Tuning hydrophobicity of highly cationic tetradecameric Gramicidin S analogues using adamantane amino acids

Article abstract of DOI:10.1016/j.bmc.2010.09.018

Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane amino acids resulted in a small set of compounds with varying

Full text of DOI:10.1016/j.bmc.2010.09.018

Bioorganic & Medicinal Chemistry 18 (2010) 8403–8409
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tuning hydrophobicity of highly cationic tetradecameric Gramicidin
S analogues using adamantane amino acids
Annemiek D. Knijnenburg a, Varsha V. Kapoerchan a, Emile Spalburg b, Albert J. de Neeling b,
Roos H. Mars-Groenendijk c, Daan Noort c, Gijs A. van der Marel a, Herman S. Overkleeft a, Mark Overhand a,
⇑
a Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
b National Institute of Public Health and the Environment, Laboratory for Infectious Diseases PO Box 1, 3720 BA Bilthoven, The Netherlands
c TNO Defense, Security and Safety, Business unit Biological and Chemical Protection, Prins Maurits Laboratory, PO Box 45, 2280 AA Rijswijk, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues
were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane
amino acids resulted in a small set of compounds with varying amphipathic character. It was found that
the amphipathicity of these compounds is correlated to their biological activity. Several bacterial strains
including MRSA strains were shown to be killed by the novel peptides. The most potent antibacterial pep-
tides are tetradecameric GS analogues containing six positives charges and two adamantane moieties.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 11 August 2010
Revised 3 September 2010
Accepted 7 September 2010
Available online 24 September 2010
Keywords:
Gramicidin S
Cationic antimicrobial peptides
Amphipathicity
b-Sheet
MRSA
5
D
1. Introduction
The antibiotic Gramicidin S (GS, cyclo-(PVOL F)2) is a well-stud-
ied member of the CAP family. GS adopts a cyclic b-hairpin structure
Cationic antimicrobial peptides (CAPs) are amphipathic
peptides that often contain either an -helix or a b-sheet as a
in solution that is stabilized by four intra-molecular hydrogen
bonds. The side-chains of the Val and Leu residues and the side-
chains of the Orn residues create a hydrophobic and a hydrophilic
face, giving the cyclic b-hairpin structure its amphipathic charac-
ter.6,7 GS disrupts bacterial membranes and is able to kill Gram-po-
sitive and certain Gram-negative bacteria, however its use is limited
only as topical antibiotic because of its haemolytic activity.8
a
distinguishing secondary structural element. They are produced
by prokaryotes and eukaryotes and are part of the host defence
mechanism against invading bacteria. As their mechanism of
action is not specifically based on cellular targets, but rather aimed
at targeting the cell membrane as a whole, this class of peptides are
promising leads for the development of new bactericidal agents.
The biological activity of membrane disrupting antimicrobial
peptides is frequently based on their common characteristics such
as their size, number of positive charges and amphipathicity.1 As
yet, no effective resistance mechanisms are reported against this
class of peptides.2–4
Spanning several decades, numerous derivatives based on GS
have been investigated with the aim to find new molecules with
an improved biological profile. It appears that the amphipathicity
of the GS derivative is crucial for its biological activity.9–12 We
recently reported an approach to vary the amphipathicity by the
synthesis of ‘reversed’ GS derivatives,13,14 as exemplified by the
structure of 1 (Fig. 1).15 In this compound two central hydrophobic
adamantane amino acids are flanked by four Orn residues to render
the molecule its positive charge. The molecule adopts a cyclic
b-hairpin structure dependent on the solvent. Analogue 1 proved
to be much less haemolytic as GS, while at the same time being
potently active against several bacterial strains, including certain
MRSA strains.15 Another approach we16 and others17–22 have
pursued involves the synthesis of tetradecameric GS derivatives,
such as compound 216 (Fig. 1). This compound also has four posi-
tive charges and adopts an extended cyclic b-hairpin structure. In
both of these approaches variation of the number and position of
the hydrophilic and hydrophobic side-chains to fine-tune the
Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; HMPB, 4-(4-
hydroxymethyl-3-methoxyphenoxy)butyric acid; BHA, benzhydrylamine; NMR,
nuclear magnetic resonance; CD, circular dichroism; TOCSY, total correlation
spectroscopy; TFE, 2,2,2-trifluoroethanol; MIC, minimal inhibitory concentration;
RP-HPLC, reversed phase high performance liquid chromatography; SPPS, solid
phase peptide synthesis; NMP, 1-methyl-2-pyrrolidone; DiPEA, N,N0-diisopropyl-
ethylamine; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate methanaminium; TFA, 2,2,2-trifluoroacetic acid; DCM, dichlo-
romethane; DMF, N,N0-dimethylformamide; HOBt, N-hydroxybenzotriazole; PyBOP,
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; Fmoc,
9-fluorenylmethyloxycarbonyl.
⇑
Corresponding author. Tel.: +31 715274483; fax: +31 715274307.
E-mail address: overhand@chem.leidenuniv.nl (M. Overhand).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.018

8404
A. D. Knijnenburg et al. / Bioorg. Med. Chem. 18 (2010) 8403–8409
+
+H3N
+H3N
O
NH3
+
NH3
O
O
H
N
H
N
O
O
H
N
H
N
H
N
N
H
O
N
H
N
H
O
N
H
O
N
N
N
O
O
O
O
H
O
O
O
O
O
H
H
H
H
H
N
N
O
N
N
O
N
N
N
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
+H3N
NH3
+
+H3N
+
NH3
2
+H3N
+H3N
+H3N
R1
R2
3: R1 = R4 = CHMe2; R2 = R3 = CH2CHMe2
O
4: R1 = R4 = CHMe2; R2 = CH2adamantyl; R3 = CH2CHMe2
5: R1 = adamantyl; R2 = R3 = CH2CHMe2; R4 = CHMe2
6: R1 = adamantyl; R2 = CH2adamantyl; R3 = CH2CHMe2; R4 = CHMe2
7: R1 = R4 = adamantyl; R2 = R3 = CH2CHMe2
8: R1 = CHMe2; R2 = CH2adamantyl; R3 = CH2CHMe2; R4 = adamantyl
9: R1 = R4 = CHMe2; R2 = R3 = CH2adamantyl
10: R1 = CHMe2; R2 = R3 = CH2adamantyl; R4 = adamantyl
11: R1 = R4 = adamantyl; R2 = CH2adamantyl; R3 = CH2CHMe2
12: R1 = R4 = adamantyl; R2 = R3 = CH2adamantyl
O
H
O
H
N
H
N
N
N
H
O
N
H
O
N
N
H
O
O
O
O
O
H
H
H
N
O
N
N
N
N
H
N
H
N
H
O
O
O
R3
R4
+H3N
+H3N
+H3N
Figure 1. Extended ‘reversed’ GS analogues containing adamantane amino acids and six cationic residues.
amphipathicity of the GS derivatives plays a key role. We decided
to design a series of compounds according to both approaches. For
all compounds we used Orn as cationic residue and Phe-Pro as
and a slight minimum around 220 nm in a mixture of TFE/H2O,
indicative of a b-sheet/b-hairpin structure (Fig. 2).26,27 CD spectra
recorded in methanol show the same CD curves as for TFE/H2O (re-
ported in Supplementary data).
D
two-residue turn element. Starting from extended ‘reversed’ GS
derivative 3, we systematically replaced either one or both hydro-
The cyclic peptides 3–12 were screened for antibacterial activ-
ity against several Gram-negative and Gram-positive strains,
including six MRSA bacterial strains (Table 1). In addition, their
haemolytic properties were investigated (Table 2). For comparison
our observed MIC values and haemolytic data of previously re-
ported compounds GS, GS14,18 1,15 and compound 2,16 are also in-
cluded in Tables 1 and 2. Compounds 3 and 12 showed the lowest
antibacterial activity in this series of compounds. Interestingly,
several compounds are more active than GS against both Gram-
negative and Gram-positive strains. Especially compounds 5, 7
and 9 containing two adamantane moieties are potently active
against the six MRSA strains. Compound 3 is the least haemolytic
in this series and compounds 4 and 5 are slightly less haemolytic
as compared to the natural product GS (Table 2). The other deriv-
atives are more haemolytic than GS.
Determination of retention time under controlled conditions on
RP-HPLC can be used as a measure of peptide hydrophobicity.28,29
However, it is reported30,31 that interactions of reversed-phase
matrices with hydrophobic binding domains of the peptide are also
influenced by the secondary structure of a peptide. To ensure that
all the peptides exhibit a comparable secondary structure on the
reversed-phase column, the observed retention times of peptides
3–12 were correlated with their hydrophobic moment.32 The
hydrophobic moment is a theoretical value of a peptide sequence
indicating its amphipathic character. The calculation is based on
the hydrophobicity of the constituting amino acids and secondary
structure of the peptide. The retention times correlate well (r = 1.0,
R2 = 0.99, see Supplementary data) with the hydrophobic moments
of peptides 3–12 (Table 2). As anticipated, compound 3 is the most
polar peptide in this series and compound 12 containing four
adamantane moieties the most hydrophobic. Compound 3 is not
haemolytic but also not antibacterial active, whereas compound
12 is highly haemolytic without major bactericidal activity. The
properties of these molecules are in agreement with data on other
phobic Val and Leu residues by adamantyl-
L
-glycine23 and
adamantyl-
L
-alanine23, respectively (4–12, Fig. 1) to create a varia-
tion in hydrophobicity at the a-polar face of the molecule. We here
present the synthesis, structural analysis and biological evaluation
of the compounds 3–12 and compare the outcome with their par-
ent compounds.
2. Results and discussion
All peptides were prepared using a standard automated step-
wise solid phase peptide synthesis protocol and the highly acid
sensitive HMPB–BHA resin24 preloaded with Fmoc protected orni-
thine 13 (Scheme 1). After mild acidic cleavage from the solid sup-
port, the partially protected linear peptides containing six Orn(Boc)
residues were cyclised under high dilution conditions and purified
by gel filtration to obtain the partially protected cyclised peptides
in yields ranging from 45% to 70%. Removal of the Boc protective
groups, using strong acidic conditions, was followed by preparative
HPLC purification to give the desired cyclic peptides 3–12 in
10–35% overall yield (Scheme 1).
The secondary structure of the peptides was evaluated using
NMR and CD spectroscopy. NMR spectra were recorded in various
solvents, but the 1-D spectra were not that well resolved to deter-
mine homonuclear couplings constants. In the 2-D TOCSY spectra
of analogues 3–12, the geminal coupling between the two d
protons of the Pro residues were determined. The chemical shift
perturbation value
(DdProdd,u) of these protons reflects the
deformation of the turn propensity of the Pro residue as part of a
b-hairpin structure, also called b-sheet content.25 According to this
method all synthesized peptides showed a similar amount of
b-sheet content, having a
DdPro ranging from 0.8 to 0.9 ppm. This
finding was corroborated by recording their CD spectra. Peptides
3–12 gave CD curves with negative ellipticities around 210 nm

A. D. Knijnenburg et al. / Bioorg. Med. Chem. 18 (2010) 8403–8409
8405
NH2-DF-P-O-X4-O-X3-O-DF-P-O-X1-O-X2-O-HMPB-BHA
i,ii
Fmoc-O-HMPB-BHA
13
iii, iv, v
+H3N
+H3N
+H3N
R1
R2
O
O
H
N
O
H
N
H
N
Rn
N
H
O
N
H
O
N
N
H
O
O
O
O
O
N
H
H
H
H
O
N
O
N
N
N
N
H
N
H
N
H
Xn
O
O
O
n = 1-4
R3
R4
+H3N
+H3N
+H3N
3-12
Scheme 1. Synthesis of GS analogues 3–12 using automated SPPS. Reagents and conditions: (i) deprotection: 20% pip/NMP; (ii) coupling: standard Fmoc-AA-OH (5 equiv),
HATU (90%), DiPEA, NMP, 30 min or Fmoc-Adamantyl-
L
-glycine, Fmoc-Adamantyl-
L
-alanine (1.5 equiv), HATU (90%), DiPEA, NMP, 30 min; (iii) cleavage: 1% TFA, DCM; (iv)
cyclisation: PyBOP, HOBt, DiPEA, DMF, 16 h; (v) 95% TFA, 2.5% TIS, 2.5% H2O.
A general trend in the correlation between amphipathicity of
the molecules and the haemolytic activity was observed. That is,
the most hydrophobic molecule 12 displayed the highest haemo-
lytic activity, whereas the least hydrophobic molecule 3 had negli-
gible haemolytic activity. These findings are in agreement with
other hydrophilic and hydrophobic peptides.22 An optimum in
antimicrobial activity was observed for compound series 3–12.
The outer limits of the peptide series, 3 and 12, showed low anti-
microbial activity. The relation of antimicrobial activity is complex
due to the diversity in membrane structures of microorgan-
isms.33–35 A possible explanation for the observed trend in antimi-
crobial activity might be the subtle equilibrium between
amphipathicity and overall hydrophobicity. Probably compound
3 is too hydrophilic to have any affinity with the lipid bilayers
(erythrocytes membrane or bacterial membrane) whereas com-
pound 12 is too hydrophobic and might bind strongly to bacterial
cell wall elements.35 Peptides 4 and 5 showed less haemolytic
activity compared to GS and still retained a similar antimicrobial
activity, leading to a better therapeutic profile compared to GS,
compound 216 and GS1418 (Table 2). It must be said that our pre-
viously reported ‘reversed’ adamantane compound 115 outper-
forms all these peptides, by being potently antimicrobial and
having low haemolytic activity. The peptides containing two ada-
mantane amino acids 6–9 displayed the highest antimicrobial
activity of all peptides tested and also displayed very good antimi-
crobial activity against MRSA strains. Unfortunately these mole-
cules appeared to be highly haemolytic as well, and further
modifications need to be pursued in order to discover bactericidal
GS derivatives for in vivo applications to treat systemic bacterial
infections.
Figure 2. CD-spectra of 3–12 analogs containing six basic residues. CD spectra
recorded in 0.1 mM in 50% TFE/0.01 M NaOAc (pH 5.3).
hydrophilic and hydrophobic peptides.22 The amphipathic
characteristics of compounds 3 and 12 thus form the outer limits
within this series. Compounds 4–7 are the most antibacterial
active peptides of this series having amphiphatic characteristics
corresponding to a hydrophobic moment around 10
however, these peptides are also very haemolytic.
l (Table 2),
3. Conclusion
We here presented our investigation of a series of novel antimi-
crobial peptides (3–12) in which two design approaches, ‘reversed’
GS derivatives13–15 and tetradecameric GS derivatives16–22, were
combined. These analogues of the natural product Gramicidin S
contain fourteen amino acid residues of which six are cationic
and in which the Val and Leu residues are systematically replaced
by adamantane amino acids. We found that the elongated reversed
GS analogues (3–12) adopt cyclic b-hairpin structures similar to
the tetradecameric derivatives reported previously. Having estab-
lished that the compound series exhibit similar secondary struc-
tures, we conclude that differences in biological activity are
mainly based on their varied amphipathic character.
4. Experimental section
Peptides were synthesized on an Applied Biosystems 433A
Peptide Synthesizer. LC/MS analyses (detection simultaneously at
214 and 254 nm) were performed on a LCQ Adventage Max
(Thermo Finnigan) equipped with
(250 mmL ꢀ 4.6 mmD, 5 particle size, Phenomenex). The applied
buffers were A: H2O, B: MeCN and C: 1.0% aq TFA. High resolution
mass spectra were recorded by direct injection (2 L of a 2
solution in H2O/MeCN; 50/50: v/v and 0.1% formic acid) on a mass
sp-ectrometer Thermo Finnigan LTQ Orbitrap equipped with an
electrospray ion source in positive mode (source voltage 3.5 kV,
a Gemini C18 column
l
l
lM

8406
A. D. Knijnenburg et al. / Bioorg. Med. Chem. 18 (2010) 8403–8409
Table 1
Antimicrobial activity (MIC, mg/L) of GS, GS14 and GS analogues 1–12
Analoguesa S. aureusb S. epider
E.
B. cereusb
ATCC
P.
aeruginosac ATCC
E. colic
MRSAb
MRSAb
MRSAb MRSAb MRSAb
MRSAb
ATCC
ATCC
midisb
faecalisb
1110301146 1110301981 N229
N133
ATCC
29213
ATCC 12228 ATCC
29212
11778
ATCC
27853
25922
49775
43300
GS
32
>64
8
16
64
16
16
8
8
8
8
16
16
64
8
64
4
8
8
8
4
4
8
4
8
4
8
8
32
8
8
32
8
4
32
8
8
1
2
4
64
>64
16
64
32
16
16
16
16
16
32
32
32
64
32
>64
8
32
64
16
8
16
—
8
16
—
8
—
64
8
8
16
8
16
8
16
8
8
—
8
16
—
8
—
>64
8
8
16
8
32
8
16
8
8
—
—
—
64
8
8
16
8
16
8
16
16
32
8
—
—
—
>64
8
8
16
8
16
8
16
16
32
GS1418
1
2
3
4
5
6
7
8
8
—
—
>64
32
32
8
8
16
8
8
8
32
>64
16
16
16
8
16
8
32
8
64
16
8
16
8
16
8
16
8
8
16
16
8
16
16
64
9
8
4
4
2
10
11
12
32
32
32
32
32
a
Molecular weight GS: 1369.49; GS14: 2126.23; 1: 1783.79; 2: 2038.12; 3: 2282.17; 4,5: 2374.31; 6–9: 2466.45; 10,11: 2558.59; 12: 2650.72. GS14 is an 14-meric
D
D
analogue of GS with Tyr and Lys instead of Phe and Orn (Ref. 18).
b
Gram-positive bacteria, MIC in mg/L. For detailed experimental set up: see Section 4.
Gram-negative bacteria, MIC mg/L.
c
Table 2
Correlation of physical and biological properties of peptide 3–12
Analogue
Sequencea
Observed
retention
timeb
Hydrophobic
moment (l)
Haemolytic
activityd
Antimicrobial
activitye
Gram positive
bacteria
Antimicrobial
activityf
Gram negative
bacteria
Therapeutic
indexg
S. aureus
Therapeutic
indexh
E. coli
c
D
GS
cyclo-(PVOL F)2
8.38
7.26
6.39
8.00
4.02
4.58
4.69
5.09
5.13
5.16
5.23
5.63
5.69
6.12
—
—
—
—
62.5
3.9
500
+
+/ꢁ
ꢁ
2.0
0.03
62.5
0.2
11.7
7.8
7.8
3.9
3.9
3.9
2.0
0.03
62.5
0.1
11.7
7.8
15.6
3.9
2.0
2.0
3.9
1.0
1.0
GS1418
cyclo-(PVKLKV YPLKVKL Y)
cyclo-(POXglyO F)2
cyclo-(PVOLOV FPLOVOL F)
cyclo-(POVOLO F)2
cyclo-POVOXalaO FPOVOLO F
cyclo-POXglyOLO FPOVOLO F
cyclo-POXglyOXalaO FPOVOLO F
cyclo-(POXglyOLO F)2
cyclo-POVOXalaO FPOXglyOLO F
cyclo-(POVOXalaO F)2
cyclo-POVOXalaO FPOXglyOXalaO F
cyclo-POXglyOXalaO FPOXglyOLO F
+/ꢁ
++
++
ꢁ
D
D
D
1
2
3
4
5
6
7
8
+
D
D
3.9
+/ꢁ
+/ꢁ
++
++
++
++
++
+
+
+
+/ꢁ
D
7.3
8.5
8.6
9.8
9.9
9.9
10.0
11.1
11.2
12.5
>500
125
125
31.3
31.3
31.3
31.3
15.6
15.6
7.8
D
D
+
D
D
++
++
++
++
++
++
++
+/ꢁ
D
D
D
D
D
D
9
3.9
1.0
1.0
0.1
D
D
10
11
12
D
D
D
cyclo-(POXglyOXalaO F)2
0.12
a
Linear sequences of cyclic peptides. One-letter amino acid code is used; amino acids with superscripted
ala = Adamantyl- -alanine.
Observed retention time on RP-HPLC at 25 °C (for more information see Section 4).
D represent D-amino acids. Xgly = Adamantyl-L-glycine;
X
L
b
c
The hydrophobic moment is only calculated for the homologues series 3–12. Calculated hydrophobic moment (
l) with values for Leu, Val, Orn, Pro, and Phe as 9.7, 4.1,
ꢁ9.0, ꢁ0.2 and 10, respectively. The values for Xgly and Xala were calculated as 24.1 and 28.7 (see Section 4).
d
Peptide concentration (in
l
M) required for 100% lysis of erythrocytes. Haemolytic curves reported in Supporting information.
General trend observed for Gram positive antimicrobial activity with most MIC (mg/L) values in the range of: ꢁ = 64–>64 mg/L; +/ꢁ = 32–64 mg/L; + = 8–32 mg/L, ++ = 1–
16 mg/L.
e
f
General trend observed for Gram negative antimicrobial activity with most MIC (mg/L) values in the range of: ꢁ = 64–>64 mg/L; +/ꢁ = 32–64 mg/L; + = 8–32 mg/L, ++ = 1–
16 mg/L.
g
Therapeutic index of S. aureus = haemolytic activity at 100% lysis/MIC. For calculation of the therapeutic index, values of 128 mg/L were used for MIC values of >64 mg/L,
and values of 750 were used for haemolytic activity values of >500
M.18
Therapeutic index of E. coli = haemolytic activity at 100% lysis/MIC. For calculation of the therapeutic index, values of 128 mg/L were used for MIC values of >64 mg/L, and
values of 750 were used for haemolytic activity values of >500
M.18
l
h
l
sheath gas flow 10, capillary temperature 523 K) with resolution
R = 60,000 at m/z 400 (mass range m/z = 150-2000) and dioc-
tylphthalate (m/z = 391.28428) as lock mass. HPLC purifications
were performed on a Gilson GX-281 automated HPLC system,
equipped with a preparative Gemini C18 column (150 mmL ꢀ
gradient accessory and a cryo-probe. Chemical shifts are given in
ppm (d) relative to CD3OH (3.33) ppm.
4.1. General peptide synthesis
21.20 mmD, 5
l
, particle size). The applied buffers were: A: 0.2%
(a) Peptide chain elongation: Preloaded resin (HMPB–BHA, 100-
200 mesh) with Fmoc-Orn(Boc) (133 mg, 0.75 mmol/g, 0.1 mmol)
was subjected to 13 cycles of automated Fmoc solid-phase synthe-
sis using the Fmoc based solid phase peptide synthesis protocols.
Commercially available Fmoc protected amino acids were coupled
with 90% HATU with respect to 5 equiv of amino acid in 30 min.
aq TFA, B: MeCN. CD and haemolytic curves were analyzed with
GRAPHPAD PRISM version 5.01 for Windows, GraphPad Software, San
Diego California USA, www.graphpad.com. 1H-, 13C-, Standard
DGF-COSY (512c ꢀ 2084c) and TOCSY (400c ꢀ 2048c) NMR spectra
were recorded on a Bruker DMX 600 equipped with a pulsed field

A. D. Knijnenburg et al. / Bioorg. Med. Chem. 18 (2010) 8403–8409
8407
The Fmoc adamantyl-
were coupled with 90% HATU coupling reagent with respect to
1.5 equiv amino acid in 30 min.
(b) Cleavage from resin: The peptides were released from the
resin by mild acidic cleavage (4 ꢀ 10 min, 10 mL 1% TFA in DCM).
The fractions were collected and coevaporated with toluene (3 ꢀ
50 mL) to give the crude linear peptide which was immediately
cyclised without further purification.
L
-glycine and Fmoc adamantyl-
L
-alanine
(m, 3H), 7.92 (m, 6H), 7.31 (m, 12H), 4.36 (m, 5H), 3.67 (s, 1H),
3.60 (m, 2H), 3.01 (m, 18H), 2.30–1.28 (m, 73H), 1.03–0.68 (m,
18H). 13C NMR (151 MHz, CD3OH) d 173.52, 162.99, 162.76,
130.35, 129.67, 128.49, 68.02, 61.34, 40.26, 39.57, 37.63, 29.98,
29.66, 29.31, 25.10.
D
D
4.1.4. cyclo-POXglyOXalaO FPOVOLO F (6)
Prepared and cyclised according to the general procedure. Puri-
(c) Cyclisation: The crude partially protected peptide in DMF
(20 mL) was dropwise added over 16 h to a solution of HOBt
(5 equiv), pyBOP (5 equiv) and DiPEA (15 equiv) in DMF (160 mL).
The solvent was removed under diminished pressure and the
residue applied to a Sephadex™ LH-20 size exclusion column
(50.0 mmD ꢀ 1500 mmL) and eluted with MeOH. The volatiles were
removed under diminished pressure and the protected peptides
were analyzed by LC/MS and HRMS.
fied protected yield: 129 mg, 54.13 lmol; 54%. HRMS (ESI) m/z
1191.73959 [M+H]2+, calcd 1191.73876 for C124H197N20O26; Re-
moval of the Boc group, purification by preparative RP-HPLC (linear
gradient of 46–76%, 3 CV) and lyophilisation of the combined pure
fractions furnished peptide 6 (39, 62 mg, 22.23 lmol, 22%); LCMS
Rf 5.09 min, linear gradient 10?90% B in 13.5 min; m/z = 1783.8
[M+H]+; HRMS (ESI) m/z 891.58195 [M+H]2+, calcd 891.58147 for
C
94H148N20O14;
1H NMR (600 MHz, CD3OH) d 9.00 (br s, 1H), 8.71
(d) Deprotection: The Boc-protection groups of the peptides
were removed by addition of TFA/TIS/H2O mixture (10 mL, 95/
2.5/2.5) and subsequently the peptide was purified by preparative
RP-HPLC.
(br s, 1H), 8.37 (br s, 2H), 7.93 (br s, 2H), 7.85 (br s, 2H), 7.72 (br
s, 2H), 7.43–7.13 (m, 12H), 4.44 (m, 6H), 3.67 (s, 2H), 3.54 (s,
2H), 3.15–2.81 (m, 18H), 2.70 (s, 2H), 2.18–1.43 (m, 88H), 1.39
(m, 1H), 1.30 (s, 1H), 0.89 (m, 12H). 13C NMR (151 MHz, CD3OH)
d 173.41, 163.00, 137.01, 130.33, 129.64, 128.43, 101.28, 68.02,
61.39, 45.75, 43.28, 40.25, 40.12, 37.77, 33.70, 29.90, 25.13, 19.54.
D
4.1.1. cyclo-(POVOLO F)2 (3)
Prepared and cyclised according to the general procedure. Puri-
D
fied protected yield: 103 mg, 46.8
lmol; 47%. HRMS (ESI) m/z
4.1.5. cyclo-(POXglyOLO F)2 (7)
1099.67703 [M+H]2+, calcd 1099.67616 for C110H180N20O26; Re-
moval of the Boc group, purification by preparative RP-HPLC (linear
gradient of 46–76%, 3 CV) and lyophilisation of the combined pure
Prepared and cyclised according to the general procedure. Puri-
fied protected yield: 138 mg, 57.91 lmol; 57%. HRMS (ESI) m/z
1191.73980 [M+H]2+, calcd 1191.73876 for C124H196N20O26; Re-
moval of the Boc group, purification by preparative RP-HPLC (linear
gradient of 46–76%, 3 CV) and lyophilisation of the combined pure
fractions furnished peptide 3 (18.62 mg, 8.16 lmol, 8%); LCMS Rf
4.02 min, linear gradient 10?90% B in 13.5 min; m/z = 1599.4
[M+H]+; HRMS (ESI) m/z 799.51914 [M+H]2+, calcd 799.51887 for
fractions furnished peptide 7 (56, 81 mg, 31.87 lmol, 32%); LCMS
C
80H132N20O14
;
1H NMR (600 MHz, CD3OH) d 9.07 (br s, 1H), 8.64
Rf 5.13 min, linear gradient 10?90% B in 13.5 min; m/z = 1783.8
(m, 3H), 7.91 (m, 5H), 7.74 (m, 2H), 7.46–7.10 (m, 12H), 4.68–
4.16 (m, 7H), 3.67 (s, 1H), 3.56 (s, 2H), 3.18–2.89 (m, 18H), 2.68
(m, 2H), 2.25–1.48 (m, 51H), 1.45–1.21 (m, 4H), 1.10–0.68 (m,
24H). 13C NMR (151 MHz, CD3OH) d 181.60, 181.51, 173.61,
173.58, 173.00, 172.97, 162.99, 162.77, 136.92, 130.34, 129.65,
128.46, 101.28, 61.33, 40.27, 37.80, 30.00, 29.68, 25.62, 25.10,
23.62, 19.68.
[M+H]+; HRMS (ESI) m/z 891.58179 [M+H]2+, calcd 891.58147 for
C
94H148N20O14;
1H NMR (600 MHz, CD3OH) d 9.06 (s, 2H), 8.78–
8.38 (m, 4H), 7.92 (m, 6H), 7.71 (s, 2H), 7.46–7.20 (m, 12H), 4.47
(m, 5H), 3.67 (br s, 4H), 3.58 (m, 2H), 3.20–2.86 (m, 17H), 2.22–
1.40 (m, 81H), 1.32 (m, 1H), 1.14–0.64 (m, 12H). 13C NMR
(151 MHz, CD3OH) d 173.53, 162.71, 137.38, 130.35, 129.65,
128.47, 68.02, 61.28, 43.33, 40.24, 39.65, 37.79, 37.70, 33.82,
30.11, 29.88, 29.69, 25.15.
D
D
4.1.2. cyclo-POVOXalaO FPOVOLO F (4)
D
D
Prepared and cyclised according to the general procedure. Puri-
4.1.6. cyclo-POVOXalaO FPOXglyOLO F (8)
fied protected yield: 147 mg, 64.17
l
mol; 64%. HRMS (ESI) m/z
Prepared and cyclised according to the general procedure. Puri-
1145.70846 [M+H]2+, calcd 1145.70746 for C117H188N20O26 Re-
moval of the Boc group, purification by preparative RP-HPLC (linear
gradient of 46–76%, 3 CV) and lyophilisation of the combined pure
fied protected yield: 150 mg, 62.95 lmol; 63%. HRMS (ESI) m/z
1191.73974 [M+H]2+, calcd 1191.73876 for C124H196N20O26; Re-
moval of the Boc group, purification by preparative RP-HPLC (linear
gradient of 46–76%, 3 CV) and lyophilisation of the combined pure
fractions furnished peptide 4 (40, 62 mg, 24.03 lmol, 24%); LCMS
Rf 4.58 min, linear gradient 10?90% B in 13.5 min; m/z = 1691.6
fractions furnished peptide 8 (49, 03 mg, 27.51 lmol, 28%); LCMS
[M+H]+; HRMS (ESI) m/z 845.55070 [M+H]2+, calcd 845.55017 for
Rf 5.16 min, linear gradient 10?90% B in 13.5 min; m/z = 1783.8
C
87H140N20O14;
1H NMR (600 MHz, CD3OH) d 9.05 (br s, 1H), 8.68
[M+H]+; HRMS (ESI) m/z 891.58182 [M+H]2+, calcd 891.58147 for
(m, 2H), 8.53 (br s, 2H), 7.91 (m, 5H), 7.75 (br s, 1H), 7.39–7.20
(m, 12H), 4.44 (m, 5H), 3.67 (s, 1H), 3.55 (s, 2H), 3.18–2.85 (m,
18H), 2.15–1.35 (m, 72H), 1.30 (s, 1H), 1.04–0.74 (m, 18H). 13C
NMR (151 MHz, CD3OH) d 173.51, 172.53, 162.99, 162.76, 130.33,
129.65, 128.46, 68.02, 61.35, 43.29, 40.26, 40.11, 37.78, 33.67,
30.02, 29.89, 25.11, 19.58.
C94H148N20O14;
1H NMR (600 MHz, CD3OH) d 9.07 (br s, 1H), 8.67
(br s, 2H), 8.60 (br s, 1H), 7.94 (br s, 5H), 7.87 (br s, 2H), 7.31 (m,
12H), 4.45 (m, 5H), 3.67 (s, 3H), 3.57 (s, 2H), 3.01 (m, 17H), 2.11–
1.40 (m, 85H), 1.30 (s, 1H), 0.98–0.79 (m, 12H). 13C NMR
(151 MHz, CD3OH) d 162.98, 162.75, 130.33, 129.65, 128.47,
68.02, 61.34, 43.30, 40.25, 39.52, 37.79, 37.63, 29.89, 29.68, 25.11.
D
D
D
4.1.3. cyclo-POXglyOLO FPOVOLO F (5)
4.1.7. cyclo-(POVOXalaO F)2 (9)
Prepared and cyclised according to the general procedure. Puri-
Prepared and cyclised according to the general procedure. Puri-
fied protected yield: 164 mg, 71.59
l
mol; 71%. HRMS (ESI) m/z
fied protected yield: 161 mg, 67.56 lmol; 68%. HRMS (ESI) m/z
1145.70824 [M+H]2+, calcd 1145.70746 for C117H189N20O26; Re-
moval of the Boc group, purification by preparative RP-HPLC (linear
gradient of 46–76%, 3 CV) and lyophilisation of the combined pure
1191.73976 [M+H]2+, calcd 1191.73876 for C124H196N20O26; Re-
moval of the Boc group, purification by preparative RP-HPLC (linear
gradient of 46–76%, 3 CV) and lyophilisation of the combined pure
fractions furnished peptide 5 (32, 58 mg, 19.28
lmol, 19%); LCMS
fractions furnished peptide 9 (63, 46 mg, 35.61 lmol, 36%); LCMS
Rf 4.69 min, linear gradient 10?90% B in 13.5 min; m/z = 1690.6
Rf 5.23 min, linear gradient 10?90% B in 13.5 min; m/z = 1783.8
[M+H]+; HRMS (ESI) m/z 845.55045 [M+H]2+, calcd 845.55017 for
[M+H]+; HRMS (ESI) m/z 891.58183 [M+H]2+, calcd 891.58147 for
C87H140N20O14
;
1H NMR (600 MHz, CD3OH) d 9.09 (m, 2H), 8.62
C94H148N20O14;
1H NMR (600 MHz, CD3OH) d 9.08 (br s, 1H), 8.58

8408
A. D. Knijnenburg et al. / Bioorg. Med. Chem. 18 (2010) 8403–8409
(br s, 2H), 8.17 (br s, 1H), 7.92 (m, 4H), 7.62 (br s, 2H), 7.31 (m,
12H), 4.39 (m, 7H), 3.67 (s, 4H), 3.58 (s, 2H), 3.01 (m, 17H), 2.19–
1.35 (m, 83H), 1.30 (s, 1H), 0.87 (m, 12H). 13C NMR (151 MHz,
CD3OH) d 162.89, 162.66, 130.35, 129.64, 128.47, 68.02, 61.62,
61.26, 47.88, 40.26, 39.45, 37.64, 32.07, 29.82, 29.66, 29.26,
25.26, 25.17, 21.26.
(pH 5.3). Ellipticity is reported as mean residue ellipticity [h], with
approximate errors of 10% at 220 nm.
4.3. Antimicrobial assays
The following bacterial strains were used: Staphylococcus aureus
(ATCC 29213), Staphylococcus epidermidis (ATCC 12228), Enterococ-
cus faecalis (ATCC 29212), Bacillus cereus (ATCC 11778), Escherichia
coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and the
MRSA strains MRSA-USA300-1110301146 PVL+ (community ac-
quired MRSA), and MRSA-NT 1110301981-spa/t034-PVL+, MRSA-
NT N133-spa/t034-PVL-, MRSA-NT N229-spat/034-PVL- (cattle-re-
lated strains), MRSA (ATCC 49775) en MRSA (ATCC 43300). Bacte-
ria were stored at ꢁ70 °C and grown at 35 °C on Columbia Agar
with sheep blood (Oxoid, Wesel, Germany) suspended in physio-
logical saline until an optical density of 0.1 AU (at 595 nm, 1 cm
cuvette). The suspension was diluted (100ꢀ) with physiological
D
D
4.1.8. cyclo-POVOXalaO FPOXglyOXalaO F (10)
Prepared and cyclised according to the general procedure. Puri-
fied protected yield: 139 mg, 56.16 lmol; 56%. HRMS (ESI) m/z
1237.77097 [M+H]2+, calcd 1237.77006 for C131H204N20O26; Re-
moval of the Boc group, purification by preparative RP-HPLC (linear
gradient of 46–76%, 3 CV) and lyophilisation of the combined pure
fractions furnished peptide 10 (12, 02 mg, 6.41 lmol, 6%); LCMS Rf
5.63 min, linear gradient 10?90% B in 13.5 min; m/z = 1875.4
[M+H]+; HRMS (ESI) m/z 937.61366 [M+H]2+, calcd 937.61277 for
C101H156N20O14;
1H NMR (600 MHz, CD3OH) d 9.06 (br s, 1H),
8.68 (br s, 1H), 8.36 (br s, 2H), 8.13 (br s, 2H), 7.93 (br s, 2H),
7.70 (br s, 2H), 7.31 (m, 12H), 4.51 (s, 2H), 4.41 (m, 3H), 4.12 (m,
1H), 3.87–3.76 (m, 1H), 3.57 (m, 2H), 3.44 (m, 1H), 3.16–2.78 (m,
18H), 2.69 (m, 1H), 2.37 (s, 1H), 2.21–1.37 (m, 96H), 1.32 (m,
2H), 1.09–0.75 (m, 6H). 13C NMR (151 MHz, CD3OH) d 162.99,
130.32, 129.65, 128.46, 68.02, 61.37, 43.33, 40.24, 37.77, 33.80,
30.74, 29.90, 29.71.
saline, and 2 lL of this inoculum was added to 50 lL growth med-
ium, Cation Adjusted Mueller Hinton II Broth (BBL Ref. No.
212322), in microtiter plates (96 wells).
All peptides including GS were dissolved in methanol (4 g/L)
and diluted in distilled water (1000 mg/L and two-fold diluted in
the broth (64, 32, 16, 8, 4 and 1 mg/L). The plates were incubated
at 35 °C (24–48 h) and the MIC was determined as the lowest con-
centration inhibiting bacterial growth.
D
D
4.1.9. cyclo-POXglyOXalaO FPOXglyOLO F (11)
Prepared and cyclised according to the general procedure. Puri-
4.4. Haemolytic assays
fied protected yield: 133 mg, 53.73 lmol; 54%. HRMS (ESI) m/z
1237.77107 [M+H]2+, calcd 1237.77006 for C131H204N20O26; Re-
moval of the Boc group, purification by preparative RP-HPLC (linear
gradient of 46–76%, 3 CV) and lyophilisation of the combined pure
Freshly drawn heparinized blood was centrifuged for 10 min at
1000g at 10 °C, 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 evalu-
ated 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 sus-
pension was sonicated for a few seconds. A 1% Triton-X solution
fractions furnished peptide 11 (50, 87 mg, 27.14 lmol, 27%); LCMS
Rf 5.69 min, linear gradient 10?90% B in 13.5 min; m/z = 1875.4
[M+H]+; HRMS (ESI) m/z 937.61316 [M+H]2+, calcd 937.61277 for
C
101H156N20O14;
1H NMR (600 MHz, CD3OH) d 9.08 (br s, 1H),
8.63 (br s, 2H), 8.20 (m, 1H), 7.95 (br s, 4H), 7.64 (br s, 2H), 7.31
(m, 12H), 4.67–4.07 (m, 8H), 3.67 (s, 3H), 3.60 (m, 2H), 3.00 (m,
17H), 2.72 (s, 1H), 2.14–1.34 (m, 96H), 1.30 (s, 1H), 1.05–0.77 (m,
was prepared. Subsequently, 100
dispensed in columns 1–11 of a microtiter plate, and 100
1% Triton solution was dispensed in column 12. To wells A1–C1,
100 L of the peptide was added and mixed properly. 100 L of
wells A1–C1 was dispensed into wells A2–C2. This process was
repeated until wells A10–C10, followed by discarding 100 L of
wells A10–C10. These steps were repeated for the other peptides.
Subequently, 50 L of the red blood cell solution was added to
l
L
of saline solution was
l
L of
6H). 13C NMR (151 MHz, CD3OH)
d
173.51, 162.95, 162.72,
l
l
130.35, 129.65, 101.26, 68.02, 44.06, 43.35, 40.25, 37.70, 29.85,
29.67, 25.16, 21.31.
l
D
4.1.10. cyclo-(POXglyOXalaO F)2 (12)
l
Prepared and cyclised according to the general procedure. Puri-
the wells and the plates were incubated at 37 °C for 4 h. After
incubation, the plates were centrifuged at 1000g at 10 °C for
4 min. In a new microtiter plate, 50 lL of the supernatant of each
well was dispensed into a corresponding well. The absorbance at
405 nm was measured and the percentage of haemolysis was
determined.
fied protected yield: 169 mg, 65.83 lmol; 66%. HRMS (ESI) m/z
1283.80267 [M+H]2+, calcd 1283.80136 for C138H212N20O26; Re-
moval of the Boc group, purification by preparative RP-HPLC (linear
gradient of 46–76%, 3 CV) and lyophilisation of the combined pure
fractions furnished peptide 12 (57, 53 mg, 29.25 lmol, 29%); LCMS
Rf 6.12 min, linear gradient 10?90% B in 13.5 min; m/z = 1967.6
[M+H]+; HRMS (ESI) m/z 983.64407 [M+H]2+, calcd 983.64420 for
C
4.5. Calculation peptide hydrophobic moment
108H164N20O14;
1H NMR (600 MHz, CD3OH) d 9.41 (br s, 1H),
9.10 (br s, 1H), 8.67 (br s, 2H), 8.22 (br s, 2H), 7.94 (br s, 2H),
7.64 (br s, 2H), 7.42–7.22 (m, 12H), 7.07 (m, 1H), 4.47 (m, 6H),
4.31–3.97 (m, 1H), 3.67 (s, 9H), 3.57 (s, 2H), 3.00 (m, 19H), 2.52–
2.22 (m, 1H), 2.21–1.34 (m, 102H), 1.30 (s, 1H). 13C NMR
(151 MHz, CD3OH) d 162.75, 130.35, 129.65, 68.02, 43.35, 40.24,
37.69, 29.89, 29.70.
The hydrophobic moment of peptides 3–12 was calculated
using the hydrophobic moment relationship of Eisenberg et al.32
To determine the hydrophobicity scale of the non-proteinogenic
amino acids Adamantyl-L-glycine and Adamantyl-L-alanine, the
method was used of Tossi et al.36 The retention time of a collection
of Fmoc-N-capped amino acids were determined using HPLC (Val:
D
7.83 min; Met: 7.76; Phe: 8.34; Ile: 8.32; Phe: 8.35; Leu: 8.37; Ala:
4.2. Circular dichroism spectroscopy
6.98; Gly: 6.58; Pro: 7.32; 13.5 min run time with 1 mL/min flow
rate, using a Gemini C18 column, the applied buffers were A:
H2O, B: MeCN and C: 1.0% aq TFA). The observed retention times
of the Fmoc protected amino acids agreed well with the consensus
hydrophobicity scale and showed a good correlation (r = 0.98).36
CD spectra were recorded at 298 K on a Jasco J-815 spectropo-
larimeter using 0.1 cm path length quartz cells The CD spectra
are averages of four scans, collected at 0.1 nm intervals between
190 and 250 nm with scanning speed 50 nm/min. The peptides
were prepared at concentrations 0.1 mM in 50% TFE/0.01 M NaOAc
The retention times of Fmoc-N-Adamantyl-L-glycine and Adaman-
tyl- -alanine are 10.43 and 11.07 min, respectively. The values for
L

A. D. Knijnenburg et al. / Bioorg. Med. Chem. 18 (2010) 8403–8409
8409
15. Kapoerchan, V. V.; Knijnenburg, A. D.; Niamat, M.; Spalburg, E.; De Neeling, A.
J.; Nibbering, P. H.; Mars-Groenendijk, R.; Noort, D.; Otero, J. M.; Llamas-Saiz, A.
L.; Van Raaij, M. J.; Van der Marel, G. A.; Overkleeft, H. S.; Overhand, M., Chem.
Eur. J., in press. doi:10.1002/chem.201001686.
16. Knijnenburg, A. D.; Spalburg, E.; de Neeling, A. J.; Mars-Groenendijk, R. H.;
Noort, D.; Grotenbreg, G. M.; van der Marel, G. A.; Overkleeft, H. S.; Overhand,
M. ChemMedChem 2009, 4, 1976.
17. Ando, S.; Takiguchi, H.; Izumiya, N. Bull. Chem. Soc. Jpn. 1983, 56, 3781.
18. Kondejewski, L. H.; Farmer, S. W.; Wishart, D. S.; Kay, C. M.; Hancock, R. E. W.;
Hodges, R. S. J. Biol. Chem. 1996, 271, 25261.
Adamantyl-L-glycine and Adamantyl-L-alanine were calculated as
24.1 and 28.7. The hydrophobicity scale values used for Leu, Val,
Orn, Pro, and DPhe were 9.7, 4.1, ꢁ9.0, ꢁ0.2 and 10, respectively.36
To account for the two b-turns in the molecule the hydrophobic
moment (
l
) was calculated for each half of the molecule (e.g., for
D
D
2 OVOLO YP and OX1OLO YP) and averaged out. A value of
d = 180° was used to define the angle of the backbone b-sheet.20
19. Gibbs, A. C.; Kondejewski, L. H.; Gronwald, W.; Nip, A. M.; Hodges, R. S.; Sykes,
B. D.; Wishart, D. S. Nat. Struct. Biol. 1998, 5, 284.
Acknowledgments
20. Kondejewski, L. H.; Jelokhani-Niaraki, M.; Farmer, S. W.; Lix, B.; Kay, C. M.;
Sykes, B. D.; Hancock, R. E. W.; Hodges, R. S. J. Biol. Chem. 1999, 274, 13181.
21. Jelokhani-Niaraki, M.; Kondejewski, L. H.; Wheaton, L. C.; Hodges, R. S. J. Med.
Chem. 2009, 52, 2090.
22. Kondejewski, L. H.; Lee, D. L.; Jelokhani-Niaraki, M.; Farmer, S. W.; Hancock, R.
E. W.; Hodges, R. S. J. Biol. Chem. 2002, 277, 67.
This work was supported by the Leiden Institute of Chemistry.
Assistance in performing NMR measurements by C. Erkelens and
A. W. M. Lefeber is kindly acknowledged.
23. Kapoerchan, V. V.; Wiesner, M.; Hillaert, U.; Drijfhout, J. W.; Overhand, M.;
Alard, P.; van der Marel, G. A.; Overkleeft, H. S.; Koning, F. Mol. Immunol. 2010,
47, 1091.
24. Grotenbreg, G. M.; Kronemeijer, M.; Timmer, M. S. M.; El Oualid, F.; van Well, R.
M.; Verdoes, M.; Spalburg, E.; van Hooft, P. A. V.; de Neeling, A. J.; Noort, D.; van
Boom, J. H.; van der Marel, G. A.; Overkleeft, H. S.; Overhand, M. J. Org. Chem.
2004, 69, 7851.
Supplementary data
Supplementary data (HRMS, 1H NMR, CD, haemolytic curves,
correlation of retention time and hydrophobic moment are reported
in the supporting information) associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2010.09.018.
25. Gibbs, A. C.; Bjorndahl, T. C.; Hodges, R. S.; Wishart, D. S. J. Am. Chem. Soc. 2002,
124, 1203.
26. Blanco, F. J.; Jimenez, M. A.; Pineda, A.; Rico, M.; Santoro, J.; Nieto, J. L.
Biochemistry 1994, 33, 6004.
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