Synthesis, preferred conformation, and membrane activity of medium-length peptaibiotics: Tylopeptin B

Article abstract of DOI:10.1111/j.1747-0285.2009.00920.x

The solid-phase synthesis and full chemical characterization of the medium-length (14-amino acid residues) peptaibol with antibiotic properties of tylopeptin B, originally extracted from the fruiting body of the mushroom Tylopilus neofelleus, are described. These data are accompanied by the results on the solution-phase synthesis via the segment condensation approach of a selected, side-chain protected, analog. A solution conformational analysis, performed by the combined use of FTIR absorption, circular dichroism, and 2D-NMR (the latter technique coupled to molecular dynamics calculations), favors the conclusion that the 3D-structure of tylopeptin B is largely helical with a preference for the α- or the 3₁₀-helix type depending upon the nature of the solvent. Helix topology and (partial) amphiphilic character are responsible for the observed membrane-modifying properties of this peptaibiotic.

Full text of DOI:10.1111/j.1747-0285.2009.00920.x

ª 2009 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2009.00920.x
Chem Biol Drug Des 2010; 75: 169–181
Research Article
Synthesis, Preferred Conformation, and
Membrane Activity of Medium-Length
Peptaibiotics: Tylopeptin B
not find rapidly new classes of compounds able to kill the opportu-
nistic 'superbug' invaders (1).
Marina Gobbo*, Claudia Poloni, Marta De
Zotti, Cristina Peggion, Barbara Biondi,
Gema Ballano, Fernando Formaggio and
Claudio Toniolo*
Peptaibiotics are a unique class of proteolytic enzyme-resistant,
membrane active, linear, and cyclopeptides with antibiotic activity
of fungal origin (2) which may represent a useful scaffold for the
construction of novel antibacterial drugs with promising properties.
ICB, Padova Unit, CNR, Department of Chemistry, University of
Padova, 35131 Padova, Italy
*Corresponding authors: Marina Gobbo, marina.gobbo@unipd.it and
Claudio Toniolo, claudio.toniolo@unipd.it
These peptides are characterized by
a linear a-amino acid
sequence, a high population of the strongly helicogenic, Ca-tetra-
substituted a-amino acids Aib (a-aminoisobutyric acid or Ca,a-dim-
ethylglycine) (3–5) and Iva (isovaline or Ca-ethyl, Ca-methylglycine)
(6–8), and an N-terminal acyl moiety. Linear peptaibiotics have been
classified into three groups (2) according to the number of amino
acids in their sequence: (i) short (from 4- to 10-mers), the prototype
of which is trichogin (9–12); (ii) medium-length (from 14- to 16-
mers), a less investigated subclass; and (iii) long (from 17- to 21-
mers), the most extensively studied group of which is represented
by the alamethicins (13–15). While the latter peptaibiotics have
been shown to form voltage-dependent membrane pores, the
detailed mechanism of action of short- and medium-length peptaibi-
otics is still largely unknown.
The solid-phase synthesis and full chemical char-
acterization of the medium-length (14-amino acid
residues) peptaibol with antibiotic properties of
tylopeptin B, originally extracted from the fruiting
body of the mushroom Tylopilus neofelleus, are
described. These data are accompanied by the
results on the solution-phase synthesis via the
segment condensation approach of
a selected,
side-chain protected, analog. A solution conforma-
tional analysis, performed by the combined use of
FTIR absorption, circular dichroism, and 2D-NMR
(the latter technique coupled to molecular dynam-
ics calculations), favors the conclusion that the
3D-structure of tylopeptin B is largely helical with
In this paper, we describe our recent results on tylopeptin B. A few
years ago, a Korean group determined the amino acid sequences of
two new medium-length peptaibiotics extracted from the fruiting
body of the mushroom (basidiomycete) Tylopilus neofelleus that they
termed tylopeptins A and B (16). Both tylopeptins were found to be
active against Gram-positive bacteria, but inactive against several
yeasts, fungi, and Gram-negative bacteria.
a
preference for the a- or the 310-helix type
depending upon the nature of the solvent. Helix
topology and (partial) amphiphilic character are
responsible for the observed membrane-modifying
properties of this peptaibiotic.
Key words: antibiotic, conformation analysis, membrane activity,
peptaibol, peptide synthesis
Tylopeptin A contains as many as one D-Iva and four Aib residues
(the former at position 4), while five Aib residues occur in its con-
gener B. More recently, tylopeptins were found in a strain of the
mycoparasitic Sepedonium chalcipori, suggesting an ascomycete ori-
gin for these peptaibiotics (17). The amino acid sequence of tylo-
peptin B is given below.
Received 30 October 2009, revised 12 November 2009 and accepted for
publication 12 November 2009
Drug resistance is rapidly emerging as a dramatic issue in human
medicine. Antibiotics are the most successful drugs, but the major
gains in life expectancy may be reversed if medicinal chemists will
Specifically, this 14-mer peptide is Na-acetylated (Ac) and C-terminal
amidated with the chiral 1,2-amino alcohol L-Lol (leucinol). In addi-
Editor’s invited manuscript to celebrate the 4th Anniversary of Chemical Biology & Drug Design.
169

Gobbo et al.
tion to the residues typical of peptaibiotics (Ala, Val, Leu, and Gln), it
is characterized by two residues unusual for this class of compounds,
namely Trp (bearing the indole moiety, a useful intrinsic spectro-
scopic probe) and Ser (with a reactive hydroxyl side-chain group).
determined at 25 ꢀC, in the indicated solvent, using a Perkin-Elmer
(Norwalk, CT, USA) 241 polarimeter and 1-dm thermostatted cell. H
1
NMR spectra were recorded on a Bruker (Karlsruhe, Germany)
AC200 or an AM 400 spectrometer in the indicated solvents: CDCl3
(99.96% d, Aldrich), dimethylsulphoxide (DMSO) (99.96% d6; Acros
Organics, Geel, Belgium), or CD3OH (99% d3, Aldrich). Chemical
shifts (d) are in ppm relative to SiMe4 as internal standard, and J
in Hz. Spectral analyses were carried out on a Mariner API-ToF
workstation (PerSeptive Biosystems, Framingham, MA, USA), operat-
ing in positive mode. Solid-state FTIR absorption spectra (KBr pellet
technique) were recorded with a Perkin-Elmer model 1720X spectro-
photometer, nitrogen flushed, equipped with a sample-shuttle
device, at 2 cm)1 nominal resolution, averaging 100 scans.
We synthesized tylopeptin B by the solid-phase methodology. A
full-length, side-chain protected, synthetic precursor was also pre-
pared by the segment condensation, solution-phase approach. We
planned to exploit its segments for our currently ongoing, easily
tunable, syntheses of a number of analogs bearing laterally
positioned spectroscopic probes. The preferred conformation of the
peptaibiotic was also analyzed in different environments by Fourier
transform infra red (FTIR) absorption, circular dichroism (CD), and
2D-nuclear magnetic resonance (NMR) ⁄ molecular dynamics (MD)
calculations. Permeabilization experiments were performed to
assess its activity on model membranes. A preliminary report of
part of these data has been recently published (18).
Chemistry
tert-Butyloxycarbonyl-L-leucinol (Boc-Lol). A solution of Boc-Leu-OH
(8.53 g, 37 mmol) in 40 mL of tetrahydrofuran (THF) was cooled at
)10 ꢀC. Then, N-methylmorpholine (NMM) (4 mL, 37 mmol) and ethyl
chloroformate (3.53 mL, 37 mmol) were added. The reaction mixture
was stirred at )10 ꢀC for 30 min followed by addition of a 2 M solu-
tion of LiBH4 in THF (75 mL, 150 mmol). Over a period of 30 min,
250 mL of methanol (MeOH) was slowly added to the reaction mix-
ture, which was then stirred at 0 ꢀC for 3 h. The reaction mixture
was neutralized by addition of 1 M HCl, then the organic solvents
were evaporated. The remaining residue was distributed between di-
chloromethane (DCM) and water, and the aqueous phase extracted
again with DCM. The combined organic layers were dried over
Na2SO4 and evaporated to dryness. The crude product was purified by
flash chromatography (3% MeOH in DCM). Yield: 80%. Rf: 0.95 (I ); Rf:
Materials and Methods
General
L-Leucinol-2-chlorotrityl resin (200–400 mesh, loading 0.45 mmol ⁄ g
resin) was purchased from Iris Biotech (Marktredwitz, Germany).
Fmoc-amino acids were supplied from Novabiochem (Merck Bio-
sciences, La Jolla, CA, USA), and all other amino acid derivatives and
reagents for peptide synthesis were purchased from Sigma–Aldrich
(St. Louis, MO, USA). O-[7-Azabenzotriaz-1-olyl]-tetramethyluronioum
hexafluorophosphate (HATU) was purchased from GLS (Shangai,
China). 5,6-Carboxyfluorescein (CF), cholesterol and L-a-phosphatidyl-
choline (PC) (from egg yolk, type XVI-E) were Sigma–Aldrich products.
20
0.85 (II); Rf: 0.50 (III); ½aꢀD )25.1 (c = 0.5, MeOH); IR(KBr): 3337, 1687,
1
1525 cm)1. H NMR (200 MHz, DMSO-d6): d 6.40 (d, 1H, NH Lol),
Ascending thin-layer chromatographies were routinely performed on
TLC plates of silica gel F254 (Merck, Darmstadt, Germany) using the
following eluant systems: 10% ethanol ⁄ CHCl3 (I); 10% acetic acid,
10% water ⁄ butan-1-ol (II); 15% ethanol ⁄ toluene (III); and UV detec-
tion (254 nm) for all compounds. Flash chromatography was per-
formed on silica gel 60 (0.040–0.063 mm; Merck) and a 15-cm long
column; eluant monitoring via TLC. Analytical HPLC separations
were carried out on a Dionex (Sunnyvale, CA, USA) Summit Dual-
Gradient HPLC apparatus, equipped with a four-channel UV–Vis
detector, using a Vydac C18 or C4 column (250 · 4.6 mm, 5 lm,
flow rate at 1.5 mL ⁄ min) from W. R. Grace (Columbia, MD, USA).
To prepare the mobile phase, deionized water was further purified
using a milliQ reagent grade water system from Millipore (Bedford,
MA, USA). The eluants A [aqueous 0.1% trifluoroacetic acid (TFA)]
and B (90% aqueous acetonitrile containing 0.1% TFA) were used
for preparing binary gradients. Analyses were carried out under the
following elution conditions: A – 3 min isocratic 35% B; linear gra-
dient 35–90% B in 30 min; B – 3 min isocratic 50% B; linear gradi-
ent 50–90% B in 30 min. Semi-preparative HPLC was carried out
on a Shimadzu (Kyoto, Japan) series LC-6A chromatographic appara-
tus, equipped with two independent pump units, an UV–Vis detec-
tor, and a Vydac C18 column (250 · 22 mm, 10 lm, flow rate at
15 mL ⁄ min). Elutions were carried out with the same mobile phases
described earlier, using a linear gradient from 35% to 90% B in
20 min. Melting points were taken on a Bꢀchi (Flawil, Switzerland)
150 apparatus and were not corrected. Optical rotations [a]D were
4.54 (t, 1H, OH Lol), 3.45 (m, 1H, aCH Lol), 3.34–3.10 (m, 2H, CH2OH
Lol), 1.58 (m, 1H, cCH Lol), 1.36 (s, 9H, CH3 Boc), 1.22 (m, 2H, bCH2
Lol), 0.86 (d, 3H, dCH3 Lol), 0.83 [d, 3H, dCH3 Lol).
tert-Butyloxycarbonyl-L-leucinol benzylether (Boc-Lol-Bzl). A solu-
tion of Boc-Lol (4.87 g, 22 mmol) in anhydrous THF was cooled at
0 ꢀC and NaH (60% dispersion in oil, 1 g, 25 mmol) was added.
When the evolution of gas ceased, Bzl-Br (2.73 mL, 23 mmol) and
tetrabutylammonium iodide (0.8 g, 2.3 mmol) were added and the
reaction mixture was stirred overnight at room temperature. The
solvent was removed in vacuo, the residue was taken up with ethyl
acetate (EtOAc) and washed with water. The organic phase was
dried over Na2SO4 and evaporated to dryness. The crude product
was purified by flash chromatography (10% EtOAc in petroleum
ether). Yield: 68%. Rf: 0.95 (I ); Rf: 0.90 (II ); Rf: 0.80 (III );
20
½aꢀD = )34.6 (c = 1, MeOH); IR (KBr): 3351, 1712 cm)1
;
1H NMR
(200 MHz, DMSO-d6): d 7.31 (m, 5H, CH arom Bzl), 6.62, (d, 1H, Lol
NH), 4.45 (s, 2H, Bzl CH2), 3.66 (m,1H, aCH), 3.28 (m, 2H, Lol
CH2OH), 1.59 (m, 1H, Lol cCH), 1.36 (s, 9H, Boc CH3), 1.24 (m, 2H,
Lol bCH2), 0.86 (d, 3H, Lol dCH3), 0.83 (d, 3H, Lol dCH3).
Synthesis of segments A, B, and C
A general procedure for the coupling reactions is given below. To a
solution of the appropriately N a-protected a-amino acid or peptide
carboxylic acid (4.33 mmol) in DCM, 1-hydroxybenzotriazole (HOBt),
170
Chem Biol Drug Des 2010; 75: 169–181

Medium-Length Peptaibiotic
or 7-aza-1-hydroxybenzotriazole (HOAt) (4.33 mmol) was added, and
the solution was cooled to 0 ꢀC in an ice bath. N-[3-(Dimethylamino)-
propyl]-N¢-ethylcarbodiimide hydrochloride (EDC)ÆHCl (4.33 mmol)
was added, followed by the solution of the amino component
(4.33 mmol) in DCM, obtained after acidolytic removal of the Boc-pro-
tecting group and NMM (4.33 mmol). The reaction mixture was stir-
red 1 h at 0 ꢀC, then at room temperature for 2–3 days, by keeping
the pH (moistened pH paper) to 8. The solvent was removed in vacuo,
the residue was dissolved in EtOAc and repeatedly washed with
0.5 M citric acid, water, 5% NaHCO3, and water. The organic phase
was dried over Na2SO4 and evaporated to dryness. The peptide prod-
uct was crystallized from the appropriate solvent mixture or purified
by flash chromatography. Physical data and analytical properties for
the newly prepared peptides are listed in Table 1.
EtOAc, and repeatedly washed with 0.5 M citric acid, water, 5%
NaHCO3, and water. The organic phase was dried over Na2SO4 and
concentrated in vacuo to a small volume. The peptide product was
precipitated by addition of petroleum ether. Yield: 84%.
20
M. p. 94–96 ꢀC; Rf 0.95 (I ); Rf 0.95 (II ); Rf 0.25 (III ); ½aꢀD )20.9
(c = 0.5, MeOH); IR (KBr): 3327, 1739, 1660, 1533 cm)1 1H-NMR
;
(400 MHz, CDCl3): d 8.19 (d, 1H, 1NH), 7.81 (m, 2H, 2NH), 7.63 (d, 1H,
1NH), 7.55 (d, 1H, 1NH), 7.49 (d, 1H, 1NH), 7.32 (m, 13H, 3NH, 5CH
arom Ser(Bzl), 5CH arom Lol-Bzl), 7.10 (d, 1H, 1NH), 5.15 (s, 1H, 1NH
Boc), 4.57 (m, 4H, 2 · CH2 Bzl), 4.32 (m, 1H, 1aCH), 4.17 (m, 2H,
2aCH), 4.02 (m, 5H, 5aCH), 3.85 (m, 2H, bCH2 Ser(Bzl)), 3.74 (s, 3H,
OCH3 Glu(OMe)), 3.70 (s, 3H, OCH3 Glu(OMe)), 3.54 (m, 1H, b¢CH2 Lol),
3.42 (m, 1H, b¢CH2 Lol), 2.45 (m, 5H, 2 · 2cCH2 and 1bCH2 Glu(OMe)),
2.45 (m, 1H, bCH2 Glu(OMe)), 2.01 (m, 2H, bCH2 Glu(OMe)), 1.87–1.40
(m, 42H, bCH2 and cCH Lol, bCH2 and cCH Leu, 3 CH3 Boc, 6 CH3
3Aib, 3CH3 3Ala), 0.89 (m, 12H, 2dCH3 Lol, 2dCH3 Leu).
Boc-Ala-Glu(OMe)-Ala-Aib-Ser(Bzl)-Aib-Ala-Leu-Aib-
Glu(OMe)-Lol-Bzl (segment B-C). Boc-Ala-Glu(OMe)-Ala-Aib-OH
(obtained by catalytical hydrogenolysis of fragment B) (0.27 g,
0.55 mmol) and HOAt (0.08 g, 0.55 mmol) were dissolved in DCM.
The mixture was cooled to 0 ꢀC and EDCÆHCl (0.10 g, 0.55 mmol)
was added, followed by a solution of H-Ser(Bzl)-Aib-Ala-Leu-Aib-
Glu(OMe)-Lol-Bzl (obtained as trifluoroacetate salt after acidolytic
deprotection of segment C) (0.50 g, 0.51 mmol) in DCM and NMM
(0.06 mL, 0.51 mmol). The reaction mixture was stirred 1 h at 0 ꢀC,
then at room temperature for 2–3 days, by keeping the pH to 8.
The solvent was removed in vacuo, the residue was dissolved in
Ac-Trp-Val-Aib-Aib-Ala-Glu(OMe)-Ala-Aib-Ser(Bzl)-Aib-
Ala-Leu-Aib-Glu(OMe)-Lol-Bzl (segment A-B-C). Ac-Trp-Val-
Aib-Aib-OH (obtained by catalytical hydrogenolysis of segment A)
(0.13 g, 0.27 mmol) was dissolved in DCM-dimethylformamide
(DMF) (1:1 v ⁄ v) and 0.04 g of HOAt (0.27 mmol) was added. The
mixture was cooled to 0 ꢀC and EDCÆHCl (0.052 g, 0.27 mmol) was
added, followed by a solution of the Na-amino deprotected segment
B-C, as trifluoroacetate salt, (0.24 g, 0.18 mmol) in DCM and
Table 1: Physical data and analytical properties for the newly synthesized peptide segments (the one letter code for amino acids is used,
where U stands for Aib)
TLC Rf valuesd
Yield M. p.
Crystallization
solventsb
20
c
Peptide
(%)a
(ꢀC)
½aꢀD
I
II
III
IR absorption (cm)1 e
)
Segment A
Boc-UU-OBzl
Boc-VUU-OBzl
Fmoc-WVUU-OBzl
Ac-WVUU-OBzl
Ac-WVUU-OH
78
68
71
92
99
122–123 EtOAc ⁄ PE
102–104 EtOAc ⁄ PE
122–124 EtOAc ⁄ PE
208–210 DE
–
0.90 0.95 0.55
0.95 0.85 0.50
0.95 0.90 0.40
3421, 3308, 1734, 1699, 1650, 1537
3324, 1741, 1686, 1660, 1524
3408, 3302, 1717, 1665, 1641, 1516
)6.9
)12.7
)7.1
0.95 0.95 0.50 3444, 3310, 3261, 1736, 1668, 1630, 1543, 1518
159–161
–
)12.3f 0.05 0.85 0.05
3416, 1657, 1521
Segment B
Boc-AU-OBzl
Boc-E(OMe)AU-OBzl
Boc-AE(OMe)AU-OBzl
Boc-AE(OMe)AU-OH
Segment C
g
80
86
80
99
122–123
)34
)39.2
)48.9
0.95 0.85 0.60
0.90 0.85 0.45
0.85 0.80 0.45
3318, 1739, 1661, 1522
3344, 3315, 3241, 1734, 1687,1652, 1540, 1525
3400, 3298, 1738, 1718,1663, 1634, 1526
3319, 1720, 1653, 1525
107–109 EtOAc ⁄ PE
147–148 EtOAc ⁄ PE
102–104
–
)36.6f 0.30 0.80 0.15
Boc-E(OMe)Lol-Bzl
Boc-UE(OMe)Lol-Bzl
Boc-LUE(OMe)Lol-Bzl
Boc-ALUE(OMe)Lol-Bzl
Boc-UALUE(OMe)Lol-Bzl
71
91
74
94
71
72–74
98–100 EtOAc ⁄ PE
114–115 EtOAc ⁄ PE
61–63
71–73
66–68
EtOAc ⁄ PE
)28.7
0.95 0.85 0.65
3336, 3300, 1731, 1685, 1649, 1553, 1526
3365, 3308, 1744, 1690, 1650, 1543, 1522
3306, 1741, 1684, 1644, 1521
)26.9f 0.95 0.90 0.50
)29.2f 0.95 0.90 0.45
)33.1f 0.95 0.90 0.45
)14.6f 0.70 0.80 0.35
)14.1f 0.95 0.95 0.40
EtOAc ⁄ PE
3311, 1740, 1722, 1646, 1530
3307, 1740, 1651, 1531
3310, 1738, 1722, 1661, 1530
EtOAc ⁄ PE
h
Boc-S(Bzl)UALUE(OMe)Lol-Bzl 65
DCM, dichloromethane.
aYields refer to the last step.
bEtOAc, ethyl acetate; PE, petroleum ether; DE, diethyl ether.
cc = 1, MeOH.
dFor eluants, see Materials and Methods.
eFor the solid-state IR absorption spectra, only bands in the 3500–3250 and 1800–1500 cm)1 regions are reported.
fc = 0.5, MeOH.
gFlash chromatography with 25% EtOAc in n-hexane.
hFlash chromatography with 4% MeOH in DCM.
Chem Biol Drug Des 2010; 75: 169–181
171

Gobbo et al.
NMM (0.05 mL, 0.30 mmol). The reaction mixture was stirred 1 h
at 0 ꢀC, then at room temperature for 2–3 days, by keeping the pH
to 8. The solvent was removed in vacuo, the residue was dissolved
in EtOAc, and repeatedly washed with 0.5 M citric acid, water, 5%
NaHCO3, and water. The organic phase was dried over Na2SO4 and
evaporated to dryness in vacuo. The crude product was purified by
flash chromatography (8% MeOH in DCM), and the desired peptide
was isolated in 40% yield.
treatments with 30% 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) in
DCM for 30 min. The filtrates were combined and evaporated to
dryness providing the crude protected peptide. Removal of the side-
chain-protecting groups was achieved by treatment with reagent K
(TFA⁄ phenol ⁄ thioanisole ⁄ water ⁄ 1,2-ethanedithiol, 82:5:5:5:2.5 by
vol) for 90 min. The cleavage mixture was concentrated under
reduced pressure. The product was precipitated by addition of
diethyl ether and lyophilized from a mixture of acetonitrile in 5%
aqueous acetic acid. The crude peptide (70 mg, 70% purity based
on HPLC) was purified by semi-preparative HPLC to afford the
desired product with a 95% purity in 43% yield. HPLC: (C18 gradient
20
M. p. 149–151 ꢀC; Rf 0.60 (I); Rf 0.85 (II); Rf 0.10 (III); ½aꢀD )29.8
(c = 0.5, MeOH); HPLC: (C18 gradient A) Rt 32.5 min, (C4 gradient B)
Rt 25.1 min. electrospray ionization mass spectrometry (ESI-MS)
(m ⁄ z): calcd for C88H132N16O21 1748.94; found 875.48 [M + 2H]2+. IR
20
A) Rt 18.24 min; ½aꢀD )15.0 (c = 0.1, MeOH); IR (KBr): 3418, 1658,
1538 cm)1; ESI-MS (m ⁄ z): calcd for C72H118N18O19 1538.85; found
770.42 [M+2H]2+, 781.90 [M+H+Na]2+, and 789.39 [M+H+K]2+. 1H-
NMR (600 MHz, CD3OH): d 10.42 (d, 1H, e-NH Trp1); 8.25 (s and d,
2H, NH Gln6 and Aib8); 8.19–8.17 (m, 2H, NH Leu12 and Trp1); 8.15
(s, 1H, NH Aib10) 8.02 (s and d, 2H, NH Aib13 and Ala5); 7.89 (s,
1H, NH Aib4); 7.87 (d, 1H, NH Ala11); 7.86 (d, 2H, NH Ala7 and
Ser9); 7.60–7.59 (m, 2H, 2 NH Val2 and Lol); 7.57 (s, 1H, NH Aib3);
7.52–7.50 (m, 3H, NH and dNH Gln14, H-4 Trp1); 7.42 (s, 1H, dNH
Gln6); 7.33–7.32 (d, 1H, H-7 Trp1); 7.22 (d, 1H, H-2 Trp1); 7.10–7.08
(t, 1H, H-6 Trp1); 7.00–6.97 (t, 1H, H-5 Trp1); 6.82 (s, 1H, dNH
Gln14); 6.72 (s, 1H, dNH Gln6); 5.35 (m, 1H, OH Lol); 5.19–5.14 (t,
1H, OH Ser9); 4.51–4.48 (m, 1H, a-CH Trp1); 4.20–4.15 (m, 1H, a-CH
Gln14); 4.18–3.98 (m, 8H, a-CH Ala5, a-CH Ala7, a-CH Gln6, a-CH
Ala11, a-CH Leu12, a-CH Lol15, a-CH and b-CH Ser9); 3.57–3.54 (m,
3H, a-CH Val2 and b2-CH2 Lol); 3.26–3.24 (m, 2H, b-CH2 Trp1);
2.59–2.46 (m, 3H, c-CH2 Gln6 and c-CH Gln14); 2.43–2.37 (m, 1H, c-
CH Gln14); 2.33–2.25 (m, 2H, b-CH Gln6 and b-CH Gln14); 2.21–2.18
(m, 1H, b-CH Gln6); 2.16–2.10 (m, 1H, b-CH Gln14); 2.04 (s, 3H, CH3
Ac); 1.94–1.85 (m, 3H, b-CH Val2, b-CH and c-CH Leu12); 1.79–1.72
(m, 1H, c-CH Lol); 1.65–1.60 (m, 2H, b-CH Leu12 and b1-CH Lol);
1.58 (s, 3H, b-CH3 Aib10); 1.57 (s, 3H, b-CH3 Aib8); 1.55 (s, 3H, b-
CH3 Aib13); 1.52–1.51 (m, 15H, b-CH3 Aib4, Ala7, Aib8, Ala11 and
Aib13); 1.50–1.496 (d, 3H, b-CH3 Ala5); 1.49 (s, 3H, b-CH3 Aib10);
1.48 (s, 3H, b-CH3 Aib4); 1.43 (s, 3H, b-CH3 Aib3); 1.42 (s, 3H, b-
CH3 Aib3); 1.37–1.31 (m, 1H, b1-CH Lol); 0.94–0.89 (m, 15H, 2 d-
CH3 Leu12, 2 d-CH3 Lol and c-CH3 Val2); 0.72–0.70 (d, 3H, c-CH3
Val2).
1
(KBr): 3328, 1740, 1660, 1538 cm)1. H-NMR (400 MHz, CD3OH): d
10.415 (d, 1H, e-NH Trp1); 8.25 (s, 1H, NH Aib10); 8.18–8.16 (m, 1H,
NH Trp1); 8.10–8.08 (d, 1H, NH Glu(OMe)6]; 8.04 (s and d, 2H, NH
Aib8 and Ser(Bzl)9); 7.98–7.96 (m, 2H, NH Ala5 and Leu12); 7.87–
7.86 (d, 1H, NH Ala11); 7.81 (s, 1H, NH Aib4); 7.79–7.78 (d, 1H, NH
Ala7); 7.73 (s, 1H, NH Aib13); 7.63–7.61 (d, 1H, NH Glu(OMe)14);
7.59–7.57 (d, 1H, NH Val2); 7.52–7.50 (m, 2H, NH Aib14, H-4 Trp1);
7.49–7.47 (d, 1H, NH Lol); 7.36–7.24 (m, 11H, H-7 Trp1, 2 · 5CH
arom Bzl); 7.22 (d, 1H, H-2 Trp1); 7.11–7.07 (t, 1H, H-6 Trp1); 7.01–
6.97 (t, 1H, H-5 Trp1); 5.19 (s, 2H, CH2-Ph Ser(Bzl)9); 4.52 (s, 2H,
CH2-Ph Lol-Bzl); 4.50–4.49 (m, 1H, a-CH Trp1); 4.24–3.19 (m, 1H, a-
CH Glu(OMe)14); 4.18–4.12 (m, 3H, a-CH Lol, a-CH and b-CH
Ser(Bzl)9); 4.11–3.94 (m, 5H, a-CH Ala5, a-CH Glu(OMe)6, a-CH Ala7,
a-CH Ala11 and a-CH Leu12); 3.89–3.88 (d, 1H, b-CH Ser(Bzl)9); 3.62
or
or
(s, 3H, CH3 Glu(OMe)6
14); 3.56 (s, 3H, CH3 Glu(OMe)6
14);
3.49–3.41 (m, 3H, a-CH Val2 and b2-CH2 Lol); 3.26–3.24 (m, 2H, b-
CH2 Trp1); 2.65–2.40 (m, 4H, c-CH2 Glu(OMe)6 and c-CH2
Glu(OMe)14); 2.35–2.22 (m, 2H, b-CH Glu(OMe)6 and b-CH
Glu(OMe)14); 2.15–2.06 (m, 2H, b-CH Glu(OMe)6 and b-CH
Glu(OMe)14); 2.04 (s, 3H, CH3 Ac); 1.94–1.85 (m, 3H, b-CH Val2, b-
CH and c-CH Leu12); 1.81–1.71 (m, 1H, c-CH Lol); 1.65–1.60 (m, 2H,
b-CH Leu12 and b1-CH Lol); 1.58 (s, 6H, b-CH3 Aib8 and Aib10); 1.53
(s, 3H, b-CH3 Aib13); 1.52–1.49 (m, 18H, b-CH3 Aib4, Ala7, Aib8,
Aib10, Ala11 and Aib13); 1.46 (s, 3H, b-CH3 Aib4); 1.42 (s, 6H, b-CH3
Ala5 and b-CH3 Aib3); 1.37–1.39 (bs, 4H, b-CH3 Aib3 and b1-CH
Lol); 1.02–1.01 (m, 1H); 0.91–0.87 (m, 15H, 2 d-CH3 Leu12, 2 d-CH3
Lol and c-CH3 Val2); 0.72–0.70 (d, 3H, c-CH3 Val2).
FTIR absorption
Solid-phase peptide synthesis (SPPS)
The solution FTIR spectra were recorded at 293 K using a Perkin-
Elmer model 1720X FTIR spectrophotometer, nitrogen flushed,
equipped with a sample-shuttle device, at 2 cm)1 nominal resolu-
tion, averaging 100 scans. Solvent (baseline) spectra were recorded
under the same conditions. For spectral elaboration, the software
SPECTRACALC provided by Galactic (Salem, MA, USA) was employed.
Cells with path lengths of 1.0 and 10 mm (with CaF2 windows)
were used. Spectrograde deuterated chloroform (99.8%, d2) was
purchased from Merck.
Tylopeptin B was synthesized in a 0.05 mmol scale, using an
Advanced Chemtech (Louisville, KY, USA) 348X peptide synthesizer,
starting from H-L-Lol-2-chlorotrityl resin. The trityl (Trt) group was
used for masking the Gln side chain, and Boc and tert-butyl groups
were used to protect the Trp and Ser side chains, respectively. 9-
Fluorenylmethoxycarbonyl (Fmoc) deprotection was achieved with
20% piperidine in DMF (5 + 15 min). Couplings were performed in
the presence of HATU ⁄ diisopropylethylamine (DIPEA) (reaction time
45–60 min or 120 min for Aib on Aib), using an excess of 4 equiva-
lents of the carboxyl component. A single coupling protocol was
used to acylate Lol, Leu12, Ala7, Gln(Trt)6, and Val2; in all other
cases, the coupling step was repeated. N-terminal acetylation of
the peptide-resin was performed by a repeated treatment with ace-
tic anhydride (0.75 mmol) and DIPEA (0.2 mmol) in DMF for 30 min.
The 1,2-aminoalcohol peptide was cleaved from the resin upon 3–4
Nuclear magnetic resonance
Nuclear magnetic resonance experiments were carried out on a
Bruker Avance model DMX-600 spectrometer. The peptide concen-
tration was 6.6 mM in CD3OH. The alcohol –OH signal was sup-
pressed by presaturation during the relaxation delay. All
172
Chem Biol Drug Des 2010; 75: 169–181

Medium-Length Peptaibiotic
homonuclear spectra were acquired by collecting 512 experiments,
each one consisting of 64 scans and 2K data points. The spin sys-
tems of protein amino acid residues were identified using standard
correlated spectroscopy (COSY) (19) and total correlation spectro-
scopy (TOCSY) (20) experiments. In the latter case, the spin-lock
pulse sequence was 70 ms long. The assignment of methyl groups,
belonging to the same Aib residue, was obtained by means of 2D
1H-13C correlation spectra. To optimize the digital resolution in the
carbon dimension, heteronuclear multiple quantum coherence
(HMQC) and heteronuclear multiple bond correlation (HMBC) experi-
ments were acquired using selective excitation by means of Gauss-
ian-shaped pulses with 1% truncation (21,22).
the interactions between the two b-geminal protons of the Leu and
Gln residues, set to a distance of 1.78 ꢁ. When peaks could not be
integrated because of partial overlap, a distance corresponding to
the maximum limit of detection of the experiment (4.0 ꢁ) was
assigned to the corresponding proton pair.
Distance geometry and MD calculations were carried out using the
simulated annealing (SA) protocol of the XPLOR-NIH 2.9.6 program
(26). For distances involving equivalent or non-stereo-assigned pro-
tons, r)6 averaging was used. The MD calculations involved a mini-
mization stage of 100 cycles, followed by SA and refinement
stages. The SA consisted of 30 ps of dynamics at 1500 K (10 000
cycles, in 3 fs steps) and of 30 ps of cooling from 1500 to 100 K in
50 K decrements (15 000 cycles, in 2 fs steps). The SA procedure,
in which the weights of nuclear Overhauser enhancement (NOE)
and non-bonded 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 (20 000
cycles, in 1 fs steps). Finally, the calculations were completed with
200 cycles of energy minimization using a NOE force constant of
50 kcal ⁄ mol. The generated structures were visualized using the
MOLMOL (27) (version 2K.2) program.
The Cb-selective HMQC experiments with gradient coherence selec-
tion (23) were recorded with 400 t1 increments, of 100 scans and
1K points each (24). A spectral width of 13 ppm centered at
24.5 ppm in F1 was used, yielding a digital resolution of 1.91 Hz ⁄ pt
prior to zero filling. HMBC experiments (25) with selective excitation
in the CO region were performed using a long-range coupling con-
stant of 7.5 Hz, a spectral width in F1 of 14 ppm centered at
175.5 ppm, 256 t1 experiments of 220 scans, and 2K points in F2.
The digital resolution in F1, prior to zero filling, was 2.21 Hz ⁄ pt.
rotating frame Overhauser enhancement (ROESY) experiments were
used for sequence-specific assignment. The mixing time of the
ROESY experiment used for inter-proton distance determination was
250 ms. Inter-proton distances were obtained by integration of the
ROESY spectra using SPARKY 3.111. The calibration was based on
the average of the integration values of the cross peaks because of
Circular dichroism
Circular dichroism spectra were recorded at 293 K using a Jasco
(Tokyo, Japan) model J-715 spectropolarimeter, equipped with a
Haake thermostat (Thermo Fisher Scientific, Waltham, MA, USA),
A
B
C
3
1
Trp
2
Val
4
13
Aib
5
Ala
6
8
9
10
Aib
11
Ala
12
14
7
Leu
Aib Aib
Lol
Glu(OMe)
Glu(OMe) Ala Aib
Ser(Bzl)
Boc
Bzl
i
Boc OH H Bzl
ii
Boc
Bzl
Bzl
Bzl
Bzl
Boc OH H
ii
Boc
Figure 1: Scheme for the
synthesis of the [Glu(OMe)6,14
,
Boc
OHH
Ser(Bzl)9, Lol-Bzl] (A-B-C) tylopep-
tin B analog. Reagents and condi-
tions: (i) 50% trifluoroacetic acid
in CH2Cl2, 1 h at room tempera-
ture. These conditions were used
to remove the Boc group through-
out synthesis; (ii) EDC ⁄ HOBt,
NMM, 1 h at 0 ꢀC, then room
temperature; (iii) EDC ⁄ HOAt,
NMM, 1 h at 0 ꢀC, then room
temperature; (iv) H2, Pd ⁄ C, 1 h at
room temperature; (v) 20% diethyl-
amine in CH2Cl2, 3 h; (vi) acetic
anhydride (10 equiv) and diisopro-
pylamine (five equiv), 5 h at room
temperature. NMM, N-methyl-
morpholine.
iii
Boc OBzl
Boc OBzl
Boc
Bzl
Bzl
Boc OHH OBzl
Boc OHH OBzl
Boc OH H
ii
ii
ii
Boc
OBzl
OBzl
OBzl
Boc
OBzl
OBzl
OBzl
Bco
Bzl
Bzl
Boc OH
H
Boc OHH
Boc OHH
ii
iii
iii
Boc
Bzl
Bzl
Bzl
Bzl
Boc
Boc
Fmoc OH H
Boc OHH
OBzlBoc OHH
OBzl
ii
iii
ii
Boc
OBzl
OBzl Boc
Fmoc
v
H
iv
OH
OBzlBoc
H
vi
Ac
iii
OBzlBoc
iv
Bzl
Bzl
Bzl
H
Ac
Ac
OH
iii
Chem Biol Drug Des 2010; 75: 169–181
173

Gobbo et al.
averaging eight scans. Baselines were corrected by subtracting the
solvent contribution. Cylindrical, fused quartz cells of 0.5 and
0.2 mm path length (Hellma, Mꢀllheim, Germany) were employed.
The data are expressed in terms of [h]R, the residue molar ellipticity
(deg ⁄ cm2 ⁄ dmol). MeOH 99.9%, spectrophotometric grade, and
2,2,2-trifluoroethanol (TFE) were purchased from Acros Organics.
Sodium dodecyl sulphate (SDS), 99% purity, was a Pierce Chem.
Co. (Rockford, IL, USA) product and was used without recrystalliza-
tion. Deionized water was further purified using a milliQ reagent
grade water system from Millipore.
Boc ⁄ OBzl-protecting groups for the peptide a-amino and a-carbox-
ylic functions, respectively. For the assembling of segment A,
instead of coupling directly Ac-Trp-OH to the tripeptide, we used
a urethane Na-amino protected derivative of this residue (Fmoc-
Trp-OH), to avoid any risk of racemization. After removal of the
base labile Fmoc group, Na-acetylation of segment
A was
achieved by treatment with acetic anhydride and DIPEA. The N-
protected, C-terminal 1,2-aminoalcohol Lol was prepared in a good
yield starting from Boc-Leu-OH, slightly modifying a literature pro-
cedure (36). The Ser and Lol primary alcoholic functions were pro-
tected as benzyl ethers by treatment with Bzl-Br ⁄ NaH (37) to
suppress any undesired acylation during the coupling reactions
and to improve the solubility of the peptide segments in solvents
of low polarity. Couplings of segments B to C (affording the seg-
ment B-C) and of segment A to B-C (giving the protected ana-
log of tylopeptin B) were performed by aid of EDC ⁄ HOAt. With
very few exceptions, the isolated yields of all coupling steps,
including those involving two peptide segments, were from moder-
ate to good (in the range 40–90%). The newly prepared final pep-
tides and their synthetic intermediates were characterized by
melting point determination, optical rotatory power, TLC in three
eluant systems, solid-state IR absorption (Table 1), and by RP-HPLC
Liposome leakage assay
Peptide-induced leakage from egg PC vesicles was measured at
293 K using the CF-entrapped vesicle technique (28) and a Perkin
Elmer model MPF-66 spectrofluorimeter. CF-encapsulated small unil-
amellar vesicles (egg PC ⁄ cholesterol, 7:3) were prepared by sonica-
tion in Hepes buffer, pH 7.4. The phospholipid concentration was
kept constant (0.06 mM), and increasing [peptide] ⁄ [lipid] molar ratios
(R)1) were obtained by adding aliquots of MeOH solutions of pep-
tides, keeping the final MeOH concentration below 5% by volume.
After rapid and vigorous stirring, the time–course of fluorescence
change corresponding to CF escape was recorded at 520 nm (6 nm
band pass) with kexc 488 nm (3 nm band pass). The percentage of
released CF at time t was determined as (Ft ) F0) ⁄ (FT ) F0) · 100,
with F0 = fluorescence intensity of vesicles in the absence of pep-
tide, Ft = fluorescence intensity at time t in the presence of pep-
tide, and FT = total fluorescence intensity determined by disrupting
the vesicles by addition of 50 lL of a 10% Triton X-100 solution.
The kinetics experiments were stopped at 20, 30, or 50 min.
1
(Figure 2) and H NMR.
Among the medium-length peptaibols, tylopeptin B is the most
accessible by automatic SPPS. Its sequence does not include any
acid-sensitive Aib-Pro ⁄ Hyp bond, as most of these peptaibols (2,14).
Furthermore, there are only two consecutive Ca-tetrasubstituted
amino acids in the sequence -Aib3-Aib4-, while all other Aib resi-
dues are flanked by proteinogenic amino acids. For the SPPS of
tylopeptin B, we employed a standard Fmoc ⁄ tBu approach (38),
based on HATU (35) as the coupling reagent. We used a double
acylation cycle for couplings involving an N-terminal Aib residue
and, only in the coupling involving the two consecutive Aib resi-
dues, we extended the reaction time from one to two hours. Using
this protocol, the final peptide-resin was obtained in more than
90% yield. The peptide-alcohol was cleaved from the chlorotrityl
resin by a mild acidic treatment (30% HFIP in DCM) (39), and the
Results and Discussion
Peptide synthesis
For the synthesis of the [Glu(OMe)6,14, Ser(Bzl)9, Lol-Bzl] analog of
tylopeptin B in solution, we planned a segment (A-B-C) conden-
sation strategy (Figure 1) on the basis of the following consider-
ations: (i) The availability of several segments will allow us to
prepare rapidly each desired analog of the peptaibol without the
need for a complete resynthesis of the 14-amino acid residue
peptide. (ii) The reactivity of the a-amino function of the Ca-tet-
rasubstituted a-amino acid Aib is remarkably lower than those of
protein residues (29,30). (iii) The significant risk of epimerization,
intrinsic to the segment condensation approach, should be
reduced as much as possible by planning N-terminal and central
segments with the achiral Aib residue at their C-terminus (30,31).
(iv) The presence of a Trp residue in the sequence suggests to
avoid an acidolytic removal of the protecting groups from the
final peptide (32,33).
A
B
We decided to split the sequence of the target analog into three
parts: segment A corresponding to the N-terminal sequence 1–4,
segment B spanning the sequence 5–8 (both segments bear an
Aib residue at the C-terminus), and segment C corresponding to
the peptide C-terminal sequence 9-14-Lol. The segments were syn-
thesized by a stepwise approach, using the EDC ⁄ HOBt (34) or the
EDC ⁄ HOAt (35) method for C-activation and adopting orthogonal
0
10
20
30
0
10
20
30
Time (min)
Time (min)
Figure 2: RP-HPLC profiles of synthetic tylopeptin B (A) and its
[Glu(OMe)6,14, Ser(Bzl)9, Lol-Bzl] (A-B-C) analog (B).
174
Chem Biol Drug Des 2010; 75: 169–181

Medium-Length Peptaibiotic
side-chain-protecting groups on Ser(tBu), Gln(Trt), and Trp(Boc) were
smoothly removed by reagent K, without damaging the acid-sensi-
tive Trp indole (40). After HPLC purification, tylopeptin B was
obtained in over 40% isolated yield. This result is comparable or
even better than those recently reported for tylopeptin A and other
peptaibols (41). The RP-HPLC profile of synthetic tylopeptin B is
shown in Figure 2.
Conformational analysis
A
A detailed analysis of the conformational preferences of synthetic
tylopeptin B and its [Glu(OMe)6,14, Ser(Bzl)9, Lol-Bzl] (A-B-C) analog
was carried out by use of FTIR absorption, CD, and 2D NMR
combined with MD calculations.
The FTIR absorption spectra in CDCl3 solution (Figure 3) are domi-
nated by a very intense absorption near 3305 cm)1 assigned to the
N-H stretching modes of strongly H-bonded amide groups (42–44).
Additional, but very weak bands (or shoulders) are seen at about
3410, 3430, and 3480 cm)1. The 3410 and 3430 cm)1 bands are
attributed to the N-H stretching modes of free (solvated) amide
groups, while the 3480 cm)1 band to the indole N-H stretching
mode of the Trp side-chain indole moiety. At the lowest concentra-
tion examined (0.1 mM), Figures 3A,B indicate that the 3305 cm)1
band is still quite intense, supporting the view that these two pep-
tides are characterized by an extensive set of intramolecular
B
A
15
10
a
5
0
b
-5
c
-10
200
220
240
Wavelength (nm)
C
B
10
5
0
a
-5
b
-10
200
220
240
Wavelength (nm)
Figure 3: FTIR absorption spectra in the N-H stretching (amide
A) region of: (A) synthetic tylopeptin B and (B) its [Glu(OMe)6,14
,
Figure 4: (A) Far-UV CD spectra of synthetic tylopeptin B in
MeOH (a), 2,2,2-trifluoroethanol (b), and H2O (c). (B) Far-UV CD
spectra of synthetic tylopeptin B (a) and its [Glu(OMe)6,14, Ser(Bzl)9,
Lol-Bzl] (A-B-C) analog (b) in 30 mM aqueous sodium dodecyl sul-
phate. Peptide concentration: 0.1 mM. CD, circular dichroism.
Ser(Bzl)9, Lol-Bzl] (A-B-C) analog in CDCl3 solution. Peptide concen-
trations: (a) 1.0 mM, (b) 0.1 mM. Part (C) FTIR absorption spectra of
the tylopeptin analog (A-B-C) and its shorter synthetic precursors
B-C and C. Peptide concentration: 1.8 mM in CDCl3.
Chem Biol Drug Des 2010; 75: 169–181
175

Gobbo et al.
C=OÆÆÆH–N H-bonds, typical of an overwhelmingly helical conforma-
tion. In contrast to the spectrum of the lipophilic analog (Figure 3B)
that of tylopeptin B (Figure 3A) does not change significantly upon
increasing peptide concentration, which suggests that self-aggrega-
tion does not play a major role in the behavior of this peptaibol.
Also, as expected for Aib-rich peptides (43,44), the intensity of the
3305 cm)1 band relative to those of the bands above 3400 cm)1
increases steadily upon peptide main-chain elongation from seg-
ment C to the longer peptide B-C and, eventually, to the tylopeptin
analog A-B-C (Figure 3C). This finding implies that the amount of
the folded forms for this class of peptides depends markedly on the
backbone length.
In three different environments (MeOH, TFE, and H2O), the tylopeptin
B CD spectrum exhibits two negative Cotton effects of moderate
intensities, located near 222 nm (n fi p* transition of the peptide
chromophore) (45,46) and 205 nm (parallel component of the split
peptide p fi p* transition) (Figure 4A). A third, positive Cotton
effect is apparent between 190 and 195 nm and attributed to the
perpendicular component of the split peptide p fi p* transition.
Overall, this general pattern is reminiscent of those shown by right-
handed, predominantly helical, peptides (45,46). Because the
observed ratio (R) between the ellipticities of the two negative
bands, [h]222 ⁄ [h]205, in MeOH and TFE solutions is in the range 0.8–
0.9 (47–49), the amount of a-helix structure is largely prevailing over
A
B
Figure 5: Amide NH proton
region of the ROESY spectra of
synthetic tylopeptin B (A) and its
[Glu(OMe)6,14, Ser(Bzl)9, Lol-Bzl]
(A-B-C) analog (B) in MeOH,d3
solution. Peptide concentration:
5.0 mM.
176
Chem Biol Drug Des 2010; 75: 169–181

Medium-Length Peptaibiotic
that of the 310-helix (50–52) in these two solvents. On the other
hand, the R value is about 0.4 in water, which points to the 310-heli-
cal conformation being much more populated than the a-helical con-
formation under these conditions. The spectrum of the tylopeptin
analog in aqueous SDS, which mimics a membrane environment, is
close to that of the parent compound and indicative of an a-helix
(Figure 4B). Our helical CD spectra of tylopeptin B, especially those
in aqueous solutions, are not surprising because this peptide,
despite being relatively short, is heavily based (about 40%) on the
non-proteinogenic, very strongly helicogenic, Aib residue (3–5).
A 2D-NMR investigation was performed in MeOH,d3 solution for
both tylopeptin B and its analog. The spin systems of the peptide
residues were identified using COSY and TOCSY spectra, while
HMQC and HMBC experiments were used for the assignment of
the Aib residues (53). The sequential assignments were performed
using ROESY and CO-selective HMBC experiments (the proton chem-
ical shift assignments are reported in Tables S1 and S2, Supporting
Information). A summary of the conformationally significant inter-
residue NOE connectivities, found in the ROESY spectrum of tylo-
peptin B, is reported in Figure S1.
A
B
Figure 6: Fingerprint region of
the ROESY spectra of synthetic
tylopeptin B (A) and its
[Glu(OMe)6,14, Ser(Bzl)9, Lol-Bzl]
(A-B-C) analog (B) in MeOH,d3
solution. Peptide concentration:
5.0 mM. The CaH(i) fi NH(i + 2),
CaH(i) fi NH(i + 3), and
CaH(i) fi NH(i + 4) medium-
range connectivities are high-
lighted.
Chem Biol Drug Des 2010; 75: 169–181
177

Gobbo et al.
A
Ser9
Gln14
Gln6
Gln14
B
Ser9
Figure 7: Representations of
the lowest-energy 3D-structure
obtained for synthetic tylopeptin
B. The views perpendicular (A) to
and parallel (B) to the peptide
helix axis are shown. In both
views, the three hydrophilic
residues are labeled.
Gln6
The pattern of NH(i) fi NH(i + 1) connectivities (Figure 5) strongly
suggests the onset of an overall helical structure for both peptides.
More relevant 3D-structural information was extracted from the
fingerprint region of the ROESY spectra (Figure 6). Both spectra in
Figure 6 show evidence for da,N (i, i + 2), (i, i + 3), and (i, i + 4)
medium-range connectivities, diagnostic of the presence of both
310- and a-helical conformations (53).
methyl carbons if 2 ppm or higher has been ascribed to the pres-
ence of a helical conformation. In Table S3 (Supporting Information),
the CNE values for the five Aib residues of tylopeptin B are
reported. All values are significantly higher than 2 ppm, confirming
the occurrence of a helical structure in MeOH solution. The stereo-
specific assignment of the two diastereotopic methyl groups was
achieved from the Cb-selective HMQC spectrum, using the method
reported by Bellanda et al. (54).
The two side-chain methyl groups in Aib residues belonging to chi-
ral peptides are diastereotopic. Consequently, the carbon atoms of
the two methyl groups (labeled as b and b¢) are magnetically non-
equivalent and therefore have two different chemical shifts, above
and below 25 ppm. The observed difference in their 13C chemical
shifts (termed chemical non-equivalence, CNE) of the two pro-chiral
The conformational properties of tylopeptin B were further investi-
gated by distance geometry and restrained MD calculations. A total
of 118 inter-proton distance restraints, derived from the ROESY
spectrum (Table S4A, Supporting Information), were used in the SA
protocol. Of the 150 structures that were generated, 78 had viola-
Val2
Aib13
Val2
Ser9
Ala5
Lol15
Gln6
Leu12
Aib8
Ala5
Ser9
Ala11
Aib10
Aib3
Leu12
Trp1
Gln14
Trp1
Gln6
Gln14
Aib3
Aib8
Aib4
Figure 8: Schiffer-Edmundson
helical wheel projections of tylo-
peptin B: a-helix (A) and 310-helix
(B). The hydrophilic residues
Ala7
Aib13
Lol15
Ala7
Aib10
Aib4
Ala11
Gln6,14 and Ser9 are highlighted.
A
B
178
Chem Biol Drug Des 2010; 75: 169–181

Medium-Length Peptaibiotic
tions to the NOE restraints lower than 0.5 ꢁ. The 10 structures
with a total energy <270 kcal ⁄ mol were selected. All these struc-
tures converge to a well-defined, right-handed, largely predominant
a-helical conformation throughout the sequence, especially in the
segment 1–12, with a backbone average pairwise root-mean-square
deviation of 0.47 € 0.12 ꢁ (Table S4A, Supporting Information).
Unfortunately, because of heavily overlapped NOE signals, the heli-
cal structures in correspondence of the last three residues of the
sequence are not well defined. In any case, the residues belonging
to the central part of the sequence show torsion angle values cor-
responding to those characteristic of right-handed a-helices
(Table S4B, Supporting Information). The helical structures are stabi-
lized by intramolecular C=OÆÆÆH–N H-bonds (Table S5, Supporting
Information) of the a- (i ‹ i + 4) and 310- (i ‹ i + 3) helical
types. The latter H-bonds were found only near the C-terminus. The
lowest-energy 3D-structure of tylopeptin B, shown in Figure 7,
exhibits an (partially) amphiphilic character with the hydrophilic
Gln6, Ser9, and Gln14 residues being aligned on the same face of
the helix. However, it is worth pointing out that the orientation of
the Gln14 side chain is compatible with neither of the two Schiffer-
Edmunson helical wheel projections (full a- or full 310-) for tylopep-
tin B (Figure 8). The most reasonable explanation for this side-chain
arrangement is probably associated with the marked distortion
observed for the peptide helical structure near the C-terminus.
gin GA IV would be superior, even if slightly, with respect to that of
tylopeptin B. A modest increase is also observed if the fluorescence
analysis is stopped at times longer than the usual 20 min (28).
Conclusions
There is a perpetual need for new antibiotics. Undoubtedly, the
inevitable rise of resistant microbial strains will erode the utility of
modern antibiotic drugs. A current, promising and counteracting
approach deals with the search for new scaffolds, including proteo-
lytic enzyme-resistant, naturally occurring, peptide antibiotics (1). In
this connection, we are currently investigating the class of peptaibi-
otics (2).
In this work, we synthesized tylopeptin B, a medium-length pep-
taibiotic active against Gram-positive bacteria (16), and a selected,
more lipophilic, analog thereof. A detailed 3D-structural investiga-
tion, carried out by FTIR absorption, CD, and in particular
2D-NMR spectroscopic techniques, clearly demonstrated that this
14-mer peptide is mostly helical in solution. A micellar environ-
ment optimally supports this (partially) amphiphilic secondary struc-
ture. With regard to the details of its mode of action, although
the present data suggest that tylopeptin B would act directly on
the bacterial membrane, further studies are needed. To this end,
the easily tunable segment condensation approach (55,56) utilized
in this work for the solution-phase synthesis of the tylopeptin
analog will certainly prove to be useful. Indeed, in our laboratory,
we are presently working on the preparation of a variety of
appropriately selected tylopeptin B analogs, each characterized by
a site-specific replacement with an amino acid residue containing
a probe for electron paramagnetic resonance (EPR), fluorescence,
or solid-state NMR spectroscopy. Recently, we followed the same
approach in the study of long (alamethicin) and short (trichogin)
peptaibiotics (57–61).
Membrane-modifying properties
The membrane-modifying properties of synthetic tylopeptin B and its
[Glu(OMe)6,14, Ser(Bzl)9, Lol-Bzl] analog were preliminarily tested in
comparison with our typical reference compound for this type of
study, i.e. trichogin GA IV (10), by measuring the induced leakage of
CF entrapped in egg PC ⁄ cholesterol (7:3) small unilamellar vesicles
(Figure 9). It is worth noting that the membrane activity increases,
but to a limited extent, with the peptide hydrophobicity enhancement
(from the more hydrophilic, side-chain unprotected, tylopeptin B to
the more hydrophobic, side-chain protected, analog). In any case,
both compounds exhibit remarkable activity, although that of tricho-
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Figure S1: Conformationally significant inter-residue connectivi-
ties found in the ROESY spectrum of synthetic tylopeptin B
(MeOH,d3; T = 298 K).
Table S1: Proton chemical shift assignments for synthetic tylo-
peptin B (MeOH,d3; T = 298 K)
Table S2: Proton chemical shift assignments for the synthetic
tylopeptin B analogue [Glu(OMe)6,14, Ser(Bzl)9, Lol-Bzl] (MeOH,d3;
T = 298 K)
Table S3: Chemical shifts of the 13Cb carbon of the methyl
groups of the Aib residues of synthetic tylopeptin B and the corre-
sponding CNE values (b¢: proR bCH3)
Table S4: (A) NOE constraints, deviations from the idealized
geometry, and mean energies for the NMR-based structure of syn-
thetic tylopeptin B. (B) Average values [ꢀ] of backbone torsion
angles um and wm and the relative standard deviations resulting
from the 10 lowest-energy structures (energy < 270 kcal ⁄ mol) calcu-
lated for synthetic tylopeptin B
Table S5: Average distances and H–NÆÆÆO angles (ꢀ) and their
standard deviations measured for the intramolecular H-bonds of the
10 lowest-energy structures (energy < 270 kcal ⁄ mol) calculated for
synthetic tylopeptin B
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Peggion E., Galvez J.A., Diaz-de-Villegas M.D., Cativiela C., Toni-
olo C. (2003) Lipopeptaibol metabolites of Tolypocladium geodes:
total synthesis, preferred conformation, and membrane activity.
Chemistry;9:3567–3576.
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of alamethicin F50 ⁄ 5 by an easily tunable segment condensation
approach. Biopolymers;76:485–493.
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corresponding author for the article.
Chem Biol Drug Des 2010; 75: 169–181
181