Gramicidin S derivatives containing cis- and trans-morpholine amino acids (MAAS) as turn mimetics

Article abstract of DOI:10.1002/chem.200902984

The cyclic decapeptide gramicidin S (GS) was used as a model for the evaluation of four turn mimetics. For this purpose, one of the D-Phe-Pro two-residue turn motifs in the rigid cyclic β-hairp0in structure of GS was replaced with morpholine amino acids (MAA 2-5), differing in stereochemistry and length of the side-chain. The conformational properties of the thus obtained GS analogues (6-9) was assessed by using NMR spectroscopy and X-ray crystallography, and correlated with their biological properties (antimicrobial and hemolytic activity). We show that compound 8, containing the dipeptide isostere trans-MAA 4, has an apparent high structural resemblance with GS and that its antibacterial activity against a panel of Gram positive and -negative bacterial strains is better than the derivatives 6, 7 and 9.

Full text of DOI:10.1002/chem.200902984

FULL PAPER
DOI: 10.1002/chem.200902984
Gramicidin S Derivatives Containing cis- and trans-Morpholine Amino Acids
(MAAs) as Turn Mimetics
Varsha V. Kapoerchan,[b] Emile Spalburg,[c] Albert. J. de Neeling,[c]
Roos H. Mars-Groenendijk,[d] Daan Noort,[d] Josꢀ M. Otero,[e] Patricia Ferraces-Casais,[e]
Antonio L. Llamas-Saiz,[f] Mark J. van Raaij,[e, g] Joop van Doorn,[h]
Gijs A. van der Marel,[b] Herman S. Overkleeft,[b] and Mark Overhand*[a]
Abstract: The cyclic decapeptide gra-
micidin S (GS) was used as a model for
the evaluation of four turn mimetics.
For this purpose, one of the d-Phe-Pro
two-residue turn motifs in the rigid
cyclic b-hairpin structure of GS was re-
placed with morpholine amino acids
(MAA 2–5), differing in stereochemis-
try and length of the side-chain. The
conformational properties of the thus
obtained GS analogues (6–9) was as-
sessed by using NMR spectroscopy and
X-ray crystallography, and correlated
with their biological properties (antimi-
crobial and hemolytic activity). We
show that compound 8, containing the
dipeptide isostere trans-MAA 4, has an
apparent high structural resemblance
with GS and that its antibacterial activ-
ity against a panel of Gram positive
and -negative bacterial strains is better
than the derivatives 6, 7 and 9.
Keywords: antibiotics · conforma-
tion analysis
·
gramicidin S
·
peptides · peptidomimetics
Introduction
itive and to a lesser extent Gram-negative bacteria, but is
toxic to human red blood cells (hemolytic activity).[2] GS
kills bacteria by disrupting the bacterial cell membrane.[3]
This property is attributed[4] to both the basic and amphi-
philic nature of the rigid cyclic b-hairpin molecular structure
The cyclic decapeptide gramicidin S (GS, 1, cyclo-(Pro-Val-
Orn-Leu-d-Phe)2, Figure 1a), produced by Bacillus brevis,[1]
displays potent antibacterial activity against both Gram-pos-
[a] Dr. M. Overhand
[f] Dr. A. L. Llamas-Saiz
Bioorganic Synthesis
Unidad de Rayos X (RIAIDT)
Leiden Institute of Chemistry, Leiden University
P.O. Box 9502, 2300 RA Leiden (The Netherlands)
E-mail: overhand@chem.leidenuniv.nl
Laboratorio Integral de dinꢁmica y Estructura
de Biomolꢂculas “Josꢂ R. Carracido”
Edificio CACTUS, Campus Sur
Universidad de Santiago de Compostela
15782 Santiago de Compostela (Spain)
[b] Dr. V. V. Kapoerchan, Prof. Dr. G. A. van der Marel,
Prof. Dr. H. S. Overkleeft
Bioorganic Synthesis, Leiden Institute of Chemistry
Leiden University, P.O. Box 9502
2300 RA Leiden (The Netherlands)
[g] Dr. M. J. van Raaij
Departamento de Biologꢀa Estructural
Instituto de Biologꢀa Molecular de Barcelona
Consejo Superior de Investigaciones Cientꢀficas, Baldiri Reixac 10
08028 Barcelona (Spain)
[c] E. Spalburg, Dr. A. J. de Neeling
Laboratory for Infectious Diseases
National Institute of Public Health and the Environment
P.O. Box 1, 3720 BA Bilthoven (The Netherlands)
[h] Dr. J. van Doorn
Applied Plant Research, P.O. Box 85
2160 AB Lisse (The Netherlands)
[d] R. H. Mars-Groenendijk, Dr. D. Noort
Defense, Security and SafetyTNO, P.O. Box 45
2280 AA Rijswijk (The Netherlands)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902984.
[e] J. M. Otero, Dr. P. Ferraces-Casais, Dr. M. J. van Raaij
Departamento de Bioquꢀmica y Biologꢀa Molecular
Faculta de Farmacia Campus Sur
Universidad de Santiago de Compostela
15782 Santiago de Compostela (Spain)
Chem. Eur. J. 2010, 16, 4259 – 4265
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4259

of GS[5–8] that is stabilized by four intramolecular hydrogen-
bonding interactions formed between the C=O and NH of
the amides of opposing Val and Leu residues (Figure 1b).
The side chains of the two Orn residues are on one side of
the molecule and the hydrophobic side chains of the two
Val and Leu residues reside on the opposing face, making
the molecule amphiphilic (Figure 1c).
morpholine amino acid (MAA). Their synthesis, structural
and biological (antibacterial and hemolytic) properties are
evaluated and compared to those of GS.
Results and Discussion
Synthesis of the target compounds: The synthesis of MAAs
2, 3 and 4 as their azide derivatives was reported previously
by us (Figure 2).[18] The synthesis of the novel MAA 5, as its
azide derivative, is described in the Supporting Information.
MAAs 2 and 4 represent dipeptide isosteres (d-amino
acids), MAAs 3 and 5 are one carbon atom longer (e-amino
acids). The difference between MAAs 2 and 3 and MAAs 4
and 5 concerns the cis or trans relationship between the ami-
nomethyl and carboxyl groups of the morpholino ring, re-
spectively. All four MAAs have an N-benzyl functionality to
mimic the hydrophobic characteristics of the d-Phe-Pro se-
quence.
Figure 2. Morphilino amino acids (MAAs) investigated in this study as
replacement of one of the type two b-turns in gramicidin S. MAAs 2 and
3 are cis configured and MAAs 4 and 5 are trans configured d- and e-
morpholine amino acids, respectively.
Figure 1. Structure of GS. a) Structural formula of the GS molecule. b)
Top view of the GS molecule (Val, Orn and Leu side chains omitted).
Hydrogen bonds (stabilizing the secondary structure) are depicted by
thin green lines. c) Side view of the GS molecule. Hydrophobic side
chains of the Val and Leu residues reside on one face of the molecule,
the positively charged side-chains of the two Orn residues are on the op-
posing face, providing its amphiphilic characteristics.[22]
The peptides were assembled by adaptation of a standard
Fmoc SPPS method, starting with preloaded Fmoc-Leu-
HMBP-BHA resin. In the final stage of the syntheses com-
pounds 2–5 were incorporated as terminal residues as their
azide derivatives. Reduction of the azide functionality[18] was
followed by mildly acidic cleavage from the resin. Cycliza-
tion with PyBOP, HOBt and DIPEA as base, deprotection
of the Boc protecting groups with strong acid and subse-
quent HPLC purification was straightforward, yielding pep-
tide 6–9 (Figure 3) in an overall yield of 25–65%, as their
TFA salts.
Because of its well-defined secondary structure, GS has
served as a model to probe the effect of turn mimetics on
the b-cyclic hairpin structure. The d-Phe-Pro sequence of
GS has been replaced by bicyclic thioindolizine deriva-
tives[9,10] as well as the structurally related 5,6-fused azabicy-
cloalkane[11] and indolizines.[12,13] In a related approach, sev-
eral sugar amino acids (SAA) have been incorporated and
their structural and biological properties studied.[14–16] C-
functionalized morpholines are found frequently both in
natural products and in drugs.[17] Various synthetic strategies
towards these compounds have been developed, allowing to
obtain morpholines with different substitution patterns. Be-
cause of its rigid structure, a morpholine ring incorporated
in a GS molecule can possibly invoke structural changes
which may affect the antibacterial and hemolytic properties.
Here, four GS analogues are presented in which one of the
d-Phe-Pro turn motifs is replaced by a cis- or trans-, d- or e-
Structural analysis of synthesized GS analogues 6–9: The
effect on the incorporation of the MAAs on the cyclic b-
hairpin secondary structure was determined by using 1D
and 2D NMR techniques and X-ray crystal structure deter-
mination. The observed JNH–Ha values of the residues of
compounds 6–9 were compared to those of GS (Figure 4).
In the amide region of the 1H NMR spectra eight signals
were observed for all analogues, with the exception of com-
4260
www.chemeurj.org
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 4259 – 4265

Gramicidin S Derivatives
FULL PAPER
Figure 4. Comparison of JNH–Ha values of the analogues 6–9 to those of
GS. In analogue 9, the CONH signals of the V2 and L9 residues were ob-
served as broad signals, so no coupling constant could be determined.
The JNH–Ha values for analogue 7 were taken from ref. [18].
Figure 3. GS analogues containing the morpholine-amino acid residues.
1
pound 9. Certain amide signals in the H spectrum of 9 were
observed as broad singlets instead of doublets and the
amide signal of the MAA residue was not observed at all,
indicating structural flexibility in this region of the molecule.
The JNH–Ha values of the ornithine, leucine and valine resi-
dues were all between 8 and 12 Hz, which is a strong indica-
tion of a b-strand conformation.[19] The JNH–Ha values of the
d-Phe residues were in the range of 2–4 Hz, which is typical
for an amino acid that is part of a b-turn.[19]
These features indicate that the cyclic peptides 6–8 and to
a lesser extent compound 9, adopt overall secondary struc-
tures similar to that of GS. A second NMR analysis tech-
nique compares the chemical shift of the a-proton of an
amino acid residue to the average value of this residue as
part of a random coil. The results of this chemical shift per-
turbation analysis method[20,21] are depicted in Figure 5. The
valine, ornithine and leucine residues show positive pertur-
bations indicating that these residues are part of a b-strand
and all proline and d-phenylalanine residues display nega-
tive perturbations, consistent with their presence in a turn.
Figure 5. Chemical shift perturbation of Ha protons in GS analogues 6–9
as compared to GS. For ornithine residues, the reported[20] d(Ha) random
coil value of lysine was taken. The chemical shift perturbation was calcu-
lated as DdHa =dH
of 9 no difference between the observed and reported value was seen.
The Ha of V7 in 7 was not observed.
G
Overall, the chemical shift perturbation indicates a cyclic b-
hairpin-like secondary structure. However, specific differen-
ces in perturbation values are found when the analogues are
compared to GS.
To obtain detailed molecular information nuclear Over-
hauser effect (NOE) experiments were performed for all an-
Chem. Eur. J. 2010, 16, 4259 – 4265
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4261

M. Overhand et al.
alogues at 298 K. (see Figure S1 in the Supporting Informa-
tion). Several sequential NOEs are observed in the NH
region of the 2D spectra of compounds 6–9, however, the
observation of specific long-range NOEs in the turn regions
are crucial. For peptide 6, a long range NOE crosspeak be-
tween Val2-NH and Leu9-NH (peak B in Figure S1, panel I)
is observed. This implies that these NH protons are in close
proximity to each other. As such correlations are also found
for GS itself, the presence of this NOE crosspeak in 6 may
indicate a conformation similar to that of GS. Interestingly,
in the NOE spectrum of compound 8 (the trans isomer of
compound 6) also this long-range NOE crosspeak between
Val2-NH and Leu9-NH is observed (B, Figure S1, panel
III).
were assayed against a panel of Gram-positive and Gram-
negative bacterial strains. The results are shown in Table 1.
As can be seen, none of the four analogues are signifi-
cantly more potent than GS. Peptides 6 and 7 show de-
creased inhibitory activity for all Gram-positive bacteria
tested, especially for E. faecalis. Compound 8 performs
almost as well as GS, with the exception of the E. faecalis
and the S. epidermis strains. Peptide 9 has similar antimicro-
bial activity as the compounds 6 and 7. Peptides 6–9 were
also evaluated for their toxicity towards human red blood
cells (Figure 7). All peptides are less hemolytic when com-
pared to GS, with 8 displaying the most hemolytic activity
and 6 being the least toxic.
Two long-range crosspeaks in the turn region between
Val2-NH and Leu9-NH and between MAA-NH and Val2-
NH of peptide 7 indicates an al-
tered turn conformation. Pep-
tide 9 did not show NOE cross-
peaks around the MAA turn, in
agreement with partial flexibili-
ty in this region as observed in
the 1D spectra.
Crystallization trials were
performed with analogues 6–9,
only yielding suitable crystals
for X-ray analysis of peptides 7
and 8. Peptide 7 adopts overall
a similar secondary structure in
the crystal as GS, with a distort-
ed b-turn conformation. As a
consequence, only three instead
of the usual four intramolecular
hydrogen-bonding interaction
are observed, due to the distort-
ed orientation (“flip” of the
amine bond)[14,16] connection of
this MAA residue with the
neighboring Leu9 residue (Fig-
ure 6a). This altered turn
region of compound 7 is in
agreement with the findings of
Figure 6. a) X-ray analysis of peptide 7: stereoview of the peptide monomer. In the top view, side chains are
omitted for clarity. Intramolecular hydrogen-bonds are indicated with dotted lines. b) Top and side stereoview
of the monomer of 8.
the NMR experiments. Interest-
ingly, the cyclic b-hairpin secon-
dary structure as observed in
the X-ray structure of com-
pound 8 is very similar to that
of GS (Figure 6b), with all four
intramolecular hydrogen-bonds
in place. This X-ray structure of
Table 1. Antibacterial activity of compounds 6–9 as compared to GS. MIC values (MIC=minimal inhibitory
concentration) are given in mg per mL and were measured after 24 h of incubation. The experiments were con-
ducted once. The experimental error is one MIC interval.
8
is in agreement with the
Gram+
S. aureus
Gram+
S. epidermidis
Gram+
E. faecalis
Gram+
B. cereus
GramÀ
GramÀ
NMR data. (See Figure S2 for
X-ray assembly of compound
8).
E. coli
aeruginosa
GS
6
7
8
9
8
4
16
8
8
8
8
8
32
64
64
32
64
64
16
16
8
64
64
16
64
16
16
8
>64
>64
64
Evaluation of biological charac-
teristics: GS analogues 6–9
32
16
>64
4262
www.chemeurj.org
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 4259 – 4265

Gramicidin S Derivatives
FULL PAPER
Table 2. Structural data extracted from the crystal structures from GS
and compound 8.
GS[39]
Peptide 8
Leu9 O···H-N Val2
Leu9 O···N Val2
angle O···H-N
2.15 ꢄ
2.99 ꢄ
161.58
3.31 ꢄ
3.86 ꢄ
122.78
an intramolecular hydrogen bond in the MAA-containing
turn region of 8 the geometry of the two peptidic bonds in-
volved in the turn is quite similar to the usual b-turn motive
in GS.
Figure 7. Hemolytic activity of GS analogues 6–9. Experiments were car-
ried out in duplo. Multiple blood donors (both male and female) were
used. A maximum of 10% experimental error was found.
The positioning of the N-benzyl group of the MAA resi-
due is different in comparison with the benzyl side-chain of
d-Phe residue of GS (Figure 8). Interestingly, compound 8 is
a much better antimicrobial agent than compound 6, with
comparable activity as GS and slightly less hemolytic.
Discussion and Conclusion
In this work, four GS analogues 6–9 are presented, in which
a d-Phe-Pro motif of one of the b-turns was replaced with
cis- and trans, d- and e-morpholine amino acids (MAAs 2–
5). The secondary structures of these analogues and their
biological properties (antibacterial and hemolytic activity)
were evaluated and compared with those of GS.
Compound 7 adopts a distorted b-turn as result of the
presence of the cis e-MAA 3. This distortion is characterized
by a repositioning of one of the turn amides (amide flip) in
such a way that no intra-molecular hydrogen-bonding inter-
action is possible (see Figure 6a). A similar distorted b-turn
arrangement has been observed by us in a series of related
GS analogues containing sugar amino acids (SAAs).[14,16]
The b-turn of compound 9, containing the trans e-MAA 5, is
flexible. The isomeric compounds 7 and 9 have a compara-
ble biological profile.
The macrocycles 6 and 8, containing cis d-MAA 2 and
trans d-MAA 4, respectively, both agree well with the cyclic
b-hairpin secondary structural characteristics of GS. In case
of compound 6 this is the expected outcome since we and
others have designed and synthesized related turn mimetics,
such as sugar amino acids (SAAs), having cis configured
aminomethyl- and carboxyl groups functionalities.[23–37] How-
ever, in general trans isomers are considered as mimetics of
extended conformations and we were thus somewhat sur-
prised to observe that also compound 8 assumes a secondary
structure strongly resembling that of GS, with all four intra-
molecular hydrogen bonds apparently in place. It can be as-
sumed that the octapeptide sequence originating from GS
overrules a possible intrinsic conformational preference that
MAA 4 would transpose to a linear oligopeptide.
Figure 8. X-ray structure of GS (white) overlayed to compound
(green). Intramolecular hydrogen-bonds are indicated with dotted lines
in red for GS and black for 8. Side chains are omitted for clarity.
8
In conclusion, we have demonstrated that morpholine
amino acids are useful building blocks in the design of new
amphiphilic cationic cyclic peptides. Specifically, GS ana-
logue 8, incorporating trans-configured 4, most closely re-
sembles GS itself both in secondary structure and biological
activity. Compound 8 has antibacterial activity comparable
to that of GS and is less hemolytic. From a structural point
this finding is interesting, since related trans-substituted di-
peptide isosteres are reputed to induce linear configura-
tions.[23,35–37] With respect to the identification of GS deriva-
tives that are bactericidal with reduced hemolytic activity
that a strategy in which the morpholine secondary amine is
alkylated or acylated with a range of lipophilic functionali-
ties is well worth pursuing.
Experimental Section
The X-ray structure of compound 8 overlaid with GS is
depicted in Figure 8. Comparison of the distance informa-
tion with respect to the turn region of both compounds
(Table 2) reveals that the determined distance between
Val2-NH···O=Leu9 is more than 1 ꢄ longer than the corre-
sponding distance in GS and the N-H···O angle is very small
(122.7 versus 161.58). Whether this is considered a hydrogen
bond is therefore doubtful[38] but fact is that the Val2-
NH···O=Leu9 functionalities are rigidly positioned in close
proximity in compound 8. Therefore, in spite of the lack of
Antibacterial assays: The following bacterial strains were used: Staphylo-
coccus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228),
Enterococcus faecalis (ATCC 29212), Bacillus cereus (ATCC 11778), Es-
cherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853).
Bacteria were stored at À708C and grown at 308C on Columbia Agar
with sheep blood (Oxoid, Wesel, Germany) suspended in physiological
saline until an optical density of 0.1 AU (at 595 nm, 1 cm cuvette). The
suspension was diluted (10ꢅ) with physiological saline, and 2 mL of this
inoculum was added to 100 mL growth medium, Nutrient Broth from
Difco (ref. nr. 234000, lot nr. 6194895) with yeast extract (Oxoid LP
Chem. Eur. J. 2010, 16, 4259 – 4265
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4263

M. Overhand et al.
Table 3. Selected crystallographic data of the crystals obtained from 7
and 8.
X-ray crystallography
Crystallization: Suitable colorless prism-shaped crystals were obtained
after slow evaporation of 2 mL droplets of 8.8 mgmLÀ1 7 in an 80% solu-
tion of MeOH in H2O plus 2 mL of 0.5m NaOH in H2O under paraffin
oil in Terasaki plates. Colorless prism-shaped crystals were obtained after
slow evaporation of 2 mL droplets of 7.0 mgmLÀ1 8 in 50% solution of
MeOH in H2O plus 2 mL of 0.5m CaCl2 in MeOH under paraffin oil in
Terasaki plates.
7
8
formula
2(C60H97N12O10)·O17 2(C59H95N12O10), C2F3O2, O11
2565.00
0.939
orthorhombic
P212121
19.175(3)
24.656(4)
32.061(8)
90
90
90
15152(5)
1.124
4
formula weight
wavelength [ꢄ]
crystal system
space group
a [ꢄ]
b [ꢄ]
c [ꢄ]
a [8]
b [8]
2553.96
0.939
hexagonal
P65
28.781(2)
28.781(2)
34.911(2)
90
90
120
25044(3)
1.016
6
Crystal structure determination of 7 and 8A crystal was mounted in air
and then rapidly transferred to liquid nitrogen. Synchrotron data were
collected at beamline ID14-2 at the ESRF (Grenoble, France). Images
were collected with DNA software,[40] processed with MOSFLM[41] and
scaled with POINTLESS and SCALA.[42] Both structures could be solved
by direct methods using the SHELXD[43] program and were refined by
full-matrix least-squares methods on F2 with SHELXL[42] included in the
WinGX[44] package. All hydrogen positions were calculated and refined
using a riding atom model. There are two crystallographically independ-
ent molecules per asymmetric unit in both crystal structures and there
are several disordered parts in all molecules. All non-hydrogen atoms
were refined using anisotropic refinement. Selected crystallographic data
is reported in Table 3.
g [8]
cell volume [ꢄ3]
1calcd [gcmÀ3
]
Z
m [mmÀ1
]
0.084
5512
0.29ꢅ0.12ꢅ0.12
100(2)
1.4–28.0
0.077
8214
F
N
crystal size [mm3]
T [K]
0.32ꢅ0.24ꢅ0.15
100(2)
1.3–28.0
39521
8373
CCDC 751340 (7), 751341 (8) contain the supplementary crystallographic
data for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif
q range [8]
measured reflections 31070
unique reflections
completeness [%]
redundancy
7530
88.0
4.1
93.7
4.7
R(merge)
data in refinement
0.058
7514
0.065
8342
data with Fo >4s(Fo) 6657
7721
average s(I)
no. of parameters
15.0
1928
18.8
1811
Acknowledgements
extinction coefficient 0.032(3)
0.26(2)
0.3856
0.1579
0.1609
2.070
We are grateful to Kees Erkelens and Fons Lefeber for assistance in
measuring NMR spectra. We thank Pablo Guardado-Calvo and Bruno
Dacunha-Marinho for help with crystallographic software. This work was
wR2
0.3352
0.1415
0.1344
1.726
R1 (obs data)
R1 (all data)
Gof=S
supported by
a grant from the Leiden Institute of Chemistry and
BFU2008-01588/BMC of the Spanish Ministry of Science and Innovation.
J.M.O. is supported by an “Angeles AlvariÇo” contract of the Xunta de
Galicia.
DF peak/hole [eꢄÀ3
]
0.80/À0.47
0.40/À0.28
0021, lot nr. 900711, 2 g per 400 mL broth), in microtiter plates (96
wells). The peptides GS and 6–9 were dissolved in ethanol (4 gLÀ1) and
diluted in distilled water (1000 mgLÀ1), and two-fold diluted in the broth
(64, 32, 16, 8, 4 and 1 mgLÀ1). The plates were incubated at 308C (24–
96 h) and the MIC was determined as the lowest concentration inhibiting
bacterial growth. The experiments were conducted once, the experimen-
tal error is one MIC interval (a factor two).
[1] G. F. Gause, M. G. Brazhnikova, Nature 1944, 153, 703.
[2] L. H. Kondejewski, S. W. Farmer, D. S. Wishart, R. E. W. Hancock,
R. S. Hodges, Int. J. Pept. Protein Res. 1996, 47, 460–466.
[3] G. M. J. Schmidt, D. C. Hodgkin, B. M. Oughton, Biochem. J. 1957,
65, 744–750.
[4] E. J. Prenner, R. N. A. H. Lewis, R. N. McElhaney, Biochim. Bio-
phys. Acta Biomembr. 1999, 1462, 201–221.
Hemolytic assays: Freshly drawn heparinized blood was centrifuged for
10 min at 1000 g at 108C. Subsequently, the erythrocyte pellet was
washed three times with 0.85% saline solution and diluted with saline to
a 1/25 packed volume of red blood cells. The peptides to be evaluated
(6–9 and GS) were dissolved in a 30% DMSO/0.5 mm saline solution to
give a 1.5 mm solution of peptide. If a suspension was formed, the suspen-
sion was sonicated for a few seconds. A 1% Triton-X solution was pre-
pared. Subsequently, 100 mL of saline solution was dispensed in columns
1–11 of a microtiter plate, and 100 mL of 1% Triton solution was dis-
pensed in column 12. To wells A1–C1, 100 mL of the peptide was added
and mixed properly. 100 mL of wells A1–C1 was dispensed into wells A2–
C2. This process was repeated until wells A10–C10, followed by discard-
ing 100 mL of wells A10–C10. These steps were repeated for the other
peptides. Subsequently, 50 mL of the red blood cell solution was added to
the wells and the plates were incubated at 378C for 4 h. After incubation,
the plates were centrifuged at 1000 g at 108C for 4 min. In a new micro-
titer plate, 50 mL of the supernatant of each well was dispensed into a
corresponding well. The absorbance at 405 nm was measured and the
percentage of hemolysis was determined. Experiments were carried out
in duplo. Multiple blood donors (both male and female) were used. A
maximum of 10% experimental error was found.
[5] A. Stern, W. A. Gibbons, L. C. Craig, Proc. Natl. Acad. Sci. USA
1968, 61, 734–741.
[6] S. E. Hull, R. Karlsson, P. Main, M. M. Woolfson, E. J. Dodson,
Nature 1978, 275, 206–207.
[7] A. Liquori, P. De Santis, Int. J. Biol. Macromol. 1980, 2, 112–115.
[8] S. Rachovsky, H. A. Scheraga, Proc. Natl. Acad. Sci. USA 1980, 77,
6965–6967.
[9] K. Sato, U. Nagai, J. Chem. Soc. Perkin Trans. 1 1986, 1231–1234.
[10] A. C. Bach, J. A. Markwalder, W. C. Ripka, Int. J. Pept. Protein Res.
1991, 38, 314–323.
[11] S. Roy, H. G. Lombart, W. D. Lubell, R. E. W. Hancock, S. W.
Farmer, J. Pept. Res. 2002, 60, 198–214.
[12] N. de La Figuera, I. Alkorta, M. T. Garcia-Lopez, R. Herranz, R.
Gonzalez-Muniz, Tetrahedron 1995, 51, 7841–7856.
[13] W. C. Ripka, G. V. de Lucca, A. C. Bach, R. S. Pottorf, J. M. Blaney,
Tetrahedron 1993, 49, 3609–3628.
[14] G. M. Grotenbreg, M. S. M. Timmer, A. L. Llamas-Saiz, M. Verdoes,
G. A. van der Marel, M. J. van Raaij, H. S. Overkleeft, M. Over-
hand, J. Am. Chem. Soc. 2004, 126, 3444–3446.
[15] G. M. Grotenbreg, M. Kronemeijer, M. S. M. Timmer, F. El Oualid,
R. M. van Well, M. Verdoes, E. Spalburg, P. A. V. van Hooft, A. J. de
4264
www.chemeurj.org
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 4259 – 4265

Gramicidin S Derivatives
FULL PAPER
Neeling, D. Noort, J. H. van Boom, G. A. van der Marel, H. S. Over-
kleeft, M. Overhand, J. Org. Chem. 2004, 69, 7851–7859.
[16] G. M. Grotenbreg, A. E. M. Buizert, A. L. Llamas-Saiz, E. Spalburg,
P. A. V. van Hooft, A. J. de Neeling, D. Noort, M. J. van Raaij, G. A.
van der Marel, H. S. Overkleeft. M. Overhand, J. Am. Chem. Soc.
2006, 128, 7559–7565.
[17] R. Wijtmans, M. K. S. Vink, H. E. Schoemaker, F. L. van Delft,
R. H. Blaauw, F. P. J. T. Rutjes, Synthesis 2004, 641–662.
[18] G. M. Grotenbreg, A. E. Christina, A. E. M. Buizert, G. A. van der
Marel, H. S. Overkleeft, M. Overhand, J. Org. Chem. 2004, 69,
8331–8339.
[19] K. Wꢆthrich, in NMR of proteins and nucleic acids, Wiley-Inter-
science, New York, 1986, pp. 162–175.
[20] D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 1992, 31,
1647–1651.
[21] A. J. Maynard, G. J. Sharman, M. S. Searle, J. Am. Chem. Soc. 1998,
120, 1996–2007.
[22] E. G. von Roedern, E. Lohof, G. Hessler, M. Hoffmann, H. Kessler,
J. Am. Chem. Soc. 1996, 118, 10156–10167.
[23] C. Kꢆhn, G. Lindeberg, A. Gogoll, A. Hallberg, B. Schmidt, Tetrahe-
dron 1997, 53, 12497–12504.
[24] L. Szano, B. L. Smith, K. D. McReynolds, A. L. Parrill, E. R. Morris,
J. Gervay, J. Org. Chem. 1998, 63, 1074–1078.
[25] M. D. Smith, T. D. W. Claridge, G. E. Tranter, M. S. P. Sansom,
G. W. J. Fleet, Chem. Commun. 1998, 2041–2042.
[26] T. K. Chakraborty, S. Javaprakash, P. V. Diwan, R. Nagarai, S. R. B.
Jampani, A. C. Kunwar, J. Am. Chem. Soc. 1998, 120, 12962–12963.
[27] A. B. Smith III, S. Sasho, B. A. Barwis, P. Sprengeler, J. Barbosa, R.
Hirshmann, B. S. Cooperman, Bioorg. Med. Chem. Lett. 1998, 8,
3133–3136.
[28] H. S. Overkleeft, S. H. L. Verhelst, E. Pieterman, W. J. Meeuwe-
noord, M. Overhand, L. H. Cohen, G. A. van der Marel, J. H. van
Boom, Tetrahedron Lett. 1999, 40, 4103–4106.
[30] U. Koert, J. Prakt. Chem./Chem.-Ztg. 2000, 342, 325–333.
[31] R. M. van Well, H. S. Overkleeft, M. Overhand, E. V. Carstenen,
G. A. van der Marel, J. H. van Boom, Tetrahedron Lett. 2000, 41,
9331–9335.
[32] E. Lohof, E. Planker, C. Mang, F. Burkhart, M. A. Haubner, H. J.
Wester, M. Schwaiger, G. Hçlzemann, S. L. Goodman, H. Kessler,
Angew. Chem. 2000, 112, 2868–2871; Angew. Chem. Int. Ed. 2000,
39, 2761–2764.
[33] B. Aguilera, G. Siegal, H. S. Overkleeft, N. J. Meeuwenoord,
F. P. J. T. Rutjes, J. C. M. van Hest, H. E. Schoemaker, G. A. van der
Marel, J. H. van Boom, M. Overhand, Eur. J. Org. Chem. 2001, 8,
1541–1547.
[34] F. Schweizer, Angew. Chem. 2002, 114, 240–264; Angew. Chem. Int.
Ed. 2002, 41, 230–253.
[35] S. A. W. Gruner, E. Locardi, E. Lohof, H. Kessler, Chem. Rev. 2002,
102, 491–514.
[36] T. K. Chakraborty, S. Ghosh, S. Javaprakash, Curr. Med. Chem.
2002, 9, 421–435.
[37] M. D. P. Risseeuw, M. Overhand, G. W. J. Fleet, M. I. Simone, Tetra-
hedron: Asymmetry 2007, 18, 2001–2010.
[38] S. Aravinda, V. V. Harini, N. Shamala, C. Das, P. Balaram, Biochem-
istry 2004, 43, 1832.
[39] A. L. Llamas-Saiz, G. M. Grotenbreg, M. Overhand, M. J. van Raaij,
Acta Crystallogr. 2007, D63, 401–407.
[40] A. G. W. Leslie, H. R. Powell, G. Winter, O. Svensson, D. Spruce, S.
McSweeney, D. Love, S. Kinder, E. Duke, C. Nave, Acta Crystallogr.
Sect. D 2002, 58, 1924–1928.
[41] A. G. W. Leslie, Acta Crystallogr. Sect. D 2006, 62, 48–57.
[42] P. Evans, Acta Crystallogr. Sect. D 2006, 62, 72–82.
[43] G. M. Sheldrick, Acta Crystallogr. Sect. A 2008, 64, 112–122.
[44] L. J. Farrugia, J. Appl. Crystallogr. 1999, 32, 837–838.
Received: October 28, 2009
Revised: December 21, 2009
Published online: March 5, 2010
[29] K. Brickmann, Z. Q. Yuan, I. Sethson, P. Somfai, J. Kihlberg, Chem.
Eur. J. 1999, 5, 2241–2253.
Chem. Eur. J. 2010, 16, 4259 – 4265
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4265