Therapeutic index of gramicidin S is strongly modulated by D-phenylalanine analogues at the β-turn

Article abstract of DOI:10.1021/jm800886n

Analogues of the cationic antimicrobial peptide gramicidin S (GS), cyclo(Val-Orn-Leu-D-Phe-Pro)₂, with D-Phe residues replaced by different (restricted mobility, mostly) surrogates have been synthesized and used in SAR studies against several p

Full text of DOI:10.1021/jm800886n

664
J. Med. Chem. 2009, 52, 664–674
Therapeutic Index of Gramicidin S is Strongly Modulated by D-Phenylalanine Analogues at the
ꢀ-Turn
†
‡
§
|
|
§
´
Concepcio´n Solanas, Beatriz G. de la Torre, Mar´ıa Ferna´ndez-Reyes, Clara M. Santiveri, M. Angeles Jime´nez, Luis Rivas,
Ana I. Jime´nez,† David Andreu,*,‡ and Carlos Cativiela*,†
Departamento de Qu´ımica Orga´nica, Instituto de Ciencia de Materiales de Arago´n, UniVersidad de Zaragoza-CSIC, 50009 Zaragoza, Spain,
Departament de Cie`ncies Experimentals i de la Salut, UniVersitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain, Centro de
InVestigaciones Biolo´gicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain, and Instituto de Qu´ımica-F´ısica Rocasolano, CSIC, Serrano 119,
28006 Madrid, Spain
ReceiVed July 17, 2008
Analogues of the cationic antimicrobial peptide gramicidin S (GS), cyclo(Val-Orn-Leu-D-Phe-Pro)2, with
D-Phe residues replaced by different (restricted mobility, mostly) surrogates have been synthesized and
used in SAR studies against several pathogenic bacteria. While all D-Phe substitutions are shown by NMR
to preserve the overall ꢀ-sheet conformation, they entail subtle structural alterations that lead to significant
modifications in biological activity. In particular, the analogue incorporating D-Tic (1,2,3,4-tetrahydroiso-
quinoline-3-carboxylic acid) shows a modest but significant increase in therapeutic index, mostly due to a
sharp decrease in hemolytic effect. The fact that NMR data show a shortened distance between the D-Tic
aromatic ring and the Orn δ-amino group may help explain the improved antibiotic profile of this analogue.
Introduction
A remarkable increase in bacterial resistance to classical
antibiotics has in recent years emerged as a major global health
concern and spurred intense efforts toward the development of
new antimicrobial chemotherapy approaches. A number of
peptides with antimicrobial activity have been identified in a
wide variety of organisms1,2 and recognized as promising
candidates against multidrug resistant bacteria. An attractive
feature of these peptides is that, by acting mostly through
interaction with plasma membrane phospholipids, their develop-
ing resistance is less likely than for other mechanisms of action
because it would involve major changes in phospholipid
composition that would in turn require substantial overhauling
Figure 1. ꢀ-Sheet structure of gramicidin S.
of membrane-based enzymatic and transport systems.
Gramicidin S (GSa) [cyclo(Val-Orn-Leu-D-Phe-Pro)2] (Figure
1), a cyclic decapeptide isolated from Bacillus breVis3 and active
against bacteria and fungi,4,5 is one of the most widely studied
antimicrobial peptides. GS adopts a full ꢀ-sheet pleated
structure6-8 where the Val, Orn, and Leu residues align to form
two antiparallel ꢀ-strands while D-Phe and Pro form type II′
ꢀ-turns. The structure is stabilized by four interstrand hydrogen
bonds between the Leu and Val residues and displays C2
symmetry. The ꢀ-sheet places the hydrophobic Val and Leu
side chains on a nonpolar face of the GS molecule and across
from the cationic Orn side chains, thus creating an amphiphilic
structure that is thought to be essential for bioactivity.5,9
Although not fully unveiled, the mechanism of action of GS
sets out with a peptide-membrane interaction, leading to the
disruption of the lipid bilayer and concomitant enhancement of
its permeability,10 eventually resulting in cell death. Unfortu-
nately, GS not only affects bacterial membranes but also
mammalian cells such as erythrocytes. This hemolytic activity
hampers its use as a systemic antibiotic and confines it to topical
administration.5
Significant efforts have been devoted to rationalize the
biological behavior of GS with the ultimate goal of generating
derivatives with an improved therapeutic index. In this respect,
both the ꢀ-strand11-13 and ꢀ-turn14-23 regions of the molecule
have been the subject of modification. SAR studies on these
GS analogues have identified several factors underlying the
biological properties of GS, cationic charge,10,23 amphipathic
character,24,25 and ꢀ-sheet structure,23,24,26 are believed to be
* To whom correspondence should be addressed. For D.A.: phone, +34-
933160868; fax, +34-933160901; e-mail, david.andreu@upf.edu. For C.C.:
phone, +34-976761210; fax, +34-976761210; e-mail, cativiela@unizar.es.
†
Universidad de Zaragoza-CSIC.
‡
Universitat Pompeu Fabra.
§
Centro de Investigaciones Biolo´gicas, CSIC.
Instituto de Qu´ımica-F´ısica Rocasolano, CSIC.
|
a Abbreviations: ATCC, American Type Culture Collection; Boc, tert-
butoxycarbonyl; CECT, Spanish Type Culture Collection; COSY, correlated
spectroscopy; DIEA, N,N-diisopropylethylamine; Dip, ꢀ,ꢀ-diphenylalanine;
DMF, N,N-dimethylformamide; DSS, 2,2-dimethyl-2-silapentane-5-sul-
fonate; Flg, fluorenylglycine; For, formyl; GS, gramicidin S; HATU, 2-(7-
aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;
HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate; HC50, 50% hemolytic concentration; HOBt, N-hydroxybenzo-
triazole; Hpa, homophenylalanine; HSQC, heteronuclear single quantum
coherence spectra; IM, inner membrane; LPS, lipopolysacharide; MALDI-
TOF, matrix-assisted laser desorption ionization-time-of-flight; MIC50,
minimal inhibitory concentration; 1-Nal, 1-naphthylalanine; 2-Nal, 2-naph-
thylalanine; NOESY, nuclear Overhauser enhancement spectroscopy; OM,
outer membrane; Orn, ornithine; Phg, phenylglycine; PXE, polymyxin E;
rmsd, root-mean-square deviation; 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, triisopropylsilane; TOCSY, total correlated spectroscopy.
10.1021/jm800886n CCC: $40.75
2009 American Chemical Society
Published on Web 01/08/2009

Gramicidin S Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 665
Figure 2. Amino acids selected as D-Phe replacements for GS analogues 1-6.
Scheme 1. Synthetic Strategy for GS and Its Analoguesa
essential for an interaction of the peptide with lipid bilayers
with some degree of specificity. In addition, global hydropho-
bicity provided not only by the ꢀ-sheet27 but also by the
ꢀ-turn14-16,23,28 appears to be strongly related to the biological
properties of GS. The ꢀ-turn region of GS can be successfully
replaced by a variety of peptidomimetics, provided the sur-
rogateshavehydrophobiccharacterandadequategeometry.5,15,29-31
This paper decribes a GS analogue series generated by
replacing the D-Phe residue in both ꢀ-turn regions by hitherto
unexplored, Phe-related residues. Mutations at these two posi-
tions have received considerable attention14,23,32,33 as particularly
promising in the search for GS analogues with improved
pharmacological profiles. We have examined the antimicrobial
and hemolytic activity of these analogues along with their
secondary structures by NMR and found that, aside from the
ꢀ-sheet conformation, side chain packing appears to play an
important role in biological activity. Thus, the analogue with
the best antimicrobial-to-hemolytic activity ratio (peptide 6,
Figure 2) in the series displays rather distinctive packing
between its cationic (Orn) and aromatic (D-Tic) sites.
Results
Peptide Design and Synthesis. Amino acid residues selected
as D-Phe replacements in GS are shown in Figure 2. All of them
have D configuration and include various modifications at the
aromatic side chain such as insertion of a methylene group (Hpa,
analogue 1) or further aromatic rings in diverse arrangements
(analogues 2-5), including one (5) where the fluorenyl system
imposes coplanarity of the two phenyl rings, with presumable
conformational restriction. In a final analogue (6), we explored
the behavior of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
(Tic) in which a methylene unit connects the aromatic substituent
to the backbone.
a Reagents and conditions: (a) standard solid-phase peptide synthesis (Boc
chemistry); (b) TFA/TFMSA/TIS 10:1:1, rt, 90 min; (c) HBTU/HOBt/DIEA
(3:3:5 equiv), DMF, rt, 1 h; (d) 20% HCl in MeOH, 37 °C, 21 h. D-Phe*
stands for D-Phe or the corresponding substitute.
analogues each contain two Pro residues, minor NMR signals
from the cis conformation were observed, as expected in Pro-
containing peptides. Still, the trans-trans rotamers were
confirmed in all cases as the major species by the observation
of Pro 13Cꢀ-13Cγ chemical shift differences (∆δPro ) δCꢀ
-
δCγ, ppm) in the 5.0-5.9 ppm range characteristic of trans, vs
∆δPro ∼ 10 ppm for cis Pro residues.36
Solid-phase Boc chemistry protocols18,22 were used to prepare
GS and the six analogues in Figure 2. Noncommercially
available Boc-D-Flg-OH was obtained in enantiomerically pure
form following our previously reported methodology.34 In
addition, the side chain of N-Boc ornithine was protected as a
formamide as described by Kitagawa.35 For each peptide, the
synthetic route in Scheme 1 was followed. Once sequence
assembly was complete, the linear decapeptide was released
from the polymeric support by TFMSA acidolysis and submitted
to cyclization under high-dilution conditions. Deprotection of
the Orn side chains by acid hydrolysis furnished the target
peptides (GS and analogues 1-6), which were purified by
reversed-phase HPLC in satisfactory yields and high purity. All
peptides were further characterized by MS and NMR.
The first evidence that the analogues adopt the same structure
1
as GS comes from the similarity of their H, 13C, and 15N
chemical shifts with those of the parent peptide (Supporting
Information Table ST1). The significant differences (g0.2 ppm)
observed for some protons are explained by anisotropy effects
from the aromatic ring currents. Analogues 5 and 6 are those
1
with larger H chemical shift differences relative to GS, and
the only ones for which some 13C and 15N chemical shifts also
deviate considerably from GS (g1.0 ppm; Supporting Informa-
tion Table ST1).
Chemical shifts of CRH protons and CR carbons, given their
dependence on φ and ψ backbone torsion angles, are good indica-
tors of secondary structure formation. For GS (Figure 1), ∆δCRH
NMR Structural Studies. Peptides 1-6 were investigated
in aqueous solution by NMR in search for characteristic GS
traits such as ꢀ-sheet conformation. Because GS and all
observed
random coil
conformational shifts (∆δCRH ) δCRH
- δCRH
,
ppm) expected for ꢀ-strand residues Val1/1′, Orn2/2′, and

666 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Solanas et al.
Figure 3. Histograms showing the ∆δCRH and ∆δCR profiles (∆δ ) δobserved - δrandom coil, ppm) for GS (black bars) and its analogues (patterned or
colored bars as indicated in the corresponding insets) in aqueous solution at pH 3.0 and 5 °C. Lys δrandom coil values40 were used to calculate the ∆δ
values for the Orn residues.
Leu3/3′ are positive (∆δCRH ) +0.40 ppm on average in
very negative ∆δCR values displayed by Leu3/3′ in analogue 6
are probably due to the cyclic nature of D-Tic4/4′, leading to
effects equivalent to those described for Pro-preceding resi-
dues.40 In summary, these data confirm that peptides 1-4 and
6 in aqueous solution adopt the same backbone conformation
as GS. The resemblance is not so clear-cut in the case of
analogue 5, for which ∆δCRH values for other residues than Val1/
1′ also deviate from the expected behavior (Figure 3). Again,
deviations are attributable to aromatic ring current effects, which
in this particular peptide might differ from the rest, as the phenyl
ꢀ-sheets)37,38 and ∆δCR conformational shifts (∆δCR ) δCR
observed
random
- δCR
coil, ppm) negative (-1.6 ppm on average in
ꢀ-sheets)37. Turn residues were excluded from this analysis, as
no references are available for random coil values of D-Phe*4/
4′. The ∆δCRH and ∆δCR profiles of GS and peptides 1-4 and
6 are very similar and fit the expected pattern, except for Val1/
1′ (Figure 3), whose anomalous CRH chemical shifts can be
explained by the strong anisotropy of the aromatic ring, as
reported for some ꢀ-hairpin peptides.39 The larger ∆δCRH and

Gramicidin S Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 667
Table 1. Summary of NOEs Observed for GS and Peptides 1-6a
NOE
GS D-Phe
1 D-Hpa
2b D-1-Nal
3 D-2-Nal
4 D-Dip
5 D-Flg
6 D-Tic
NH Val1-NH Leu3′/ NH Val1′-NH Leu3
Val1/1′-Leu3/3′
+
+
+
+
+(vw)
Ov
+
+
+
+
+
Orn2/2′-DPhe*4/4′
+
+(vw)
Leu3/3′-DPhe*4/4′
DPhe*4/4′-Pro5/5′
+
+
+
+
+(w)
+
+
+
+
+
+
a Observation of one or more NOE involving at least one side chain proton of the indicated residues is shown by a “+”. D-Phe* stands for D-Phe or the
corresponding substitute. “Ov” indicates signal overlapping. “w” and “vw” indicates weak and very weak peak intensities, respectively. b Signal-to-noise
ratio is poor in NOESY spectra of peptide 2 as a consequence of its low solubility.
rings in D-Flg (Figure 2) are necessarily coplanar. Further
In either case, the different behavior of Val1/1′ vs Leu3/3′ NH
protons would arise from their proximity to the turn region
(Figure 1) and the ensuing disruption of the antiparallel ꢀ-sheet
regularity. Such deviation from canonical ꢀ-sheet for Val1/1′
residues would also account for their anomalous ∆δCRH and
∆δCR values (see above; Figure 3).
confirmation about the ꢀ-sheet conformation of residues 1/1′
3
to 3/3′ came from their large JCRH-NH coupling constants
(8.2-10.0 Hz; Supporting Information Table ST2). Also, the
3
small JCRH-NH coupling constants of residues 4/4′ corroborate
their involvement in a turn.
Indeed, the strongest evidence about the conformational
features of the peptides came from NOE data. The GS ꢀ-sheet
structure (Figure 1) is characterized by three NOEs involving
backbone protons CRH Orn2-CRH Orn2′ (distance in a canoni-
cal antiparallel ꢀ-sheet: 2.2 Å), NH Val1-NH Leu3′, and NH
Val1′-NH Leu3 (distance in a canonical antiparallel ꢀ-sheet:
3.3 Å). Because GS and peptides 1-6 are symmetrical, the first
NOE is not observable, while the last two must overlap into a
single cross-peak corresponding to both NOEs. Such a cross-
peak is present in the NOESY spectra of GS and peptides 1, 3,
and 6 (Table 1), demonstrating that the three analogues have
GS-like ꢀ-sheet backbones. The failure to detect such a NOE
in 4 and 5 precludes its use as a proof of ꢀ-sheet backbone
structure, but does not necessarily exclude the presence of this
conformational trait.
Aside from NOEs involving backbone protons, NOEs be-
tween Val and Leu side chain protons were observed for GS
and analogues (Table 1 and Figure 4), except for 2, probably
due to low solubility. Because Val and Leu side chains pack
together on the same face of the GS ꢀ-sheet, these NOEs provide
additional proof of the peptides adopting GS-like ꢀ-sheet
structures.
The cross-strand hydrogen bonding network in the ꢀ-sheet
(NH Val1-O Leu3′, NH Val1′-O Leu3, NH Leu3-O Val1′,
and NH Leu3′-O Val1; see Figure 1) can be also examined by
NMR. Experimental evidence for the involvement of NH amide
protons in intramolecular hydrogen bonds can be obtained from
NH/ND exchange rates and from temperature coefficients of
the amide protons (∆δ/∆T). The latter are easier to measure
and have long been recognized as related to solvent exposure.
Accessible amide protons usually have ∆δ/∆T values within
the -6.0 to -9.5 ppb K-1 range, while values above -4.5 ppb
K-1 indicate low solvent accessibility and hence involvement
in intramolecular hydrogen bonds. As expected, solvent-exposed
NH amide protons of Orn2/2′ and D-Phe*4/4′ displayed tem-
perature coefficients below -6.5 ppb K-1, with peptide 5 as
single exception (Table 2). More interestingly, the low ∆δ/∆T
absolute values for the NH amide protons of Leu3/3′ in all
peptides, as well as for the NH of Val1/1′ in 6, suggested
participation in intramolecular hydrogen bonds, as expected for
GS. In contrast, Val1/1′ NH amide protons in both GS and
peptides 1-5 had ∆δ/∆T values in the range typical of solvent-
accessible protons, with values larger (in absolute value) than
the -9.5 ppb K-1 limit for peptides 3-5. Two explanations
for this behavior can be given: either (i) hydrogen bonding in
Val1/1′ NH is nonexistent or weaker than that of the NH of
Leu3/3′, or (ii) anisotropy effects from D-Phe4/4′ (or D-Phe*4/
4′) affect the Val1/1′ NH chemical shifts in both GS and 1-5.
To discern between the two alternatives, the NH/ND exchange
rates, which provide more definitive evidence about solvent
protection and are unaffected by aromatic ring currents, were
measured. Thus, the amide NH signals of Orn2/2′ and D-Phe4/
4′ in GS at 5 °C disappeared shortly after dissolving the sample
in D2O (see Experimental Section), indicating fast solvent
exchange. In contrast, NH protons of Val1/1′ and Leu3/3′ were
slow-exchanging, their NMR signals remaining after >18 h.
This is in agreement with the NH protons of both Val1/1′ and
Leu3/3′ being hydrogen-bonded and thus suggests that the large
∆δ/∆T absolute values observed for Val1/1′ NH protons are
due to magnetic anisotropy. Their variability among GS and
the six analogues stems from their distinctive D-Phe*4/4′
residues, each giving rise to characteristic ring current effects.
In this regard, the fact that the ∆δ/∆T coefficient for Val1/1′ in
peptide 6 has the expected value for hydrogen-bonded amide
protons suggests that the side chain of D-Tic freezes the phenyl
ring in an orientation that minimizes its anisotropy effects over
the Val1/1′ NH proton.
Analogue 5, for which the Val1/1′ temperature coefficient is
the most negative (-23.4 ppb K-1; Table 2), and that of Orn2/
2′ less negative than expected for an exposed amide proton,
merits further comment (Table 2 and Figure 1). The most likely
explanation is that the anisotropy effects of the large, rigid
aromatic D-Flg side chain strongly influence the chemical shifts
of both NH protons. This is supported by the fact that the
remaining analogues show very similar chemical shifts for the
NH protons of Val1/1′ and Orn2/2′ (7.52-7.82 and 8.64-8.74
ppm, respectively; Supporting Information Table ST1), while
those of 5 are quite different (8.25 and 8.31 ppm, respectively).
Having shown that analogues 1-6 adopt the same ꢀ-sheet
backbone structure as the parent GS (perhaps a bit distorted for
peptide 5), we next investigated side chain packing, which is
responsible for the polar/hydrophobic distribution seemingly
essential for membrane interaction and hence antimicrobial and
hemolytic activity. A qualitative examination of NOEs from
D-Phe*4/4′ (Table 1 and Figure 4) indicates that the aromatic
rings interact with the adjacent Leu and Pro residues, both in
GS and in 1-5. For analogue 6, in contrast, NOE data suggest
that the aromatic rings interact with Orn2/2′ and retain the
proximity to Leu3/3′ but not to Pro5/5′. This is confirmed by
large chemical shift differences (relative to GS) of some protons
of Orn2/2′ (0.4-0.6 ppm; Supporting Information Table ST1)
and of Pro5/5′ (1.18 and 0.33 ppm for the Cδδ′H protons;
Supporting Information le ST1), while for the side chain protons
of Leu3/3′, the differences do not exceed 0.22 ppm. Proximity
between the D-Tic and Orn side chains may be explained by a
π-cation interaction.

668 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Solanas et al.
Figure 4. Selected NOESY spectral regions for peptides 1 (A) and 6 (B) in H2O/D2O 9:1 v/v at pH 3 and 5 °C. Relevant sequential and nonsequential
NOEs are boxed.

Gramicidin S Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 669
Table 2. Temperature Coefficientsa (∆δ/∆T, ppb ·K-1) for Amide
against both PXE-sensitive and resistant strains, suggesting it
is impervious to modifications in lipopolysacharide (LPS)
structure.
Protons in GS and Its Analogues
peptide D-Phe*b Val1,1′(NH) Orn2,2′(NH) Leu3,3′(NH) D-Phe*4,4′(NH)
The hemolytic effect of the peptides was evaluated on sheep
erythrocytes (Table 3) as an indicator of toxicity against
mammalian cells. In this assay, five (1-5) out of six analogues
turned out to be more toxic than the parent GS. The finding
that the D-Flg analogue (5) was more cytotoxic and yet less
potent than the parent antibiotic underscores the fact that subtle
structural changes in GS may translate into different abilities
to damage prokaryotic and eukaryotic cells. An opposite, though
similar and more promising, segregation of antibacterial and
hemolytic activities was found for 6, which has GS-like MIC50
values for Gram-positives (Table 3) but is essentially harmless
toward erythrocytes.
Beyond adequate intrinsic microbicidal activity, a convenient
assessment of the clinical potential of an antibiotic is provided
by its therapeutic index (TI, defined as HC50/MIC50, see Table
3), where the cytotoxic and microbicidal effects are combined.
As found in previous SAR studies on GS,23,25,42,43 improved
TIs are more often due to a decrease in cytotoxicity than to an
increase in antibiotic potency. In the present series, the above-
mentioned (higher than for GS) toxicity values, coupled with
the minute, if any, improvements in microbicidal activity,
resulted in TI values slightly lower or clearly worse than that
of the parent structure. The sole exception to this pattern was
the D-Tic analogue (6), for which a modest but significant
improvement in TI was obtained (Table 3).
GS
1
2
3
4
5
6
D-Phe
D-Hpa
D-1-Nal
D-2-Nal
D-Dip
D-Flg
-7.4
-6.1
-7.1
-7.8
-7.0
-6.6
-7.3
-3.6
-8.0
-0.8
-1.2
-1.5
0.2
-1.7
0.0
-8.6
-11.0
-8.0
-7.6
-10.1
c
-8.0
-11.4
-13.4
-23.4
-1.3
D-Tic
-3.9
b
a Measured in H2O/D2O 9:1 (v/v) at pH 3.0. D-Phe* stands for D-Phe
or the corresponding substitute. c Not determined; NH protons not observed
at 25 °C probably because of fast exchange with water.
To further visualize the side chain packing of peptides 1-6
relative to GS, structure calculations were performed. Because
of the symmetry of GS and analogues, every observed NOE
has four possible assignments. However, after our NMR analysis
demonstrated that the peptides adopt the GS ꢀ-sheet structure
(Figure 1), some NOE ambiguities could be solved. Thus the
restraints used for structure calculation combined (i) theoretical
restraints for the backbone of strand residues Val1/1′, Orn2/2′,
and Leu3/3′ derived from those expected for the GS ꢀ-sheet
(Figure 1; more details in the Experimental Section and the
Supporting Information) and (ii) experimental distance restraints
derived from the complete set of NOEs observed for each
peptide. For every nonintraresidual NOE, all the existing
possibilities were taken into account, and the NOE assignments
compatible with the GS ꢀ-sheet were identified by iterative
structure calculations. The final model structures obtained for
GS and peptides 1-6 (Figures 5 and Supporting Information
Figure SF3) are well defined, particularly for 6 (see rmsd values
in Supporting Information Table ST3). A general examination
of the structures corroborates the main conformational traits
deduced from the qualitative analysis of NMR parameters.
Concerning the orientation of the aromatic side chain at positions
4/4′, the phenyl rings in 1 are quite disordered and explore a
much wider space than in GS, while the aromatic systems of
peptides 4 and 5 adopt quite well-defined positions. Also
noteworthy is the different relative orientation of the two phenyl
rings in D-Flg and D-Dip, in the latter case with the rings almost
perpendicular to each other, and one in an orientation close to
that of the D-Phe ring in GS (Supporting Information Figure
SF3). Even more remarkable are the differences between GS
and analogue 6 (Figure 5) in the relative orientation of the
aromatic rings and of the positively charged Orn side chains,
with noticeable consequences on their respective biological
activities (see below).
Biological Activity. Microbicidal activities were determined
in solution because, in solid media, they tend to be undermea-
sured, especially for Gram-negatives.4 Two Gram-positive
(Staphylococcus aureus and Listeria monocytogenes) and a
Gram-negative (Acinetobacter baummanii ATCC 19606 and its
isogenic strain resistant to polymyxin E (PXE)) bacterial strains
were tested. Although GS is mostly active against Gram-
positives, the occurrence of cutaneous infection by A. baumannii
and the increasing resistance to PXE, the last universal drug
active against these bacteria,41 prompted us to include both of
them in the panel. Results are listed in Table 3.
Within the Gram-positive group, the tested set of analogues
had better and comparable activities, respectively, for Staphy-
lococcus and Listeria than the parental GS. In contrast, against
the Gram-negative A. baumannii, activity in the analogue series
tended to deteriorate relative to GS, more markedly so for the
isogenic PXE-resistant strain. The sole exception was the D-Hpa-
containing peptide 1, which performed slightly better than GS
Discussion
The type II′ ꢀ-turn is a structural motif especially suited to
induce alignment of its two side sequence segments into an
antiparallel ꢀ-sheet. The quintessential example of this situation
is GS, whose type II′ ꢀ-turns have been a testing ground for
extensive D-Phe-Pro replacements by a variety of peptidomi-
metics, with variable success (see Introduction and refs 29-31,
44-46). In comparison, fewer studies have addressed the
substitution of either L-Pro17,47 or D-Phe33,46,48-50 by appropriate
surrogates. The goal of this work was to explore and structurally
interpret the fine-tuning of GS antibiotic activity by replacement
of D-Phe with a novel set of noncoded aromatic D-amino acids
already tested as turn-promoters.
The activity of GS is assumed to be somehow related to the
permeabilization of bacterial membranes, although no unified
opinion on the nature of such process exists.51 Hydrophobicity
has been cited as a relevant parameter,25,42,43 and inspection of
the current set of D-Phe analogues (Figure 2) shows considerable
variation in the number and size of aromatic side chains, hence
in overall hydrophobicity at this position. The presence of a
sizable hydrophobic domain is closely related to hemolytic
activity in antimicrobial peptides, as it seemingly promotes a
higher partition of the peptide into biological membranes
regardless of their zwitterionic or anionic character.24,25,42
Accordingly, the hemolytic and Gram-positive microbicidal
activities run somewhat parallel, provided hydrophobicity does
not drastically reduce peptide solubility in aqueous medium.52
For Gram-negatives, however, the situation changes in that an
external barrier, i.e., the outer membrane (OM), must be
disrupted prior to the “lethal hit” i.e., permeabilization of the
inner membrane (IM). In this scenario, while high hydrophobic-
ity improves the affinity of the antibiotic peptide for LPS (the
major component of the OM), excess affinity may sequester
the peptide into the OM, hence precluding its crucial interaction
with the IM target. Therefore, a rather subtle balance between

670 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Solanas et al.
Figure 5. Model structures for GS (A) and peptide 6 (B) in two different views. Backbone atoms are shown in black. Side chains for aromatic
D-Phe and D-Tic are colored in green, for Pro in cyan, for Orn in blue, and for Leu and Val in magenta.
Table 3. Cytotoxic (Hemolytic) and Antimicrobial Activity of GS and Analogues
erythrocytes
S. aureus
L. monocytogenes
A. baumanni S
A. baumannii Re
b
c
peptide
D-Phe*a
HC50 (µM)
MIC50 (µM)
TId
MIC50 (µM)
TI
MIC50 (µM)
TI
MIC50 (µM)
TI
GS
1
2
3
4
5
6
D-Phe
D-Hpa
D-1-Nal
D-2-Nal
D-Dip
D-Flg
21.1 ((2.6)
7.0 ((0.3)
5.0 ((0.1)
7.1 ((3.5)
8.6 ((1.1)
6.2 ((0.3)
>50
7.9 ((0.8)
3.5 ((0.1)
2.9 ((0.5)
4.6 ((0.6)
3.4 ((0.0)
8 ((0.0)
2.7 (1)
2 (0.7)
6.7 ((0.2)
2.4 ((0.0)
8.6 ((0.6)
7.8 ((0.4)
7.7((0.6)
9.2((1.4)
8.3 ((0.5)
3.1 (1)
10.1 ((0.4)
4.6 ((0.1)
35.9 ((0.0)
39.6 ((0.0)
9.6 ((0.9)
15.4 ((1.3)
>40
2.1 (1)
13.1 ((0.0)
6.2 ((0.1)
>40
1.6 (1)
1.1 (0.7)
>0.1
2.9 (0.9)
0.6 (0.2)
0.9 (0.3)
1.1 (0.4)
0.7 (0.2)
>6 (2.0)
1.5 (0.7)
0.1 (0.0)
0.2 (0.1)
0.9 (0.4)
0.4 (0.2)
>ND
1.7 (0.6)
1.5 (0.6)
2.5 (0.9)
0.8 (0.3)
>10.2 (3.8)
>40
>0.2
17.8 ((0.0)
>40
>40
0.5 (0.3)
>0.1
ND
D-Tic
4.9 ((0.4)
a
D-Phe* stands for D-Phe or the corresponding substitute. b HC50: peptide concentration required for 50% lysis of sheep erythrocytes. c MIC50: peptide
concentration required for 50% inhibition of bacterial growth after 24 h, relative to a control culture. d TI (therapeutic index) ) HC50/MIC50. e An A.
baumannii strain resistant to polymyxin E (colistin). ND, not determined. Standard deviations (SD) are shown in parentheses.
hydrophobicity and cationic character is desirable for activity
against Gram-negatives.18 These considerations broadly agree
with the finding that peptides 1-5, more toxic than GS toward
erythrocytes (Table 3), are also more hydrophobic than GS by
an accepted criterion,23 namely RP-HPLC retention times
consistently longer than those of the parent peptide (see
Supporting Information, Figure SF1). On the other hand, the
D-Tic (6) analogue combines decreased cytotoxicity with higher
hydrophobicity (longer HPLC retention time) than GS (Table
3). Likewise, the similar HPLC retention times for peptides 1-4
(10.3-10.6 min, Supporting Information Figure SF1) do not
correlate well with their antimicrobial profiles. Thus, while 1
improved on GS against all bacteria tested, Nal-containing
analogues 2 and 3, with presumably comparable hydrophobicity,
were similar to GS against Gram-positives but practically
inactive against Gram-negatives. In conclusion, it appears that,
at least for this series, hydrophobicity has limited value in
explicating antibiotic performance.
consequently a wider repertoire of membrane (either prokaryotic
or eukaryotic) interaction modes than the D-Phe-containing GS,
thus explaining its increased activity toward both bacterial and
mammalian cells. In contrast, the naphthalene rings in 2 and 3
have conformational freedoms similar in principle to the phenyl
group of GS but their bulkiness is likely to pose a limitation on
their interaction with LPS and ensuing OM disruption in Gram-
negative bacteria. For Gram-positives, on the other hand, no
comparable tightly packed external barrier exists, which would
agree with both the preservation of antibiotic activity in both
analogues as well as their hemolytic properties. Similar con-
siderations on the orientation and/or rotational freedom of the
aromatic rings can be applied to the contrasting activities of 4
(D-Dip) and 5 (D-Flg). In the latter analogue, the two aromatic
rings are fastened into a bulky planar tricylic system, whose
predictable sluggishness may help to explain its significantly
worse profile than 4.
By far the most striking result in the present series is the
improved therapeutic profile of the D-Tic analogue 6. With an
apparent hydrophobicity (based on RP-HPLC retention time)
slightly higher than GS, it is basically equipotent to GS against
Gram-positives but has considerably lower toxicity, hence a
much better TI (Table 3). From the structural point of view, 6
has two distinctive features over the other analogues in the
series: first, the severe conformational restriction on the (proline-
NMR results shed additional light on the structure-activity
analysis of the peptides. In particular, they reveal a varying
degree of conformational freedom in their side chains that can
be reasonably related to differences in antimicrobial activity.
For instance, the aromatic ring of the D-Hpa-containing analogue
(1), separated from the backbone by an extra methylene group,
can plausibly explore a larger conformational space and

Gramicidin S Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 671
was purchased from Bachem (Bubendorf, Switzerland) and its side
chain amino group was protected as a formamide by reaction with
formic acid and 1,1′-oxalyldiimidazole.35 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
Innovation (Montreal, Quebec). Solvents and other chemicals were
from SDS (Peypin, France). Mass determination by the MALDI-
TOF technique was done in a Voyager DE-RP spectrometer
(Applied Biosystems, Foster City, CA) using 2,5-dihydroxybenzoic
acid as a matrix. 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 (+0.036% TFA, v/v) into H2O (+0.045% TFA, v/v) over
15 min at 1 mL/min flow rate, with UV detection at 220 nm.
Preparative RP-HPLC purification was done on a Phenomenex Luna
C8 column (10 µm, 250 mm × 10 mm) running linear gradients of
CH3CN (+0.1% TFA, v/v) into H2O (+0.1% 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-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 coupling of the 4/4′ residues. To avoid diketopiperazine
formation, the third amino acid of each sequence was incorporated
by the in situ neutralization method.57 Coupling and deprotection
reactions were monitored by the nynhidrin58 or p-nitrophenylester59
colorimetric 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. The residue was dissolved in
DMF to a final concentration of 2 mg/mL and stirred for 1 h at
room temperature in the presence of HBTU/HOBt/DIEA (3:3:5
equiv). After solvent removal and lyophilization, the cyclized
peptide was deformylated 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 by preparative reversed-
phase HPLC as indicated in each case. HPLC-homogeneous
fractions were combined and lyophilized to give white powders of
g99% HPLC purity (see Supporting Information, Figure SF2).
cyclo(Val-Orn-Leu-D-Phe-Pro)2 (GS). RP-HPLC, linear 35-70%
CH3CN gradient into H2O for 30 min (33 mg, 0.029 mmol, 25%
like) imino acid D-Tic imposed by the cyclization between the
phenyl group and the backbone and borne out by the observation
of practically a single conformer by NMR. Interestingly, the
hydrogen-bonding interaction between the δ-amino group of
Orn and the backbone carbonyl of D-Phe reported for GS and
some analogues53,54 would thus not be present in analogue 6.
The absence of this favorable effect would most likely be
compensated by the stabilizing contribution that the increased
rigidity of D-Tic -compared with D-Phe- imparts to the ꢀ-turn
structure. As a rigid side chain is conceivably more difficult to
insert into lipid bilayers than a flexible one (i.e., that of GS),
this could account for the lack of hemolytic effect of 6 and
+
thus its improved antibiotic profile. Second, in 6, the δ-NH3
groups of Orn are positioned close to the aromatic ring of D-Tic,
possibly favored by a π-cation interaction. According to NMR
and MD studies on the membrane-bound structure of GS,43,55,56
the peptide lies flat relative to the bilayer plane, near the interface
+
between polar and apolar regions, so that the δ-NH3 groups
of Orn can interact with phospholipid polar head groups. Now,
if a similar contact model is assumed for analogue 6, the
aforementioned π-cation interaction between D-Tic and Orn
must disappear. The thermodynamic penalty thus incurred would
be higher for the interaction with (zwitterionic) eukaryotic than
for prokaryotic membranes, where the loss of the D-Tic-Orn
interaction would be compensated by electrostatic attraction
+
between Orn δ-NH3 and phosphate head groups. Again, this
may account for the higher relative loss in hemolytic than in
bactericidal effect.
Conclusions
Analogues of the antimicrobial peptide GS with different
aromatic replacements at both D-Phe residues have been
synthesized in order to explore how the conformational freedom
and the size of the aromatic moiety modulate both biological
and structural properties. NMR studies confirmed that the overall
molecular shape of the GS framework is maintained in all
analogues (some distortion cannot be discarded for the D-Flg
derivative), so that variations in antibiotic profile can be ascribed
to the different D-Phe replacements. The results suggest that
both bulkiness and orientation of the aromatic system at the
4/4′ position are essential for biological activity, to the point
that slight modifications in these parameters bring about
significant changes in antibacterial activity as well as in the
desirable specificity for bacterial over mammalian cells. An
interesting finding is that analogue 6, with D-Tic replacing D-Phe,
retains antibiotic potency against Gram-positive bacteria but is
hardly cytotoxic. Our NOE data clearly indicate that in this
analogue the Orn side chain orients toward the D-Tic aromatic
rings. A plausible explanation for this orientation is the existence
overall yield). [M + H]+
) 1141.7; [M + H]+ ) 1141.1.
calcd
obs
cyclo(Val-Orn-Leu-D-Hpa-Pro)2 (1). RP-HPLC, linear 45-75%
CH3CN gradient into H2O for 30 min (51 mg, 0.044 mmol, 41%
overall yield). [M + H]+
) 1169.7; [M + H]+ ) 1169.5.
calcd
obs
+
of a cation-π interaction between the δ-NH3 groups of the
cyclo(Val-Orn-Leu-D-1-Nal-Pro)2 (2). RP-HPLC, linear 45-75%
Orn side chain and the aromatic ring of D-Tic. In any event,
and regardless of the nature of the interaction, the specific
orientation of the D-Tic aromatic ring relative to the Orn side
chain allows explanation of the distinct biological behavior
exhibited by this peptide. While the increase in therapeutic index
relative to GS is only 5-fold, it is nonetheless remarkable
because, in contrast to other studies,25,42 it has been achieved
with minimal alteration of the GS structure, including chirality
and cycle size. Modifications of this type may be helpful in
understanding the mechanism of action of GS and developing
peptide antibiotics with improved pharmaceutical applications.
CH3CN gradient into H2O for 30 min (34 mg, 0.027 mmol, 26%
overall yield). [M + H]+
) 1241.7; [M + H]+ ) 1241.8.
calcd
obs
cyclo(Val-Orn-Leu-D-2-Nal-Pro)2 (3). RP-HPLC, linear 45-75%
CH3CN gradient into H2O for 30 min (26 mg, 0.021 mmol, 20%
overall yield). [M + H]+
) 1241.7; [M + H]+ ) 1241.6.
calcd
obs
cyclo(Val-Orn-Leu-D-Dip-Pro)2 (4). RP-HPLC, linear 50-80%
CH3CN gradient into H2O for 30 min (22 mg, 0.017 mmol, 15%
overall yield). [M + H]+
) 1293.8; [M + H]+ ) 1293.6.
calcd
obs
cyclo(Val-Orn-Leu-D-Flg-Pro)2 (5). RP-HPLC, linear 55-85%
CH3CN gradient into H2O for 30 min (18 mg, 0.015 mmol, 14%
overall yield). [M + H]+
) 1289.7; [M + H]+ ) 1289.8).
calcd
obs
cyclo(Val-Orn-Leu-D-Tic-Pro)2 (6). RP-HPLC, linear 45-65%
CH3CN gradient into H2O for 30 min (23 mg, 0.020 mmol, 19%
Experimental Section
overall yield). [M + H]+
) 1165.7; [M + H]+ ) 1165.8.
calcd
obs
Chemicals and Instrumentation for Peptide Synthesis. Fluo-
renylglycine was prepared as previously reported.34 N-Boc ornithine
NMR Spectroscopy. Samples for NMR experiments were
prepared at 1-2 mM peptide concentration in 0.5 mL of H2O/D2O

672 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Solanas et al.
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 spectrometer
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 60 and 150 ms,
was measured by turbidimetry at 600 nm in a model 680 microplate
reader (Bio-Rad Laboratories, Hercules. CA). MIC50 was defined
as the lowest peptide concentration inhibiting bacterial growth by
50%, relative to untreated control, and was calculated using the
SigmaPlot (Systat Software, San Jose, CA) software, v. 9.0.
Hemolytic Activity Assay. Hemolytic activity of the peptides
was determined by triplicate. Defibrinated sheep blood (Biomedics,
Madrid, Spain) was centrifuged 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 erythrocytes 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, and hemoglobin release was read 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.
60
respectively. The H-13C and H-15N HSQC spectra 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) software. Sodium 2,2-dimethyl-
2-silapentane-5-sulfonate (DSS) was used as an internal reference
for 1H chemical shifts. The 13C and 15N chemical shifts were
indirectly calibrated by multiplying the spectrometer frequency that
corresponds to 0 ppm in the 1H spectrum, assigned to internal DSS
reference, by 0.25144954 and 0.101329118, respectively.61
The 1H NMR signals of the peptides were assigned by sequential
assignment methods.62 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.
To measure the NH/ND exchange rates, a freeze-dried sample
of peptide GS was solved in pure D2O at pH 3.0, and after the
10-15 min required to set up the NMR experiment, consecutive
1D and 2D 20 ms-TOCSY spectra were recorded at 5 °C.
Structure Calculation. Structure calculations were performed
by using the CYANA program63 and an annealing strategy. Since
the nonproteinogenic amino acids were not included in the standard
CYANA libraries, we built them using MOLMOL64 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 retrieved from the Cambridge Structural
Database.65 Theoretical constraints for the GS ꢀ-sheet incorporated
for structure calculation included φ and ψ angle restraints, lower
and upper-limit distance restraints for the four characteristic cross-
strand hydrogen-bonds, and upper-limit distance restraints for the
backbone atoms of strand residues (Supporting Information Table
ST4). Experimental distance constraints were derived from 2D
NOESY spectra recorded in H2O, also in D2O for analogue 6. 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.63 Given
the symmetrical nature of the peptides, for structure calculations
residues were renumbered from 1 to 10 starting at Leu3 (Figure
1). Lower and upper limit restraints required for peptide backbone
cyclization were also introduced (see Supporting Information). For
each peptide, a total of 50 conformers were generated, and the 20
conformers with the lowest target function were analyzed. Model
structures for GS and analogues 1-6 were examined with MOL-
MOL. 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°.
1
1
Acknowledgment. This work was supported by Ministerio
de Educacio´n y Ciencia (BIO2005-07592-CO2-02 to D.A.,
BFU2005-01855 and CTQ2008-0080 to M.A.J., CTQ2007-
62245 to C.C.), Fondo de Investigaciones Sanitarias (PI061125
and RD 06/0021/0006 to L.R., PI040885 to D.A.), by the
regional governments of Arago´n (research group E40), Cata-
lunya (SGR2005-00494), and Madrid (S-BIO-0260/2006). This
project has been funded in whole or in part with Federal funds
from the National Cancer Institute, National Institutes of Health,
under contract N01-CO-12400. C.S. and C.M.S. thank Minis-
terio de Educacio´n y Ciencia and Consejo Superior de Inves-
tigaciones Cient´ıficas-European Social Fund for an FPU and
I3P fellowship, respectively. 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.
Supporting Information Available: NMR data, details on
structure calculations, and analytical data on GS and analogues 1-6.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Hirsch, T.; Jacobsen, F.; Steinau, H. U.; Steinstraesser, L. Host defense
peptides and the new line of defence against multiresistant infections.
Protein Pept. Lett. 2008, 15, 238–243.
(2) Parisien, A.; Allain, B.; Zhang, J.; Mandeville, R.; Lan, C. Q. Novel
alternatives to antibiotics: bacteriophages, bacterial cell wall hydrolases,
and antimicrobial peptides. J. Appl. Microbiol. 2008, 104, 1–13.
(3) Gause, G. F. Gramicidin S and its use in the treatment of infected
wounds. Nature 1944, 154, 703.
(4) Kondejewski, L. H.; Farmer, S. W.; Wishart, D. S.; Hancock, R. E.;
Hodges, R. S. Gramicidin S is active against both gram-positive and
gram-negative bacteria. Int. J. Pept. Protein. Res. 1996, 47, 460–466.
(5) Prenner, E. J.; Lewis, R. N.; McElhaney, R. N. The interaction of the
antimicrobial peptide gramicidin S with lipid bilayer model and
biological membranes. Biochim. Biophys. Acta 1999, 1462, 201–221.
(6) Hull, S. E.; Karlsson, R.; Main, P.; Woolfson, M. M.; Dodson, E. J.
The crystal structure of a hydrated gramicidin S-urea complex. Nature
1978, 275, 206–207.
(7) Schmidt, G. M.; Hodgkin, D. C.; Oughton, B. M. A crystallographic
study of some derivatives of gramicidin S. Biochem. J. 1957, 65, 744–
750.
(8) Tishchenko, G. N.; Andrianov, V. I.; Vainstein, B. K.; Woolfson,
M. M.; Dodson, E. Channels in the gramicidin S-with-urea structure
and their possible relation to transmembrane ion transport. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 151–159.
(9) Ovchinnikov, Y. A.; Ivanov, V. T. Conformational states and biological
activity of cyclic peptides. Tetrahedron 1975, 31, 2177–2209.
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 concen-
tration) 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.41
Bacterial cells were harvested at exponential growth phase, washed
twice with phosphate buffered saline (PBS, 10 mM Na2HPO4, 1
mM KH2PO4, 140 mM NaCl, 3 mM KCl, pH 7.0) and resuspended
in MBH, at 5 × 105 CFU/mL. Aliquots (100 µL) from this
suspension 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 analogue concentration. Afterward, growth

Gramicidin S Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 673
(10) Katsu, T.; Kobayashi, H.; Fujita, Y. Mode of action of gramicidin S
on Escherichia coli membrane. Biochim. Biophys. Acta 1986, 860,
608–619.
(11) Afonin, S.; Glaser, R. W.; Berditchevskaia, M.; Wadhwani, P.; Guhrs,
K. H.; Mollmann, U.; Perner, A.; Ulrich, A. S. 4-Fluorophenylglycine
as a label for 19F NMR structure analysis of membrane-associated
peptides. ChemBioChem 2003, 4, 1151–1163.
(31) Xiao, J.; Weisblum, B.; Wipf, P. Trisubstituted (E)-alkene dipeptide
isosteres as beta-turn promoters in the gramicidin S cyclodecapeptide
scaffold. Org. Lett. 2006, 8, 4731–4734.
(32) Grotenbreg, G. M.; Spalburg, E.; de Neeling, A. J.; van der Marel,
G. A.; Overkleeft, H. S.; van Boom, J. H.; Overhand, M. Synthesis
and biological evaluation of novel turn-modified gramicidin S
analogues. Bioorg. Med. Chem. 2003, 11, 2835–2841.
(12) Arai, T.; Imachi, T.; Kato, T.; Ogawa, H. I.; Fujimoto, T.; Nishino,
N. Synthesis of [hexafluorovalyl1,1′]gramicidin S. Bull. Chem. Soc.
Jpn. 1996, 69, 1383–1389.
(13) Waki, M.; Abe, O.; Okawa, R.; Kato, T.; Makisumi, S.; Izumiya, N.
Studies of peptide antibiotics. XII. Syntheses of [2,2′-R,γ-diaminobu-
tyric acid] and [2,2′-lysine]-gramicidin S. Bull. Chem. Soc. Jpn. 1967,
40, 2904–2909.
(14) Aimoto, S. The synthesis of a heavy-atom derivative of gramicidin S
(GS), [D-Phe(4-Br) 4,4′]-GS, by a novel method. Bull. Chem. Soc. Jpn.
1988, 61, 2220–2222.
(15) Andreu, D.; Ruiz, S.; Carren˜o, C.; Alsina, J.; Albericio, F.; Jime´nez,
M. A.; de la Figuera, N.; Herranz, R.; Garc´ıa-Lo´pez, M. T.; Gonza´lez-
Mun˜iz, R. IBTM-containing gramicidin S analogues: Evidence for
IBTM as a suitable type II′ ꢀ-turn mimetic. J. Am. Chem. Soc. 1997,
119, 10579–10586.
(33) Shimohigashi, Y.; Kodama, H.; Imazu, S.; Horimoto, H.; Sakaguchi,
K.; Waki, M.; Uchida, H.; Kondo, M.; Kato, T.; Izumiya, N. [4,4′-
(Z)-dehydrophenylalanine]gramicidin S with stabilized bioactive con-
formation and strong antimicrobial activity. FEBS Lett. 1987, 222,
251–255.
(34) Royo, S.; Jime´nez, A. I.; Cativiela, C. Synthesis of enantiomerically
pure ꢀ,ꢀ-diphenylalanine (Dip) and fluorenylglycine (Flg). Tetrahe-
dron: Asymmetry 2006, 17, 2393–2400.
(35) Kitagawa, T.; Arita, J.; Nogahata, A. Convenient one-pot method for
formylation of amines and alcohols using formic acid and 1,1′-
oxalyldiimidazole. Chem. Pharm. Bull. (Tokyo) 1994, 42, 1655–1657.
(36) Schubert, M.; Labudde, D.; Oschkinat, H.; Schmieder, P. A software
tool for the prediction of Xaa-Pro peptide bond conformations in
proteins based on 13C chemical shift statistics. J. Biomol. NMR 2002,
24, 149–154.
(37) Santiveri, C. M.; Rico, M.; Jime´nez, M. A. 13CR and 13Cꢀ chemical
shifts as a tool to delineate ꢀ-hairpin structures in peptides. J. Biomol.
NMR 2001, 19, 331–345.
(38) Wishart, D. S.; Sykes, B. D.; Richards, F. M. Relationship between
nuclear magnetic resonance chemical shift and protein secondary
structure. J. Mol. Biol. 1991, 222, 311–333.
(16) 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. ꢀ-Turn modified
gramicidin S analogues containing arylated sugar amino acids display
antimicrobial and hemolytic activity comparable to the natural product.
J. Am. Chem. Soc. 2006, 128, 7559–7565.
(17) 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 hemolytic activity. J.
Pept. Res. 2005, 65, 98–104.
(18) Lee, D. L.; Hodges, R. S. Structure-activity relationships of de novo
designed cyclic antimicrobial peptides based on gramicidin S. Biopoly-
mers 2003, 71, 28–48.
(19) Ripka, W. C.; Delucca, G. V.; Bach, A. C.; Pottorf, R. S.; Blaney,
J. M. Protein ꢀ-turn mimetics II: design, synthesis and evaluation in
the cyclic peptide gramicidin S. Tetrahedron 1993, 49, 3609–3628.
(20) Sato, K.; Kato, R.; Nagai, U. Studies on ꢀ-turn of peptides. XII.
Synthetic conformation of weak activity of [D-Pro5,5′]-gramicidin S
predicted from ꢀ-turn preference of its partial sequence. Bull. Chem.
Soc. Jpn. 1986, 59, 535–538.
(21) Tamaki, M.; Okitsu, T.; Araki, M.; Sakamoto, H.; Takimoto, M.;
Muramatsu, I. Synthesis and properties of gramicidin S analogs
containing Pro-D-Phe sequence in place of D-Phe-Pro sequence in the
ꢀ-turn part of the antibiotic. Bull. Chem. Soc. Jpn. 1985, 58, 531–
535.
(39) Santiveri, C. M.; Rico, M.; Jime´nez, M. A. Position effect of cross-
strand side chain interactions on beta-hairpin formation. Protein Sci.
2000, 9, 2151–2160.
(40) Wishart, D. S.; Bigam, C. G.; Holm, A.; Hodges, R. S.; Sykes, B. D.
1H, 13C, and 15N random coil NMR chemical shifts of the common
amino acids. I. Investigations of nearest-neighbor effects. J. Biomol.
NMR 1995, 5, 67–81.
(41) Saugar, J. M.; Rodr´ıguez-Herna´ndez, M. J.; de la Torre, B. G.; Pacho´n-
Iba´n˜ez, M. E.; Ferna´ndez-Reyes, M.; Andreu, D.; Pacho´n, J.; Rivas,
L. Activity of cecropin A-melittin hybrid peptides against colistin-
resistant clinical strains of Acinetobacter baumannii: molecular basis
for the differential mechanisms of action. Antimicrob. Agents Chemoth-
er. 2006, 50, 1251–1256.
(42) Kondejewski, L. H.; Lee, D. L.; Jelokhani-Niaraki, M.; Farmer, S. W.;
Hancock, R. E. W.; Hodges, R. S. Optimization of microbial specificity
in cyclic peptides by modulation of hydrophobicity within a defined
structural framework. J. Biomol. Chem. 2002, 1, 67–74.
(43) McInnes, C.; Kondejewski, L. H.; Hodges, R. S.; Sykes, B. D.
Development of the structural basis for antimicrobial and hemolytic
activities of peptides based on gramicidin S and design of novel
analogs using NMR spectroscopy. J. Biol. Chem. 2000, 275, 14287–
14294.
(22) Wishart, D. S.; Kondejewski, L. H.; Semchuk, P. D.; Sykes, B. D.;
Hodges, R. S. A method for the facile solid-phase synthesis of
gramicidin S and its analogs. Lett. Pept. Sci. 1996, 3, 53–60.
(23) Yamada, K.; Shinoda, S. S.; Oku, H.; Komagoe, K.; Katsu, T.; Katakai,
R. Synthesis of low-hemolytic antimicrobial dehydropeptides based
on gramicidin S. J. Med. Chem. 2006, 49, 7592–7595.
(44) Bach, A. C.; Markwalder, J. A.; Ripka, W. C. Synthesis and NMR
conformational analysis of a ꢀ-turn mimic incorporated into gramicidin
SsA general approach to evaluate ꢀ-turn peptidomimetics. Int. J. Pept.
Protein Res. 1991, 38, 314–323.
(45) Estiarte, M. A.; Rubiralta, M.; D´ıez, A.; Thormann, M.; Giralt, E.
Oxazolopiperidin-2-ones as type II′ ꢀ-turn mimetics: synthesis and
conformational analysis. J. Org. Chem. 2000, 65, 6992–6999.
(46) Gibbs, A. C.; Bjorndahl, T. C.; Hodges, R. S.; Wishart, D. S. Probing
the structural determinants of type II′ ꢀ-turn formation in peptides
and proteins. J. Am. Chem. Soc. 2002, 124, 1203–1213.
(47) Matsuura, S.; Waki, M.; Izumiya, N. Studies of peptide antibiotics.
Bull. Chem. Soc. Jpn. 1972, 45, 863–866.
(48) Higashijima, T.; Miyazawa, T.; Kawai, M.; Nagai, U. Gramicidin S
analogs with a D-Ala, Gly, or L-Ala residue in place of the D-Phe
residue: molecular conformations and interactions with phospholipid
membrane. Biopolymers 1986, 25, 2295–2307.
(49) Aarstad, K.; Zimmer, T. L.; Laland, S. G. Replacement of phenyla-
lanine in gramicidin S by other amino acids. FEBS Lett. 1979, 103,
118–121.
(50) Ando, S.; Aoyagi, H.; Waki, M.; Kato, T.; Izumiya, N. Studies of
peptide antibiotics. XLIII. Syntheses of gramicidin S analogs contain-
ing D-serine or dehydroalanine in place of D-phenylalanine and
asymmetric hydrogenation of the dehydroalanine residue. Int. J. Pept.
Protein Res. 1983, 21, 313–321.
(51) Wu, M.; Maier, E.; Benz, R.; Hancock, R. E. Mechanism of interaction
of different classes of cationic antimicrobial peptides with planar
bilayers and with the cytoplasmic membrane of Escherichia coli.
Biochemistry 1999, 38, 7235–7242.
(52) Lee, D. L.; Powers, J. P.; Pflegerl, K.; Vasil, M. L.; Hancock, R. E.;
Hodges, R. S. Effects of single D-amino acid substitutions on disruption
of beta-sheet structure and hydrophobicity in cyclic 14-residue
antimicrobial peptide analogs related to gramicidin S. J. Pept. Res.
2004, 63, 69–84.
(24) Jelokhani-Niaraki, M.; Kondejewski, L. H.; Farmer, S. W.; Hancock,
R. E.; Kay, C. M.; Hodges, R. S. Diastereoisomeric analogues of
gramicidin S: structure, biological activity and interaction with lipid
bilayers. Biochem. J. 2000, 349, 747–755.
(25) Kondejewski, L. H.; Jelokhani-Niaraki, M.; Farmer, S. W.; Lix, B.;
Kay, C. M.; Sykes, B. D.; Hancock, R. E.; Hodges, R. S. Dissociation
of antimicrobial and hemolytic activities in cyclic peptide diastereomers
by systematic alterations in amphipathicity. J. Biol. Chem. 1999, 274,
13181–13192.
(26) Abraham, T.; Marwaha, S.; Kobewka, D. M.; Lewis, R. N.; Prenner,
E. J.; Hodges, R. S.; McElhaney, R. N. The relationship between the
binding to and permeabilization of phospholipid bilayer membranes
by GS14dK4, a designed analog of the antimicrobial peptide grami-
cidin S. Biochim. Biophys. Acta 2007, 1768, 2089–2098.
(27) Abe, O.; Izumiya, N. Studies of peptide antibiotics. Analogs of
gramicidin S containing glycine or alanine in place of leucine. Bull.
Chem. Soc. Jpn. 1970, 43, 1202–1207.
(28) Prenner, E. J.; Lewis, R. N.; Kondejewski, L. H.; Hodges, R. S.;
McElhaney, R. N. Differential scanning calorimetric study of the effect
of the antimicrobial peptide gramicidin S on the thermotropic phase
behavior of phosphatidylcholine, phosphatidylethanolamine and phos-
phatidylglycerol lipid bilayer membranes. Biochim. Biophys. Acta
1999, 1417, 211–223.
(29) Graciani, N. R.; Tsang, K. Y.; McCutchen, S. L.; Kelly, J. W. Amino
acids that specify structure through hydrophobic clustering and
histidine-aromatic interactions lead to biologically active peptidomi-
metics. Bioorg. Med. Chem. 1994, 2, 999–1006.
(30) Kee, K. S.; Jois, S. D. Design of ꢀ-turn based therapeutic agents. Curr.
Pharm. Des. 2003, 9, 1209–1224.

674 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Solanas et al.
(53) Krauss, E. M.; Chan, S. I. Intramolecular hydrogen bonding in
gramicidin S. 2. Ornithine. J. Am. Chem. Soc. 1982, 104, 6953–6961.
(54) Yamada, K.; Unno, M.; Kobayashi, K.; Oku, H.; Yamamura, H.; Araki,
S.; Matsumoto, H.; Katakai, R.; Kawai, M. Stereochemistry of
protected ornithine side chains of gramicidin S derivatives: X-ray
crystal structure of the bis-Boc-tetra-N-methyl derivative of gramicidin
S. J. Am. Chem. Soc. 2002, 124, 12684–12688.
(55) Salgado, J.; Grage, S. L.; Kondejewski, L. H.; Hodges, R. S.;
McElhaney, R. N.; Ulrich, A. S. Membrane-bound structure and
alignment of the antimicrobial beta-sheet peptide gramicidin S derived
from angular and distance constraints by solid state 19F-NMR.
J. Biomol. NMR 2001, 21, 191–208.
(56) Mihailescu, D.; Smith, J. C. Atomic detail peptide-membrane
interactions: molecular dynamics simulation of gramicidin S in a
DMPC bilayer. Biophys. J. 2000, 79, 1718–1730.
(57) Schno¨lzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. In
situ neutralization in Boc-chemistry solid phase peptide synthesis.
Rapid, high yield assembly of difficult sequences. Int. J. Pept. Protein
Res. 1992, 40, 180–193.
visual detection of solid phase bound amines. Eur. J. Org. Chem. 1999,
2787–2791.
(60) Bax, A.; Lerner, L. Two-dimensional nuclear magnetic resonance
spectroscopy. Science 1986, 232, 960–967.
(61) Markley, J. L.; Bax, A.; Arata, Y.; Hilbers, C. W.; Kaptein, R.; Sykes,
B. D.; Wright, P. E.; Wu¨thrich, K. Recommendations for the
presentation of NMR structures of proteins and nucleic acidssIUPAC-
IUBMB-IUPAB Inter-Union Task Group on the standardization of
data bases of protein and nucleic acid structures determined by NMR
spectroscopy. J. Biomol. NMR 1998, 12, 1–23.
(62) Wu¨thrich, K.; Billeter, M.; Braun, W. Polypeptide secondary structure
determination by nuclear magnetic resonance observation of short
proton-proton distances. J. Mol. Biol. 1984, 180, 715–740.
(63) Gu¨ntert, P. Automated NMR structure calculation with CYANA.
Methods Mol. Biol. 2004, 278, 353–378.
(64) Koradi, R.; Billeter, M.; Wu¨thrich, K. MOLMOL: a program for
display and analysis of macromolecular structures. J. Mol. Graph 1996,
14, 29–32; 51-55.
(65) Allen, F. H. The Cambridge Structural Database: a quarter of a million
crystal structures and rising. Acta Crystallogr., Sect. B: Struct. Sci.
2002, 58, 380–388.
(58) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color test
for detection of free terminal amino groups in solid-phase synthesis
of peptides. Anal. Biochem. 1970, 34, 595–598.
(59) Madder, A.; Farcy, N.; Hosten, N. G. C.; De Muynck, H.; De Clercq,
P. J.; Barry, J.; Davis, A. P. A novel sensitive colorimetric assay for
JM800886N