Sequence inversion and phenylalanine surrogates at the β-Turn enhance the antibiotic activity of gramicidin S

Article abstract of DOI:10.1021/jm100143f

A series of gramicidin S (GS) analogues have been synthesized where the Phe (i + 1) and Pro (i + 2) residues of the β-turn have been swapped while the respective chiralities (d-, l-) at each position are preserved, and Phe is replaced by surrogates with aromatic side chains of diverse size, orientation, and flexibility. Although most analogues preserve the β-sheet structure, as assessed by NMR, their antibiotic activities turn out to be highly dependent on the bulkiness and spatial arrangement of the aromatic side chain. Significant increases in microbicidal potency against both Gram-positive and Gram-negative pathogens are observed for several analogues, resulting in improved therapeutic profiles. Data indicate that seemingly minor replacements at the GS β-turn can have significant impact on antibiotic activity, highlighting this region as a hot spot for modulating GS plasticity and activity.

Full text of DOI:10.1021/jm100143f

J. Med. Chem. 2010, 53, 4119–4129 4119
DOI: 10.1021/jm100143f
Sequence Inversion and Phenylalanine Surrogates at the β-Turn Enhance the Antibiotic Activity of
Gramicidin S
†
Concepcion Solanas, Beatriz G. de la Torre, Marıa Fernandez-Reyes, Clara M. Santiveri, M. Angeles Jimenez, Luis Rivas,
‡
§
§
ꢀ
ꢀ
ꢀ
ꢀ
Ana I. Jimenez, David Andreu,*,‡ and Carlos Cativiela*,†
†
ꢀ
†
´
ꢀ
ꢀ
Departamento de Quımica Organica, Instituto de Ciencia de Materiales de Aragon, Universidad de Zaragoza-CSIC, Pedro Cerbuna 12, 50009
Zaragoza, Spain, ‡Departament de Cieꢁncies Experimentals i de la Salut, Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain,
§
´
Centro de Investigaciones Bioloꢀgicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain, and Instituto de Quımica-Fısica Rocasolano,
´
CSIC, Serrano 119, 28006 Madrid, Spain
Received February 3, 2010
A series of gramicidin S (GS) analogues have been synthesized where the Phe (i þ 1) and Pro (i þ 2)
residues of the β-turn have been swapped while the respective chiralities (D-, L-) at each position are
preserved, and Phe is replaced by surrogates with aromatic side chains of diverse size, orientation, and
flexibility. Although most analogues preserve the β-sheet structure, as assessed by NMR, their antibiotic
activities turn out to be highly dependent on the bulkiness and spatial arrangement of the aromatic side
chain. Significant increases in microbicidal potency against both Gram-positive and Gram-negative
pathogens are observed for several analogues, resulting in improved therapeutic profiles. Data indicate
that seemingly minor replacements at the GS β-turn can have significant impact on antibiotic activity,
highlighting this region as a hot spot for modulating GS plasticity and activity.
Introduction
Gramicidin S (GS), one of the best-studied cationic AMPs,
is synthesized nonribosomally by Bacillus brevis3 and active
against bacteria and fungi.4,5 GS is a cyclic symmetrical
decapeptide, cyclo(Val-Orn-Leu-D-Phe-Pro)2 (Figure 1), that
adopts a pleated β-sheet structure6-8 in which the Val, Orn,
and Leu residues align to form two antiparallel β-strands.
Two type-II0 β-turns centered at the D-Phe-Pro sequences
flank the β-strands, stabilized by four cross-strand hydrogen
bonds (Figure 1). Amphipaticity of the β-sheet structure is
achieved by positioning of the hydrophobic side chains of Leu
and Val facing one side of the molecule, whereas the two basic
Orn residues project to the opposite side.5,9
Increasing resistance of bacteria to conventional antibiotics
has posed a serious threat to human health and generated a
growing need for new drugs to combat microbial infection.
Cationic antimicrobial peptides (AMPsa) from either micro-
bial or eukaryotic sources have been proposed as an alter-
native to conventional antibiotics because their lethal
mechanism, based on disruption of the pathogen’s cytoplas-
mic membrane, differs from those of most conventional
antibiotics in clinical use and makes induction of resistance
rather unlikely.1,2
Although the mechanism of action of GS is not completely
understood,10 it is generally accepted that the peptide kills
bacterial cells through destabilization and permeabilization of
their cytoplasmic membranes.11 Unfortunately, GS displays
poor selectivity between microbial and mammalian cells, a fact
that restricts its use to topical applications.5 In recent years,
considerable effort has been devoted to developing GS analo-
gues with improved therapeutical index where the antimicrobial
and cytotoxic (e.g., hemolytic) activities are dissociated. In this
quest, both the β-strand12-14 and the β-turn15-25 regions have
been extensively modified in SAR studies that have shed light
on factors governing GS bioactivity such as cationic nature,11,24
amphipathic character,26,27 β-sheet structure,24,26,27 ring size,28
and global hydrophobicity.26,27 The latter property results not
only from the hydrophobic domain defined by the Val and Leu
side chains at the β-strands but also from the presence of an
aromatic side chain at the two β-turn regions that is regarded
as essential for bioactivity.15-17,24,29-32 Indeed, replacement of
D-Phe by either Gly, D-Ala, or D-cyclohexylalanine leads to less
active or inactive compounds,29,30 thus evidencing that an
aromatic group at this particular position is needed for full
*To whom correspondence should be addressed. For D.A.: phone,
þ34-933160868; fax, þ34-933160901; e-mail, david.andreu@upf.edu.
For C.C.: phone, þ34-976761210; fax, þ34-976761210; e-mail, cativiela@
unizar.es.
aAbbreviations: Ac3c, 1-aminocyclopropanecarboxylic acid; AMP, anti-
microbial peptide; ATCC, American Type Culture Collection; (RMe)Phe,
R-methylphenylalanine; Boc, tert-butoxycarbonyl; CECT, Spanish Type
Culture Collection; COSY, correlated spectroscopy; c3diPhe, 1-amino-c-2,
t-3-diphenylcyclopropane-r-1-carboxylic acid; c3Phe, 1-amino-2-phenylcy-
clopropanecarboxylic acid; Dbg, dibenzylglycine; DIEA, N,N-diisopropy-
lethylamine; Dip, β,β-diphenylalanine; DMF, N,N-dimethylformamide;
DSS, sodium 2,2-dimethyl-2-silapentane-5-sulfonate; ESI, electrospray
ionization; Flg, fluorenylglycine; For, formyl; GS, gramicidin S; HATU,
2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; HC50, 50% hemolytic concentration; HOAt, N-7-aza-
hydroxybenzotriazole; HRMS, high resolution mass spectrometry; HSQC,
heteronuclear single quantum coherence spectra; LPS, lipopolysacharide;
MIC50, minimal inhibitory concentration; NOESY, nuclear Overhauser
enhancement spectroscopy; Orn, ornithine; rmsd, root-mean-square devia-
tion; SAR, structure-activity relationship; TFA, trifluoroacetic acid;
TFMSA, trifluoromethanesulfonic acid; TI, therapeutic index (HC50/
MIC50); Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; TIS, triiso-
propylsilane; TOCSY, total correlated spectroscopy.
r
2010 American Chemical Society
Published on Web 04/22/2010
pubs.acs.org/jmc

4120 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Solanas et al.
bactericidal potency. Moreover, by replacing the D-Phe residues
in GS by a variety of noncoded aromatic amino acids, we have
recently shown25 that the pharmacological profile changes
dramatically with the size and spatial arrangement of the
aromatic side chain at the i þ 1 position of the β-turns.
β-turn. In line with this, we hypothesized that the i þ 1 and i þ
2 positions could be swapped without dramatic alteration of
GS overall architecture (i.e., the β-turns and the whole β-sheet
structure) as long as the chirality of the said i þ 1 and i þ 2
positions (D- and L-, respectively) remained unaltered, as in
peptide 1 (Figure 1). The hypothesis is supported on the
known requirement for a D-amino acid (or glycine) at the
i þ 1 position33 oftypeII0 β-turnssuchasthat inGSand on the
fact that Pro is the amino acid residue with the highest pro-
pensity to occupy an i þ 1 position at a type-II β -turn;33-35
therefore, a D-Pro at the i þ 1 position would appear to be an
optimal choice for nucleating a type II0 β-turn. Herein we
report on a family of GS analogues bearing double modifica-
tions based on the aforementioned considerations. Specifi-
cally, in the lead analogue 1, cyclo(Val-Orn-Leu-D-Pro-Phe)2
(Figure 1), both D-Phe-Pro sequences in GS have been
replaced by D-Pro-Phe. In the remaining analogues of the
series, Phe is replaced by a variety of nonproteinogenic
counterparts bearing aromatic side chains of different size,
orientation, and flexibility. The structure of these GS ana-
logues has been analyzed by NMR spectroscopy and related
to their antibiotic and hemolytic activities.
These latter findings have led us to question whether an
aromatic moiety is strictly necessary at the i þ 1 position of the
Results
Peptide Design and Synthesis. The amino acids selected as
Phe replacements (Phe*) in the GS analogue 1 (Figure 1) are
shown in Figure 2. β,β-Diphenylalanine (Dip), fluorenylgly-
cine (Flg), dibenzylglycine (Dbg), 1,2,3,4-tetrahydroisoqui-
noline-3-carboxylic acid (Tic), and the two enantiomers of
the cyclopropane amino acid c3diPhe were initially consid-
ered. Most of them incorporate two phenyl rings exhibiting
diverse orientations and degrees of conformational freedom.
In Dbg, the aromatic substituents can freely rotate, whereas
in c3diPhe they are tightly held. Both Dip and Flg bear two
phenyl groups on the same β carbon that in the latter case are
forced to be coplanar. The orientation of the aromatic
moiety in Tic is fixed by the methylene unit linking it to the
backbone. In addition, it should be noted that Dbg is achiral
while c3diPhe is characterized by an achiral R carbon and two
chiral β carbons. To evaluate separately the structural effect
associated exclusively to the strained three-membered cycle
Figure 1. Structure of gramicidin S (top) and the analogue gener-
ated (1) by sequence inversion at the β-turn (bottom). In both cases,
the peptide adopts a β-sheet structure.
Figure 2. Amino acids selected as L-Phe replacements (Phe*) in 1 (left). Sequence and numbering of the corresponding gramicidin S analogues
generated (2-13) (right). For amino acids containing two chiral centers, configurations are given for the 2,3 (in c3diPhe) or 1,2 (in c3Phe) carbon
atoms.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4121
Scheme 1. Synthetic Strategy for the GS Analoguesa
following the general route presented in Scheme 1. Unsuc-
cessful coupling of the Dbg residue prevented the synthesis of
the linear precursor of peptide 4. For the remaining peptides,
the solid-phase synthetic protocols were completed satisfac-
torily. The linear decapeptides were released from the poly-
meric support by treatment with TFMSA and then cyclized
in solution under high-dilution conditions. Finally, depro-
tection of the Orn side chains by acid hydrolysis followed
by reversed-phase HPLC purification furnished the target
peptides (1-3, 5-13) in good overall yields and high purity
(g99%, see Supporting Information). All peptides were
satisfactorily characterized by MS and NMR.
NMR Structural Studies. The structure of GS analogues
1-3 and 5-13 was evaluated in aqueous solution by NMR
as previously done for GS and a first series of analogues.25 A
set of minor NMR signals coexisting with those of the major
species were present in the spectra of all analogues, except for
peptide 5 (Figure 3, see also Supporting Information, Figure
SF2, and Table ST1). In all cases, the major conformer was
found to contain both Leu-D-Pro bonds in a trans arrange-
ment, as inferred from the presence of an NOE between the
0
Leu HR proton and the Pro Hδδ protons (Figure 3 and Table
ST2, Supporting Information), as well as from the
13Cβ-13Cγ chemical shift differences in the 2.9-5.2 ppm
range observed for the Pro residues (ΔδPro = δCβ - δCγ ppm;
see Supporting Information, Table ST2).44 For peptides
1-3, as expected for Pro-containing peptides, the minor
species were assigned to the cis rotamer of at least one
Leu-D-Pro bond, as deduced from the presence of an NOE
between the HR protons of Leu and Pro residues and from a
ΔδPro value near 10 ppm.44 The most populated of the two
minor species is an aymmetric conformer in which one Leu-
D-Pro bond corresponds to the trans rotamer and the other to
the cis rotamer, and the least populated is a symmetric
conformer with the two Leu-D-Pro bonds in cis (Figure 3A,
and Supporting Information, Table ST3). In contrast, ana-
logues 6-13 showed broad NMR signals and, except for
peptides 11 and 13, exchange cross-peaks between signals
belonging to the major and minor species, as well as between
signals of different minor species, were observed in the
150 ms NOESY spectra (Supporting Information, Figure
SF2 and Table ST1). This indicates that the conformational
equilibrium in peptides 6-13 is faster than that usually
associated to the isomerization of the Leu-D-Pro bonds.
More strikingly, the minor species observed in peptides 6,
7, and 9 could not be attributed to the cis-trans isomerism of
the amide bonds preceding Pro residues, as they exhibit
a Reagents and conditions: (a) standard solid-phase peptide synthesis
(Boc chemistry); (b) TFA/TFMSA/TIS 10:1:1, rt, 90 min; (c) HATU/
HOAt/DIEA (3:3:5 equiv), DMF, rt, 1 h; (d) 20% HCl in MeOH, 37 °C,
21 h. Phe* stands for Phe or the corresponding substitute.
present in c3diPhe, the unsubstituted cyclopropane amino
acid Ac3c was also considered for this study. Cyclopropane
amino acids have been shown to exert a strong stabilizing
effect on β-turns when incorporated into Pro-Xaa dipep-
tides36 and are therefore expected to stabilize the β-sheet
structure of GS, provided no steric conflict between the
cyclopropane substituents and the contiguous residues
arises.
The different antibiotic properties found for the peptides
incorporating the two c3diPhe enantiomers (see below)
prompted us to evaluate the effect produced by the cyclo-
propane amino acid bearing a single phenyl substituent,
c3Phe (Figure 2). In this case, four stereoisomeric forms
are possible, each of them with a different orientation of
the aromatic ring. It is worth noting that each c3diPhe
residue can be regarded as formally containing the aromatic
side chain of two c3Phe stereoisomers. Additionally, R-
methylphenylalanine [(RMe)Phe] was included in this study
as the open-chain counterpart of c3Phe, in the same way as
Dbg can be viewed as the acyclic equivalent of c3diPhe. Both
(RMe)Phe and Dbg keep the R-tetrasubstituted character
of c3Phe and c3diPhe but not the restriction imposed by
the three-membered ring. As an R-methylated amino acid,
(RMe)Phe is also particularly suitable to occupy the i þ 2
position of a β-turn.37
0
NOEs between the Leu HR proton and the Pro Hδ and Hδ
protons, and ΔδPro values below 5.0 ppm (Supporting
Information, Figure SF2 and Table ST4), both typical of
trans rotamers. Hence, the minor species observed for pep-
tides 6-13 could not be attributed to the cis-trans isomerism
of the amide bonds preceding Pro. Because these compounds
(6-13) incorporate an R-tetrasubstituted amino acid, the
observed conformational equilibrium could be somehow
related to the presence of such nonproteinogenic residues.
It should be noted that peptides 6, 7, and 9-13 contain Phe
surrogates that are not only R-tetrasubstituted but also bear
one (or more) bulky, rigidly held phenyl group(s) that could
further hamper rotation about the bonds in the neighbor-
hood of the R carbon. The presence of minor species, broad
signals, and exchange NOE cross-peaks made the spectral
analysis of some of these peptides rather complex (see
Supporting Information, Tables ST2 and ST4). Results
Solid-phase Boc chemistry protocols19,23 were applied to
the synthesis of peptides 1-13 (Figures 1,2), as done for GS
and the first series of analogues in our previous work.25 All
stereoisomers of c3diPhe, c3Phe, and Flg shown in Figure 2
were obtained as N-Boc derivatives in enantiomerically pure
form following previously reported procedures.38-40 Achiral
Boc-Dbg-OH was synthesized according to the protocol
described by Kotha.41 Boc-(RMe)Phe-OH was obtained
from commercially available H-(RMe)Phe-OH, and the side
chain of N-Boc ornithine was protected as a formamide as
described by Kitagawa.42 Each peptide was synthesized

4122 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Solanas et al.
Figure 3. Selected NOESY spectral regions for peptides 1 (A) and 5 (B) in D2O (top panels) and in H2O/D2O 9:1 v/v (all other panels) at
pH 3 and 5 °C. Green, blue, and red labels correspond, respectively, to symmetrical major, asymmetrical minor, and symmetrical minor species.
The top panel for peptide 1 shows the sequential NOEs between the Leu HR proton and the Pro Hδ and Hδ protons, indicative of the trans
0
rotamery for the two Leu-Pro bonds in the major species, and for one Leu-Pro bond in the aymmetrical minor species, as well as the sequential
NOE between the Leu and Pro HR protons demonstrating the cis rotamery for one Leu-Pro bond in the aymmetrical minor species.
Nonsequential NOEs, as those between the amide protons of Val1 and Leu30, and Val10 and Leu3 (Figure 1 and Table 2), are boxed.
described below correspond, in all cases, to the major
trans-trans species.
and adopt either positive or negative values. This anomalous
behavior may be explained by the ring current effects gene-
rated by the aromatic groups in the contiguous position
(Phe*5/50), as observed in our previous work on GS analo-
gues25 and some β-hairpin peptides.47 Indeed, in the absence
of aromatic substituents (peptide 8), Val1/10 shows confor-
mational shifts appropriate for a β-strand residue.
Chemical shifts of CRH protons and CR carbons are
dependent on the φ,ψ torsion angles and are, therefore, good
indicators of the secondary structure adopted by the peptide
backbone. Lys δrandom coil values43 were used to calculate the
Δδ values for the Orn residues. For all compounds investi-
3
gated (Figure 4), Orn2/20 and Leu3/30 exhibited positive
The large JCRH-NH coupling constants observed for
observed
ΔδCRH conformational shifts (ΔδCRH = δCRH
-
=
Val1/10, Orn2/20 and Leu3/30 (8.0-9.8 Hz, Supporting
Information, Table ST5) confirm that these residues adopt
a β-sheet conformation. Again, in analogue 2, the Orn2/20
deviate from the expected behavior (3JCRH-NH = 5.7 Hz).
On the other hand, in the (R,S)c3Phe derivative (12), the
3JCRH-NH values of Val1/10 and Leu3/30 could not be
determined because of signal broadening.
δCRHrandom coil, ppm) and negative ΔδCR values (ΔδCR
observed
δCR
- δCRrandom coil, ppm), as expected for β-strand
residues (ΔδCRH = þ0.40 ppm and ΔδCR = -1.6 ppm on
average in β-sheets).45,46 The Orn2/20 residues in 2 were the
sole exception to this pattern. The ΔδCRH and ΔδCR profiles
observed for Val1/10 do not seem to follow a particular trend

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4123
Figure 4. ΔδCRH and ΔδCR profiles (Δδ = δobserved - δrandom coil, ppm) for GS analogues in aqueous solution at pH 3.0 and 5 °C. Lys δrandom coil
values43 were used to calculate the Δδ values for the Orn residues. The δCRH and δCR values for the Orn residues in 12 could not be assigned.
Table 1. Temperature Coefficientsa (Δδ/ΔT, ppb K-1) for the Amide
Protons
the peptides (with the probable exception of 2 and 12) adopt
a more regular β-sheet structure, which could be attributed
to the presence of the D-Pro-Phe* instead of the D-Phe*-Pro
sequence.
3
0
0
0
0
Val1,1
(NH)
Orn2,2
(NH)
Leu3,3
(NH)
Phe*5,5
(NH)
0
peptide
Phe*5,5 b
NOE data provided further structural information on the
current set of analogues. The GS β-sheet structure (Figure 1)
should give rise to five characteristic backbone proton
NOEs: CRH Orn2-CRH Orn20 (distance in a canonical
antiparallel β-sheet: 2.2 A), CRH Orn2-NH Leu30, CRH
Orn20-NH Leu3 (distance in a canonical antiparallel
β-sheet: 3.2 A), NH Val1-NH Leu30, and NH Val10-NH
Leu3 (distance in a canonical antiparallel β-sheet: 3.3 A).
Given the molecule symmetry, the first NOE would not be
observable, the second and third ones will be undistinguish-
able from the sequential CRH Orn2/20-NH Leu3/30 NOEs,
while the last two must overlap into a single cross-peak. Such
a cross-peak is indeed observed in the NOESY spectra of all
peptides investigated (Table 2, Figure 3), with the exception
of 2 and 12, which once again deviate from the expected
behavior. NOE data are also informative on the layout of the
side chains. The backbone β-sheet conformation orients the
Val and Leu side chains toward the same side of the molecule
(Figure 1) and, indeed, NOE correlations between hydrogens
of these side chains are observed for all peptides (Table 2,
Figure 3) except 7, for which spectrum complexity precluded
analysis. The presence of such NOE cross-peaks in analogues
2 and 12 suggests that the Val and Leu side chains are
proximal. This could translate into a certain amphiphilic
character despite the fact that these GS analogues do not
seem to adopt a canonical β-sheet structure.
1
Phe
Dip
-2.2
-2.9
-0.7
-2.0
-3.2
-1.0
-1.9
-1.6
-2.1
-3.1
-7.5
-1.6
-9.1
-4.1
-9.9
-8.5
-10.5
-9.0
-9.0
-9.8
-9.0
-10.0
-8.5
-7.0
-1.8
þ0.9
þ2.6
-3.8
-4.2
-1.5
-5.1
-4.0
-3.9
-3.7
-10.0
-3.0
-8.7
-7.4
-7.3
;
2
3
Flg
Tic
5
6
(R,R)c3diPhe
(S,S)c3diPhe
Ac3c
-7.1
-9.0
-11.8
Ovc
7
8
9
(S,S)c3Phe
(R,R)c3Phe
(S,R)c3Phe
(R,S)c3Phe
(RMe)Phe
10
11
12
13
-7.1
-8.1
-15.1
-9.9
a Measured in H2O/D2O 9:1 (v/v) at pH 3.0 and temperature range
of 5-25 °C. b Phe* stands for Phe or the corresponding substitute. c Not
determined because of signal overlapping.
The involvement of the NH amide protons in intramole-
cular hydrogen bonds was deduced from the temperature
coefficients (Δδ/ΔT) (Table 1). The amide protons of Orn2/20
and Phe*5/50 displayed large negative Δδ/ΔT values
(below -7.0 ppb K-1) in all peptides, except again for
3
analogue 2, in agreement with their non-hydrogen bonded,
solvent-exposed character in the GS β-sheet (Figure 1).
In comparison, and with the exception of compound 12,
Δδ/ΔT values above -5.1 ppb K-1 were observed for the
3
NH protons of Val1/10 and Leu3/30, which are involved
in intramolecular hydrogen bonds in the GS structure
(Figure 1).
The above structural data suggest that most analogues of
this study fit into a β-sheet conformation, as does GS.6-8,25
It is worth noting that, in almost all peptides of the series, the
Val1/10 NH exhibits the typical behavior of a hydrogen-
bonded amide proton, which is not the case for either GS or
the analogues in our previous work.25 This may suggest that
Biological Activity. The 1-3 and 5-13 analogues (peptide
4 was not synthetically viable, see above) were tested along
with GS against two Gram-positive (Staphylococcus aureus
and Listeria monocytogenes) and two Gram-negative bac-
terial strains (Acinetobacter baummanii ATCC 19606 and an
isogenic strain resistant to colistin (polymyxin E)). Although

4124 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Solanas et al.
Table 2. Summary of NOEs Observeda
backbone NOEd
side-chain NOEe
0
0
0
0
0
0
0
0
0
0
0
peptideb
Phe*5,5 c
NHVal1-NHLeu3 /NHVal1 -NHLeu3
Val1,1 -Leu3,3
Orn2,2 -Phe*5,5
Val1,1 -Phe*5,5
D-Pro4,4 -Phe*5,5
1
Phe
Dip
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
-
-
-
þ
þ
þ
þ
-
þ
-
þ
þ
þ
-
þ
þ
-
þ
2
-
f
3
Flg
Tic
-
5
þ
þ
þ
þ
þ
þ
-
þ
g
6
(R,R)c3diPhe
Ac3c
þ
h
h
8
þ
þ
h
9
(S,S)c3Phe
(R,R)c3Phe
(S,R)c3Phe
(R,S)c3Phe
(RMe)Phe
þ
þ
þ
h
10
11
12
13
þ
h
-
þ
h
þ
þ
-
h
þ
þ
þ
a Observation of one or more NOEs is shown by a “þ”, and nondetected NOEs by a “-”. b Nonambiguous assignment of NOESY cross-peaks was
precluded in peptide 7 because of its very broad signals and the presence of numerous exchange cross-peaks. c Phe* stands for Phe or the corresponding
surrogate. d NOE involving backbone protons. e NOE involving at least one side chain proton of the residues indicated. f Not observed due to closeness to
diagonal. g Weak peak intensities. h Only protons of the cyclopropane ring or the R-methyl group are involved.
Table 3. Hemolytic and Antimicrobial Activitiesa (μM) of Gramicidin S (GS) Analogues with Sequence Inversion at the β-Turn
erythrocytes
HC50
S. aureus
MIC50
7.9 ((0.8) 2.7 (1.0) 8.0 ((0.0)
L. monocytogenes
A. baumanni S
MIC50 TI
11.0 ((0.4) 1.9 (1.0) 13.1 ((0.0) 1.6 (1.0)
24.8 ((1.2) 3.1 (1.7) >40 nd
10.0 (3.8) 20.3 ((0.3) 1.7 (0.9) 23.9 ((2.1) 1.4 (0.9)
A. baumannii Rd
0
peptide
Phe*5,5 b
TIc
MIC50
TI
MIC50 TI
GS
1
;
Phe
21.1 ((2.6)
78.7 ((4.2)
34.0 ((3.7)
2.1 ((1.4)
7.7 ((1.5)
26.0 ((2.4)
>60
2.6 (1.0)
15.8 ((0.0) 4.9 (1.8) 23.6 ((0.0) 3.3 (1.3)
2
Dip
Flg
3.8 ((0.8) 8.9 (3.3) 3.4 ((0.5)
2.2 ((0.1) 0.9 (0.3) 2.2 ((0.2)
2.4 ((0.2) 3.2 (1.2) 2.4 ((0.1)
3.2 ((0.5) 8.1 (3.0) 5.1 ((0.5)
25.8 ((0.0) 2.3 (0.8) >30
3
0.9 (0.3)
3.2 (1.2)
5.1 (1.9)
nd
5.0 ((0.3)
5.1 ((0.1)
>40
0.4 (0.2) 14.3 ((1.9) 0.1 (0.1)
5
Tic
1.5 (0.8) 8.5 ((2.1)
0.9 (0.6)
nd
6
(R,R)c3diPhe
(S,S)c3diPhe
Ac3c
nde
nd
nd
nd
nd
nd
nd
>40
>40
7
>40
>40
nd
nd
8
>60
>60
>30
>30
>30
>30
>30
nd
nd
nd
nd
nd
>30
>30
>30
>30
>30
nd
nd
>40
>40
9
(S,S)c3Phe
(R,R)c3Phe
(S,R)c3Phe
(R,S)c3Phe
(RMe)Phe
>40
>40
nd
nd
10
11
12
13
>60
57.0 ((3.8)
>60
nd
nd
41.0 ((11)
>40
>40
>40
>40
nd
nd
nd
8.3 (3.2)
56.5 ((3.9)
11.9 ((1.0) 4.7 (1.7) 6.8 ((0.3)
9.6 ((0.4)
5.9 (3.1) 7.0 ((1.4)
8.0 (5.0)
a HC50: peptide concentration required for 50% lysis of sheep erythrocytes; MIC50: peptide concentration required for 50% inhibition of bacterial
0
growth after 24 h, relative to a control culture; standard deviatio0ns for HC50 and MIC50 are shown in parentheses. b Phe*5,5 stands for Phe or the
corresponding surrogate (see Figures 1 and 2); GS contains D-Phe4,4 . c TI = therapeutic index = HC50/MIC50; values relative to TI of GS in parentheses.
d An A. baumannii strain resistant to colistin (polymyxin E). e nd=not determined.
GS is mostly active against Gram-positives, the last two
strains were included in view of the growing incidence of
A. baumannii infections, particularly those caused by strains
resistant to polymyxin E, which until recently has been the
last universally active drug against these pathogens.48 Micro-
bicidal activities were determined in solution, as in solid
media they tend to be underestimated, especially for Gram-
negatives.4 Pseudomonas aeruginosa, another Gram-negative,
was included earlier on in the assays, but as none of the
analogues showed growth inhibitions above 30% at 50 μM,
the highest concentration assayed, it was disregarded.
Results are given in Table 3 and show that the replacement
of the D-Phe-Pro sequences in GS by D-Pro-Phe in 1 has a
negative impact on activity. However, further modification
of the Phe residue allows the recovery and even an enhance-
ment of the microbicidal potency of the natural peptide.
Thus, the Dip (2), Flg (3), Tic (5), and (R,R)c3diPhe (6)
analogues turned out to be more active than GS against
Gram-positive bacteria, while 3 and 5 were also superior
against Gram-negatives. The improved microbicidal activity
of the Tic analogue (5) against the colistin-resistant strain is
also worth mentioning. On the other hand, the Ac3c deriva-
tive (8), lacking an aromatic moiety at the β-turn region, was,
perhaps not surprisingly, practically devoid of any antibiotic
activity.
The contrasting activity of the analogues incorporating
the c3diPhe enantiomers (6, 7) deserves some comment.
Thus, while 7 had the lowest activity against all bacterial
strains among the 1-7 subset, 6 was unique in its highest
selectivity for Gram-positives. These differences, which
might be ascribed to the rigid orientation imposed by the
cyclopropane ring on the aromatic substituents, prompted us
to expand the analogue series with the four c3Phe stereo-
isomers bearing a single phenyl group (Figure 2). Surpris-
ingly enough, all analogues containing these Phe surrogates
(9-12) showed marginal activity, if at all, against all organ-
isms. In contrast, the analogue with the closely related
(RMe)Phe residue (13) not only retained the activity of GS
but displayed the best therapeutic index (TI, see below)
against both A. baumannii strains.
The hemolytic effect of the peptides was evaluated on
sheep erythrocytes as an indicator of toxicity against mam-
malian cells. Results are summarized in Table 3, where the
therapeutic index (TI, defined as HC50/MIC50) is also given
as a combined measure of both effects. As a general rule, the
antibiotic and hemolytic activities were found to run parallel.
Thus, peptides with very low microbicidal power (1, 7-12)
were hardly harmful to erythrocytes, whereas analogues of
high antibiotic activity (3, 5) turned out to be also fairly toxic.
However, some notable deviations to this general behavior

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4125
were observed. For instance, the Dip analogue 2 was more
active against Gram-positives yet slightly less cytotoxic than
GS, which translated into a modest (about 3-fold) but
significant TI increase vs GS. A similar improvement in TI
was observed for the (R,R)c3diPhe-containing analogue (6)
regarding the S. aureus strain. The peptide incorporating
(RMe)Phe (13) was found to be essentially harmless to
erythrocytes while exhibiting an antibiotic potency similar
or even superior to that of GS, which resulted in an enhanced
TI for all pathogens considered. Particularly noteworthy is
the fact that the most significant increase in TI for this
analogue (near 5-fold that of the natural peptide) was
observed for the Gram-negative A. baumannii strand resis-
tant to conventional antibiotics. It should also be noted that
improved TIs reported in the literature for GS analogues
most often stem from lower cytotoxicity,24,25,27,49,50 whereas
the most significant increases in TI relative to GS found in
this work (g3-fold) are also associated to a higher antibiotic
potency.
Figure 5. Retention times (Rt) of GS analogues on RP-HPLC.
Elution conditions: see Experimental Section. The retention time
of GS under the same conditions is also shown for comparison.
Gram-positive bacteria, where the peptide can diffuse rela-
tively easily through the peptidoglycan layer.
For GS and the 12 analogues of the present series, however,
hydrophobicity does not provide a straightforward interpre-
tation to antimicrobial action. RP-HPLC retention times
(Figure 5), widely accepted as a criterion of relative hydro-
phobicity for structurally related compounds, clearly show
that while the least (8) and most (3) hydrophobic analogues,
respectively, show low and high activities against Gram-
positives and erythrocytes, analogues of intermediate hydro-
phobicity follow no discernible general trend in this regard.
For instance, the four c3Phe derivatives (9-12) differ signifi-
cantly in hydrophobicity but coincide in (a very poor) activity.
Again, comparing GS with 2 or 5, of similar hydrophobicity
but higher potency against Gram-positives, reveals similar
discrepancies, albeit in the opposite sense. The fact that the
antimicrobial performance of the current set of analogues
cannot be explained simply on the basis of hydrophobicity
agrees with recent data on AMPs like lactoferricin51 or again
GS,52 for which additional parameters (e.g., bulkiness) have
been invoked as crucial for activity.
Against Gram-negatives, the situation is more complex due
to the existence of an outer membrane that AMPs must
traverse before interacting with the plasma membrane. In this
context, hydrophobicity can be regarded as a two-edged
sword because, abovea certain limit, it will induce aggregation
and poor water solubility, causing the AMP to be sequestered
within the outer membrane, unable to reach and disrupt the
plasma membrane. Thus, a balanced combination of hydro-
phobicity with additional parameters such as amphipathicity,
molecular flexibility, or cationic character becomes essential
for AMP interaction with the LPS of the outer membrane
and efficacy against Gram-negatives. In the present work,
improved performance against Gram-negatives is observed
for analogues 3, 5, and 13, all of them with hydrophobicities
(i.e., retention times) equal or superior to GS and with
substantial structural diversity as Phe surrogates (i.e., Flg in
3, Tic in 5, (RMe)Phe in 13). The failure of analogues 6-12
against Gram-negatives may be explained because, in these
analogues, the bulkiness and rigidity of the cyclopropane
system may dramatically impair AMP transit through the
membrane, regardless of its potential to disrupt the plasma
membrane final target.
Discussion
Several groups, including our own,25,29-32 have shown that
the β-turn of GS is particularly suited to modulation of the TI
by subtle substitutions. In this work, we have explored the
impact of replacing Phe by residues bearing one or two phenyl
rings that impart different degrees of hydrophobicity, bulki-
ness, and rotational freedom to the GS molecule. To carry out
these substitutions with minimal perturbation of the β-turn
architecture, the native D-Phe-L-Pro sequence of GS has been
changed to D-Pro-L-Phe because it is known that simple
residue permutation (i.e., to L-Pro-D-Phe) results in practically
full loss of activity.22 This distinguishing feature of the present
series compares favorably with our earlier work,25 where the
canonical D-Phe-L-Pro β-turn was used. In absolute terms,
sequence inversion to D-Pro-L-Phe, as exemplified by analo-
gue 1, entails a certain loss of antimicrobial potency relative to
the parent GS. However, the change also involves a substan-
tial decrease in cytotoxicity (determined as hemolysis) that,
when factored into the therapeutic index (TI), actually results
in an almost 2-fold improvement against the Gram-positive
S. aureus as well as the colistin-sensitive strain of the Gram-
negative A. baumannii. A similarly improved antimicrobial
profile can be found for the 2 (Dip) and 6 [(R,R)c3diPhe]
analogues against Gram-positives, as well as for the
(RMe)Phe replacement (13) against both Gram-positives
and -negatives. The Tic replacement, which in the previous
series proved to be very advantageous,25 is now (analogue 5)
of less consequence, as the enhanced antimicrobial potency
entails a considerable penalty in cytotoxicity. In the canonical
D-Phe-L-Pro series,25 the proximity between Orn and the
aromatic side chain exclusive of the Tic analogue was invoked
to explain the TI improvement. Such interaction is not
observed in 5, neither in 3, the most hemolytic analogue
(Tables 2-3). Interestingly, however, Orn-Phe* NOEs are
observed foranalogues lesshemolytic thanGS, such as1 and2
(Tables 2-3).
Changes in antimicrobial activity patterns were evident not
only between Gram-positive and Gram-negative organisms
but also at the species or even the strain level. The case of the
Gram-negative pathogen Acinetobacter baumannii, of which
both colistin-susceptible and -resistant strains that reflect
differences in outer membrane composition were tested, is
illustrative. Against this organism, a substantially improved
TI was found for analogue 13 (3-fold relative to the parent GS
for the colistin-sensitive strain), making this peptide an opti-
mized lead within the current D-Pro-L-Phe series, with the
added incentive that the TI optimization applies also to the
Among several factors known to influence the interaction
of AMPs with bacterial or mammalian membranes, hydro-
phobicity is viewed as crucial. In general, and regardless of
the anionic or zwitterionic nature of the bilayer, a hydro-
phobic patch of size above a minimal threshold is required for
a peptide to partition into the membrane. In this scenario,
hydrophobicity correlates relatively well with AMP acti-
vity against systems such as eukaryotes (e.g., hemolysis) or

4126 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Solanas et al.
resistant strain, which constitutes a serious clinical concern53
given that colistin is the last drug universally active on
this pathogen. It has been proposed that the self-uptake
mechanism used by GS to traverse the outer membrane of
Gram-negatives differs from the more general one of other
membrane-active AMPs,54 for which high-affinity binding
to LPS (mediated by both hydrophobicity and cationic
character) entails disruption of the outer membrane and
access to the periplasmic space. For GS, instead, the mecha-
nism would involve diffuse binding to LPS and further
passage driven by hydrophobic interactions. This alternative
mechanism would not seem to apply to the current set of
analogues, because the resistant strain, with a highly modified
LPS, is significantly more insensitive to analogues such as 3 or
5, which incorporate Phe* surrogates equally or more hydro-
phobic than Phe. Also, recent experiments have shown the
permeability of the outer membrane to be quite compromised
in the resistant strain.55
In summary, although inversion of the β-turn sequence of
GS has a detrimental impact on antibiotic potency, this can be
productively offset by the incorporation of Phe surrogates
containing an additional aromatic group or a single phenyl
substituent with the adequate orientation. Because the NMR
data, with the probable exception of analogues 2 and 12 (see
Results), do not reveal substantial alterations in conforma-
tion, it is fair to assume that, as long as a robust global β-sheet
structure is preserved, sequence inversion at the β-turn of GS
is a productive source of antimicrobial leads, provided that a
modicum of hydrophobicity is preserved and that the con-
formational restriction involved is not inadequate (as in the
cyclopropane analogues except 6). As is often the case in SAR
studies of peptides, subtle changes such as the addition of a
methyl group (compare analogues 1 and 13) can have a
dramatic impact on the biological profile. Thus, as shown
by analogues 7 and 9-12, structural constraints imposing
inadequate orientation of the aromatic substituent at the
β-turn of GS can be highly detrimental for antibiotic potency.
In contrast, analogues 2, 6, and 13 show a remarkable 3-fold
boost in TI against Gram-positives relative to GS. Of these, 6
is selective for Gram-positives and, more interestingly, analo-
gue 13 is quite effective against Gram-negatives, including an
interesting 5-fold increase in TI against the colistin-resistant
Acinetobacter strain.
H2O (þ0.045% TFA, v/v) over 15 min at 1 mL/min flow rate,
with UV detection at 220 nm. Preparative RP-HPLC purifica-
tion was done on a Phenomenex Luna C8 column (10 μm,
250 mmꢀ10 mm) running linear gradients of CH3CN (þ0.1%
TFA, v/v) into H2O (þ0.1% TFA, v/v) as indicated for each
peptide, at a flow rate of 5 mL/min. The preparative system
included two Shimadzu LC-8A pumps, a Shimadzu SPD-10A
detector and a Foxy Jr. fraction collector (Teledyne Isco,
Lincoln, NE).
General Procedure for Peptide Synthesis. All peptides were
synthesized manually by solid-phase methods on a Boc-D-Pro-
Merrifield resin (0.1 mmol) using Boc chemistry. Boc-amino
acids (0.3 mmol) were coupled by HBTU/DIEA (0.3 and
0.6 mmol, respectively) for 30-45 min in DMF. HATU was
used instead of HBTU for the coupling reactions involving the
5/50 residues. To avoid diketopiperazine formation, the third
amino acid of each sequence was incorporated by the in situ
neutralization method.56 Coupling and deprotection reactions
were monitored by the nynhidrin57 or p-nitrophenylester58 col-
orimetric tests. The linear decapeptide was cleaved from the resin
by treatment with TFA/TFMSA/TIS 10:1:1 (v/v/v) (4 mL) at
room temperature for 90 min and then precipitated with cold tert-
butyl methyl ether. The peptide was redissolved in glacial acetic
acid, filtered off the resin, and lyophilized. Cyclization was
performed by dissolving the peptide in DMF to a final concen-
tration of 2 mg/mL and stirringfor 1 h at room temperature in the
presence of HATU/HOAt/DIEA (3:3:5 equiv). After solvent
removal and lyophilization, the cyclized peptide was deformy-
lated by treatment with 20% hydrochloric acid in methanol at
37 °C for 21 h. The solvent was evaporated under reduced
pressure, and the residue was taken up in glacial acetic acid and
lyophilized. Final purification was carried out by preparative
reversed-phase HPLC as indicated in each case. HPLC-homo-
geneous fractions were combined and lyophilized to give white
powders of g99% HPLC purity (see Supporting Information).
Variations to this general protocol are indicated for each peptide.
cyclo(Val-Orn-Leu-D-Pro-Phe)2 (1). RP-HPLC, linear 30-
70% CH3CN gradient into H2O for 30 min (66 mg, 0.063 mmol,
47% overall yield). HRMS (ESI) C60H93N12O10 [M þ H]þ:
calcd 1141.7132, found 1141.7088; C60H92N12O10Na [M þ
Na]þ: calcd 1163.6952, found 1163.6924.
cyclo(Val-Orn-Leu-D-Pro-Dip)2 (2). RP-HPLC, linear 45-
75% CH3CN gradient into H2O for 30 min (55 mg, 0.042 mmol,
34% overall yield). HRMS (ESI) C72H101N12O10 [M þ H]þ:
calcd 1293.7758, found 1293.7682; C72H100N12O10Na [M þ
Na]þ: calcd 1315.7578, found 1315.7521.
cyclo(Val-Orn-Leu-D-Pro-Flg)2 (3). RP-HPLC, linear 35-
65% CH3CN gradient into H2O for 30 min (57 mg, 0.044 mmol,
38% overall yield). HRMS (ESI) C72H97N12O10 [M þ H]þ:
calcd 1289.7445, found 1289.7420; C72H96N12O10Na [M þ
Na]þ: calcd 1311.7265, found 1311.7228.
Experimental Section
Chemicals and Instrumentation for Peptide Synthesis. The
different stereoisomers of c3diPhe,38 c3Phe,39 and fluorenylgly-
cine40 were prepared as previously reported. Dibenzylglycine
was obtained following the methodology described by Kotha.41
N-Boc ornithine was purchased from Bachem (Bubendorf,
Switzerland), and its side-chain amino group was protected as
a formamide by reaction with formic acid and 1,10-oxalyldiimi-
dazole.42 All other amino acids were purchased from Senn
Chemicals (Dielsdorf, Switzerland), NeoMPS (Strasbourg,
France), or Fluka (Buchs, Switzerland). Chloromethylated
Merrifield resin and TFMSA were from Fluka, HATU from
GenScript (Piscataway, NJ), and HBTU from Matrix Innova-
tion (Montreal, Quebec). Solvents and other chemicals were
from SDS (Peypin, France). Mass determination was done by
the ESI technique in a MicrOTOF-Q spectrometer (Bruker
Daltonics, Billerica, MA). Analytical RP-HPLC was performed
on an LC-2010A workstation (Shimadzu Corporation, Kyoto,
Japan) with a Luna C8 (3 μm, 50 mm ꢀ 4.6 mm) column
(Phenomenex BV, Utrecht, The Netherlands) eluted with a
linear 5-95% gradient of CH3CN (þ0.036% TFA, v/v) into
cyclo(Val-Orn-Leu-D-Pro-Tic)2 (5). The cyclized peptide was
purified by RP-HPLC (linear 60-90% CH3CN gradient into
H2O for 30 min) prior to deformylation. Final RP-HPLC, linear
40-70% CH3CN gradient into H2O for 30 min (52 mg,
0.045 mmol, 39% overall yield). HRMS (ESI) C62H93N12O10
[M þ H]þ: calcd 1165.7132, found 1165.7088; C62H92N12O10Na
[M þ Na]þ: calcd 1187.6952, found 1187.6895.
cyclo[Val-Orn-Leu-D-Pro-(R,R)c3diPhe]2 (6). Precipitation of
the linear decapeptide by addition of cold tert-butyl methyl ether
proved unsuccessful. The organic solvent was then evaporated
and the crude was redissolved in CH3CN. The peptide precipi-
tated upon addition of H2O. The cyclization mixture was
allowed to react overnight. The cyclized peptide was purified
by RP-HPLC (linear 65-95% CH3CN gradient into H2O for
30 min) prior to deformylation. Final RP-HPLC, linear 40-
75% CH3CN gradient into H2O for 30 min (24 mg, 0.018 mmol,
15% overall yield). HRMS (ESI) C74H101N12O10 [M þ H]þ:
calcd 1317.7758, found 1317.7737; C74H100N12O10Na [M þ
Na]þ: calcd 1339.7578, found 1339.7557.

Article
cyclo[Val-Orn-Leu-D-Pro-(S,S)c3diPhe]2 (7). The linear dec-
apeptide was precipitated with H2O, as described above for 6.
Final RP-HPLC, linear 40-75% CH3CN gradient into H2O
for 30 min (64 mg, 0.049 mmol, 41% overall yield). HRMS
(ESI) C74H101N12O10 [M þ H]þ: calcd 1317.7758, found
1317.7717; C74H100N12O10Na [M þ Na]þ: calcd 1339.7578,
found 1339.7559.
cyclo(Val-Orn-Leu-D-Pro-Ac3c)2 (8). RP-HPLC, linear 30-
60% CH3CN gradient into H2O for 30 min (68 mg, 0.067 mmol,
59% overall yield). HRMS (ESI) C50H85N12O10 [M þ H]þ:
calcd. 1013.6506, found 1013.6494; C50H84N12O10Na [M þ
Na]þ: calcd 1035.6326, found 1035.6309.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4127
methods.61 The 13C and 15N resonances were then assigned
following the cross-correlations observed in the HSQC spectra
between the proton and the heteronucleus to which it is bonded.
Structure Calculation. Structure calculations were performed
by using the CYANA program62 and an annealing strategy.
Because the nonproteinogenic amino acids were not included in
the standard CYANA libraries, we built them using MOL-
MOL63 and manual optimization (these libraries are available
upon request from the authors). For this purpose, X-ray data of
compounds containing these amino acids were obtained from
our laboratories or retrieved from the Cambridge Structural
Database.64 Theoretical constraints for the GS β-sheet incorpo-
rated for structure calculation included φ and ψ angle restraints,
lower and upper-limit distance restraints for the four character-
istic cross-strand hydrogen-bonds, and upper-limit distance
restraints for the backbone atoms of strand residues (see Sup-
porting Information). Experimental distance constraints for
peptides 1 and 5 were derived from 2D NOESY spectra recorded
in H2O/D2O 9:1 (v/v). The NOE cross-peaks were integrated
by using the automatic integration subroutine of the Sparky
program (T. D. Goddard and S. G. Kneller, University of
California at San Francisco) and then calibrated and converted
to upper-limit distance constraints with CYANA.62 Given the
symmetrical nature of the peptides, for structure calcula-
tions, residues were renumbered from 1 to 10 starting at Leu3
(Figure 1). Lower and upper limit restraints required for peptide
backbone cyclization were introduced with CYANA (see
Supporting Information). For each peptide, a total of 50 con-
formers were generated and the 20 conformers with the lowest
target function were analyzed. Model structures were examined
with MOLMOL.63 A side chain torsion angle was considered
as well-defined when its root-mean-square deviation between
values in the 20 best calculated structures was less than (30°.
Antimicrobial Activity. Stocks of Staphylococcus aureus
CECT 240, Listeria monocytogenes CECT 4032, Acinetobacter
baumannii ATCC 19606, and its isogenic colistin-resistant strain
19606R (obtained by continuous growing under increasing
colistin concentration) were maintained at -80 °C in freezing
medium (65% glycerol, 0.1 M MgSO4, 25 mM Tris-HCl,
pH 8.0). Two days prior to the assay for microbicidal activity,
they were thawed and grown in MBH medium (Mueller-Hinton
II Broth Cation Adjusted (Becton-Dickinson, Cockeysville,
MD)) at 37 °C; for 19606R, 64 μg/mL colistin sulfate (Sigma,
Madrid, Spain) was included.47 Bacterial cells were harvested at
exponential growth phase, washed twice with phosphate buf-
fered saline (PBS, 10 mM Na2HPO4, 1 mM KH2PO4, 140 mM
NaCl, 3 mM KCl, pH 7.0), and resuspended in MBH, at 5 ꢀ 105
colony forming units/mL. Aliquots (100 μL) from this suspen-
sion were transferred into a polypropylene 96-well plate, and
bacteria were allowed to proliferate for 24 h at 37 °C in the
presence of the corresponding peptide concentration. After-
ward, growth was measured by turbidimetry at 600 nm in a
model 680 microplate reader (Bio-Rad Laboratories, Hercules.
CA). Determinations were carried out twice on triplicated
samples. MIC50 was defined as the lowest peptide concentration
inhibiting bacterial growth by 50%, relative to untreated con-
trol, and was calculated using the SigmaPlot (Systat Software,
San Jose, CA) software, v. 9.0.
cyclo[Val-Orn-Leu-D-Pro-(S,S)c3Phe]2 (9). The linear deca-
peptide was precipitated with H2O, as described above for 6 and
purified by RP-HPLC (linear 40-70% CH3CN gradient into
H2O for 30 min) before cyclization. The cyclized peptide was
purified by RP-HPLC (linear 50-80% CH3CN gradient into
H2O for 30 min) prior to deformylation. Final RP-HPLC,
linear 40-70% CH3CN gradient into H2O for 30 min (61 mg,
0.052 mmol, 45% overall yield). HRMS (ESI) C62H93N12O10
[M þ H]þ: calcd 1165.7132, found 1165.7088; C62H92N12O10Na
[M þ Na]þ: calcd 1187.6952, found 1187.6912.
cyclo[Val-Orn-Leu-D-Pro-(R,R)c3Phe]2 (10). The linear dec-
apeptide was precipitated with H2O, as described above for 6,
and purified by RP-HPLC (linear 45-75% CH3CN gradient
into H2O for 30 min) before cyclization. The cyclized peptide
was purified by RP-HPLC (linear 50-80% CH3CN gradient
into H2O for 30 min) prior to deformylation. Final RP-HPLC,
linear 35-65% CH3CN gradient into H2O for 30 min (49 mg,
0.042 mmol, 36% overall yield). HRMS (ESI) C62H93N12O10
[M þ H]þ: calcd 1165.7132, found 1165.7102; C62H92N12O10Na
[M þ Na]þ: calcd 1187.6952, found 1187.6928.
cyclo[Val-Orn-Leu-D-Pro-(S,R)c3Phe]2 (11). RP-HPLC, line-
ar 45-65% CH3CN gradient into H2O for 30 min (26 mg,
0.021 mmol, 18% overall yield). HRMS (ESI) C62H93N12O10
[M þ H]þ: calcd 1165.7132, found 1165.7117; C62H92N12O10Na
[M þ Na]þ: calcd 1187.6952, found 1187.6947.
cyclo[Val-Orn-Leu-D-Pro-(R,S)c3Phe]2 (12). The cyclization
step required an additional amount of the HATU/HOAt/DIEA
(0.6:0.6:1.0 equiv) coupling mixture. RP-HPLC, linear 35-60%
CH3CN gradient into H2O for 30 min (67 mg, 0.057 mmol, 50%
overall yield). HRMS (ESI) C62H93N12O10 [M þ H]þ: calcd
1165.7132, found 1165.7116; C62H92N12O10Na [MþNa]þ: calcd
1187.6952, found 1187.6939.
cyclo[Val-Orn-Leu-D-Pro-(rMe)Phe]2 (13). RP-HPLC, linear
35-65% CH3CN gradient into H2O for 30 min (76 mg,
0.065 mmol, 56% overall yield). HRMS (ESI) C62H97N12O10
[M þ H]þ: calcd 1169.7445, found 1169.7458; C62H96N12O10Na
[M þ Na]þ: calcd 1191.7265, found 1191.7261.
NMR Spectroscopy. Samples for NMR experiments were
prepared at 1-2 mM peptide concentration in 0.5 mL of H2O/
D2O 9:1 (v/v) or pure D2O at pH 3.0. pH was measured with a
glass microelectrode and was not corrected for isotope effects.
NMR spectra were acquired on a Bruker AV 600 MHz spectro-
meter equipped with a z-gradient cryoprobe. A methanol sample
was used to calibrate the temperature of the NMR probe. One-
dimensional (1D) and two-dimensional (2D) spectra were
acquired by standard pulse sequences using presaturation of
the water signal. Mixing times for 2D TOCSY and NOESY were
Hemolytic Activity Assay. As above, hemolytic activity of the
peptides was also determined twice on triplicate samples. Defi-
brinated sheep blood (Biomedics, Madrid, Spain) was centri-
fuged and washed twice with Hank’s medium (136 mM NaCl,
4.2 mM Na2HPO4, 4.4 mM KH2PO4, 5.4 mM KCl, 4.1 mM
NaHCO3, pH 7.2), supplemented with 20 mM D-glucose (Hank’s-
Glc). Erythrocytes were resuspended in the same buffer at 2 ꢀ 107
erythrocytes/mL, and 100 μL aliquots of the suspension were
incubated with the peptides (4 h, 37 °C). The remaining erythro-
cytes were harvested in a Micro 200 microfuge (A. Hettich GmbH
& Co. KG, Germany) (14000 rpm, 5 min, 4 °C), 80 μL of the
supernatant were transferred into a 96-well culture microplate,
1
1
60 and 150 ms, respectively. The H-13C and H-15N HSQC
spectra59 at natural 13C and 15N abundance were recorded in
D2O and H2O/D2O 9:1 (v/v), respectively. Data were processed
using TOPSPIN (Bruker Biospin, Rheinstetten, Germany) soft-
ware. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS)
1
was used as an internal reference for H chemical shifts. The
13C and 15N chemical shifts were indirectly calibrated by multi-
plying the spectrometer frequency that corresponds to 0 ppm
in the 1H spectrum, assigned to internal DSS reference,
1
by 0.25144954 and 0.101329118, respectively.60 The H NMR
signals of the peptides were assigned by sequential assignment

4128 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Solanas et al.
and released hemoglobin was determined at 550 nm in a Bio Rad
680 (Hercules, CA) microplate reader. The asymptotic ordinate
of the GS supernatant was taken as 100% hemolysis. HC50 values
were calculated using SigmaPlot, version 9.0.
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0
N. Synthesis of [hexafluorovalyl1,1 ]gramicidin S. Bull. Chem. Soc.
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Acknowledgment. This work was supported by Ministerio
ꢀ
(15) Aimoto, S. The synthesis of a heavy-atom derivative of gramicidin
0
de Ciencia e Innovacion (BIO2008-04487-CO3-02 to D.A.,
S (GS), [D-Phe(4-Br)4,4 ]-GS, by a novel method. Bull. Chem. Soc.
Jpn. 1988, 61, 2220–2222.
CTQ2008-00080/BQU to M.A.J., CTQ2007-62245 to C.C.),
Fondo de Investigaciones Sanitarias (PI061125, PS09/1928,
and RD06/0021/0006 to L.R.), by the regional governments
ꢀ
of Aragon (research group E40), Catalonia (SGR2008-492),
and Madrid (COMBACT S-BIO-0260/2006). C.S. and
~
(16) Andreu, D.; Ruiz, S.; Carreno, C.; Alsina, J.; Albericio, F.;
Jimenez, M. A.; de la Figuera, N.; Herranz, R.; Garcıa-Lopez,
ꢀ
ꢀ
ꢀ
~
M. T.; Gonzalez-Muniz, R. IBTM-containing gramicidin S ana-
logs: evidence for IBTM as a suitable type II0 β-turn mimetic.
J. Am. Chem. Soc. 1997, 119, 10579–10586.
(17) Grotenbreg, G. M.; Buizert, A. E.; Llamas-Saiz, A. L.; Spalburg,
E.; van Hooft, P. A.; de Neeling, A. J.; Noort, D.; van Raaij, M. J.;
van der Marel, G. A.; Overkleeft, H. S.; Overhand, M. β-Turn
modified gramicidin S analogs containing arylated sugar amino
acids display antimicrobial and hemolytic activity comparable to
the natural product. J. Am. Chem. Soc. 2006, 128, 7559–7565.
(18) Kawai, M.; Yamamura, H.; Tanaka, R.; Umemoto, H.; Ohmizo,
C.; Higuchi, S.; Katsu, T. Proline residue-modified polycationic
analogs of gramicidin S with high antibacterial activity against
both Gram-positive and Gram-negative bacteria and low hemo-
lytic activity. J. Pept. Res. 2005, 65, 98–104.
(19) Lee, D. L.; Hodges, R. S. Structure-activity relationships of de
novo designed cyclic antimicrobial peptides based on gramicidin S.
Biopolymers 2003, 71, 28–48.
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J. M. Protein β-turn mimetics II: Design, synthesis and evaluation
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ꢀ
C.M.S. thank Ministerio de Educacion y Ciencia and Consejo
Superior de Investigaciones Cientıficas-European Social
Fund for FPU and I3P fellowships, respectively. This project
has been funded in whole or in part with federal funds from
the National Cancer Institute, National Institutes of Health,
under contract number HHSN261200800001E. The content
of this publication does not necessarily reflect the view of the
policies of the Department of Health and Human Services,
nor does mention of trade names, commercial products, or
organization imply endorsement by the U.S. Government.
This research was supported (in part) by the Intramural
Research Program of the NIH, National Cancer Institute,
Center for Cancer Research.
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0
Synthetic conformation of weak activity of [D-Pro5,5 ]-Gramicidin
S predicted from β-turn preference of its partial sequence. Bull.
Chem. Soc. Jpn. 1986, 59, 535–538.
Supporting Information Available: NMR data, details on
structure calculations, and analytical data on the peptides
synthesized. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Muramatsu, I. Synthesis and properties of gramicidin S analogs
containing Pro-D-Phe sequence in place of D-Phe-Pro sequence in
the β-turn part of the antibiotic. Bull. Chem. Soc. Jpn. 1985, 58,
531–535.
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