Trichogin GA IV: A versatile template for the synthesis of novel peptaibiotics

Article abstract of DOI:10.1039/c1ob06178j

Trichogin GA IV, isolated from the fungus Trichoderma longibrachiatum, is the prototype of lipopeptaibols, the sub-class of short-length peptaibiotics exhibiting membrane-modifying properties. This peptaibol is predominantly folded in a mixed 3₁₀-/α-helical conformation with a clear, albeit modest, amphiphilic character, which is likely to be responsible for its capability to perturb bacterial membranes and to induce cell death. In previous papers, we reported on the interesting biological properties of trichogin GA IV, namely its good activity against Gram positive bacteria, in particular methicillin-resistant S. aureus strains, its stability towards proteolytic degradation, and its low hemolytic activity. Aiming at broadening the antimicrobial activity spectrum by increasing the peptide helical amphiphilicity, in this work we synthesized, by solution and solid-phase methodologies, purified and fully characterized a set of trichogin GA IV analogs in which the four Gly residues at positions 2, 5, 6, 9, lying in the poorly hydrophilic face of the helical structure, are substituted by one (position 2, 5, 6 or 9), two (positions 5 and 6), three (positions 2, 5, and 9), and four (positions 2, 5, 6, and 9) Lys residues. The conformational preferences of the Lys-containing analogs were assessed by FT-IR absorption, CD and 2D-NMR techniques in aqueous, organic, and membrane-mimetic environments. Interestingly, it turns out that the presence of charged residues induces a transition of the helical conformation adopted by the peptaibols (from 3 ₁₀-to α-helix) as a function of pH in a reversible process. The role played in the analogs by the markedly increased amphiphilicity was further tested by fluorescence leakage experiments in model membranes, protease resistance, antibacterial and antifungal activities, cytotoxicity, and hemolysis. Taken together, our biological results provide evidence that some of the least substituted among these analogs are good candidates for the development of new membrane-active antimicrobial agents. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c1ob06178j

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PAPER
Trichogin GA IV: A versatile template for the synthesis of novel
peptaibiotics†‡
Marta De Zotti,a Barbara Biondi,a Cristina Peggion,a Fernando Formaggio,a Yoonkyung Park,b,c
Kyung-Soo Hahmb,d and Claudio Toniolo*a
Received 15th July 2011, Accepted 8th November 2011
DOI: 10.1039/c1ob06178j
Trichogin GA IV, isolated from the fungus Trichoderma longibrachiatum, is the prototype of
lipopeptaibols, the sub-class of short-length peptaibiotics exhibiting membrane-modifying properties.
This peptaibol is predominantly folded in a mixed 310-/a- helical conformation with a clear, albeit
modest, amphiphilic character, which is likely to be responsible for its capability to perturb bacterial
membranes and to induce cell death. In previous papers, we reported on the interesting biological
properties of trichogin GA IV, namely its good activity against Gram positive bacteria, in particular
methicillin-resistant S. aureus strains, its stability towards proteolytic degradation, and its low
hemolytic activity. Aiming at broadening the antimicrobial activity spectrum by increasing the peptide
helical amphiphilicity, in this work we synthesized, by solution and solid-phase methodologies, purified
and fully characterized a set of trichogin GA IV analogs in which the four Gly residues at positions 2, 5,
6, 9, lying in the poorly hydrophilic face of the helical structure, are substituted by one (position 2, 5, 6
or 9), two (positions 5 and 6), three (positions 2, 5, and 9), and four (positions 2, 5, 6, and 9) Lys
residues. The conformational preferences of the Lys-containing analogs were assessed by FT-IR
absorption, CD and 2D-NMR techniques in aqueous, organic, and membrane-mimetic environments.
Interestingly, it turns out that the presence of charged residues induces a transition of the helical
conformation adopted by the peptaibols (from 310- to a-helix) as a function of pH in a reversible
process. The role played in the analogs by the markedly increased amphiphilicity was further tested by
fluorescence leakage experiments in model membranes, protease resistance, antibacterial and antifungal
activities, cytotoxicity, and hemolysis. Taken together, our biological results provide evidence that some
of the least substituted among these analogs are good candidates for the development of new
membrane-active antimicrobial agents.
diverse mechanism of action makes them less susceptible to
resistance.1–3
Introduction
Conventional antibiotics target definite sites, such as specific
enzymes or DNA, in bacteria. For this reason, microorganism
resistance may occur with high probability. The clinical impact
of antibiotic resistance implies high cost and mortality. Plant
and animal kingdoms are rich sources of antimicrobial peptides
(AMPs) physically disrupting bacterial cell membranes. This
Among AMPs, there is a group of natural antibacterial peptides,
the lipopeptaibols (short-length peptaibiotics4), characterized
by an N-terminal long-chain fatty acid protecting group, high
amount of the noncoded amino acid Aib (a-aminoisobutyric
acid) and a C-terminal 1,2-amino alcohol. Most lipopeptaibols
exhibit remarkably valuable properties, such as well developed
helicity, amphiphilicity, stability to proteolytic degradation, and
short main-chain length.5 These properties render them intriguing
lead compounds in the search of candidates as novel, potent
antibacterial agents. Trichogin GA IV6 is considered the prototype
of lipopeptaibols. It was isolated in 1992 as the main component of
the natural (Trichoderma longibrachiatum) trichogin microhetero-
geneous mixture and sequenced by Bodo and coworkers.7,8 The 10-
mer amino acid primary structure of trichogin GA IV is blocked
at the N-terminus by an n-octanoyl (n-Oct) moiety and has a
chiral (S) 1,2-aminoalcohol, Lol (leucinol), at the C-terminus. The
sequence is as follows:
aICB, Padova Unit, CNR, Department of Chemistry, University of Padova,
35131, Padova, Italy. E-mail: claudio.toniolo@unipd.it; Fax: +39 049
8275829; Tel: +39 049 8275247
bResearch Center for Proteinous Materials, Chosun University, Gwangju,
501-759, Korea
cDepartment of Biotechnology, Chosun University, Gwangju, 501-759, Korea
dDepartment of Cellular Molecular Medicine, School of Medicine, Chosun
University, Gwangju, 501-759, Korea
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob06178j
‡ This article is part of an Organic & Biomolecular Chemistry web theme
issue on Foldamer Chemistry.
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n-Oct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Lol
are extremely promising agents in the framework of modern anti-
infective therapeutic strategies,1–3 we decided: (i) to take advantage
of the trichogin GA IV sequence as a template for the preparation
of novel antibacterial peptaibols with improved bioproperties
and (ii) to enhance the weak hydrophilicity of one of its faces
(characterized by four Gly) by incorporation of one, two, three
or four side-chain positively charged Lys residues. In one of these
analogs, we aimed at reinforcing helicity upon replacement of one
central Gly with an Aib residue.
On the basis of the results from a number of conformational
investigations, including X-ray diffraction analyses, on synthetic
trichogin GA IV and selected analogs, mostly carried out by some
of us (C.P., F.F., and C.T.), a right-handed helical structure was
suggested.6,7,9–19 This conclusion is not surprising in view of the
heavy presence (30%) of the strongly helicogenic Aib residues
in the sequence.20–23 It was also proposed that the flexible -Gly5-
Gly6- dipeptide would create a hinge point between two helical
regions. The overall mixed 310-/a- helical structure24,25 is largely
amphiphilic, with the hydrophobic face including the n-Oct group
and the Leu, Ile, and Lol side chains, while the opposite face
is characterized by the four Gly residues strategically located at
positions 2, 5, 6, and 9. In refs. 12 and 18 the minimal sequence
requirements for trichogin GA IV activity were also reported.
The experimental data accumulated on synthetic analogs con-
siderably expanded our knowledge of the minimal structural
requirements for membrane activity of this lipopeptaibol. We
found that the substitution of the C-terminal 1,2-amino alcohol
(Lol) by the corresponding a-amino methyl ester (Leu-OMe)
affects only marginally the biological properties of trichogin GA
IV.12 Conversely, the Na-blocking fatty acyl moiety was shown to
play a major role in its membrane modifying properties.12 More
specifically, at least six carbon atoms in the aliphatic chain are
required for a significant membrane activity. By use of a set of
Na-octanoylated peptides of increasing main-chain length (from
the C-terminus), it was established that membrane permeability
properties, although weak, begin at a main-chain length as short
as the tetrapeptide, and progressively increase up to the full length
of the peptide.18 A significant influence of the three Aib residues is
also evident. It is clear that the extent of membrane activity and the
amount of folding run roughly parallel, in the sense that largely
folded, but not helical, conformers are typically present in the
short sequences, while the longest peptides exhibit regular helical
structures. We are currently actively investigating details of the self-
aggregation process and the mechanism of action of trichogin GA
IV in the membranes under a variety of experimental conditions,
using not only sensitive fluorescence and EPR techniques26–44 but
solid-state NMR45 and other biophysical methods46,47 as well.
Recently, using a large set of proteolytic enzymes, we also provided
clear evidence that trichogin GA IV is remarkably stable towards
biocatalyzed hydrolysis.48
Results and discussion
Peptide synthesis and characterization
In this work, we firstly synthesized by solution methods two
Lys-monosubstituted, C-terminal [Leu11-OMe] trichogin GA IV
analogs, herein referred to as K2-OMe and K9-OMe (Table 1),
in which a Gly residue (at position 2 or 9, respectively) was
replaced by Lys. The synthetic approach in solution phase was
initially preferred because of the well known poor reactivity of
the Aib residues (three of them occur in the trichogin GA IV
sequence) when located at the N-terminus in the peptide coupling
reactions.49 To perform these syntheses, we employed the segment
condensation approach, which allows a faster preparation of
multiple analogs than the step-by-step procedure. An important
feature, which makes trichogin GA IV a versatile synthetic
platform, is the presence of as many as seven achiral (Aib or Gly)
residues spread all over its primary structure, which allow one to
design suitable segments with an achiral residue at their C-termini,
thus reducing the risk of epimerization during coupling reactions.
Schemes 1 and 2 describe our synthetic strategy. To synthesize the
target peptide sequence K2-OMe (Scheme 1), we split it in three
short segments. Segments C (1–4), containing Lys2(Z) where Z is
benzyloxycarbonyl, and B (5–8) were designed as to have an achiral
Aib residue at their C-termini. Then, segment B was condensed
on segment A (9–11) and segment C was reacted on the resulting
segment B–A (5–11). To synthesize peptide K9-OMe (Scheme
2), we followed a similar strategy [via segments E(1–4), B(5–8),
and D(9–11), the latter containing Lys9(Z), and E–B(1–8)]. The B
segment is common to both synthetic strategies. The two K2-OMe
and K9-OMe analogs were obtained in an overall yield of 18 and
25%, respectively. For the details of the synthetic procedures and
characterizations of these analogs (and their synthetic precursor
segments as well), see the Experimental section. Table 1 and Fig.
1 give details of their RP-HPLC profiles.
Since it is widely recognized that short, membrane active, resis-
tant to protease degradation, cationic and amphiphilic peptides
Table 1 Amino acid sequences and HPLC retention times of trichogin GA IV and its analogs investigated in this worka
Abbreviation
Sequence
tR/minb
TRIC
K5
n-Oct-Aib1-Gly2-Leu3-Aib4-Gly5-Gly6-Leu7-Aib8-Gly9-Ile10-Lol
n-Oct-Aib-Gly-Leu-Aib-Lys5-Gly-Leu-Aib-Gly-Ile-Lol
n-Oct-Aib-Gly-Leu-Aib-Gly-Lys6-Leu-Aib-Gly-Ile-Lol
n-Oct-Aib-Gly-Leu-Aib-Lys5-Lys6-Leu-Aib-Gly-Ile-Lol
n-Oct-Aib-Lys2-Leu-Aib-Lys5-Aib6-Leu-Aib-Lys9-Ile-Lol
n-Oct-Aib-Lys2-Leu-Aib-Lys5-Lys6-Leu-Aib-Lys9-Ile-Lol
n-Oct-Aib-Lys2-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe
n-Oct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Lys9-Ile-Leu-OMe
17.7
17.6
16.1
13.9
15.2
9.5
K6
K56
K259U6
K2569
K2-OMe
K9-OMe
14.8
18.3
a Single-letter amino acid abbreviations include: lysine (K) and a-aminoisobutyric acid (U). b tR: retention time in RP-HPLC, using the conditions
described in the Experimental section.
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Scheme 1 Synthetic strategy adopted for the synthesis of peptide K2-OMe: (i) HOBt, EDC in distilled CH2Cl2, pH kept at 8 by adding NMM; (ii) H2,
Pd/C (20%) in distilled CH2Cl2. (iii) HOAt, EDC in distilled CH2Cl2, pH kept at 8 by adding NMM; (iv) H2, Pd/C (10%) in CH3OH; (v) DEA (20 eq.) in
distilled CH2Cl2; (vi) TFA (10 eq.) in distilled CH2Cl2; (vii) n-Oct-OH, HOAt, EDC, in distilled CH2Cl2, pH kept at 8 by adding NMM; (viii) H2, Pd/C
(10%) in CH3OH, HCl 3 M in CH3OH.
Scheme 2 Synthetic strategy adopted for the synthesis of peptide K9-OMe. Solvents and reagents are those reported in Scheme 1. Segment B is common
to both strategies.
Fig. 1 RP-HPLC profiles obtained for the purified SPPS-produced trichogin GA IV and its K5, K6, K56, K259U6, and K2569 analogs (A) and for the
K2-OMe and K9-OMe analogs produced by peptide synthesis in solution (B). [Leu11-OMe]-trichogin GA IV (Tric-OMe)12 is reported as reference.
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Fig. 2 FT-IR absorption spectra (3500–3200 cm-1 region) in CDCl3 solution of the Ne-Lys Z-protected K2-OMe and K9-OMe trichogin GA IV analogs
at 1 mM [curves labeled K2(Z) in A and K9(Z) in B] and 0.1 mM [curves labeled K2(Z)¢ in A and K9(Z)¢ in B] peptide concentrations. The spectra of the
shorter peptides (C and B–A in A, and E and E–B in B) at 0.1 mM peptide concentration are also shown.
Although the solution-phase procedure afforded the two desired
analogs in good amounts, and allowed us to study the confor-
mational properties of their synthetic segments as well, it was
significantly time consuming. For this reason, we decided to make
use of our improved protocol for automatic solid-phase peptide
synthesis (SPPS), developed for the synthetically difficult medium-
length peptaibiotic heptaibin,50 to prepare trichogin GA IV and its
five analogs K5, K6, K56, K259U6 and K2569 (Table 1) where the
four Gly at position 2, 5, 6 and 9 were partially or fully replaced
by Lys residues (in one of them Gly6 was additionally substituted
by Aib).
These syntheses, performed in parallel on the Lol-substituted
2-chlorotrityl resin51,52 as described in the Experimental section,
required 30 h only. After cleavage of the peptides from the resin, we
collected the crude products, protected as tert-butyloxycarbonyl
(Boc) derivatives at the side-chain of Lys residues, in about 90%
purity. After purification by flash chromatography, the peptides
were subjected to Boc removal using HCl in methanol. The desired
final compounds were isolated in 42–50% yield with a purity
>98%, as highlighted by RP-HPLC (Table 1 and Fig. 1). The
peptides were additionally characterized by ESI-MS and NMR
spectrometry (see Experimental section). The recently reported
SPPS of trichogin GA IV, carried out according to a procedure
partially different from that described here, resulted in a 20%
isolated yield.53
3325 cm-1, assigned to the N–H stretching mode of H-bonded
peptide groups54–56 (Fig. 2). An additional weak band is visible
at about 3450 cm-1, attributed to free (solvated) amide/peptide
groups. A remarkable dilution effect is observed in the spectra of
both peptides between 1.0 and 0.1 mM concentration (Fig. 2). We
ascribe this difference to a strong tendency of the two analogs
to self-aggregate above 0.1 mM concentration in this solvent.
The spectra observed at the lowest concentration examined can
be reasonably interpreted as arising almost exclusively from
intramolecular C O ◊ ◊ ◊ H–N interactions. We conclude that these
FT-IR absorption spectra are consistent with the hypothesis that in
CDCl3, a solvent of low polarity, at low concentration the preferred
conformation of these two analogs, rich in the helix-supporting
Aib residues,20–23 would be highly folded and extensively stabilized
by intramolecular H-bonds. These results parallel closely those
already published for natural trichogin GA IV and its [Leu11-OMe]
analog.12,15,16,18
Fig. 2 also shows the spectra (peptide concentration: 1.0 mM)
of the shorter segments C(1–4) and B–A (5–11) utilized for the
synthesis of the Ne-Lys Z-protected K2-OMe (part A), and E(1–
4) and E–B(1–8) employed to prepare the Ne-Lys Z-protected
K9-OMe (part B). It is clear that in both series peptide main-
chain elongation induces a substantial enhancement of the N–H
stretching band of the H-bonded peptide groups relative to that
of free peptide groups. Concomitantly, the frequency maximum
of the former band shifts to lower wavenumbers. These findings
represent an unambiguous evidence for the organization of the
peptide chain into a secondary structure stabilized by intramolec-
ular C O ◊ ◊ ◊ H–N H-bonds. Interestingly, among the four shorter
segments, only E–B exhibits the concentration dependent effect
(between 1.0 and 0.1 mM; Fig. S1†) which characterizes the IR
absorption properties of the two mono-substituted trichogin GA
IV Lys(Z) analogs. This result suggests that the tendency to self-
associate is promoted by the presence of the N-terminal, long-
chain, n-octanoyl amide group.
Conformational analysis
A detailed analysis of the conformational preferences of the
seven Lys-containing trichogin GA IV analogs synthesized in this
work was performed using FT-IR absorption, CD, and 2D-NMR
spectroscopies in different solvents and environments.
As the K2-OMe and K9-OMe free Lys-containing peptides are
only sparingly soluble in CDCl3, we carried out the FT-IR absorp-
tion study on their synthetic precursors, namely the corresponding
Ne-Lys Z-protected compounds. This spectroscopic technique,
although not providing definitive conformational conclusions,
is able to offer useful initial hints. In the conformationally
informative 3500–3200 cm-1 region at 1.0 mM concentration, the
two spectra are dominated by an intense absorption at 3330–
The far-UV CD spectra of the seven Lys-based trichogin
GA IV analogs recorded in methanol (MeOH) and in water
containing 100 mM sodium dodecylsulphate (SDS) at 0.1 mM
peptide concentration are shown in Fig. 3. The corresponding
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Fig. 3 Far-UV CD spectra of trichogin GA IV and the seven Lys-based analogs in MeOH solution (A) and in aqueous solution containing 100 mM
SDS (B). Peptide concentration: 0.1 mM.
spectra for the native peptaibol are reported for comparison
(see also ref. 7,12,15,16,18). In both environments all peptides
display a negative maximum at approximately 205 nm (parallel
component of the exciton split p → p* amide transition57) and a
pronounced negative shoulder located at about 225 nm (n→p *
amide transition). These features are characteristic of peptides
mainly structured in a right-handed helix. Although the shapes of
all of the curves are similar, the ellipticity values vary significantly.
This finding does not necessarily indicate a much higher helicity
for the more substituted analogs (although this conclusion might
also apply) because in these compounds one or more achiral Gly
are replaced by chiral Lys residues. In this connection, it is worth
noting that the ellipticity values exhibited by the K2-OMe and K9-
OMe monosubstituted analogs are lower than those of trichogin
GA IV. This latter result may suggest that the replacement of a
Gly residue at either the N- or the C- terminus of the sequence
is not beneficial for folding. However, it is worth remembering
that the difference in the C-terminal moiety between the K2-OMe
and K9-OMe analogs (Leu11-OMe carboxyl ester) and the other
Lys-containing analogs (Lol 1,2-amino alcohol) might play a role
as well.7,12,15,16,18 Moreover, if the ratio (R) between the ellipticity
values at 225 and 205 nm is considered,58–60 it turns out clearly
that for all peptides this parameter is enhanced (from 0.3–0.5
to 0.6–0.7) from MeOH to the membrane mimetic environment
(SDS). On the basis of literature data, we interpret this result as
arising from a contribution of mixed 310-/a-helical conformations
with increasing a-helix population in SDS micelles. However,
increasing the temperature from 20 to 40 ◦C in aqueous SDS,
the contribution of the a-helix to the conformational equilibrium
mixture of the Lys tetrasubstituted analog K2569 decreases (R
value at 40 ◦C: 0.43, Fig. S2†). This result, while underscoring
the thermal stability of the overall helical structure of this short
peptide containing four cationic residues (37% of the overall
sequence), also highlights its uncommon capability to undergo
a transition between the 310- and a- helical conformations.
is not soluble in water), all similar in shape (R values between 0.4
and 0.5) and with only a limited variation in the ellipticity values,
reflect the propensity of the peptides to adopt a helical conforma-
tion endowed with a predominant 310-helical character. The only
exception is represented by K5 which seems to adopt a slightly
more populated a-helical conformation (R = 0.65). Interestingly,
in phosphate buffer the CD curves of the analogs containing two
or more Lys residues change substantially from pH 3 to 11 (Fig.
4B). In particular, a dramatic variation is experienced by the K2569
analog. Its CD profile at pH 3 (R value 0.3), where all four Lys side
chains are protonated,61,62 is clearly indicative of a well developed
310-helical structure (for this analog the 310-helix extent at pH 3 is
even higher than that in MeOH). However, at pH 11 (R value 0.95),
where all four Lys side chains are essentially uncharged, the peptide
is folded in a fully developed a-helical structure. Fig. 5 highlights
the intriguing phenomenon of a pH controlled, reversible 310-helix
to a-helix conversion (from acidic to highly basic pH values and
vice versa). At this point, it should be emphasized that this finding,
never authenticated before in the literature for any peptaibol or
˚
analog thereof, implies a significant overall contraction (about 4 A)
of the peptide backbone following the conformational transition
from the longer 310-helix24 to the shorter a-helix and an associated
generation of a biomolecular spring.
To study the lipopeptaibol conformation in more detail, an
NMR analysis was performed. The 2D-NMR spectra of the three
Lys-containing trichogin GA IV analogs, K56, K2569, K259U6
were first acquired in CD3OH. The complete assignments of
the proton resonances were obtained following the Wu¨thrich
procedure.63 The ROESY spectra exhibit all of the sequential
NHi→NHi+1 cross peaks characteristic of the presence of an he-
lical structure. In particular, the fingerprint region of the ROESY
spectrum of K2569 in CD3OH (Fig. 6A) shows four Ca Hi→NHi+3
cross peaks, typical of the presence of a helical conformation,
along with three Ca Hi→NHi+4 and four Ca Hi→NHi+2 cross
peaks, which denote the presence of an a- and a 310- helical
conformation, respectively. The NMR analysis was extended to
an aqueous solution (pH 3) (Fig. 6B) and a 100 mM SDS
We extended our CD analysis to aqueous solutions. In water at
pH 5–6 (Fig. 4A) the spectra of the seven analogs (trichogin GA IV
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Fig. 4 Far-UV CD spectra of the seven trichogin GA IV analogs in aqueous solution. (A) Spectra in water (pH 5–6). (B) Spectra of the K56, K259U6,
and K2569 analogs in phosphate buffer at pH 3 and 11. Peptide concentration: 0.1 mM.
Fig. 5 Far-UV CD spectra of the K2569 trichogin GA IV analog in
aqueous solution at pH 3 and 11. Repeated cycles of helix conversion were
performed, the order of pH switches being pH 3 (1), pH 11 (1), pH 3 (2),
and pH 11 (2). Peptide concentration: 0.015 mM.
Fig. 6 Fingerprint region of the ROESY/NOESY spectra of the K2569
membrane-mimetic environment (pH 2.83) (Fig. 6C). Here, the
trichogin GA IV analog under three different conditions: (A) 5.6 mM in
310-helix contribution to the overall conformation is larger with
CD3OH (298 K, 600 MHz); (B): 1.8 mM in 9 : 1 H2O/D2O, pH 3 (303 K,
respect to that of the a-helix (four Ca Hi→NHi+2 cross peaks
600 MHz); (C) 3.5 mM in 9 : 1 H2O/D2O containing 100 mM SDS-d25, pH
versus only one Ca Hi→NHi+4). The present findings highlight
2.83 (313 K, 600 MHz). Medium-range interactions, diagnostic of the pres-
ence of a mixed 310-/a-helical structure: in blu CaHi→NHi+2 (310-helix); in
the occurrence of a mixed 310-/a-helical conformation in the
green: CaHi→NHi+4 (a-helix); in red: CaHi→NHi+3 (310-/a-helix).
three solvents examined, which is more markedly 310-helical in
the aqueous acidic environment. These results nicely confirm the
CD observation discussed above. In particular, the CD spectra
recorded at different temperatures explain the NMR observation
of a prevailing 310-helical structure in the membrane-mimicking
environment at 40 ◦C (Fig. 6C). The NMR spectrum of this
analog was not recorded at pH 11, where it is known to be fully
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a-helical (see above), because of the rapid exchange rate of the
amide protons under basic conditions.
K5 and K6 have the same net charge, they possess different
hydrophobicities. Also in this case, the hemolytic activities follow
the same trend, with K5 being less toxic than K6. A particular
effect of the C-terminal methyl ester is probably at the basis of
the behavior of K2-OMe that, although less hydrophobic than
the other monosubstituted analogs, exhibits a remarkably low
hemolytic activity.
Biological activity
The hemolytic activity of the seven Lys-containing trichogin
GA IV analogs, a chronic handicap for most AMPs (including
peptaibols),64 was tested. The lytic concentrations causing 50%
hemolysis (LC50) after one hour incubation with 4% human red
blood cells (hRBCs) in PBS, are reported in Table 2. From the
biophysical properties summarized in Table 2, it is clear that the
capability of trichogin GA IV analogs to kill hRBCs is correlated
to the peptide hydrophobicity rather than to the total number of
charges. Notably, the presence of an Aib residue at position 6 of
K259U6 analog seems to balance out the net charge, enhancing
the compound hydrophobicity to a value greater than that of the
K56 analog. Not surprisingly, the hemolytic activity of these two
analogs follows the same trend. This observation brings us to
conclude that it might be possible to modulate the effect of cationic
residues by strengthening the overall helical peptide conformation.
From the RP-HPLC retention times of the monosubstituted
analogs (Table 1), K9-OMe results to be even more hydrophobic
than trichogin GA IV, probably because of the presence of the
C-terminal methyl ester functionality. Moreover, even though
The antimicrobial activity of the seven Lys-containing trichogin
GA IV analogs was investigated against two strains of Gram-
positive bacteria (Staphylococcus aureus and Staphylococcus epi-
dermidis), two strains of Gram-negative bacteria (Escherichia coli
and Pseudomonas aeruginosa), and two types of fungi (Candida
albicans and Trichosporon beigelli). The minimum inhibitory
concentration (MIC) values are reported in Table 3, together with
the values found for the well-known antimicrobial peptide melittin
under the same experimental conditions. In general, the presence
of the positively charged Lys residues increases the antibacterial
activity and broadens the spectrum of action of the analogs with
respect to those of the native peptide. Among all analogs, K56
shows a remarkable increased activity against all strains tested,
while K259U6 exhibits a specific, very strong activity against S.
aureus. Remarkably, the presence of one or more Lys residues
induces antifungal properties to all of the analogs of trichogin
GA IV, which is a fungal peptide. The monosubstituted analogs
allowed us to study the influence of the position of the Lys residue
in the sequence on the biological properties. From Table 3 it
is clear that the Lys position plays an important role. Indeed,
while the antimicrobial action of K2-OMe closely parallels that
of the native peptide and K5 and K6 show the same selectivity
as that of trichogin GA IV, though somewhat enhanced, the K9-
OMe analog exhibits a broader antibacterial spectrum, with a
remarkable activity against both the Gram-positive strains tested
and the Gram-negative E. coli. This analog also shows a very
promising selectivity for bacteria with respect to hRBCs. We also
tested the cytotoxicity of K9-OMe against HaCaT (Human adult
low-Calcium high Temperature keratinocyte cells) and found little
toxicity (> 64 mg ml-1).
Table 2 Biophysical properties of trichogin GA IV and its analogs
Abbreviation
MWcalc
MWobs
Qa
H (%)b
LC50 (mg ml-1)c
TRIC
1065.72 1065.74
1136.79 1136.77
1136.79 1136.86
1207.86 1207.86
1306.97 1306.95
1350.01 1350.00
1093.71 1093.72
1164.78 1164.82
1164.78 1164.81
2846.46 2846.46
0
1
1
2
3
4
0
1
1
5
100
>32
16
8
5.4
10
5.4
>32
>32
>32
2
K5
99.4
91.0
78.5
85.9
53.7
100
96.8
78.3
n.d.e
K6
K56
K259U6
K2569
Tric-OMe12
K9-OMe
K2-OMe
Melittind
a Q: total charge. b H: molecular hydrophobicity estimated from elution
time in reversed-phase HPLC with respect to the native compound (TRIC
or Tric-OMe). c LC50: peptide concentration responsible for 50% hemolysis
after 3 h incubation with 4% red blood cells in PBS. d Antimicrobial,
hemolytic peptide used as standard (ref. 87). e n.d.: not determined.
Analyzing in depth the MIC values reported in Table 3, we
can make an attempt to establish a relationship between the
number of Lys residues and the overall antimicrobial activity of the
trichogin GA IV analogs. The substitution of either Gly5 or Gly6
Table 3 Antimicrobial activities (MIC values) of trichogin GA IV and its analogsa
Bacteria
Gram-positive
Gram-negative
Fungi
Peptide
S. aureus
S. epidermidis
E. coli
P. aeruginosa
C. albicans
T. beigelli
TRIC
8–16
4
4
4
2
8
4
4
>64
64
64
4
8
8
64
4
>64
2
32
4
4
4
8
8
32
8
32
2
>64
64
64
4
8
8
32
64
>64
4
>64
8
16
4
16
8
64
64
n.d.c
8
>64
8
8
4
4
K5
K6
K56
K259U6
K2569
K2-OMe
K9-OMe
Tric-OMe12,48
Melittinb
8
32
64
n.d.c
8
16
4
a All values are in mg ml-1. b Antimicrobial peptide used as standard. c n.d.: not determined.
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with Lys dramatically increases the toxicity of this peptaibiotic
against S. aureus and E. coli. The concomitant substitution of
both those Gly residues does not improve the activity against
the two above mentioned strains, thus excluding the presence of
a cooperative effect between the two positive charged residues
in the peptide mechanism of action against these two bacteria.
The picture changes if we look at the MIC values obtained
against the other bacteria tested. Indeed, it seems that, in order
to broaden the spectrum of the antimicrobial action of trichogin
GA IV, at least two Gly residues must be replaced by Lys. This
conclusion is further confirmed by the selectivity shown by the
other monosubstituted analogs K2-OMe and K9-OMe. Moreover,
the presence of additional positively charged residues does not
further reduce the MIC values, which, for the K259U6 and K2569
analogs, are almost comparable to those of K56.
In summary: (i) the insertion of an Aib residue may modulate
the biological properties of cationic peptides; (ii) it is not only
the net charge that is responsible for the enhanced antimicrobial
properties of trichogin GA IV analogs, but the position of the
Lys residue(s) in the primary structure as well; (iii) the K9-OMe
analog is a very promising antimicrobial agent, as it possesses a
7-fold greater activity against bacteria than against hRBCs.
action of K2-OMe against S. aureus as compared to that against
E. coli is reflected by its higher ability to induce depolarization
of the related, former membrane. These results underscore the
important role played by peptide ◊ ◊ ◊ membrane interactions on the
antimicrobial mechanism of the trichogin GA IV analogs. Indeed,
the ability of these compounds to interact with the membrane
bilayer is likely to be mainly responsible for their biological activity
and makes them promising candidates as new antimicrobial agents
with reduced resistance-induction risks.
Proteolytic stability
We assessed the resistance of trichogin GA IV analogs to the
action of several proteolytic enzymes.65–72 The Lol-containing
analogs proved to be resistant towards the proteolysis of pronase
E, subtilisin and papain for more than four hours. However,
the C-terminal methyl esters of the monosubstituted K2-OMe
and K9-OMe analogs were hydrolyzed to the corresponding
free carboxylic acids within 15 min, as proved by isolation and
characterization of the products by ESI-MS. The resulting free
carboxylic acid peptides were stable towards further proteolysis
for more than four hours. All of the analogs were extensively
digested by trypsin within 15 min, except K2-OMe and K6 which
are surprisingly resistant to the action of this enzyme, with an
half-life time of two and three hours, respectively (Fig. 8).
Membrane activity
The membrane permeability properties of most of the syn-
thetic analogs were at first tested in comparison with the na-
tive lipopeptaibol trichogin GA IV by measuring the induced
leakage of 5(6)-carboxyfluorescein (CF) entrapped in small
unilamellar vesicles (SUVs). For this investigation, the over-
all neutral, zwitter-ionic 1,2-dioleyl-sn-glycero-3-phosphocholine
(DOPC)/cholesterol (Ch) model membrane was exploited. The
permeability effect of the Lys-containing trichogin GA IV analogs
is quite remarkable and close to that of the native compound (Fig.
S3, Supporting Information†).7,12
To further investigate the mechanism of antimicrobial action
of these novel peptaibiotics, we performed depolarization leakage
assays on S. aureus and E. coli membranes. As shown in Fig.
7, the trend of their ability to induce membrane depolarization
parallels their antimicrobial activities. In particular, the selective
Conclusions
The most extensively studied short peptaibiotic is trichogin GA
IV, a 10-amino acid peptide characterized by the presence of a
fatty acid chain (n-octanoyl) at its N-terminus and a C-terminal
1,2-amino alcohol. Its mechanism of action on the membranes is
under debate, even if it is clearly driven by its stable, mixed 310-/
a- helical conformation. In this paper, we enhanced the natural
modest amphiphilic character of the trichogin GA IV 3D-structure
by replacing one or more Gly, lying on the same face of the helix,
by Lys residues. To determine the conformational preferences
in solution of the peptaibiotics synthesized, we subjected them
to a thorough investigation by means of IR absorption, CD,
and 2D NMR in various solvents and in a membrane-mimetic
Fig. 7 Peptide-induced depolarization of bacterial membranes. Each peptide was added to S. aureus (A) or E. coli (B) bacteria previously pre-equilibrated
for 60 min with DiSC3-5. Fluorescence recovery was measured in continuous after peptide incorporation. The maximum fluorescence recovery values are
shown for each peptide.
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Fig. 8 RP-HPLC profiles obtained for the K2-OMe trichogin GA IV analog in presence of the proteolytic enzyme trypsin, checked every 15 min. Elution
-1
˚
conditions: column: Phenomenex Kromasil 100 A, 5 mm; flux: 1 ml min ; gradient: 70–90% B in 10 min; eluant A: 9 : 1 H2O/CH3CN + 0.05% TFA;
eluant B: 9 : 1 CH3CN/H2O + 0.05% TFA. The initial peak for K2-OMe is starred.
and are uncorrected. Optical rotations [a]2D0 (given in units of 10-1
environment. The Lys-containing analogs exhibit a stable helical
conformation, which can be intriguingly switched from 310- to
deg cm2 g-1) were measured at 20 ◦C on a Perkin-Elmer PE241
a-helix by changing the pH from 3 to 11. The critical pH for
the transition, which is located between 9 and 10 (far above
the physiological pH conditions), seems to exclude a possible
role for this conformational switch on the mechanism of the
antimicrobial action of our analogs. The fluorescence and depo-
larization leakage experiments reveal that the novel peptaibiotics
retain the native capability to interact with model membranes.
The sensitivity assays in vitro against a collection of bacteria
(both Gram-positive and Gram-negative) and fungal species of
clinical relevance assessed on the synthetic analogs underscored
their broad antimicrobial spectrum. However, the presence of
more that one Lys residue increases the hemolytic activity and
shorten the resistance toward trypsin digestion. To summarize,
the Lys-containing, short peptides described herein (particularly
the most substituted ones) could find applications as molecular
switches,73 for their uncommon ability to undergo pH-mediated,
reversible, conformational transitions. More importantly, in our
view, two of the least substituted analogs, K9-OMe for its broad
antimicrobial spectrum and low cytotoxicity and K2-OMe for
its uncommon tryptic resistance, may represent promising leads
for the development of new membrane-disrupting antimicrobial
agents.
polarimeter using a 1 dm path length cell, at the D-wavelength
of sodium (589 nm). The concentration of each compound (c)
is given in mg cl-1. Mass spectra (electrospray ionization, ESI-
MS) was performed by using a PerSeptive Biosystem Mariner
instrument (Framingham, MA). Analytical TLC and preparative
column chromatography were performed on Kieselgel F 254 and
Kieselgel 60 (0.040–0.063 mm) (Merck), respectively. The reten-
tion factor (Rf) values were determined using four solvent mixtures
as eluants: Rf1: chloroform/ethanol 9 : 1; Rf2: 1-butanol/acetic
acid/water 3 : 1 : 1; Rf3: toluene/ethanol 7 : 1; Rf4: methylene
chloride/ethanol 9 : 1.
Fmoc(9-fluorenylmethyloxycarbonyl)-amino acids were sup-
plied by Novabiochem (Merck Biosciences, La Jolla, CA). All
other amino acid derivatives and reagents for peptide synthesis
were purchased from Sigma-Aldrich (St. Louis, MO). The final
products were characterized by analytical RP-HPLC on a Vydac
˚
C18 column (4.6 ¥ 250 mm, 5 m, 300 A) using a Dionex (Sunnyvale,
CA) P680 HPLC pump with an ASI-100 automated sample
injector. The binary elution system used was: A, 0.1% TFA in
H2O; B, 0.1% TFA (trifuoroacetic acid) in CH3CN/H2O (9 : 1
v/v); gradient 50%–90% B in 20 min (flow rate 1.5 ml min-1);
spectrophotometric detection at l = 216 nm.
Solution-phase peptide synthesis. The peptide segments were
obtained step-by-step using orthogonal, terminal- and side-chain
protecting groups (Scheme 1). Removals of the Z and Fmoc Na-
protections were achieved using Pd-catalyzed hydrogenation and
diethylamine in methylene chloride followed by flash chromatog-
raphy, respectively. In either the Xxx-Aib or the Aib-Xxx peptide
Experimental
Peptide synthesis and characterization
General methods. Melting points were measured by means of
a capillary tube immersed in an oil bath (Tottoli apparatus, Bu¨chi)
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bond formation, C-activation was carried out using EDC, N-[3-
(dimethylamino)-propyl]-N¢-ethylcarbodiimide/HOAt (7-aza-1-
hydroxy-1,2,3-benzotriazole)74 procedure, while the EDC/HOBt
(1-hydroxy-1,2,3-benzotriazole)75 procedure, although less effi-
cient for difficult couplings, was appropriate for the formation of
the other peptide bonds. Every coupling reaction was performed
with a 10% excess of the incoming residue. The pH of the
coupling mixtures were kept at 8 by adding N-methylmorpholine.
The C-terminal tert-butyl (OtBu) esters were converted to free
carboxylic acids upon treatment with diluted TFA in methylene
chloride. The segment condensation reactions were obtained by
EDC/HOAt C-activation, using a 10% excess of the C-terminal
carboxyl free segment. The yields of the segment condensation
reactions were about 50%. After each coupling reaction, the
product was purified by flash chromatography using suitable ratios
of methylene chloride/methanol or ethyl acetate/hexane mixtures.
[m, 2H, urethane CH2], 3.16 [d, 2H], 1.85 [s, 6H], 1.64 [m, 3H,
Leu b-CH2, Leu g-CH], 1.45 [m, 12H, Aib b-CH3], 1.43 [s, 9H,
OtBu], 0.82 [m, 6H, Leu d-CH3]. 13C NMR (100 MHz, CDCl3):
d 175.349, 173.548, 171.460, 171.383 [4s, ester and amide C O],
157.139, 156.015 [2s, urethane C O], 143.513, 143.383, 141.387,
136.607 [4s, aromatic quaternary C], 128.598, 128.101, 127.917,
127.626, 127.160, 127.092, 124.920, 124.700, 120.123 [9s, aromatic
C], 80.537 [s, OtBu quaternary C], 66.875, 66.403 [2s, urethane
CH2], 56.984, 56.416, 54.983, 51.926 [4s, aC], 47.056 [s, Fmoc
tertiary C], 39.943, 39.781 [2s, Leu bC and Lys eC], 30.247, 29.709,
29.624 [3s, Lys bC, Lys dC], 27.843 [s, OtBu CH3], 26.548, 25.081,
g
24.938, 24.532, 24.091 [5s, Leu C, 4 Aib bC], 23.263, 22.741,
21.012 [3s, Lys g C and Leu dC].
Segment D [Fmoc-Lys(Z)-Ile-Leu-OMe]. Overall yield: 69%.
Melting p◦oint: 192–194 ◦C. Rf2: 0.95, Rf3: 0.65, Rf4: 0.95.
[a]2D0:-43.8 (c = 0.5, MeOH). IR (KBr): 3294, 1693, 1642, 1536
cm-1. ESI-MS m/z: calculated for C42H54N4O8 742.40, found
742.37. 1H NMR (600 MHz, CDCl3): d 7.75 [d, 2H, Fmoc], 7.58
[t, 2H, Fmoc], 7.49 [m, 2H, Fmoc], 7.32 [m, 5H, Z phenyl CH],
6.64 [d, 1H, NH], 6.36 [d, 1H, NH], 5.96 [d, 1H, NH], 5.07 [m,
2H, urethane CH2], 4.60 [s, 1H, Lys e-NH], 4.39 [m, 2H, urethane
CH2], 4.29 [t, 1H, a-CH], 4.20 [m, 2H, a-CH], 3.70 [s, 3H, -OMe
CH3], 3.20 [d, 2H], 1.80–1.38 [m, 5H, Leu b-CH2, Leu g-CH and
Ile g-CH2], 0.89 [m, 12H, Leu d-CH3, Ile g-CH3 and d-CH3]. 13C
NMR (100 MHz, CDCl3): d 173.069, 171.813, 170.790 [3s, ester
and amide C O], 159.557, 156.735 [2s, urethane C O], 143.776,
141.294, 136.608 [3s, aromatic quaternary C] 128.505, 127.750,
127.101, 125.037, 119.995 [5s, aromatic C], 67.156, 66.637 [2s,
urethane CH2], 57.950, 54.874, 53.540 [3s, aC], 52.279 [s, -OCH3],
47.129 [s, Fmoc tertiary C], 41.184 [s, Leu bC], 40.209 [s, Lys eC],
37.020 [s, Ile bC], 31.844 [s, Lys dC], 29.334 [s, Lys bC], 25.377 [s,
Ile g CH], 24.864 [s, Leu g C], 22.766 [s, Leu dC], 22.274 [s, Lys g C],
21.809 [s, Leu dC], 15.386, 11.254 [2s, Ile g CH3 and dC].
Segment A (Z-Gly-Ile-Leu-OMe):12. Overall yield: 76%. Melt◦-
ing point: 119-120 ◦C. Rf1: 0.85, Rf2: 0.95, Rf3: 0.30. [a]D20: -45.9
(c = 0.5, MeOH). IR (KBr): 3290, 1745, 1703, 1676, 1637, 1537
cm-1. ESI-MS m/z: calculated for C23H35N3O6 449.25, found
1
449.25. H NMR (200 MHz, CDCl3): d 7.35 [m, 5H, Z phenyl
CH], 6.64 [d, 1H, Ile NH], 6.35 [d, 1H, Leu NH], 5.48 [t, 1H,
Gly NH], 5.13 [s, 2H, Z CH2], 4.58 [m, 1H, Leu a-CH], 4.34 [m,
1H, Ile a-CH], 3.90 [m, 2H, Gly a-CH2], 3.72 [s, 3H, OMe CH3],
1.85 [m, 1H, Ile b-CH], 1.70–1.40 [m, 4H, Leu b-CH2, Leu g-CH,
Ile 1 g-CH], 1.13 [m, 1H, Ile 1 g-CH], 1.00–0.80 [m, 12H, Leu
2 d-CH3, Ile g-CH3, Ile d-CH3]. 13C NMR (150 MHz, CDCl3):
d 173.042, 171.051, 169.055 [3s, ester and amide C O], 156.557
[s, urethane C O], 136.223 [s, aromatic quaternary C], 128.449,
128.420, 128.097, 128.001 [4s, aromatic C], 66.999 [s, urethane
CH2], 57.507, 52.144, 50.803, 44.339 [4s, aC and -OCH3], 40.996
[s, Leu bC], 37.518 [s, Ile bC], 24.840, 23.236, 22.618, 21.872 [4s, Ile
g C, Leu g C and dC], 15.072, 11.176 [2s, Ile g C and dC].
Segment B (Z-Gly-Gly-Leu-Aib-OtBu). Overall yield: 52%.
Segment E (Z-Aib-Gl◦y-Leu-Aib-OtBu). Overall yield: 71%.
Melting point: 165–166 C. Rf1: 0.50, Rf2: 0.80, Rf3: 0.30. [a]2D0:
-3.2◦ (c = 0.5, MeOH). IR (KBr): 3316, 1732, 1701, 1659, 1536
cm-1. ESI-MS m/z: calculated for C28H44N4O7 548.32, found
◦
Melting point: 67–68 C. Rf2: 0.95, Rf3: 0.40, Rf4: 0.75. [a]2D0:
-22.1◦ (c = 0.5, MeOH). IR (KBr): 3312, 1732, 1711, 1651,
1535 cm-1. ESI-MS m/z: calculated for C26H40N4O7 520.29, found
1
1
520.29. H NMR (400 MHz, CDCl3): d 7.35 [m, 5H, Z phenyl
548.31. H NMR (200 MHz, CDCl3): d 7.36 [m, 6H, Z phenyl
CH], 6.94 [t, 1H, Gly2 NH], 6.81 [s, 1H, Aib NH], 6.67 [d, 1H,
Leu NH], 5.61 [t, 1H, Gly1 NH], 5.13 [s, 2H, Z CH2], 4.40 [m, 1H,
Leu a-CH], 4.00–3.80 [m, 4H, Gly a-CH2], 1.63 [m, 3H, Leu b-
CH2, Leu g-CH], 1.50 [s, 6H, Aib b-CH3], 1.43 [s, 9H, OtBu], 0.92
[m, 6H, Leu d-CH3]. 13C NMR (100 MHz, CDCl3): d 173.469,
170.981, 169.598, 168.642 [4s, amide and ester C O], 156.737
[s, urethane C O], 136.063 [s, aromatic quaternary C], 128.531,
128.237, 128.073 [3s, aromatic C], 81.524 [s, OtBu quaternary C],
67.228 [s, urethane CH2], 56.904, 51.929, 44.455, 43.000 [4s, aC],
CH, 1H NH], 6.93 [m, 1H, NH], 6.87 [s, 1H, Aib NH], 5.37 [s,
1H, Aib NH], 5.10 [2d, 2H, Z CH2], 4.42 [m, 1H, Leu a-CH], 4.0–
3.80 [m, 4H, Gly a-CH2], 1.68 [m, 3H, Leu b-CH2, Leu g-CH],
1.49–1.51 [m, 12H, Aib 4 b-CH3], 1.43 [s, 9H, OtBu], 0.92 [m, 6H,
Leu d-CH3]. 13C NMR (100 MHz, CDCl3): d 174.893, 173.513,
171.315, 169.209 [4s, amide and ester C O], 155.833 [s, urethane
C
O], 135.528 [s, aromatic quaternary C], 128.137, 127.946 [2s,
aromatic C], 80.824 [s, OtBu quaternary C], 67.366 [s, urethane
a
b
CH2], 57.021, 51.930, 43.647 [3s, 4 C], 39.849 [s, C], 27.751 [s,
b
g
41.496 [s, C], 27.791 [s, OtBu CH3], 24.706, 24.534, 24.425 [3s,
OtBu CH3], 24.782, 24.696, 24.617, 24.529, 24.350 [5s, Leu C,
Leu g C, Aib bC], 22.894, 22.006 [2s, Leu dC].
Aib bC], 22.853, 21.952 [2s, Leu dC].
Segment C [Fmoc-Aib-Lys(Z)-Leu-Aib-OtBu]. Overall yield:
50%. Oil. Rf2: 0.95, Rf3: 0.50, Rf4: 0.85. [a]2D0: -21.5◦ (c = 0.5,
MeOH). IR (KBr): 3317, 1700, 1658, 1525 cm-1. ESI-MS m/z:
Segment
B-A
(Z-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe):12.
Overall yield: 60%. Melting p◦oint: 152–153 ◦C. Rf1: 0.40,
Rf2: 0.90, Rf3: 0.10. [a]2D0: -39.1 (c = 0.5, MeOH). IR (KBr):
3318, 1734, 1706, 1651, 1540 cm-1. ESI-MS m/z: calculated for
C37H59N7O10 761.43, found 761.43. 1H NMR (400 MHz, CDCl3):
d 8.19 [t, 1H, Gly NH], 7.68 [t, 1H, Gly NH], 7.59 [d, 1H, Ile
NH], 7.45 [d, 1H, Leu NH], 7.35 [m, 5H, Z phenyl CH], 6.76 [m,
2H, Aib NH, Leu NH], 5.94 [t, 1H, Gly NH], 5.12 [m, 2H, Z
1
calculated for C47H63N5O9 841.46, found 841.46. H NMR (400
MHz, CDCl3): d 7.77 [d, 2H, Fmoc], 7.56 [t, 2H, Fmoc], 7.40 [t,
2H, Fmoc], 7.31 [m, 5H, Z phenyl CH], 7.26 [m, 1H, NH], 6.87
[s, 1H, NH], 6.67 [m, 1H, NH], 5.00 [m, 3H, 1 Lys e-NH and
urethane CH2], 4.58 [m, 1H, a-CH], 4.37 [m, 1H, a-CH], 4.13
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◦
CH2], 4.31–3.26 [m, 2H, Gly a-CH2], 4.23 [m, 1H, Leu a-CH],
4.21–3.45 [m, 2H, Gly a-CH2], 4.05 [m, 1H, Leu a-CH], 4.02
[m, 1H, Ile a-CH], 3.91 [m, 2H, Gly a-CH2], 3.66 [s, 3H, OMe
CH3], 2.10 [m, 1H, Ile b-CH], 1.80–1.60 [m, 7H, Leu b-CH2
and g-CH, Ile 1 g-CH], 1.57 [s, 3H, Aib b-CH3], 1.44 [s, 3H,
Aib b-CH3], 1.13 [m, 1H, Ile 1 g-CH], 1.00–0.80 [m, 18H, Leu
d-CH3, Ile g-CH3 and d-CH3]. 13C NMR (150 MHz, CDCl3): d
174.743, 173.608, 173.483, 172.915, 171.949, 170.573, 170.398 [7s,
ester and amide C O], 157.586 [s, urethane C O], 136.110 [s,
aromatic quaternary C], 128.478, 128.160, 127.779 [3s, aromatic
C], 67.230 [s, urethane CH2], 58.378, 57.174, 53.749, 52.221,
K9-OMe: Overall yield: 25%. Melting point: 152–153 C. Rf2:
0.70, Rf3: 0.05 Rf4: 0.05. [a]2D0: -14.6◦ (c = 0.2, MeOH). IR (KBr):
3317, 1743, 1657, 1537 cm-1. ESI m/z: calculated for C57H104N12O13
1164.78, found 1164.82. 1H NMR (600 MHz, H2O/D2O 9 : 1): d
8.66 [t, 1H, Gly2 NH], 8.43 [t, 1H, Gly5-NH], 8.35 [s, 1H, Aib1-
NH], 8.08 [t, 1H, Gly6-NH], 8.04 [d, 1H, Leu3-NH], 7.85 [d, 1H,
Leu7-NH], 7.84 [s, 1H, Aib8-NH], 7.82 [s, 1H, Aib4-NH], 7.60–7.59
[d, 1H, Ile10 NH], 7.55 [d, 1H, Leu11-NH], 7.37 [d, 1H, Lys9 NH],
4.36 [m, 1H, Leu11 a-CH], 4.20 [m, 1H, Leu3 a-CH], 4.13–4.12 [m,
2H, Ile10 a-CH and Lys9 a-CH], 4.04 [m, 1H, Leu7 a-CH], 3.96 [m,
1H, Gly6 1 a-CH], 3.92 [m, 1H], 3.89 [m, 1H, Gly2 1 a-CH], 3.84
[m, 1H, Gly5 1 a-CH], 3.76–3.71 [m, 3H, Gly5 a-CH, Gly6 a-CH
and Gly2 a-CH], 3.65 [s, 3H, OMe CH3], 2.92 [m, 2H, Lys9 e-CH2],
2.17 [m, 2H, nOct CH2], 1.85 [m, 1H, Ile10 b-CH], 1.74 [m, 2H, Lys9
b-CH2], 1.68 [m, 2H, Leu3 b-CH2], 1.61 [m, 2H, Leu11 b-CH2], 1.55
[m, 1H, Leu3 1 g-CH], 1.44 [m, 2H, nOct b-CH2], 1.42 [s, 8H, Lys9 g-
CH2, Ile10 1 g-CH and Aib4 b-CH3], 1.36 [s, 6H, Aib1 2 b-CH3], 1.35
and 1.32 [2s, 6H, 2 Aib8 b-CH3], 1.20–1.04 [m, 11H, Ile10 1 g-CH
and nOct (CH2)5], 0.83–0.79 [m, 9H, Leu3 2 d-CH3, Ile10 g-CH3],
0.76–0.72 [m, 15H, Leu7 2 d-CH3, Ile10 d-CH3 and Leu11 2 d-CH3],
0.67 [t, 2H, nOct CH3]. 13C NMR (150 MHz, H2O/D2O 9 : 1):
d 190.053, 178.580, 175.148, 175.088, 174.851, 174.782, 174.693,
174.276, 174.248, 174.216, 173.528, 173.490 [12s, ester and amide
a
a
51.951 [5s, C and -OCH3], 45.033, 43.387, 42.611 [3s, C Gly],
40.036, 39.239, 35.376 [3s, Ile and Leu bC], 24.834, 24.654, 24.607,
b
g
g
22.614, 22.467, 22.484, 21.549 [7s, Aib C, Ile CH, Leu C and
dC], 15.317, 10.632 [2s, Ile g CH3 and dC].
Segment E-B [Z-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-OtBu].
Overall yield: 61%. Melting point: 112–114 ◦C. Rf2: 0.95, Rf3:
0.20, Rf4: 0.45. [a]2D0: -10.2◦ (c = 0.5, MeOH). IR (KBr): 3316,
1733, 1660, 1535 cm-1. ESI-MS m/z: calculated for C42H68N8O11
860.50, found 860.48. 1H NMR (400 MHz, CDCl3): d 7.90 [t, 1H,
Gly NH], 7.81 [d, 1H, Leu NH], 7.52 [t, 1H, Gly NH], 7.49 [t,
1H, Gly NH], 7.35 [m, 5H, Z phenyl CH], 7.26 [s, 1H, Aib NH],
7.11 [d, 1H, Leu NH], 6.90 [s, 1H, Aib NH], 5.82 [s, 1H, Aib
NH], 5.10 [m, 2H, Z CH2], 4.38 [m, 1H, a-CH], 4.05 [m, 2H, 2
a-CH], 3.86 [m, 4H, 4 a-CH], 3.69 [m, 1H, a-CH], 1.72 [m, 6H,
Leu b-CH2 and g-CH], 1.45 [m, 27H, OtBu, Aib b-CH3], 0.93
[m, 12H, Leu d-CH3]. 13C NMR (100 MHz, CDCl3): d 176.866,
174.689, 173.414, 172.198, 171.982, 171.081, 170.361 [7s, 8 amide
and ester C O], 156.593 [s, urethane C O], 135.744 [s, aromatic
quaternary C], 128.658, 128.339, 127.612 [3s, aromatic C], 80.842
[s, OtBu quaternary C], 67.030 [s, urethane CH2], 56.868, 56.570,
C
O], 56.777, 56.398, 56.308, 54.828, 54.132, 53.504, 53.080,
52.781, 50.754 [9s, aC and -OCH3], 44.062, 43.775, 42.912 [3s, aC
Gly], 39.447, 36.507, 35.279, 31.463 [4s, n-Oct, Lys C and C,
Ile and Leu C], 29.044, 28.826, 26.320, 25.839, 25.220, 25.163,
25.037, 24.392, 24.067, 23.624, 23.598, 22.925, 22.832, 22.585,
22.368, 22.321, 21.349, 21.126, 20.484 [19s, n-Oct, Lys g C and bC,
Aib C, Ile CH, Leu C and C], 15.122, 15.060, 13.641, 11.062
e
d
b
b
g
g
d
[4s, n-Oct, Ile g CH3 and dC].
Solid-phase peptide synthesis. Assembly of peptides on the
Advanced ChemTech (Louisville, KY) 348X peptide synthe-
sizer was performed on a 0.05 mmol scale by the FastMoc
methodology, as described below, starting with the Lol-substituted
2-chlorotrityl resin51,52 (Iris Biotech, Marktredwitz, Germany)
(110 mg, checked loading 0.40 mmol g-1). The Lys side chain
was protected with the Boc group, while the removal of the
Na-Fmoc protection was performed with 20% piperidine in
N,N-dimethylformamide. The total syntheses were achieved by
using the very efficient HATU [2-(1H-7-aza-1,2,3-benzotriazol-
1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate]74 double
coupling C-activation procedure for the formation of peptide
bonds involving the three poorly reactive Aib49 residues in the
native sequence. All other coupling reactions were conducted us-
ing the HBTU [2-(1H-1,2,3-benzotriazol-1-yl)-1,1,3,3-tetramethyl
uronium hexafluorophosphate]/HOBt procedure76 for activation
of the carboxylic function. Each coupling step was carried out by
use of a threefold excess of the Fmoc Na-protected a-amino acid in
the presence of a sevenfold excess of N,N¢-diisopropylethylamine
for 60 min. Final on-resin Na-n-octanoylation was achieved using
a threefold excess of n-Oct-OH preactivated with EDC/HOAt74
in the presence of N-methylmorpholine. Cleavage of the peptide
from 2-chlorotrityl resin was performed by repeated treatments
with 30% HFIP (1,1,1,3,3,3,-hexafluoropropan-2-ol) in distilled
CH2Cl2 (45 min each).52 The peptide cleaved from the resin was
filtered off and collected. Then, the solution was concentrated
under a flow of nitrogen. The crude peptides, protected as Boc
a
54.481, 52.511 [4s, 5 C Leu and Aib],44.573, 44.454, 43.536 [3s,
aC Gly], 40.191, 39.192 [2s, Leu bC], 27.830 [s, OtBu CH3], 26.542,
g
26.248, 25.089, 24.803, 24.509, 24.376, 24.095 [7s, Leu C, 6 Aib
bC], 23.860, 23.010, 21.357, 21.234 [4s, Leu dC].
◦
K2-OMe: Overall yield: 18%. Melting point: 148–150 C. Rf2:
0.70, Rf3: 0.05, Rf4: 0.05. [a]2D0: -24.8◦ (c = 0.2, MeOH). IR
(KBr): 3321, 1743, 1655, 1538 cm-1. ESI-MS m/z: calculated
1
for C57H104N12O13 1164.78, found 1164.81. H NMR (400 MHz,
H2O/D2O 9 : 1): d 8.37 [m, 2H, 2 NH], 8.31 [s, 1H NH], 8.08 [t,
1H, NH], 7.96 [m, 2H, 2 NH], 7.85 [s, 1H, 1 NH], 7.80–7.77 [m,
2H, 2 NH], 7.64–7.60 [m, 2H 2 NH], 7.48 [bs, 1H], 4.36 [m, 1H],
4.20–3.68 [m, 10H 10 a-CH], 3.64 [s, 3H, OMe CH3], 3.11 [m, 2H,
Lys2 e-CH2], 2.23–2.11 [m, 2H, nOct CH2], 1.98–1.51 [m, 30H],
1.39, 1.37. 1.35, 1.33, 1.32, [5s, 18H, b-CH3 Aib], 1.20–1.03 [m,
11H], 0.83–0.70 [m, 24H, d-CH3 Leu, d-CH3 and g-CH3 Ile], 0.69–
0.65 [t, 2H, nOct CH3]. 13C NMR (100 MHz, H2O/D2O 9 : 1):
d 183.497, 177.329, 175.247, 175.103, 173.887, 173.689, 173.626,
173.401, 173.095, 173.043, 172.158, 172.040 [12s, ester and amide
C
O], 56.751, 56.461, 56.372, 55.248, 53.381, 52.732, 51.458,
50.908, 49.247 [9s, aC and -OCH3], 44.486, 43.462, 42.645 [3s, aC
e
d
Gly], 39.429, 36.401, 35.347, 31.439 [4s, n-Oct, Lys C and C,
Ile and Leu C], 29.585, 28.986, 28.781, 26.327, 25.212, 25.093,
b
24.761, 24.541, 24.105, 23.538, 23.211, 22.803, 22.667, 22.574,
g
22.471, 22.342, 21.212, 20.942, 20.575 [19s, n-Oct, Lys C and
g
d
bC, Aib bC, Ile g CH, Leu C and C], 14.975, 13.634, 11.044 [3s,
n-Oct, Ile g CH3 and dC].
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derivatives at side-chain of Lys residues, were purified by flash
chromatography using chloroform-methanol (13 : 1) mixture as
eluant. The purified fractions were subjected to Boc removal using
3 M HCl in methanol, yielding (without further purification steps)
the desired products in a >98% purity (Fig. 1).
TRIC ESI-MS m/z: calculated for C52H95N11O12 1065.72, found
1065.75
identified using standard DQF-COSY77 and CLEAN-TOCSY78,79
spectra. In the latter case, the spin-lock pulse sequence was 70
ms long. The assignment of the two methyl groups belonging
1
to the same Aib residue was obtained by means of H-13C 2D
correlation spectra. To optimize the digital resolution in the
carbon dimension, heteronuclear multiple quantum coherence
(HMQC80) and heteronuclear multiple bond coherence (HMBC81)
experiments were acquired using selective excitation by means of
Gaussian-shaped pulses with 1% truncation.82,83
K5 ESI-MS m/z: calculated for C56H104N12O12 1136.79, found
1136.77
K6 ESI-MS m/z: calculated for C56H104N12O12 1136.79, found
1136.86
K56 ESI-MS m/z: calculated for C60H113N13O12 1207.86, found
1207.86
K259U6 ESI-MS m/z: calculated for C66H126N14O12 1306.97,
found 1306.95
K2569 ESI-MS m/z: calculated for C68H131N15O12 1350.01,
found 1350.00
The Cb-selective HMQC experiments with gradient coherence
selection84 were recorded with 320 t1 increments, of 300 scans and
2 K points each.80 A spectral width of 16 ppm centered at 22 ppm
in F1 was used, yielding a digital resolution of 2.36 Hz/pt prior
to zero filling. HMBC experiments with selective excitation in the
CO region were performed using a long-range coupling constant
of 7.5 Hz, a spectral width in F1 of 15 ppm centered at 176 ppm,
250 t1 experiments of 640 scans, and 4 K points in F2. The digital
resolution in F1, prior to zero filling, was 2.2 Hz/pt. NOESY
experiments were used for sequence specific assignment.
FT-IR absorption
The FT-IR absorption spectra were recorded at 293 K using a
Perkin-Elmer model 1720X FT-IR spectrophotometer, nitrogen
flushed, equipped with a sample-shuttle device, at 2 cm-1 nominal
resolution, averaging 100 scans. Solvent (baseline) spectra were
recorded under the same conditions. For spectral elaboration, the
software SPECTRACALC provided by Galactic (Salem, MA) was
employed. Cells with path lengths of 1.0 and 10 mm (with CaF2
windows) were used. Spectrograde deuterated chloroform (99.8%,
d) was purchased from Merck (Darmstadt, Germany).
Proteolytic stability
To evaluate their proteolytic stability, the peptides were dissolved
in a buffer solution (50 mM Tris·HCl, pH 7.8). The enzymes
trypsin, pepsin, chymotrypsin, endoprotease Glu-c V8, pronase
E, a protease from Streptomyces griseus, elastase (from porcine
pancreas), and subtilisin A (from Bacillus sp.) were obtained from
Sigma (St. Louis, MO). The enzyme was added to the peptide
in the ratio 1 : 100 (w/w) and the mixture was incubated at 37◦
C for 4 h. The samples were analyzed using a C18 reverse-phase
HPLC column. Appropriate programmed gradients from 40 to
95% B solution for 20 or 50 min were applied using eluants A
(H2O/0.05% TFA) and B (CH3CN/0.075% TFA).
Circular dichroism
The CD spectra were measured on a Jasco (Tokyo, Japan)
model J-715 spectropolarimeter equipped with a Haake thermo-
stat (Thermo Fisher Scientific, Waltham, MA). Baselines were
corrected by subtracting the solvent contribution. Fused quartz
cells of 1.0 mm and 10.0 mm path lengths (Hellma, Mu¨hlheim,
Germany) were used. The values are expressed in terms of [q]T, the
total molar ellipticity (deg ¥ cm2 x dmol-1). Spectrograde methanol
99.9% (Acros Organic, Geel, Belgium) was used as solvent.
SUV preparation
DOPC was purchased from Avanti Polar Lipids, Inc. (Alabaster,
AL), while Ch was a Sigma-Aldrich (St. Louis, MO) product.
The lipid mixture, DOPC/Ch (7 : 3) was dissolved in CHCl3 in
a test tube, dried under N2, and lyophilized overnight. The lipid
film was reconstituted with a solution of CF in 30 mM Hepes
buffer (pH 7.4) at room temperature for 1 h. To make SUVs, the
resulting multilamellar vesicle suspension was sonicated (GEX 400
Ultrasonic Processor, Sigma) on ice until the initially cloudy lipid
dispersion became translucent. The excess of fluorescent dye was
eliminated by gel filtration on Sephadex G-75 (Sigma). SUVs were
diluted to a concentration of 0.06 mM with Hepes buffer (5 mm
Hepes, 100 mM NaCl, pH 7.4). The SUVs were stored at 4 ◦C and
used within 24 h.
Nuclear magnetic resonance
1
All H NMR and 13C NMR experiments were performed on a
Bruker AVANCE DMX-600, DRX-400 or AC200 spectrometer
operating at 600 MHz, 400 MHz or 200 MHz, respectively, using
the TOPSPIN software package. For the characterizations, the
solvent was used as the internal standard: CDCl3 (1H: d = 7.26
ppm; 13C: d = 77.00 ppm). Splitting patterns are abbreviated as
follows: (s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multi-
plet. Samples for NMR conformational studies were dissolved in
water (9 : 1 H2O/D2O), in water (9 : 1 H2O/D2O) containing SDS-
d25 (100 mM) or in MeOH-d3 solution (peptide concentrations:
1.8 mM, 3.5 mM and 5.6 mM, respectively). The pH of the SDS-
containing solution was adjusted at 2.83 by adding 1 ml of 3 M
HCl. The spectra were recorded at 313 K. Presaturation of the
H2O solvent signal was obtained using a WATERGATE gradient
program. All homonuclear spectra were acquired by collecting
512 experiments, each one consisting of 64–80 scans and 2 K
data points. The spin systems of protein amino acid residues were
Leakage from lipid vesicles
The peptide-induced leakage from SUVs was measured at 293 K
using the CF-entrapped vesicle technique85 and a Perkin Elmer
model MPF-66 spectrofluorimeter. The SUVs composition used
was DOPC/CH 7 : 3. SUVs were prepared as described above.
The phospholipid concentration was kept constant (0.06 mM),
and increasing [peptide]/[lipid] molar ratios (R-1) were obtained by
adding aliquots of water solutions of peptides, except for the native
non hydrosoluble trichogin GA IV, used as reference compound,
1296 | Org. Biomol. Chem., 2012, 10, 1285–1299
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which was added as a methanol solution, 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 lexc
488 nm (3-nm band pass). The percentage of released CF at time t
was determined as (Ft-F0)/(FT-F0) x 100, with F0 = fluorescence
intensity of vesicles in the absence of peptide, Ft = fluorescence
prepared and tested by mixing 100 ml of each solution with 100
ml of the organism suspension in a microtiter plate well. The test
for each peptide was repeated three times at every concentration.
The plates were incubated for 18 h at 37 ◦C. The MIC value
was defined as the lowest concentration of peptide that gave no
visible growth on the plate. The endpoint was detected by use of a
spectrophotometer.
intensity of vesicles at time t in the presence of peptide, and FT
=
total fluorescence intensity determined by disrupting the vesicles
by addition of 50 mL of a Triton X-100 solution. The kinetics
experiments were stopped at 20 min.
Hemolytic activity
The hemolytic activity of each peptide was determined using
hRBCs from healthy donors that were collected on heparin. Fresh
hRBCs were washed three times in PBS (phosphate buffered
saline) by centrifugation for 10 min at 800¥ g and resuspension in
PBS. The peptides dissolved in PBS were then added to 100 mL
of stock hRBCs suspended in PBS (final hRBCs concentration:
8%, v/v). The samples were incubated with agitation for 1 h at
37 ◦C and centrifuged at 800¥g for 10 min. The absorbance of the
supernatant was measured at 414 nm. Controls for zero hemolysis
(blank) and 100% hemolysis consisted of hRBCs suspended in
PBS and 1% Triton X-100, respectively. The percentage hemolysis
was calculated using the following equation:% hemolysis = [(A414 nm
with protein solution- A414 nm in PBS)/(A414 nm with 0.1% Triton
X-100- A414 nm in PBS)] x 100. Each measurement was made in
triplicate.87,88 The hemolytic peptide melittin was synthesized in
the Chosun University laboratories.
Membrane depolarization assay
The peptide ability to dissipate the membrane potential was deter-
mined using intact S. aureus, E. coli, and the membrane potential-
sensitive, fluorescent dye 3,3¢-dipropylthiadicarbocyanine iodide
[DiSC3-5, from Molecular Probes (Eugene, OR)]. S. aureus and
E. coli bacteria were grown to mid-logarithmic phase at 37 ◦C.
The cells were washed three times in buffer A (20 mM glucose,
5 mM Hepes, pH 7.2) and resuspended to an OD600 nm of 0.05 in
buffer A containing 0.1 M KCl. The cells were then incubated
with DiSC3-5 (final concentration 0.5 mM) until stable baseline
fluorescence was achieved (ca. 60 min). The experiments were
conducted in sterile 96-well plates at a final volume of 200 ml.
Then, the peptides dissolved in buffer A were added to achieve the
desired concentration. Membrane depolarization was detected as
an increase in the DiSC3-5 fluorescence (excitation wavelength,
622 nm; emission wavelength, 670 nm).86
Cytotoxicity
HaCaT (Human adult low-Calcium high Temperature ker-
atinocyte cells) were cultivated in Dulbeccos’s Minimal Eale
Medium (DMEM) at 37 ◦C and 5% CO2 for 5–7 days. Assays
were performed by incubation of 2 ¥ 103 cells with various
concentrations of peptide for 19 h at 37 ◦C and 5% CO2 in
a 96-well plate. The Triton X-100 (0.1% final concentration)
was used as negative control and pure medium assay was used
as positive control. Proliferation and viability were determined
by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium
bromide] assay. The plate was then incubated for 24 h before
adding toeachwell50 ml of MTT solution. The medium containing
MTT was removed and 100 ml of DMSO were added. Cells were
Bacterial strains
Escherichia coli (ATCC 25 922), Listeria monocytogenes (ATCC
19 115), and Staphylococcus aureus (ATCC 25 923) were ob-
tained from American Type Culture Collection. Bacillus subtilis
(KCTC 1918), Staphylococcus epidermidis (KCTC 3096), and
Pseudomonas aeruginosa (KCTC 1637) were from the Korean
Collection for Type Cultures (KCTC), Korean Research Institute
of Bioscience and Biotechnology (KRIBB), Taejon, Korea. To as-
sess the antimicrobial activities of our peptides against antibiotic-
resistant bacteria, clinically isolated multidrug-resistant bacterial
strains were obtained from the Culture Collection of Antibiotic-
Resistant Microbes (CCARM) at Seoul Women’s University,
Korea. These included five strains of methicillin-resistant S.
aureus (MRSA) (CCARM 3089, CCARM 3090, CCARM 3108,
CCARM 3114, and CCARM 3126).
◦
incubated for 10 min at 37 C under gentle shaking. The optical
density was read at 550 nm in an enzyme-linked immunosorbent
assay plate reader after 2 h of incubation. Cell viability was
determined relative to the control. Studies were performed in
triplicate.
Antibacterial activity
Antifungal activity
To determine the MIC values for the tested peptides, serial
microdilution assays were performed. Cells were grown to log-
phase in 10 g l-1 bactotryptone, 5 g l-1 yeast extract, and 10 g l-1
NaCl, pH 7.0, then passed through a 0.22 mm filter and stepwise-
diluted in a medium of 1% bactopeptone. Each organism to be
tested was suspended at 2 ¥ 106 colony formation units (CFU) ml-1
in growth medium. A stock solution of each peptide was prepared
by dissolving it in the minimum amount of dimethylsulfoxide
(DMSO) and diluting with PBS (phosphate buffered saline). The
maximum amount of DMSO in each solution was 1.45%. Then,
eight solutions, obtained by successive two-fold dilutions, were
Microdilution assays were performed to establish the MIC values
for the peptides. Candida albicans was grown at 28 ◦C in YPD (2%
dextrose, 1% peptone, and 0.5% yeast extract, pH 5.5) for 3 h. Cell
densities were counted with a hemocytometer. The fungal cells
(2 ¥ 103/well) were seeded on the wells of a flat-bottom 96-well
microtiter plate (Greiner, Nurtingen,Germany) containing YPD
(100 ml/well). Serial dilutions of the peptide solution were added to
each well, and the cell suspension was incubated at 28 ◦C for 24 h. A
10 ml of MTT solution (5 mg ml-1) was added to each well, and the
plates were incubated at 37 ◦C for 4 h. Then, absorbance at 570 nm
was measured using an Emax microtiter plate reader (Molecular
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Devices, California, USA). All assays were performed in triplicate.
The assays for antifungal activity against Fusarium oxysporum,
Rhizoctonia solani, Aspergillus awamori, Aspergillus parasiticus,
and Astragalus flavus were carried out in 100 ¥ 15 mm Petri dishes
containing YPD. After the mycelial colony had developed, sterile
blank paper disks (8 mm diameter) were placed 5 mm from the
leading edge of the mycelial colony. An aliquot of the peptide test
sample in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (20
mM, pH 6.0) was added to each disk, and the plates were incubated
at 28 ◦C for 72 h. Antifungal activity was shown as a clear zone of
growth inhibition.
28 V. Monaco, F. Formaggio, M. Crisma, C. Toniolo, P. Hanson and G.
L. Millhauser, Biopolymers, 1999, 50, 239–253.
29 A. D. Milov, Yu. D. Tsvetkov, F. Formaggio, M. Crisma, C. Toniolo
and J. Raap, J. Am. Chem. Soc., 2001, 123, 3784–3789.
30 A. D. Milov, Yu. D. Tsvetkov, F. Formaggio, M. Crisma, C. Toniolo, G.
L. Millhauser and J. Raap, J. Phys. Chem. B, 2001, 105, 11206–11213.
31 A. D. Milov, Yu. D. Tsvetkov, F. Formaggio, S. Oancea, C. Toniolo and
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32 A. D. Milov, Yu. D. Tsvetkov, F. Formaggio, M. Crisma, C. Toniolo
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33 A. D. Milov, Yu. D. Tsvetkov, F. Formaggio, S. Oancea, C. Toniolo and
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34 L. Stella, C. Mazzuca, M. Venanzi, A. Palleschi, M. Didone`, F.
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35 A. D. Milov, D. A. Erilov, E. S. Salnikov, Yu. D. Tsvetkov, F. Formaggio,
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36 C. Mazzuca, L. Stella, M. Venanzi, F. Formaggio, C. Toniolo and B.
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37 E. S. Salnikov, D. A. Erilov, A. D. Milov, Yu. D. Tsvetkov, C. Peggion,
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