Scheme 1a
a (a) Fmoc-OSu, Et3N, DMF/MeOH; (b) H2N(CH2)6NH2, DIPEA,
HOBt, PyBOP, DMF; (c) piperidine.
of C-linked dimer 2 was prepared by acetylating an amino
group with N-succinimidyl acetate followed by the same
coupling reaction with diamine (5% yield from 1). Low
yields of the dimers were mainly due to poor solubility in
solvents and low recovery during HPLC purification.
Figure 1. 31P NMR spectra of liposome-entrapped phosphate for
AmB 1 and dimers 2 and 3. The peak around δ 1.2 corresponds to
-
H2PO4 at pH 5.5 (initial pH) and that around δ 3.1 corresponds
to HPO42- at pH 7.5. PC-only: liposomes were prepared with PC.
PC-Cho: with 10% cholesterol in PC. PC-Erg: with 10% ergosterol
in PC. AmB or dimer was dissolved in DMF and added to liposome
suspensions. For all experiments, molar ratios between AmB or an
AmB equivalent within dimers and lipids were 1:1000.
activity; as reported by Gary-Bobo et al.,5 once an ion
channel with high activity is formed, K+ concentration in
liposomes instantly reaches equilibrium, resulting in a rapid
rise of lumen pH to give rise to these two peaks. This spectral
pattern is characteristic of “all-or-none” type of ion flux.
It was reported that the CD spectra of AmB in liposomes
give rise to very small Cotton effects in these AmB-lipid
ratios, while those in aqueous media show large CD splits.4,7
In the CD spectra of dimers 2 and 3 (Figure 2), however,
large Cotton effects were observed both in liposomes and
in buffer; CD for liposomes were partly due to aqueous
micelles of dimers in an aqueous phase.4 For all conditions
tested, the magnitudes of CD extrema in buffer were
significantly smaller than those in both ergosterol- and
cholesterol-containing liposomes. Moreover, unlike AmB,6
the dependence of the CD magnitude of 2 and 3 on dimer/
lipid ratios was not evident (see Supporting Information).
These observations suggest that dimers 2 and 3 bind to
membrane effectively to form a stable assemblage even in
low concentrations.
The positions of CD extrema of dimer 2 are virtually
identical with those of AmB,6 demonstrating that inter- and
intramolecular interactions among the heptaene chro-
mophores mimic that of AmB.4 Conversely, the spectra of
dimer 3 gave rise to large positive Cotton effects both in
liposomes and in MeOH, suggesting that 3 forms different
assemblage from that of AmB or 2. Significant CD spectral
changes for dimer 3 with ergosterol liposomes, particularly
three prominent positive peaks, resembling the UV absorp-
tion characteristic of AmB, suggest that 3 has some interac-
tion with ergosterol.
Membrane permeabilizing activity of the dimers was
evaluated by two methods, hemolysis tests and K+ flux
assays using liposomes. Dimer 2 revealed powerful hemolytic
activity, which exceeded that of AmB by six times.5
Conversely, N-acetyl dimer 3 showed virtually no activity.
It has been reported that cation current across liposomal
membrane can be monitored by pH-dependent changes in
the 31P NMR chemical shift of phosphate.6 In the spectra of
1 and 3 for PC-Erg (Figure 1), a signal appearing at δ 3.1
was derived from phosphate entrapped in liposomes where
AmB or dimer 3 formed ion-permeable channels and H+
leaked out via a proton transporter, FCCP, at the expense of
K+ influx. This pH rise was detected as a downfield shift of
a
31P NMR signal; background resonance due to external
phosphate was quenched by Mn2+ (see Supporting Informa-
tion for experimental details). The spectra of 3 (Figure 1)
demonstrated two peaks for three liposome preparations,
while dimer 2 gave rise to no clear peak at δ 3.1. These
results indicate that dimer 3 forms K+ channels with high
(5) Hemolytic activity was measured for 1% human erythrocytes. EC50
value of 2 was 0.25 µM, whereas that of N-acetyl derivative 3 was over 30
µM. As was the case with the N-linked dimers,3 antifungal activity of 2
and 3 should be weaker than that of AmB as a result of their poor solubility.
(6) (a) Herve´, M.; Cybulska, B.; Gary-Bobo, C. M. Eur. Biophys. J. 1985,
12, 121-128. (b) Herve´, M.; Debouzy, J. C.; Borowski, E.; Cybulska, B.;
Gary-Bobo, C. M. Biochim. Biophys. Acta 1989, 980, 261-272.
(7) Balakrishnan, A. R.; Easwaran, K. R. K. Biochemistry 1993, 32,
4139-4144.
2088
Org. Lett., Vol. 4, No. 12, 2002
Yamaji, Nahoko
