Effect of N(α)-acyl chain length on the membrane-modifying properties of synthetic analogs of the lipopeptaibol trichogin GA IV

Article abstract of DOI:10.1021/ja954081o

Trichogin GA IV, an1-residue lipopeptaibol blocked at the N-terminus by an n-octanoyl group and at the C-terminus by a 1,2-amino alcohol (L-leucinol), extracted from the fungus Trichoderma longibrachiatum, exhibits remarkable membrane-modifying properties

Full text of DOI:10.1021/ja954081o

4952
J. Am. Chem. Soc. 1996, 118, 4952-4958
Effect of NR-Acyl Chain Length on the Membrane-Modifying
Properties of Synthetic Analogs of the Lipopeptaibol Trichogin
GA IV
C. Toniolo,1a M. Crisma,1a F. Formaggio,1a C. Peggion,1a V. Monaco,1a
C. Goulard,1b S. Rebuffat,1b and B. Bodo*,1b
Contribution from the Biopolymer Research Center, CNR, Department of Organic Chemistry,
UniVersity of PadoVa, 35131 PadoVa, Italy, and the Laboratoire de Chimie,
URA 401 CNRS, GDR 1153 CNRS, Muse´um National d’Histoire Naturelle,
75231 Paris 05, France
ReceiVed December 5, 1995X
Abstract: Trichogin GA IV, an 11-residue lipopeptaibol blocked at the N-terminus by an n-octanoyl group and at
the C-terminus by a 1,2-amino alcohol (L-leucinol), extracted from the fungus Trichoderma longibrachiatum, exhibits
remarkable membrane-modifying properties. We have synthesized trichogin GA IV and several [L-Leu-OMe11]
analogs carrying at the N-terminus an acyl chain of variable length (C2-C8, C10, C12, C14, C16, C18). A succinoylated
head-to-head dimer was also prepared. A conformational analysis, carried out by FTIR absorption, CD, and NMR,
showed that the right-handed helical structure of the natural lipopeptaibol is essentially preserved in all its analogs.
Permeability measurements revealed that at least six carbon atoms in the NR-blocking fatty acyl moiety are required
for the onset of significant membrane-modifying properties. Also the head-to-head dimer is remarkably active.
Possible models for the mechanism of membrane permeability of trichogin GA IV are discussed.
nOc-Aib1-Gly-L-Leu-Aib-Gly-Gly-L-Leu-Aib-Gly-L-
Ile-L-Lol11
Introduction
Peptaibols2 are a unique group of membrane-active peptides
biosynthesized by molds, mainly of the genus Trichoderma.
These antibiotic peptides are usually characterized by a linear
sequence of 15-19 amino acid residues, a high proportion of
Despite having a shorter amino acid sequence than the classical
peptaibols, this lipopeptaibol exhibits membrane activity. On
the basis of a CD and NMR conformational investigation of
the natural compound, a right-handed helical structure was
proposed. More recently, this 3D-structural assignment was
fully supported by an X-ray diffraction analysis on synthetic,
racemic trichogin GA IV.9 These conformational properties are
not surprising in view of its relatively high (30%) content in
Aib, the strongest known helix-forming amino acid.10-12
C
R,R-disubstituted glycines (R-aminoisobutyric acid, Aib, and
isovaline, Iva), an Ν-terminal acetyl group, and a C-terminal
1,2-amino alcohol.3 These long-sequence peptaibols, such as
alamethicin, are known to form voltage-dependent membrane
channels and to modify the membrane permeability even in the
absence of voltage.4
Recently, from several Trichoderma species we isolated the
following 11-residue peptaibols with an N-terminal n-octanoyl
group: trichogin GA IV from T. longibrachiatum5,6 and
trikoningins KB I and KB II from T. koningii.7 The name
lipopeptaibol was proposed for such compounds.6 Subse-
quently, the list of lipopeptaibols was expanded by Fujita et
al.8 who reported the sequences of the 7-residue trichodecenins
I and II isolated from T. Viride.
In order to examine the role of the N-terminal fatty acyl chain
length on the membrane-modifying properties of trichogin GA
IV, the prototype of lipopeptaibols, we synthesized trichogin
GA IV itself and a number of [L-Leu-OMe11] analogs with
variable acyl chain length, including the C2-C8, C10,C12, C14,
C16, and C18 acyl groups. A succinoylated head-to-head dimer
was also prepared. In this paper, in addition to the synthesis
and characterization, we describe the results of a comparative
conformational study (using FTIR, CD, and NMR) and the
ability of these peptides to modify membrane permeability as
measured on liposomes and erythrocytes. Preliminary data on
part of this work have been reported.13,14
Trichogin GA IV,5,6 the main component of the natural
trichogin microheterogeneous mixture, is blocked by an n-
octanoyl moiety at the N-terminus and has a 1,2-amino alcohol,
L-Lol (leucinol), at the C-terminus with the following sequence:
X Abstract published in AdVance ACS Abstracts, May 1, 1996.
(1) (a) University of Padova. (b) Muse´um National d’Histoire Naturelle.
(2) Benedetti, E.; Bavoso, A.; Di Blasio, B.; Pavone, V.; Pedone, C.;
Toniolo, C.; Bonora, G. M. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 7951-
7954.
(9) Toniolo,C.; Peggion, C.; Crisma, M.; Formaggio, F.; Shui, X.;
Eggleston, D.S. Nature: Struct. Biol. 1994, 1, 908-914.
(3) Nagaraj, R.; Balaram, P. Acc. Chem. Res. 1981, 14, 356-362.
(4) Sansom, M. S. P. Prog. Biophys. Mol. Biol. 1991, 55, 139-236.
(5) Auvin-Guette, C.; Rebuffat, S.; Prigent, Y; Bodo, B. In Peptides 1990;
Giralt, E., Andreu, D., Eds.; ESCOM: Leiden, 1991; pp 428-429.
(6) Auvin-Guette, C.; Rebuffat, S.; Prigent, Y; Bodo, B. J. Am. Chem.
Soc. 1992, 114, 2170-2174.
(10) Marshall, G. R. In Intra-Science Chemistry Reports; Kharasch, N.,
Ed.; Gordon and Breach: New York, 1971; Vol. 5, pp 305-316.
(11) Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747-6756.
(12) Benedetti, E.; Toniolo,C. Macromolecules 1991, 24, 4004-4009.
(13) Toniolo, C.; Crisma, M.; Formaggio, F.; Pirrone, L.; Bonora, G.
M.; Mammi, S.; Peggion, E. In Peptides 1992; Schneider, C. H., Eberle,
A. N., Eds.; ESCOM: Leiden, 1993; pp 613-614.
(14) Bodo, B.; Rebuffat, S.; Goulard, C.; Peggion, C.; Monaco, V.;
Formaggio, F.; Crisma, M.; Toniolo, C. In Peptides 1994; Maia, H. L. S.,
Ed.; ESCOM: Leiden, 1995; pp 749-750.
(7) Auvin-Guette, C.; Rebuffat, S.; Vuidepot, I.; Massias, M.; Bodo, B.
J. Chem. Soc., Perkin Trans.1 1993, 249-255.
(8) Fujita, T.; Wada, S. I.; Iida, A.; Nishimura, T.; Kanai, M.; Toyama,
N. Chem. Pharm. Bull. 1994, 42, 489-494.
S0002-7863(95)04081-9 CCC: $12.00 © 1996 American Chemical Society

Trichogin GA IV Analogs
J. Am. Chem. Soc., Vol. 118, No. 21, 1996 4953
RPMI containing 7.5% serum to a final concentration of 107 cells/mL.
The peptide samples were dissolved in DMSO such as to give a 20
mM solution. Other concentrations were obtained by successive
dilutions with DMSO. The peptide solutions (10 µL) were diluted with
the 7.5% serum containing RPMI (490 µL), keeping the final DMSO
concentration below 1%. The final peptide concentration range was
3-200 µM. A 500 µL sample of the erythrocyte suspension was then
added with stirring and the mixture incubated at 37 °C for 3 or 27 h.
Absorbance of the supernatant (200 µL) was measured at 405 nm. The
0% hemolysis (OD0) was determined by incubating 1 mL of the
erythrocyte suspension containing 1% DMSO, and the 100% hemolysis
(OD100) was measured by incubating the erythrocyte suspension (500
µL) with 490 µL of RPMI and 10 µL of a dilute (1:10) Triton X-100
solution. The percentage of lysis was calculated according to % lysis
) (ODi - OD0)/(ODi - OD100) x 100, where ODi is the optical density
for the peptide concentration i.
Experimental Section
Peptide Synthesis. Melting points were determined using a Leitz
Model Laborlux 12 apparatus and are not corrected. Optical rotations
were measured using a Perkin-Elmer Model 241 polarimeter equipped
with a Haake Model D thermostat. Thin-layer chromatography was
performed on Merck Kieselgel 60-F254 precoated plates using the
following solvent systems: (I) CHCl3/EtOH, 9:1; (II) nBuOH/AcOH/
H2O, 3:1:1; (III) toluene/EtOH, 7:1. The chromatograms were devel-
oped by quenching of UV fluorescence, chlorine-starch-potassium
iodide or ninhydrin chromatic reaction as appropriate. All the new
compounds were obtained in a chromatographically homogeneous state.
The amino acid analyses were performed on a C. Erba Model 3A27
amino acid analyzer. The Aib color yield with ninhydrin is about 20
times lower than those of protein amino acids. Elution of Aib was
observed immediately after the Ala peak. HPLC analyses were
performed on a Pharmacia Model LKB-LCC 2252 liquid chromatograph
equipped with an UVICORD Model SD UV detector (226 nm) and a
reversed-phase C18 Vydac Model 218 TP54 column.
Results
FTIR Absorption. The FTIR absorption spectra were recorded
using a Perkin-Elmer Model 1720X FTIR spectrophotometer, nitrogen-
flushed, equipped with a sample-shuttle device, at 2 cm-1 nominal
resolution, averaging 100 scans. Solvent (baseline) spectra were
obtained under the same conditions. Cells with path lengths of 0.1,
1.0, and 10 mm (with CaF2 windows) were used. Spectrograde
deuteriochloroform (99.8% D) was purchased from Fluka.
Circular Dichroism. The CD spectra were obtained on a JASCO
Model J-710 spectropolarimeter. Cylindrical fused quartz cells of 10
and 1 mm path lengths were used. The values are expressed in terms
of [Θ]T, the total molar ellipticity (deg cm dmol-1). MeOH (Riedel-
de-Ha¨en) was used as the solvent.
Peptide Synthesis. The syntheses of trichogin GA IV and
its [L-Leu-OMe11] undecapeptide analogs were performed step-
by-step in solution, beginning from the C-terminal H-L-Leu-
OMe residue, Via the mixed anhydride method with isobutyl
chloroformate to incorporate the protein amino acids and the
symmetrical anhydride method to incorporate the internal Aib
residues (Chart 1). The acylated N-terminal Aib residue was
added using the 5(4H)-oxazolone method. However, more
recently higher yields for the insertion of the N-terminal Aib
residue were obtained by treatment of the C-terminal decapep-
tide with (Z-Aib)2O (Z ) benzyloxycarbonyl), followed by NR-
deprotection and treatment with the appropriate carboxylic acid
preactivated in CH2Cl2 solution with N-ethyl-N′-[3-(dimethyl-
amino)propyl]carbodiimide (EDC), and 1-hydroxybenzotriazole.
In the last step the NR-octanoyl undecapeptide methyl ester was
reduced using LiBH4 to afford the synthetic trichogin GA IV,
which was shown by chromatographic and physical methods
to be identical to the natural lipopeptaibol.
The Z-protected derivatives were obtained by reacting the
pertinent free amino acid with Z-OSu (1-hydroxysuccinimido
ester). The L-Leu methyl ester hydrochloride was prepared by
the methanol/thionyl chloride method. Removal of the Z group
was carried out by catalytic hydrogenation. The Aib 5(4H)-
oxazolones were synthesized from their NR-acylated free acid
precusors and 1 equiv of EDC. Owing to the difficulties in the
isolation of the 5(4H)-oxazolone from Ac-Aib-OH,16 for the
synthesis of the NR-acetylated undecapeptide methyl ester a
different route was designed, namely, the synthesis of the NR-
benzyloxycarbonylated undecapeptide analog Via (Z-Aib)2O,
followed by deprotection of the Z group and acetylation of the
NR-free derivative with acetic anhydride. The stable, crystalline
derivative (Z-Aib)2O17 was obtained by reacting Z-Aib-OH with
0.5 equiv of thionyl chloride in ethyl acetate.
NMR Spectroscopy. NMR spectra were recorded on a Bruker AC
300 spectrometer equipped with an Aspect 3000 computer. Peptide
solutions in CD3OH (CEA, Saclay, France) were 20 and 50 mM for
1H and 13C NMR spectra, respectively. Unless otherwise specified,
all experiments were run at a temperature of 23 °C. 1H NMR spectra
were obtained by solvent presaturation and referred to the central
component of the quintet of the CHD2 resonance of methanol at 3.313
ppm downfield from TMS. 13C NMR spectra were collected at 75.47
MHz, and chemical shifts were referred to internal CD3OH taken at
49.00 ppm relative to TMS. The 1D spectra were obtained with 300-
700 scans. Standard methods were used to perform the 2D experiments,
and pulsed programs were taken from the Bruker software library.
Liposome Leakage Assay. Peptide-induced leakage from egg
phosphatidylcholine (PC) vesicles was measured at 20 °C using the
carboxyfluorescein (CF)-entrapped vesicle technique as previously
described.15 CF-encapsulated small unilamellar vesicles (egg PC/
cholesterol, 7:3) were prepared by sonication in Hepes buffer, pH 7.4.
The phospholipid concentration was kept constant (0.6 mM), and
increasing [peptide]/[lipid] molar ratios (Ri-1) were obtained by adding
aliquots of methanolic solutions of peptides, keeping the final methanol
concentration below 5% by volume. After rapid and vigorous stirring,
the time course of fluorescence change corresponding to CF escape
was recorded at 520 nm (1 nm band pass) with λexc 488 nm (1 nm
band pass). The percentage of released CF at time t was determined
as (Ft - Fo)/(FT - Fo) × 100, with Fo ) fluorescence intensity of
vesicles in the absence of peptide, Ft ) fluorescence intensity at time
t in the presence of peptide, and FT ) total fluorescence intensity
determined by disrupting the vesicles by addition of 30 µL of a 10%
Triton X-100 solution. The kinetics were stopped at 20 min.
Antimicrobial Activity. The antibacterial activity of trichogin GA
IV and some of its NR-acyl-[Leu-OMe11] analogs were examined against
Staphylococcus aureus (strain 209 P) by the agar diffusion test using
the Muller-Hinton culture medium and 6 mm diameter pits. The
peptide samples were dissolved in DMSO such as to give a 4 mg/mL
solution. Eight other concentrations were obtained by successive
dilutions, and 50 µL of each solution was deposited into the pits (1.2-
200 µg). Inhibition zones were measured after 24 h of incubation at
37 °C.
The synthesis of the NR-succinoylated [L-Leu-OMe11] tri-
chogin GA IV dimer was achieved by reacting a large excess
of the NR-deprotected undecapeptide methyl ester with succinoyl
chloride in acetonitrile in the presence of N-methylmorpholine.
The chemical and optical purities of all the intermediates and
final synthetic products were assessed by polarimetry, TLC in
three different solvent systems, and amino acid analysis (data
1
listed in Table 1), solid-state IR absorption, H NMR, and
HPLC, and for the synthetic trichogin GA IV by chiral
chromatography and mass spectrometry. It is worth noting that
the reversed-phase HPLC retention times of the [L-Leu-OMe11]
(16) Levene, P. A.; Steiger, R. E. J. Biol. Chem.1931, 93, 581-604.
(17) Valle, G.; Formaggio, F.; Crisma, M.; Bonora, G. M.; Toniolo, C.;
Bavoso, A.; Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C. J. Chem.
Soc., Perkin Trans. 2 1986, 1371-1376.
(18) Okumura, Y.; Sakurai, A. Bull. Chem. Soc. Jpn. 1973, 46, 2190-
2193.
Hemolytic Activity. Human erythrocytes (O+) were centrifuged at
600 g for 5 min, washed three times with RPMI 1640, and diluted in
(15) El Hajji, M.; Rebuffat, S.; Le Doan, T.; Klein, G.; Satre, M.; Bodo,
B.; Biochim. Biophys. Acta 1989, 978, 97-104.

4954 J. Am. Chem. Soc., Vol. 118, No. 21, 1996
Toniolo et al.
Chart 1a
a Conditions: (1) iBuOCOCl, NMM. (2) H2, Pd/C. (3) 0.5 equiv of SOCl2, TEA. (4) EDC (HCl). (5) LiBH4, THF.
trichogin GA IV analogs with an even number of carbon atoms
in the acyl chain increase linearly as the number of carbon atoms
increases (Figure 1).
Conformational Analysis. A detailed analysis of the
preferred conformation of the [L-Leu-OMe11] trichogin GA IV
analogs with variable NR-acyl chain and the succinoylated head-
to-head dimer was performed using FTIR absorption, CD, and
NMR in different solvents and compared with that of the parent
peptaibol.6
The IR absorption spectra of the undecapeptide esters and
trichogin GA IV in CHCl3 solution are dominated by strong
bands at 3323-3328 cm-1 (NsH stretching mode of H-bonded
amide groups) and 1655-1657 cm-1 (CdO stretching mode
of H-bonded amide groups).19 Additional, but very weak, bands
are seen in the 3426-3453 cm-1 region (free, solvated amide
NH groups) and at 1738-1739 cm-1 (ester carbonyl). Obvi-
ously, the latter band is absent in the peptaibol. In the NsH
stretching region all peptides examined show a more or less
pronounced concentration effect in the range 10-0.1 mM,
indicating the onset of a nonmarginal amount of intermolecular
H bonds. However, at the lowest concentration (0.1 mM) the
3323-3328 cm-1 band is still very intense, supporting the view
that all these peptides are characterized by an extensive set of
intramolecular NsH‚‚‚OdC H-bonds. The position of the
strong CdO stretching band is also in favor of this conclusion.
Auvin-Guette et al.6 have reported the CD spectrum of
trichogin GA IV in methanol. The lipopeptaibol exhibits a weak
negative maximum at 223 nm (amide n f π* transition),
followed by more intense amide π f π* exciton split dichroic
bands at 205 nm (negative) and 192 nm (positive). This pattern
is reminiscent of those shown by (right-handed) predominantly
helical peptides.20 Figure 2 illustrates the CD curves for three
representative [L-Leu-OMe11] trichogin GA IV analogs in
methanol. Although the spectrum of trichogin GA IV is in
general more intense, a similar pattern is found also in all the
undecapeptide ester analogs.
The 1H NMR spectra of the various analogs in methanol are
very similar, differing mainly by an increase in the intensity of
the polymethylene chain signal at 1.27 ppm. In general, the
amide proton and carbonyl carbon chemical shifts and thermal
coefficients, and the 3JNH-RCH coupling constants in the 1H and
13C NMR spectra of the C2, C8, C14, and C18 NR-acyl analogs
(19) Palumbo, M.; Da Rin, S.; Bonora, G. M.; Toniolo, C. Makromol.
Chem. 1976, 177, 1477-1492.
(20) Beychok, S. In Poly-R-Amino Acids; Fasman, G. D., Ed.; Dekker:
New York, 1967; pp 293-337.

Trichogin GA IV Analogs
J. Am. Chem. Soc., Vol. 118, No. 21, 1996 4955

4956 J. Am. Chem. Soc., Vol. 118, No. 21, 1996
Toniolo et al.
Table 2. Chemical Shift Values at 296 K (δ, ppm) and Vicinal Coupling Constants (3J, Hz) of Amide Protons for Trichogin GA IV and Its
[L-Leu-OMe11] Analogs
amino acid
GA IV
C2
C8
C14
C18
dimer
Aib1
8.510(s)
8.541(s)
8.496(s)
8.499(s)
8.540(s)
8.495(s)
Gly2
8.544(t, 5.6)
8.091(d, 7.5)
7.980(s)
8.327(t, 5.5)
8.138(t, 5.8)
7.926(d, 6.4)
7.987(s)
8.523(t, 5.7)
8.048(d, 6.8)
7.999(s)
8.261(t, 5.6)
8.097(t, 6.1)
7.932(d, 6.8)
8.009(s)
8.037(t, 5.7)
7.752(d, 8.4)
8.089(d, 7.5)
8.528(t, 5.5)
8.080(d, 7.8)
7.963(s)
8.289(t, 5.6)
8.110(t, 5.8)
7.914(d, 6.8)
7.996(s)
8.044(t, 5.8)
7.749(d, 8.4)
8.096(d, 7.3)
8.530(t, 5.6)
8.080(d, 8.1)
7.964(s)
8.293(t, 5.6)
8.114(t, 5.8)
7.915(d, 6.9)
7.995(s)
8.046(t, 6.0)
7.752(d, 8.5)
8.092(d, 7.3)
8.495(t, br)
8.091(d, 7.5)
7.965(s)
8.287(t, 5.6)
8.110(t, 5.8)
7.916(d, 6.6)
8.001(s)
8.045(t, 6.0)
7.751(d, 8.5)
8.087(d, 7.8)
8.393(t, 5.8)
8.063(d, 7.0)
8.190(s)
8.179(t, 5.6)
8.126(t, 5.9)
8.004(d, 6.9)
8.168(s)
8.062(t, 6.0)
7.761(d, 8.4)
8.202(d, 7.5)
Leu3
Aib4
Gly5
Gly6
Leu7
Aib8
Gly9
8.053(t, 5.7)
7.699(d, 8.4)
Ile10
Leu-OMe11
Lol11
7.309(d, 9.0)
Figure 2. CD spectra in methanol of the [L-Leu-OMe11] trichogin GA
IV analogs with C4, C8, and C12 atoms in the NR-acyl chain (concentra-
tion 1 mM).
Figure 1. (a) Reversed-phase HPLC chromatography and (b) plot of
HPLC retention times of the NR-acylated [L-Leu-OMe11] trichogin GA
IV analogs as a function of n, the number of carbon atoms in the acyl
chain (only even numbers are reported). Mobile phase: gradient from
50% to 100% of B in A during 40 min (A is 0.05% TFA in H2O; B is
0.05% TFA in a 9:1 MeCN/H2O mixture). Flow rate: 1 mL/min.
Column: reversed-phase C4 Vydac Model 214TP54 (250 × 4.6 mm).
Detection: UV, 226 nm.
and the succinoyl dimer are close to those of trichogin GA IV
(Tables 2 and 3 and Figure 3). However, some differences are
seen near the C-terminus: (i) The JNH-RCH value is smaller
Figure 3. Amide proton region of the 1H NMR spectrum of trichogin
GA IV (a) and the [L-Leu-OMe11] trichogin GA IV analogs with C8
(b) and C2 (c) atoms in the NR-acyl chain (300 MHz, 295 K, CD3OH,
20 mM).
3
for the C-terminal residue of the [Leu-OMe11] analogs as
compared to the value for Lol11 in trichogin GA IV. (ii) The
thermal coefficients of the Lol11 NH proton and Ile10 carbonyl
carbon in trichogin GA IV are lower than the corresponding
values for Leu11 and Ile10 in the three [Leu-OMe11] peptides.
These findings can be interpreted in terms of a preferential
structure with a more extensive set of intramolecular H bonds
(C-terminal helical cap) for the natural antibiotic. Interestingly,
the spectrum of the succinoyl dimer exhibits signals for only
half of the protons, and the succinoyl methylene gives an AB
system centered at 2.54 ppm, thus indicating that this molecule
is symmetrical. This result is confirmed by the 13C NMR
spectrum which displays only 12 carbonyl carbon signals (Table
3). More generally, the chemical shifts for the carbonyl groups
of the C2 NR-acyl analog and the succinoyl dimer, which were
1
assigned from H-13C long-range COSY experiments, show
great similarities with those of trichogin GA IV. All these data,
taken together, strongly suggest that all the peptide analogs
should have similar helical conformations. In summary, the
helical structure, previously determined for trichogin GA IV in
methanol solution6 and in the crystal state,9 appears to be
maintained in the synthetic analogs.

Trichogin GA IV Analogs
J. Am. Chem. Soc., Vol. 118, No. 21, 1996 4957
Table 3. Temperature Dependence (-∆δ/∆T, ppb/K) of Amide Protons, Chemical Shift Values at 296 K (δ, ppm), and Temperature
Dependence (-∆δ/∆T, ppb/K) of Carbonyl Carbons for Trichogin GA IV and Its [L-Leu-OMe11] Analogs
-∆δ/∆T NH
δ CO
-∆δ/∆T CO
amino acid
GA IV
C2
C8
dimer
GA IV
C2
dimer
GA IV
C2
dimer
O/A/ Sa
Aib1
Gly2
Leu3
Aib4
Gly5
Gly6
Leu7
Aib8
Gly9
Ile10
176.6O
178.5
173.1
175.8
178.3
173.4
173.0
175.3
178.1
172.5
173.6
173.5 A
178.2
173.0
175.7
178.1
173.1
172.7
174.8
177.6
172.1
173.8
174.2
175.0S
178.0
172.7
175.3
177.9
172.9
172.5
174.8
177.5
172.1
173.9
174.2
0.2
1.5
0.8
2.7
-0.8
1.8
0.8
0.9
1.6
3.0
2.0
1.0
0.1
1.3
-1.0
0.9
-1.2
2.2
1.8
0.9
5.6
1.7
3.1
7.1
6.0
2.0
4.9
5.7
2.4
4.7
7.4
3.8
2.3
8.2
6.8
2.5
6.1
6.2
2.5
5.1
8.6
5.6
3.7
8.8
8.6
6.9
2.6
4.9
6.2
2.7
4.9
8.8
5.5
3.7
9.0
7.7
5.7
3.2
6.6
5.1
3.0
5.1
9.6
5.5
3.4
9.1
-0.1
-0.9
-2.3
-1.8
-0.5
3.3
2.4
0.0
6.0
0.1
2.3
Leu11
Lol11
0.2
4.8
a O, A, and S refer to octanoyl, acetyl and succinoyl groups, respectively.
Figure 4. Peptide-induced CF leakage at 20 min for different ratios
Ri-1 ) [peptide]/[lipid] from egg PC/cholesterol (70:30) vesicles. The
number of carbon atoms (2-8) in the NR- acyl chain of the [L-Leu-
OMe11] trichogin GA IV analogs is indicated.
Figure 6. Kinetic profile for the peptide-induced CF leakage from
egg PC/cholesterol (70:30) vesicles. The Ri-1 ) [peptide]/[lipid] values
are listed for each peptide. The number of carbon atoms (3, 7, 12, 14,
16) in the NR- acyl chain of the [L-Leu-OMe11] trichogin GA IV analogs
is indicated.
most active analog is the C10 peptide which induces, at 20 min,
50% leakage for Ri-1 ) 1.8 × 10-3, and 100% for Ri-1 ) 4 ×
10-3. Upon further increasing the chain length from C10 to C14
atoms, a slight decrease in the membrane activity is noticed.
Surprisingly, the activity dramatically falls for the C16 and C18
analogs, which are found inactive, even for Ri-1 ratios higher
than 4 × 10-3. This apparently lower or null activity for the
C14-C18 analogs can be explained on the basis of a comparison
of their kinetic profiles with those of the more active peptides
(Figure 6). While the natural peptide (not shown) and the
efficient analogs exhibit kinetics with steep slopes at the origin,
the C14-C18 analogs develop a lag time (possibly due to self-
association of these lipopeptides) which increases when increas-
ing the lipid chain length. The succinoyl dimer is more efficient
than all the undecapeptides (Figure 5).
Biological Activities. The antibacterial activity of the C2,
C8, and C16 NR-acyl and succinoyl dimer analogs were examined
against S. aureus by the agar diffusion method using 6 mm pits
and compared to the natural lipopeptide (Table 4). The C8 [Leu-
OMe11] analog has activity similar to that of the natural
compound, whereas the observed inactivities of the C2 and C16
analogs are explained by the absence of a lipidic chain for the
former analog and by a lack of diffusion (probably due to a
greater hydrophobicity) for the latter analog.
Figure 5. Peptide-induced CF leakage at 20 min for different ratios
Ri-1 ) [peptide]/[lipid] from egg PC/cholesterol (70:30) vesicles. The
number of carbon atoms (8, 10, 12, 14, 16, 18) in the NR- acyl chain
of the [L-Leu-OMe11] trichogin GA IV analogs is indicated. T and D
refer to trichogin GA IV and the [L-Leu-OMe11] trichogin GA IV
succinoyl dimer, respectively.
Membrane-Modifying Properties from Liposome Leakage
Measurements. The membrane-modifying properties of the
different analogs were tested in comparison to those of the
natural lipopeptide by measuring the induced leakage of CF
entrapped in egg PC/cholesterol (7:3) small unilamellar vesicles
(Figures 4 and 5). The C8 [Leu-OMe11] analog has similar
activity as the natural lipopeptaibol, both compounds leading
to 50% CF leakage at 20 min for Ri-1 ) 4.2 × 10-3 and 100%
for Ri-1 ) 7 × 10-3. The C2 analog is almost inactive. By
increasing the lipid chain length from C3 to C10, a continuous
increase in the activity is observed. A threshold in the increase
is observed from an acyl chain length of six carbon atoms. The
The hemolytic activity of trichogin GA IV and its C2, C8,
and C16 [Leu-OMe11] analogs was examined on human eryth-
rocytes. The natural compound (not shown) and its C8 analog
exhibit similar hemolytic activity, showing a threshold effect
for a concentration around 1.6 × 10-5 M after 3 h of incubation
(Figure 7). The C12 analog is slightly more active, whereas

4958 J. Am. Chem. Soc., Vol. 118, No. 21, 1996
Toniolo et al.
Table 4. Antibacterial Activity of Trichogin GA IV and Its [L-Leu-OMe11] Analogs against S. aureus As Determined by the Agar Diffusion
Method (Inhibition Diameters in Millimeters)
peptide
200 µg/pits
100 µg/pits
50 µg/pits
25 µg/pits
12.5 µg/pits
6.25 µg/pits
3 µg/pits
1.5 µg/pits
trichogin GA IV
C2 [Leu-OMe11]
C8 [Leu-OMe11]
17
-
17
-
16
-
15
-
15
-
14
-
13
-
9
-
8
-
-
17
-
16
-
15
-
15
-
16
-
14
-
12
-
C
16 [Leu-OMe11]
succinoyl dimer
-
-
-
-
-
-
-
through which water molecules and ions may readily pass.3,4,6
Very different patterns of helix aggregation have been observed.
For these long peptaibols the helix length is sufficient to span
the lipid bilayer. In contrast, the helix length of trichogin GA
IV is approximately half that of alamethicin.9 It is reasonable
to conclude that, if the mechanism of action for this latter class
of peptaibols involves channel formation in bilayers, the
arrangement of molecules required to bring about such disrup-
tions must be quite different from the longer sequences. The
crystal structure observed for the trichogin GA IV racemate was
revealing in light of this unique requirement. Trichogin GA
IV is able to pack with other like molecules to form a channel
in which water molecules can clearly be accommodated.
Molecular aggregation is enhanced by the amphiphilic nature
of the helical structure. Noncovalent linkage of two trichogin
molecules (“dimer” formation) at the membrane surface Via the
orthogonally-oriented fatty acyl chains covalently bound to the
R-amino groups, and subsequent insertion into the membrane,
could lead to a molecular aggregate with Gly-rich faces at its
interior to generate a hydrophilic channel of sufficient length
to span the bilayer. The results described in this paper, in
particular the requirement for a minimal aliphatic chain length
for membrane activity and the full activity exhibited by the head-
to-head dimer, give some support to this hypothesis. In this
model the succinoyl covalent linker of the trichogin GA IV
dimer may replace the two sticky fatty acyl chains.
However, it is fair to point out that the orientation of a peptide
in the liposome lipid is not necessarily the same as in a voltage-
dependent channel; i.e., a peptide chain is not necessarily parallel
to the normal to the bilayer. With liposomes, a situation where
the trichogin GA IV undecapeptide would float on the bilayer,
with the hydrophobic moieties (octanoyl group and Leu, Ile,
and Lol side chains) buried into the membrane and the Gly-
rich face exposed to the bulk water, cannot be discarded on the
basis of the results presented in this work. Alternatively, an
oblique disposition of the peptide, with the anchoring octanoyl
chain deeply buried into the membrane, may also represent a
plausible model. A study is currently underway in our
laboratories aiming at discriminating among the different
possibilities discussed above by exploiting two series of
synthetic analogs of trichogin GA IV, each designed to
incorporate at selected positions of the peptide chain either a
paramagnetic or a fluorescent amino acid residue. We are also
examining the properties of trichogin GA IV and selected
analogs under the conditions of voltage-controlled experiments,
a prerequisite to probe the nature of a voltage-dependent
channel. Due to their short chain length and low dipole moment,
one would expect a burstlike behavior with short-living single
pores, as already found for sequential Ala/Aib model peptides
of comparable length.21,22
Figure 7. Hemolytic activity of selected [L-Leu-OMe11] trichogin GA
IV analogs at 3 h (a) and 27 h (b). The number of carbon atoms in the
NR- acyl chain is indicated.
the C16 analog is found much less active and the C2 analog
completely inefficient. Incubating for 27 h allows a charac-
teristic distinction between the C16 and the C2 analogs, as the
hemolytic activity increases with increasing incubation time only
for the C16 analog. These results parallel those observed with
liposomes, suggesting that the trichogin GA IV analogs, having
a lipidic chain with more than 15 carbon atoms, take a much
longer time to reach the membrane target.
Discussion
In addition to the first total synthesis of the lipopeptaibol
trichogin GA IV, we have reported here the preparation and
characterization of 12 [L-Leu-OMe11] undecapeptide analogs and
the succinoylated head-to-head dimer. We have also demon-
strated that a right-handed helix is a characteristically common
structural element for all these peptide molecules. The NR-
blocking fatty acyl moiety has been shown to play a major role
in the membrane-modifying properties of the undecapeptide
esters. More specifically, at least six carbon atoms in the
aliphatic chain are required for a significant activity. Full
membrane activity is also exhibited by the succinoylated head-
to-head dimer.
JA954081O
(21) Menestrina, G.; Voges, K. P.; Jung, G.; Boheim, G. J. Membr. Biol.
1986, 93, 111-132.
(22) Vogel, H.; Nilsson, L.; Rigler, R.; Meder, S.; Boheim, G.; Beck,
W.; Kurth, H. H.; Jung, G. Eur. J. Biochem. 1993, 212, 305-313.
A considerable body of evidence has been accumulated to
support the view that 15-19 amino acid peptaibols are helical
and capable of forming channels in biological membranes