Lipopeptaibol metabolites of tolypocladium geodes: Total synthesis, preferred conformation, and membrane activity

Article abstract of DOI:10.1002/chem.200304756

We have synthesized by solution methods and characterized the lipopeptaibol metabolite LP237-F8 extracted from the fungus Tolypocladium geodes and five selected analogues with the Etn → Aib or Etn → Nva replacement at position 8 and/or a triple Gln → Glu(OMe) replacement at positions 5, 6, and 9 (Etn = Cα-ethylnorvaline, Aib = α-aminoisobutyric acid, Nva = norvaline). Conformation analysis, performed by FT-IR absorption, NMR, and CD techniques, strongly supports the view that the six terminally blocked decapeptides are highly helical in solution. Helix topology and amphiphilic character are responsible for their remarkable membrane activity. At position 8 the combination of high hydrophobicity and Ca tetrasubstitution, as in the Etn-containing LP237-F8 metabolite, has a positive effect on membrane interaction.

Full text of DOI:10.1002/chem.200304756

FULL PAPER
Lipopeptaibol Metabolites of Tolypocladium geodes: Total Synthesis, Pre-
ferred Conformation, and Membrane Activity
Mario Rainaldi,[a] Alessandro Moretto,[a] Cristina Peggion,[a] Fernando Formaggio,[a]
Stefano Mammi,[a] Evaristo Peggion,[a] Jose¬ Antonio Galvez,[b]
Maria Dolores DÌaz-de-Villegas,[b] Carlos Cativiela,[b] and Claudio Toniolo*[a]
Abstract: We have synthesized by sol-
ution methods and characterized the
lipopeptaibol metabolite LP237-F8 ex-
tracted from the fungus Tolypocladium
geodes and five selected analogues with
the Etn ! Aib or Etn ! Nva replace-
ment at position 8 and/or a triple Gln !
Glu(OMe) replacement at positions 5, 6,
and 9 (Etn ˆ Ca-ethylnorvaline, Aib ˆ
a-aminoisobutyric acid, Nva ˆ norva-
line). Conformation analysis, performed
by FT-IR absorption, NMR, and CD
techniques, strongly supports the view
that the six terminally blocked decapep-
tides are highly helical in solution. Helix
topology and amphiphilic character are
responsible for their remarkable mem-
brane activity. At position 8 the combi-
nation of high hydrophobicity and Ca
tetrasubstitution, as in the Etn-contain-
ing LP237-F8 metabolite, has a positive
effect on membrane interaction.
Keywords: antitumor agents ¥ con-
formation analysis
¥ peptaibols ¥
peptides ¥ total synthesis
mixtures contain a series of closely related peptides with a
limited number of conservative variations in the sequence.
In particular, in the course of a recent screening program
for fungal metabolites exhibiting antitumor activity, the highly
cytotoxic peptides LP237-F5, -F7, and -F8 were isolated and
sequenced[5, 6] (Scheme 1). The n-octanoyl (Oc) Na-blocked
and leucinol (Lol) Ca-blocked 10-mer peptide F8 was found
to be the main component of the bioactive mixture. In
addition to the frequently observed achiral Aib, another
member of the family of Ca,a-disubstituted glycines, the
chiral Ca-ethylnorvaline (Etn) residue, was shown to occur in
Introduction
Peptaibols are a unique class of membrane-active metabolites
of fungal origin.[1, 2] These antibiotic peptides are character-
ized by a linear sequence of a-amino acids, a high population
of the Ca,a-disubstituted glycine a-aminoisobutyric acid
(Aib), an N-terminal acetyl group, and a C-terminal 1,2-
amino alcohol. In the last fewyears a variety of peptides have
been sequenced that bear a fatty acyl moiety linked to the
N-terminal amino acid as a newcharacteristic feature for
peptaibols.[3, 4] These peptides are referred to lipopeptaibols
because of the lipophilic character of the N-terminal group.
They were isolated from cultures of the fungi Trichoderma
longibrachiatum (trichogin), Trichoderma koningii (trikonin-
gins), Trichoderma viride (trichodecenins), Trichoderma
polysporum (trichopolyns), Mycogone rosea (helioferins),
and Tolypocladium geodes (metabolites LP237). Typically,
most of them exhibit microheterogeneity, that is, the natural
[a] Prof. C. Toniolo, M. Rainaldi, Dr. A. Moretto, Dr. C. Peggion, Prof. F.
Formaggio, Prof. S. Mammi, Prof. E. Peggion
Institute of Biomolecular Chemistry, CNR
Department of Organic Chemistry, University of Padova
via Marzolo 1, 35131 Padova (Italy)
Fax : (‡39)49-827-5239
Scheme 1. Amino acid sequences of the lipopeptaibol metabolites LP237-
F5, -F7, and -F8. All the a-amino acids and the 1,2-amino alcohol have the S
configuration. At the N-terminal acyl moiety and at positions 3 and 8,
variations in the sequence are indicated in bold. The chemical formulae of
Nva and the two Ca-tetrasubstituted a-amino acids are also shown. Aib ˆ
a-aminoisobutyric acid, Dec ˆ n-decanoyl, Etn ˆ Ca-ethylnorvaline, Lol ˆ
leucinol, Nva ˆ norvaline, Oc ˆ n-octanoyl.
E-mail: claudio.toniolo@unipd.it
[b] Dr. J. A. Galvez, Dr. M. D. DÌaz-de-Villegas, Prof. C. Cativiela
Departamento de Quimica Organica y Quimica Fisica
Instituto de Ciencia de Materiales de Aragon
Universidad de Zaragoza, CSIC
50009 Zaragoza (Spain)
Chem. Eur. J. 2003, 9, 3567 3576
DOI: 10.1002/chem.200304756
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3567

C. Toniolo et al.
FULL PAPER
the F5 and F8 primary structures. To our knowledge, this latter
a-amino acid has not previously been reported as a compo-
nent of a terrestrial product. However, it has been found in
extraterrestrial sediments such as the Murchison meteorite.[7]
Unfortunately, in none of these works has the absolute
configuration of the Etn residue been assessed. Etn has been
synthesized (in the racemic form) and shown to behave as a
competitive inhibitor of methionine.[8] The F5, F7, and F8
metabolites differ by the N-terminal acyl moiety (Oc or
decanoyl (Dec)) and the a-amino acids in positions 3 (Tyr or
Phe) and 8 (Etn or Aib).
decided to incorporate Etn as the S enantiomer because of its
greater availability; the asymmetric synthesis of this enan-
tiomer is also reported here. In addition, we have examined
the effects induced by hydrophobicity (higher in Etn and
norvaline (Nva) than in Aib) and Ca substitution[9] (higher in
Aib and Etn than in Nva) at position 8 (analogues a' and b' in
Scheme 2). Finally, the spectroscopic and biophysical proper-
ties of peptides a' c' have been compared with those of their
synthetic precursors (peptides a c in Scheme 2), which are
characterized by three Glu(OMe) residues with an ester side-
chain functionality in place of the three Gln residues with a
primary amide side-chain functionality at positions 5, 6, and 9.
Herein we describe the total synthesis, conformation
analysis (by FT-IR absorption, NMR, and CD techniques)
in solution, and membrane permeability measurements of the
lipopeptaibol metabolite LP237-F8 (c' in Scheme 2). We
Results and Discussion
Synthesis and characterization: Preparation of (S)-Etn was
performed by extension of our general chemical methodology
developed for the synthesis of Ca-tetrasubstituted a-amino
acids.[10] Diastereoselective alkylation of chiral (1'S,2'R,4'R)-
10'-(dicyclohexylsulfamoyl)isobornyl 2-cyanobutanoate (1)
with allyl bromide according to our previously described
methodology[11] cleanly affords the corresponding allylated
compound (2; Scheme 3) in 92% yield as a 90:10 mixture in
which the diastereoisomer with S configuration at the C2
position predominates; this diastereoisomer is derived from
the formation of a chelated Z enolate and attack by the
electrophile from the Ca-Re side, opposite to the 10'-
(dicyclohexylsulfamoyl) group. The diastereomeric ratio of
the product was determined in the crude reaction mixture by
integration of the 13C NMR absorptions (75 MHz) of the
vinylic carbon atoms at about d ˆ 130.7 ppm. The major
diastereoisomer can be easily isolated from the mixture by
crystallization from MeOH and further elaborated to afford
(S)-Etn (5) according to Scheme 3. Hydrogenation of the
alkene moiety of 2 under 1 atm of hydrogen with palladium on
charcoal yielded the corresponding saturated compound 3.
Basic hydrolysis of the ester group with potassium hydroxide
in MeOH followed by synthesis of the a-cyanoacylazide and
subsequent Curtius rearrangement gave the corresponding
urethane 4. Finally, acidic hydrolysis afforded (S)-Etn (5) as
the hydrochloride salt in enantiomerically pure form and with
a 68% yield from the S cyanoester. Ion-exchange chromatog-
raphy released the free amino acid.
Scheme 2. Amino acid sequences of the peptides investigated in this work.
Peptide c' corresponds to the lipopeptaibol LP237-F8. All a-amino acids
and the 1,2-amino alcohol have the S configuration.
Abstract in Italian: Abbiamo sintetizzato in soluzione e
caratterizzato il metabolita lipopeptaibolico LP237-F8 estratto
dal fungo Tolypocladium geodes e cinque suoi analoghi recanti
la sostituzione Etn ! Aib o Etn ! Nva in posizione 8 e la tripla
sostituzione Gln ! Glu(OMe) nelle posizioni 5, 6 e 9. La
nostra indagine conformazionale, condotta con le tecniche
dell'assorbimento IR, NMR e CD, indica chiaramente che i sei
decapeptidi bloccati alle funzioni N- e C- terminali assumono
un'alta percentuale di forma elicoidale in soluzione. La natura
elicoidale e il carattere anfifilico sembrano essere i fattori
¡
responsabili della loro elevata attivita sulle membrane. In
¡
posizione 8 la combinazione di una elevata idrofobicita e della
tetrasostituzione all'atomo Ca ha un effetto positivo sull'inte-
razione con le membrane.
Since we expected potentially difficult peptide-coupling
steps, particularly those required for the incorporation of the
Ca-tetrasubstituted a-amino acids Etn and Aib,[12] the total
chemical syntheses of the metabolite LP237-F8 (peptide c')
and its Aib (peptide a') and Nva (peptide b') analogues were
carried out either by the EDC/HOBt method[13] or the EDC/
HOAt method[14] (Scheme 4). Originally, we decided to take
advantage of the racemization-free, step-by-step synthetic
strategy. However, we were forced to limit such a strategy to
the protected hexapeptide level because of the relatively
small amount of (S)-Etn available. In any case, by using this
approach we were able to exploit a series of short sequences
which proved to be useful to investigate the role of main-chain
lengthening on peptide conformation (see below). The methyl
ester group of the Z-Ala-Leu-OMe dipeptide was reduced to
¬
Abstract in Spanish: Utilizando metodos sinteticos en disolu-
¬
cion se ha preparado y caracterizado el metabolito lipopeptai-
bol LP237-F8 extraÌdo del hongo Tolypocladium geodes, asÌ
como cinco analogos en los que el residuo Etn en posicion 8 ha
¬
¬
sido sustituido por Aib o Nva y una triple Gln ! Glu(OMe)
¬
sustitucion ha sido introducida en las posiciones 5, 6 y 9. El
¬
analisis conformacional realizado mediante FT-IR, NMR y
CD pone claramente de manifesto que los seis decapeptidos
muestran una alta helicidad en solucion. La topologÌa helicoi-
dal y el caracter amfifÌlico son responsables de su importante
actividad de membrana. Como ocurre en el metabolito F8 que
contiene Etn, la combinacion de la alta hidrofobicidad y la
tetrasustitucion en Ca del residuo en posicion 8 tiene un efecto
¬
¬
¬
¬
¬
¬
¬
positivo en la interaccion con la membrana.
3568
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2003, 9, 3567 3576

Peptaibol Metabolites
3567 3576
based analogues of peptides
a' c', namely peptides a c.
However, this strategy forced
us to reduce the C-terminal
ester function at the dipeptide
stage immediately before incor-
poration of the first Glu(OMe)
residue. To avoid the possible
competition between the N-ter-
minal amino group of the grow-
ing peptide chain and the C-ter-
minal Lol alcohol function dur-
ing the various peptide bond
formation steps, we took ad-
vantage of the higher nucleo-
philicity of the amino function.
To this end we slowly added the
solution containing a slight ex-
cess of the acylating reagent to
the solution of the a-amino
peptide alcohol. The strategy
for the syntheses of the protect-
ed tetrapeptide fragment and
the decapeptides a c was chos-
en in such a way as to minimize
racemization. In particular:
1) the C-terminal Pro residue
of the dipeptide carboxylic acid,
being N-alkylated, cannot form
the 5(4H)-oxazolone inter-
mediate and 2) the C-terminal
Aib residue of the tetrapeptide
carboxylic acid is achiral. Con-
version of the Glu(OMe) de-
capeptides a c into the corre-
sponding Gln peptides a' c'
was obtained by triple ester
Scheme 3. Asymmetric synthesis of (S)-Etn (5). Cy ˆ cyclohexyl.
aminolysis
in
anhydrous
MeOH.[16] The progress of this
latter reaction was monitored
by reverse-phase HPLC. The
reaction was slow and took
several days for completion.
The overall yields for the syn-
thesis of decapeptides a', b', and
c' were 13, 18, and 10%, re-
spectively.
The purities of the newly
synthesized intermediates and
final synthetic products were
Scheme 4. Strategy for the synthesis of the lipopeptaibol LP237-F8 (c') and its five analogues (a c, a', and b').
Boc ˆ tert-butyloxycarbonyl, Bzl ˆ benzyl, EDC ˆ N-ethyl-N'-[3-(dimethylamino)propyl]carbodiimide, HOAt ˆ
7-aza-1-hydroxy-1,2,3-benzotriazole, HOBt ˆ 1-hydroxy-1,2,3-benzotriazole, TFA ˆ trifluoroacetic acid, Z ˆ
benzyloxycarbonyl.
assessed by TLC in three differ-
ent solvent systems, solid-state
primary alcohol (Z-Ala-Lol) by using LiBH4, which does not
interfere with the peptide and urethane functions.[15] Removal
of the Boc and Z Na-protecting groups was achieved by mild
acidolysis (diluted TFA) and catalytic hydrogenolysis, respec-
tively. During the course of the synthesis, the difficult-to-
handle Gln residues were replaced by Glu(OMe) residues.
This approach also allowed us to synthesize three Glu(OMe)-
IR absorption spectroscopy, ESI-TOF mass spectrometry (the
latter technique was only used for peptides a c and a' c'),
and H NMR spectroscopy (Table 1).
1
Conformation analysis in solution: A detailed investigation of
the preferred conformation in solution of the lipopeptaibol
metabolite LP237-F8 (peptide c') and its analogues (a', b', and
Chem. Eur. J. 2003, 9, 3567 3576
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3569

C. Toniolo et al.
FULL PAPER
Table 1. Physical properties and analytical data for the newly synthesized peptides.
‡
‡
[f]
Compound[a]
Yield M.p. [8C][b] Purifi-
[%]
TLC[d]
[M ‡ H]
/ ]
IR nÄ [cmÀ1
calcd
[e]
cation[c]
Rf(I) Rf(II) Rf(III) [M ‡ H]
exp
Z-Etn-OH
Z-Ala-Lol
Boc-Glu(OMe)-Ala-Lol
Z-Aib-Glu(OMe)-Ala-Lol
Z-Etn-Glu(OMe)-Ala-Lol
Z-Nva-Glu(OMe)-Ala-Lol
Z-Aib-Aib-Glu(OMe)-Ala-Lol
Z-Aib-Etn-Glu(OMe)-Ala-Lol
Z-Aib-Nva-Glu(OMe)-Ala-Lol
Boc-Glu(OMe)-Aib-Aib-Glu(OMe)-Ala-Lol
Boc-Glu(OMe)-Aib-Etn-Glu(OMe)-Ala-Lol
Boc-Glu(OMe)-Aib-Nva-Glu(OMe)-Ala-Lol
Boc-[Glu(OMe)]2-Aib-Aib-Glu(OMe)-Ala-Lol 68
Boc-[Glu(OMe)]2-Aib-Etn-Glu(OMe)-Ala-Lol 69
Boc-[Glu(OMe)]2-Aib-Nva-Glu(OMe)-Ala-Lol 76
79
89
87
79
69
88
76
76
81
74
69
72
oil
90 91
EtOAc/PE 0.40 0.90
EtOAc/PE 0.65 0.90
0.25
0.40
0.30
0.25
0.20
0.35
0.15
0.15
0.25
0.10
0.10
0.15
0.15
0.10
0.20
0.45
0.55
0.35
0.30
3472, 3410, 1704, 1678, 1522
3317, 1711, 1690, 1653, 1531
3308, 1745, 1711, 1682, 1666, 1522
3305, 1751, 1721, 1654, 1540
3309, 1745, 1724, 1658, 1533
3313, 1738, 1666, 1537
3290, 1747, 1704, 1678, 1637, 1543
3310, 1734, 1714, 1653, 1644, 1541
3307, 1751, 1708, 1662, 1639, 1514
3311, 1731, 1664, 1657, 1538
3317, 1740, 1664, 1660, 1532
3315, 1739, 1660, 1542
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
0.60 0.90
0.60 0.85
0.55 0.85
0.65 0.90
0.60 0.90
0.60 0.90
0.65 0.90
0.65 0.85
0.60 0.85
0.70 0.90
0.65 0.85
0.60 0.85
0.65 0.90
0.90 0.95
3314, 1745, 1659, 1541
3308, 1751, 1712, 1664, 1521
3312, 1745, 1708, 1657, 1531
3308, 1736, 1664, 1525
3311, 1744, 1655, 1537
3308, 1746, 1712, 1661, 1649, 1524
3309, 1743, 1655, 1529
Oc-Aib-Pro-OBzl
Boc-Phe-Aib-OBzl
78
87
75
66
oil
91 92
oil
f.c.
EtOAc/PE 0.90 0.95
Oc-Aib-Pro-Phe-Aib-OBzl
Oc-Aib-Pro-Phe-Aib-[Glu(OMe)]2-Aib-Aib-
Glu(OMe)-Ala-Lol
Oc-Aib-Pro-Phe-Aib-[Glu(OMe)]2-Aib-Etn-
Glu(OMe)-Ala-Lol
Oc-Aib-Pro-Phe-Aib-[Glu(OMe)]2-Aib-Nva-
Glu(OMe)-Ala-Lol
Oc-Aib-Pro-Phe-Aib-(Gln)2-Aib-Aib-
Gln-Ala-Lol
Oc-Aib-Pro-Phe-Aib-(Gln)2-Aib-Etn-
Gln-Ala-Lol
Oc-Aib-Pro-Phe-Aib-(Gln)2-Aib-Nva-
Gln-Ala-Lol
f.c.
0.70 0.90
0.70 0.90
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
waxy solid f.c.
1328.8463/
1328.8494
1370.8955/
1370.8986
1342.8718/
1342.8749
642.9157/
642.9172[g]
1325.9617/
1325.9648
1297.8457/
1297.8488
58
65
98
97
98
0.70 0.90
0.65 0.85
0.45 0.80
0.50 0.85
0.40 0.85
0.20
0.25
0.10
0.15
0.10
3313, 1740, 1662, 1537
3307, 1741, 1649, 1519
3311, 1669, 1661, 1537
3315, 1661, 1657, 1527
3322, 1656, 1647, 1525
[a] All a-amino acids and the 1,2-amino alcohol have the S configuration. [b] Determined on a Leitz model Laborlux 12 apparatus (Wetzlar, Germany).
[c] Purification was either by recrystallization from the given solvent(s) (EtOAc ˆ ethyl acetate, PE ˆ petroleum ether) or by flash chromatography (f.c.;
silica gel 60, 40 63 mesh, Merck, Darmstadt, Germany). [d] TLC on silica gel 60F plates (Merck) in the following solvent systems: I) chloroform/ethanol
(9:1), II) butan-1-ol/water/acetic acid (3:1:1), III) toluene/ethanol (7:1); the plates were developed with a UV lamp or with the hypochlorite/starch/iodide
chromatic reaction, as appropriate; a single spot was observed in each case. [e] Determined on a Perseptive Biosystems model Mariner ESI-TOF mass
spectrometer (Foster City, CA). [f] Determined in KBr pellets on a Perkin-Elmer 580B spectrophotometer (Norwalk, CT) equipped with a Perkin
Elmer 3600 IR data station. [g] [(M ‡ 2H)/2]calcd/[(M ‡ 2H)/2]exp
.
a
c) was carried out by using FT-IR absorption, two-dimen-
sional NMR, and CD techniques in solvents of different
polarity.
The conformational preferences of the C-terminal, Na-
protected peptides (up to the hexamer) were investigated in a
structure-supporting solvent (CDCl3) by FT-IR absorption
spectroscopy over the peptide concentration range 10
0.1 mm. Representative spectra (for short sequences of
À
peptide a) in the most informative N H stretching (amide A)
region (peptide concentration: 1 mm) are illustrated in Fig-
ure 1. The curves are characterized by two bands, at about
3425 cmÀ1 (free, solvated NH groups) and 3355 3310 cmÀ1
(strongly hydrogen-bonded NH groups of folded conforma-
tions).[17 19] The intensity of the low-frequency band relative to
that of the high-frequency band increases significantly as
main-chain length increases. We have also been able to
demonstrate that, even at a concentration of 10 mm, self-
Figure 1. FT-IR absorption spectra in the 3500 3200 cmÀ1 region for the
C-terminal short sequences Z-Ala-Lol (6), Boc-Glu(OMe)-Ala-Lol (7),
Z-Aib-Glu(OMe)-Ala-Lol (8), Z-(Aib)2-Glu(OMe)-Ala-Lol (9), Boc-
Glu(OMe)-(Aib)2-Glu(OMe)-Ala-Lol (10), and Boc-[Glu(OMe)]2-
(Aib)2-Glu(OMe)-Ala-Lol (11) of peptide a in CDCl3 solution. Peptide
concentration: 1 mm. Spectrum 11' is the spectrum of the hexapeptide at
10 mm peptide concentration.
À
ˆ
association through intermolecular N H ¥¥¥ O C hydrogen
bonding is negligible (less than 5%) for all oligomers.
(Figure 1 shows the close similarity between the spectra of
the longest peptide in this series at the two concentrations.)
Therefore, the observed hydrogen bonding should be inter-
preted as arising almost exclusively from intramolecular
À
ˆ
N H ¥¥¥ O C interactions. As the low-frequency band is
clearly seen in the Na-protected dipeptide amino alcohol
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Chem. Eur. J. 2003, 9, 3567 3576

Peptaibol Metabolites
3567 3576
(7) but not in the shorter Z-Ala-Lol (6), it is reasonable to
exclude the possibility that the preferred folded conformation
would be of the g-bend type.[20]
band is still very intense in all of these decapeptides, which
supports the viewthat they are characterized by an extensive
ˆ
À
set of intramolecular C O ¥¥¥ H N hydrogen bonds. From our
FT-IR absorption analysis it may be concluded that in CDCl3
solution all the lipopeptaibols examined tend to fold in an
intramolecularly hydrogen-bonded structure. However, this
technique alone did not allowus to unambiguously determine
the nature of the helix that is formed.
The FT-IR absorption analysis provided evidence that
25]
intramolecular hydrogen bonding typical of a[21, 22] or b[23
-
turn conformations is the predominant feature of the C-ter-
minal sequences of these lipopeptaibols in CDCl3 solution.
The FT-IR absorption spectra of the lipopeptaibols a c and
a' c' in CDCl3 solutions (peptide concentrations: 1 and
0.1 mm) are shown in Figure 2. They are dominated by a very
strong band at about 3325 cmÀ1. Additional bands (shoulders)
are barely visible at wavenumbers >3400 cmÀ1. A modest
decrease in the relative intensity of the 3325 cmÀ1 band is
observed between the spectra recorded at 1 and 0.1 mm
concentrations; this is indicative of the contribution of a
minor amount of intermolecular hydrogen bonds. In any case,
at the lower concentration examined (0.1 mm) the 3325 cmÀ1
The NMR spectroscopic investigation of the conformation
of lipopeptaibol a' was carried out in an aqueous sodium
dodecylsulfate (SDS) solution. The combined analysis of
TOCSY and NOESY spectra led to the complete assignment
of all proton resonances. In spite of the presence of a Pro
residue at position 2, no evidence of cis/trans isomerism about
the Aib1 Pro2 bond was found in the NMR spectra. The
complete proton assignment is reported in Table 2. The
assignment of the methyl groups of the Aib residues is based
Figure 2. FT-IR absorption spectra in the 3500 3200 cmÀ1 region for peptides a c and a' c' in CDCl3 solution at peptide concentrations of 1 mm (I) and
0.1 mm (II).
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C. Toniolo et al.
FULL PAPER
Table 2. Proton chemical shift values (ppm) at 313 K relative to tetramethylsilane
and temperature dependencies of amide proton chemical shift values (ppbKÀ1) for
peptide a'.
Residue aH
bH
gH
dH
eH
H aromatic HN Dd/DT
8.48 5.6 Æ 0.2
Aib1
1.60, 1.61
Pro2
Phe3
Aib4
Gln5
Gln6
Aib7
Aib8
Gln9
Ala10
Lol
4.47 2.32, 1.43 2.00, 1.88 4.04, 3.47
4.66 3.35, 3.19
7.28, 7.41 8.13 3.8 Æ 0.1
7.96 1.9 Æ 0.1
1.52, 1.69
2.19
4.11
4.16 2.18, 2.25
1.66
2.58, 2.51
2.51
7.47, 6.83
7.42, 6.78
8.02 6.7 Æ 0.3
7.88 4.1 Æ 0.1
8.01 4.5 Æ 0.1
1.57, 1.64
2.28
1.62
7.86 5.6 Æ 0.1
4.14
4.41
4.16 1.49, 1.38
3.69, 3.60
2.70, 2.60
1.81
7.37, 6.76
7.73 4.0 Æ 0.1
7.95 2.4 Æ 0.1
0.98
7.08 3.0 Æ 0.1
on the analysis of the NOESY spectrum alone, on the
assumption that the helix formed (see below) would be right-
handed. The summary of the interresidue NOESY connectiv-
ities is presented in Figure 3. The presence of strong NH(i)
NH(i ‡ 1) peaks almost throughout the sequence, accompa-
nied by weak aH(i) NH(i ‡ 1) peaks, is an indication that the
Figure 4. Section of the NOESY spectrum of peptide a'. The medium-
range daN(i, i ‡ 4) and daN(i, i ‡ 2) cross-peaks are indicated.
dependence of the amide protons is also reported in Table 2.
The protons of residues Phe3, Aib4, Gln9, Ala10, and Lol show
a
small temperature dependence (Dd/DTꢀ 4.0 ppbKÀ1),
which is usually interpreted as indicating the presence of
intramolecular hydrogen bonds.
Extensive molecular dynamics calculations resulted in a
family of 32 accepted structures with violations to the NOE
restraints lower than 0.4 ä. A superimposition of these
structures is shown in Figure 5. The average values of the
Figure 3. Interresidue NOEs found for peptide a'. The thickness of the bar
reflects the integrated intensity of the peak, as the scale at the bottom
shows.
peptide might adopt a helical conformation. Of the four
possible aH(i) NH(i ‡ 3) peaks, only one was detected
(Figure 4). In a right-handed helical conformation, the pro-
R methyl protons b' of the Aib residues are in close proximity
with the NH protons of residues one turn away.[26] Again, of
the four possible b'H(i) NH(i ‡ 3) peaks, only one was
detected (outside the section shown in Figure 4). These
findings support the presence of a helical conformation only
in part and indicate that such an arrangement is probably in
equilibrium with other conformers. Two aH(i) NH(i ‡ 4)
connectivities suggest that the a-helix conformation is indeed
populated. Two b'H(i) NH(i ‡ 2) peaks were also found. The
first one is around Pro2 and could arise from a b-turn
conformation. The other one, toward the C terminus, could be
due to a transient 310-helical arrangement. The temperature
Figure 5. Overlay of the 32 minimized structures of peptide a'. Amino acid
numbering is indicated. Sinde chains are omitted.
3572
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3567 3576

Peptaibol Metabolites
3567 3576
the Aib4 NH proton, which exhibits the lowest temperature
dependence in the entire sequence, is not found to form
intramolecular hydrogen bonds in the dynamics calculations.
A possible explanation for this result could be that this
particular proton is in part shielded from the solvent for
different reasons, for example by the Phe ring or by the n-
octanoyl N-terminal chain.
The far-UV CD spectra of the six lipopeptaibols a c and
a' c' in MeOH solution (Figure 6) are very similar, with a
negative maximum at 208 nm (peptide p ! p* exciton split
Table 3. Average values of f,y backbone torsion angles, their relative
standard deviations, and order parameters resulting from the 32 calculated
structures of peptide a'.
Residue
Angle
Average value and
standard deviation
Order
parameter
Aib1
Aib1
Pro2
Pro2
Phe3
Phe3
Aib4
Aib4
Gln5
Gln5
Gln6
Gln6
Aib7
Aib7
Aib8
Aib8
Gln9
Gln9
Ala10
Ala10
Lol
f
y
f
y
f
y
f
y
f
y
f
y
f
y
f
y
f
y
f
y
f
À 80.2 Æ 5.4
À 53.2 Æ 3.5
À 84.1 Æ 3.8
13.5 Æ 1.4
0.996
0.998
0.998
1
0.989
1
0.999
0.995
0.99
0.978
0.993
0.998
0.997
0.994
0.88
À 91.7 Æ 8.6
À 9.6 Æ 1.4
À 83.1 Æ 2
À 48.3 Æ 6
À 86.3 Æ 8.3
À 12.6 Æ 2.1
À 77.1 Æ 6.7
À 26.4 Æ 4
À 75.7 Æ 4.4
À 42 Æ 6.4
À 89 Æ 30.9
À 38.5 Æ 7.8
À 77.2 Æ 15.2
À 15.5 Æ 11.5
À 83.2 Æ 19.2
À 8.5 Æ 12.5
108.4 Æ 59.4
0.991
0.967
0.981
0.946
0.977
0.655
f,y angles recovered from the dynamics are reported in
Table 3 together with their standard deviations. These devia-
tions are very small, as is also indicated by the values of the
order parameter, which are very close to one. This is evidence
for a strong tendency toward the average structure, which is a
slightly distorted a helix from residue Aib4 to Ala10. The
distortion is mainly due to the unusual values of the f angles,
which vary from À768 to À898. The difference from the
canonical value of À638 is nevertheless not high. As expected,
the C terminus experiences a higher degree of conformational
averaging, as indicated by the lower order parameters already
observed at the level of Aib8. At the N terminus, the distortion
is greater, very likely because of the presence of the Pro
residue at position 2. The angles of residues Aib1 and Pro2 are
compatible with the presence of a type-I b turn.
Table 4 summarizes the intramolecular hydrogen bonds
found in the 32 accepted structures. The amide protons
significantly involved in such bonds are those of Phe3, Gln9,
Ala10, and Lol. The agreement with the results of the variable
temperature study is quite reasonable. Indeed, the temper-
ature coefficient of these NH protons is ꢀ4.0 ppbKÀ1. Only
Figure 6. Far-UV CD spectra of the lipopeptaibol LP237-F8 (c') and its
five analogues (a c, a', and b') in MeOH solution.
component) and a pronounced negative shoulder in the
vicinity of 222 nm (peptide n ! p* transition).[27] The con-
formationally sensitive [q]2T22/[q]T208 ratio R is approximately
0.7, a fact indicating that the right-handed helix adopted in this
30]
solvent is predominantly of the a type.[28
In 1,1,1,3,3,3-
hexafluoroisopropanol solution (spectra not shown) the R
value (ꢁ0.95) suggests a further increase in the a-helix
population. By applying the conventional criteria for the
determination of the percentage of helicity of polypeptides
containing protein amino acids from ellipticity values,[31] the
helical content of these decapeptides can be calculated to be
in the range of 25 35%. We believe that these values
underestimate the actual amounts of helicity of our peptides
because: 1) three (or four) out of ten amino acids in the
sequences are achiral (Aib) residues, and 2) the main-chain
length of the sequences (10 amino acids) is rather limited,
thereby increasing the relative impact of terminal fraying (end
effects).[28, 32, 33]
ˆ
À
Table 4. Intramolecular C O ¥¥¥ H N hydrogen bonds found in the
32 calculated structures of peptide a'.[a]
NH donor
O acceptor
Occurrence
Membrane permeability properties: Trichogin GA IV, the
Phe3
Aib7
Aib8
Gln9
Ala10
Ala10
Lol
Oc
12
2
1
8
10
1
prototypical lipopeptaibol, is known to exhibit a considerable
Phe3
Gln5
Gln5
Gln6
Aib7
Aib8
37]
membrane-perturbing activity.[3, 4, 34
It is even more de-
structive to small unilamellar vesicles of phosphatidyl choline
and cholesterol than several longer nonlipidated peptaibols
are. However, the details of the mechanism by which
trichogin GA IV is able to release the aqueous content of
small unilamellar liposomes are not clear yet.[3, 4, 36, 37] The
membrane-modifying properties of the six 10-mer lipo-
6
[a] The maximum hydrogen acceptor distance was set to 2.4 ä. The
donor hydrogen acceptor angle was 1458.
Chem. Eur. J. 2003, 9, 3567 3576
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3573

C. Toniolo et al.
FULL PAPER
peptaibols a c and a' c', tested by use of a liposome leakage
assay, are compared in Figure 7 with those of the 10-mer
trichogin GA IV (Oc-Aib-Gly-Leu-Aib-(Gly)2-Leu-Aib-Gly-
Ile-Lol) and its nonlipidated analogue (with Ac-Aib0 and
Furthermore, carefully tailored analogues of lipopeptaibols
such as metabolite LP237-F8 are excellent tool components
for biophysical studies of peptide membrane interac-
tions.[3, 4, 36]
Experimental Section
General for the synthesis and characterization of (S)-Etn: Melting points
were determined on a B¸chi SMP-20 capillary apparatus and are not
corrected. IR absorption spectra were registered on a Perkin-Elmer 1600
FT-IR spectrophotometer. Optical rotations were measured in a cell with
10-cm pathlength on a Jasco P-1020 polarimeter. 1H NMR (300 MHz) and
13C NMR (75 MHz) spectra were recorded either with a Varian Unity-300
or Bruker ARX-300 spectrometer. Elemental analyses were obtained by
using a Perkin-Elmer 2400 analyser.
(2S)-(1'S,2'R,4'R)-10'-(Dicyclohexylsulfamoyl)isobornyl-2-cyano-2-ethyl-
4-pentenoate (2): Anhydrous potassium carbonate (3.45 g, 25 mmol) was
added to a well-stirred solution of (2RS)-(1'S,2'R,4'R)-10'-(dicyclohexyl-
sulfamoyl)isobornyl-2-cyanobutanoate (1)[39] (2.46 g, 5 mmol) and allyl
bromide (1.21 g, 10 mmol) in dry acetone (60 mL). The resulting mixture
was stirred at room temperature for 24 h and filtered, and the solid residue
was washed with diethyl ether. The combined filtrates were concentrated in
vacuo and the residue was dissolved in diethyl ether, washed with water,
dried over anhydrous MgSO4, filtered, and concentrated in vacuo to afford
the corresponding (1'S,2'R,4'R)-10'-(dicyclohexylsulfamoyl)isobornyl-2-cy-
ano-2-ethyl-4-pentenoate as a 90:10 mixture of diastereoisomers. Recrys-
tallization from MeOH afforded the diastereomerically pure 2S compound
in 62% yield. M.p. 1248C; 1H NMR (300 MHz, CDCl3, 258C): d ˆ 0.85 (s,
3H; CH3), 1.03 (s, 3H; CH3), 1.12 (t, 3J(H,H) ˆ 7.5 Hz, 3H; CH3), 1.10 2.20
Figure 7. Peptide-induced carboxyfluorescein (CF) leakage after 20 min
for different ratios RiÀ1 ˆ [peptide]/[lipid] (Â103) from egg phosphatidyl
choline/cholesterol (70:30) vesicles for the lipopeptaibol LP237-F8 (c') and
its five analogues (a c, a', and b'). A comparison is also made with the
membrane activities of the prototypical lipopeptaibol trichogin GA IV (T)
and the negative control analogue of trichogin GA IV (with Ac-Aib0 and
Leu-OMe11; NC).[38]
À
(m, 29H; ethyl, isobornyl, (Cy)2), 2.50 2.58 (m, 2H; CH2 allyl), 2.57 (d,
2J(H,H) ˆ 13.2 Hz, 1H; CHSO2), 3.22 3.36 (m, 2H; isobornyl), 3.38 (d,
2J(H,H) ˆ 13.2 Hz, 1H; CHSO2), 4.94 (dd, 3J(H,H) ˆ 7.8, 3.0 Hz, 1H;
Leu(OMe)11); the latter was taken as a negative control.[38]
Notably, all of the compounds examined (except the negative
control) are able to modify the artificial membrane. The
enhanced capability of peptides c and c' for lysis should be
associated with the increased hydrophobicity and helix
stabilization imparted by the Etn8 residue. The
Glu(OMe) ! Gln triple replacement seems beneficial, at
least for peptides a' and b'. The naturally-occurring peptide
LP237-F8 (peptide c') is more active in the membranes than
trichogin GA IV even though they have the same main-chain
length. Thus, the observed difference might be related to the
higher amphiphilic properties characterizing the 3D-structure
of the former lipopeptaibol.
À
CH O), 5.15 5.26 (m, 2H; allyl); 5.74 5.90 (m, 1H; allyl) ppm;
13C NMR (75 MHz, CDCl3): d ˆ 9.6, 19.9, 20.3, 22.9, 25.2, 26.2, 26.4, 27.0,
29.9, 30.7, 32.1, 33.5, 39.5, 41.4, 44.4, 49.3, 49.6, 49.7, 53.5, 57.3, 80.7, 119.0,
À1
ꢂ
ˆ
120.8, 130.8, 167.4 ppm; IR (Nujol): nÄ ˆ 2244 (C N), 1737 (C O) cm
;
elemental analysis calcd (%) for C30H48N2O4S: C 67.63, H 9.08, N 5.26, S
6.02; found: C 67.59, H 9.06, N 5.31, S 5.96.
(2S)-(1'S,2'R,4'R)-10'-(Dicyclohexylsulfamoyl)isobornyl-2-cyano-2-ethyl-
pentanoate (3): 1H NMR (300 MHz, CDCl3, 258C): d ˆ 0.86 (s, 3H; CH3),
3
0.93 (t, J(H,H) ˆ 7.2 Hz, 3H; CH3), 1.05 (s, 3H; CH3), 1.12 (t, 3J(H,H) ˆ
7.5 Hz, 3H; CH3), 1.16 2.20 (m, 31H; ethyl, isobornyl, (Cy)2), 2.58 (d,
2J(H,H) ˆ 13.3 Hz, 1H; CHSO2), 3.20 3.36 (m, 2H; isobornyl), 3.40 (d,
2J(H,H) ˆ 13.3 Hz, 1H; CHSO2), 4.95 (dd, 3J(H,H) ˆ 7.8, 2.9 Hz, 1H;
CH O) ppm; 13C NMR (75 MHz, CDCl3): d ˆ 9.7, 13.9, 18.8, 20.0, 20.4,
À
25.2, 26.2, 26.4, 27.0, 30.4, 30.7, 32.0, 33.5, 39.3, 39.6, 44.4, 49.4, 49.6, 50.1,
À1
ꢂ
ˆ
53.5, 57.4, 80.5, 119.6, 168.2; IR (Nujol): nÄ ˆ 2240 (C N), 1737 (C O) cm
;
elemental analysis calcd (%) for C30H50N2O4S: C 67.38, H 9.42, N 5.24, S
6.00; found: C 67.45, H 9.29, N 5.36, S 6.07.
(S)-2-Methoxycarbonylamino-2-ethylpentanonitrile (4): Oil; 1H NMR
Conclusion
3
(300 MHz, CDCl3, 258C): d ˆ 0.95 (t, J(H,H) ˆ 7.2 Hz, 3H; CH3), 1.02 (t,
3J(H,H) ˆ 7.2 Hz, 3 H ; CH3), 1.40 1.53 (m, 2H; CH2), 1.79 1.88 (m, 2H;
CH2), 1.90 2.01 (m, 2H; CH2), 3.70 (s, 3H; OCH3), 4.81 (brs, 1H;
NH) ppm; 13C NMR (CDCl3, 75 MHz): d ˆ 9.7, 13.9, 16.3, 27.1, 33.7, 52.5,
In this work, by using the (S)-Etn residue obtained by
asymmetric chemical synthesis, we prepared by solution
methods the terminally blocked, 10-mer lipopeptaibol metab-
olite LP237-F8. Two analogues in which the Etn residue is
replaced either by an Aib or a Nva residue have also been
prepared. In solution, the three decapeptides are highly
folded in helical structures. By combining high hydrophobic-
ity and helix-stabilizing Ca-tetrasubstitution[9] at position 8, as
in the naturally occurring, Etn-containing F8 metabolite,
increased amphiphilicity and an associated, significantly
higher capability for membrane lysis are obtained.
ꢂ
57.4, 119.4, 155.2 ppm; IR (Nujol): nÄ ˆ 3330 (NH), 2238 (C N), 1715
‡
(C O) cmÀ1; HR-MS (EI): calcd for C9H16N2O2: 184.1212; m/z: 184.1215 [M] .
ˆ
(S)-2-Amino-2-ethylpentanoic acid (5): M.p. >3008C (decomp); [a]D22
ˆ
‡6.9 (c ˆ 1 in H2O); 1H NMR (300 MHz, D2O, 25 8C): d ˆ 0.79 (t,
3J(H,H) ˆ 7.2 Hz, 3 H ; CH3), 0.79 (t, 3J(H,H) ˆ 7.5 Hz, 3H; CH3), 1.08
1.10 (m, 1H; CH2), 1.20 1.32 (m, 1H; CH2), 1.50 1.82 (m, 4H;
2CH2) ppm; 13C NMR (75 MHz, D2O): d ˆ 5.91, 12.0, 15.3, 27.9, 36.8,
À1
ˆ
64.6, 174.9 ppm; IR (neat): nÄ ˆ 3500 2000 (NH), 1712 (C O) cm
;
elemental analysis calcd (%) for C7H15NO2: C 57.90, H 10.41, N 9.65;
found: C 57.83, H 10.48, N 9.57.
Synthesis and characterization of amino acid derivatives and peptides: The
physical properties and analytical data for the newly synthesized amino
acid derivatives and peptides are listed in Table 1. The syntheses and
It is likely that additional lipopeptaibol mixtures with
potent cytotoxic activity will be discovered in the near future.
3574
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3567 3576

Peptaibol Metabolites
3567 3576
43]
characterization of Z-Aib-OH,[40 Oc-Aib-OH,[35] Boc-Aib-OBzl,[42] and
Acknowledgements
Z-Ala-Leu-OMe[44] have already been reported.
The authors gratefully acknowledge financial support for this research from
the Spain Italy exchange program ™Accion Integrada∫ HI 97 20/™Azioni
Integrate∫ 1997 1999.
FT-IR absorption spectroscopy: FT-IR absorption spectra were recorded
with
a Perkin-Elmer 1720X spectrophotometer, nitrogen flushed and
equipped with a sample-shuttle device, at a nominal resolution of 2 cmÀ1
,
averaging 100 scans. Solvent (baseline) spectra were recorded under the
same conditions. Cells with path lengths of 0.1, 1.0, and 10 mm (with CaF2
windows) were used. Spectrograde CDCl3 (99.8% D) was purchased from
Fluka.
[1] E. Benedetti, A. Bavoso, B. Di Blasio, V. Pavone, C. Pedone, C.
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NMR studies: NMR experiments were carried out on a Bruker Avan-
ce DMX-600 spectrometer. The peptide concentration was 3.5 mm in 0.3 m
SDS-d25 and the sample temperature was 313 K. The water signal was
suppressed by use of the WATERGATE pulse sequence. In all two
dimensional experiments the spectra were acquired by collecting 500 512
runs, each one consisting of 64 scans and 4000 data points. The spin systems
of protein amino acid residues were identified by using standard DQF-
COSY and TOCSY experiments. In the latter case the spin-lock pulse
sequence was 70 ms long. The stereospecific assignment of the Aib methyl
group (except for Aib8) was obtained by means of semisoft NOESY
experiments with digital resolution of 0.8 Hz per point in F1. A G4-shaped
pulse, centered in the bH region, was used to replace the first 908 hard pulse.
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2419 2425.
Normal NOESY experiments were used for specific sequence assignment.
The mixing time of the NOESY experiments used for interproton distance
determinations was 120 ms. Interproton distances were obtained by
integration of the NOESY spectrum with the AURELIA software
package.[45] Distances were calibrated on the peak between the two Phe
bH protons, set to a distance of 1.78 ä. When peaks could not be integrated
because of partial overlap, a distance corresponding to the maximum limit
of detection of the experiment (4 ä) was assigned to the corresponding
proton pair.
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Structure calculations: Distance geometry (DG), molecular dynamics
(MD), and simulated annealing (SA) protocol calculations were carried out
by using the X-PLOR 3.1 program.[46] For distances involving equivalent or
nonstereo assigned protons, rÀ6 averaging was used. The MD calculations
involved a minimization stage of 100 cycles, followed by SA and refinement
stages. The SA consisted of 30 ps of dynamics at 1500 K (10000 cycles in
3 fs steps) and of 30 ps of cooling from 1500 to 100 K in 50 K decrements
(15000 cycles in 2 fs steps). The SA procedure, in which the weights of the
NOE and nonbonded terms were gradually increased, was followed by 200
cycles of energy minimization. In the SA refinement stage the system was
cooled from 1000 to 100 K in 50 K decrements (20000 cycles in 1 fs steps).
Finally, the calculations were completed with 200 cycles of energy
minimization with an NOE force constant of 50 kcalmolÀ1. The generated
structures were visualized by using the MOLMOL 2.6 program[47] with a
Silicon Graphics O2R 10000 workstation.
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mm pathlength (Hellma) were used. The values are expressed in terms of
[q]T, the total molar ellipticity (deg  cm2  dmolÀ1). Spectrograde MeOH
(Baker) and 1,1,1,3,3,3-hexafluoroisopropanol (Acros Organics) were used
as solvents. The peptide concentrations were determined by weight and the
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1
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the absence of peptide, Ft ˆ fluorescence intensity at time t in the presence
of peptide, and FT ˆ total fluorescence intensity determined by disrupting
the vesicles by addition of 30 mL of a 10% Triton X-100 solution. The
kinetics studies were stopped at 20 min.
Chem. Eur. J. 2003, 9, 3567 3576
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Received: January 22, 2003
Revised: April 16, 2003 [F4756]
3576
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3567 3576