Peptaibolin: Synthesis, 3D-structure, and membrane modifying properties of the natural antibiotic and selected analogues

Article abstract of DOI:10.1016/S0040-4020(01)00124-7

We synthesized by solution methods and fully characterized the naturally occurring, tetrapeptide antibiotic peptaibolin and selected analogues with replacements at the N- and C-terminal groups and the C^α-tetrasubstituted α-amino acids. The preferred conformation of all of the peptides was assessed in solution by using FT-IR absorption and ¹H NMR techniques. Results of the X-ray diffraction analyses of peptaibolin itself and three analogues are also presented. Permeability measurements of such multiple turn forming, very short peptides indicate that peptaibolin is devoid of membrane activity because a lipoyl N-terminal blocking group is an essential requisite.

Full text of DOI:10.1016/S0040-4020(01)00124-7

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 2813±2825
Peptaibolin: synthesis, 3D-structure, and membrane modifying
properties of the natural antibiotic and selected analogues
Marco Crisma,a Alessandra Barazza,a Fernando Formaggio,a Bernard Kaptein,b
Quirinus B. Broxterman,b Johan Kamphuisc and Claudio Tonioloa,p
aDepartment of Organic Chemistry, University of Padova, Biopolymer Research Centre, C.N.R., Via Marzolo 1, 35131 Padova, Italy
bDSM Research, Organic Chemistry and Biotechnology Section, P.O. Box 18, 6160 MD Geleen, The Netherlands
cDSM Food Specialties, Nutritional Ingredients, P.O. Box 1, 2600 MA Delft, The Netherlands
Received 13 November 2000; revised 20 December 2000; accepted 18 January 2001
AbstractÐWe synthesized by solution methods and fully characterized the naturally occurring, tetrapeptide antibiotic peptaibolin and
selected analogues with replacements at the N- and C-terminal groups and the Ca-tetrasubstituted a-amino acids. The preferred conformation
of all of the peptides was assessed in solution by using FT-IR absorption and 1H NMR techniques. Results of the X-ray diffraction analyses of
peptaibolin itself and three analogues are also presented. Permeability measurements of such multiple turn forming, very short peptides
indicate that peptaibolin is devoid of membrane activity because a lipoyl N-terminal blocking group is an essential requisite. q 2001 Elsevier
Science Ltd. All rights reserved.
1. Introduction
alamethicin) to as low as six amino acids (in the lipopeptai-
bol trichodecenin). Peptaibols are known to induce leakage
of the cytoplasmic material and eventually to lead to cell
death. It has been demonstrated that the long-sequence
peptaibols form voltage-dependent channels,2 whereas the
short-sequence (lipo)peptaibols tend to ¯oat on the lipid
bilayer (carpet-like mechanism).8
Peptaibols1 are a unique class of membrane active peptides
biosynthesized by fungi.2 These antibiotics are character-
ized by a high proportion of Aib (a-aminoisobutyric acid
or C a,a-dimethylglycine), a strong a/310-helix3 supporting
Ca-tetrasubstituted a-amino acid,4±7 and a C-terminal 1,2-
amino alcohol. Some peptaibols typically contain Iva
(isovaline or C a-methyl, C a-ethylglycine), another repre-
sentative of the Aib family. An additional common feature
is represented by an Na-terminal acyl group (more speci®c-
ally, an acetyl group in the longest members of the class,
while a fatty acyl group of eight or ten carbon atoms in the
shortest members). Sequences of these peptides range from
19 amino acids (e.g. in the extensively investigated
In the course of a screening for fungal peptides HuÈlsmann et
al.9 have recently reported on the isolation and primary
structure of peptaibolin, an unusual representative of
the peptaibol class in that it contains only four amino
acids in combination with the small acetyl blocking
group at the N-terminus. The sequence of peptaibolin
is as follows:
where Ac is acetyl and Phol is the 1,2-amino alcohol
phenylalaninol. This compound exhibits antimicrobial
activity, albeit moderate, against Gram-positive bacteria
and yeasts.
Keywords: conformation; membranes; peptides; X-ray crystallography.
Corresponding author. Tel.: 139-049-827-5247; fax: 139-049-827-
5239; e-mail: biop02@chor.unipd.it
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)00124-7

2814
M. Crisma et al. / Tetrahedron 57 (2001) 2813±2825
Figure 1. (I) FT-IR absorption spectra (3500±3200 cm21 region) in CDCl3 solution (peptide concentration: 1.0 mM) of Z-Aib-l-Phe-OMe (2), Z-l-Leu-Aib-l-
Phe-OMe (3), Z-Aib-l-Leu-Aib-l-Phe-OMe (4), and Z-(l-Leu-Aib)2-l-Phe-OMe (5). (II) FT-IR absorption spectra (3500±3200 cm21 region) in CDCl3
solution of Ac-(l-Leu-Aib)2-l-Phol (peptaibolin) at the peptide concentrations 10 mM (A), 1.0 mM (B), and 0.1 mM (C).
In an attempt to shed light on the relationship between
3D-structure and mechanism of action of peptaibolin we
have carried out the total synthesis of this terminally
blocked tetrapeptide, assessed its preferred conformation
in a structure-supporting solvent (CDCl3) by FT-IR absorp-
of the benzyloxycarbonyl (Z) Na-protecting group was
achieved by catalytic hydrogenation.
The chemical and optical purities of all intermediates and
®nal synthetic compounds were assessed by TLC in three
different solvent systems, polarimetry, solid-state IR
absorption, analytical HPLC, 1H NMR, high-resolution
mass spectrometry and, in selected cases, by amino acid
analysis as well.
1
tion and H NMR techniques and in the crystal state by
X-ray diffraction, and analyzed its tendency to disrupt
membrane organization. The results obtained are compared
with those (also reported in this paper) of carefully selected
analogues which include modi®cations at the N-terminus
(from the short native acetyl group to the lipophilic n-octa-
noyl group), the C-terminus (from the native 1,2-amino
alcohol to the a-amino acid methyl ester), and the two
internal Ca-tetrasubstituted a-amino acids (from the native
Aib to the slightly more hydrophobic Iva residues).
2.2. Solution conformational analysis
The conformational preferences of Ac-(l-Leu-Aib)2-l-Phe-
OMe (OMe, methoxy) and Ac-(l-Leu-l-Iva)2-l-Phe-OMe,
and their synthetic precursors Z-(l-Leu-Aib)2-l-Phe-OMe
and Z-(l-Leu-l-Iva)2-l-Phe-OMe and short sequences,
along with those of n-Oct-(l-Leu-Aib)2-l-Phe-OMe
(n-Oct, n-octanoyl), n-Oct-(l-Leu-l-Iva)2-l-Phe-OMe,
Ac-(l-Leu-Aib)2-l-Phol (peptaibolin), n-Oct-(l-Leu-Aib)2-
l-Phol, Ac-(l-Leu-l-Iva)2-l-Phol, and n-Oct-(l-Leu-l-
Iva)2-l-Phol were examined in a solvent of low polarity
(CDCl3) at different peptide concentrations by using
FT-IR absorption. A more detailed solution conformational
analysis of two representative peptides was performed by 1H
NMR.
2. Results and discussion
2.1. Peptide synthesis
For the production of the enantiomerically pure l-Iva we
exploited an economically attractive and generally applic-
able chemo-enzymatic synthesis developed by the DSM
Research group a few years ago.10 It involves a combination
of organic syntheses for preparation of the racemic amino
acid amide followed by the use of a broadly speci®c amino-
peptidase to achieve optical resolution on a large scale.
Fig. 1(I) shows the FT-IR absorption spectra in the N±H
stretching region of the Z-protected Aib/Phe-OMe series
from di- to pentapeptide. The curves are characterized by
two bands at 3428^6 cm21, assigned to free (solvated) NH
groups, and at 3364^14 cm21, assigned to H-bonded NH
groups.13±15 The intensity of the low-frequency band, rela-
tive to the high-frequency band, increases as the peptide
main-chain length increases. Concomitantly, the absorption
maximum markedly shifts to lower wavenumbers. A
remarkably similar trend was observed for the Z-protected
l-Iva/Phe-OMe series (results not shown). All of the
members of these two peptide series tend to self-associate
only marginally in the concentration range examined (10±
0.1 mM) (not shown). Therefore, the observed band at
Peptide coupling reactions were performed step-by-step in
solution beginning from the C-terminal amino acid ester by
using the EDC [N-ethyl,N0-(3-dimethylaminopropyl)-
carbodiimide]/HOAt (1-hydroxy,7-aza-1,2,3-benzotriazole)
method11 in CH2Cl2 in the presence of N-methylmorpholine
(NMM). Na-Acetylation and Na-n-octanoylation were
achieved by treatment of the Na-deprotected peptide with
acetic anhydride and n-octanoic acid/EDC/HOAt, respec-
tively. In the last step of the synthesis the methyl group
was reduced to primary alcohol by using LiBH4.12 Removal

M. Crisma et al. / Tetrahedron 57 (2001) 2813±2825
2815
technique alone did not allow us to unambiguously deter-
mine the nature of the folded structures that were formed.
To get more detailed, local information on the preferred
conformations of the Phe-OMe and Phol C-terminating
peptides in CDCl3 solution we performed a 400 MHz H
1
NMR investigation of two representative examples, n-Oct-
(l-Leu-Aib)2-l-Phe-OMe and Ac-(l-Leu-Aib)2-l-Phol
(peptaibolin). The delineation of inaccessible (or intra-
molecularly H-bonded) NH groups was carried out by
using solvent dependences of NH chemical shifts by adding
increasing amounts of the H-bonding acceptor DMSO
(dimethylsulphoxide) to the CDCl3 solutions.18,19 Unam-
biguous assignments of the NH protons were performed
via analysis of their multiplicities and 2D ROESY experi-
ments. In particular, sequential assignments were conducted
beginning from the N-terminal amide NH proton, which was
identi®ed by means of its NOE effects with either the
CH3CO± protons of the Ac group or the ±CH2CO± protons
of the n-Oct group.
Figure 2. Plot of NH chemical shifts as a function of DMSO added to the
CDCl3 solution (v/v) from the 1H NMR titrations of n-Oct-(l-Leu-Aib)2-l-
Phe-OMe (A) and Ac-(l-Leu-Aib)2-l-Phol (peptaibolin) (B). Peptide
concentration: 0.7 mM.
3364^14 cm21, quite intense for the longest peptides,
should be ascribed almost entirely to intramolecular
CvO´´´H±N interactions. Replacement of the Z urethane
Na-protecting group with the n-Oct and, in particular, the Ac
acyl groups signi®cantly promoted peptide self-association,
but only above 1.0 mM concentration (not shown). Remark-
ably, the Ac-blocked Iva/Phe-OMe peptide is sparingly
soluble in CDCl3 solution at 10 mM concentration.
From an analysis of the spectra of the Phe-OMe peptide as a
function of concentration (in the range 10±1.0 mM) (not
shown) we were able to conclude that dilution induces a
signi®cant shift (.0.2 ppm) to higher ®elds of the N(1)H
and N(2)H protons only. More speci®cally, the N(1)H
proton is more sensitive (0.55 ppm) than the N(2)H proton
(0.30 ppm). We also found that peptaibolin is much more
strongly aggregated than the related Phe-OMe peptide.
Indeed, upon dilution the signals of its NH protons were
signi®cantly broadened to a concentration as low as
0.7 mM, where only the N(1)H and N(2)H were still some-
what broad (but both clearly visible).
As for the FT-IR spectra of the Phol C-terminating peptides
(Fig. 1(II)), an additional broad band was seen at 3460±
3450 cm21, which we assigned to the H-bonded alcoholic
OH group.16 Further, the curves below 3350 cm21 showed
that all of the C-terminal Phol peptides tend to self-associate
more extensively than their Phe-OMe ester counterparts,
again particularly above 1.0 mM concentration. However,
besides the contribution of H-bonded NH groups, it is not
safe to exclude the absorption of the H-bonded OH group
also in that spectral region.16
For each peptide in the CDCl3±DMSO solvent mixtures at
0.7 mM concentration, where self-association is of minor
signi®cance, two classes of NH protons were observed
(Fig. 2). Class (i) (N(1)H and N(2)H protons) included
protons whose chemical shifts are sensitive to the addition
of DMSO. For both compounds the sensitivity of the N(1)H
proton was markedly higher than that of the N(2)H proton.
Class (ii) (N(3)H to N(5)H protons) included those dis-
playing a behaviour characteristic of shielded protons (rela-
tive insensitivity of chemical shifts to solvent composition).
In the 1800±1600 cm21 region ester, urethane, amide and
peptide CvO groups are responsible for the observed bands
(results not shown).15,16 Clearly, the band at about
1745 cm21 (ester CvO stretching) was absent in the spectra
of the C-terminal Phol peptides. The urethane CvO stretch-
ing band (1727±1712 cm21) was seen only in the Z-
protected peptides. The amide and peptide CvO stretching
bands (amide I) were observed between 1690 and
1657 cm21. Additionally, the C-terminal Phol peptides
exhibited a shoulder at approximately 1635 cm21 (but
only at the highest concentration examined). The intensity
of the amide I band increased with increasing peptide main-
chain length and its maximum progressively shifted to lower
wavenumbers. In the longest peptides the position of this
maximum (1663±1657 cm21) was close to the canonical
wavelengths for the 310-helical (1662 cm21) and a-helical
(1658 cm21) structures.17
These 1D-1H NMR ®ndings, which agree well with the FT-
IR absorption data discussed above, allowed us to conclude
that in CDCl3 solution self-association of peptaibolin and its
n-Oct/Phe-OMe analogue is promoted by the N(1)H and
N(2)H protons, while the N(3)H to N(5)H protons are
involved in the intramolecular H-bonding scheme.
However, on the basis of these results alone it was pre-
mature to distinguish between a regular 310-helix3 (with
three consecutive b-turns20±22) and a mixed helix formed
by the co-existence of b- and a-turns.21,23±25 This confor-
mational problem was solved for the n-Oct/Phe-OMe
peptide by a thorough analysis of its ROESY spectrum.
Fig. 3 clearly illustrates that, in addition to cross-peaks
corresponding to the daN(i,i11) and daN(i,i13) short
distances, a cross-peak corresponding to the daN(i,i12)
short distance, diagnostic of the 310-helix,26 was observed.
Conversely, the typical cross-peak for an a-turn (a-helix),
daN(i,i14),26 was not seen.
From our FT-IR absorption analysis it may be concluded
that in the structure-supporting solvent CDCl3 in the
absence of self-association the longest peptides examined
tend to adopt highly folded structures extensively stabilized
by intramolecular CvO´´´H±N±H bonds. However, this

2816
M. Crisma et al. / Tetrahedron 57 (2001) 2813±2825
Figure 3. Partial ROESY spectrum of n-Oct-(l-Leu-Aib)2-l-Phe-OMe in CDCl3 solution.
Figure 4. X-Ray diffraction structures of n-Oct-(l-Leu-Aib)2-l-Phe-OMe (top) and Ac-(l-Leu-l-Iva)2-l-Phe-OMe (bottom) with numbering of the atoms.
The intramolecular H-bonds are represented by dashed lines.

M. Crisma et al. / Tetrahedron 57 (2001) 2813±2825
2817
Figure 5. X-Ray diffraction structures of Ac-(l-Leu-Aib)2-l-Phol (peptaibolin) (top) and Ac-(l-Leu-l-Iva)2-l-Phol (bottom) with numbering of the atoms.
The intramolecular H-bonds are represented by dashed lines.
2.3. Crystal-state conformational analysis
Aib)2-l-Phe-OMe, Ac-(l-Leu-l-Iva)2-l-Phe-OMe and Ac-
(l-Leu-l-Iva)2-l-Phol were determined by X-ray diffrac-
tion. The molecular structures of the four peptides with
the atomic numbering schemes are illustrated in Figs. 4
The molecular and crystal structures of peptaibolin, Ac-(l-
Leu-Aib)2-l-Phol, and the three analogues n-Oct-(l-Leu-

2818
M. Crisma et al. / Tetrahedron 57 (2001) 2813±2825
Table 1. Selected backbone and side-chain torsion angles (the torsion angles for rotation about bonds of the peptide backbone (f,c,v) and side chains (x) are
described in Ref. 27) (deg) for the four peptides investigated
Torsion
angle
n-Oct-(l-Leu-Aib)2-l-Phe-OMe
Ac-(l-Leu-l-Iva)2-l-Phe-OMe
Ac-(l-Leu-Aib)2-l-Phol
(peptaibolin)
Ac-(l-Leu-l-Iva)2-l-Phol
v0
f1
c1
v1
f2
c2
v2
f3
c3
v3
f4
c4
v4
f5
c5
v5
x11
2172.8 (8)
255.2 (10)
238.7 (10)
2176.1 (6)
255.7 (9)
237.1 (9)
2175.6 (6)
272.7 (9)
223.8 (9)
2175.4 (5)
257.2 (8)
233.0 (9)
172.7 (7)
273.2 (8)
262.1 (7)b
176.7 (6)d
270.8 (8)
274.2 (11)
163.6 (8)
±
2178.2 (9)
253.6 (12)
241.8 (11)
2176.4 (7)
252.4 (10)
236.6 (10)
2174.2 (7)
266.6 (10)
224.9 (12)
2179.8 (8)
260.8 (11)
233.2 (11)
179.7 (8)
2172.2 (8)
254.1 (12)
247.5 (11)
2177.9 (8)
251.2 (11)
245.7 (10)
2174.8 (8)
274.8 (10)
241.0 (11)
2177.4 (9)
268.1 (11)
225.9 (11)
2166.8 (8)
2161.3 (8)a
56.7 (10)c
±
2178.5 (9)
59.9 (12)
2176.3 (11)
±
2176.7 (9)
60.0 (14)
2175.7 (9)
±
176.9 (7)
253.4 (9)
238.2 (9)
2178.4 (6)
253.1 (8)
242.1 (8)
2178.9 (6)
282.1 (7)
240.2 (8)
177.0 (5)
250.1 (7)
240.4 (7)
175.7 (6)
275.2 (11)
256.4 (10)b
175.4 (9)d
294.2 (8)a
60.6 (8)c
±
284.8 (14)e/2158.5 (13)e
46(2)f/172.5 (12)f
262.3 (19)f/141.8(8)f
66.8 (12)e/274 (2)e
263.7 (11)
260.4 (14)
172.6 (10)
280.2 (11)e/2147.6 (13)e
246.6 (16)f/178.8 (14)f
52 (3)f/2174.3 (10)f
46.3 (13)e/266(2)e
261.2 (8)
x21;1
x21;2
x21
x31
276.5 (10)
263.7 (11)
174.6 (8)
±
178.6 (6)
82.6 (6)
292.7 (6)
x23;1
x23;2
x41
255.3 (10)
2176.0 (8)
2179.8 (6)
256.7 (8)
225.9 (8)
158.0 (5)
179.3 (8)
179.6 (8)
75.0 (9)
2101.9 (9)
x51
2175.6 (9)
59.3 (11)
2120.9 (8)
x25;1
x25;2
a
C-N-CA-C(sp3).
N-CA-C-OT.
b
c
d
e
f
N-CA-C(sp3)-O.
CA-C-OT-CT.
The CG atom is disordered over two sites.
The CD atom is disordered over two sites.
and 5. Selected backbone and side-chain torsion angles27 are
given in Table 1. In Table 2, the intra- and intermolecular H-
bond parameters are listed.
C00vO0 is the common acceptor of the two H-bonds,
with the N3±H and N4±H as the donor groups. This
structural motif develops into an additional a-turn
(C01vO1´´´H±N5), giving rise to an incipient a-helix, an
uncommon observation for such short peptides.42,43 Near the
C-terminus both compounds are folded in a unusual oxy-
analogue of a turn motif,21 but peptaibolin is characterized
by the oxy-analogue of a wider a-turn,44 while Ac-(l-Leu-
l-Iva)2-l-Phol by the oxy-analogue of a narrower b-
turn.45 The related intramolecular C02vO2´´´H±O5 and
C03vO3´´´H±O5 H-bonds have (carbonyl)O´´´O(alcohol)
Bond lengths and bond angles are in general agreement with
previously reported values for the geometry of the n-octa-
noyl moiety,28 the amide,29 peptide30,31 and ester32 groups,
the Aib/Iva33±35 and Leu/Phe36,37 residues, and the Phol
unit.28
The backbone of the two Na-blocked pentapeptide esters is
very similar (Fig. 4) in that both adopt a regular right-
handed 310-helical structure.3 Peptide groups N3±H to
N5±H and O0vC00 to O2vC02 participate in three con-
secutive 1Ã4 (type-III b-turn20±22) C0vO´´´H±N intra-
molecular H-bonds, appropriate for a 310-helix. The range
46,47
Ê
distances of 2.745 (9) and 2.875 (7) A, respectively.
In the four peptides investigated only one signi®cant devia-
tion of the v torsion angles (uDvu.88) from the ideal value
of the trans planar amide, peptide, and ester units (1808) is
observed, namely the peptide v4 torsion angle of peptai-
bolin, 2166.8 (8)8. In the two pentapeptides the methyl
ester conformation with respect to the preceding Ca±N
bond is intermediate between the anticlinal and antiperi-
planar conformations.48
Ê
of observed N´´´O distances is 2.935 (8)±3.290 (8) A, while
that of N±H´´´O angles is 135.8±148.68.38±40 In both
structures the C-terminal l-Phe residue is also right-handed
helical.
With the exception of the C-terminal part, the backbone of
the two Na-blocked tetrapeptide amino alcohols is very
similar (Fig. 5), but somewhat different from that of the
two Na-blocked pentapeptide esters discussed above. At
the N-terminus the conformation of the peptide chain is
characterized by an intramolecular three-centre double
H-bond,41 forming a type-III b-turn (C10-ring structure)
fused with an a-turn (C13-ring structure).21,23±25 The acyl
The Iva side-chain conformations of both Iva-containing
peptides con®rm the lack of a clear bias,35 in that they are
g1 (g2) in the disordered residue 2, while t in residue 4. The
absence of the side-chain g1 rotamer (x1 torsion angle) in
the Leu, Phe and Phol residues is not unexpected in view of
the signi®cant steric hindrance experienced (destabilization
due to repulsive interaction of the Cg atom with the N and C0

M. Crisma et al. / Tetrahedron 57 (2001) 2813±2825
Table 2. Intra- and intermolecular H-bond parameters for the four peptides investigated
2819
Ê
Ê
Distance (A)
H´´´A
Peptide
Donor
D±H
Acceptor
A
Symmetry
operation
Distance (A)
D´´´A
Angle (deg)
D±H´´´A
n-Oct-(l-Leu-Aib)2-l-Phe-OMe
N3±H
N4±H
N5±H
N1±H
N2±H
O0
O1
O2
O4
O5
x, y, z
x, y, z
x, y, z
x, y21, z
x, y21, z
2.935 (8)
3.082 (7)
3.290 (8)
2.879 (8)
2.959 (7)
2.23
2.41
2.61
2.02
2.11
138.6
135.8
136.4
176.2
169.9
Ac-(l-Leu-l-Iva)2-l-Phe-OMe
N3±H
N4±H
N5±H
N1±H
N2±H
O0
O1
O2
O4
O5
x, y, z
x, y, z
x, y, z
x11, y, z
x11, y, z
2.947 (10)
3.003 (9)
3.216 (10)
2.883 (10)
2.989 (9)
2.19
2.24
2.48
2.04
2.13
146.8
148.6
143.4
167.7
174.6
Ac-(l-Leu-Aib)2-l-Phol (peptaibolin)
N3±H
N4±H
N5±H
O5±H
N1±H
O0
O0
O1
O2
O5
x, y, z
x, y, z
x, y, z
x, y, z
x21, y,
z21
3.053 (10)
3.062 (9)
2.963 (9)
2.745 (9)
2.955 (9)
2.46
2.22
2.33
1.92
2.16
126.4
167.3
130.7
178.4
154.1
N2±H
O4
x, y, z21
2.878 (9)
2.05
161.0
Ac-(l-Leu-l-Iva)2-l-Phol
N3±H
N4±H
N5±H
O5±H
N1±H
N2±H
O0
O0
O1
O3
O4
O5
x, y, z
x, y, z
x, y, z
x, y, z
x21, y, z
x21, y, z
2.975 (7)
2.995 (7)
2.989 (7)
2.875 (7)
2.880 (8)
3.339 (8)
2.33
2.14
2.15
2.06
2.04
2.49
131.8
174.3
164.6
171.7
167.4
168.5
atoms).36,37 The two known overwhelming combinations of
side-chain conformers for the Leu residue, g2 (tg2) and
t(g1 t),36 are those exclusively observed in these four
peptides. The usual de®nite preference of the Phe residues
for the x2 angle ù ^90836 is observed. The difference of x2
from ^908 is wider for the Phol residues, in particular when
x1 is g2 (in the Iva/Phol peptide).
gated by following ¯uorimetrically the induced carboxy-
¯uorescein (CF) leakage from small unilamellar vesicles
(egg phosphatidycholine (PC)/cholesterol 70:30) for differ-
ent R21ˆ[peptide]/[lipid] molar ratios.49,50
In Fig. 6, the membrane modifying properties of peptaibolin
and its derivatives with modi®cations at either the N- or
C-terminus are compared to some of their Iva-containing
analogues. It is evident that only the Na-n-octanoylated
tetrapeptides are signi®cantly active. Neither C-terminal
modi®cation nor Aib!Iva double replacement induced
any marked variation of membrane activity in the Na-acetyl-
ated peptides.
The molecules of the two 310-helical pentapeptide esters are
held together in the crystal in a head-to-tail fashion along
the y-direction (for the Aib compound) or the x-direction
(for the Iva compound) in rows stabilized by (amide) N1±
H´´´O4vC04 (peptide) and (peptide) N2±H´´´O5vC05
(ester) intermolecular H-bonds of normal strength.38±40
3. Conclusions
The same intermolecular H-bonding motif as above
characterizes the crystal packing mode of the Iva/Phol
peptide, with rows along the z-direction. The only remark-
able variation is given by the nature of the acceptor of the
H-bond with the (peptide) N2±H donor, namely the unusual
O5±C5 (alcohol) group replacing the O5vC05 (ester)
group.
In the present work, we have described the ®rst total chemi-
cal synthesis of the natural antibiotic peptaibolin, the
shortest peptaibol, and some analogues with Na-terminal
Ac!n-Oct, C-terminal Phe-OMe!Phol, and internal
Aib!Iva replacements, using the step-by-step solution
approach. The results of the solution and crystal-state
conformational analyses, also reported in this paper,
strongly favour the conclusion that all of these peptides
are highly folded. These 3D-structural conclusions are in
excellent agreement with those already published by our
and other groups on Aib-4±7,33 and Iva-rich35,51 peptides.
In the crystal packing of the Aib/Phol peptide (peptaibolin)
the donor and acceptor groups of the two intermolecular
H-bonds are the same as those of the Iva/Phol analogue,
but they are cross-exchanged: (amide) N1±H´´´O5±C5
(alcohol) and (peptide) N2±H´´´O4vC04 (peptide)
H-bonds, generating rows of molecules along the x,z-direc-
tion and the z-direction, respectively.
Finally, our permeability measurements clearly indicate that
peptaibolin is membrane inactive. However, we were able to
show that a signi®cantly bene®cial effect can be induced by
replacing the small Na-blocking group with a fatty acid
characterized by a markedly longer (n-octanoyl) alkyl
chain. Conversely, the effects produced by the C-terminal
ester!alcohol modi®cation or by slightly increasing amino
2.4. Membrane permeability measurements
Peptaibols bind to phospholipid bilayers and are able to
modify their permeability. Therefore, the permeability
properties of peptaibolin and its analogues were investi-

2820
M. Crisma et al. / Tetrahedron 57 (2001) 2813±2825
nmax (KBr) 3302, 1740, 1672, 1526 cm21; dH (250 MHz,
CDCl3) 7.33±7.10 (10H, Z and Phe aromatic CH), 6.80
(d, 1H, Jˆ6.9 Hz, Phe NH), 6.53 (s, 1H, Aib NH), 5.07
(3H, Leu NH and Z CH2), 4.80 (m, 1H, Phe aCH), 4.06
(m, 1H, Leu aCH), 3.69 (s, 3H, OMe CH3), 3.11 (m, 2H,
Phe bCH2), 1.63 (m, 3H, Leu bCH2 and gCH), 1.47 and
1.45 (2s, 6H, Aib bCH3), 0.92 (m, 6H, Leu dCH3); HRMS
(FAB): M1, found 512.2762. C28H38N3O6 requires
512.2761.
4.1.2. Z-Aib-l-Leu-Aib-l-Phe-OMe. To a solution of
Z-Aib-OH54±56 (2.3 g, 10 mmol) in CH2Cl2 cooled to 08C,
HOAt (1.2 g, 9 mmol) and EDC´HCl (1.8 g, 10 mmol) were
added. After 10 min H-l-Leu-Aib-l-Phe-OMe (obtained by
catalytic hydrogenation of the above-mentioned Z-deriva-
tive (4.4 g, 9 mmol) in MeOH) and NMM (2.0 mL,
17 mmol) were added and the reaction was stirred at room
temperature for 24 h. Then, EtOAc was added and the solu-
tion was extracted with 10% KHSO4, H2O, 5% NaHCO3,
H2O, and dried over anhydrous Na2SO4. The title compound
was puri®ed by ¯ash chromatography (ICN silica 32±63,
60A; eluant: EtOAc/PE (2:1)) and obtained as a white solid
(3.70 g, 69%), mp 62±638C; RfI 0.90, RfII 0.95, RfIII 0.40;
Figure 6. Peptide-induced CF leakage at 20 min for different ratios R-1ˆ
[peptide]/[lipid] from egg PC/cholesterol (70:30) unilamellar vesicles:
(A) Ac-(l-Leu-Aib)2-l-Phol (peptaibolin); (B) Ac-(l-Leu-Aib)2-l-Phe-
OMe; (C) n-Oct-(l-Leu-Aib)2-l-Phol; (D) n-Oct-(l-Leu-Aib)2-l-Phe-
OMe; (E) Ac-(l-Leu-l-Iva)2-l-Phe-OMe; (F) n-Oct-(l-Leu-l-Iva)2-l-
Phe-OMe.
acid side-chain hydrophobicity (Aib!Iva) were found to be
of minor signi®cance. These results agree well with those
published in previous studies on longer peptaibols.50 In
conclusion, the moderate antimicrobial activity reported
for peptaibolin9 does not seem to be ascribable to a mecha-
nism involving membrane modi®cation. It remains to be
seen whether this activity might be related to the tendency
of peptaibolin for self-association, unusually high for a
peptide of such limited size.
20
[a]D ˆ223.6 (c 0.5, MeOH); HPLC: Rt 17.55 min
(Phenomenex-Rp C18; gradient: 30±80% B in 20 min;
eluants: A, 0.05% TFA in H2O, and B, 0.05% TFA in
CH3CN/H2O (9:1)); nmax (KBr) 3337, 1735, 1703, 1662,
1524 cm21; dH (250 MHz, CDCl3) 7.35 (m, 5H, Z aromatic
CH), 7.25±7.13 (6H, Phe aromatic CH and Phe NH), 7.02
(s, 1H, Aib3 NH), 6.39 (d, 1H, Jˆ7.0 Hz, Leu NH), 5.22 (s,
1H, Aib1 NH), 5.08 (s, 2H, Z CH2), 4.80 (m, 1H, Phe aCH),
4.21 (m, 1H, Leu aCH), 3.67 (s, 3H, OMe CH3), 3.12 (m,
2H, Phe bCH2), 1.77±1.57 (3H, Leu bCH2 and gCH), 1.51,
1.48, 1.47, 1.46 (4s, 12H, 2Aib bCH3), 0.94±0.82 (m, 6H,
Leu dCH3); HRMS (FAB): M1, found 597.3269.
C32H45N4O7 requires 597.3288.
4. Experimental
4.1. Peptide characterization
Melting points were determined using a Leitz model Labor-
lux 12 apparatus and are not corrected. Optical rotations
were measured using a Perkin±Elmer model 241 polari-
meter equipped with a Haake model D thermostat. Thin-
layer chromatography was performed on Merck Kieselgel
60/F254 precoated plates. The solvent systems used were: I,
chloroform/ethanol (9:1); II, 1-butanol/acetic acid/water
(3:1:1); III, toluene/ethanol (7:1). The chromatograms
were developed by quenching of UV ¯uorescence, chlor-
ine/starch/potassium iodide or ninhydrin chromatic reac-
tion, as appropriate. Amino acid analyses were performed
on a C. Erba model 3A 30 analyzer. The Aib and Iva colour
yields with ninhydrin are about 1/15 of those of protein
amino acids.52
4.1.3. Z-l-Leu-Aib-l-Leu-Aib-l-Phe-OMe. To a solution
of Z-l-Leu-OH (1.4 g, 5.4 mmol) in CH2Cl2 cooled to 08C,
HOAt (0.7 g, 4.9 mmol) and EDC´HCl (1.0 g, 5.4 mmol)
were added. After 10 min H-Aib-l-Leu-Aib-l-Phe-OMe
(obtained by catalytic hydrogenation of the above-
mentioned Z-derivative (3.0 g, 4.9 mmol) in MeOH) and
NMM (1.1 mL, 9.9 mmol) were added and the reaction
was stirred at room temperature for 24 h. Then, the solvent
was evaporated and the residue dissolved in EtOAc. The
solution was extracted with 10% KHSO4, H2O, 5%
NaHCO3, H2O, and dried over anhydrous Na2SO4. The
title compound was puri®ed by ¯ash chromatography
(ICN silica 32±63, 60A; eluant: CHCl3/EtOH (98:2)) and
crystallized as a white solid from EtOAc/PE (2.54 g, 73%),
4.1.1. Z-l-Leu-Aib-l-Phe-OMe. To a solution of Z-l-Leu-
OH (3.7 g, 14 mmol) in CH2Cl2 cooled to 08C, HOAt (1.7 g,
13 mmol) and EDC´HCl (2.7 g, 14 mmol) were added. After
10 min H-Aib-l-Phe-OMe (obtained by catalytic hydro-
genation of the corresponding Z-derivative53 (5.1 g,
13 mmol) in MeOH) and NMM (2.9 mL, 26 mmol) were
added and the reaction was stirred at room temperature for
12 h. Then, EtOAc was added and the solution was
extracted with 10% KHSO4, H2O, 5% NaHCO3, H2O, and
dried over anhydrous Na2SO4. The title compound was puri-
®ed by ¯ash chromatography (ICN silica 32±63, 60A;
eluant: CHCl3/EtOH (98:2)) and crystallized as a white
solid from EtOAc/PE (4.99 g, 75%), mp 96±978C; RfI
20
mp 166±1678C; RfI 0.85, RfII 0.95, RfIII 0.35; [a]D ˆ28.8
(c 0.5, MeOH); HPLC: Rt 11.31 min (Phenomenex-Rp C18;
gradient: 70±90% B in 20 min; eluants: A, 0.05% TFA in
H2O, and B, 0.05% TFA in CH3CN/H2O (9:1)); nmax (KBr)
3351, 3321, 1742, 1669, 1641, 1529 cm21; dH (250 MHz,
CDCl3) 7.38±7.18 (m, 12H, Phe NH, Aib4 NH, and Phe and
Z aromatic CH), 7.13 (d, 1H, Jˆ6.9 Hz, Leu3 NH), 6.59 (s,
1H, Aib2 NH), 5.48 (d, 1H, Jˆ4.6 Hz, Leu1 NH), 5.05 (m,
2H, Z CH2), 4.81 (m, 1H, Phe aCH), 4.24 (m, 1H, Leu3
aCH), 3.98 (m, 1H, Leu1 aCH), 3.65 (s, 3H, OMe CH3),
3.11 (m, 2H, Phe bCH2), 1.76±1.60 (6H, 2Leu bCH2 and
gCH), 1.58, 1.55, 1.52 and 1.44 (4s, 12H, 2Aib bCH3),
20
0.95, RfII 0.95, RfIII 0.45; [a]D ˆ27.0 (c 0.5, MeOH);

M. Crisma et al. / Tetrahedron 57 (2001) 2813±2825
2821
1.00±0.88 (12H, 2Leu dCH3); HRMS (FAB): M1, found
710.4097. C38H56N5O8 requires 710.4129.
EtOAc was added and the excess of LiBH4 was neutralized
by addition of 10% KHSO4. The organic layer was washed
with 10% KHSO4, H2O, 5% NaHCO3 and H2O, and dried
over anhydrous Na2SO4. The title compound was puri®ed by
¯ash chromatography (ICN silica 32±63, 60A; eluant:
CHCl3/EtOH (91:9)) and crystallized as a white solid from
CHCl3/PE (0.23 g, 77%), mp 224±2258C; RfI 0.50, RfII
4.1.4. Ac-l-Leu-Aib-l-Leu-Aib-l-Phe-OMe. A solution
of H-l-Leu-Aib-l-Leu-Aib-l-Phe-OMe (obtained by cata-
lytic hydrogenation of the above-mentioned Z-derivative
(0.5 g, 0.7 mmol) in MeOH) in CH2Cl2 (2 mL) was treated
with an excess of Ac2O. The reaction was stirred at room
temperature for 3 h. Then, the solvent was removed and the
residue ¯ushed several times with toluene. The crude title
compound was puri®ed by ¯ash chromatography (ICN silica
32±63, 60A; eluant: CHCl3/EtOH (92:8)) and crystallized
as a white solid from CHCl3/PE (0.41 g, 94%), mp 208±
20
0.90, RfIII 0.10; [a]D ˆ226.0 (c 0.5, MeOH); HPLC: Rt
4.23 min (Phenomenex-Rp C18; gradient: 70±90% B in
20 min; eluants: A, 0.05% TFA in H2O, and B, 0.05%
TFA in CH3CN/H2O (9:1)); nmax (KBr) 3326, 1653,
1539 cm21; dH (250 MHz, CDCl3) 7.91 (d, 1H, Jˆ4.5 Hz,
Leu1 NH), 7.75 (s, 1H, Aib2 NH), 7.52 (s, 1H, Aib4 NH),
7.39 (d, 1H, Jˆ6.3 Hz, Leu3 NH), 7.17 (m, 5H, Phol
aromatic CH), 6.97 (d, 1H, Jˆ8.9 Hz, Phol NH), 4.04 (m,
1H, Phol aCH), 4.01 (m, 1H, Leu1 aCH), 3.95 (m, 1H, Leu3
aCH), 3.60 (d, 2H, Jˆ5.3 Hz, Phol CH2OH), 2.81 (m, 2H,
Phol bCH2), 2.02 (s, 3H, Ac CH3), 1.75 (m, 2H, 2Leu gCH),
1.57±1.55 (m, 4H, 2Leu bCH2), 1.51, 1.43, 1.40 and 1.34
(4s, 12H, 2Aib bCH3), 0.99 (d, 3H, Jˆ6.5 Hz, Leu1 dCH3),
0.96 (d, 3H, Jˆ7.7 Hz, Leu3 dCH3), 0.94 (d, 3H, Jˆ6.8 Hz,
Leu1 dCH3), 0.88 (d, 3H, Jˆ6.1 Hz, Leu3 dCH3); Amino
acid analysis: Aib 1.98, Leu 2.02; HRMS (FAB): M1, found
590.3954. C31H52N5O6 requires 590.3918.
20
2098C; RfI 0.60, RfII 0.90, RfIII 0.25; [a]D ˆ218.0 (c 0.5,
MeOH); HPLC: Rt 5.03 min (Phenomenex-Rp C18;
gradient: 70±90% B in 20 min; eluants: A, 0.05% TFA in
H2O, and B, 0.05% TFA in CH3CN/H2O (9:1)); nmax (KBr)
3321, 1732, 1666, 1530 cm21; dH (250 MHz, CDCl3) 7.40
(d, 1H, Jˆ10.0 Hz, NH), 7.31 (s, 1H, Aib NH), 7.21 (m, 5H,
Phe aromatic CH), 7.03 (d, 1H, Jˆ7.5 Hz, NH), 6.69 (s, 1H,
Aib NH), 6.54 (d, 1H, Jˆ5.0 Hz, NH), 4.76 (m, 1H, aCH),
4.23 (m, 1H, aCH), 4.05 (m, 1H, aCH), 3.63 (s, 3H, OMe
CH3), 3.09 (m, 2H, Phe bCH2), 1.99 (s, 3H, Ac CH3), 1.80±
1.65 (6H, 2Leu bCH2 and gCH), 1.51, 1.49, 1.44 and 1.41
(4s, 12H, 2Aib bCH3), 0.98±0.88 (m, 12H, 2Leu dCH3);
Amino acid analysis: Aib 1.88, Leu 2.10, Phe 1.02; HRMS
(FAB): M1, found 618.3906. C32H52N5O7 requires
618.3867.
4.1.7. n-Oct-l-Leu-Aib-l-Leu-Aib-l-Phol. To a solution of
n-Oct-l-Leu-Aib-l-Leu-Aib-l-Phe-OMe (0.4 g, 0.5 mmol)
in dry THF (freshly distilled over LiAlH4), a solution of
LiBH4 in THF (3 mL) was added. The reaction was stirred
at room temperature for 20 min. Then, cold EtOAc was
added and the excess of LiBH4 was neutralized by addition
of 10% KHSO4. The organic layer was washed with 10%
KHSO4, H2O, 5% NaHCO3 and H2O, and dried over
anhydrous Na2SO4. The title compound was puri®ed by
¯ash chromatography (ICN silica 32±63, 60A; eluant:
CH2Cl2/EtOH (93:7)) and crystallized as a white solid
from CHCl3/PE (0.29 g, 87%), mp 185±1868C; RfI 0.65,
4.1.5. n-Oct-l-Leu-Aib-l-Leu-Aib-l-Phe-OMe. To a solu-
tion of n-Oct-OH (0.12 mL, 0.8 mmol) in CH2Cl2 cooled to
08C, HOAt (0.1 g, 0.7 mmol) and EDC´HCl (0.15 g,
0.8 mmol) were added. After 10 min H-l-Leu-Aib-l-Leu-
Aib-l-Phe-OMe (obtained by catalytic hydrogenation of the
above-mentioned Z-derivative (0.5 g, 0.7 mmol) in MeOH)
and NMM (0.16 mL, 1.4 mmol) were added and the reaction
was stirred at room temperature for 24 h. Then, the solvent
was removed and the residue dissolved in EtOAc. The solu-
tion was extracted with 10% KHSO4, H2O, 5% NaHCO3,
H2O, and dried over anhydrous Na2SO4. The title compound
was crystallized as a white solid from CHCl3/PE (0.42 g,
85%), mp 188±1898C; RfI 0.80, RfII 0.95, RfIII 0.30;
20
RfII 0.95, RfIII 0.20; [a]D ˆ219.0 (c 0.5, MeOH);
HPLC: Rt 15.40 min (Phenomenex-Rp C18; gradient:
70±90% B in 20 min; eluants: A, 0.05% TFA in H2O, and
B, 0.05% TFA in CH3CN/H2O (9:1)); nmax (KBr) 3290,
1652, 1537 cm21; dH (250 MHz, CDCl3) 7.90 (m, 3H,
3NH), 7.37 (d, 1H, Jˆ5.0 Hz, NH), 7.26±7.13 (6H, Phol
aromatic CH and 1NH), 4.30 (m, 1H, aCH), 4.13±3.95
(m, 1H, aCH), 3.66 (m, 1H, aCH), 2.77 (m, 2H, CH2),
2.37 (m, 2H, CH2), 1.82±1.63 (8H, 2Leu bCH2 and gCH
and n-Oct aCH2), 1.52, 1.48, 1.38 and 1.35 (4s, 12H, 2Aib
bCH3), 1.27±1.18 (m, 10H, n-Oct (CH2)5), 1.00±0.85 (m,
15H, 2Leu dCH3 and n-Oct vCH3); HRMS (FAB): M1,
found 674.4860. C37H64N5O6 requires 674.4857.
20
[a]D ˆ215.6 (c 0.5, MeOH); HPLC: Rt 16.70 min
(Phenomenex-Rp C18; gradient: 70±90% B in 20 min;
eluants: A, 0.05% TFA in H2O, and B, 0.05% TFA in
CH3CN/H2O (9:1)); nmax (KBr) 3330, 1730, 1658,
1532 cm21; dH (250 MHz, CDCl3) 7.40 (d, Jˆ8.1 Hz, 1H,
Phe NH), 7.37 (s, 1H, Aib4 NH), 7.26±7.18 (m, 5H, Phe
aromatic CH), 7.17 (d, 1H, Jˆ7.6 Hz, Leu3 NH), 6.69 (s,
1H, Aib2 NH), 6.56 (broad s, 1H, Leu1 NH), 4.74 (m, 1H,
Phe aCH), 4.23 (m, 1H, Leu3 aCH), 4.05 (m, 1H, Leu1
aCH), 3.62 (s, 3H, OMe CH3), 3.10 (m, 2H, Phe bCH2),
2.23 (m, 2H, n-Oct aCH2), 1.82±1.55 (6H, 2Leu bCH2 and
gCH), 1.53 and 1.39 (2s, 6H, Aib2 bCH3), 1.51 and 1.45 (2s,
6H, Aib4 bCH3), 1.27 (10H, n-Oct (CH2)5), 0.99±0.84 (m,
15H, 2Leu dCH3 and n-Oct vCH3); HRMS (FAB): M1,
found 702.4796. C38H64N5O7 requires 702.4806.
4.1.8. Z-l-Iva-l-Phe-OMe. To a solution of Z-l-Iva-OH35
(3.2 g, 13 mmol) in CH2Cl2 cooled to 08C, HOAt (1.7 g,
13 mmol) and EDC´HCl (2.7 g, 14 mmol) were added.
After 10 min HCl´H-l-Phe-OMe (2.8 g, 13 mmol), neutra-
lized with NMM (2.9 mL, 26 mmol), was added and the
reaction mixture stirred at room temperature for 12 h.
Then, EtOAc was added and the solution was extracted
with 10% KHSO4, H2O, 5% NaHCO3, H2O, and dried
over anhydrous Na2SO4. The title compound was puri®ed
by ¯ash chromatography (ICN silica 32±63, 60A; eluant:
EtOAc/PE (2:3)) and crystallized as a white solid from
EtOAc/PE (3.91 g, 73%), mp 95±968C; RfI 0.95, RfII
4.1.6. Ac-l-Leu-Aib-l-Leu-Aib-l-Phol (peptaibolin). To
a solution of Ac-l-Leu-Aib-l-Leu-Aib-l-Phe-OMe (0.3 g,
0.5 mmol) in dry THF (freshly distilled over LiAlH4), a
solution of LiBH4 in THF (3 mL) was added. The reaction
was stirred at room temperature for 20 min. Then, cold

2822
M. Crisma et al. / Tetrahedron 57 (2001) 2813±2825
20
0.95, RfIII 0.65; [a]D ˆ28.2 (c 0.5, MeOH); nmax (KBr)
3319, 1747, 1658, 1535 cm21; dH (250 MHz, CDCl3) 7.34
(m, 5H, Z aromatic CH), 7.25 (m, 5H, Phe aromatic CH),
6.46 (d, 1H, Jˆ7.7 Hz, Phe NH), 5.56 (s, 1H, Iva NH), 5.07
(s, 2H, Z CH2), 4.88 (m, 1H, Phe aCH), 3.72 (s, 3H, OMe
CH3), 3.12 (m, 2H, Phe CH2), 2.08 and 1.70 (2m, 2H, Iva
bCH2), 1.45 (m, 3H, Iva bCH3), 0.77 (t, 3H, Jˆ7.4 Hz, Iva
gCH3); HRMS (FAB): M1, found 413.2070. C23H29N2O5
requires 413.2076.
4.1.11. Z-l-Leu-l-Iva-l-Leu-l-Iva-l-Phe-OMe. To a
solution of Z-l-Leu-OH (1.4 g, 5.3 mmol) in CH2Cl2 cooled
to 08C, HOAt (0.7 g, 4.9 mmol) and EDC´HCl (1.0 g,
5.3 mmol) were added. After 10 min H-l-Iva-l-Leu-l-Iva-
l-Phe-OMe (obtained by catalytic hydrogenation of the
above-mentioned Z-derivative (3.0 g, 4.9 mmol) in
MeOH) and NMM (1.1 mL, 9.7 mmol) were added and
the reaction was stirred at room temperature for 12 h.
Then, the solvent was evaporated and the residue dissolved
in EtOAc. The solution was extracted with 10% KHSO4,
H2O, 5% NaHCO3, H2O, and dried over anhydrous
Na2SO4. The title compound was puri®ed by ¯ash chroma-
tography (ICN silica 32±63; eluant: CHCl3/EtOH (97:3))
and crystallized as a white solid from EtOAc/PE (2.82 g,
78%), mp 156±1578C; RfI 0.90, RfII 0.95, RfIII 0.35;
4.1.9. Z-l-Leu-l-Iva-l-Phe-OMe. To a solution of Z-l-
Leu-OH (2.6 g, 9.9 mmol) in CH2Cl2 cooled to 08C, HOAt
(1.2 g, 9.0 mmol) and EDC´HCl (1.9 g, 9.9 mmol) were
added. After 10 min H-l-Iva-l-Phe-OMe (obtained by cata-
lytic hydrogenation of the above-mentioned Z-derivative
(3.7 g, 9.0 mmol) in MeOH) and NMM (2.0 mL,
18.1 mmol) were added and the reaction was stirred at
room temperature for 12 h. Then, EtOAc was added and
the solution was extracted with 10% KHSO4, H2O, 5%
NaHCO3, H2O, and dried over anhydrous Na2SO4. The
title compound was puri®ed by ¯ash chromatography
(ICN silica 32±63, 60A; eluant: CHCl3/EtOH (99:1)) and
crystallized as a white solid from EtOAc/PE (3.45 g, 73%),
20
[a]D ˆ219.2 (c 0.5, MeOH); HPLC: Rt 15.15 min
(Phenomenex-Rp C18; gradient: 70±90% B in 20 min;
eluants: A, 0.05% TFA in H2O, and B, 0.05% TFA in
CH3CN/H2O (9:1)); nmax (KBr) 3322, 1743, 1658,
1528 cm21; dH (250 MHz, CDCl3) 7.34 (m, 5H, Z aromatic
CH), 7.20 (m, 7H, 2NH and Phe aromatic CH), 7.07 (d, 1H,
Jˆ6.9 Hz, NH), 6.51 (s, 1H, Iva NH), 5.39 (d, 1H, Jˆ
3.1 Hz, Leu1 NH), 5.08 (m, 2H, Z CH2), 4.80 (m, 1H,
aCH), 4.24 (m, 1H, aCH), 3.98 (m, 1H, aCH), 3.66 (s,
3H, OMe CH3), 3.09 (m, 2H, Phe bCH2), 1.92±1.63 (m,
10H, 2Iva bCH2, 2Leu bCH2 and gCH), 1.52 and 1.46
(2s, 6H, 2Iva bCH3), 0.99±0.89 (m, 12H, 2Leu dCH3),
0.81 (t, 3H, Jˆ7.4 Hz, Iva gCH3), 0.71 (t, 6H, Jˆ7.5 Hz,
Iva gCH3); HRMS (FAB): M1, found 738.4456.
C40H60N5O8 requires 738.4442.
20
mp 51±528C; RfI 0.95, RfII 0.95, RfIII 0.50; [a]D ˆ226.2
(c 0.5, MeOH); nmax (KBr) 3328, 1737, 1731, 1672, 1651,
1527 cm21; dH (250 MHz, CDCl3) 7.35 (m, 5H, Z aromatic
CH), 7.12 (m, 5H, Phe aromatic CH), 6.80 (s, 1H, Iva NH),
6.52 (d, 1H, Jˆ9.2 Hz, Phe NH), 5.11 (m, 3H, Leu NH and
Z CH2), 4.85 (m, 1H, Phe aCH), 4.11 (m, 1H, Leu aCH),
3.72 (s, 3H, OMe CH3), 3.13 (m, 2H, Phe bCH2), 2.21 and
1.64 (2m, 2H, Iva bCH2), 1.62±1.44 (m, 3H, Leu bCH2 and
gCH), 1.47 (s, 3H, Iva bCH3), 0.96±0.93 (m, 6H, Leu
dCH3), 0.72 (t, 3H, Jˆ9.2 Hz, Iva gCH3); HRMS (FAB):
M1, found 526.2940. C29H40N3O6 requires 526.2917.
4.1.12. Ac-l-Leu-l-Iva-l-Leu-l-Iva-l-Phe-OMe. A solu-
tion of H-l-Leu-l-Iva-l-Leu-l-Iva-l-Phe-OMe (obtained
by catalytic hydrogenation of the above-mentioned Z
derivative (0.6 g, 0.8 mmol) in MeOH) in CH2Cl2 (2 mL)
was treated with an excess of Ac2O. The reaction was stirred
at room temperature for 3 h. Then, the solvent was removed
and the residue ¯ushed several times with toluene. The
crude title compound was puri®ed by ¯ash chromatography
(ICN silica 32±63, 60A; eluant: CHCl3/EtOH (97:3)) and
4.1.10. Z-l-Iva-l-Leu-l-Iva-l-Phe-OMe. To a solution of
Z-l-Iva-OH (1.5 g, 6.1 mmol) in CH2Cl2 cooled to 08C,
HOAt (0.8 g, 5.5 mmol) and EDC´HCl (1.2 g, 6.1 mmol)
were added. After 10 min H-l-Leu-l-Iva-l-Phe-OMe
(obtained by catalytic hydrogenation of the above-
mentioned Z-derivative (2.9 g, 5.5 mmol) in MeOH) and
NMM (1.2 mL, 11.1 mmol) were added and the reaction
was stirred a room temperature for 24 h. Then, EtOAc
was added and the solution was extracted with 10%
KHSO4, H2O, 5% NaHCO3, H2O, and dried over anhydrous
Na2SO4. The title compound was puri®ed by ¯ash chroma-
tography (ICN silica 32±63, 60A; eluant: CHCl3/EtOH
(98:2)) and crystallized as a white solid from EtOAc/PE
(3.02 g, 88%), mp 60±618C; RfI 0.90, RfII 0.95, RfIII 0.40;
crystallized as a white solid from CH2Cl2/PE (0.45 g, 87%),
mp 215±2168C; RfI 0.65, RfII 0.90, RfIII 0.25; [a]D
20
ˆ
237.6 (c 0.5, MeOH); HPLC: Rt 6.93 min (Phenomenex-
Rp C18; gradient: 70±90% B in 20 min; eluants: A, 0.05%
TFA in H2O, and B, 0.05% TFA in CH3CN/H2O (9:1)); nmax
(KBr) 3313, 1732, 1658, 1531 cm21; dH (250 MHz, CDCl3)
7.22±7.17 (6H, 1 NH and Phe aromatic CH), 7.11 (s, 1H,
Iva NH), 6.99 (d, 1H, Jˆ7.5 Hz, NH), 6.53 (s, 1H, Iva NH),
6.23 (d, 1H, Jˆ5.0 Hz, NH), 4.79 (m, 1H, aCH), 4.23 (m,
1H, aCH), 4.13 (m, 1H, aCH), 3.66 (s, 3H, OMe CH3), 3.10
(m, 2H, Phe bCH2), 2.02 (s, 3H, Ac CH3), 1.96±1.63 (10H,
2Iva bCH2, 2Leu b-CH2 and gCH), 1.49 and 1.44 (2s, 6H,
2Iva bCH3), 0.98±0.85 (15H, 2Leu dCH3 and Iva gCH3),
0.71 (t, 3H, Jˆ7.5 Hz, Iva gCH3); Amino acid analysis: Iva
2.10, Leu 1.92, Phe 0.95; HRMS (FAB): M1, found
646.4166. C34H56N5O7 requires 646.4180.
20
[a]D ˆ237.6 (c 0.5, MeOH); HPLC: Rt 14.07 min
(Phenomenex-Rp C18; gradient: 65±85% B in 20 min;
eluants: A, 0.05% TFA in H2O, and B, 0.05% TFA in
CH3CN/H2O (9:1)); nmax (KBr) 3333, 1733, 1705, 1659,
1523 cm21; dH (250 MHz, CDCl3) 7.35 (m, 5H, Z aromatic
CH), 7.25 (m, 5H, Phe aromatic CH), 6.99 (m, 2H, Phe NH
and Iva3 NH), 6.36 (d, 1H, Jˆ8.9 Hz, Leu NH), 5.29 (s, 1H,
Iva1 NH), 5.08 (s, 2H, Z CH2), 4.84 (m, 1H, Phe aCH), 4.30
(m, 1H, Leu aCH), 3.68 (s, 3H, OMe CH3), 3.14 (m, 2H,
Phe bCH2), 1.86±1.39 (m, 7H, 2Iva bCH2, Leu bCH2 and
gCH), 1.52 and 1.44 (2s, 6H, 2Iva bCH3), 0.90 (m, 9H, Leu
dCH3 and Iva gCH3), 0.71 (t, 3H, Jˆ9.3 Hz, Iva gCH3);
HRMS (FAB): M1, found 625.3606. C34H49N4O7 requires
625.3601.
4.1.13. n-Oct-l-Leu-l-Iva-l-Leu-l-Iva-l-Phe-OMe. To a
solution of n-Oct-OH (0.14 mL, 0.9 mmol) in CH2Cl2
cooled to 08C, HOAt (0.1 g, 0.8 mmol) and EDC´HCl
(0.12 g, 0.9 mmol) were added. After 10 min H-l-Leu-l-
Iva-l-Leu-l-Iva-l-Phe-OMe (obtained by catalytic hydro-
genation of the above-mentioned Z-derivative (0.6 g,

M. Crisma et al. / Tetrahedron 57 (2001) 2813±2825
2823
0.8 mmol) in MeOH) and NMM (0.2 mL, 1.7 mmol) were
added and the reaction was stirred at room temperature for
24 h. Then, the solvent was removed and the residue
dissolved in EtOAc. The solution was extracted with 10%
KHSO4, H2O, 5% NaHCO3, H2O, and dried over anhydrous
Na2SO4. The title compound was crystallized as a white
solid from CHCl3/PE (0.47 g, 81%), mp 198±1998C; RfI
from CHCl3/PE (0.23 g, 81%), mp 218±2198C; RfI 0.60,
RfII 0.95, RfIII 0.20; [a]D ˆ225.8 (c 0.5, MeOH);
20
HPLC: Rt 14.01 min (Phenomenex-Rp C18; gradient: 80±
100% B in 20 min; eluants: A, 0.05% TFA in H2O, and B,
0.05% TFA in CH3CN/H2O (9:1)); nmax (KBr) 3287, 1650,
1538 cm21; dH (250 MHz, CDCl3) 7.97 (broad s, 1H, NH),
7.84 (m, 2H, 2 NH), 7.38 (d, 1H, Jˆ5.0 Hz, NH), 7.22 (m,
5H, Phol aromatic CH), 7.05 (d, 1H, Jˆ7.7 Hz, NH), 4.42±
3.60 (m, 5H, 3 aCH and Phol CH2OH), 2.75 (m, 2H, Phol
bCH2), 2.38 (t, 2H, Jˆ6.9 Hz, n-Oct aCH2), 1.74±1.68
(10H, 2Leu bCH2 and gCH, 2Iva bCH2), 1.52 and 1.39
(2s, 6H, Iva bCH3), 1.27 (m, 10H, n-Oct (CH2)5), 1.00±
0.81 (m, 18H, 2Leu dCH3, n-Oct vCH3 and Iva gCH3),
0.60 (t, 3H, Jˆ7.3 Hz, Iva gCH3); HRMS (FAB): M1,
found 702.5188. C39H68N5O6 requires 702.5170.
20
0.85, RfII 0.95, RfIII 0.30; [a]D ˆ228.6 (c 0.5, MeOH);
HPLC: Rt 14.41 min (Phenomenex-Rp C18; gradient: 80±
100% B in 20 min; eluants: A, 0.05% TFA in H2O, and B,
0.05% TFA in CH3CN/H2O (9:1)); nmax (KBr) 3305, 1732,
1657, 1532 cm21; dH (250 MHz, CDCl3) 7.40 (d, 1H, Jˆ
7.1 Hz, NH), 7.37 (s, 1H, Iva NH), 7.32 (m, 5H, Phe
aromatic CH), 7.23 (d, 1H, Jˆ2.9 Hz, NH), 6.75 (d, 1H,
Jˆ5.0 Hz, NH), 6.73 (s, 1H, Iva NH), 4.72 (m, 1H, aCH),
4.23 (m, 1H, aCH), 4.05 (m, 1H, aCH), 3.66 (s, 3H, OMe
CH3), 3.12 (m, 2H, Phe bCH2), 2.26 (t, 2H, Jˆ7.5 Hz, n-Oct
aCH2), 2.02±1.63 (10H, 2Iva bCH2, 2Leu bCH2 and gCH),
1.51 and 1.47 (2s, 6H, 2Iva bCH3), 1.27 (m, 10H, n-Oct
(CH2)5), 0.99±0.86 (m, 18H, 2Leu dCH3, n-Oct vCH3
and Iva gCH3), 0.74 (t, 3H, Jˆ7.9 Hz, Iva gCH3); HRMS
(FAB): M1, found 730.5089. C40H68N5O7 requires
730.5119.
4.2. FT-IR absorption
The solid-state infrared absorption spectra (KBr disk tech-
nique) were recorded with a Perkin±Elmer model 580 B
spectrophotometer equipped with a Perkin±Elmer model
3600 IR data station. The solution IR absorption spectra
were recorded using a Perkin±Elmer model 1720 X FT-IR
spectrophotometer, nitrogen-¯ushed, equipped with
a
sample-shuttle device, at 2 cm21 nominal resolution,
averaging 100 scans. Solvent (baseline) spectra were
obtained under the same conditions. Cell with path lengths
of 0.1, 1.0 and 10 mm (with CaF2 windows) were used.
Spectrograde deuterochloroform (99.8% D) was purchased
from Fluka.
4.1.14. Ac-l-Leu-l-Iva-l-Leu-l-Iva-l-Phol. To a solution
of Ac-l-Leu-l-Iva-l-Leu-l-Iva-l-Phe-OMe (0.2 g, 0.3 mmol)
in dry THF (freshly distilled over LiAlH4), a solution of
LiBH4 in THF (3 mL) was added. The reaction was stirred
at room temperature for 20 min. Then, cold EtOAc was
added and the excess of LiBH4 was neutralized by addition
of 10% KHSO4. The organic layer was washed with 10%
KHSO4, H2O, 5% NaHCO3 and H2O, and dried over anhy-
drous Na2SO4. The title compound was puri®ed by ¯ash
chromatography (ICN silica 32±63, 60A; eluant: CH2Cl2/
EtOH (95:5)) and crystallized as a white solid from CH2Cl2/
PE (0.14 g, 78%), mp 235±2368C; RfI 0.50, RfII 0.90, RfIII
4.3. Nuclear magnetic resonance
1
The H NMR spectra were recorded either with a Bruker
model AC 250 spectrometer or with a Bruker model AM
400 spectrometer. Measurements were carried out in deutero-
chloroform (99.96% D; Aldrich) and deuterated DMSO
(99.96% D6; Acros Organics) with tetramethylsilane as
the internal standard.
20
0.10; [a]D ˆ231.0 (c 0.5, MeOH); HPLC: Rt 5.90 min
(Phenomenex-Rp C18; gradient: 70±90% B in 20 min;
eluants: A, 0.05% TFA in H2O, and B, 0.05% TFA in
CH3CN/H2O (9:1)); nmax (KBr) 3298, 1652, 1537 cm21
;
dH (250 MHz, CDCl3) 8.17 (d, 1H, Jˆ2.5 Hz, NH), 7.92
(s, 1H, Iva NH), 7.78 (s, 1H, Iva NH), 7.38 (d, 1H, Jˆ
7.5 Hz, NH), 7.22 (m, 5H, Phol aromatic CH), 7.09 (d,
1H, Jˆ10.0 Hz, NH), 4.36±3.67 (m, 5H, 3 aCH and Phol
CH2OH), 2.75 (m, 2H, Phol bCH2), 2.12 (s, 3H, Ac CH3),
1.85±1.60 (10H, 2Iva bCH2, 2Leu bCH2 and gCH), 1.52
and 1.39 (2s, 6H, 2Iva bCH3), 0.97±0.87 (12H, 2Leu
dCH3), 0.81 (t, 3H, Jˆ7.8 Hz, Iva gCH3), 0.63 (t, 3H,
Jˆ7.5 Hz, Iva gCH3); Amino acid analysis: Iva 2.08, Leu
1.96; HRMS (FAB): M1, found 618.4221. C33H56N5O6
requires 618.4231.
4.4. Mass spectrometry
Fast Atom Bombardment (FAB) mass spectrometry was
carried out using a JEOL SX/SX model 102A four-sector
tandem mass spectrometer (B1E1B2E2 geometry), coupled to
a JEOL MS/MP9021D/UPD data system. The samples were
loaded in a nitrobenzyl alcohol solution onto a stainless steel
probe and bombarded with xenon atoms with an energy of
8 keV.
During the high-resolution FABMS measurements a resolv-
ing power of 5,000±10,000 (10% valley de®nition) was
used. Polyethyleneglycols (PEG) 300 and 600 were used
to calibrate the mass spectrometer.
4.1.15. n-Oct-l-Leu-l-Iva-l-Leu-l-Iva-l-Phol. To a solu-
tion of n-Oct-l-Leu-l-Iva-l-Leu-l-Iva-l-Phe-OMe (0.3 g,
0.4 mmol) in dry THF (freshly distilled over LiAlH4), a
solution of LiBH4 in THF (3 mL) was added. The reaction
was stirred at room temperature for 20 min. Then, cold
EtOAc was added and the excess of LiBH4 was neutralized
by addition of 10% KHSO4. The organic layer was washed
with 10% KHSO4, H2O, 5% NaHCO3 and H2O, and dried
over anhydrous Na2SO4. The title compound was puri®ed by
¯ash chromatography (ICN silica 32±63, 60A; eluant:
CH2Cl2/EtOH (97:3)) and crystallized as a white solid
4.5. X-Ray diffraction
Single crystals of n-Oct-(l-Leu-Aib)2-l-Phe-OMe, Ac-(l-
Leu-l-Iva)2-l-Phe-OMe, Ac-(l-Leu-Aib)2-l-Phol (peptai-
bolin), and Ac-(l-Leu-l-Iva)2-l-Phol were obtained by
slow evaporation at room temperature from an MeOH/
H2O (the former peptide) or an MeOH (the other three
peptides) solution. Intensity data collection was performed

2824
M. Crisma et al. / Tetrahedron 57 (2001) 2813±2825
by using a Philips PW 1100 four-circle diffractometer.
Ê
H atom of the C-terminal alcoholic function was selected as
the one able to form the best H-bond among the three pos-
sible staggered orientations relative to the C5±O5 bond.
During the re®nement all H atoms were allowed to ride on
their carrying atom, with Uiso set equal to 1.2 (or 1.5 for the
hydroxyl and methyl groups) times the Uequiv. of the parent
atom.
Graphite-monochromated Cu Ka radiation (lˆ1.54178 A)
and u/2u scan mode were used. Cell parameters were
obtained by least-squares re®nements of the angular setting
of 48 carefully centred high angle re¯ections. The structures
were solved by direct methods (shelxs 9757 program).
Re®nement was carried out by the full-matrix block least-
squares procedure on F2, with all non-H atoms anisotropic,
by application of the shelxl 9758 program.
The trial structure of Ac-(l-Leu-l-Iva)2-l-Phol having the
best combined ®gure of merit allowed the location of all
non-H atoms with the exception of those belonging to the
disordered parts of the side chains of Leu(1) and Iva(2)
residues. The positions of the latter atoms were recovered
from subsequent difference Fourier maps. The positional
parameters and the anisotropic displacements of the non-H
atoms were allowed to re®ne at alternate cycles. The dis-
ordered Cg, Cd1 and Cd2 atoms of the Leu(1) side chain were
re®ned on two sets of positions (atoms C1G, C1D1, C1D2,
and atoms C1G0, C1D3, C1D4) with population parameters
of 0.65 and 0.35, respectively. The phenyl ring of the Phol
residue was constrained to the idealized geometry.
Restraints were applied to most of the bond distances, the
chiral volume of the carbonyl carbon atoms, and the aniso-
tropic displacement parameters to approach isotropic beha-
viour. H atoms were calculated at idealized positions.
During the re®nement they were allowed to ride on their
carrying atom, with Uiso set equal to 1.2 (or 1.5 for the
hydroxyl and methyl groups) times the Uequiv. of the parent
atom.
The crystals of n-Oct-(l-Leu-Aib)2-l-Phe-OMe did not
Ê
signi®cantly diffract above uˆ508 (1.0 A resolution). The
positional parameters and the anisotropic displacements of
the non-H atoms were allowed to re®ne at alternate cycles.
H atoms were calculated at idealized positions. During the
re®nement the H atoms were allowed to ride on their carry-
ing atom, with Uiso set equal to 1.2 (or 1.5 for methyl
groups) times the Uequiv. of the parent atom.
The trial structure of Ac-(l-Leu-l-Iva)2-l-Phe-OMe with
the best combined ®gure of merit allowed the location of
41 (out of 46) non-H atoms. The positions of the remaining
non-H atoms were recovered from subsequent difference
Fourier maps. The positional parameters and the anisotropic
displacements of the non-H atoms were allowed to re®ne at
alternate cycles. The side chain of the Leu(1) residue is
disordered. Its Cg and Cd atoms were re®ned on two sets
of positions (atoms C1G, C1D1, C1D2, and atoms C1G0,
C1D3, C1D4) with population parameters of 0.65 and 0.35,
respectively. A disorder was also observed at the Cg atom of
the Iva(2) residue, which was re®ned on two positions
(atoms C2G and C2G0) with population parameters of
0.77 and 0.23, respectively. A number of restraints were
applied to the 1±2 and 1±3 interatomic distances involving
atoms of the disordered side chains. The anisotropic dis-
placement parameters of the same atoms were also
restrained to approach isotropic behaviour. The phenyl
ring of the Phe(5) residue was constrained to the idealized
geometry. H atoms were calculated at idealized positions
and re®ned by allowing them to ride on their carrying atom,
with Uiso set equal to 1.2 (or 1.5 for methyl groups) times the
Uequiv. of the parent atom.
4.6. Liposome leakage assay
Peptide-induced leakage from egg PC vesicles was
measured at 208C using the CF-entrapped vesicle tech-
nique.49,50 CF-encapsulated small unilamellar vesicles (egg
PC/cholesterol, 70:30) were prepared by sonication in
Hepes buffer, pH 7.4. The phospholipid concentration was
kept constant (0.06 mM), and increasing [peptide]/[lipid]
molar ratios (R21) were obtained by adding aliquots of
MeOH solutions of peptides, keeping the ®nal MeOH
concentration below 5% by volume. After rapid and
vigorous stirring the time course of ¯uorescence change
corresponding to CF escape was recorded at 520 nm
(6 nm band pass) with lexc 488 nm (3 nm band pass). The
percentage of released CF at time t was determined as
(Ft2F0)/(FT2F0)£100, with F0 the ¯uorescence intensity
of vesicles in the absence of peptide, Ft the ¯uorescence
intensity at time t in the presence of peptide, and FT the
total ¯uorescence intensity determined by disrupting the
vesicles by addition of 50 mL of a 10% Triton X-100 solu-
tion. The kinetics were stopped at 20 min.
The solution of the structure of Ac-(l-Leu-Aib)2-l-Phol was
somewhat complicated by the low diffracting power of the
crystal, which in turn has to be ascribed to the reduced
crystal thickness (only 0.05 mm). Indeed, the fraction of
re¯ections having I$4s(I) was 25% in the resolution
Ê
Ê
range 1.2±1.1 A, while it was 5% between 1.1 and 1.0 A.
Eventually, the structure was solved by using 422 re¯ections
for the phasing process and 554 re¯ections (E$1.1) for the
structure expansion with the tangent formula. The trial
structure with the best combined ®gure of merit allowed
the location of 23 out of 42 non-H atoms. The remaining
non-H atoms were recovered from subsequent difference
Fourier maps. The positional parameters and the anisotropic
displacements of the non-H atoms were allowed to re®ne at
alternate cycles. The phenyl ring of the Phol residue was
constrained to the idealized geometry. Restraints were
applied to most of the bond distances, the chiral volume
of the carbonyl carbon atoms, and the anisotropic displace-
ment parameters to approach isotropic behaviour. H atoms
were calculated at idealized positions. The position of the
Acknowledgements
The authors thank the National Research Council (CNR,
Target Project on Biotechnology) of Italy for the ®nancial
support to this research.
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