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

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PAPER

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

peptaibiotics†‡

Marta De Zotti,^aBarbara Biondi,^aCristina Peggion,^aFernando Formaggio,^aYoonkyung Park,^b,c

Kyung-Soo Hahm^b,dand 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.⁵These properties render them intriguing

lead compounds in the search of candidates as novel, potent

antibacterial agents. Trichogin GA IV⁶is 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.^7,8The 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,^1–3we 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.^6,7,9–19This conclusion is not surprising in view of the

heavy presence (30%) of the strongly helicogenic Aib residues

in the sequence.^20–23It was also proposed that the ﬂexible -Gly⁵-

Gly⁶- dipeptide would create a hinge point between two helical

regions. The overall mixed 3₁₀-/a- helical structure^24,25is 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.¹²Conversely, the N^a-blocking fatty acyl moiety was shown to

play a major role in its membrane modifying properties.¹²More

speciﬁcally, at least six carbon atoms in the aliphatic chain are

required for a signiﬁcant membrane activity. By use of a set of

N^a-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.¹⁸A 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 techniques^26–44but

solid-state NMR⁴⁵and other biophysical methods^46,47as 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.⁴⁹To 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 work^a

Abbreviation

Sequence

t_R/min^b

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

^aSingle-letter amino acid abbreviations include: lysine (K) and a-aminoisobutyric acid (U). ^bt_R: 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)¹²is reported as reference.

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Fig. 2 FT-IR absorption spectra (3500–3200 cm^-1region) in CDCl₃solution of the N^e-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,⁵⁰to 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 Gly⁶was additionally substituted

by Aib).

These syntheses, performed in parallel on the Lol-substituted

2-chlorotrityl resin^51,52as 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 groups^54–56(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,^20–23would 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 N^e-Lys Z-protected K2-OMe (part A), and E(1–

4) and E–B(1–8) employed to prepare the N^e-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

N^e-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^-1region 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,^58–60it 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,^61,62is 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-helix²⁴to 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.⁶³The ROESY spectra exhibit all of the sequential

NH_i→NH_i+1cross 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 C^aH_i→NH_i+3

cross peaks, typical of the presence of a helical conformation,

along with three C^aH_i→NH_i+4and four C^aH_i→NH_i+2cross

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 C^aH_i→NH_i+2cross peaks

600 MHz); (C) 3.5 mM in 9 : 1 H₂O/D₂O containing 100 mM SDS-d₂₅, pH

versus only one C^aH_i→NH_i+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 C^aH_i→NH_i+2(3₁₀-helix); in

the occurrence of a mixed 3₁₀-/a-helical conformation in the

green: C^aH_i→NH_i+4(a-helix); in red: C^aH_i→NH_i+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),⁶⁴was 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

MW_calc

MW_obs

Q^a

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

2

3

4

0

1

5

100

>32

16

8

5.4

10

5.4

>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

Melittin^d

^aQ: total charge. ^bH: molecular hydrophobicity estimated from elution

time in reversed-phase HPLC with respect to the native compound (TRIC

or Tric-OMe). ^cLC₅₀: peptide concentration responsible for 50% hemolysis

after 3 h incubation with 4% red blood cells in PBS. ^dAntimicrobial,

hemolytic peptide used as standard (ref. 87). ^en.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 Gly⁵or Gly⁶

Table 3 Antimicrobial activities (MIC values) of trichogin GA IV and its analogs^a

Bacteria

Gram-positive

Gram-negative

Fungi

Peptide

S. aureus

S. epidermidis

E. coli

P. aeruginosa

C. albicans

T. beigelli

TRIC

8–16

4

2

8

4

>64

64

4

8

64

4

>64

2

32

4

8

32

8

32

2

>64

64

4

8

32

64

>64

4

>64

8

16

4

16

8

64

n.d.^c

8

>64

8

4

K5

K6

K56

K259U6

K2569

K2-OMe

K9-OMe

Tric-OMe^12,48

Melittin^b

8

32

64

n.d.^c

8

16

4

^aAll values are in mg ml^-1. ^bAntimicrobial peptide used as standard. ^cn.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.^65–72The 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]²_D⁰(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 cm²g^-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,⁷³for 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

˚

C₁₈column (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 N^a-

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)⁷⁴procedure, while the EDC/HOBt

(1-hydroxy-1,2,3-benzotriazole)⁷⁵procedure, 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 ^gC 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]²_D⁰:-43.8 (c = 0.5, MeOH). IR (KBr): 3294, 1693, 1642, 1536

cm^-1. ESI-MS m/z: calculated for C₄₂H₅₄N₄O₈742.40, 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-CH₃and 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 ^gCH], 24.864 [s, Leu ^gC], 22.766 [s, Leu ^dC], 22.274 [s, Lys ^gC],

21.809 [s, Leu ^dC], 15.386, 11.254 [2s, Ile ^gCH₃and ^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 C₂₃H₃₅N₃O₆449.25, 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

^gC, Leu ^gC and ^dC], 15.072, 11.176 [2s, Ile ^gC 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]²_D⁰:

-3.2^◦(c = 0.5, MeOH). IR (KBr): 3316, 1732, 1701, 1659, 1536

cm^-1. ESI-MS m/z: calculated for C₂₈H₄₄N₄O₇548.32, found

◦

Melting point: 67–68 C. Rf₂: 0.95, Rf₃: 0.40, Rf₄: 0.75. [a]²_D⁰:

-22.1^◦(c = 0.5, MeOH). IR (KBr): 3312, 1732, 1711, 1651,

1535 cm^-1. ESI-MS m/z: calculated for C₂₆H₄₀N₄O₇520.29, found

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, Gly²NH], 6.81 [s, 1H, Aib NH], 6.67 [d, 1H,

Leu NH], 5.61 [t, 1H, Gly¹NH], 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 ^gC, 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]²_D⁰: -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]²_D⁰: -39.1 (c = 0.5, MeOH). IR (KBr):

3318, 1734, 1706, 1651, 1540 cm^-1. ESI-MS m/z: calculated for

C₃₇H₅₉N₇O₁₀761.43, 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 C₄₇H₆₃N₅O₉841.46, 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-CH₃and 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]²_D⁰: -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, Gly²NH], 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, Ile¹⁰NH], 7.55 [d, 1H, Leu¹¹-NH], 7.37 [d, 1H, Lys⁹NH],

4.36 [m, 1H, Leu¹¹a-CH], 4.20 [m, 1H, Leu³a-CH], 4.13–4.12 [m,

2H, Ile¹⁰a-CH and Lys⁹a-CH], 4.04 [m, 1H, Leu⁷a-CH], 3.96 [m,

1H, Gly⁶1 a-CH], 3.92 [m, 1H], 3.89 [m, 1H, Gly²1 a-CH], 3.84

[m, 1H, Gly⁵1 a-CH], 3.76–3.71 [m, 3H, Gly⁵a-CH, Gly⁶a-CH

and Gly²a-CH], 3.65 [s, 3H, OMe CH₃], 2.92 [m, 2H, Lys⁹e-CH₂],

2.17 [m, 2H, nOct CH₂], 1.85 [m, 1H, Ile¹⁰b-CH], 1.74 [m, 2H, Lys⁹

b-CH₂], 1.68 [m, 2H, Leu³b-CH₂], 1.61 [m, 2H, Leu¹¹b-CH₂], 1.55

[m, 1H, Leu³1 g-CH], 1.44 [m, 2H, nOct b-CH₂], 1.42 [s, 8H, Lys⁹g-

CH₂, Ile¹⁰1 g-CH and Aib⁴b-CH₃], 1.36 [s, 6H, Aib¹2 b-CH₃], 1.35

and 1.32 [2s, 6H, 2 Aib⁸b-CH₃], 1.20–1.04 [m, 11H, Ile¹⁰1 g-CH

and nOct (CH₂)₅], 0.83–0.79 [m, 9H, Leu³2 d-CH₃, Ile¹⁰g-CH₃],

0.76–0.72 [m, 15H, Leu⁷2 d-CH₃, Ile¹⁰d-CH₃and Leu¹¹2 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

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

22.614, 22.467, 22.484, 21.549 [7s, Aib C, Ile CH, Leu C and

^dC], 15.317, 10.632 [2s, Ile ^gCH₃and ^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]²_D⁰: -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-CH₂and 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 ^gC and ^bC,

Aib C, Ile CH, Leu C and C], 15.122, 15.060, 13.641, 11.062

e

d

b

g

d

[4s, n-Oct, Ile ^gCH₃and ^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 resin^51,52(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

N^a-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]⁷⁴double

coupling C-activation procedure for the formation of peptide

bonds involving the three poorly reactive Aib⁴⁹residues 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 procedure⁷⁶for activation

of the carboxylic function. Each coupling step was carried out by

use of a threefold excess of the Fmoc N^a-protected a-amino acid in

the presence of a sevenfold excess of N,N¢-diisopropylethylamine

for 60 min. Final on-resin N^a-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).⁵²The 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]²_D⁰: -24.8^◦(c = 0.2, MeOH). IR

(KBr): 3321, 1743, 1655, 1538 cm^-1. ESI-MS m/z: calculated

1

for C₅₇H₁₀₄N₁₂O₁₃1164.78, 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,

Lys²e-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-CH₃Aib], 1.20–1.03 [m,

11H], 0.83–0.70 [m, 24H, d-CH₃Leu, d-CH₃and g-CH₃Ile], 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 ^gCH, Leu C and C], 14.975, 13.634, 11.044 [3s,

n-Oct, Ile ^gCH₃and ^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 C₅₂H₉₅N₁₁O₁₂1065.72, found

1065.75

identiﬁed using standard DQF-COSY⁷⁷and CLEAN-TOCSY^78,79

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 C₅₆H₁₀₄N₁₂O₁₂1136.79, found

1136.77

K6 ESI-MS m/z: calculated for C₅₆H₁₀₄N₁₂O₁₂1136.79, found

1136.86

K56 ESI-MS m/z: calculated for C₆₀H₁₁₃N₁₃O₁₂1207.86, found

1207.86

K259U6 ESI-MS m/z: calculated for C₆₆H₁₂₆N₁₄O₁₂1306.97,

found 1306.95

K2569 ESI-MS m/z: calculated for C₆₈H₁₃₁N₁₅O₁₂1350.01,

found 1350.00

The C^b-selective HMQC experiments with gradient coherence

selection⁸⁴were recorded with 320 t₁increments, of 300 scans and

2 K points each.⁸⁰A 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 t₁experiments 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^-1nominal

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 C₁₈reverse-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 ¥ cm²x 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 CHCl₃in

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-d₃solution (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 technique⁸⁵and 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 l_exc

488 nm (3-nm band pass). The percentage of released CF at time t

was determined as (F_t-F₀)/(F_T-F₀) x 100, with F₀= ﬂuorescence

intensity of vesicles in the absence of peptide, F_t= 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 F_T

=

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 = [(A_{414 nm}

with protein solution- A_{414 nm}in PBS)/(A_{414 nm}with 0.1% Triton

X-100- A_{414 nm}in PBS)] x 100. Each measurement was made in

triplicate.^87,88The 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 OD_{600 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% CO₂for 5–7 days. Assays

were performed by incubation of 2 ¥ 10³cells with various

concentrations of peptide for 19 h at 37 ^◦C and 5% CO₂in

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^-1bactotryptone, 5 g l^-1yeast 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 ¥ 10⁶colony 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

J. Raap, J. Phys. Chem. B, 2003, 107, 13719–13727.

32 A. D. Milov, Yu. D. Tsvetkov, F. Formaggio, M. Crisma, C. Toniolo

and J. Raap, J. Pept. Sci., 2003, 9, 690–700.

33 A. D. Milov, Yu. D. Tsvetkov, F. Formaggio, S. Oancea, C. Toniolo and

J. Raap, Phys. Chem. Chem. Phys., 2004, 6, 3596–3603.

34 L. Stella, C. Mazzuca, M. Venanzi, A. Palleschi, M. Didone`, F.

Formaggio, C. Toniolo and B. Pispisa, Biophys. J., 2004, 86, 936–

945.

35 A. D. Milov, D. A. Erilov, E. S. Salnikov, Yu. D. Tsvetkov, F. Formaggio,

C. Toniolo and J. Raap, Phys. Chem. Chem. Phys., 2005, 7, 1794–1799.

36 C. Mazzuca, L. Stella, M. Venanzi, F. Formaggio, C. Toniolo and B.

Pispisa, Biophys. J., 2005, 88, 3411–3421.

37 E. S. Salnikov, D. A. Erilov, A. D. Milov, Yu. D. Tsvetkov, C. Peggion,

F. Formaggio, C. Toniolo, J. Raap and S. A. Dzuba, Biophys. J., 2006,

91, 1532–1540.

38 M. Venanzi, E. Gatto, G. Bocchinfuso, A. Palleschi, L. Stella, F.

Formaggio and C. Toniolo, ChemBioChem, 2006, 7, 43–45.

39 E. Gatto, C. Mazzuca, L. Stella, M. Venanzi, C. Toniolo and B. Pispisa,

J. Phys. Chem. B, 2006, 110, 22813–22818.

40 M. Venanzi, E. Gatto, G. Bocchinfuso, A. Palleschi, L. Stella, C.

Baldini, F. Formaggio and C. Toniolo, J. Phys. Chem. B, 2006, 110,

22834–22841.

References

1 R. M. Epand and R. F. Epand, J. Pept. Sci., 2011, 17, 298–305.

2 H. Duclohier, Curr. Pharm. Des., 2010, 16, 3212–3223.

3 R. E. W. Hancock and H.-G. Sahl, Nat. Biotechnol., 2006, 24, 1551–

1557.

4 C. Toniolo and H. Bru¨ckner, Peptaibiotics, Wiley/VCH, Wein-

heim/Zu¨rich, 2009.

5 C. Toniolo, M. Crisma, F. Formaggio, C. Peggion, R. F. Epand and R.

M. Epand, Cell. Mol. Life Sci., 2001, 58, 1179–1188.

6 C. Peggion, F. Formaggio, M. Crisma, R. F. Epand, R. M. Epand and

C. Toniolo, J. Pept. Sci., 2003, 9, 679–689.

7 C. Auvin-Guette, S. Rebuffat, Y. Prigent and B. Bodo, J. Am. Chem.

Soc., 1992, 114, 2170–2174.

41 M. Venanzi, G. Bocchinfuso, E. Gatto, A. Palleschi, L. Stella, F.

Formaggio and C. Toniolo, ChemBioChem, 2009, 10, 91–97.

42 V. N. Syryamina, N. P. Isaev, C. Peggion, F. Formaggio, C. Toniolo, J.

Raap and S. A. Dzuba, J. Phys. Chem. B, 2010, 114, 12277–12283.

43 C. Mazzuca, B. Orioni, M. Coletta, F. Formaggio, C. Toniolo, G.

Maulucci, M. De Spirito, B. Pispisa, M. Venanzi and L. Stella, Biophys.

J., 2010, 99, 1791–1800.

44 S. Smeazzetto, M. De Zotti and M. R. Moncelli, Electrochem.

Commun., 2011, 13, 834–836.

45 C. Heuber, F. Formaggio, C. Baldini, C. Toniolo and K. Mu¨ller, Chem.

Biodiversity, 2007, 4, 1200–1217.

46 R. F. Epand, R. M. Epand, V. Monaco, S. Stoia, F. Formaggio, M.

Crisma and C. Toniolo, Eur. J. Biochem., 1999, 266, 1021–1028.

47 R. F. Epand, R. M. Epand, F. Formaggio, M. Crisma, H. Wu, R. I.

Lehrer and C. Toniolo, Eur. J. Biochem., 2001, 268, 703–712.

48 M. De Zotti, B. Biondi, F. Formaggio, C. Toniolo, L. Stella, Y. Park

and K.-S. Hahm, J. Pept. Sci., 2009, 15, 615–619.

49 F. Formaggio, Q. B. Broxterman and C. Toniolo, in Houben-Weyl:

Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimet-

ics, Vol. E22c, ed. M. Goodman, A. Felix, L. Moroder and C. Toniolo,

Thieme, Stuttgart, Germany, 2003, pp. 292–310.

50 M. De Zotti, B. Biondi, C. Peggion, Y. Park, K.-S. Hahm, F. Formaggio

and C. Toniolo, J. Pept. Sci., 2011, 17, 585–594.

51 K. Barlos, O. Chatzi, D. Gatos and G. Stavropoulos, Int. J. Pept. Protein

Res., 1991, 37, 513–520.

8 S. Rebuffat, C. Goulard, B. Bodo and M.-F. Roquebert, Recent Res.

Devel. Org. Biorg. Chem., 1999, 3, 65–91.

9 C. Toniolo, C. Peggion, M. Crisma, F. Formaggio, X. Shui and D. S.

Eggleston, Nat. Struct. Biol., 1994, 1, 908–914.

10 R. Gurunath and P. Balaram, Biopolymers, 1995, 35, 21–29.

11 V. Monaco, F. Formaggio, M. Crisma, C. Toniolo, X. Shui and D. S.

Eggleston, Biopolymers, 1996, 39, 31–42.

12 C. Toniolo, M. Crisma, F. Formaggio, C. Peggion, V. Monaco, C.

Goulard, S. Rebuffat and B. Bodo, J. Am. Chem. Soc., 1996, 118, 4952–

4958.

13 P. Scrimin, A. Veronese, P. Tecilla, U. Tonellato, V. Monaco, F.

Formaggio, M. Crisma and C. Toniolo, J. Am. Chem. Soc., 1996, 118,

2505–2506.

14 M. Crisma, V. Monaco, F. Formaggio, C. Toniolo, C. George and J. L.

Flippen-Anderson, Lett. Pept. Sci., 1997, 4, 213–218.

15 V. Monaco, E. Locardi, F. Formaggio, M. Crisma, S. Mammi, E.

Peggion, C. Toniolo, S. Rebuffat and B. Bodo, J. Pept. Res., 2009,

52, 261–272.

16 E. Locardi, S. Mammi, E. Peggion, V. Monaco, F. Formaggio, M.

Crisma, C. Toniolo, B. Bodo, S. Rebuffat, J. Kamphuis and Q. B.

Broxterman, J. Pept. Sci., 1998, 4, 389–399.

17 C. Peggion, V. Moretto, F. Formaggio, M. Crisma, C. Toniolo, J.

Kamphuis, B. Kaptein and Q. B. Broxterman, J. Pept. Res., 2001, 58,

317–324.

18 F. Formaggio, C. Peggion, M. Crisma and C. Toniolo, J. Chem. Soc.,

52 R. Bollhagen, M. Schmiedberger, K. Barlos and E. Grell, J. Chem. Soc.,

Chem. Commun., 1994, 2559–2560.

53 C. U. Hjørringgaard, J. M. Pedersen, T. Vosegaard, N. C. Nielsen and

T. Skrydstrup, J. Org. Chem., 2009, 74, 1329–1332.

54 L. J. Bellamy, The Infrared Spectra of Complex Molecules, 2nd edn,

Methuen, London, 1966.

Perkin Trans. 2, 2001, 1372–1377.

19 M. Saviano, R. Improta, E. Benedetti, B. Carrozzini, G. L. Cascarano,

C. Didierjean, C. Toniolo and M. Crisma, ChemBioChem, 2004, 5,

541–544.

20 I. L. Karle and P. Balaram, Biochemistry, 1990, 29, 6747–6756.

21 E. Benedetti, B. Di Blasio, V. Pavone, C. Pedone, C. Toniolo and M.

Crisma, Biopolymers, 1992, 32, 453–456.

22 C. Toniolo, M. Crisma, F. Formaggio, G. Valle, G. Cavicchioni, G.

Pre´cigoux, A. Aubry and J. Kamphuis, Biopolymers, 1993, 33, 1061–

1072.

55 M. T. Cung, M. Marraud and J. Ne´el, Ann. Chim. (Paris), 1972, 183–

209.

56 E. S. Pysh and C. Toniolo, J. Am. Chem. Soc., 1977, 99, 6211–6219.

57 S. Beychok, in Poly-a-Amino Acids: Protein Models for Conformational

Studies, ed. G. D. Fasman, Dekker, New York, 1967, pp. 293–337.

58 M. C. Manning and R. W. Woody, Biopolymers, 1991, 31, 569–585.

59 C. Toniolo, A. Polese, F. Formaggio, M. Crisma and J. Kamphuis, J.

Am. Chem. Soc., 1996, 118, 2744–2745.

60 F. Formaggio, M. Crisma, P. Rossi, P. Scrimin, B. Kaptein, Q. B.

Broxterman, J. Kamphuis and C. Toniolo, Chem.–Eur. J., 2006, 6, 4498–

4504.

61 P. M. Hardy, in Chemistry and Biochemistry of the Amino Acids; ed. G.

C. Barrett, Chapman and Hall, London, 1985, pp. 6–24.

62 I. Andre´, S. Linse and F. A. A. Mulder, J. Am. Chem. Soc., 2007, 129,

15805–15813.

23 C. Toniolo, M. Crisma and F. Formaggio, Biopolymers, 2001, 60, 396–

419.

24 C. Toniolo and E. Benedetti, Trends Biochem. Sci., 1991, 16, 350–353.

25 K. A. Bolin and G. L. Millhauser, Acc. Chem. Res., 1999, 32, 1027–

1033.

26 V. Monaco, F. Formaggio, M. Crisma, C. Toniolo, P. Hanson, G.

Millhauser, C. George, J. R. Deschamps and J. L. Flippen-Anderson,

Bioorg. Med. Chem., 1999, 7, 119–131.

27 D. J. Anderson, P. Hanson, J. Mc Nulty, G. Millhauser, V. Monaco, F.

Formaggio, M. Crisma and C. Toniolo, J. Am. Chem. Soc., 1999, 121,

6919–6927.

1298 | Org. Biomol. Chem., 2012, 10, 1285–1299

This journal is

The Royal Society of Chemistry 2012

©

View Article Online

63 K. Wu¨thrich, NMR of Proteins and Nucleic Acids, Wiley, New York,

1986.

64 G. Irmscher and G. Jung, Eur. J. Biochem., 1977, 80, 165–174.

65 H.-H. Nguyen, D. Imhof, M. Kronen, B. Schlegel, A. Hartl, U. Gra¨fe,

L. Gera and S. Reissmann, J. Med. Chem., 2002, 45, 2781–2787.

66 R. Banerjee, G. Basu, P. Che`ne and S. Roy, J. Pept. Res., 2002, 60,

88–94.

67 H. Yamaguchi, H. Kodama, S. Osada, F. Kato, M. Jelokhani-Niaraki

and M. Kondo, Biosci. Biotechnol. Biochem., 2003, 67, 2269–2272.

68 S. Zikou, A.-I. Koukkou, P. Mastora, M. Sakarellos-Daitsiotis, C.

Sakarellos, C. Drainas and E. Panou-Pomonis, J. Pept. Sci., 2007, 13,

481–486.

76 L. A. Carpino, A. El-Faham, C. A. Minor and F. Albericio, J. Chem.

Soc. Chem. Commun., 1994, 201–203.

77 M. Rance, O. W. Sørensen, G. Bodenhausen, G. Wagner, R. R. Ernst

and K. Wu¨thrich, Biochem. Biophys. Res. Commun., 1983, 117, 479–

485.

78 A. Bax and D. G. Davis, J. Magn. Reson., 1985, 65, 355–360.

79 C. Griesinger, G. Otting, K. Wu¨thrich and R. R. Ernst, J. Am. Chem.

Soc., 1988, 110, 7870–7872.

80 A. Bax and S. Subramanian, J. Magn. Reson., 1986, 67, 565–569.

81 A. Bax and M. F. Summers, J. Am. Chem. Soc., 1986, 108, 2093–2094.

82 C. Bauer, R. Freeman, T. Frenkiel, J. Keeler and A. J. Shak, J. Magn.

Reson., 1984, 58, 442–457.

69 J. D. Sadowsky, J. K. Murray, Y. Tomita and S. H. Gellman,

ChemBioChem, 2007, 8, 903–916.

70 J. Svenson, W. Stensen, B.-O. Brandsdal, B. E. Haug, J. Monrad and J.

S. Svendsen, Biochemistry, 2008, 47, 3777–3788.

71 L. P. Miranda, K. A. Winters, C. V. Gegg, A. Patel, J. Aral, J. Long, J.

Zhang, S. Diamond, M. Guido, S. Stanislaus, M. Ma, H. Li, M. J. Rose,

L. Poppe and M. M. Ve´niant, J. Med. Chem., 2008, 51, 2758–2765.

72 J. Taira, Y. Kida, H. Yamaguchi, K. Kuwano, Y. Higashimoto and H.

Kodama, J. Pept. Sci., 2010, 16, 607–612.

83 L. Emsley and G. Bodenhausen, J. Magn. Reson., 1989, 82, 211–221.

84 A. Bax, R. H. Griffey and B. L. Hawkins, J. Magn. Reson., 1983, 55,

301–315.

85 M. El Hajji, S. Rebuffat, T. Le Doan, G. Klein, M. Satre and B. Bodo,

Biochim. Biophys. Acta, 1989, 978, 97–104.

86 S. C. Park, M. H. Kim, M. A. Hossain, S. Y. Shin, Y. Kim, L. Stella, J.

D. Wade, Y. Park and K.-S. Hahm, Biochim. Biophys. Acta, 2008, 1778,

229–241.

87 Y. Park, S. N. Park, S. C. Park, J. Y. Park, Y. H. Park, J. S. Hahm

and K.-S. Hahm, Biochem. Biophys. Res. Commun., 2004, 321, 631–

637.

73 B. L. Feringa, Molecular Switches, Wiley-VCH, Weinheim, Germany,

2003.

74 L. A. Carpino, J. Am. Chem. Soc., 1993, 115, 4397–4398.

75 W. Ko¨nig and R. Geiger, Chem. Ber., 1970, 103, 788–798.

88 M. T. Tosteson, S. J. Holmes, M. Razin and D. C. Tosteson, J. Membrane

Biol., 1985, 87, 35–44.

This journal is

The Royal Society of Chemistry 2012

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

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