Synthesis and biological evaluation of gramicidin S dimers

Article abstract of DOI:10.1039/b414618b

The design and synthesis of analogues of the cyclic β-sheet peptide gramicidin S (GS), having additional functionalities in their turn regions, is reported. The monomeric GS analogues were transformed into dimers and their activities towards biological me

Full text of DOI:10.1039/b414618b

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A R T I C L E
Synthesis and biological evaluation of gramicidin S dimers†
Gijsbert M. Grotenbreg,a Martin D. Witte,a Peter A. V. van Hooft,b Emile Spalburg,c
Philipp Reiß,d Daan Noort,b Albert J. de Neeling,c Ulrich Koert,d Gijsbert A. van der Marel,a
Herman S. Overkleeft*a and Mark Overhand*a
a Leiden Institute of Chemistry, Gorlaeus Laboratories, P. O. Box 9502, 2300, RA Leiden, The
Netherlands. E-mail: overhand@chem.leidenuniv.nl; Fax: +31(0)71-5274307
b TNO Prins Maurits Laboratory, P.O. Box 45, 2280, AA Rijswijk, The Netherlands
c National Institute of Public Health and the Environment, Research Laboratory for Infectious
Diseases, P.O. Box 1, 3720, BA Bilthoven, The Netherlands
d Philipps-Universita¨t Marburg, Fachbereich Chemie, Hans-Meerwein-Strasse, 35032,
Marburg, Germany
Received 22nd September 2004, Accepted 10th November 2004
First published as an Advance Article on the web 8th December 2004
The design and synthesis of analogues of the cyclic b-sheet peptide gramicidin S (GS), having additional
functionalities in their turn regions, is reported. The monomeric GS analogues were transformed into dimers and
their activities towards biological membranes, through antimicriobial and hemolytic assays, were evaluated. Finally,
conductivity measurements have been performed to elucidate ion channel forming properties.
peptide gramicidin S (GS)9 and analogues thereof. GS is a cyclic
Introduction
decapeptide of the primary sequence cyclo-(DPhe-Pro-Val-Orn-
Cationic antimicrobial peptides (CAPs), a class of structurally
diverse peptide-based compounds, are ubiquitously present in
Leu)2 that adopts a stable C2-symmetric b-sheet structure.10 The
valine, ornithine and leucine residues of the two opposing b-
nature and are often important compounds of innate immune
strands are positioned on alternate sides of the pleated sheet
systems.1 CAPs exert their biological activity by disturbing the
and are flanked by two type IIꢀ b-turns consisting of a DPhe-
integrity of the cell membrane of pathogenic organisms, and
Pro dipeptide sequence, thus creating an amphipathic structure
are thought to do so through direct interaction with the lipid
that is thought to be at the basis of the cell-permeabilization
bilayer.2 From the thought that, in most cases, there is no specific
activity of GS. Through the synthesis of novel GS analogues,
subcellular protein target involved in CAP-mediated cell lysis,
we ultimately aim to gain insight into the mechanism of its
it follows that the occurrence of pathogens with an acquired
lytic effects, induce membrane specificity in order to curb its
CAP-resistance may be unlikely. These considerations, to a large
undesirable erythrocytic toxicity and to see whether defined
extent, explain current interest in CAPs and research efforts are
channels can be resolved. In line with these studies, we here
focussed both on the elucidation of the molecular mechanisms
present our results in the design, synthesis and biological
that are at the basis of CAP-mediated membrane disturbance,
evaluation of a set of dimeric GS analogues.
and on the development of CAPs towards therapeutic agents.
Gramicidin A (GA) is a CAP that has attracted consider-
able attention over the years.3 This pentadecapeptide exerts
Results and discussion
its antibacterial properties by adopting a b-helical secondary
The synthesis of GS dimer 6 was accomplished as follows
(Scheme 1). Standard Fmoc-based solid-phase synthesis us-
ing commercially available building blocks and the readily
accessible Fmoc-2S,4R-azidoproline (Azp) and starting from
4-(4-hydroxymethyl-3-methoxyphenoxy)butanoic acid (HMPB)
structure that associates into a dimer, thereby forming an active
ion channel that traverses lipid bilayers. Schreiber and coworkers
elegantly demonstrated that covalently linked GA monomers
result in unimolecular channels spanning the membrane.4 More
recently, unimolecular membrane-spanning ion channels that
have preference for specific ions were obtained by linking two
GA monomers through tetrahydrofuran (THF)-based dipeptide
isosters.5 The potential of synthetic dimers is further under-
scored by the requirement that accumulation of CAPs onto
the lipid bilayer precedes pore formation. In this way, syn-
thetic dimers can induce a shift in the dissociation/association
equilibrium, resulting in favouring of the conducting over the
non-conducting states of ion-channels. This enables the study
of the molecular architecture of ion channels and creates an
understanding of the dynamics involved. For example, Woolley
et al. showed that tethered alamethicin monomers selectively
stabilize specific conductance states,6 while Murata and cowork-
ers have prepared bioactive dimers of the polyene antibiotic
amphotericin B that enabled the study of pore assemblage.7
As part of an ongoing research program,8 we have developed
a practical strategy towards the synthesis of the antimicrobial
functionalized 4-methylbenzhydrylamine (MBHA) resin 1 that
8a,11
had been esterified with Fmoc-protected leucine,
was fol-
lowed by mild acidolytic cleavage from the solid support
8b,c
and ensuing solution-phase cyclization,
to provide Boc-
protected cyclic peptide 2 (86%). Reduction of the azide moiety
under Staudinger conditions gave free amine 3, that was
treated with succinic anhydride and subsequently condensed
with pentafluorophenol (Pfp) under the agency of 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to
furnish 4 in 80% over three steps. The activated ester in the
monomeric GS analogue was next reacted with a slight excess
of 3, to produce GS dimer 5. Acidolysis of the Boc-protection
groups still present on the Orn-residues followed by reversed-
phase HPLC purification, yielded the succinyl-tethered dimer 6
in 69%, the identity of which was confirmed by LC/MS analysis.
Encouraged by these results, we set out to connect turn-
modified GS analogues directly through newly introduced
amino acid side-chain functionalities. For this purpose, a set
of monomeric GS analogues, in which DPhe- and Pro-residues
residing in the same or opposing turn regions are replaced
† Electronic supplementary information (ESI) available: Selected NMR
spectra. See http://www.rsc.org/suppdata/ob/b4/b414618b/
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 3 3 – 2 3 8
2 3 3
©

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Scheme 1 Reagents and conditions: (i) ref. 8, 86%; (ii) PMe3 (1 M in toluene, 8 equiv), 1,4-dioxane/MeCN (1/1 v/v), 6 h; (iii) (a) succinic anhydride
(4 equiv), TEA (4 equiv), DMF, 16 h. (b) pentafluorophenol (2 equiv), EDC (2 equiv), DCM, 2 h, 80% in 3 steps; (v) 3 (1.1 equiv), DiPEA (2 equiv),
DMF, 48 h, 92%; (iv) TFA/DCM (1/1 v/v), 30 min, 69%.
by DGlu- and aminoproline (Amp)-residues, respectively, were
prepared as follows (Scheme 2). Commencing from solid support
1, fully protected cyclic peptides 7 and 8 were constructed as
described for 2 in 82 and 92% yield, respectively. Staudinger
reduction of the azide in 7 and 8 afforded the amines 9 and
10, respectively. Saponification of the ester moiety in 7 and 8
revealed their carboxylic acid counterparts that were directly
esterified with pentafluorophenol employing EDC to furnish 11
and 12. GS monomers 9–12 were used without further purifica-
tion in the following reaction steps. To facilitate characterization
by LC/MS analysis and to evaluate the biological profile of
the monomers, small aliquots of 7 and 8 were deprotected and
purified by HPLC to produce 13 and 14 in their respective yields
of 68 and 89%.
Having set the stage for coupling of the separate monomeric
building blocks, equimolar amounts of amine 9 and activated
ester 11 were reacted to furnish dimer 15 in 45%, as is depicted
in Scheme 3. Similarly, acylation of GS analogue 10 with Pfp-
ester 12 gave side-chain linked dimer 16 in 85%. The remaining
Boc groups in 15 and 16 were subsequently removed to provide,
after HPLC purification, their respective unprotected dimers 17
(66%) and 18 (31%). The azide and ester moieties in dimers 15
and 16 were also quantitatively transformed into the amine and
carboxylic acid functionalities present in 19 and 20 and were
subsequently deprotected to give dimers 21 (53%) and 22 (53%)
as gauged by LC/MS analysis.12
be effected by dropwise addition of the peptide solution to
a mixture of benzotriazole-1-yloxytris(dimethylamino)-phos-
phonium hexafluorophosphate (BOP), N-hydroxybenzotriazole
(HOBt) and N,N-diisopropylethylamine (DiPEA) in DMF. Lib-
eration of the Boc-protection groups yielded the rigid GS dimer
23 as the major product, albeit in 25% yield over two steps.
A similar intramolecular cyclization was also performed on
13a
peptide 20. Previous observations of b-sheet alignment in GS,
13b
and the proposed ‘cross-b’ aggregates by Goodman et al., were
expected to facilitate this cyclization reaction. However, only the
reaction conditions described for the cyclization of 19 proved to
be marginally effective in yielding product 24 (15%; Scheme 4).14
Having the various GS dimers in hand, attention was
focussed on the evaluation of their antimicrobial, hemolytic
and conductance-increasing properties. The capacity of the GS
dimers, and the monomers from which they are assembled, to
arrest the proliferation of several Gram-positive and -negative
bacterial strains was examined using a standard minimal
inhibitory concentration (MIC) test (Table 1).15 From these
results, it can be concluded that the modifications in the reverse
turn, an Azp- for a Pro-residue and a DGlu- for DPhe-residue in
monomers 13 and 14, have no adverse effect on the antimicrobial
properties compared to native GS. Conversely, the dimers had
lost virtually all activity towards the tested Gram-positive and
-negative strains with only dimer 22 displaying limited activity
against Staphylococcus epidermidis.
As a final synthetic objective, we investigated whether in-
tramolecular cyclization of 19 and 20 was feasible. Condensation
of the carboxylic acid and amine functionalities in 19 could
A standard assay was applied to assess the hemolytic activity
of the GS analogues towards human erythrocytes using a two-
fold dilution series of the appropriate peptide and interpolating
Scheme 2 Reagents and conditions: (i) ref. 8, 7; 82% and 8; 92%; (ii) PMe3 (1 M in toluene, 8 equiv), 1,4-dioxane/MeCN (1/1 v/v), 6 h; (iii) 1 M
NaOH, 1,4-dioxane, 4 h, then Amberlite IR-120 (H+); (iv) pentafluorophenol (2 equiv), EDC (2 equiv), DCM, 2 h; (v) TFA/DCM (1/1 v/v), 30 min,
13; 68% and 14; 89%.
2 3 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 3 3 – 2 3 8

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Scheme 3 Reagents and conditions: (i) DMF, 15; 45% and 16; 85%; (ii) TFA/DCM (1/1 v/v), 30 min, 17; 66%, 18; 31%, 21; 53% and 22; 53%;
(iii) PMe3 (1 M in toluene, 8 equiv), 1,4-dioxane/MeCN (1/1 v/v), 6 h; (iv) 1 M NaOH, 1,4-dioxane, 4 h, then Amberlite IR-120 (H+) 19; quant. and
20; quant. (two steps).
Scheme 4 Reagents and conditions: (i) BOP (5 equiv), HOBt (5 equiv), DiPEA (15 equiv), DMF, 16 h. (ii) TFA/DCM (1/1 v/v), 30 min, 23; 25%
and 24; 15% (two steps).
a
Table 1 Antimicrobial activity (MIC in lg ml−1
)
S. aureusb
S. epidermidisb
E. faecalisb
B. cereusb
E. colic
P. aeruginosac
Peptide
25Wd
MTe
25Wd
MTe
25Wd
MTe
25Wd
MTe
25Wd
MTe
25Wd
MTe
GS
6
8
>64
8
8
>64
8
4
64
4
4
>64
64–>64
>64
64
4
>64
8
n.d.
>64
16
8
>64
4–8
>64
8
4
>64
8
>64
>64
64
>64
>64
>64
>64
>64
>64
32
>64
64–>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
13
14
17
18
21
22
23
8–16
8–16
>64
>64
>64
>64
>64
8
8
8
8
8
8
>64
64–>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
a Measurements were executed using standard agar two-fold dilution techniques (n.d. = not determined). b Gram-positive. c Gram-negative d 3 ml/25
well plates e 100 ll/96 microtiter plates.
between 100% lysis induced by 1% Triton X-100 in saline and
a blank measurement. As can be gauged from the results in
Table 2, all tested peptides, both GS monomers and GS dimers,
cause considerable hemolysis in the 60 to 30 lM range, making
them similarly or even more (i.e. 17) toxic as the native peptide.
However, most GS dimers in this assay display the propensity to
lyse erythrocytes over a broader range of concentrations starting
as low as 1.0 to 0.5 lM but often reaching 100% lysis only at 250
to 125 lM.
Finally, we have conducted studies concerning the pore-
forming properties of GS, succinyl-tethered GS dimer 6 and
rigid GS dimer 23 (Fig. 1). Events of this type are commonly
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 3 3 – 2 3 8
2 3 5

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Table 2 Hemolytic activity (% hemolysis)a
Peptide concentration (lM)
Peptide
250.0
125.0
62.5
31.3
15.6
7.8
3.9
2.0
1.0
0.5
0.2
GS
6
100
50
92
2
3
3
1
80
62
52
35
100
70
76
52
64
3
5
7
7
1
9
2
6
4
50
65
21
25
93
62
52
41
51
1
3
3
2
3
7
2
5
3
24
49
3
0
6
1
0
2
32
0
2
5
0
0
2
3
2
1
1
0
8
0
0
4
0
1
3
3
1
0
1
0
8
0
0
14
6
3
1
1
1
1
0
0
3
1
4
0
4
0
3
0
0
6
0
0
0
0
0
3
0
0
2
0
1
1
0
n.d.
n.d.
n.d.
n.d.
0
n.d.
0
100
80
5
5
79
90
100
9
3
5
13
14
17
18
21
22
23
66
8
0
0
90 31
77
44
18
14
26
29
18
10
2
0
0
100
98
100
99
2
2
1
4
87
87
78
82
1
7
3
4
66
26
23
39
2
0
2
4
100
96
0
1
n.d.
n.d.
11
a Measurements were executed using standard two-fold dilution techniques. (n.d. = not determined).
Experimental
General
Reactions were monitored by TLC-analysis using DC-
fertigfolien (Schleicher & Schuell, F1500, LS254) with detection
by spraying with 20% H2SO4 in EtOH, (NH4)6Mo7O24·4H2O
(25 g L−1) and (NH4)4Ce(SO4)4·2H2O (10 g L−1) in 10% sulfuric
acid or by spraying with a solution of ninhydrin (3 g L−1) in
EtOH/AcOH (20/1 v/v), followed by charring at ≈150 ◦C. Size
exclusion chromatography was performed on SephadexTM LH-
20. For LC/MS analysis, a Jasco HPLC-system (detection
simultaneously at 214 and 254 nm) equipped with an analytical
Alltima C18 column (Alltech, 4.6 × 250 mm (id × l), 5 lm
particle size) in combination with buffers A: H2O, B: MeCN
and C: 0.5% aq. TFA and coupled to a Perkin Elmer Sciex
API 165 mass instrument with a custom-made Electronspray
Interface (ESI) was used. For reversed-phase HPLC purification
of the peptides, a BioCAD ‘Vision’ automated HPLC system
(PerSeptive Biosystems, inc.) supplied with a semi-preperative
Alltima C18 column (Alltech, 10.0 × 250 mm (id × l), 5 l particle
size) was used. The applied buffers were A: H2O, B: MeCN and
C: 1.0% aq. TFA.
Monomer 2. From resin 1 (200 mg, 0.1 mmol), the peptide
was constructed following a literature procedures to give the
Fig. 1 Conductivity traces for GS (A), dimer 6 (B) and dimer 23 (C)
8b,c
performed with 1 M KCl in a DPhPC/DPhPGlycerol 4 : 1 membrane.
title compound 2 (120 mg, 86 lmol) in 86% as a white amorphous
solid. To confirm the identity of 2, an aliquot was dissolved in
DCM (1 mL), cooled to 0 ◦C and TFA (1 mL) was added slowly.
After 30 min of stirring, the solvents were removed in vacuo
and the identity of peptide 2 was established by LC/MS: Rt
13.94 min; linear gradient 10→90% B in 20 min.; m/z = 1183.0
[M + H]+, 592.4 [M + H]2+.
Monomer 3. Azide 2 (26 mg, 19 lmol) was dissolved in 1,4-
dioxane (2 mL) and acetonitrile (2 mL), to which trimethylphos-
phine (0.15 mL, 0.15 mmol, 8 equiv, 1 M in toluene) was added.
The mixture was stirred for 3 h, water (0.1 mL) was added and
stirring was continued for another 3 h. Next, all solvents were
removed in vacuo and the crude peptide was thrice coevaporated
with dry toluene.
described as bursts and depending on the concentration, there is
a minimal voltage required for these bursts. Below 10−6 molar, no
effects were observed for GS and dimer 6, but at 10−6 molar they
induced rapid changes in conductances. Dimer 23 was slightly
more active, displaying conductance-increasing effects at 10−7
molar concentrations. Although all three compounds showed
conductance-increasing properties,16 no series of discrete single
channels could be resolved.
In summary, we have described the synthesis of GS analogues
that have been modified in the b-turn region, with DPhe residues
being replaced by DGlu(OEt) and Pro-residues by Azp-residues.
These GS monomers have subsequently been covalently linked
either via a succinyl-tether or directly through their side-chains
to produce several differently functionalized GS dimers. In-
tramolecular cyclization also produced more conformationally
restricted dimers, albeit in moderate yields. As was previously
observed with a GS dimer,17 no bactericidal effect against either
Gram-positive or -negative strains could be detected. However,
the GS dimers displayed a significant increase in hemolytic
activity. Moreover, these compounds proved hemolytic over
a broader range of concentrations, which might suggest a
different mode of action on the lipid bilayer. Upon studying
the conductance-increasing properties of selected GS-dimers,
they showed membrane disruptive properties but no discrete
channels could be observed.
Monomer 4. Crude peptide 3 was dissolved in DMF
(2 mL) and succinic anhydride (76 mg, 76 lmol, 4 equiv)
and triethylamine (76 mg, 76 lmol, 4 equiv) were added.
After stirring for 16 h, the solvent was evaporated and the
mixture was directly applied to a LH-20 column that was eluted
with MeOH. The peptide-containing fractions were pooled,
evaporated and redissolved in DCM (2 mL). To the solution were
added pentafluorophenol (6.3 mg, 34 lmol, 2 equiv) and EDC
(6.5 mg, 34 lmol, 2 equiv) and stirring was continued for 2 h. The
mixture was subsequently concentrated and partitioned between
0.1 M HCl and CHCl3. The organic layer was dried (MgSO4),
filtered and concentrated, to furnish the activated ester 4 (22 mg,
14 lmol) in 80% over 3 steps as amorphous white solid.
2 3 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 3 3 – 2 3 8

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Dimer 5. To Pfp-ester 4 (22 mg, 14 lmol) in DMF (0.5 mL),
a fresly prepared batch of amine 3 (22 mg, 16 lmol, 1.1 equiv) in
DMF (0.5 mL) was added and the resulting mixture was stirred
for 48 h. The solvents were removed under reduced pressure and
the mixture separated by size-exclusion chromatography using
MeOH as eluent. The fractions containing peptide were pooled
and evaporated to dryness to produce the title compound 5
(36 mg, 13 lmol, 92%) as amorphous white solid.
771.8 [M + H]3+, 597.4 [M + H]4+ and 18: Rt 18.71 min; linear
gradient 10→90% B in 30 min; m/z = 1157.6 [M + H]2+, 772.0
[M + H]3+, 597.4 [M + H]4+. Subsequent purification by RP-
HPLC of 17: linear gradient of 3.0 CV; 55→70% B; Rt 2.2
CV and 18: linear gradient of 3.0 CV; 50→60% B; Rt 2.9 CV,
followed by freeze-drying, furnished 17 (7.4 mg, 3.2 lmol, 66%)
and 18 (10.4 mg, 4.5 lmol, 31%) as white amorphous powders.
Dimer 19 and 20. Peptide 15 (55 mg, 20 lmol) was dissolved
in 1,4-dioxane (2 mL) and MeCN (2 mL) and PMe3 (0.16 mL,
0.16 mmol, 8 equiv, 1 M in toluene) was added. The solution
was stirred for 3 h, water (0.1 mL) was added and stirring
was continued for another 3 h. All solvents were evaporated
and the crude peptide was redissolved in EtOH (2 mL) and
1 M aq. NaOH (0.5 mL) was added. After stirring for 2 h,
Amberlite IR-120 (H+) was added and the neutral solution
was subsequently concentrated and coevaporated thrice with
dry toluene to quantitatively provide 19 (53 mg, 20 lmol)
as amorphous solid. Dimer 16 (20 mg, 7.5 lmol) treated as
described above to quantitatively furnish 20 (20 mg, 7.5 lmol).
Dimer 6. The dimer 5 (18 mg, 6.4 lmol) was deprotected as
described for monomer 2 to give crude 6, that was analyzed by
LC/MS (Rt 15.26 min; linear gradient 10→90% B in 20 min.;
m/z = 2395.8 [M + H]+, 1198.6 [M + H]2+, 799.6 [M + H]3+)
and purified by reversed-phase HPLC (linear gradient of 3.0 CV;
50→60% B; Rt 3.1 CV) to give dimer 6 (10.6 mg, 4.4 lmol, 69%)
as a fluffy white solid.
Monomer 7 and 8. From resin 1 (500 mg, 0.25 mmol), the
8b,c
peptides were assembled according to literature procedure
to furnish cyclic peptides 7 (299 mg, 0.21 mmol, 82%) and 8
(320 mg, 0.23 mmol, 92%) as a white solids.
Monomer 9 and 10. Azides 7 (125 mg, 90 lmol) and 8
(125 mg, 90 lmol) were individually treated with PMe3, as
described for monomer 3, to furnish crude amines 9 and 10
as white solids that were directly used in the next reaction step.
Dimer 21 and 22. Dimers 19 (15 mg, 5.4 lmol) and 20 (20 mg,
7.5 lmol) were treated as described for 13, to give crude 21 and
22, respectively, and were analyzed by LC/MS; 21: Rt 17.10 min;
linear gradient 10→90% B in 30 min; m/z = 1130.5 [M + H]2+,
754.0 [M + H]3+, 556.7 [M + H]4+ and 22: Rt 16.40 min; linear
gradient 10→90% B in 30 min; m/z = 1130.6 [M + H]2+, 754.0
[M + H]3+, 565.7 [M + H]4+. Subsequent purification by RP-
HPLC of 21: linear gradient of 3.0 CV; 40→60% B; Rt 2.7
CV and 22: linear gradient of 3.0 CV; 40→55% B; Rt 2.1 CV,
followed by freeze-drying, furnished 21 (6.5 mg, 2.9 lmol, 53%)
and 22 (8.9 mg, 3.9 lmol, 53%) as white amorphous powders.
Monomer 11 and 12. Ethyl esters 7 (125 mg, 90 lmol) and 8
(125 mg, 90 lmol) were individually dissolved in EtOH (5 mL)
and 1 M aq. NaOH (0.5 mL) was added. After stirring the
mixtures for 2 h, TLC analysis showed completed conversion
of starting material and Amberlite IR-120 (H+) was added. The
neutral solutions were subsequently concentrated, coevaporated
thrice with dry toluene and the crude acids were individually
redissolved in DCM. To these were added pentafluorophenol
(33 mg, 0.18 mmol, 2 equiv) and EDC (35 mg, 0.18 mmol,
2 equiv) and stirring was continued for 2 h. The mixture was
diluted with CHCl3 and extracted with 0.1 M HCl after which
the organic layer was dried (MgSO4), filtered and concentrated,
to furnish the crude Pfp-esters 11 and 12 that were directly used
in the next reaction step.
Dimer 23. The crude 19 (45 mg, 20 lmol) was taken up in
DMF (3 mL) and slowly added to a solution of BOP (52 mg,
100 lmol, 5 equiv), HOBt (14 mg, 100 lmol, 5 equiv) and DiPEA
(50 lL, 300 lmol, 15 equiv) in DMF (8 mL). The reaction was
then stirred overnight, concentrated and the resulting mixture
applied to a size-exclusion column that was eluted with MeOH,
after which the peptide-containing fractions were combined and
evaporated to dryness. Deprotection, as descibed for 13, was
followed by LC/MS analysis (Rt 19.10 min, linear gradient
10→90% B in 30 min; m/z = 1121.4 [M + H]2+, 747.8 [M +
H]3+, 561.1 [M + H]4+ of the crude product, purification by RP-
HPLC (linear gradient of 4.0 CV; 50→65% B; Rt 3.4 CV) and
lyophilization, to produce title compound 23 (8.4 mg, 4.5 lmol,
25%) as white amorphous powder over 2 steps.
Monomer 13. To a cooled solution of cyclic peptide 7 (25 mg,
18 lmol) in DCM (4 mL) was added TFA (4 mL) and the mixture
was stirred for 30 min after which it was evaporated to dryness.
The crude product was analyzed by LC/MS (Rt 18.53 min, linear
gradient 10→90% B in 30 min; m/z = 1193.1 [M + H]+, 597.0
[M + H]2+), purified by RP-HPLC (linear gradient of 3.0 CV;
50→60% B; Rt 2.0 CV) and lyophilized, to produce peptide 13
(14.7 mg, 12 lmol, 68%) as white amorphous powder.
Dimer 24. The crude 20 (20 mg, 7.5 lmol) was treated as
described for 23, to give the crude product (LC/MS analysis:
Rt 17.80 min, linear gradient 10→90% B in 30 min; m/z =
1121.8 [M + H]2+, 748.4 [M + H]3+) that was purified by RP-
HPLC (linear gradient of 4.0 CV; 40→65% B; Rt 3.2 CV) and
freeze-dried, to furnish in 2 steps the title compound 24 (2.5 mg,
1.1 lmol, 15%) as white amorphous powder.
Monomer 14. The cyclic peptide 8 (30 mg, 22 lmol) was
similarly deprotected as for 13, to give the crude peptide that was
analyzed by LC/MS (Rt 18.48 min; linear gradient 10→90% B
in 30 min; m/z = 1193.1 [M + H]+, 597.0 [M + H]2+), purified
by RP-HPLC (linear gradient of 3.0 CV; 50→60% B; Rt 2.3 CV)
and lyophilized, to produce 14 (23.1 mg, 19 lmol, 89%) as white
amorphous powder.
Antimicrobial activity. The following bacterial strains were
used: Staphylococcus aureus (ATCC 29213), Staphylococcus epi-
dermidis (ATCC 12228), Enterococcus faecalis (ATCC 29212),
Bacillus cereus (ATCC 11778), Escherichia coli (ATCC 25922)
and Pseudomonas aeruginosa (ATCC 27853). Bacteria were
Dimer 15 and 16. Pfp-ester 11 was dissolved in DMF (2 mL)
and a solution of amine 9 in DMF (2 mL) was slowly added. To
this mixture, DiPEA (29 lL, 0.18 mmol, 2 equiv) was added and
the reaction was stirred 48 h. The solvents were subsequently
removed in vacuo and the resulting mixture applied to a size-
exclusion column that was eluted with MeOH. Pooling of the
peptide-containing fractions gave dimer 15 (109 mg, 40 lmol,
45%) as white amorphous solid. Monomer 12 was treated in
an equal manner with amine 10 to furnish dimer 16 (207 mg,
76 lmol, 85%) as white amorphous solid.
◦
◦
stored at −70 C and grown at 35 C on Columbia Agar with
sheep blood (Oxoid, Wesel, Germany) overnight and diluted in
0.9% NaCl. Microtitre plates (96 wells of 100lL) as well as large
plates (25 wells of 3 mL) were filled with Mueller Hinton II
Agar (Becton Dickinson, Cockeysvill, USA) containing serial
two-fold dilutions of the peptides. To the wells were added 3 lL
of bacteria, to give a final inoculum of 104 colony forming units
Dimer 17 and 18. Dimers 15 (13 mg, 4.8 lmol) and 16 (40 mg,
14.7 lmol) were treated as described for 13, to give crude 17 and
18, respectively, and were analyzed by LC/MS; 17: Rt 15.97 min;
linear gradient 10→90% B in 20 min; m/z = 1157.5 [M + H]2+,
◦
(CFU) per well. The plates were incubated overnight at 35 C
and the MIC was determined as the lowest concentration
inhibiting bacterial growth.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 3 3 – 2 3 8
2 3 7

View Article Online
Hemolytic activity. The hemolytic activity of the peptides
was determined in quadruplo. Human blood was collected
into EDTA-tubes and centrifuged to remove the buffy coat.
The residual erythrocytes were washed three times in 0.85%
saline. Serial two-fold dilutions of the peptides in saline were
prepared in sterilized round-bottom 96-well plates (polystyrene,
U-bottom, Costar) using 100 lL volumes (500–0.5 lM). Red
blood cells were diluted with saline to 1/25 packed volume of
cells and 50 lL of the resulting cell suspension was added to each
well. Plates were incubated while gently shaking at 37 ◦C for 4 h.
Next, the microtiter plate was quickly centrifuged (1000 g, 5 min)
and 50 lL supernatant of each well was transported into a flat-
bottom 96-well plate (Costar). The absorbance was measured at
405 nm using a mQuant microplate spectrophotometer (Bio-Tek
Instruments). The Ablank was measured in the absence of additives
and 100% hemolysis (Atot) in the presence of 1% Triton X-100
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in saline. The percentage hemolysis is determined as (Apep
−
Ablank)/(Atot − Ablank)100.
Conductivity measurements. Planar lipid membranes were
prepared by painting a solution of diphytanoylphosphatidyl-
cholin (DPhPC, Avanti Polar Lipids, Alabaster, AL) in n-decane
(25 mg ml−1) over the aperture of a polystyrene cuvette with a di-
ameter of 0.15 mm.18 All experiments were performed at ambient
temperature. The used electrolyte solutions at a concentration
of 1 M each were unbuffered. Probes were dissolved in methanol
and added to the trans or cis side (containing the measuring
electrode) of the cuvette. Current detection and recording was
performed with a patch-clamp amplifier Axopatch 200B, a Digi-
data A/D converter and pClamp6 software (Axon Instruments,
Foster City, MA). The acquisition frequency was 5 kHz. The
data were filtered with an digital filter at 50 Hz for further
analysis.
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J. Am. Chem. Soc., 2002, 124, 1203–1213; (b) K. Yamada, M. Unno,
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Katakai and M. Kawai, J. Am. Chem. Soc., 2002, 124, 12684–12688;
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Acknowledgements
This work was financially supported by the Council for Chemical
Sciences of the Netherlands Organization for Scientific Research
(CW-NWO), the Netherlands Technology Foundation (STW)
and DSM Research. We thank Nico Meeuwenoord and Hans
van den Elst for their technical assistance. Kees Erkelens and
Fons Lefeber are gratefully acknowledged for assistance with
NMR experiments.
12 The sequence of reactions proved to be essential, as LC/MS analysis
of the crude mixtures of 21 and 22 indicated that the remaining
Glu-residues were unexpectedly converted into their methyl ester
counterparts when Staudinger reduction of the azides was performed
following the saponification. Fortunately, this side-reaction was not
observed when the order of reactions was reversed.
13 (a) K. Yamada, H. Ozaki, N. Kanda, H. Yamamura, S. Araki and
M. Kawai, J. Chem. Soc. Perkin Trans. 1, 1998, 3999–4004; (b) R. T.
Ingwall, C. Gilon and M. Goodman, J. Am. Chem. Soc., 1975, 97,
4356–4362.
14 Variation of the reaction conditions (e.g. the reaction sequence,
coupling reagents and their order of addition) did not improve the
cyclization results.
15 The set-up used for antimicrobial testing allows for an experimental
error that can be approximately one dilution range, and the MIC
values therefore need to be referenced to the native peptide GS. This
also explains the deviation from earlier reported MIC values of GS.
16 (a) R. E. W. Hancock and A. Rozek, FEMS Microbiol. Lett., 2002,
206, 143–149; (b) M. Wu, E. Maier, R. Benz and R. E. W. Hancock,
Biochemistry, 1999, 38, 7235–7242.
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O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 3 3 – 2 3 8