0.89 (t, 6H, 2CH2CH3, J 6.6). Anal. Calcd 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
calcd: 823.7 (M + H+). Found: 824. ADL6: dH(270 MHz, CDCl3 and
CD3OD) 4.75 (t, 1H, CH, J 5.6), 4.64 (t, 1H, CH, J 6.1), 3.10–3.22 (m, 4H,
2NHCH2), 2.78 (dd, 1H, CHCHAHB, J 5.8 and 15.3), 2.75 (dd, 1H,
CHCHAHB, J 5.8 and 15.3), 2.63 (dd, 1H, CHCHAHB, J 5.9 and 15.0), 2.53
(dd, 1H, CHCHAHB, J 6.3 and 15.0), 2.30 (t, 2H, CH2CH2CO, J 7.4), 2.24
(t, 2H, CH2CH2CO, J 7.9), 1.62 (m, 4H, 2CH2CH2CO), 1.48 (m, 4H,
2NHCH2CH2), 1.33 (m, 4H, (CH2)2CH2CH2CO), 1.26 (br s, 52H,
2(CH2)13CH3), 0.89 (t, 6H, 2CH2CH3, J 6.8). Anal. Calcd for C48H90N4O8:
C, 67.73; H, 10.66; N, 6.58. Found: C, 67.60; H, 10.69; N, 6.40%. FAB MS
calcd: 851.7 (M + H+). Found: 852. ADL8: dH(270 MHz, CDCl3 and
CD3OD) 4.76 (t, 1H, CH, J 5.6), 4.64 (t, 1H, CH, J 5.9), 3.10–3.22 (m, 4H,
2NHCH2), 2.79 (dd, 1H, CHCHAHB, J 5.6 and 15.2), 2.75 (dd, 1H,
CHCHAHB, J 5.6 and 15.2), 2.64 (dd, 1H, CHCHAHB, J 5.9 and 14.6), 2.53
(dd, 1H, CHCHAHB, J 6.3 and 14.6), 2.29 (t, 2H, CH2CH2CO, J 7.6), 2.23
(t, 2H, CH2CH2CO, J 8.3), 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, J 6.6). Anal. Calcd for C50H94N4O8:
C, 68.29; H, 10.78; N, 6.37. Found: C, 68.26; H, 10.78; N, 6.21%. FAB MS
calcd: 879.7 (M + H+), Found: 880. ADL10: dH(270 MHz, CDCl3 and
CD3OD) 4.76 (t, 1H, CH, J 5.6), 4.63 (t, 1H, CH, J 5.9), 3.10–3.24 (m, 4H,
2NHCH2), 2.74–2.84 (m, 2H, CHCH2CO), 2.64 (dd, 1H, CHCHAHB, J 5.9
and 14.7), 2.52 (dd, 1H, CHCHAHB, J 5.9 and 14.7), 2.29 (t, 2H,
CH2CH2CO, J 7.2), 2.23 (t, 2H, CH2CH2CO, J 8.6), 1.61 (m, 4H,
2CH2CH2CO), 1.48 (m, 4H, 2NHCH2CH2), 1.29 (br s, 12H,
(CH2)6CH2CH2CO), 1.26 (br s, 52H, 2(CH2)13CH3), 0.88 (t, 6H, 2CH2CH3,
J 6.4). Anal. Calcd for C52H98N4O8·H2O: C, 67.49; H, 10.89; N, 6.05.
Found: C, 67.85; H, 10.77; N, 5.84%. FAB MS calcd: 907.7 (M + H+),
Found: 908. ADL12: dH(270 MHz, CDCl3 and CD3OD) 4.76 (t, 1H, CH, J
5.6), 4.63 (t, 1H, CH, J 6.1), 3.10–3.22 (m, 4H, 2NHCH2), 2.70–2.84 (m,
2H, CHCH2), 2.63 (dd, 1H, CHCHAHB, J 5.8 and 14.9), 2.52 (dd, 1H,
CHCHAHB, J 6.3 and 14.9), 2.29 (t, 2H, CH2CH2CO, J 7.4), 2.23 (t, 2H,
Fig. 1 Dependence of fusion % after 5 min reaction upon alkyl chain lengths
of head groups of ADLn; pH 4.0, 37 °C; composition (mol%) of ADLn in
liposomes is described inside the figure.
CH2CH2CO,
J 8.6), 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, J 6.4). Anal. Calcd for C54H102N4O8·H2O: C, 68.03;
H, 10.99; N, 5.88. Found: C, 68.52; H, 10.83; N, 5.67%. FAB MS calcd:
935.8 (M + H+), Found: 936.
‡ Egg PC was dissolved in CHCl3–MeOH (3:1, v/v) with various amounts
of ADLn for fusion liposome (Fusion-Lip), and with 0.5 mol% of NBD-PE
and 0.5 mol% of Rh-PE for fluorescence-labeled liposome (Label-Lip).
Lipid solutions were dried under a N2 gas stream followed by the removal
of residual solvent under high vacuum for 3 h. The resulting lipid films were
hydrated by vortex-mixing with HEPES buffer (10 mM HEPES, 100 mM
NaCl, pH 7.2) to make multilamellar vesicles (MLVs). MLVs were
sonicated at 65 °C for 5 min by a probe-type sonicator and the resulting clear
suspension (SUVs) was used in the experiments. After Fusion-Lip and
Label-Lip (10:1, v/v) were mixed at 37 °C the lipid mixing assay was started
by adding the necessary amount of citric acid (0.4 M) to adjust the pH.
Fig. 2 pH profile of fusion % after 5 min reaction at 37 °C; [ADLn] = 20
mol%.
between ADL6 and ADL10. The reason for the very poor 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, consequently, ADL12 may be
embedded in the membrane even at neutral pH. In contrast, the
head alkyl chains of ADL2 and ADL4 are considered too short
and too hydrophilic to migrate to another membrane, which may
also result in low fusion %.
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Notes and references
† All new lipids were fully characterized by 1H NMR and mass
spectroscopy and elemental analysis as follows. ADL2: dH(270 MHz,
CDCl3 and CD3OD) 4.76 (t, 1H, CH, J 5.5), 4.62 (t, 1H, CH, J 5.9),
3.10–3.22 (m, 4H, 2NHCH2), 2.78 (d, 2H, CH(COOH)CH2, J 5.6),
2.46–2.73 (m, 6H, CHCH2 and (CH2)2CO), 1.48 (br s, 4H, 2NHCH2CH2),
1.27 (br s, 52H, 2(CH2)13CH3), 0.89 (t, 6H, 2CH2CH3, J 6.4). Anal. Calcd
for C44H82N4O8·0.7CF3COOH: C, 62.32; H, 9.53; N, 6.40. Found: C,
62.00; H, 9.41; N, 6.16. FAB MS calcd: 795.6 (M + H+). Found: 796.
ADL4: dH(270 MHz, CDCl3 and CD3OD); 4.76 (t, 1H, CH, J 5.8), 4.64 (t,
1H, CH, J 6.1), 3.11–3.22 (m, 4H, 2NHCH2), 2.79 (dd, 1H, CHCHAHB, J
5.9 and 15.7), 2.74 (dd, 1H, CHCHAHB, J 6.6 and 15.7), 2.64 (dd, 1H,
CHCHAHB, J 5.9 and 15.2), 2.54 (dd, 1H, CHCHAHB, J 6.3 and 15.2), 2.33
(t, 2H, CH2CH2CO, J 6.9), 2.27 (t, 2H, CH2CH2CO, J 7.3), 1.67 (m, 4H,
2CH2CH2CO), 1.48 (m, 4H, 2NHCH2CH2), 1.27 (br s, 52H, 2(CH2)13CH3),
9 D. K. Struck, D. Hoekstra and R. E. Pagano, Biochemistry, 1981, 20,
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10 A. L. Bailey and P. R. Cullis, Biochemistry, 1997, 36, 1628.
Communication 9/01419E
824
Chem. Commun., 1999, 823–824