Anion transport in liposomes responds to variations in the anchor chains and the fourth amino acid of heptapeptide ion channels

Article abstract of DOI:10.1039/b417808b

Seven heptapeptide derivatives have been prepared. The peptide structure is (Gly)₃Xxx(Gly)₃ in which Xxx stands for a variable amino acid. The amino acid variations include azetidine carboxylic acid, pipecolic acid, meta-aminobenzoic

Full text of DOI:10.1039/b417808b

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P A P E R
Anion transport in liposomes responds to variations in the anchor
chains and the fourth amino acid of heptapeptide ion channels
a
a
a
a
ˇ
Riccardo Ferdani, Robert Pajewski, Natasha Djedovic, Jolanta Pajewska,
Paul H. Schlesingerc and George W. Gokel*ab
a Department of Molecular Biology and Pharmacology, Washington University School of
Medicine, 660 South Euclid Ave., Campus Box 8103, St Louis, MO 63110, USA.
E-mail: ggokel@wustl.edu; Fax: þ1 314/362-9298; Tel: þ1 314/362-9297
b Department of Chemistry, Washington University, 1 Brookings Drive, St Louis, MO 63130,
USA
c Department of Cell Biology and Physiology, Washington University School of Medicine,
660 South Euclid Ave., St Louis, MO 63110, USA
Received (in Durham, UK) 24th November 2004, Accepted 17th March 2005
First published as an Advance Article on the web 13th April 2005
Seven heptapeptide derivatives have been prepared. The peptide structure is (Gly)3Xxx(Gly)3 in which Xxx
stands for a variable amino acid. The amino acid variations include azetidine carboxylic acid, pipecolic acid,
meta-aminobenzoic acid, proline, and leucine. All seven compounds have a C-terminal benzyl group. In all
cases, the heptapeptide’s N-terminus was linked to diglycolic acid and a dialkylamine. In five cases, the
N-terminal group was didecylamine and in two cases, N-ethyl-N-decyl. Chloride and carboxyfluorescein
release from phospholipid vesicles was studied with the result that C10H21N(C2H5)COCH2OCH2CO–NH–
(Gly)3Leu(Gly)3–OCH2Ph was the most active. Hill analysis showed that this compound involves pore
formation by four monomer units rather than two, as previously found for other members of this family.
interact with the phospholipid’s fatty acyl chains. The hydro-
Introduction
carbon anchors are connected to the heptapeptide by diglycolic
acid. This diacid was chosen for convenience and because its
polar residues mimic the midpolar regime of phospholipid
monomers. Finally, the C-terminus was esterified by benzyl
alcohol. The latter was used to prevent ionization of the
C-terminal carboxyl and to permit future manipulations be-
cause it can be removed hydrogenolytically.
The amphiphilic peptide7 (C18H37)2NCOCH2OCH2CONH–
(Gly)3Pro(Gly)3OCH2Ph8 showed remarkable properties when
it inserted into phospholipid bilayers.9 In a previous study
reported in this Journal, we showed that changing the central
amino acid in the heptapeptide from proline to pipecolic acid
dramatically altered the ion transport profile of this com-
pound.10 We now report the results of systematic variation
of that fourth residue and its effect on transport efficacy.
The recent appearance of protein channel crystal structures has
dramatically increased the amount of structural information
available about these ion and molecule transporters. The
importance of such structures is apparent from the award of
the 2003 Nobel Prize in Chemistry for the accomplishment.1
Important as such structures are, they do not provide direct
information about interactions with the membrane or the
mechanism(s) of ion transport. Synthetic organic channels2
are more amenable to structure–activity studies because of
their smaller size and the fact that the most successful ones
were designed to be modular.3
In previous work, we developed a successful cation-trans-
porting channel compound.4 Although it was designed to be a
model for proteins, it is a true channel. Members of this
‘‘hydraphile’’ family insert in the phospholipid bilayer and
transport ions effectively and with some selectivity. The syn-
thetic channels lack the highly developed selectivity filters and
rectification behavior of protein channels5 but the synthetic
versions transport ions and show open–closed behavior6 char-
acteristic of proteins. These synthetic models are, in fact,
functioning ion channels although they are clearly less evolved
than their protein counterparts.
Quite recently, we designed an amphiphilic peptide intended
to be a chloride ion transporter. It was constructed from four
key modules. The central element is a heptapeptide that has the
sequence (Gly)3Pro(Gly)3. The sequence was based on the
conserved Gly-Xxx-Xxx-Pro sequence identified in the CLC
family of chloride transporters. The simplest such sequence is
Gly-Gly-Gly-Pro (GGGP). Addition of three glycine residues
gives a heptapeptide with the sequence GGGPGGG. Two
modules anchored the N-terminal end of this peptide. The
N-terminus comprises a dialkylamide, R2NCO–. The initial
anchors chosen were octadecyl chains. These were intended to
Results and discussion
Pipecolic acid and proline are homologs. They have the same
structural features and stereochemistry and differ only in ring
size. Our expectation was that changing the (Gly)3-Pro-(Gly)3
sequence to (Gly)3-Pip-(Gly)3 would lead to a minor variation
in the peptide’s bend angle and a commensurate small change
in anion release activity. Instead, we found that release from
phospholipid vesicles of the fluorescent dye carboxyfluorescein
was significantly diminished when a single methylene group
expanded the proline ring.
Compounds studied
The compounds prepared for the present report are shown as
1–7. In all cases, the compounds possess four ‘‘modules.’’ The
N-terminus comprises a pair of hydrocarbon chains [either
ethyl and decyl (1 and 4) or didecyl (2, 3 and 5–7)]. These
T h i s j o u r n a l i s & T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 5
N e w J . C h e m . , 2 0 0 5 , 2 9 , 6 7 3 – 6 8 0
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hydrocarbon chains are expected to insert in the phospholipid
bilayer and anchor the compound therein. The dialkylamino
group is linked to the heptapeptide by a diglycolic acid residue.
The C-terminus of the heptapeptide is benzyl in 1–7.
The principal variations in structure are changes in the
fourth amino acid residue of the heptapeptide chain. They
are: leucine (Leu, L) in 1 and 2, azetedinecarboxylic acid in 3,
proline (Pro, P) in 4 and 5, pipecolic (Pip) acid in 6, and 3-
aminobenzoic acid in 7. The stereochemistry in the central
amino acid of compounds 1–6 is the same. Although 3-
aminobenzoic acid is not one of the common natural amino
acids, it has amino and carboxyl groups disposed at 1201 to
each other. The use of this compound also permitted us to
assess the influence, if any, of an aromatic residue within the
heptapeptide chain.
Scheme 1
3-phosphate (DOPA) (20 mg, 7 : 3 w/w) by the lipid film
method.12 The vesicle suspension was filtered through a 200 nm
filter membrane (5 times) using a mini-extruder. The suspen-
sion was then passed through a Sephadex G25 column, which
had been equilibrated with external buffer (400 mM K2SO4
:
10 mM HEPES, pH adjusted to 7.0). Analysis by light
scattering showed that the vesicle diameter was consistently
B200 nm. The final lipid concentration was confirmed by
colorimetric determination of the phospholipid–ammonium
ferrothiocyanate complex.13
Chloride release was determined by recording the voltage
output of a chloride selective (Accumet Chloride Combination)
electrode. The electrode and vesicle suspension were allowed to
equilibrate for five minutes, after which aliquots containing the
compound of interest (B9 mM in 2-propanol) were added. Ion
release was monitored for approximately 30 min in each case;
data for 1500 s are presented in the graphs. After 0.5 h,
complete lysis of the vesicles was induced by addition of a
2% aqueous solution of Triton X-100 (100 mL) and the data
previously recorded were normalized to this maximal value.
The measured transport rates are shown in Fig. 1. Each curve
shown is the average of 3–7 different experiments done with
[lipids] ¼ 0.31 mM and [compound] ¼ 65 mM.
The amphiphilic peptides were prepared in a series of steps
beginning at the heptapeptide’s N-terminus. The dialkylamine
and diglycolic anhydride were heated at reflux in toluene or
THF for 48 h. The resulting acid, R2NCOCH2OCH2COOH,
was coupled to triglycine by standard carbodiimide methods to
give R2NCOCH2OCH2CONH–Gly-Gly-Gly–OR0. The sec-
ond segment, which contained various amino acids (Aaa) in
The upper panel of Fig. 1 shows chloride release data for
compounds 1, 4, and 5. In compounds 1 and 4, the dialkyl-
amino groups are CH3CH2N(CH2)9CH3 and in 5 it is didecyl-
amino. Amphiphiles 1 and 4 are identical except for the fourth
amino acid (Leu vs. Pro) and 4 and 5 are identical except
for the dialkylamino group. A comparison of 1 and 4 reveals
that release of chloride ion when 1 is present is much greater
than that induced by a comparable amount of 4. In both
cases, however, the initial portions of the curve are steep,
suggesting that insertion occurs quite readily. This result
contrasts with the more gradual initial rate observed for 5.
The flat portions of the release curves for 1 and 4 are clearly
offset but nearly parallel. Although Clꢀ release begins more
gradually with 5, the rate of increase is greater at 1500 s
than for 4. Essentially complete Clꢀ release is observed for 1
at 1500 s.
the incipient fourth position,
H(1,2)N–Aaa-Gly-Gly-Gly–
OCH2Ph was then coupled to the deprotected fragment. The
compounds obtained had spectral properties that corres-
ponded to their assigned structures and the following melting
points: 1, 179–181 1C; 2, 186–188 1C; 3, 117–119 1C; 4, 195
(dec) 1C; 5, 127–128 1C; 6, 78–80 1C; and 7, 4205 (dec) 1C. The
synthetic sequence used for the preparation of 4 is shown
in Scheme 1.
Chloride release from phospholipid vesicles
The efficacy of ion transport was assayed by using a chloride
selective electrode to detect efflux directly from phospholipid
vesicles.11 The liposomes were prepared from 1,2-dioleoyl-sn-
glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-
We note two technical issues with respect to the chloride ion
release study. First, the response of the chloride electrode is
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Release of the carboxyfluorescein anion from vesicles
In previous studies, we found that chloride release from
phospholipid vesicles was mediated by (C18H37)2NCO-
CH2OCH2CO–NH–GGGPGGG–OCH2Ph (8) and by its –
GGGLGGG– analog (9). Anion transport by the latter was
about 6-fold lower at an identical concentration than when the
central amino acid was proline.10 This led us to assess the effect
of pipecolic acid in the center of the heptapeptide. In those
studies, transport of carboxyfluorescein (CF) through a phos-
pholipid bilayer was also measured. Carboxyfluorescein is an
anionic dye that self quenches when it is encapsulated at
sufficiently high concentrations within liposomes. If the anion
is released from the vesicles, its external concentration is lower
and fluorescence is readily and quantitatively detected. The
concentration-dependent release of CF by 1 is shown in Fig. 2.
In the earlier study,10 we found about a 3-fold difference in
CF transport by the bis(octadecyl) heptapeptides when the
central amino acid was proline (faster) or pipecolic acid
(slower). A nearly 30-fold difference in CF transport between
10,10-Pro (5) and 10,10-Pip (6, slower) was also noted. The
results presented here for chloride transport show much less
difference between 5 and 6 than was observed when the anion
was CF. Indeed, there is relatively little difference in the
chloride transport rates for 2–7, although 1 is dramati-
cally different. The high chloride transport rate exhibited
by C10H21NEtCOCH2OCH2CONH–(Gly)3Leu(Gly)3OCH2Ph
(1) compared to 2–7 demanded that a concentration depen-
dence study be undertaken. This was done and is shown in Fig.
2. A reasonable concentration dependence of CF transport
by 1 was observed for a B7-fold concentration range from
25.9–189 mM.
By using the concentration dependent fluorescence data for 1
(Fig. 2) we prepared a Hill plot to ascertain if pores formed
from 1 involved more than 2 monomers, as found for previous
compounds in this series. The data are shown in Fig. 3.
The slope of the Hill plot is 3.8 ꢁ 0.7, suggesting that twice
as many monomers are involved in pore formation by 1 than
the dimer assembly found for either 5 or previously for 8. It
seems reasonable that if more monomers associate to form a
pore, the pore would be larger than if fewer monomers were
present. This, in turn, should afford a larger pore, resulting in a
higher ion flux. Overall, we expect leucine-containing 1 and 2
to be the most flexible and adaptable. Of these, only 1
combines a single decyl chain with the flexible leucine. We
have already shown in previous work that anchor chain length
is an important variable in pore-forming ability by these
compounds.15 It appears that 1 has a unique combination of
flexibility and insertion dynamics to permit facile pore forma-
tion and complete anion release.
Fig. 1 (Upper panel) Fractional chloride release from phospholipid
vesicles by 1, 4, and 5. (Lower panel) Fractional chloride release from
phospholipid vesicles by 2–7. See text for concentration details.
slow. Ion release is most rapid at the initial stage when the
system is equilibrating. This presumably most affects the
exponential portion of the release curve. Second, the linear
portion of the curve has a shallow slope at the lowest iono-
phore concentrations. Following these profiles to full release
would require considerable time and greater liposome stability
than the system currently exhibits. We further note that the
graphical representations shown in the figures are either
averages of three or more independently acquired data sets
or representative data sets that clearly portray the average
result. In all cases, more data were obtained than are shown in
the figures and none contradicts the representations included
herein.
Ion release from phospholipid vesicles occurs as soon as a
pore is formed in the bilayer.14 However pore formation
occurs, it seems reasonable that the amphiphiles having a
single long chain require less pre-organization to insert into
the bilayer. A comparison of the initial release profiles com-
ports with this expectation. It also seems reasonable that the
ultimate efficacy of the pore would vary when the alkyl chains
differ. The difference in Clꢀ release observed for 4 and 5
suggests that while insertion and pore formation is apparently
more rapid for 4, the resulting pore is ultimately superior for 5.
The data presented in the lower panel of Fig. 1 compare the
five N,N-didecyl heptapeptide derivatives. Compound 5 is
included in both graphs and permits comparison even though
the ordinates differ. As in the upper panel of this figure, we
note that there are differences in curve shape. Overall, however,
the five compounds all show similar Clꢀ release rates. The
inclusion of 5 in the upper panel shows that Clꢀ release is
similar for it and for 4. The similarity in Clꢀ release rates seems
reasonable considering the small structural differences that
exist in the fourth amino acids. The key exception is compound
1, which is by far the best Clꢀ transporter in this study. It is
discussed further below.
Fig. 2 Concentration dependent, fractional carboxyfluorescein release
from phospholipid vesicles (see text for details). Concentrations of 1
are, from bottom to top, 25.9, 64.5, 99.0, 127.0, and 189 mM.
N e w J . C h e m . , 2 0 0 5 , 2 9 , 6 7 3 – 6 8 0
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mmol) was added dropwise at 0 1C. The reaction was stirred at
room temperature overnight. The reaction mixture was washed
with dil. aq. HCl (2 ꢂ 50 mL), dried over MgSO4, and
evaporated. The crude product was recrystallized from hexanes
to give a white solid (7.30 g, 98%) mp 50–51 1C, Lit.16 39–41 1C.
1H-NMR: 0.87 (3H, t, J ¼ 6.6 Hz, CH3(CH2)7CH2CH2N),
1.21 (14H, pseudo-s, CH3(CH2)7CH2CH2N), 1.44 (2H, m,
CH3(CH2)7CH2CH2N), 1.93 (3H, s, COCH3), 3.16 (1H, t,
J ¼ 7.0 Hz CH3(CH2)7CH2CH2N), 3.18 (1H, t, J ¼ 7.0 Hz
CH3(CH2)7CH2CH2N), 5.98 (1H, bs, NH). 13C-NMR: 14.0,
22.5, 23.1, 29.2, 29.4, 29.5, 31.8, 39.6, 170.1. IR (CHCl3): 3284,
2925, 2916, 2871, 2850, 1641, 1566, 1466, 1380, 1298, 1171,
1110, 1001, 984, 957, 910, 852, 812, 786, 723 cmꢀ1
.
N-Ethyl-N-decylamine. To LiAlH4 (1.60 g, 42.2 mmol) sus-
pended in dry THF (40 mL), C10H21NHCOCH3 (5.6 g, 28.4
mmol) dissolved in THF (30 mL) was added dropwise and the
reaction was refluxed for 48 h. The reaction was cooled down
and ethyl acetate was added to decompose excess of LiAlH4.
The mixture was poured into dil. aq. H2SO4 (250 mL) and
extracted with Et2O (2 ꢂ 100 mL). The aqueous phase was
adjusted to pH 10 with NaOH pellets and the mixture was
extracted with Et2O (3 ꢂ 50 mL), dried over MgSO4, and the
solvent was evaporated in vacuo. Bulb-to-bulb distillation
using a Kugelrohr apparatus (85–89 1C, 0.35 mmHg) gave
the product (8.2 g, 78%) as a colorless oil. 1H-NMR: 0.87 (3H,
t, J ¼ 6.6 Hz, CH3(CH2)7CH2CH2N), 1.01 (1.5H, t, J ¼ 7.2 Hz,
NHCH2CH3), 1.11 (1.5H, t, J ¼ 7.2 Hz, NHCH2CH3), 1.25
(14H, pseudo-s, CH3(CH2)7CH2CH2N), 1.48 (2H, m,
CH3(CH2)7CH2CH2N), 1.85 (1H, bs, NH), 2.35–2.70 (4H,
overlapping signals due to CH3(CH2)7CH2CH2N) and
CH3CH2N. 13C-NMR (75 MHz, CDCl3): 11.6, 14.1, 15.2,
22.7, 26.9, 27.4, 27.7, 29.3, 29.5, 29.6, 30.1, 31.9, 44.1, 46.9,
49.9, 53.0. IR (neat): 3310, 3263, 3093, 2959, 2925, 2854, 2811,
Fig. 3 Hill plot showing a slope indicating an aggregation of four
monomers active in pore formation. See text for details.
Conclusion
Seven heptapeptides have been prepared that have either two
decyl chains or a decyl and an ethyl chain at their N-terminal
ends. The fourth (central) amino acid in the heptapeptide
sequence has been varied from cyclic to acyclic (leucine). The
cyclic amino acids include proline and its 4- and 6-membered ring
analogs. In addition, the unnatural amino acid meta-aminoben-
zoic acid has been incorporated into the peptide chain. Chloride
release mediated by each of these compounds was measured and
found generally to be similar, except for the –GGGLGGG–
derivative having ethyl and decyl N-terminal groups. When
carboxyfluorescein release by unusually active compound 1 was
studied, Hill plot analysis showed that four monomers were
involved in pore formation. Previous studies with identical,
twin-chained analogs showed that only two amphiphiles were
involved in pore formation. We speculate that the larger number
of monomers leads to a larger pore and a higher anion release
rate. Whether the difference in monomer number affects ion
selectivity was not addressed and will be part of a future effort to
better understand pore formation by these ionophores.
1720, 1645, 1574, 1466, 1379, 1298, 1204, 1134, 1069, 722 cmꢀ1
.
N-Decyl-N-ethylcarbamoyl)methoxyacetic acid ((10,2)DGAOH).
A solution of C10H21NHCH2CH3 (7.11 g, 38.4 mmol) and digly-
colic anhydride (4.45 g, 38.4 mmol) in THF (60 mL) was refluxed
for 24 h. The solvent was evaporated and the crude product was
dissolved in Et2O (150 mL) and washed with 10% aqueous HCl
(2ꢂ20 mL). The acid was extracted with 10% NaOH (3 ꢂ30 mL),
and the water layer was acidified with conc. HCl to pH 2, extracted
with CH2Cl2 (4 ꢂ 30 mL), dried (MgSO4) and evaporated to give
the product (6.48 g, 56%) as a yellow oil, which was used directly
General methods
All reaction solvents were freshly distilled and reactions were
conducted under N2 unless otherwise stated. Et3N was distilled
from KOH and stored over KOH. CH2Cl2 was distilled from
CaH2. Column chromatography was performed on silica gel 60
(230–400 mesh). Thin layer chromatography was performed
with silica gel 60 F254 plates with visualization by UV light (254
nm) and/or by phosphomolybdic acid (PMA) spray. Starting
materials were purchased from Aldrich Chemical Co., and used
as received. 1H-NMR spectra were recorded at 300 MHz in
CDCl3 unless otherwise noted and are reported as follows:
Chemical shifts (ppm d, internal Me4Si) [integrated intensity,
multiplicity (b ¼ broad; s ¼ singlet; d ¼ doublet; t ¼ triplet; m ¼
multiplet, bs ¼ broad singlet, etc.), coupling constants in hertz,
assignment]. 13C-NMR spectra were obtained at 75 MHz in
CDCl3 unless otherwise noted and referenced to CDCl3 (d 77.0).
Infrared spectra were recorded in KBr unless otherwise noted
and were calibrated against the 1601 cmꢀ1 band of polystyrene.
Melting points were determined on a Thomas Hoover apparatus
in open capillaries. Combustion analyses were performed by
Atlantic Microlab, Inc., Atlanta, GA, and are reported as
percents. Optical rotations were recorded on a Perkin-Elmer
Model 214 polarimeter.
in the next step. 1H-NMR: 0.87 (3H, t,
J
¼
6.6 Hz,
CH3(CH2)7CH2CH2N), 1.10–1.35 (17H, overlapping signals due
to NCH2CH3 and CH3(CH2)7CH2CH2N), 1.55 (2H, m,
CH3(CH2)7CH2CH2N), 3.00–3.50 (4H, m, CH3(CH2)7CH2CH2N
and CH3CH2N), 4.21 (2H, s, COCH2), 4.38 and 4.39 (2H, s,
COCH2). 13C-NMR: 12.6, 13.8, 14.1, 14.2, 22.6, 26.8, 26.9, 27.5,
28.7, 29.2, 29.3, 29.4, 29.5, 31.8, 41.3, 41.7, 46.4, 46.6, 71.0, 71.1,
72.8, 72.9, 170.4, 171.8. IR (neat): 2927, 2855, 1742, 1619, 1461,
1439, 1378, 1216, 1135, 1047, 972, 880, 794, 722 cmꢀ1
.
{2-[(N-Decyl-N-ethylcarbamoyl)methoxy]acetylamino}digly-
cylacetic acid benzyl ester ((10,2)DGA–GGGOCH2Ph). To
(10,2)DGAOH (1.43 g, 4.8 mmol) dissolved in CH2Cl2 (30
mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (0.96 g, 5.0 mmol) and HOBt (0.70 g, 5.2 mmol) were
added and the mixture was stirred at room temperature. After
0.5 h, TsOH ꢃ GGG–OCH2Ph (2.15 g, 4.8 mmol) and Et3N (3.0
mL) were added and stirring was continued overnight. After
evaporation of the solvent, the residue was dissolved in EtOAc
(50 mL), washed with 5% citric acid (20 mL), water (20 mL),
5% NaHCO3 (20 mL), and brine (20 mL), dried (MgSO4), and
evaporated. The resulting oil was chromatographed (SiO2,
10% MeOH–CH2Cl2) to give the tripeptide derivative (1.94
{2-[(N-Decyl-N-ethylcarbamoyl)methoxy]acetylamino}
triglycyl-L-leucyldiglycyl acetic acid benzyl ester,
((10,2)DGA-GGGLGGG-OBz) 1
N-Decylacetamide. To decylamine (6.5 g, 41.3 mmol) dis-
solved in dry CH2Cl2 (50 mL), acetic anhydride (3.8 g, 37.2
1
g, 73%) as a colorless oil. H-NMR: 0.86 (3H, t, J ¼ 6.6 Hz,
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CH3(CH2)7CH2CH2N), 1.05–1.30 (17H, overlapping signals
due to NCH2CH3 and CH3(CH2)7CH2CH2N), 1.50 (2H, bs,
CH3(CH2)7CH2CH2N), 3.00–3.35 (4H, m, CH3(CH2)7CH2
CH2N and CH3CH2N), 3.95–4.05 (6H, overlapping signals
due to Gly NHCH2), 4.09 (2H, s, COCH2), 4.29 and 4.30
(2H, s, COCH2), 5.13 (2H, s, PhCH2O), 7.21 (1H, t, J ¼ 6.0 Hz,
NH), 7.30–7.40 (5H, m, HAr), 7.85–7.95 (1H, m, NH), 8.27
(1H, t, J ¼ 6.0 Hz, NH). 13C-NMR: 12.7, 13.9, 14.0, 22.6, 26.8,
27.0, 28.8, 29.2, 29.3, 29.4, 29.5, 29.6, 31.8, 41.2, 43.0, 45.9,
46.5, 67.0, 69.7, 71.7, 71.8, 128.2, 128.4, 128.6, 135.3, 168.3,
169.6, 169.7, 169.8, 169.9, 170.0, 171.3. IR (CHCl3): 3302,
2926, 2855, 1749, 1656, 1541, 1457, 1358, 1260, 1193, 1129,
J ¼ 6.0 Hz, Gly NH), 7.59 (1H, t, J ¼ 6.0 Hz, Gly NH). 13C-
NMR: 21.7, 22.8, 24.6, 28.2, 40.8, 41.2, 42.9, 43.1, 53.6, 67.1,
80.4, 128.3, 128.5, 128.7, 135.3, 156.4, 169.7, 169.8, 170.0,
174.2. Anal. Calcd for C24H36N4O7: C, 58.52; H, 7.37; N,
11.37%. Found: C, 58.54; H, 7.31; N, 11.34%.
L-Leucyldiglycylacetic acid benzyl ester hydrochloride
.
(LGGG–OCH2Ph HCl). Boc-LGGG–OCH2Ph (0.24 g, 0.48
mmol) was dissolved in 4 N HCl in dioxane (10 mL) at 5 1C
and the reaction mixture was stirred for 1 h. The solvent was
evaporated in vacuo and the residue was used in the next
reaction without further purification.
1032, 697 cmꢀ1
.
(10,2)DGA–GGGLGGG–OCH2Ph, 1. To (10,2)DGA–GGG
–OH (0.22 g, 0.46 mmol) suspended in CH2Cl2 (20 mL), 1-(3-
dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride
(0.10 g, 0.52 mmol) and HOBt (0.07 g, 0.52 mmol) was added
and reaction was stirred for 0.5 h. Then LGGG–OCH2Ph ꢃ HCl
(0.17 g, 0.48 mmol) in CH2Cl2 (20 mL) containing Et3N (1.5
mL) was added and reaction mixture was stirred for 48 h. The
mixture was evaporated and the residue washed with 5% citric
acid (20 mL), water (20 mL), 5% NaHCO3 (20 mL), brine (20
mL), dried (MgSO4), and evaporated. The oily product was
chromatographed (SiO2, 10% MeOH–CH2Cl2) to give 1 (0.39
{2-[(N-Decyl-N-ethylcarbamoyl)methoxy]acetylamino}diglyc-
ylacetic acid ((10,2)DGA–GGGOH). (10,2)DGA–GGG–
OCH2Ph (2.3 g, 3.3 mmol) was dissolved in EtOH (abs, 40
mL) and 10% Pd/C (0.13 g) was added and this mixture was
shaken under 60 psi of H2 for 3 h. The reaction mixture was
heated to reflux and filtered through a celite pad. The solvent
was evaporated under reduced pressure to afford a white solid
1
(1.50 g, 96%), mp 158–159 1C. H-NMR (CD3OD): 0.90 (3H,
t, J ¼ 6.6 Hz, CH3(CH2)7CH2CH2N), 1.05–1.45 (17H, over-
lapping signals due to NCH2CH3 and CH3(CH2)7CH2CH2N),
1.56 (2H, bs, CH3(CH2)7CH2CH2N), 3.15–3.40 (4H, m,
CH3(CH2)7CH2CH2N and CH3CH2N), 3.90–4.00 (6H, over-
lapping signals due to Gly NHCH2), 4.11 (2H, s, COCH2), 4.38
and 4.40 (2H, s, COCH2). 13C-NMR (CD3OD): 13.2, 14.2,
14.6, 23.8, 28.0, 28.2, 28.8, 29.9, 30.6, 30.7, 30.8, 33.2, 41.9,
42.2, 42.5, 43.4, 43.5, 46.9, 47.0, 70.2, 70.3, 71.7, 170.6, 170.7,
172.2, 172.3, 172.9, 173.4. IR (CHCl3): 2953, 2920, 2851, 1719,
1694, 1674, 1558, 1466, 1415, 1274, 1230, 1203, 1162, 1040,
1
g, 70%) as a white solid, mp 179–81 1C. H-NMR (CDCl3–
CD3OD B9 : 1 v/v): 0.80 (9H, m, CH3(CH2)7CH2CH2N,
CH(CH3)2), 1.00–1.24 (17H, overlapping signals due to
NCH2CH3 and CH3(CH2)7CH2CH2N), 1.46 (2H, bs,
CH3(CH2)7CH2CH2N), 1.56 (3H, m, CH2CH(CH3)2), 3.00–
3.30 (4H, overlapping signals due to CH3(CH2)7CH2CH2N
and CH3CH2N), 3.70–4.00 (14H, Gly NCH2, COCH2O), 4.21
(3H, overlapping signals due to Leu NCH and COCH2O), 5.06
(2H, s, CH2OPh), 7.24 (5H, m, HAr). 13C-NMR (CDCl3–
CD3OD B9 : 1 v/v): 12.1, 13.2, 13.4, 20.8, 22.2, 22.3, 24.4,
26.4, 26.6, 27.2, 27.4, 28.4, 28.9, 28.96, 29.0, 29.1, 29.2, 31.5,
39.6, 40.7, 40.8, 41.0, 42.0, 42.2, 42.5, 42.6, 42.8, 45.5, 46.3,
52.5, 66.7, 68.5, 68.6, 70.3, 70.4, 127.8, 128.0, 128.2, 135.1,
168.38, 168.42, 169.5, 170.38, 170.43, 170.6, 171.0, 171.4, 174.0.
Anal. Calcd for C40H62N8O11 ꢃ 1/2 H2O: C, 57.53; H, 7.89; N,
13.08%. Found: C, 57.47; H, 7.81; N, 13.10%.
1029, 908, 732, 651 cmꢀ1
.
.
Triglycine benzyl ester toluenesulfonic acid salt (TsOH
GGG–OCH2Ph). GGG (3.0 g, 15.9 mmol) and p-toluenesul-
fonic acid (monohydrate, 3.6 g, 18.9 mmol) were added to a
mixture of benzyl alcohol (20 mL) and toluene (30 mL). The
mixture was heated to reflux and water was removed by using a
Dean–Stark trap. When no more water appeared in the
distillate, the mixture was cooled, diluted with Et2O (50 mL)
and cooled (0 1C) for 2 h. The crystalline p-toluenesulfonate of
GGG–OBz was collected on a filter, washed with Et2O (50
mL), and dried. After recrystallization from methanol–ether
the salt (5.5 g, 77%) melted at 176–177 1C. 1H-NMR: 2.34 (3H,
s, CH3Ph), 3.74 (2H, s, Gly NCH2), 3.97 (4H, s, Gly NCH2),
5.14 (2H, s, PHCH2O), 7.21 (2H, d, J ¼ 8.4 Hz, Tosyl HAr),
7.30–7.35 (5H, m, Ph HAr), 7.69 (2H, d, J ¼ 8.4 Hz, Tosyl HAr).
13C-NMR: 21.4, 41.7, 42.1, 43.2, 68.1, 127.2, 129.5, 129.6,
129.9, 130.2, 137.5, 142.1, 143.7, 168.4, 171.4, 172.2. IR
(KBr): 3331, 3083, 1747, 1670, 1545, 1456, 1406, 1362, 1202,
{2-[(N,N-Didecylcarbamoyl)methoxy]acetylamino}
triglycyl-L-leucyldiglycylacetic acid benzyl ester,
(102DGA–GGGLGGG–OCH2Ph) 2.
{2-[(N,N-Didecylcarbamoyl)methoxy]acetylamino}diglycylace-
tic acid (102DGA-GGG-OH) was prepared as previously
described.10
102DGA–GGGLGGG–OCH2Ph, 2. 102DGA–GGG–OH
(0.21 g, 0.45 mmol) was suspended in CH2Cl2 (50 mL) and
cooled to 0 1C. 1-(3-Dimethylaminopropyl)-3-ethyl carbodii-
mide hydrochloride (0.10 g, 0.52 mmol), HOBt (0.08 g, 0.59
mmol), Leu–GlyGlyGly–OCH2Ph ꢃ HCl (0.19 g, 0.45 mmol)
and Et3N (0.7 mL) were added, the mixture was stirred for 0.5
h, and stirred for 48 h at room temperature. After evaporation
of the solvent, the residue was washed with 5% citric acid, 5%
NaHCO3, and water. The residue was chromatographed (SiO2,
CHCl3–CH3OH 95 : 5 - 9 : 1) to give a white solid.
Crystallization from MeOH gave 1 (0.12 g, 28%), mp 186–
1125, 1035, 1011, 913, 817, 736, 685 cmꢀ1
.
N-Benzyloxycarbonyl-L-leucyldiglycyl acetic acid benzyl ester
(Boc-LGGG–OCH2Ph). Boc-L-Leucine (0.13 g, 0.56 mmol)
and TsOH ꢃ GGG–OCH2Ph (0.25 g, 0.56 mmol) were dissolved
in CH2Cl2 (20 mL) containing Et3N (0.2 mL). This mixture was
cooled to 5 1C and 1-(3-dimethylaminopropyl)-3-ethyl carbo-
diimide hydrochloride (0.118 g, 0.62 mmol) was added fol-
lowed by 1-hydroxybenzotriazole (HOBt, hydrate, 0.085 g,
0.63 mmol). Reaction was stirred at room temperature over-
night. The solvent was evaporated and the residue was chro-
matographed (SiO2 column, 5% MeOH–CHCl3) to give Boc-
LGGG–OCH2Ph as a colorless solid (0.24 g, 88%), mp 63–
64 1C. 1H-NMR: 0.88–0.95 (6H, m, Leu CH(CH3)2), 1.39 (9H,
s, C(CH3)3), 1.45–1.70 (3H, overlapping signals due to Leu
CH2CH(CH3)2), 3.80–4.10 (7H, overlapping signals due to Leu
NCH and Gly NCH2), 5.14 (2H, s, OCH2Ph), 5.35 (1H, d, J ¼
6.3 Hz, Leu NH), 7.30–7.35 (5H, m, HAr), 7.41 (1H, t,
1
188 1C. H-NMR (CDCl3–CD3OD 9 : 1 v/v): 0.68 (12H, m,
CH3), 1.05 (28H, m, CH3(CH2)7CH2CH2N), 1.32 (4H, m,
CH3(CH2)7CH2CH2N), 1.46 (3H, m, Leu CH2CH(CH3)2),
2.89 (2H, t, J ¼ 7.5 Hz, CH3CH2(CH2)7CH2N), 3.09 (2H, t,
J ¼ 7.5 Hz, CH3CH2(CH2)7CH2N), 3.57–3.91 (14H, overlap-
ping signals due to Gly CH2 and COCH2O), 4.07 (2H, s,
COCH2O), 4.10 (1H, m, Leu CH), 4.94 (2H, s, OCH2Ph),
7.14 (5H, m, HAr). 13C-NMR (CDCl3–CD3OD 9 : 1 v/v): 13.9,
21.2, 22.5, 22.7, 24.7, 26.8, 26.9, 27.4, 28.6, 29.17, 29.2, 29.3,
N e w J . C h e m . , 2 0 0 5 , 2 9 , 6 7 3 – 6 8 0
677

View Article Online
29.4, 29.5, 31.8, 39.6, 41.0, 42.4, 42.6, 42.8, 43.0, 43.2, 46.3,
46.8, 52.7, 67.0, 68.7, 70.7, 128.1, 128.4, 128.5, 135.2, 168.5,
169.9, 170.5, 170.9, 171.1, 171.2, 171.5, 174.1. Elem. Analysis
Calcd for C49H82O11N8 ꢃ 1/2 H2O: C 60.79, H 8.64, N 11.57%.
Found: C 60.85, H 8.60, N 11.46%.
washed with 5% citric acid (20 mL), water (20 mL), 5%
NaHCO3 (20 mL), brine (20 mL), dried (MgSO4), and evapo-
rated. The residue was chromatographed (SiO2, 10% MeOH-
CH2Cl2) to give 4 (0.32 g, 75%) as a white solid, mp 195 1C
decomposition. [a]2D3 ꢀ17.7 deg cm3 gꢀ1 dmꢀ1 (c 1.325,
MeOH). 1H-NMR: 0.87 (3H, t, J ¼ 6.6 Hz, CH3(CH2)7
CH2CH2N), 1.05–1.35 (17H, overlapping signals due to
NCH2CH3 and CH3(CH2)7CH2CH2N), 1.51 (2H, bs, CH3
(CH2)7CH2CH2N), 1.95–2.15 (4H, m, Pro NCH2CH2CH2),
3.00–3.40 (4H, m, CH3(CH2)7CH2CH2N and CH3CH2N), 3.40
–3.55 (2H, m, Pro NCH2CH2CH2), 3.70–4.40 (17H, overlap-
ping signals due to Gly NCH2, Pro NCH and COCH2O), 5.05–
5.20 (2H, m, OCH2Ph), 7.30–7.40 (5H, m, HAr), 7.45–7.55 (2H,
m, Gly NH), 7.85–8.00 (2H, m, Gly NH), 8.12 (1H, t, J ¼ 6.0
Hz, Gly NH), 8.30–8.40 (1H, bs, Gly NH). 13C-NMR: 13.0,
14.2, 14.3, 22.8, 25.3, 27.1, 27.3, 27.9, 29.2, 29.5, 29.6, 29.7,
32.1, 41.3, 41.5, 42.1, 42.3, 43.1, 43.2, 43.7, 46.0, 46.8, 47.1,
61.4, 67.3, 69.8, 71.6, 71.7, 128.5, 128.6, 128.8, 135.6, 168.5,
169.0, 170.3, 170.4, 170.5, 171.0, 171.1, 171.3, 173.6, 173.7.
Anal. Calcd for C40H62N8O11: C, 57.82; H, 7.52; N, 13.48%.
Found: C, 57.61; H, 7.56; N, 13.66%.
{2-[(N,N-Didecylcarbamoyl)methoxy]acetylamino}
triglycyl-L-azetidinyldiglycylacetic acid benzyl ester,
(102DGA-(Gly)3Aze(Gly)3–OCH2Ph) 3
N-Benzyloxycarbonyl-L-azetidinyldiglycylacetic acid benzyl
ester (Boc–Aze–GGG–OCH2Ph). A solution of Boc-L-azeti-
dine-2-carboxylic acid (0.25 g, 1.24 mmol) and GGG–OCH2Ph
tosylate (0.56 g, 1.24 mmol), 1-(3-dimethylaminopropyl)-3-
ethyl carbodiimide hydrochloride (0.27 g, 1.41 mmol), HOBt
(0.19 g, 1.41 mmol) and Et3N (0.6 mL) in CH2Cl2 (40 mL) was
stirred at 5 1C for 1 h and 25 1C for 3 days. The solvent was
evaporated and the residue was chromatographed (SiO2,
CH2Cl2–MeOH 97 : 3 - 95 : 5) to give the ester as a
deliquescent solid (0.52 g, 90%). 1H-NMR: 1.39 (9H, s,
C(CH3)3), 2.32 (2H, m, Aze NCH2CH2CH), 3.85 (2H, m,
Aze NCH2CH2CH), 3.95–4.01 (6H, overlapping signals due
to Gly CH2), 4.62 (1H, t, J ¼ 7.8 Hz, NCH2CH2CH), 5.10 (2H,
s, OCH2Ph), 7.30 (5H, HAr), 7.42 (1H, t, J ¼ 5.4 Hz, Gly NH),
7.62 (1H, bs, Gly NH), 7.82 (2H, bs, Gly NH). 13C-NMR: 19.9,
28.4, 41.3, 42.9, 43.1, 47.8, 62.0, 67.2, 77.4, 81.2, 128.4, 128.5,
128.7, 135.3, 169.8, 169.9, 172.6.
{2-[(N,N-Didecylcarbamoyl)methoxy]acetylamino}triglycyl-
L-prolyldiglycylacetic acid benzyl ester, (102DGA-GGGPG-
GG–OBz)
acetylamino}triglycyl-L-pipecolyldiglycylacetic acid benzyl
5
and {2-[(N,N-didecylcarbamoyl)methoxy]
ester, (102DGA–GGGPipGGG–OBz)
reported.10
6 were previously
102DGA–GGGAzeGGG–OCH2Ph, 3. 102DGA–GGG–OH
(0.51 g, 0.87 mmol) was suspended in CH2Cl2 (50 mL) and
cooled to 0 1C. 1-(3-Dimethylaminopropyl)-3-ethyl carbodi-
imide hydrochloride (0.20 g, 1.04 mmol), HOBt (0.14 g, 1.04
mmol), Aze–GGG–OBz ꢃ HCl (0.35 g, 0.88 mmol) and Et3N
(0.8 mL) were added and the mixture was stirred for 0.5 h at
0 1C and 48 h at 25 1C. The solvent was removed and the
residue chromatographed (silica, CHCl3–CH3OH 98 : 2 - 9 :
1) to give a white solid that was crystallized from methanol to
{2-[(N,N-Didecylcarbamoyl)methoxy]acetylamino}
triglycyl-m-aminobenzoyldiglycylacetic acid benzyl ester,
(102DGA–GGGAbaGGG–OBz) 7
N-Benzyloxycarbonyl-3-aminobenzoic acid (Boc-Aba). 3-
Aminobenzoic acid (2.47 g, 18.01 mmol) and di-tert-butyl
dicarbonate (5.30 g, 24.28 mmol) were dissolved in dioxane
(18 mL) and 2 M KOH (18 mL) and the mixture was stirred for
20 h at room temperature. The dioxane was evaporated and the
remaining solution was diluted with 1 M KOH (35 mL) and
washed twice with ethyl ether; the water phase was then
acidified with conc. HCl and a brown solid precipitated from
the solution. The crude product was filtrated, washed with
water and then dissolved in ethyl acetate and washed twice
with water; the solvent was removed and the remaining solid
recrystallized in the freezer from CH2Cl2–MeOH (just enough
methanol to dissolve all the solid) and then triturated with
hexanes to give a white solid (3.40 g, 80%), mp 195–197 1C.
1H-NMR (DMSO-d6): 1.46 (9H, s, C(CH3)3), 7.34 (1H, t, J ¼
8.1 Hz, HAr), 7.52 (1H, d, J ¼ 6.3 Hz, HAr), 7.60 (1H, d, J ¼ 7.8
Hz, HAr), 8.13 (1H, s, HAr), 9.54 (1H, s, ArNH), 12.88 (1H, bs,
COOH). 13C-NMR (DMSO-d6): 28.1, 79.3, 118.8, 122.3, 122.9,
128.8, 131.3, 139.8, 152.8, 167.3.
1
give 3 (0.08 g, 10%), mp 117–119 1C. H-NMR: 0.79 (6H, m,
CH3), 1.17 (28H, m, CH3(CH2)7CH2CH2N), 1.42 (4H, m,
CH3(CH2)7CH2CH2N), 2.38 (2H, m, Aze NCH2CH2CH),
2.99 (2H, t, J ¼ 7.8 Hz, CH3CH2(CH2)7CH2N), 3.17 (2H, t,
J ¼ 7.5 Hz, CH3CH2(CH2)7CH2N), 3.55–4.10 (16H, overlap-
ping signals due to Aze NCH2CH2, Gly CH2, and COCH2O),
4.19 (2H, s, COCH2O), 4.73 (1H, m, Aze CH), 5.05 (2H, m,
OCH2Ph), 7.25 (5H, m, HAr), 7.50 (2H, m, Gly NH), 7.69 (1H,
t, J ¼ 5.4 Hz, Gly NH), 7.83 (1H, t, J ¼ 5.4 Hz, Gly NH), 8.19
(1H, t, J ¼ 5.7 Hz, Gly NH), 8.28 (1H, t, J ¼ 5.7 Hz, Gly NH).
13C-NMR: 14.2, 19.8, 22.8, 27.0, 27.2, 27.8, 29.0, 29.4, 29.5,
29.7, 32.0, 39.5, 41.4, 43.0, 43.5, 46.4, 47.0, 49.0, 61.9, 67.2,
69.7, 71.6, 128.4, 128.5, 128.7, 135.5, 168.5, 170.0, 170.1, 170.2,
170.3, 170.5, 170.5, 171.2, 173.4. Elem. Analysis Calcd for
C47H76O11N8 ꢃ 1.5H2O: C 59.04, H 8.33, N 11.71%. Found: C
59.07, H 8.37, N 11.62%.
N-Benzyloxycarbonyl-aminobenzoyldiglycyl acetic acid ben-
zyl ester (Boc–AbaGGG–OBz). Boc-Aba (0.31 g, 1.31 mmol)
was suspended in CHCl3 (20 mL) and added to 1-(3-dimethyl-
aminopropyl)-3-ethyl carbodiimide hydrochloride (0.26 g, 1.36
mmol). The mixture was stirred for 0.5 h then TsOH ꢃ GGG–
OBz (0.59 g, 1.31 mmol) in CHCl3 (10 mL) and Et3N (0.3 mL)
was added and the reaction stirred for 48 h at room tempera-
ture. The solvent was then evaporated and the residue purified
by column chromatography (silica gel, CHCl3–CH3OH 95 : 5 -
90 : 10) to give a white solid (0.20 g, 83%), mp 135–137 1C.
1H-NMR: 1.45 (9H, s, C(CH3)3), 3.91 (2H, d, J ¼ 5.1 Hz, Gly
CH2), 3.97 (2H, d, J ¼ 5.7 Hz, Gly CH2), 4.02 (2H, d, J ¼ 5.4
Hz, Gly CH2), 5.04 (2H, s, OCH2Ph), 7.23 (8H, overlapping
signals due to HAr and NH), 7.39 (1H, d, J ¼ 7.8 Hz, HAr), 7.65
(1H, bs, HAr), 7.71 (1H, bm, NH), 7.90 (1H, bt, NH), 7.97 (1H,
bt, NH). 13C-NMR: 28.4, 41.4, 43.0, 43.9, 67.3, 80.8, 117.7,
121.6, 122.2, 128.4, 128.6, 128.7, 129.3, 133.9, 135.3, 139.3,
{2-[(N-Decyl-N-ethylcarbamoyl)methoxy]acetylamino}
triglycyl-L-prolyldiglycylacetic acid benzyl ester,
((10,2)DGA-GGGPGGG–OCH2Ph) 4
L-Prolyldiglycyl acetic acid benzyl ester hydrochloride (PGGG-
OCH2Ph ꢃ HCl) was prepared as previously described.10
(10,2)DGA–GGGPGGG–OCH2Ph, 4
To (10,2)DGA-GGG-OH (0.25 g, 0.52 mmol) suspended in
CH2Cl2 (20 mL), 1-(3-dimethylaminopropyl)-3-ethyl carbodi-
imide hydrochloride (0.11 g, 0.57 mmol) and HOBt (0.08 g,
0.59 mmol) was added and reaction was stirred for 0.5 h. Then
PGGG–OCH2Ph ꢃ HCl (0.22 g, 0.52 mmol) in CH2Cl2 (20 mL)
containing Et3N (0.15 mL) was added and reaction mixture
was stirred for 48 h at 25 1C. After evaporation, the residue was
678
N e w J . C h e m . , 2 0 0 5 , 2 9 , 6 7 3 – 6 8 0

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153.3, 168.4, 170.2, 170.3, 170.7. Anal. Calcd for C25H30N4O7 ꢃ
1/2 H2O: C, 59.16; H, 6.16; N, 11.03%. Found: C, 59.42; H,
6.12; N, 10.96%.
obtained after lysis. The data were collected using a DigiData
1322A series interface and Axoscope 9.0 software.
Carboxyfluorescein dequenching from unilamellar liposomes
The reverse phase procedure of Szoka and Papahadjopoulos18
was used to prepare the liposomes used in this study. A mixture
(10 mg) composed of 30% (w/w) 1,2-dioleoyl-sn-glycero-3-
phosphate and 70% 1,2-dioleoyl-sn-glycero-3-phosphocholine,
(Avanti Polar Lipids, AL) was dissolved in 0.5 mL of Et2O. To
this was added 0.5 mL of 20 mM carboxyfluorescein in 100
mM KCl : 10 mM HEPES (pH ¼ 7.0) and the final pH was
adjusted to 7.0 with KOH. This mixture was sonicated at 1200
W for 3 ꢂ 20 s at 20 1C to produce a stable emulsion. The Et2O
was removed (water aspirator, 15 1C) in a round bottom flask
rotating at 40 rpm. The 0.5 mL suspension was supplemented
with an additional 0.5 mL of 20 mM carboxyfluorescein
solution. This mixture was passed (five times) through a
200 nm nucleopore filter. The extra-vesicular carboxyfluores-
cein was removed by passing the liposome–dye mixture over a
1 ꢂ 20 cm Sephacryl G-25 column (Sigma) in 100 mM KCl :
10 mM HEPES (pH ¼ 7.0). The liposome peak was collected
and analyzed by dynamic light scattering, diameter ¼ 182 ꢁ
12 nm. Phospholipid concentration in this fraction was deter-
102DGA–GGGAbaGGG–OBz, 7
Boc–AbaGGG–OBz (0.30 g, 0.60 mmol) was dissolved in
dioxane (5 mL) and cooled to 0 1C then a 4 N HCl solution
in dioxane (5.0 mL, 20 mmol) was added and the resulting
solution was stirred at room temperature under nitrogen atmos-
phere for 3 hours; the solvent was removed and the product
carefully dried and used right away for the next reaction. This
compound, HClꢃ 3-aminobenzoic acid-GGG–OBz (0.26 g, 0.60
mmol) was mixed with 102DGA–GGG–OH (0.42 g, 0.72
mmol), PyCloP (0.30 g, 0.72 mmol) and CH2Cl2 (35 mL). The
mixture was cooled to 0 1C then diisopropylethylamine (0.26
mL, 2.01 mmol) was added and the resulting solution was stirred
for 1 h. The reaction was then continued at room temperature
for 4 days. The solvent was removed and the product purified by
column chromatography (silica gel, CHCl3–CH3OH 95:5 - 8 :
2) to give a white solid (0.20 g, 35%). Mp: dec. 1H-NMR
(DMSO-d6): 0.83 (6H, t, J ¼ 5.4 Hz, CH3), 1.21 (28H, m,
NCH2CH2(CH2)7CH3), 1.43 (4H, bs, NCH2CH2(CH2)7CH3),
3.09 (2H, t, J ¼ 7.5 Hz, NCH2CH2(CH2)7CH3), 3.17 (2H, t, J ¼
7.5 Hz, NCH2CH2(CH2)7CH3), 3.75–3.90 (12H, overlapping
signals due to Gly CH2), 3.97 (2H, s, COCH2O), 4.26 (2H, s,
COCH2O), 5.11 (2H, s, OCH2Ph), 7.34 (6H, m, HAr), 7.56 (1H,
d, J ¼ 7.5 Hz, HAr), 7.78 (1H, d, J ¼ 7.5 Hz, HAr), 8.07 (1H, s,
mined to be 3.3 mg mlꢀ1 17
.
Carboxyfluorescein-containing vesicles were diluted to 0.66
mg mlꢀ1 in a reaction volume of 500 mL. The fluorescence
(excitation 497 nm: emission 520 nm at 2 nm bandpass) was
monitored at 25 1C. Compounds were added as a 5 mM
solution in 2-propanol with mixing to the indicated concentra-
tions. Data were digitized at 10 points per second and subse-
HAr), 8.25 (5H, m, NH), 8.71 (1H, s, NH), 9.98 (1H, s, ArNH).
13C-NMR (DMSO-d6): 13.9, 22.1, 26.2, 26.4, 27.1, 28.3, 28.7,
28.8, 29.0, 31.3, 40.7, 41.7, 41.8, 42.2, 42.7, 42.8, 45.1, 46.1, 65.9,
68.8, 70.3, 118.7, 122.0, 122.1, 127.9, 128.0, 128.1, 128.6, 134.7,
135.9, 138.9, 166.5, 167.8, 168.1, 169.26, 169.32, 169.4, 169.5,
169.59, 169.62. Anal. Calcd for C50H76N8O11 ꢃ 2 H2O: C, 59.98;
H, 8.05; N, 11.19%. Found: C, 59.66; H, 7.71; N, 10.80%.
quently decimated to one point per second. Dequenching, F520
was computed as the fraction of total release upon addition of
,
1% Triton X-100:
F ꢀ F0
F520
¼
ð1Þ
FTriton ꢀ F0
where F0 is the fluorescence at zero time and FTriton is the
fluorescence after Triton X-100 treatment. Dequenching data
were fit using a nonlinear least squares method based upon the
Levenberg–Marquardt algorithm, which generated c2 values
indicating the goodness of fit, for each group of averaged data
sets. The number of individual trials generating the data set
(degrees of freedom) was used to obtain the p value for the
individual fits and kinetic constants. In all cases, the c2 values
for individual fits were less than 0.05 and the resulting p values
varied from 0.05 to 0.001 depending upon the number of trials.
Data were fit to the equation,
Formation of vesicles
Liposomes were prepared from 1,2-dioleoyl-sn-glycero-3-phos-
phocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphate
(DOPA), both obtained from Avanti Polar Lipids as CHCl3
solutions (vials with 100 mg in 4.0 mL). Dry lipid films of
DOPC–DOPA (20 mg, 7 : 3 w/w) were dissolved in Et2O (0.5
mL) and then 0.5 mL of internal buffer (600 mM KCl : 10 mM
N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic
acid
(HEPES), pH adjusted to 7.0) were added. Sonication of the
mixture for B10 s gave an opalescent dispersion. Evaporation
under mild vacuum (30 1C) removed the organic solvent and
the remaining suspension was filtered (200 nm filter membrane,
5ꢂ, using a mini extruder) and then passed through a Sephadex
G25 column (previously equilibrated with external buffer (400
mM K2SO4 : 10 mM HEPES, pH adjusted to 7.0). Analysis by
laser light scattering (Coulter N4MD submicron particle anal-
yzer) showed that the vesicular diameter was consistently
B200 nm. The final lipid concentration was assessed through
colorimetric determination of the phospholipid–ammonium
ferrothiocyanate complex.17
F520 ¼ F0 þ A1(1 ꢀ etime/t) þ m ꢃ time
(2)
as described previously.19 In this equation, F0 is the fluores-
cence at time zero, A1 is the size of the exponential component,
t is the time constant for pore activation, and m is the slope of
the linear dequenching resulting from multiple pores per
vesicle.
Acknowledgements
We thank the NIH for a grant, GM-63190, that supported this
work.
Chloride release from liposomes
A chloride selective electrode (Accumet Chloride Combination
Electrode) was used to assay B200 nm, 0.31 mM phospholipid
vesicles. The electrode was inserted in the vesicle suspension
(2 mL), equilibrated, the voltage output was recorded, and
after five minutes, aliquots of the ionophore (B9 mM in 2-
propanol) were added. The amount of 2-propanol added was
limited to r20 mL to avoid a solvent effect on the liposomes.
Liposomal lysis was induced by 2% aqueous Triton X-100
(100 mL) and the data collected were normalized to the value
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