses of ADLs, where only
ordinarily available reagents are used. The mole-
cular weights of the synthetic lipids are smaller
than PEG and fusion proteins of viruses by 2 or 3
orders, and these lipids are quite easy to synthe-
size. Also the synthetic lipids, which induce pH-
dependent fusion, are more stable than fusion
proteins of viruses against temperature and acidic
and basic conditions. It should be specially em-
phasized that the simple structures of ADL6,
4.1. Material
Egg phosphatidylcholine (EPC) was obtained
from Kawahara Petrochemical Co., Ltd. (Osaka,
Japan). N-(7-Nitro-2,1,3-benzoxadiazol-4-yl)-1,2-
dipalmitoyl-sn-phosphatidylethanolamine (NBD-
PE) and N-(lissamine rhodamine B sulfonyl)-1,2-
dipalmitoyl-sn-phosphatidylethanolamine
PE) were purchased from Molecular Probes, Inc.
(Eugene, OR). N-tert-Butyloxycarbonyl- -aspar-
tic acid (Boc-Asp), N-tert-butyloxycarbonyl- -as-
partic acid-a-benzyl ester (Boc-Asp(aBzl)), 1-
hydroxybenzotriazole hydrate (HOBT) and 2-
(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluro-
nium tetrafluoroborate (TBTU) were commer-
cially available from Watanabe Chemical
Industries, Ltd. (Hiroshima, Japan). 4-Dimetyla-
minopyridine (DMAP) was obtained from Nacalai
Tesque, Inc. (Kyoto, Japan). 1-Hexadecylamine
was purchased from Sigma Chemical Co. (St.
Louis, MO). 1-Ethyl-3-(3-dimethylaminopropyl)-
carbodiimide monohydrochloride (WSCI), succi-
nic acid, adipic acid, suberic acid, sebacic acid and
2-methyl-2-propanol were obtained from Wako
Pure Chemical Industries, Ltd. (Osaka, Japan).
Di-n-dodecylamine, 1-hexadecylamine, 1,10-de-
canedicarboxylic acid and 1,12-dodecanedicar-
boxylic acid were purchased from Aldrich
Chemical Company, Inc. (Milwaukee, WIS). Cal-
cein was purchased from Dojindo Laboratories,
Inc. (Tokyo, Japan).
(Rh-
L
L
4.2. Synthesis of ADLs
As shown in Schemes 2 and 3, we synthesized
the final lipids (ADLs) via two routes, viz. route-I
using benzyl group and route-II using tert-butyl

Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á
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68
59
group to protect the b-carboxyl group of the
aspartic acid residue.
4.2.4. Sebacic acid mono-tert-butyl ester (4a)
Sebacic acid (1.0 g, 4.9 mmol), 2-methyl-2-
propanol (10 ml, 106 mmol), WSCI (0.95 g, 4.9
mmol) and DMAP (0.60 g, 4.9 mmol) were
dissolved in 10 ml of CH2Cl2. Compound 4a was
4.2.1. Succinic acid mono-tert-butyl ester (1a)
Succinic acid (1.0 g, 8.5 mmol), 2-methyl-2-
propanol (10 ml, 106 mmol), WSCI (1.6 g, 8.5
mmol) and DMAP (1.0 g, 8.5 mmol) were
dissolved in 10 ml of chloroform (CHCl3). The
solution was stirred at room temperature for 9 h.
The reaction mixture was diluted with ether (100
ml) and washed with 0.01 N HCl and distilled
water. After the organic phase was dried over
Na2SO4 and concentrated, the residue was purified
by column chromatography on silica gel using
obtained in a similar manner as 1a (TLC, Rfꢀ
/
1
0.4). Yield: 0.43 g (37%). H NMR (CDCl3): d
2.34 (t, 2H, CH2CO, Jꢀ7.4 Hz), 2.20 (t, 2H,
CH2CO, Jꢀ7.6 Hz), 1.60 (m, 4H, 2CH2CH2CO),
/
/
1.44 (s, 9H, C(CH3)3), 1.30 (br s, 8H,
(CH2)4CH2CH2CO). Anal. Calc. for C14H26O4:
C, 65.09; H, 10.14. Found: C, 65.23; H, 10.32%.
FAB MS calc.: 259.2 (Mꢁ
Hꢁ). Found: 259%.
/
ethyl acetateÁ
Rfꢀ0.2). Yield: 0.24 g (16%). H NMR (CDCl3):
2.51Á2.65 (m, 4H, (CH2)2), 1.45 (s, 9H,
/
hexane (4:6) as the eluant (TLC,
4.2.5. 1,10-Decanedicarboxylic acid mono-tert-
butyl ester (5a)
1,10-Decanedicarboxylic acid (1.0 g, 4.3 mmol),
2-methyl-2-propanol (10 ml, 106 mmol), WSCI
(0.83 g, 4.3 mmol) and DMAP (0.53 g, 4.3 mmol)
were dissolved in 10 ml of CHCl3. Compound 5a
1
/
d
/
C(CH3)3). Anal. Calc. for C8H14O4: C, 55.16; H,
8.10. Found: C, 55.11; H, 8.28%. FAB MS calc.:
175.1 (Mꢁ
Hꢁ). Found: 175%.
/
was obtained in a similar manner as 1a (TLC,
1
4.2.2. Adipic acid mono-tert-butyl ester (2a)
Adipic acid (1.0 g, 6.8 mmol), 2-methyl-2-
propanol (10 ml, 106 mmol), WSCI (1.30 g, 6.8
mmol) and DMAP (0.83 g, 6.8 mmol) were
dissolved in 10 ml of CHCl3. Compound 2a was
Rfꢀ
d 2.37 (t, 2H, CH2CO, Jꢀ
CH2CO, Jꢀ7.6 Hz), 1.60 (m, 4H, 2CH2CH2CO),
/
0.6). Yield: 0.54 g (44%). H NMR (CDCl3):
/
7.6 Hz), 2.20 (t, 2H,
/
1.44 (s, 9H, C(CH3)3), 1.28 (br s, 12H,
(CH2)6CH2CH2CO). Anal. Calc. for C16H30O4:
C, 67.10; H, 10.56. Found: C, 67.48; H, 10.66%.
FAB MS calc.: 287.2 (Mꢁ
Hꢁ). Found: 287%.
obtained in a similar manner as 1a (TLC, Rfꢀ
/
1
0.3). Yield: 0.40 g (29%). H NMR (CDCl3): d
2.38 (t, 2H, CH2CO, Jꢀ6.9 Hz), 2.24 (t, 2H,
CH2CO, Jꢀ2.5 Hz), 1.65 (m, 4H, CH2CH2CO),
/
/
/
4.2.6. 1,12-Dodecanedicarboxylic acid mono-tert-
butyl ester (6a)
1.44 (s, 9H, C(CH3)3), 1.30 (br s, 8H, (CH2)4).
Anal. Calc. for C10H18O4: C, 59.39; H, 8.97.
Found: C, 59.40; H, 9.14%. FAB MS calc.: 203.1
1,12-Dodecanedicarboxylic acid (1.5 g, 5.8
mmol), 2-methyl-2-propanol (10 ml, 106 mmol),
WSCI (1.13 g, 5.8 mmol) and DMAP (0.71 g, 5.8
mmol) were dissolved in 10 ml of CH2Cl2.
Compound 6a was obtained in a similar manner
(Mꢁ
Hꢁ). Found: 203%.
/
4.2.3. Suberic acid mono-tert-butyl ester (3a)
Suberic acid (1.0 g, 5.7 mmol), 2-methyl-2-
propanol (10 ml, 106 mmol), WSCI (1.09 g, 5.7
mmol) and DMAP (0.70 g, 5.7 mmol) were
dissolved in 10 ml of CHCl3. Compound 3a was
as 1a (TLC, Rfꢀ
NMR (CDCl3): d 2.35 (t, 2H, CH2CO, Jꢀ
Hz), 2.20 (t, 2H, CH2CO, Jꢀ7.4 Hz), 1.60 (m, 4H,
/
0.5). Yield: 0.54 g (30%). 1H
7.4
/
/
2CH2CH2CO), 1.44 (s, 9H, C(CH3)3), 1.26 (br s,
16H, (CH2)8CH2CH2CO). Anal. Calc. for
C18H34O4: C, 68.75; H, 10.90. Found: C, 69.18;
H, 10.95%. FAB MS calc.: 315.3 (Mꢁ
Hꢁ).
Found: 315%.
obtained in a similar manner as 1a (TLC, Rfꢀ
/
1
0.3). Yield: 0.39 g (30%). H NMR (CDCl3): d
2.35 (t, 2H, CH2CO, Jꢀ7.4 Hz), 2.21 (t, 2H,
CH2CO, Jꢀ7.4 Hz), 1.61 (m, 4H, 2CH2CH2CO),
/
/
/
1.44 (s, 9H, C(CH3)3), 1.36 (m, 4H,
(CH2)2CH2CH2CO). Anal. Calc. for C12H22O4:
C, 62.58; H, 9.63. Found: C, 62.79; H, 9.82%. FAB
4.2.7. Compound b
Boc-Asp (1.0 g, 4.3 mmol), 1-hexadecylamine
(2.0 g, 8.6 mmol), HOBT (1.5 g, 10 mmol), TBTU
MS calc.: 231.2 (Mꢁ
Hꢁ). Found: 231%.
/

60
Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á68
/
(3.2 g, 10 mmol) and DMAP (1.1 g, 8.6 mmol)
were dissolved in 30 ml of DMFÁCHCl3 (1:9). The
mmol) were dissolved in 20 ml of N,N-dimethyl-
formamide (DMF)ÁCH2Cl2 (1:9). The solution
/
/
solution was stirred at room temperature for 3 h.
The reaction mixture was diluted with CHCl3 (100
ml) and washed with 1 N NaOH aqueous solution
and distilled water. After the organic phase was
dried over Na2SO4 and concentrated, the residue
was purified by column chromatography on silica
was stirred at room temperature for 5 h. The
reaction mixture was washed with distilled water,
and d was isolated from the washed organic layer
by column chromatography on silica gel using
MeOHÁ
0.4). Yield: 1.2 g (46%). 1H NMR (CDCl3): d
7.44 (d, 1H, NHCH, Jꢀ6.6 Hz), 7.34 (m, 5H,
C6H5 and 1H, NHCH2), 5.85 (br s, 1H, NHCH2),
5.64 (d, 1H, BocNH, Jꢀ8.3 Hz), 5.17 (d, 1H,
CHAHBC6H5, Jꢀ12.3 Hz), 5.19 (d, 1H,
CHAHBC6H5, Jꢀ12.3 Hz), 4.58 (m, 2H, 2a-
CH), 3.20 (m, 4H, 2NHCH2), 2.27Á2.88 (m, 4H,
/
CHCl3 (5:95) as the eluant (TLC, Rfꢀ
/
gel using methanol (MeOH)Á
/
CHCl3 (5:95) as the
0.7). Yield: 2.6 g (88%). 1H
/
eluant (TLC, Rfꢀ
/
NMR (CDCl3): d 6.94 (br s, 1H, NHCH), 6.21 (d,
1H, NHCH), 5.97 (br s, 1H, NHCH2), 4.39 (m,
1H, CH), 3.20 (m, 4H, 2NHCH2), 2.82 (dd, 1H,
/
/
/
CHAHBCO, Jꢀ
CHAHBCO, Jꢀ
2NHCH2CH2), 1.45 (s, 9H, C(CH3)3), 1.25 (br s,
52H, 2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ
/3.6 and 15.0 Hz), 2.48 (dd, 1H,
/
/6.4 and 15.0 Hz), 1.47 (m, 4H,
2CHCH2), 1.48 (m, 4H, 2 NHCH2CH2), 1.42 (s,
9H, C(CH3)3), 1.25 (br s, 52H, 2(CH2)13CH3), 0.88
/
(t, 6H, 2CH2CH3, Jꢀ6.8 Hz). Anal. Calc. for
/
6.6 Hz). Anal. Calc. for C41H81N3O4: C, 72.41; H,
12.00; N, 6.18. Found: C, 72.58; H, 12.06; N,
C52H92N4O7: C, 70.55; H, 10.47; N, 6.33. Found:
C, 70.72; H, 10.67; N, 6.26%. FAB MS calc.: 885.7
6.07%. FAB MS calc.: 680.6 (Mꢁ
/
Hꢁ). Found:
(Mꢁ
Hꢁ). Found: 886%.
/
681%.
4.2.8. Compound c
4.2.10. Compound e
Compound b (2.6 g, 3.8 mmol) was dissolved in
CHCl3 (10 ml) and then trifluoroacetic acid (TFA)
(2.0 ml) was added. The solution was stirred at
room temperature for 2 h. The reaction mixture
was washed with 1 N NaOH aqueous solution and
distilled water. Compound c was isolated from the
washed organic layer by column chromatography
Compound d (1.0 g, 1.1 mmol) was dissolved in
CHCl3 (30 ml) and then TFA (5.0 ml) was added.
The solution was stirred at room temperature for 1
h. The reaction mixture was washed with 10% of
NaHCO3 aqueous solution. Compound e was
isolated from the washed organic layer by column
chromatography on silica gel using MeOHÁ
CHCl3 (5:95) as the eluant (TLC, Rfꢀ0.3). Yield:
0.68 g (81%). H NMR (CDCl3): d 8.00 (d, 1H,
NHCH, Jꢀ7.3 Hz), 7.35 (m, 7H, NHCH2,
NHCH and C6H5), 5.98 (t, 1H, NHCH2, Jꢀ5.5
Hz), 5.17 (s, 2H, CH2C6H5), 4.66 (ddd, 1H,
NHCH, Jꢀ6.8, 6.9 and 3.6 Hz), 3.91 (dd, 1H,
NH2CH, Jꢀ7.8 and 5.3 Hz), 3.19 (m, 4H,
2NCH2), 2.85 (dd, 1H, NHCHCHAHB, Jꢀ14.9
and 3.5 Hz), 2.42 (dd, 1H, NHCHCHAHB, Jꢀ
14.9 and 6.3 Hz), 2.72 (dd, 1H, NH2CHCHAHB,
Jꢀ15.2 and 5.3 Hz), 2.58 (dd, 1H,
NH2CHCHAHB, Jꢀ15.2 and 7.6 Hz), 1.78 (br
s, 2H, NH2), 1.47 (m, 4H, 2NHCH2CH2), 1.25 (br
s, 52H, 2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ
/
on silica gel using MeOHÁ
eluant (TLC, Rfꢀ
0.2). Yield: 1.7 g (78%). 1H
NMR (CDCl3): d 7.49 (t, 1H, NHCH2, Jꢀ5.0
Hz), 6.12 (t, 1H, NHCH2, Jꢀ5.0 Hz), 3.65 (t, 1H,
NHCH, Jꢀ5.6 Hz), 3.21 (m, 4H, 2NHCH2), 2.62
(dd, 1H, CHAHBCO, Jꢀ
(dd, 1H, CHAHBCO, Jꢀ
/
CHCl3 (5:95) as the
/
1
/
/
/
/
/
/
/5.0 and 14.2 Hz), 2.55
/
/6.6 and 14.2 Hz), 1.86
/
(br s, 2H, NH2), 1.48 (m, 4H, 2NHCH2CH2), 1.26
(br s, 52H, 2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3,
/
/
Jꢀ
H, 12.69; N, 7.25. Found: C, 74.49; H, 13.01; N,
7.16%. FAB MS calc.: 580.6 (Mꢁ
Hꢁ). Found:
/6.4 Hz). Anal. Calc. for C36H73N3O2: C, 74.55;
/
/
/
581%.
/
4.2.9. Compound d
6.8 Hz). Anal. Calc. for C47H84N4O5: C, 71.89; H,
10.78; N, 7.14. Found: C, 70.90; H, 11.11; N,
Compound c (1.7 g, 3.0 mmol), HOBT (0.54 g,
4.0 mmol), Boc-Asp(aBzl) (1.1 g, 3.5 mmol),
WSCI (0.77 g, 4.0 mmol) and DMAP (0.49 g, 3.0
7.33%. FAB MS calc.: 785.7 (Mꢁ
/
Hꢁ). Found:
786%.

Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á
/
68
61
4.2.11. Compound 1f
4.2.13. Compound 2f
Compound 1a (59 mg, 0.34 mmol), HOBT (54
mg, 0.35 mmol), TBTU (112 mg, 0.35 mmol), e
(250 mg, 0.33 mmol) and DMAP (43 mg, 0.35
Compound 2a (69 mg, 0.34 mmol), HOBT (54
mg, 0.35 mmol), TBTU (112 mg, 0.35 mmol), e
(250 mg, 0.33 mmol) and DMAP (43 mg, 0.35
mmol) were dissolved in 20 ml of DMFÁ
(1:9). Compound 2f was obtained in a similar
manner as 1f (TLC, Rfꢀ0.3). Yield: 190 mg
(59%). H NMR (CDCl3): d 7.34 (m, 7H, C6H5,
NHCH, and NHCH2), 6.78 (d, 1H, NHCH, Jꢀ
8.2 Hz), 5.91 (t, 1H, NHCH2, Jꢀ5.6 Hz), 5.19 (d,
1H, CHAHBC6H5, Jꢀ12.2 Hz), 5.15 (d, 1H,
CHAHBC6H5, Jꢀ12.2 Hz), 4.91 (ddd, 1H, CH,
Jꢀ8.3, 5.0 and 4.8 Hz), 4.57 (ddd, 1H, CH, Jꢀ
7.6, 6.9 and 3.1 Hz), 3.19 (m, 4H, 2NHCH2), 2.85
(dd, 1H, CHCHAHBCO, Jꢀ15.5 and 5.0 Hz),
2.77 (dd, 1H, CHCHAHBCO, Jꢀ15.5 and 5.0
Hz), 2.67 (dd, 1H, CHCHAHBCO, Jꢀ
Hz), 2.30 (dd, 1H, CHCHAHBCO, Jꢀ
/
CHCl3
mmol) were dissolved in 20 ml of DMFÁCHCl3
/
(1:9). The solution was stirred at room tempera-
ture for 8 h. The reaction mixture was diluted with
CHCl3 (100 ml) and washed with distilled water.
After the organic phase was dried over Na2SO4
and concentrated, the residue was purified by
column chromatography on silica gel using
/
1
/
/
/
/
MeOHÁ
0.4). Yield: 0.19 g (61%). H NMR (CDCl3): d
7.44 (d, 1H, NHCH, Jꢀ6.9 Hz), 7.34 (m, 6H,
C6H5 and NHCH2), 6.98 (d, 1H, NHCH, Jꢀ8.3
Hz), 5.99 (t, 1H, NHCH2, Jꢀ5.6 Hz), 5.18 (d, 1H,
CHAHBC6H5, Jꢀ12.2 Hz), 5.16 (d, 1H,
CHAHBC6H5, Jꢀ7.3 Hz), 4.88 (ddd, 1H, CH,
Jꢀ8.3, 5.0 and 5.0 Hz), 4.60 (ddd, 1H, CH, Jꢀ
4.0, 3.6 and 3.6 Hz), 3.19 (m, 4H, 2NHCH2), 2.32Á
/
CHCl3 (5:95) as the eluant (TLC, Rfꢀ
/
1
/
/
/
/
/
/
/
/
8.1 and 3.1
8.1 and 7.6
/
/
/
Hz), 2.23 and 2.20 (t, 4H, 2CH2CH2CO), 1.61 (m,
4H, 2CH2CH2CO), 1.47 (m, 4H, 2NHCH2CH2),
1.43 (br s, 9H, C(CH3)3), 1.25 (br s, 52H,
/
/
/
2.88 (m, 8H, 2CHCH2CO and 2CH2CH2CO), 1.47
(m, 4H, 2CH2), 1.43 (s, 9H, C(CH3)3), 1.25 (br s,
2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ6.8
/
Hz). Anal. Calc. for C57H100N4O8: C, 70.62; H,
10.40; N, 5.78. Found: C, 70.62; H, 10.50; N,
52H, 2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ
/
6.6 Hz). Anal. Calc. for C55H96N4O8: C, 70.17; H,
10.28; N, 5.95. Found: C, 70.21; H, 10.47; N,
5.68%. FAB MS calc.: 969.8 (Mꢁ
Hꢁ). Found:
970%.
/
5.95%. FAB MS calc.: 941.7 (Mꢁ
Hꢁ). Found:
942%.
/
4.2.14. Compound 2g
Compound 2f (190 mg, 0.196 mmol) was
dissolved in 8.0 ml of CHCl3 and then TFA (2.0
ml) was added. Compound 2g was obtained in a
similar manner as 1g. Yield: 181 mg (99%). H
NMR (CDCl3 and CD3OD): d 7.34 (m, 5H,
C6H5), 5.16 (s, 2H, CH2C6H5), 4.82 (t, 1H, CH,
4.2.12. Compound 1g
Compound 1f (190 mg, 0.202 mmol) was
dissolved in CHCl3 (8.0 ml) and then TFA (2.0
ml) was added. The solution was stirred at room
temperature for 2 h. The reaction mixture was
diluted with CHCl3 (100 ml) and washed with
distilled water. The organic phase was dried over
Na2SO4 and concentrated. Yield: 176 mg (99%).
1H NMR (CDCl3 and CD3OD): d 7.34 (m, 5H,
C6H5), 5.17 (s, 2H, CH2C6H5), 4.82 (t, 1H, CH,
1
Jꢀ
2NHCH2), 2.81 (dd, 1H, CHCHAHBCO, Jꢀ
and 7.6 Hz), 2.76 (dd, 1H, CHCHAHBCO, Jꢀ
13.2 and 7.3 Hz), 2.70 (dd, 1H, CHCHAHBCO,
Jꢀ15.0 and 5.8 Hz), 2.63 (dd, 1H,
CHCHAHBCO, Jꢀ15.0 and 6.4 Hz), 2.28 and
/
5.6 Hz), 4.61 (t, 1H, CH, 5.9 Hz), 3.15 (m, 4H,
/
13.2
/
/
Jꢀ
/
5.6 Hz), 4.62 (t, 1H, CH, Jꢀ
/
6.1 Hz), 3.16 (m,
/
4H, 2NHCH2), 2.44Á
and 2CH2CH2CO), 1.48 (m, 4H, 2NHCH2CH2),
1.27 (br s, 52H, 2(CH2)13CH3), 0.88 (t, 6H,
/
2.85 (m, 8H, 2CHCH2CO
2.24 (m, 4H, 2CH2CH2CO), 1.62 (m, 4H,
2CH2CH2CO), 1.47 (br s, 4H, 2NHCH2CH2),
1.26 (br s, 52H, 2(CH2)13CH3), 0.88 (t, 6H,
2CH2CH3, Jꢀ6.6 Hz). Anal. Calc. for
/
2CH2CH3, Jꢀ6.8 Hz). Anal. Calc. for
/
C51H88N4O8: C, 69.19; H, 10.02; N, 6.33. Found:
C, 69.31; H, 10.21; N, 6.23%. FAB MS calc.: 885.7
C53H92N4O8: C, 69.70; H, 10.15; N, 6.13. Found:
C, 69.63; H, 10.41; N, 6.07%. FAB MS calc.: 913.7
(Mꢁ
/
Hꢁ). Found: 886%.
(Mꢁ
Hꢁ). Found: 914%.
/

62
Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á68
/
4.2.15. Compound 3f
10.28; N, 5.95. Found: C, 70.21; H, 10.61; N,
5.80%. FAB MS calc.: 941.7 (Mꢁ
Hꢁ). Found:
942%.
Compound 3a (69 mg, 0.30 mmol), HOBT (50
mg, 0.33 mmol), TBTU (103 mg, 0.32 mmol), e
(200 mg, 0.27 mmol) and DMAP (37 mg, 0.30
/
mmol) were dissolved in 20 ml of DMFÁ
(1:9). Compound 3f was obtained in a similar
manner as 1f (TLC, Rfꢀ0.4). Yield: 142 mg
(53%). 1H NMR (CDCl3): d 7.41 (d, 1H,
NHCH, Jꢀ6.9 Hz), 7.34 (m, 6H, C6H5 and
NHCH2), 6.75 (d, 1H, NHCH, Jꢀ8.3 Hz), 5.99
(t, 1H, NHCH2, Jꢀ5.8 Hz), 5.19 (d, 1H,
CHAHBC6H5, Jꢀ12.2 Hz), 5.15 (d, 1H,
CHAHBC6H5, Jꢀ12.2 Hz), 4.91 (ddd, 1H, CH,
Jꢀ7.9, 5.0 and 4.7 Hz), 4.58 (ddd, 1H, CH, Jꢀ
7.3, 7.0 and 3.0 Hz), 3.20 (m, 4H, 2NHCH2), 2.85
(dd, 1H, CHCHAHBCO, Jꢀ15.5 and 5.0 Hz),
2.78 (dd, 1H, CHCHAHBCO, Jꢀ15.5 and 5.0
Hz), 2.67 (dd, 1H, CHCHAHBCO, Jꢀ15.2 and
3.3 Hz), 2.32 (dd, 1H, CHCHAHBCO, Jꢀ15.0
and 7.6 Hz), 2.21 (t, 2H, CH2CH2CO, Jꢀ5.9 Hz),
2.18 (t, 2H, CH2CH2CO, Jꢀ7.6 Hz), 1.57 (m, 4H,
/
CHCl3
4.2.17. Compound 4f
Compound 4a 89 mg (0.3 mmol), HOBT (54
mg, 0.35 mmol), TBTU (112 mg, 0.35 mmol), e
(250 mg, 0.33 mmol) and DMAP (43 mg, 0.35
/
/
mmol) were dissolved in 20 ml of DMFÁ
(1:9). Compound 4f was obtained in a similar
manner as 1f (TLC, Rfꢀ0.4). Yield: 175 mg
(51%). H NMR (CDCl3): d 7.34 (m, 7H, C6H5,
NHCH, and NHCH2), 6.70 (d, 1H, NHCH, Jꢀ
7.9 Hz), 5.83 (t, 1H, NHCH2, Jꢀ5.6 Hz), 5.20 (d,
1H, CHAHBC6H5, Jꢀ12.2 Hz), 5.15 (d, 1H,
CHAHBC6H5, Jꢀ12.2 Hz), 4.92 (ddd, 1H, CH,
Jꢀ7.9, 5.0 and 4.3 Hz), 4.57 (ddd, 1H, CH, Jꢀ
7.3, 7.0 and 2.8 Hz), 3.20 (m, 4H, 2NHCH2), 2.85
(dd, 1H, CHCHAHBCO, Jꢀ15.5 and 5.0 Hz),
2.77 (dd, 1H, CHCHAHBCO, Jꢀ15.5 and 4.6
Hz), 2.67 (dd, 1H, CHCHAHBCO, Jꢀ15.0 and
3.3 Hz), 2.30 (dd, 1H, CHCHAHBCO, Jꢀ15.0
/
CHCl3
/
/
/
1
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
2CH2CH2CO), 1.47 (m, 4H, 2NHCH2CH2), 1.44
(br s, 9H, C(CH3)3), 1.25 (br s, 56H,
(CH2)2CH2CH2CO and 2(CH2)13CH3), 0.88 (t,
/
and 7.3 Hz), 2.19 (m, 4H, 2CH2CH2CO), 1.67 (m,
4H, 2CH2CH2CO), 1.53 (m, 4H, 2NHCH2CH2),
1.44 (br.s, 9H, C(CH3)3), 1.25 (br.s, 60H,
(CH2)4CH2CH2CO and 2(CH2)13CH3), 0.88 (t,
6H, 2CH2CH3, Jꢀ6.6 Hz). Anal. Calc. for
/
C59H104N4O8: C, 71.04; H, 10.51; N, 5.62. Found:
C, 71.11; H, 10.65; N, 5.47%. FAB MS calc.: 997.8
6H, 2CH2CH3, Jꢀ6.4 Hz). Anal. Calc. for
/
(Mꢁ
/
Hꢁ). Found: 998%.
C61H108N4O8: C, 71.44; H, 10.62; N, 5.46. Found:
C, 71.61; H, 10.87; N, 5.38%. FAB MS calc.:
4.2.16. Compound 3g
1025.8 (Mꢁ
Hꢁ). Found: 1026%.
/
Compound 3f (106 mg, 0.098 mmol) was
dissolved in CHCl3 (8.0 ml) and then TFA (2.0
ml) was added. Compound 3g was obtained in a
similar manner as 1g. Yield: 103 mg (99%). H
NMR (CDCl3 and CD3OD): d 7.34 (m, 5H,
4.2.18. Compound 4g
Compound 4f (175 mg, 0.171 mmol) was
dissolved in CHCl3 (8.0 ml) and then TFA (2.0
ml) was added. Compound 4g was obtained in a
similar manner as 1g. Yield: 164 mg (99%). H
NMR (CDCl3 and CD3OD): d 7.34 (m, 5H,
1
1
C6H5), 5.16 (s, 2H, CH2C6H5, Jꢀ
(t, 1H, CH, Jꢀ5.8 Hz), 4.62 (t, 1H, CH, Jꢀ
Hz), 3.15 (m, 4H, 2NHCH2), 2.81 (dd, 1H,
CHCHAHBCO, Jꢀ15.7 and 5.9 Hz), 2.76 (dd,
1H, CHCHAHBCO, Jꢀ15.4 and 5.9 Hz), 2.60
(dd, 1H, CHCHAHBCO, Jꢀ14.9 and 5.6 Hz),
2.48 (dd, 1H, CHCHAHBCO, Jꢀ14.9 and 6.3
Hz), 2.28 (t, 2H, CH2CH2CO, Jꢀ7.4 Hz), 2.21 (t,
2H, CH2CH2CO, Jꢀ7.4 Hz), 1.59 (m, 4H,
/3.3 Hz), 4.82
/
/6.1
C6H5), 5.16 (s, 2H, CH2C6H5, Jꢀ
(t, 1H, CH, Jꢀ5.5 Hz), 4.62 (t, 1H, CH, Jꢀ
Hz), 3.16 (m, 4H, 2NHCH2), 2.82 (dd, 1H,
CHCHAHBCO, Jꢀ15.0 and 5.9 Hz), 2.76 (dd,
1H, CHCHAHBCO, Jꢀ15.4 and 5.9 Hz), 2.60
(dd, 1H, CHCHAHBCO, Jꢀ15.0 and 5.9 Hz),
2.48 (dd, 1H, CHCHAHBCO, Jꢀ14.9 and 6.3
Hz), 2.28 (t, 2H, CH2CH2CO, Jꢀ7.4 Hz), 2.21 (t,
2H, CH2CH2CO, Jꢀ7.4 Hz), 1.59 (m, 4H,
/
3.3 Hz), 4.82
/
/
/
5.5
/
/
/
/
/
/
/
/
/
2CH2CH2CO), 1.47 (br s, 4H, 2NHCH2CH2),
1.26 (br s, 56H, (CH2)2CH2CH2CO and
/
/
2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ
Hz). Anal. Calc. for C55H96N4O8: C, 70.17; H,
/
6.6
2CH2CH2CO), 1.47 (br s, 4H, 2NHCH2CH2),
1.27 (br s, 60H, (CH2)4CH2CH2CO and

Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á
/
68
63
2(CH2)13CH3), 0.89 (t, 6H, 2CH2CH3, Jꢀ
/
6.4 Hz).
2CH2CH2CO), 1.47 (br.s, 4H, 2NHCH2CH2),
1.26 (br.s, 64H, (CH2)6CH2CH2CO and
Anal. Calc. for C57H100N4O8: C, 70.62; H, 10.40;
N, 5.78. Found: C, 70.84; H, 10.79; N, 5.73%.
2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ6.3
/
FAB MS calc.: 969.7 (Mꢁ
/
Hꢁ). Found: 970%.
Hz). Anal. Calc. for C59H104N4O8: C, 71.04; H,
10.51; N, 5.62. Found: C, 70.99; H, 10.81; N,
4.2.19. Compound 5f
5.62%. FAB MS calc.: 997.8 (Mꢁ
Hꢁ). Found:
998%.
/
Compound 5a (97 mg, 0.34 mmol), HOBT (54
mg, 0.35 mmol), TBTU (112 mg, 0.35 mmol), e
(250 mg, 0.33 mmol) and DMAP (43 mg, 0.35
4.2.21. Compound 6f
mmol) were dissolved in 20 ml of DMFÁ
(1:9). Compound 5f was obtained in a similar
manner as 1f (TLC, Rfꢀ0.5). Yield: 237 mg
(68%). 1H NMR (CDCl3): d 7.46 (d, 1H,
NHCH, Jꢀ6.6 Hz), 7.34 (m, 6H, C6H5 and
NHCH2), 6.72 (d, 1H, NHCH, Jꢀ8.3 Hz), 5.90
(t, 1H, NHCH2, Jꢀ5.6 Hz), 5.19 (d, 1H,
CHAHBC6H5, Jꢀ12.5 Hz), 5.15 (d, 1H,
CHAHBC6H5, Jꢀ12.5 Hz), 4.92 (ddd, 1H, CH,
Jꢀ8.2, 5.0 and 5.0 Hz), 4.57 (ddd, 1H, CH, Jꢀ
7.3, 7.0 and 2.8 Hz), 3.20 (m, 4H, 2NHCH2), 2.85
(dd, 1H, CHCHAHBCO, Jꢀ15.8 and 5.0 Hz),
2.78 (dd, 1H, CHCHAHBCO, Jꢀ15.8 and 5.0
Hz), 2.67 (dd, 1H, CHCHAHBCO, Jꢀ15.0 and
3.3 Hz), 2.31 (dd, 1H, CHCHAHBCO, Jꢀ15.0
/
CHCl3
Compound 6a (110 mg, 0.35 mmol), HOBT (61
mg, 0.40 mmol), TBTU (128 mg, 0.40 mmol), e
(250 mg, 0.33 mmol) and DMAP (49 mg, 0.40
/
mmol) were dissolved in 20 ml of DMFÁ
(1:9). Compound 6f was obtained in a similar
manner as 1f (TLC, Rfꢀ0.5). Yield: 150 mg
(42%). 1H NMR (CDCl3): d 7.39 (d, 1H,
NHCH, Jꢀ6.9 Hz), 7.34 (m, 6H, C6H5 and
NHCH2), 6.70 (d, 1H, NHCH, Jꢀ8.3 Hz), 5.84
(t, 1H, NHCH2, Jꢀ5.6 Hz), 5.20 (d, 1H,
CHAHBC6H5, Jꢀ12.5 Hz), 5.15 (d, 1H,
CHAHBC6H5, Jꢀ12.5 Hz), 4.92 (ddd, 1H, CH,
Jꢀ8.3, 4.9 and 4.7 Hz), 4.57 (ddd, 1H, CH, Jꢀ
7.3, 7.0 and 2.8 Hz), 3.20 (m, 4H, 2NHCH2), 2.85
(dd, 1H, CHCHAHBCO, Jꢀ15.7 and 5.0 Hz),
2.78 (dd, 1H, CHCHAHBCO, Jꢀ16.0 and 5.0
Hz), 2.67 (dd, 1H, CHCHAHBCO, Jꢀ15.2 and
3.3 Hz), 2.30 (dd, 1H, CHCHAHBCO, Jꢀ15.2
/
CHCl3
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
and 7.3 Hz), 2.19 (m, 4H, 2CH2CH2CO), 1.57 (m,
4H, 2CH2CH2CO), 1.44 (m, 11H, 2NHCH2CH2
and C(CH3)3), 1.25 (br s, 64H, (CH2)6CH2CH2CO
/
/
/
and 2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ
/
6.6
and 7.3 Hz), 2.20 (m, 4H, 2CH2CH2CO), 1.58 (m,
4H, 2CH2CH2CO), 1.44 (m, 13H, 2NHCH2CH2
and C(CH3)3), 1.25 (br s, 68H, (CH2)8CH2CH2CO
Hz). Anal. Calc. for C63H112N4O8: C, 71.82; H,
10.72; N, 5.32. Found: C, 71.82; H, 10.86; N,
5.23%. FAB MS calc.: 1053.9 (Mꢁ
/
Hꢁ). Found:
and 2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ6.8
/
1054%.
Hz). Anal. Calc. for C65H116N4O8: C, 72.18; H,
10.81; N, 5.18. Found: C, 72.56; H, 11.02; N,
4.2.20. Compound 5g
5.14%. FAB MS calc.: 1081.9 (Mꢁ
Hꢁ). Found:
1082%.
/
Compound 5f (160 mg, 0.152 mmol) was
dissolved in CHCl3 (8.0 ml) and then TFA (2.0
ml) was added. Compound 5g was obtained in a
similar manner as 1g. Yield: 154 mg (99%). H
NMR (CDCl3 and CD3OD): d 7.34 (m, 5H,
C6H5), 5.16 (s, 2H, CH2C6H5, Jꢀ
(t, 1H, CH, Jꢀ5.5 Hz), 4.60 (t, 1H, CH, Jꢀ
Hz), 3.15 (m, 4H, 2NHCH2), 2.81 (dd, 1H,
CHCHAHBCO, Jꢀ14.5 and 5.6 Hz), 2.78 (dd,
1H, CHCHAHBCO, Jꢀ15.2 and 5.9 Hz), 2.60
(dd, 1H, CHCHAHBCO, Jꢀ15.0 and 5.9 Hz),
2.45 (dd, 1H, CHCHAHBCO, Jꢀ14.9 and 6.3
Hz), 2.29 (t, 2H, CH2CH2CO, Jꢀ7.4 Hz), 2.20 (t,
2H, CH2CH2CO, Jꢀ7.4 Hz), 1.60 (m, 4H,
4.2.22. Compound 6g
1
Compound 6f (137 mg, 0.127 mmol) was
dissolved in CHCl3 (8.0 ml) and then TFA (2.0
ml) was added. Compound 6g was obtained in a
/
3.3 Hz), 4.83
1
/
/
5.8
similar manner as 1g. Yield: 119 mg (91%). H
NMR (CDCl3 and CD3OD): d 7.34 (m, 5H,
/
C6H5), 5.16 (s, 2H, CH2C6H5, Jꢀ
(t, 1H, CH, Jꢀ5.6 Hz), 4.61 (t, 1H, CH, Jꢀ
Hz), 3.16 (m, 4H, 2 NHCH2), 2.82 (dd, 1H,
CHCHAHBCO, Jꢀ15.7 and 5.6 Hz), 2.76 (dd,
1H, CHCHAHBCO, Jꢀ16.2 and 5.3 Hz), 2.59
(dd, 1H, CHCHAHBCO, Jꢀ15.0 and 5.9 Hz),
/
3.3 Hz), 4.82
/
/
/
5.9
/
/
/
/
/
/
/

64
Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á68
/
2.48 (dd, 1H, CHCHAHBCO, Jꢀ
Hz), 2.29 (t, 2H, CH2CH2CO, Jꢀ7.4 Hz), 2.21 (t,
2H, CH2CH2CO, Jꢀ7.4 Hz), 1.60 (m, 4H,
2CH2CH2CO), 1.47 (br s, 4H, 2NHCH2CH2),
1.26 (br s, 64H, (CH2)6CH2CH2CO and
/
14.9 and 6.3
1H, NHCH), 3.76 (dd, 1H, NH2CH, Jꢀ
8.1 Hz), 3.18 (m, 4H, 2NHCH2), 2.90 (dd, 1H,
/
5.1 and
/
/
CHCHAHB, Jꢀ
CHCHAHB, Jꢀ
CHCHAHB, Jꢀ
CHCHAHB, Jꢀ2.6 and 15.1 Hz), 1.77 (br s, 2H,
/
3.6 and 15.0 Hz), 2.68 (dd, 1H,
5.0 and 15.1 Hz), 2.49 (dd, 1H,
5.0 and 14.8 Hz), 2.46 (dd, 1H,
/
/
2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ
/6.6
/
Hz). Anal. Calc. for C61H108N4O8: C, 71.44; H,
10.62; N, 5.46. Found: C, 71.55; H, 10.90; N,
NH2), 1.48 (br s, 4H, 2NHCH2CH2), 1.47 (s, 9H,
C(CH3)3), 1.25 (br s, 52H, 2(CH2)13CH3), 0.88 (t,
5.42%. FAB MS calc.: 1025.8 (Mꢁ
/
Hꢁ). Found:
6H, 2CH2CH3, Jꢀ6.6 Hz). Anal. Calc. for
/
1026%.
C44H86N4O5: C, 70.35; H, 11.54; N, 7.46. Found:
C, 70.23; H, 11.99; N, 7.27%. FAB MS calc.: 751.7
4.2.23. Compound h
(Mꢁ
Hꢁ). Found: 752%.
/
Compound c (2.39 g, 2.4 mmol), HOBT (0.40 g,
2.6 mmol), Fmoc-Asp(a-t-Bu) (1.0 g, 2.4 mmol),
TBTU (0.84 g, 2.6 mmol) and DMAP (0.32 g, 2.6
4.2.25. Compound 1j
Compound 1a (24 mg, 0.14 mmol), HOBT (23
mg, 0.15 mmol), WSCI (29 mg, 0.15 mmol), i (100
mg, 0.13 mmol) and DMAP (16 mg, 0.13 mmol)
were dissolved in CHCl3 (30 ml) including DMF
(1.0 ml). The solution was stirred at room tem-
perature for 10 h. The reaction mixture was diluted
with CHCl3 (100 ml) and washed with distilled
water. After the organic phase was dried over
Na2SO4 and concentrated, the residue was purified
by column chromatography on silica gel using
mmol) were dissolved in 150 ml of DMFÁCHCl3
/
(1:1). The solution was stirred at room tempera-
ture for 4 h. After the reaction mixture was washed
with distilled water, h was isolated from the
washed organic layer by column chromatography
on silica gel using MeOHÁ
eluant (TLC, Rfꢀ0.6). Yield: 2.17 g (92%). H
NMR (CDCl3): d 7.76 (d, 2H, fluorene, Jꢀ7.3
Hz), 7.61 (d, 2H, fluorene, Jꢀ7.3 Hz), 7.48 (d,
1H, NHCH, Jꢀ6.3 Hz), 7.40 (t, 2H, fluorene,
Jꢀ7.3 Hz), 7.31 (t, 2H, fluorene, Jꢀ7.3 Hz), 7.29
(br s, 1H, NHCH2), 5.94 (br s, 1H, NHCH2), 5.93
(d, 1H, NHCH, Jꢀ6.9 Hz), 4.65 (m, 1H, CH),
4.55 (m, 1H, CH), 4.37 (m, 2H, CHCH2OCO),
4.23 (t, 1H, CHCH2OCO, Jꢀ6.9 Hz), 3.19 (m,
4H, 2NHCH2), 2.37Á2.88 (m, 4H, 2CHCH2), 1.47
/
CHCl3 (5:95) as the
1
/
/
/
/
MeOHÁ
0.3). Yield: 97 mg (81%). H NMR (CDCl3): d
7.44 (d, 1H, NHCH, Jꢀ7.3 Hz), 7.32 (t, 1H,
NHCH2, Jꢀ5.9 Hz), 6.81 (d, 1H, NHCH, Jꢀ7.9
Hz), 6.01 (t, 1H, NHCH2, Jꢀ5.9 Hz), 4.72 (m,
1H, CH), 4.64 (m, 1H, CH), 3.20 (m, 4H,
2NHCH2), 2.40Á2.82 (m, 8H, 2CHCH2CO and
/
CHCl3 (5:95) as the eluant (TLC, Rfꢀ
/
1
/
/
/
/
/
/
/
/
/
/
(s, 9H, C(CH3)3), 1.46 (br s, 4H, 2NHCH2CH2),
1.25 (br s, 52H, 2(CH2)13CH3), 0.88 (t, 6H,
2CH2CH2CO), 1.47 (br s, 4H, 2NHCH2CH2), 1.46
(s, 9H, C(CH3)3), 1.44 (s, 9H, C(CH3)3), 1.25 (br s,
2CH2CH3, Jꢀ/6.6 Hz). Anal. Calc. for
52H, 2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ
/
C59H96N4O7: C, 72.80; H, 9.94; N, 5.76. Found:
C, 72.86; H, 10.22; N, 5.70%. FAB MS calc.: 973.7
6.6 Hz). Anal. Calc. for C52H98N4O8: C, 68.83; H,
10.89; N, 6.18. Found: C, 68.73; H, 10.71; N,
(Mꢁ
/
Hꢁ). Found: 974%.
6.04%. FAB MS calc.: 907.7 (Mꢁ
Hꢁ). Found:
908%.
/
4.2.24. Compound I
Compound h (0.70 g) was dissolved in CHCl3
(30 ml) and then piperidine (10 ml) was added. The
solution was stirred at room temperature for 4 h.
The reaction mixture was purified by column
4.2.26. ADL2
Compound 1j (55 mg, 0.060 mmol) was dis-
solved in CHCl3 (1.0 ml) and then TFA (1.0 ml)
was added. The reaction solution was stirred at
room temperature for 10 h. The solvent was
evaporated and the residue was dried under
vacuum at 70 8C for 10 h (Yield: 90%). Alter-
natively, ADL2 was also obtained from 1g by
removing the benzyl group with H2/Pd (Yield:
chromatography on the silica gel using MeOHÁ
CHCl3 (5:95) as the eluant (TLC, Rfꢀ0.3). Yield:
0.39 g (73%). H NMR (CDCl3): d 8.14 (d, 1H,
NHCH, Jꢀ7.6 Hz), 7.48 (t, 1H, NHCH2, Jꢀ5.0
Hz), 6.08 (t, 1H, NHCH2, Jꢀ5.0 Hz), 4.70 (m,
/
/
1
/
/
/

Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á
/
68
65
1
92%). H NMR (CDCl3 and CD3OD): d 4.76 (t,
1H, CH, Jꢀ5.5 Hz), 4.62 (t, 1H, CH, Jꢀ5.9 Hz),
3.10Á3.22 (m, 4H, 2NHCH2), 2.78 (d, 2H,
CH(COOH)CH2, Jꢀ5.6 Hz), 2.46Á2.73 (m, 6H,
4.2.29. Compound 3j
Compound 3j was obtained from 3a (0.14
mmol) in a similar manner as 1j (TLC, Rfꢀ0.4).
Yield: 109 mg (85%). 1H NMR (CDCl3): d 7.42 (d,
1H, NHCH, Jꢀ7.3 Hz), 7.32 (t, 1H, NHCH2,
Jꢀ5.3 Hz), 6.61 (d, 1H, NHCH, Jꢀ8.2 Hz), 6.01
(t, 1H, NHCH2, Jꢀ5.3 Hz), 4.75 (m, 1H, CH),
4.63 (m, 1H, CH), 3.20 (m, 4H, 2NHCH2), 2.81
(dd, 1H, CHCHAHBCO, Jꢀ3.0 and 14.5 Hz),
2.74 (d, 2H, CHCH2, Jꢀ5.3 Hz), 2.42 (dd, 1H,
CHCHAHBCO, Jꢀ7.3 and 14.5 Hz), 2.19 (m, 4H,
/
/
/
/
/
/
CHCH2 and (CH2)2CO), 1.48 (br s, 4H,
2NHCH2CH2), 1.27 (br s, 52H, 2(CH2)13CH3),
0.89 (t, 6H, 2CH2CH3, Jꢀ
C44H82N4O8×0.7CF3COOH: C, 62.32; H, 9.53; N,
6.40. Found: C, 62.00; H, 9.41; N, 6.16%. FAB MS
calc.: 795.6 (Mꢁ
Hꢁ). Found: 796%.
/
/
/
/
6.4 Hz). Anal. Calc. for
/
/
/
/
/
/
4.2.27. Compound 2j
Compound 2j was obtained from 2a (0.14
2CH2CH2CO), 1.58 (m, 4H, 2CH2CH2CO), 1.46
(m, 4H, 2NHCH2CH2), 1.46 (s, 9H, C(CH3)3),
1.44 (s, 9H, C(CH3)3), 1.32 (m, 4H,
mmol) in a similar manner as 1j (TLC, Rfꢀ
/
1
0.3).Yield: 107 mg (86%). H NMR (CDCl3): d
7.41 (d, 1H, NHCH, Jꢀ6.9 Hz), 7.35 (t, 1H,
NHCH2, Jꢀ5.6 Hz), 6.66 (d, 1H, NHCH, Jꢀ7.9
Hz), 6.04 (t, 1H, NHCH2, Jꢀ5.6 Hz), 4.75 (m,
1H, CH), 4.63 (m, 1H, CH), 3.20 (m, 4H,
2NHCH2), 2.81 (dd, 1H, CHCHAHBCO, Jꢀ2.4
and 15.0 Hz), 2.74 (d, 2H, CHCH2, Jꢀ5.0 Hz),
2.42 (dd, 1H, CHCHAHBCO, Jꢀ7.3 and 14.9
(CH2)2CH2CH2CO),
2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ
1.25
(br
s,
52H,
6.6
/
/
/
/
Hz). Anal. Calc. for C56H106N4O8: C, 69.81; H,
11.09; N, 5.82. Found: C, 69.99; H, 11.08; N,
/
5.67%. FAB MS calc.: 963.8 (Mꢁ
Hꢁ). Found:
964%.
/
/
/
/
4.2.30. ADL6
ADL6 was obtained from 3j or 3g in a similar
manner as ADL2. 1H NMR (CDCl3 and CD3OD):
Hz), 2.23 (m, 4H, 2CH2CH2CO), 1.63 (m, 4H,
2CH2CH2CO), 1.49 (m, 4H, 2NHCH2CH2), 1.46
(s, 9H, C(CH3)3), 1.44 (s, 9H, C(CH3)3), 1.26 (br s,
d 4.75 (t, 1H, CH, Jꢀ
Jꢀ6.1 Hz), 3.10Á3.22 (m, 4H, 2NHCH2), 2.78
(dd, 1H, CHCHAHB, Jꢀ
(dd, 1H, CHCHAHB, Jꢀ
(dd, 1H, CHCHAHB, Jꢀ
(dd, 1H, CHCHAHB, Jꢀ
2H, CH2CH2CO, Jꢀ7.4 Hz), 2.24 (t, 2H,
CH2CH2CO, Jꢀ7.9 Hz), 1.62 (m, 4H,
/5.6 Hz), 4.64 (t, 1H, CH,
52H, 2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ
/
/
/
6.8 Hz). Anal. Calc. for C54H102N4O8: C, 69.34; H,
10.99; N, 5.99. Found: C, 69.34; H, 10.98; N,
/
5.8 and 15.3 Hz), 2.75
5.8 and 15.3 Hz), 2.63
5.9 and 15.0 Hz), 2.53
/
5.89%. FAB MS calc.: 935.8 (Mꢁ
/
Hꢁ). Found:
/
936%.
/6.3 and 15.0 Hz), 2.30 (t,
/
4.2.28. ADL4
ADL4 was obtained from 2j or 2g in a similar
manner as ADL2. 1H NMR (CDCl3 and CD3OD):
/
2CH2CH2CO), 1.48 (m, 4H, 2NHCH2CH2), 1.33
(m, 4H, (CH2)2CH2CH2CO), 1.26 (br s, 52H,
d 4.76 (t, 1H, CH, Jꢀ
Jꢀ6.1 Hz), 3.11Á3.22 (m, 4H, 2NHCH2), 2.79
(dd, 1H, CHCHAHB, Jꢀ
(dd, 1H, CHCHAHB, Jꢀ
(dd, 1H, CHCHAHB, Jꢀ
(dd, 1H, CHCHAHB, Jꢀ
2H, CH2CH2CO, Jꢀ6.9 Hz), 2.27 (t, 2H,
CH2CH2CO, Jꢀ7.3 Hz), 1.67 (m, 4H,
/
5.8 Hz), 4.64 (t, 1H, CH,
2(CH2)13CH3), 0.89 (t, 6H, 2CH2CH3, Jꢀ6.8
Hz). Anal. Calc. for C48H90N4O8: C, 67.73; H,
/
/
/
/
5.9 and 15.7 Hz), 2.74
6.6 and 15.7 Hz), 2.64
5.9 and 15.2 Hz), 2.54
10.66; N, 6.58. Found: C, 67.60; H, 10.69; N,
6.40%. FAB MS calc.: 851.7 (Mꢁ
Hꢁ). Found:
852%.
/
/
/
/
6.3 and 15.2 Hz), 2.33 (t,
/
4.2.31. Compound 4j
/
Compound 4j was obtained from 4a (0.14
mmol) in a similar manner as 1j (TLC, Rfꢀ0.4).
Yield: 111 mg (84%). 1H NMR (CDCl3): d 7.44 (d,
1H, NHCH, Jꢀ6.9 Hz), 7.31 (t, 1H, NHCH2,
Jꢀ5.6 Hz), 6.59 (d, 1H, NHCH, Jꢀ7.9 Hz), 6.00
(t, 1H, NHCH2, Jꢀ5.3 Hz), 4.75 (m, 1H, CH),
4.64 (m, 1H, CH), 3.20 (m, 4H, 2NHCH2), 2.81
2CH2CH2CO), 1.48 (m, 4H, 2NHCH2CH2), 1.27
(br s, 52H, 2(CH2)13CH3), 0.89 (t, 6H, 2CH2CH3,
/
Jꢀ
/
6.6 Hz)). Anal. Calc. for C44H82N4O8×
/
/
0.5CF3COOH: C, 64.13; H, 9.91; N, 6.37. Found:
/
/
C, 63.83; H, 9.83; N, 6.13%. FAB MS calc.: 823.7
(Mꢁ
Hꢁ). Found: 824%.
/
/

66
Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á68
/
(dd, 1H, CHCHAHBCO, Jꢀ
2.74 (d, 2H, CHCH2, Jꢀ5.0 Hz), 2.42 (dd, 1H,
CHCHAHBCO, Jꢀ6.8 and 14.2 Hz), 2.19 (m, 4H,
/
3.1 and 15.0 Hz),
C60H114N4O8: C, 70.68; H, 11.27; N, 5.50. Found:
C, 70.78; H, 11.28; N, 5.38%. FAB MS calc.:
/
/
1019.9 (Mꢁ
Hꢁ). Found: 1020%.
/
2CH2CH2CO), 1.57 (m, 4H, 2CH2CH2CO), 1.52
(br s, 4H, 2NHCH2CH2), 1.46 (s, 9H, C(CH3)3),
1.43 (s, 9H, C(CH3)3), 1.28 (br s, 8H,
4.2.34. ADL10
ADL10 was obtained from 5j or 5g in a similar
manner as ADL2. 1H NMR (CDCl3 and CD3OD):
(CH2)4CH2CH2CO),
2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ
1.25
(br
s,
52H,
6.6
/
d 4.76 (t, 1H, CH, Jꢀ
Jꢀ5.9 Hz), 3.10Á3.24 (m, 4H, 2NHCH2), 2.74Á
2.84 (m, 2H, CHCH2CO), 2.64 (dd, 1H,
CHCHAHB, Jꢀ5.9 and 14.7 Hz), 2.52 (dd, 1H,
CHCHAHB, Jꢀ5.9 and 14.7 Hz), 2.29 (t, 2H,
CH2CH2CO, Jꢀ7.2 Hz), 2.23 (t, 2H,
CH2CH2CO, Jꢀ8.6 Hz), 1.61 (m, 4H,
/5.6 Hz), 4.63 (t, 1H, CH,
Hz). Anal. Calc. for C58H110N4O8: C, 70.26; H,
11.18; N, 5.65. Found: C, 70.46; H, 11.17; N,
/
/
/
5.47%. FAB MS calc.: 991.8 (Mꢁ
/
Hꢁ). Found:
/
992%.
/
/
4.2.32. ADL8
ADL8 was obtained from 4j or 4g in a similar
manner as ADL2. 1H NMR (CDCl3 and CD3OD):
/
2CH2CH2CO), 1.48 (m, 4H, 2NHCH2CH2), 1.29
(br s, 12H, (CH2)6CH2CH2CO), 1.26 (br s, 52H,
d 4.76 (t, 1H, CH, Jꢀ
Jꢀ5.9 Hz), 3.10Á3.22 (m, 4H, 2NHCH2), 2.79
(dd, 1H, CHCHAHB, Jꢀ
(dd, 1H, CHCHAHB, Jꢀ
(dd, 1H, CHCHAHB, Jꢀ
(dd, 1H, CHCHAHB, Jꢀ
2H, CH2CH2CO, Jꢀ
CH2CH2CO, Jꢀ8.3), 1.61 (m, 4H, 2CH2CH2CO),
1.48 (m, 4H, 2NHCH2CH2), 1.32 (m, 8H,
(CH2)4CH2CH2CO), 1.26 (br s, 52H,
2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ6.6
/
5.6 Hz), 4.64 (t, 1H, CH,
2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3, Jꢀ
Anal. Calc. for C52H98N4O8×H2O: C, 67.49; H,
10.89; N, 6.05. Found: C, 67.85; H, 10.77; N,
5.84%. FAB MS calc.: 907.7 (Mꢁ
Hꢁ). Found:
/
6.4 Hz).
/
/
/
/
5.6 and 15.2 Hz), 2.75
5.6 and 15.2 Hz), 2.64
5.9 and 14.6 Hz), 2.53
/
/
/
908%.
/
6.3 and 14.6 Hz), 2.29 (t,
7.6), 2.23 (t, 2H,
/
4.2.35. Compound 6j
/
Compound 6j was obtained from 6a (0.14
mmol) in a similar manner as 1j (TLC, Rfꢀ0.5).
Yield: 127 mg (91%). 1H NMR (CDCl3): d 7.47 (d,
1H, NHCH, Jꢀ7.3 Hz), 7.31 (t, 1H, NHCH2,
Jꢀ5.3 Hz), 6.60 (d, 1H, NHCH, Jꢀ7.9 Hz), 6.05
(t, 1H, NHCH2, Jꢀ5.3 Hz), 4.75 (m, 1H, CH),
4.63 (m, 1H, CH), 3.20 (m, 4H, 2NHCH2), 2.81
(dd, 1H, CHCHAHBCO, Jꢀ3.3 and 15.0 Hz),
2.75 (d, 2H, CHCH2, Jꢀ5.0 Hz), 2.42 (dd, 1H,
CHCHAHBCO, Jꢀ6.8 and 14.6 Hz), 2.20 (m, 4H,
/
/
/
Hz). Anal. Calc. for C50H94N4O8: C, 68.29; H,
/
/
10.78; N, 6.37. Found: C, 68.26; H, 10.78; N,
6.21%. FAB MS calc.: 879.7 (Mꢁ
Hꢁ). Found:
880%.
/
/
/
/
4.2.33. Compound 5j
/
Compound 5j was obtained from 5a (0.14
mmol) in a similar manner as 1j (TLC, Rfꢀ0.4).
Yield: 115 mg (85%). 1H NMR (CDCl3): d 7.44 (d,
1H, NHCH, Jꢀ6.9 Hz), 7.31 (t, 1H, NHCH2,
Jꢀ5.6 Hz), 6.59 (d, 1H, NHCH, Jꢀ7.9 Hz), 6.00
(t, 1H, NHCH2, Jꢀ5.6 Hz), 4.75 (m, 1H, CH),
4.63 (m, 1H, CH), 3.20 (m, 4H, 2NHCH2), 2.81
(dd, 1H, CHCHAHBCO, Jꢀ3.0 and 15.0 Hz),
2.74 (d, 2H, CHCH2, Jꢀ5.3 Hz), 2.42 (dd, 1H,
CHCHAHBCO, Jꢀ6.8 and 14.4 Hz), 2.20 (m, 4H,
2CH2CH2CO), 1.60 (m, 4H, 2CH2CH2CO), 1.47
(br s, 4H, 2NHCH2CH2), 1.46 (s, 9H, C(CH3)3),
1.44 (s, 9H, C(CH3)3), 1.25 (br s, 68H,
(CH2)8CH2CH2CO and 2(CH2)13CH3), 0.88 (t,
/
/
/
/
6H, 2CH2CH3, Jꢀ6.6 Hz). Anal. Calc. for
/
/
C62H118N4O8: C, 71.08; H, 11.35; N, 5.35. Found:
C, 71.09; H, 11.13; N, 5.14%. FAB MS calc.:
/
1047.9 (Mꢁ
Hꢁ). Found: 1048%.
/
/
/
4.2.36. ADL12
ADL12 was obtained from 6j or 6g in a similar
manner as ADL2. 1H NMR (CDCl3 and CD3OD):
2CH2CH2CO), 1.58 (m, 4H, 2CH2CH2CO), 1.51
(br s, 4H, 2NHCH2CH2), 1.46 (s, 9H, C(CH3)3),
1.44 (s, 9H, C(CH3)3), 1.25 (br s, 64H,
(CH2)6CH2CH2CO and 2(CH2)13CH3), 0.88 (t,
d 4.76 (t, 1H, CH, Jꢀ
Jꢀ6.1 Hz), 3.10Á3.22 (m, 4H, 2NHCH2), 2.70Á
2.84 (m, 2H, CHCH2), 2.63 (dd, 1H, CHCHAHB,
/5.6 Hz), 4.63 (t, 1H, CH,
/
/
/
6H, 2CH2CH3, Jꢀ6.4 Hz). Anal. Calc. for
/

Y. Ogawa et al. / Chemistry and Physics of Lipids 119 (2002) 51Á
/
68
67
Jꢀ
Jꢀ
Jꢀ
1.62 (m, 4H, 2CH2CH2CO), 1.48 (m, 4H,
2NHCH2CH2), 1.26 (br s, 68H,
(CH2)8CH2CH2CO and 2(CH2)13CH3), 0.88 (t,
6H, 2CH2CH3, Jꢀ6.4 Hz). Anal. Calc. for
C54H102N4O8×H2O: C, 68.03; H, 10.99; N, 5.88.
Found: C, 68.52; H, 10.83; N, 5.67%. FAB MS
calc.: 935.8 (Mꢁ
Hꢁ). Found: 936%.
/
5.8 and 14.9 Hz), 2.52 (dd, 1H, CHCHAHB,
4.5. Liposome stability
/6.3 and 14.9 Hz), 2.29 (t, 2H, CH2CH2CO,
/
7.4 Hz), 2.23 (t, 2H, CH2CH2CO, Jꢀ
/8.6 Hz),
Lipid films were prepared with EPC and 20
mol% of various ADLs (ADL2, ADL8 and
ADL12). The lipid films were then rehydrated in
the buffer (20 mM Calcein, 10 mM HEPES, 100
mM NaCl, pH 7.2) by vortexing and the resulting
suspensions of 1.5 mmol/ml were sonicated at
40 8C for 5 min. The resulting SUV was applied
to the freeze-thaw cycles five times (Hope et al.,
1985). The liposome fractions were purified by Gel
filtration (Sephadex G-50 column) with HEPES
buffer (10 mM HEPES, 100 mM NaCl, pH 7.2).
The time course of calcein leakage was measure on
/
/
/
4.3. Preparation of small unilamellar vesicles
EPC was dissolved in CHCl3ÁMeOH (3:1, v/v)
/
a fluorescence spectrometer (lexꢀ
/
490, lem
ꢀ520
/
with various amounts of ADLs for fusion liposome
(Fusion-Lip), and with 0.5 mol% of NBD-PE and
0.5 mol% of Rh-PE for fluorescence-labeled lipo-
some (Label-Lip). The lipid solutions were dried
under nitrogen gas steam followed by the removal
nm, at 37 8C) (Allen and Cleland, 1980). The
percentage of calcein leakage was calculated using
Eq. (10),
leakage %ꢀ100[G(t)ꢂG(0)]=[G(Triton)ꢂG(0)]; (10)
of residual solvent under high vacuum for 8Á12 h.
/
where G(0), G(t) and G(Triton) signify the fluo-
rescence intensities at time 0, time t and after
adding Triton X-100 (Connor et al., 1986), respec-
tively.
The resulting lipid films were hydrated by vortex-
mixing with HEPES buffer (10 mM HEPES, 100
mM NaCl, pH 7.2) to produce multilamellar
vesicles (MLVs). MLVs were sonicated at 65 8C
for 5 min with a probe-type sonicator and the
resulting clear suspension (small unilamellar vesi-
cles, SUVs) was used in the experiments.
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