Spectroscopically labeled peptaibiotic analogs: The 4-nitrophenylalanine infrared absorption probe inserted at different positions into trichogin GA IV

Article abstract of DOI:10.1002/psc.2475

A set of three analogs of the 10-residue, membrane-active lipopeptaibiotic trichogin GA IV, labeled with the promising 4-nitrophenylalanine IR absorption probe for local polarity, was synthesized by the solid-phase methodology, chromatographically purified, and extensively characterized. A single residue modification was inserted near the N-terminus, in the central region, or at the C-terminus. A solution conformational analysis, carried out by FT-IR absorption, CD, and 2D-NMR combined with molecular dynamics calculations, indicates that the mono-labeled analogs maintain the overall helical properties of the parent compound. Membrane permeabilization measurements and antimicrobial tests revealed that they possess membrane-modifying properties and in vitro antibacterial activities analogous to those of the natural lipopeptaibiotic. Our IR absorption and attenuated total reflectance investigations in various environments, from which water was excluded for solubility reasons, showed that the 1350cm^-1 4-nitrobenzyl band is a reporter group of rather limited sensitivity.

Full text of DOI:10.1002/psc.2475

Special Issue Article
Received: 30 October 2012
Revised: 22 November 2012
Accepted: 23 November 2012
Published online in Wiley Online Library: 27 December 2012
(wileyonlinelibrary.com) DOI 10.1002/psc.2475
Spectroscopically labeled peptaibiotic analogs:
the 4-nitrophenylalanine infrared absorption
probe inserted at different positions into
trichogin GA IV{
Cristina Peggion,a* Barbara Biondi,a Marta De Zotti,a Simona Oancea,b
Fernando Formaggioa and Claudio Tonioloa
A set of three analogs of the 10-residue, membrane-active lipopeptaibiotic trichogin GA IV, labeled with the promising
4-nitrophenylalanine IR absorption probe for local polarity, was synthesized by the solid-phase methodology, chromato-
graphically purified, and extensively characterized. A single residue modification was inserted near the N-terminus, in
the central region, or at the C-terminus. A solution conformational analysis, carried out by FT-IR absorption, CD, and
2D-NMR combined with molecular dynamics calculations, indicates that the mono-labeled analogs maintain the overall
helical properties of the parent compound. Membrane permeabilization measurements and antimicrobial tests revealed
that they possess membrane-modifying properties and in vitro antibacterial activities analogous to those of the natural
lipopeptaibiotic. Our IR absorption and attenuated total reflectance investigations in various environments, from which
water was excluded for solubility reasons, showed that the 1350 cmꢀ1 4-nitrobenzyl band is a reporter group of rather
limited sensitivity. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: antibacterial activitiy; conformation analysis; infrared absorption; membrane activity; 4-nitrophenylalanine; peptaibol synthesis
aim at explaining in detail the mechanism of interactions of this
lipopeptaibiotic with membrane model systems [15–40].
Introduction
Peptaibiotics are members of a unique class of membrane-active
compounds of fungal origin [1]. These antibiotic peptides are
characterized by a linear sequence of up to 19 a-amino acids,
a very high percentage of the Ca-tetrasubstituted and strong
helical inducer Aib (a-aminoisobutyric acid) residue [2–4], an N-
terminal acyl group, and a C-terminal 1,2-(or b-)amino alcohol.
The long-sequence peptaibiotics, e.g. alamethicin, generate
channels in the membranes thereby modifying their permeability
even in the absence of a voltage [5,6]. The short-sequence
peptaibiotics, which possess a fatty acyl chain at the N-terminus,
are referred to lipopeptaibiotics [7–9].
To further expand this ongoing project, in this work, we
synthesized, characterized, and investigated the 3D structural,
biological, and spectroscopic properties of three Phe(NO2)
(4-nitrophenylalanine)-containing analogs of trichogin GA IV, each
labeled with a side-chain nitrobenzyl IR absorption probe [41,42].
These chromophoric residues were incorporated at position 3, 7,
or 10, i.e. near the N-terminus, near the center, or near the C-
terminus of the trichogin GA IV sequence, where they replace other
hydrophobic residues, namely Leu, Leu, or Ile, respectively.
Phe(NO2) is a remarkable residue due to its recently described
properties: (i) It can be genetically, position-specifically, incorpo-
rated with high yield and fidelity into proteins [43–45] and
synthetically into peptides. (ii) Its nitroaromatic moiety is able
to quench the intrinsic fluorescence of nearby Trp residues
or the artificially generated fluorescence of 1-pyrenyl-alanine
Trichogin GA IV, isolated by Auvin-Guette et al. [10] from
Trichoderma longibrachiatum, is the most extensively investigated
lipopeptaibiotic. Its amino acid sequence is given in the following,
where nOct is n-octanoyl and Lol is leucinol.
1
5
10
nOct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Lol
*
Correspondence to: Cristina Peggion, Department of Chemistry, University of
Padova, via Marzolo 1, 35131 Padova, Italy. E-mail: cristina.peggion@unipd.it
In a series of collaborative efforts, we have recently shown that
trichogin GA IV, and its easier-to-synthesize [Leu11-OMe] (OMe,
methoxy) analog as well, are slightly amphiphilic, right-handed,
mixed 310-/a-helical [11–13] peptides with a remarkable capability
to alter significantly the membrane permeability despite having a
peptide main-chain too short to span it completely [14–16].
In recent years, we and others performed a systematic study of a
large set of synthetic trichogin GA IV analogs and short sequences
thereof by use of a variety of physico-chemical techniques with the
{
Special issue devoted to contributions presented at the 13th Naples Workshop
on Bioactive Peptides, June 7–10, 2012, Naples.
a ICB, Padova Unit, CNR, Department of Chemistry, University of Padova, via
Marzolo 1, 35131, Padova, Italy
b Department of Biochemistry and Toxicology, University ‘Lucian Blaga’, 550012,
Sibiu, Romania
J. Pept. Sci. 2013; 19: 246–256
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

TRICHOGIN GA IV
or para-anthraniloyl-a,b-diaminopropionic acid inserted into
proteins via site-directed mutagenesis [43–45]. (iii) It can serve
as an efficient IR absorption reporter for vibrational energy arrival
(heat transfer) to this amino acid from a distant, excited moiety in
the peptide chain [42]. (iv) It represents a potentially useful IR
absorption sensor of the environment polarity if inserted into
a globular protein [41] or, by extrapolation, into a membrane-
active peptide. In this article, we describe our results on the
trichogin GA IV analogs mentioned earlier where we examined
this last Phe(NO2) property.
(Waters, Milford, MA) C18 column using eluants with different
composition from pure water to 100% CH3CN. The fractions
collected were characterized by analytical RP-HPLC on a Jupiter
Phenomenex (Torrance, CA) C18 column (4.6ꢁ 250 mm, 5 m,
300 Å) using an Agilent (Santa Clara, CA) 1200 HPLC pump. The
binary elution system used was (A) 0.05% TFA (trifluoroacetic acid)
in H2O; (B) 0.05% TFA in CH3CN/H2O (9:1 v/v); gradient 60–90% B
in 20 min (flow rate 1 ml/min); spectrophotometric detection
at l = 216 nm. Electrospray ionization mass spectrometry was
performed by using a PerSeptive Biosystem Mariner instrument
(Framingham, MA). The amino acid sequences, mass spectral
data, and HPLC retention times of the final products are reported
in Table 1. The HPLC elution profiles are shown in Figure 1.
Complete 1H NMR (400 MHz Bruker Advance DRX model) charac-
terizations in 99.8% d3 CD3CN (Euriso-top, Saint Aubin, France)
are given in the following text. Despite various attempts, none
of the final peptides furnished any single crystal suitable for an
X-ray diffraction analysis.
Materials and Methods
Solid-Phase Peptide Synthesis and Characterization of the
Final Products
Fmoc-Phe(NO2)-OH (Fmoc, fluorenyl-9-methoxycarbonyl) was a
Bachem (Bubendorf, Switzerland) product. The other Fmoc-amino
acids were supplied from Novabiochem (Merck Biosciences, La
Jolla, CA). All other amino acid derivatives and reagents for peptide
synthesis were purchased from Sigma-Aldrich (St. Louis, MO).
Assembly of peptides on the Advanced ChemTech (Louisville, KY)
348 Ω peptide synthesizer was performed on a 0.044 mmol scale
by the FastMoc methodology starting with the Lol-substituted 2-
chlorotrityl resin (Iris Biotech, Marktredwitz, Germany) [46–48]
(110 mg, loading 0.40 mmol/g). Each of the three syntheses
required about 30 h. Most of the peptides bonds (those
involving protein amino acids only) were formed by the use
of O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HBTU) [49,50], 1-hydroxy-benzotriazole (HOBt) [51],
N,N0-diisopropylethylamine, single acylation, 45 min coupling time,
and N-methylpyrrolidin-2-one as the solvent. Five of the six
coupling steps of the three Aib residues [52], performed in the
presence of the strongly activating agent O-(7-azabenzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)
[49,50], were doubled and followed by a capping procedure
using acetic anhydride in a large excess. Removal of the Fmoc
Na-protecting group was carried out with a 20% piperidine
solution in N,N-dimethylformamide. The nOct moiety at the N-
terminus was introduced on the Aib residue at position 1 in the
last synthetic step by exploiting the preformed activated ester
of n-octanoic acid, obtained by reaction with an equivalent
amount of N-ethyl-N0-[3-(dimethylamino) propyl] carbodiimide
(EDC) and 7-aza-1-hydroxybenzotriazole (HOAt) [49,50] in the
presence of N-methylmorpholine. The yields of the final purified
products were 79–83% (40–43 mg, equivalent to 0.035–0.037 mmol,
each). The purity percentage of the final crude materials was
90–92%. The peptides cleaved from the resin were filtered and
collected. This step was repeated three times. The solutions were
concentrated under a flow of nitrogen. The crude peptides
were purified by reversed-phase chromatography on SepPak
Trichogin GA IV [Phe(NO2)]3 analog
1H NMR (400 MHz, CD3CN): 8.138 (m, 2H, Phe(NO2)3 arom.); 7.926
(d, 1H, Phe(NO2)3 NH); 7.854 (t, 1H, Gly6 NH); 7.736 (t, 1H, Gly5 NH);
7.599 (s, 1H, Aib4 NH); 7.596 (s, 1H, Aib8 NH); 7.568 (t, 1H, Gly9 NH);
7.509 (m, 2H, Phe(NO2)3 arom.); 7.496 (d, 1H, Leu7 NH); 7.482
(t, 1H, Gly2 NH); 7.335 (d, 1H, Ile10 NH); 7.258 (s, 1H, Aib1 NH);
6.839 (d, 1H, Lol11 NH); 4.494 (m, 1H, Phe(NO2)3 a-CH); 4.096
(m, 1H, Leu7 a-CH); 4.037 (m, 1H, Ile10 a-CH); 3.962 (m, 1H,
9 . 8 6
1 1 . 3 2
7
10
T
3
0
5
10
15
20
min
Figure 1. Analytical HPLC elution profiles (retention times) of (T)
trichogin GA IV, (3) the [Phe(NO2)]3 analog, (7) the [Phe(NO2)]7 analog,
and (10) the [Phe(NO2)]10 analog. Experimental conditions: Jupiter
Phenomenex C18 reversed phase column; gradient from 60% to 90% B
in 20min, where A = 0.05% TFA in a 9:1 (v/v) mixture of H2O/CH3CN
and B = 0.05% TFA in a 1:9 (v/v) mixture of H2O/CH3CN. The absorption
was monitored at 220 nm.
Table 1. Amino acid sequences, molecular masses, and retention times of the synthetic Phe(NO2) trichogin GA IV analogs studied
a
Peptide
Amino acid sequence
[M + H]+
[M + H]+
Retention time (min)
calcd
exptl
[Phe(NO2)]3-analog
[Phe(NO2)]7-analog
[Phe(NO2)]10-analog
nOct-Aib-Gly-Phe(NO2)-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Lol
nOct-Aib-Gly-Leu-Aib-Gly-Gly- Phe(NO2)-Aib-Gly-Ile-Lol
nOct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly- Phe(NO2)-Lol
1145.70
1145.70
1145.70
1145.69
11.32
9.86
1145.69
1145.70
15.01
aDetermined using ESI mass spectrometry (for details, see section on Materials and Methods).
J. Pept. Sci. 2013; 19: 246–256 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

PEGGION ET AL.
Lol11 a-CH); 3.8 (m, 2H, Gly6 a-CH); 3.753(dd, 1H, Gly2 a-CH);
3.742(dd, 1H, Gly9 a-CH); 3.737(m, 2H, Gly5 a-CH); 3.618 (dd, 1H,
Gly9 a-CH) 3.57 (dd, 1H, Gly2 a-CH); 3.43 (m, 2H, Lol11 b-CH2);
3.388 (m, 1H, Phe(NO2)3 b-CH); 3.188 (m, 1H, Phe(NO2)3 b-CH);
2.239 (m, 2H, nOct0 b-CH2); 1.926 (m, 1H, Ile10 b-CH); 1.647 (m, 1H,
Lol11 b-CH); 1.636 (m, 2H, Leu7 b-CH2); 1.619 (m, 1H, Leu7 g-CH);
1.597 (m, 1H, Ile10 g-CH); 1.461 (s, 6H, Aib4 b-CH3); 1.574 (m, 2H,
nOct0 g-CH2) 1.451 (s, 6H, Aib8 b-CH3) 1.428 (m, 1H, Lol11 b-CH);
1.418 (s, 6H, Aib1 b-CH3); 1.283 (m, 8H, nOct0); 1.276 (m, 1H, Ile10
g-CH); 1.198 (m, 1H, Lol11 g-CH); 0.925 (m, 3H, Ile10 g-CH3); 0.867
(m, 6H, Leu7 d-CH3); 0.867 (m, 6H, Lol11 d-CH3); 0.861 (m, 3H, nOct0
o-CH3); 0.841 (d, 3H, Ile10 d-CH3).
(Salem, MA) was employed. Cells with path lengths of 1.0 and
10 mm (with CaF2 windows) were used. Spectrograde CDCl3
(99.8%, d), CD3CN (99.8%, d3) and methanol were purchased from
Merck (Darmstadt, Germany).
The attenuated total reflectance (ATR) measurements were
obtained with a Nicolet-Nexus (Waltham, MA) instrument. A
germanium crystal and a mercury cadmium telluride detector
cooled to the liquid N2 temperature were used. Averaged 100 scans
between 4000 and 1000 cmꢀ1 were performed.
Circular Dichroism Spectroscopy
The CD spectra were measured on a Jasco (Tokyo, Japan) model
J-715 spectropolarimeter equipped with a Haake thermostat
(Thermo Fisher Scientific, Waltham, MA). Baselines were corrected
by subtracting the solvent contribution. A fused quartz cell of
1.0 mm path length (Hellma, Mühlheim, Germany) was used. The
values are expressed in terms of [θ]T, the total molar ellipticity
(degꢁ cm2 ꢁ dmolꢀ1). Spectrograde 2,2,2-trifluoroethanol (TFE)
(Acros Organics, Geel, Belgium) was employed as solvent. The CD
measurements in TFE and in 100 mM aqueous sodium dodecyl
sulfate (SDS) were carried out with a fused quartz cell of 0.5 mm
path length.
Trichogin GA IV [Phe(NO2)]7 analog
1H NMR (400 MHz, CD3CN): 8.114 (m, 2H, Phe(NO2)7 arom.); 7.867
(t, 1H, Gly6 NH); 7.727 (t, 1H, Gly5 NH); 7.701 (d, 1H, Phe(NO2)7 NH);
7.653 (d, 1H, Leu3 NH); 7.636 (t, 1H Gly9 NH); 7.586 (s, 1H, Aib8 NH);
7.516 (m, 2H, Phe(NO2)7 arom.); 7.479 (s, 1H, Aib4 NH); 7.399 (t, 1H,
Gly2 NH); 7.269 (d, 1H, Ile10 NH); 7.162 (s, 1H, Aib1 NH); 6.609
(d, 1H, Lol11 NH); 4.411 (m, 1H, Phe(NO2)7 a-CH); 4.164 (m, 1H, Leu3
a-CH); 4.015 (m, 1H, Ile10 a-CH ); 3.896 (m, 1H, Lol11 a-CH); 3.784 (dd,
1H, Gly2 a-CH); 3.757 (m, 2H, Gly5 a-CH); 3.746 (m, 2H, Gly6 a-CH);
3.668 (m, 2H, Gly9 a-CH); 3.623 (dd, 1H, Gly2 a-CH); 3.390 (m, 2H,
Lol11 b-CH2); 3.389 (m, 1H, Phe(NO2)7 b-CH); 3.184 (m, 1H, Phe
(NO2)7 b-CH); 2.211 (m, 2H, nOct0 b-CH2); 1.917 (m, 1H, Ile10 b-CH);
1.685 (m, 2H, Leu3 b-CH2); 1.660 (m, 1H, Leu3 g-CH) 1.585 (m, 2H,
nOct0 g-CH2); 1.563 (m, 1H, Lol11 b-CH2); 1.557 (m, 1H, Ile10 g-CH);
1.474 (s, 6H, Aib4 b-CH3); 1.452 (s, 6H, Aib8 b-CH3); 1.392 (s, 6H,
Aib1 b-CH3); 1.304 (m, 1H, Lol11 g-CH); 1.299 (m, 8H, nOct0 CH);
1.267 (m, 1H, Ile10 g-CH); 1.183 (m, 1H, Lol11 g-CH); 1.054 (m, 3H,
Ile10 d-CH3); 0.912 (m, 1H, Ile10 g-CH); 0.918 (m, 6H, Leu3 d-CH3);
0.912 (m, 3H, Ile10, g-CH3); 0.881 (m, 3H, nOct0 o-CH3); 0.822
(m, 6H, Lol11 d-CH3).
Nuclear Magnetic Resonance Spectroscopy
A solution of [Phe(NO2)]10-trichogin GA IV (peptide concentration:
3.3 mM; pH 5) in a 100 mM SDS-d25 (CIL, Andover, MA) aqueous so-
lution (9:1 H2O/D2O) was used for the 2D-NMR analysis. All NMR
experiments were acquired on a Bruker Avance DMX-600 spec-
trometer using the TOPSPIN 1.3 software package, and recorded
at 313 K. Suppression of the H2O solvent signal was obtained by
applying a WATERGATE gradient program. The spin systems of
the trisubstituted amino acid residues were assigned with the
use of the standard Wüthrich procedure [53]. DQF-COSY [54],
CLEAN-TOCSY (spin-lock pulse, 70 ms) [55], and NOESY spectra
were acquired by collecting 512 experiments, each one consisting
of 32, 64, and 80 scans, respectively, and 2 K data points. To
achieve the stereospecific assignment of the two diastereotopic
methyl groups of the Aib residues, a Cb-selective heteronuclear
multiple quantum coherence (HMQC) [56,57] experiment was
acquired using selective excitation by means of Gaussian-shaped
pulses with 1% truncation [58,59]. This Cb-selective HMQC experi-
ment was recorded with 128t1 increments of 200 scans each
and 2 K points. A spectral width of 16 ppm, centered at 22 ppm
in F1, was employed. The sequential assignment was performed
by means of the NOESY experiment (mixing time, 150 ms). A set
of interproton distances was obtained by integration of the NOESY
spectrum, using the SPARKY 3.111 software package, and was
calibrated on the average of the values found for the interactions
between the geminal protons of Gly and Leu residues, set to
1.78 Å. From the molecular dynamics (MD) calculations, carried
out using the random simulated annealing (rSA) protocol of the
XPLOR-NIH 2.9.6 program [60], 150 structures were generated. The
topology of the nonstandard residue Phe(NO2) was built from
the parameters reported in the literature for nitrobenzene [61].
For distances involving equivalent or nonstereoassigned protons,
an rꢀ6 averaging was used. The MD calculations involved a minimi-
zation stage of 100 cycles, followed by SA and refinement stages.
The SA consisted of 30 ps of dynamics at 1500 K (10 000cycles in
3 fs steps) and 30ps of cooling from 1500 to 100 K in 50 K decre-
ments (15 000 cycles in 2 fs steps). The SA procedure, in which the
Trichogin GA IV [Phe(NO2)]10 analog
1H NMR (400 MHz, CD3CN): 8.114 (m, 2H, Phe(NO2)10 arom.); 7.928
(t, 1H, Gly6 NH); 7.771 (t, 1H, Gly5 NH); 7.694 (d, 1H Phe(NO2)10
NH); 7.685 (d, 1H, Leu3 NH); 7.673 (t, 1H, Gly9 NH); 7.615 (m, 2H,
Phe(NO2)10 arom.); 7.598 (d, 1H, Leu7 NH); 7.499 (s, 1H, Aib4
NH); 7.456 (t, 1H, Gly2 NH); 7.193 (s, 1H, Aib1 NH); 6.953 (d, 1H,
Lol11 NH); 4.396 (m, 1H, Phe(NO2)10 a-CH); 4.222 (m, 1H, Leu7
a-CH); 4.197 (m, 1H, Leu3 a-CH); 3.957 (m, 1H, Lol11 a-CH);
3.814 (m, 2H, Gly6 a-CH); 3.788 (dd, 1H, Gly2 a-CH); 3.747 (m,
2H, Gly5 a-CH); 3.695 (dd, 1H, Gly9 a-CH); 3.653 (dd, 1H Gly2
a-CH); 3.508 (dd, 1H, Gly9 a-CH); 3.456 (m, 2H, Lol11 b-CH2);
3.362 (dd, 1H, Phe(NO2)10 b-CH); 3.169 (dd, 1H, Phe(NO2)10 b-CH);
2.230 (t, 2H, nOct0 b-CH2); 1.73 (m, 2H, Leu7 b-CH2); 1.698 (m, 2H,
Leu3 b-CH2); 1.694 (m, 1H, Leu7 g-CH); 1.687 (m, 1H, Leu3 g-CH);
1.653 (m, 1H Lol11 g-CH); 1.594 (m, 2H, nOct0 g-CH2); 1.483 (s, 6H,
Aib4 b-CH3); 1.456 (m, 1H, Lol11 b-CH); 1.403 (s 6H, Aib1 b-CH3);
1.307 (m, 8H, nOct0); 1.292 (m, 1H, Lol11 g-CH); 0.925 (m, 6H, Leu3
d-CH3); 0.910 (m, 6H, Leu7 d-CH3); 0.904 (m, 3H, nOct0 o-CH3);
0.894 (m, 6H, Lol11 d-CH3).
FT-IR Absorption Spectroscopy
The FT-IR absorption spectra were recorded at 293 K using a
Perkin-Elmer (Waltham, MA) model 1720X FT-IR spectrophotome-
ter, nitrogen flushed, equipped with a sample-shuttle device, at
2 cmꢀ1 nominal resolution, averaging 100 scans. Solvent (baseline)
spectra were obtained under the same conditions. For spectral
elaboration, the software SPECTRACALC provided by Galactic
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TRICHOGIN GA IV
weights of NOE and nonbonded terms were gradually increased,
was followed by 200 cycles of energy minimization. In the SA refine-
ment stage, the system was cooled from 1000 to 100 K in 50 K
decrements (20 000 cycles in 1 fs step). Finally, the calculations
were completed with 200 cycles of energy minimization using a
NOE force constant of 50 kcal/mol. The structures generated were
visualized using the MOLMOL [62] version 2 K.2) program.
medium, pH 7.2–7.4, recommended by the National Committee
for Clinical Laboratory Standards [65] and 6-mm diameter
disks. In the case of streptococci, a sheep-blood agar medium
was exploited. Disks were prepared by using blank paper disks
(Whatman, Maidstone, UK) previously autoclaved and saturated
with the peptide solution. The peptide samples were dissolved
in dimethylsulfoxide such as to give a 10 mg/ml solution. An
inoculum size of 105 CFU (colony forming units)/ml (109 CFU/ml
in case of Pseudomonas aeruginosa) was swabbed onto the test
medium and left to dry at room temperature for about 15 min.
The peptide-impregnated disks (100 mg/disk) were placed asepti-
cally onto inoculated plates and incubated at 37 ꢂC. The results
were read after 24 h of incubation by measuring the inhibition
zones (in millimeters). The antibacterial activity of peptides was
tested against clinical isolates of bacteria and reference bacterial
strains: Staphylococcus aureus ATCC 25923, Streptococcus pyogenes
ATCC 19615, and Escherichia coli ATCC 25922, where ATCC
stands for American Type Culture Collection strains. The well-
known antibiotic tetracycline (10 mg/disk) was used as control
test (Table 2).
Liposome Leakage Assay
Peptide-induced leakage from small unilamellar vesicles (SUV)
was measured at 293 K using the carboxyfluorescein (CF)-entrapped
vesicle technique [63] and a Perkin–Elmer model MPF-66 spec-
trofluorimeter. CF-encapsulated SUV of egg phosphatidylcholine
(PC)/cholesterol (Ch), 7:3, and phosphatidylethanolamine (PE)/
phosphatidylglycerol (PG) 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 (Rꢀ1) were
obtained by adding dimethylsulfoxide aliquots solutions of pep-
tides, keeping the final concentration below 5% by volume. After
rapid and vigorous stirring, the time course of fluorescence change
corresponding to the CF escape was recorded at 520 nm (6nm
band pass) with lexc 488 nm (3nm 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 peptide,
and FT = total fluorescence intensity determined by disrupting the
vesicles by addition of 50ml of a 10% Triton X-100 solution. The
kinetics experiments were stopped at 20 min.
Results and Discussion
Solid-Phase Peptide Synthesis
We prepared a set of three novel trichogin GA IV analogs, the
primary structures of which are given in Table 1, where the Leu3,
Leu7, and Ile10 residues, respectively, are replaced by Phe(NO2).
For the automatic SPPS of these three spectroscopically
mono-labeled peptides, we applied our protocol for the prepa-
ration of trichogin GA IV (illustrated in Figure 2) [22,24,25,66]
that is markedly improved with respect to that of Hjørringgard
et al [67]. In Ref. [67], the authors reported isolated yields for
the final trichogin GA IV of 20–24%, which should be compared
Antibacterial Activity Assays
Peptide antibacterial activity was tested against Gram-positive
and Gram-negative bacteria by the standardized disk diffusion
Bauer et al. method [64] using the Müller–Hinton culture
Table 2. Antibacterial activities of trichogin GA IV and its Phe(NO2) analogs studied
Bacterial strain
Trichogin GA IV
3
7
10
Tetracyclinea
Staphylococcus aureus ATCC 25923
Streptococcus pyogenes ATCC 19615
Escherichia coli ATCC 25922
15
10
0
7
7
0
8
9
0
12
9
31
32
28
0
aControl.
Figure 2. SPPS protocol for the preparation of the three Phe(NO2) trichogin GA IV analogs (exemplified for the [Phe(NO2)]3 analog). The B* coupling of
the nOct group was performed with nOct-OH using the EDC/HOAt (2.5 equiv) C-activating method.
J. Pept. Sci. 2013; 19: 246–256 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

PEGGION ET AL.
with the 79–83% yields obtained in the syntheses of our Phe
(NO2) trichogin GA IV analogs. We believe that the major factor
responsible for these highly diverging yields has to be found in
the different methods used to couple the next amino acid on
the a-NH2 functionality of the Aib residue (this side of Aib is well
known to be dramatically critical for peptide bond formation
[52]). Indeed, it is widely accepted that the HATU C-activation
procedure (employed by us in this work) is by far more effective
than the HBTU/HOBt procedure [49,50] used by Hjørringgard
et al. [67].
Cleavage of peptides from the resin was (partially) achieved by
treatment with 30% 1,1,1,3,3,3-hexafluoroisopropanol in distilled
CH2Cl2 [48]. Although repeated three times, the yields of peptide
recovery were rather modest (60–65%). A markedly improved
outcome in removing the peptides from the resin was achieved
by a subsequent treatment with a 1% TFA solution in distilled
CH2Cl2. However, the HPLC profile of each crude synthetic
material, in addition to the desired peptide alcohol, revealed
the presence of a byproduct (Figure 3A), which was identified
as the Lol trifluoroacetyl ester by MS (Figure 3B). The Lol primary
alcoholic function was then fully regenerated by treatment of the
peptide trifluoroacetate with 1 M HCl (gas) in diethyl ether in the
presence of methanol for 60 min (transesterification).
C═O. . .H–N H-bonds. The position of the strong amide I (C═O
stretching mode) and amide II absorption maxima (1656–1658 cmꢀ1
and 1540–1542 cmꢀ1, respectively) confirm that the conformation
adopted is (310- and/or a-)helical [68–71]. The Leu (or Ile) to
Phe (NO2) replacements do not affect the FT-IR absorption
spectrum of trichogin GA IV.
Because trichogin GA IV is known to interact with lipid
membranes [15–40], our far-UV CD analysis on the three Phe
(NO2) trichogin GA IV analogs included two membrane mimetic
environments, namely TFE and SDS micelles. The occurrence
in each spectrum of two negative maxima near 205 and 222 nm
(related to the parallel component of the p ! p* transition and
the n ! p* transition of the peptide chromophore, respectively
[72,73]) and one positive maximum at about 195 nm (related to
the perpendicular component of the p ! p* transition) (Figure 4)
suggests the presence of a well developed right-handed
helical conformation.
The ellipticity ratio R = [θ]222/[θ]205 in the CD spectra affords
a preliminary estimate of the prevailing helical type (whether
3
10- or a-) adopted by the three analogs investigated. This
parameter is known to be lower than 0.35 for a large amount
of 310-helix and higher than 0.70 for a high population of
a-helix [74,75]. The experimentally observed R values are 0.77,
0.68, and 1.22 for the three Phe(NO2) analogs at position 3, 7,
or 10, respectively, in aqueous SDS (Figure 4B), whereas they
are 0.46, 0.56, and 0.72 for the same analogs in TFE solution
(Figure 4A). From these data, we can safely conclude that a
high amount of a-helix occurs for all three analogs in aqueous
SDS, while a mixed 310-/a-helix (with a largely prevailing a-helix
component for the [Phe(NO2)]10 analog) is adopted in TFE.
These observations are in general agreement with the known
tendency of natural trichogin GA IV to fold into a well devel-
oped a-helix in aqueous SDS [16]. Interestingly, in both envir-
onments examined, the [Phe(NO2)]10 analog is remarkably
more a-helical than its two isomeric peptides. A Phe(NO2)
model compound was reported to show very low ellipticities
in the far-UV region [76,77]. As a result, nitroaromatic contri-
butions are not expected to complicate the CD spectra in
the region of the peptide Cotton effects for the Phe(NO2)
trichogin GA IV analogs investigated in this work.
Conformational Studies
The FT-IR absorption spectra of trichogin GA IV and its three Phe
(NO2) analogs in the amide A (3500–3200 cmꢀ1) region in CDCl3
solution at 1 mM concentration (not shown) are dominated by a
very intense band at 3313–3319 cmꢀ1 (N–H stretching mode of
H-bonded peptide groups [68–71]) accompanied by a very
weak absorption between 3400 and 3450 cmꢀ1 (N–H stretching
mode of free peptide groups). These latter bands disappear upon
increasing peptide concentration to 10 mM. This finding supports
the view that a modest self-aggregation of the analogs does take
place at concentration higher than 1 mM in this solvent of low
polarity. In any case, even at the lowest concentration examined
(0.1 mM), the main band at about 3315 cmꢀ1 is still very intense,
strongly suggesting that the conformation adopted by these
three peptides is stabilized by an extensive set of intramolecular
1145.7041
100
1000
90
A
B
80
800
1146.6943
70
60
50
40
30
600
400
*
200
0
20
10
0
1147.7063
1147.1913
1148.1528
1149.7092
1241.7099
1244.6263
0
1400
1000
1080
1160
1240
1320
0
5
10
15
20
25
min
mass (m/z)
Figure 3. (A) HPLC profile and (B) mass spectrum of the crude synthetic [Phe(NO2)]10 trichogin GA IV analog. In part (A), the starred peak refers to the
peptide ester byproduct. In part (B), the calculated mass difference between the peptide alcohol and its trifluoroacetyl ester (95.98 a.m.u.) is matched by
the experimentally observed peaks (96.01 a.m.u.).
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 246–256

TRICHOGIN GA IV
Figure 5. Amide proton region of the NOESY spectrum of the [Phe(NO2)]10
trichogin GA IV analog [3.3 mM in 100 mM SDS-d25 aqueous solution
(9:1 H2O/D2O), pH 5 (313 K, 600 MHz)].
Table 3. NOE constraints, deviations from idealized geometry, and
mean energies for the NMR-based structures (energy <40 kcal/mol)
of the [Phe(NO2)]10 trichogin GA IV analog [3.3 mM in 100 mM
SDS-d25 aqueous solution (9:1 H2O/D2O), pH 5 (313 K, 600 MHz)]
Number of NOEs
Total
68
25
24
19
Intraresidue
Sequential
Figure 4. Far-UV CD spectra of the three Phe(NO2) trichogin GA IV analogs
in TFE (A) and 100 mM SDS aqueous solution (B).
i, i + n, n = 2, 3, 4
Mean RMSDa from ideality of accepted structures
Bonds (Å)
0.0029
Angles (ꢂ)
0.66
NOEs (Å)
0.067
The 2D-NMR study of the representative [Phe(NO2)]10 trichogin
GA IV analog was conducted in a 100 mM SDS aqueous solution.
This membrane-mimetic environment was chosen to compare
the results with those obtained from the CD analysis described
ealier. The proton resonances were fully assigned following the
Wüthrich procedure [53]. The analysis of the heterocorrelated
Cb-selective HMQC spectrum allowed us to complete the assign-
ments of the resonances of the Aib residues, including the
stereospecific assignment of the two prochiral methyl groups.
In the NOESY spectrum, all of the nonoverlapping NHi ! NHi+1
sequential cross peaks, typical of a helical structure, are
detected (Figure 5). Moreover, in the fingerprint region (not
shown) several CaHi ! NHi+2, CaHi ! NHi+3, and CaHi ! NHi+4
cross peaks are seen, which account for a mixed 310-/a-helical
conformation, such as those already reported for natural trichogin
GA IV and some of its analogs [16,21,22,26]. However, the spec-
trum is characterized by a number of weak signals, probably
because of the concomitant presence of a minor, perhaps less
preferred, peptide conformation. These minor signals, overlap-
ping with those belonging to the main conformation, make
the NMR analysis more complicated, leading to a reduced
number of unequivocal interproton correlations suitable to be
employed in the restrained MD calculations. Yet, a total of 68
interproton distance restraints (Table 3) were derived and used
in the rSA protocol to generate 150 3D structures, 110 of which
Mean energies (kcal/mol) of accepted structures
Eoverall
Ebond
Eangle
ENOE
36.47
1.47
18.75
14.47
aRoot-mean-square deviation.
had (i) violations to the NOE restraints lower than 0.5 Å; (ii) bond
length deviation of <0.01 Å from the ideal geometry; and (iii)
bond angle deviation of <1ꢂ from the ideal geometry. The 43
structures with a total energy of <40 kcal/mol were selected
[backbone average RMSD, 0.84 (ꢃ0.27) Å]. The analysis of their
averaged torsion angles (Table 4) reasonably suggests the coex-
istence of the a-helical and the 310-helical conformations, which
nicely confirms the conclusions gained from the preliminary
analysis of the NOESY spectrum. Interestingly, the conforma-
tionally diagnostic NOE cross peaks used in the MD calculations
reported in Figure 6 seem to indicate a nonhomogeneous
distribution of the two different (a- and 310-)helical types along
the sequence. Indeed, the N-terminal part tends to be mostly
3
10-helical, whereas the C-terminal part is predominantly folded
in an a-helix.
J. Pept. Sci. 2013; 19: 246–256 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

PEGGION ET AL.
Table 4. Average values (ꢂ) for the fm and cm torsion angles and
their relative standard deviations resulting from the NMR-based
structures (energy <40kcal/mol) of the [Phe(NO2)]10 trichogin GA IV
analog [3.3mM in 100 mM SDS-d25 aqueous solution (9:1 H2O/D2O),
pH 5 (313 K, 600MHz)]
(33.72 kcal/mol) 3D structure is shown in Figure 7. Together with
the aforementioned conformational switch, also helix amphiphili-
city, a common feature of Aib-based peptaibiotics [8,66,78] is
clearly visible. The presence of the polar –NO2 group, lying on
the hydrophobic face of the helix, might affect the known ability
of the natural peptide to interact with biological membranes by
perturbing its tendency to aggregate. However, the ability of the
Phe aromatic ring, almost perpendicular to the helical axis, to stack
might balance the aforementioned effect of the nitro group.
Taken together, the results of our MD calculations are consis-
tent with the presence of an amphiphilic, mixed 310-/a-helical
structure, with a change in the helical type at the level of the
flexible Gly5–Gly6 central dipeptide. The spatial position adopted
by the Phe(NO2) residue is such as to balance its possible effects
on the peptide biological activity.
Residue
fm
Δf
cm
Δc
Aib1
ꢀ53
ꢀ155
ꢀ51
ꢀ52
ꢀ78
ꢀ65
ꢀ65
ꢀ74
ꢀ79
ꢀ64
ꢀ132
ꢃ60
ꢃ58
ꢃ6
ꢀ12
ꢀ78
ꢀ30
ꢀ22
ꢀ11
ꢀ23
ꢀ41
ꢀ21
ꢀ52
ꢀ12
—
ꢃ100
ꢃ29
ꢃ1
Gly2
Leu3
Aib4
Gly5
Gly6
Leu7
Aib8
Gly9
Phe(NO2)10
Lol11
ꢃ6
ꢃ4
ꢃ14
ꢃ16
ꢃ4
ꢃ18
ꢃ4
ꢃ3
ꢃ2
ꢃ7
ꢃ9
ꢃ9
ꢃ6
ꢃ6
ꢃ5
—
Membrane Permeability Properties
The membrane modifying properties of the three Phe(NO2)
trichogin GA IV analogs were tested in comparison with those
of the natural lipopeptaibiotic by measuring the induced leakage
of CF entrapped in PC/Ch and PE/PG SUVs [63] (Figure 8).
According to the MD calculations, the conformation switch
takes place at the level of the –Gly5–Gly6– dipeptide, already
known to be fairly flexible [22,27,28]. The lowest energy
Figure 6. Summary of the significant interresidue NOESY cross peaks obtained for the [Phe(NO2)]10 trichogin GA IV analog [3.3 mM in 100 mM SDS-d25
aqueous solution (9:1 H2O/D2O), pH 5 (313 K, 600 MHz)].
nOct0
Phe(NO2)10
Figure 7. Ribbon representation of the lowest energy (33.7 kcal/mol) 3D structure obtained for the [Phe(NO2)]10 trichogin GA IV analog [3.3 mM in
100 mM SDS-d25 aqueous solution (9:1 H2O/D2O), pH 5 (313 K, 600 MHz)].
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 246–256

TRICHOGIN GA IV
3
bacteria decreases in the order 10 > 7 > 3. However, their
efficiencies against the Gram-positive bacteria are lower, albeit
slightly, if compared with those of the natural lipopeptaibol.
The well-known nonpeptide reference antibiotic tetracycline is
markedly more active than trichogin GA IV and its analogs
against all types of bacteria examined. Our present results
confirm that trichogin GA IV is unable to penetrate the highly
selective external membrane of the Gram-negative bacterial
cells [10,22,24,25] and indicate that also its Phe(NO2) analogs
studied in this work share this property. Moreover, all four
trichogin GA IV compounds are efficient against Gram-positive
bacteria, which lack the external membrane but possess a
thicker cellular wall.
100
10
80
60
40
20
T
7
A
0
0
100
200
300
400
500
-1
3
R
·10
Analysis of the Phe(NO2) IR Absorption Probe
10
Recently, Brewer and coworkers [41] reported that Phe(NO2),
more specifically its nitroaromatic symmetric stretching mode
near 1350 cmꢀ1, is a promising IR absorption probe of the polar-
ity of local environments for peptide-based molecules. In the
present work, we first examined the FT-IR absorption properties
of our three Phe(NO2) trichogin GA IV analogs single labeled
at different positions in the peptide sequence with the aim
at unraveling a potential shift of that band in response to
modifications of the solvent polarity. To fully isolate the IR
absorption of interest from all other bands exhibited by
our peptide molecules, each of the spectra of the three Phe(F)
(4-fluoro-phenylalanine) trichogin GA IV analogs, labeled at the
same positions as those of the Phe(NO2) peptide, recently
prepared in our laboratory [79] and lacking the nitroaromatic
chromophore, was subtracted from the corresponding spec-
trum of the Phe(NO2) analog (the IR absorption maximum
of the aromatic C–F group is known to occur in the 1250–
1100 cmꢀ1 region [80,81], well below that of the aromatic
C–NO2 group). As a representative example, the difference
spectra for the [Phe(NO2)]3 analog in three solvents of
different polarities (CDCl3, MeOH, CD3CN) are shown in Figure 9
(A, B, and C). The results were only partially encouraging in
the sense that the position of the absorption maximum of
the nitroaromatic chromophore moves slightly (from 1346 to
1348 cmꢀ1) as a function of the solvent properties. The
same data were observed for the two other Phe(NO2) analogs
(spectra not shown).
Nevertheless, we decided to examine the behavior of the
three Phe(NO2) trichogin GA IV analogs in an environment
that closely mimics that of lipid membranes where this
lipopeptaibol is known to exert its bioactivity. To this end,
we exploited ATR spectroscopy. The peptide samples, dissolved
in a 4:1 CH2Cl2/MeOH solvent mixture, were incorporated in
5:1 PC/Ch liposomes. The mixture was deposited on the ATR
crystal, and the solvent was evaporated under a N2 stream.
Figure 9D shows the ATR spectrum in the 1800–1300 cmꢀ1
region of the [Phe(NO2)]3 analog with and without the contri-
bution of the liposome. In both spectra, the Phe(NO2) band
stands out clearly at 1348 cmꢀ1. However, the corresponding
absorptions for the [Phe(NO2)]7 and [Phe(NO2)]10 analogs
(not shown) move to 1347 and 1345 cmꢀ1, respectively. It is
clear that these modest spectral shifts provide only a limited
information on the different polarities experienced by the
Phe(NO2) probe of the trichogin GA IV analogs in this mem-
brane-mimetic environment.
100
80
3
T
60
40
20
7
B
0
0
100
200
300
400
500
-1
3
R
·10
Figure 8. Peptide induced CF leakage at 20 min for different Rꢀ1 =[peptide]/
[lipid] for PC/Ch (7:3) vesicles (A) and PE/PG (7:3) vesicles (B) for trichogin
GA IV (T) and its three Phe(NO2) analogs at position 3 (3),7 (7), or 10 (10).
The sigmoidal shape of the curves suggests that the permeability
mechanism is largely cooperative. In other words, the peptides
need to achieve a concentration high enough to form self-
aggregates before the modifying process of the phospholipid
bilayers could initiate. All three analogs are remarkably active
in both membrane mimetic environments. More specifically,
as compared with the activities of trichogin GA IV, in PC/Ch
vesicles, the [Phe(NO2)]3 analog is the only one more active,
although modestly, with the [Phe(NO2)]10 analog being equally
effective. However, when the PE/PG vesicles are explored, the
[Phe(NO2)]3 and [Phe(NO2)]10 analogs exhibit activities compara-
ble with that of the reference parent compound. Interestingly,
in both types of vesicles, the [Phe(NO2)]7 analog is less active,
although not dramatically.
Antibacterial Activity
As reported in Table 2, in analogy with trichogin GA IV
[10,15,22,24,25], we found that all three Phe(NO2) analogs are
active against two strains of Gram-positive bacteria, namely S.
aureus and S. pyogenes, but inactive against the Gram-negative
bacterium E. coli. The rank of activities against the Gram-positive
J. Pept. Sci. 2013; 19: 246–256 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

PEGGION ET AL.
1346
A
B
1348
1380
1360
1340
1320
1380
1360
1340
1320
Wavenumbers (cm-1)
Wavenumbers (cm-1)
1346
C
D
1348
1380
1360
1340
1320
1800
1700
1600
1500
1400
1300
Wavenumbers (cm-1)
Wavenumbers (cm-1)
Figure 9. Different FT-IR absorption spectra in the 1380–1320 cmꢀ1 region for the [Phe(NO2)]3 trichogin GA IV analog in CDCl3 (A), CD3CN (B), and
MeOH (C) solutions. Part (D) shows the ATR spectra of the same peptide analog in the 1800–1300 cmꢀ1 region with (top) and without (bottom) the
contribution of the PC/Ch liposome.
1350 cmꢀ1in the IR absorption spectrum of the Phe(NO2) label
from tetrahydrofuran to aqueous solutions [41].
Conclusions
In this paper, we reported the results of our investigation on
three analogs of the 10-mer lipopeptaibol antibiotic trichogin
GA IV, each of them mono-substituted with the IR absorption
nitroaromatic label Phe(NO2). This mixed hydrophobic/hydrophilic
References
1 Toniolo C, Brückner H. Peptaibiotics: Fungal Peptides Containing a-Dialkyl
a-Amino Acids. Verlag Helvetica Chimica Acta: Zürich, Switzerland and
Wiley-VCH: Weinheim, Germany, 2009.
2 Karle IL, Balaram P. Structural characteristics of a-helical peptide
molecules containing Aib residues. Biochemistry 1990; 29: 6747–6756.
3 Toniolo C, Crisma M, Formaggio F, Peggion C. Control of peptide
conformation by the Thorpe-Ingold effect (Ca-tetrasubstitution).
Biopolymers (Pept. Sci.) 2001; 60: 396–419.
4 Toniolo C, Crisma M, Bonora GM, Benedetti E, Di Blasio B, Pavone V,
Pedone C, Santini A. Preferred conformation of the terminally blocked
(Aib)10 homo-oligopeptide. A long, regular 310-helix. Biopolymers
1991; 31: 129–138.
5 Sansom MSP. The biophysics of peptide models of ion channels. Prog.
Biophys. Mol. Biol. 1991; 55: 139–235.
6 Crisma M, Peggion C, Baldini C, MacLean EJ, Vedovato N, Rispoli G,
Toniolo C. Crystal structure of a spin-labeled, channel-forming alamethi-
cin analogue. Angew. Chem. Int. Ed. 2007; 46: 2047–2050.
7 Rebuffat S, Goulard C, Bodo B, Roquebert M-F. The peptaibol antibiotics
from Trichoderma soil fungi: structural diversity and membrane
properties. Recent Res. Devel. Org. Biorg. Chem. 1999; 3: 65–91.
8 Toniolo C, Crisma M, Formaggio F, Peggion C, Epand RF, Epand RM.
Lipopeptaibols, a novel family of membrane active antimicrobial
peptides. Cell. Mol. Life Sci. 2001; 58: 1179–1188.
9 Peggion C, Formaggio F, Crisma M, Epand RF, Epand RM, Toniolo C.
Trichogin: a paradigm for lipopeptaibols. J. Pept. Sci. 2003; 9: 679–689.
10 Auvin-Guette C, Rebuffat S, Prigent Y, Bodo B. Trichogin A IV, an
11-residue lipopeptaibol from Trichoderma longibrachiatum. J.
Am. Chem. Soc. 1992; 114: 2170–2174.
probe was strategically introduced at position 3, 7, or 10 where it
replaces a hydrophobic residue (Leu, Leu, or Ile, respectively). Our
FT-IR absorption, CD, and 2D-NMR conformational findings support
the view that, despite their slightly modified overall hydrophobicity
and bulkiness, these analogs exhibit a solvent-dependent, mixed
310-/a-helical structure and a self-association tendency similar to
those of the parent compound. Although partly attenuated, they
also preserve its remarkable properties to disrupt model mem-
branes and to exhibit good activity against Gram-positive bacteria.
Moreover, the main motivation of the present work, namely
confirmation of the recently published, interesting application
of the Phe(NO2) residue as a potential reporter group of ambient
polarity in peptide-based molecules, was only partially successful
because in all environments tested the observed range of the
wavenumber of the absorption maximum of the symmetric –
NO2 stretching mode for the membrane-active trichogin GA IV
analogs (1346–1348 cmꢀ1) is rather narrow. We tend to attribute
this rather modest sensitivity to the absence of water in our
environments (in contrast to the measurements performed in
the original paper [41]). Indeed, effective hydrogen-bonding
interactions between the hydrophilic nitro group and the solvent
water molecules is a well-established phenomenon, which appar-
ently is largely responsible for the published blue shift near
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 246–256

TRICHOGIN GA IV
11 Toniolo C, Benedetti E. The polypeptide 310-helix. Trends Biochem. Sci.
1991; 16: 350–353.
12 Benedetti E, Di Blasio B, Pavone V, Pedone C, Toniolo C, Crisma M.
Characterization at atomic resolution of peptide helical structures.
Biopolymers 1992; 32: 453‐456.
13 Bolin KA, Millhauser GL. a and 310: the split personality of polypeptide
helices. Acc. Chem. Res. 1999; 32: 1027–1033.
14 Toniolo C, Peggion C, Crisma M, Formaggio F, Shui X, Eggleston DS.
Structure determination of racemic trichogin A IV using centrosymmetric
crystals. Nat. Struct. Biol. 1994; 1: 908–914.
15 Toniolo C, Crisma M, Formaggio F, Peggion C, Monaco V, Goulard C,
Rebuffat S, Bodo B. Effect of Na-acyl chain length on the membrane-
modifying properties of synthetic analogs of the lipopeptaibol
trichogin GA IV. J. Am. Chem. Soc. 1996; 118: 4952–4958.
16 Monaco V, Locardi E, Formaggio F, Crisma M, Mammi S, Peggion E,
Toniolo C, Rebuffat S, Bodo B. Solution conformational analysis of
amphiphilic helical, synthetic analogs of the lipopeptaibol trichogin
GA IV. J. Pept. Res. 1998; 52: 261–272.
17 Formaggio F, Peggion C, Crisma M, Toniolo C. Short-chain analogues
of the lipopeptaibol antibiotic trichogin GA IV: conformational analysis
and membrane modifying properties. J. Chem. Soc. Perkin Trans. 2
2001; 1372–1377.
18 Gurunath R, Balaram P. A nonhelical, multiple b-turn conformation
in a glycine-rich heptapeptide fragment of trichogin GA IV containing
a single central a-aminoisobutyric acid residue. Biopolymers 1995; 35:
21–29.
19 Monaco V, Formaggio F, Crisma M, Toniolo C, Shui X, Eggleston DS.
Crystallographic structure of a multiple b-turn containing, glycine-rich
heptapeptide: A synthetic precursor of the lipopeptaibol antibiotic
trichodecenin I. Biopolymers 1996; 39: 31–42.
20 Oancea S, Hilma G, Peggion C, Formaggio F, Toniolo C. Main-chain
length control of conformation, membrane activity, and antibiotic
properties of lipopeptaibol sequential analogues. Chem. Biodivers.
2008; 5: 681–692.
21 De Zotti M, Biondi B, Peggion C, De Poli M, Fathi H, Oancea S,
Toniolo C, Formaggio F. Partial thioamide scan on the lipopeptaibiotic
trichogin GA IV. Effects on folding and bioactivity. Beilstein J. Org.
Chem. 2012; 8: 1161–1171.
22 De Zotti M, Biondi B, Park Y, Hahm K-S, Crisma M, Toniolo C, Formaggio
F. Antimicrobial lipopeptaibol trichogin GA IV: role of the three Aib
residues on conformation and bioactivity. Amino Acids 2012; 43:
1761–1777.
23 Kropacheva TN, Raap J. Ion transport across a phospholipid mem-
brane mediated by the peptide trichogin GA IV. Biochim. Biophys. Acta
2002; 1567: 193–203.
32 Mazzuca C, Stella L, Venanzi M, Formaggio F, Toniolo C, Pispisa B.
Mechanism of membrane activity of the antibiotic trichogin GA IV: a
two-state transition controlled by peptide concentration. Biophys. J.
2005; 88: 3411–3421.
33 Venanzi M, Gatto E, Bocchinfuso G, Palleschi A, Stella L, Baldini C,
Formaggio F, Toniolo C. Peptide folding dynamics: a time-resolved
study from the nanosecond to the microsecond time regime. J. Phys.
Chem. B 2006; 110: 22834–22841.
34 Gatto E, Mazzuca C, Stella L, Venanzi M, Toniolo C, Pispisa B. Effect of
peptide lipidation on membrane perturbing activity: a comparative
study on two trichogin analogues. J. Phys. Chem. B 2006; 110:
22813–22818.
35 Scrimin P, Veronese A, Tecilla P, Tonellato U, Monaco V, Formaggio F,
Crisma M, Toniolo C. Metal ion modulation of membrane permeability
induced by a polypeptide template. J. Am. Chem. Soc. 1996; 118:
2505–2506.
36 Heuber C, Formaggio F, Baldini C, Toniolo C, Müller K. Multinuclear
solid-state-NMR and FT-IR absorption investigations on lipid/trichogin
bilayers. Chem. Biodivers. 2007; 4: 1200–1218.
37 Milov AD, Erilov DA, Salnikov ES, Tsvetkov YD, Formaggio F, Toniolo C,
Raap J. Structure and spatial distribution of the spin-labelled lipopep-
tide trichogin GA IV in a phospholipid membrane studied by pulsed
electron–electron double resonance (PELDOR). Phys. Chem. Chem.
Phys. 2005; 7: 1794–1799.
38 Salnikov ES, Erilov DA, Milov AD, Tsvetkov YD, Peggion C, Formaggio
F, Toniolo C, Raap J, Dzuba SA. Location and aggregation of the spin-
labeled peptide trichogin GA IV in a phospholipid membrane as
revealed by pulsed EPR. Biophys. J. 2006; 91: 1532–1540.
39 Syryamina VN, Isaev NP, Peggion C, Formaggio F, Toniolo C, Raap J,
Dzuba SA. Small-amplitude backbone motions of the spin-labeled
lipopeptide trichogin GA IV in a lipid membrane as revealed by electron
spin echo. J. Phys. Chem. B 2010; 114: 12277–12283.
40 Syryamina VN, De Zotti M, Peggion C, Formaggio F, Toniolo C, Raap J,
Dzuba SA. A molecular view on the role of cholesterol upon mem-
brane insertion, aggregation, and water accessibility of the antibiotic
lipopeptide trichogin GA IV as revealed by EPR. J. Phys. Chem. B 2012;
116: 5653–5660.
41 Smith EE, Linderman BY, Luskin AC, Brewer SH. Probing local environ-
ments with the infrared probe: L-4-nitrophenylalanine. J. Phys. Chem.
B 2011; 115: 2380–2385.
42 Haiser K, Koller FO, Huber M, Regner N, Schrader TE, Schreier WJ, Zinth
W. Nitro-phenylalanine: a novel sensor for heat transfer in peptides. J.
Phys. Chem. A 2011; 115: 2169–2175.
43 Murakami H, Hohsaka T, Ashizuka Y, Sisido M. Site-directed incorporation
of p-nitrophenylalanine into streptavidin and site-to-site photoinduced
electron transfer from a pyrenyl group to a nitrophenyl group on the
protein framework. J. Am. Chem. Soc. 1998; 120: 7520–7529.
44 Taki M, Hohsaka T, Murakami H, Taira K, Sisido M. Position-specific
incorporation of a fluorophore ꢀ quencher pair into a single streptavi-
din through orthogonal four-base codon/anticodon pairs. J. Am.
Chem. Soc. 2002; 124: 14586–14590.
45 Tsao M-L, Summerer D, Ryu Y, Schultz PG. The genetic incorporation
of a distance probe into proteins in Escherichia coli. J. Am. Chem.
Soc. 2006; 128: 4572–4573.
46 Barlos K, Chatzi O, Gatos D, Stavropoulos G. 2-Chlorotrityl chloride
resin: studies on anchoring of Fmoc-amino acids and peptide cleavages.
Int. J. Pept. Protein Res. 1991; 37: 513–520.
47 Bollhagen R, Schmiedberger M, Barlos K, Grell E. A new reagent for the
cleavage of fully protected peptides synthesized on 2-chlorotrityl
chloride resin. J. Chem. Soc., Chem. Commun. 1994; 2559–2560.
48 Kocsis L, Ruff F, Orosz G. The effect of peptide length on the cleavage
kinetics of 2-chlorotrityl resin-bound ethers. J. Pept. Sci. 2006; 12:
428–436.
24 De Zotti M, Biondi B, Formaggio F, Toniolo C, Stella L, Park Y, Hahm
K-S. Trichogin GA IV: an antibacterial and protease-resistant peptide.
J. Pept. Sci. 2009; 15: 615–619.
25 De Zotti M, Biondi B, Peggion C, Formaggio F, Park Y, Hahm K-S,
Toniolo C. Trichogin GA IV: a versatile template for the synthesis of
novel peptaibiotics. Org. Biomol. Chem. 2012; 10: 1285–1299.
26 Locardi E, Mammi S, Peggion E, Monaco V, Formaggio F, Crisma M,
Toniolo C, Bodo B, Rebuffat S, Kamphuis J, Broxterman QB. Conforma-
tion and membrane activity of an analogue of the peptaibol antibiotic
trichogin GA IV with a lipophilic amino acid at the N-terminus. J. Pept.
Sci. 1998; 4: 389–399.
27 Venanzi M, Bocchinfuso G, Gatto E, Palleschi A, Stella L, Formaggio F,
Toniolo C. Metal binding properties of fluorescent analogues of tricho-
gin GA IV: a conformational study by time-resolved spectroscopy and
molecular mechanics investigations. ChemBioChem 2009; 10: 91–97.
28 Venanzi M, Gatto E, Bocchinfuso G, Palleschi A, Stella L, Formaggio F,
Toniolo C. Dynamics of formation of a helix-turn-helix structure in
a membrane-active peptide: a time-resolved spectroscopic study.
ChemBioChem 2006; 7: 43–45.
29 Mazzuca C, Orioni B, Coletta M, Formaggio F, Toniolo C, Maulucci G,
De Spirito M, Pispisa B, Venanzi M, Stella L. Fluctuations and the
rate-limiting step of peptide-induced membrane leakage. Biophys. J.
2010; 99: 1791–1800.
30 Bocchinfuso G, Palleschi A, Orioni B, Grande G, Formaggio F, Toniolo C,
Park Y, Hahm K-S, Stella L. Different mechanisms of action of antimicro-
bial peptides: insight from fluorescence spectroscopy experiments and
molecular dynamics simulations. J. Pept. Sci. 2009; 15: 550–558.
31 Epand RF, Epand RM, Formaggio F, Crisma M, Wu H, Lehrer RI, Toniolo
C. Analogs of the antimicrobial peptide trichogin having opposite
membrane properties. Eur. J. Biochem. 2001; 268: 703–712.
49 Carpino LA. 1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling
reagent. J. Am. Chem. Soc. 1993; 115: 4397–4398.
50 Carpino LA, El-Faham A, Minor CA, Albericio F. Advantageous applica-
tions of azabenzotriazole (triazolopyridine)-based coupling reagents
to solid-phase peptide synthesis. J. Chem. Soc., Chem. Commun. 1994;
201–203.
51 König W, Geiger R. Eine neue Methode zur Synthese von Peptiden:
Aktivierung der Carboxylgruppe mit Dicyclohexylcarbodiimid unter
Zusatz von 1-Hydroxy-benzotriazolen. Chem. Ber. 1970; 103: 788–798.
52 Formaggio F, Broxterman QB, Toniolo C. In Houben-Weyl: Methods of
Organic Synthesis, Vol. E22c, Goodman M, Felix A, Moroder L, Toniolo
C (eds.). Thieme: Stuttgart, Germany, 2003; 292–310.
J. Pept. Sci. 2013; 19: 246–256 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

PEGGION ET AL.
53 Wüthrich K. NMR of Proteins and Nucleic Acids. Wiley: New York, 1986.
54 Rance M, Sørensen OW, Bodenhausen G, Wagner G, Ernst RR,
Wüthrich K. Improved spectral resolution in COSY 1H NMR spectra of
proteins via double quantum filtering. Biochem. Biophys. Res. Commun.
1983; 117: 479–485.
55 Griesinger C, Otting G, Wüthrich K, Ernst RR. Clean TOCSY for proton
spin system identification in macromolecules. J. Am. Chem. Soc.
1988; 110: 7870–7872.
69 Baron MH, De Lozé C, Toniolo C, Fasman GD. Structure in solution of
protected homo-oligopeptides of L-valine, L-isoleucine, and L-phenyl-
alanine: an infrared absorption study. Biopolymers 1978; 17: 2225–2239.
70 Moretto V, Crisma M, Bonora GM, Toniolo C, Balaram H, Balaram P.
Comparison of the effect of five guest residues on the b-sheet confor-
mation of host (L-Val)n homo-oligopeptides. Macromolecules 1989; 22:
2939–2944.
71 Kennedy DF, Crisma M, Toniolo C, Chapman D. Studies of peptides
forming 310- and a-helices and b-bend ribbon structures in organic
solution and in model biomembranes by Fourier transform infrared
spectroscopy. Biochemistry 1991; 30: 6541–6548.
56 Bax A, Subramanian S. Sensitivity-enhanced two-dimensional hetero-
nuclear shift correlation NMR spectroscopy. J. Magn. Reson. 1986; 67:
565–569.
57 Bax A, Summers MF. Proton and carbon-13 assignments from
sensitivity-enhanced detection of heteronuclear multiple-bond
connectivity by 2D multiple quantum NMR. J. Am. Chem. Soc. 1986;
108: 2093–2094.
72 Beychok S. Circular dichroism of poly-a-amino acids and proteins. In
Poly-a-Amino Acids: Protein Models for Conformational Studies, Fasman
GD (ed.). Dekker: New York, 1967; 293–337.
73 Manning MC, Woody RW. Theoretical CD studies of polypeptide
helices: examination of important electronic and geometric factors.
Biopolymers 1992; 31: 569–586.
74 Toniolo C, Polese A, Formaggio F, Crisma M, Kamphuis J. Circular
dichroism spectrum of a peptide 310-helix. J. Am. Chem. Soc. 1996;
118: 2744–2745.
58 Bauer C, Freeman R, Frenkiel T, Keeler J, Shaka AJ. Gaussian pulses. J.
Magn. Reson. 1984; 58: 442–457.
59 Emsley L, Bodenhausen G. Self-refocusing effect of 270ꢂ Gaussian pulses.
Applications to selective two-dimensional exchange spectroscopy. J.
Magn. Reson. 1989; 82: 211–221.
60 Schwieters CD, Kuszewski JJ, Tjandra N, Clore MG. The X-PLOR-NIH
NMR molecular structure determination package. J. Magn. Reson.
2003; 160: 65–73.
75 Formaggio F, Crisma M, Rossi P, Scrimin P, Kaptein B, Broxterman QB,
Kamphuis J, Toniolo C. The first water-soluble 310-helical peptides.
Chem. Eur. J. 2000; 6: 4498–4504.
61 Malde AK, Zuo L, Breeze M, Stroet M, Poger D, Nair PC, Oostenbrink C,
Mark AE. An automated force field topology builder (ATB) and repos-
itory: version 1.0. J. Chem. Theory Comput. 2011; 7: 4026–4037.
62 Koradi R, Billeter M, Wüthrich K. MOLMOL: A program for display and
analysis of macromolecular structures. J. Mol. Graph. 1996; 14: 51–55.
63 El Hajji M, Rebuffat S, Le Doan T, Klein G, Satre M, Bodo B. Interaction
of trichorzianines A and B with model membranes and with the
amoeba Dictyostalium. Biochim. Biophys. Acta 1989; 978: 97–104.
64 Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility
testing by a standardized single disk method. Am. J. Clin. Pathol.
1966; 45: 493–496.
65 National Committee for Clinical Laboratory Standards M2-A6, Wayne,
PA, 1997.
66 De Zotti M, Biondi B, Peggion C, Park Y, Hahm K-S, Formaggio F,
Toniolo C. Synthesis, preferred conformation, protease stability, and
membrane activity of heptaibin, a medium-length peptaibiotic. J.
Pept. Sci. 2011; 17: 585–594.
76 Goodman M, Toniolo C, Peggion E. Conformational aspects of
polypeptides. XXIX. Conformational assignments for some aromatic
polypeptides by far-UV Cotton effects. New results. Biopolymers
1968; 6: 1691‐1695.
77 Toniolo C, Falxa ML, Goodman M. Conformational aspects of polypep-
tides. XXV. Solvent and temperature effects on the conformations
of copolymers of benzyl and methyl L-aspartate with nitrobenzyl
L-aspartate. Biopolymers 1968; 6: 1579–1603.
78 De Zotti M, Damato F, Formaggio F, Crisma M, Schievano E, Mammi S,
Kaptein B, Broxterman QB, Felock PJ, Hazuda DJ, Singh SB, Kirschbaum
J, Brückner H, Toniolo C. Total synthesis, characterization, and
conformational analysis of the naturally occurring hexadecapeptide
integramide A and its diastereomer at positions 14 and 15. Chem.
Eur. J. 2010; 16: 316–327.
79 Battistella C. Chemistry Degree Thesis. University of Padova: Italy, 2011.
80 Silverstein RM, Webster FX. Spectrometric Identification of Organic
Compounds. Wiley: New York, 1998.
67 Hjørringgaard CU, Pedersen JM, Vosegaard T, Nielsen NC, Skrydstrup
T. An automatic solid-phase synthesis of peptaibols. J. Org. Chem.
2009; 74: 1329–1332.
81 Jackson RFW, Moore RJ, Dexter CS, Elliott J, Mowbray CE. Concise
synthesis of enantiomerically pure phenylalanine, homophenylalanine,
and bishomophenylalanine derivatives using organozinc chemistry:
NMR studies of amino acid-derived organozinc reagents. J. Org. Chem.
1998; 63: 7875–7884.
68 Bellamy M. The Infra-Red Spectra of Complex Molecules. 2nd edn,
Methuen: London, U.K., 1966.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 246–256