Properties and structure-activity studies of cyclic β-hairpin peptidomimetics based on the cationic antimicrobial peptide protegrin I

Article abstract of DOI:10.1016/j.bmc.2005.01.009

The properties and structure-activity relationships (SAR) of a macrocyclic analogue of porcine protegrin I (PG-I) have been investigated. The lead compound, having the sequence cyclo-(-Leu-Arg-Leu-Lys-Lys-Arg-Arg-Trp-Lys-Tyr- Arg-Val-d-Pro-Pro-), shows an

Full text of DOI:10.1016/j.bmc.2005.01.009

Bioorganic & Medicinal Chemistry 13 (2005) 2055–2064
Properties and structure–activity studies of
cyclic b-hairpin peptidomimetics based on the cationic
antimicrobial peptide protegrin I
John A. Robinson,a,* Sasalu C. Shankaramma,a Peter Jetter,a Ursula Kienzl,a
Reto A. Schwendener,b Jan W. Vrijbloedc and Daniel Obrechtc
aInstitute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
bBiomolecular Research, Molecular Cell Biology, Paul Scherrer Institut, 5232 Villingen-PSI, Switzerland
cPolyphor AG, Gewerbestrasse 14, 4123 Allschwil, Switzerland
Received 12 November 2004; accepted 7 January 2005
Abstract—The properties and structure–activity relationships (SAR) of a macrocyclic analogue of porcine protegrin I (PG-I) have
been investigated. The lead compound, having the sequence cyclo-(-Leu-Arg-Leu-Lys-Lys-Arg-Arg-Trp-Lys-Tyr-Arg-Val-D-Pro-
Pro-), shows antimicrobial activity against Gram-positive and -negative bacteria, but a much lower haemolytic activity and a much
reduced ability to induce dye release from phosphatidylcholine/phosphatidylglycerol liposomes, when compared to PG-I. The enan-
tiomeric form of the lead peptide shows comparable antimicrobial activity, a property shared with other cationic antimicrobial pep-
tides acting on cell membranes. SAR studies involving the synthesis and biological profiling of over 100 single site substituted
analogues, showed that the antimicrobial activity was tolerant to a large number of the substitutions tested. Some analogues showed
slightly improved antimicrobial activities (2–4-fold lowering of MICs), whereas other substitutions caused large increases in haemo-
lytic activity on human red blood cells.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
lysine and/or arginine residues as well as having multiple
hydrophobic residues. Cationic antimicrobial peptides
typically show minimal inhibitory concentrations
(MICs) in the range 0.25–16 lg/mL against Gram-posi-
tive and -negative bacteria, fungi and protozoa. They
have been heralded as a potential source of new antimi-
crobial drugs to combat the increasing threat posed by
multiple drug resistant microorganisms.4–6 Such pep-
tides are frequently classified on the basis of their pre-
ferred structures as b-sheet, a-helical, loop or extended
peptides.7
The naturally occurring cationic antimicrobial peptides
represent interesting targets for peptidomimetic re-
search. These natural products have been discovered in
all animal kingdoms where they are now recognized as
key mediators of innate immunity to bacterial infec-
tions.1–3 They are generally peptides of less than 50
amino acids, with a net positive charge due to multiple
Abbreviations: Boc, tert-butoxycarbonyl; DIEA, diisopropylethyl-
amine; DMF, N,N-dimethylformamide; Fmoc, 9-fluorenylmethoxy-
Most naturally occurring cationic antimicrobial pep-
tides target and disrupt the functions of microbial cell
membranes.8–11 The excess of negative charge associated
with the outer microbial cell membrane imparts some
selectivity in the mode of action, although at higher con-
centrations animal cell membranes are frequently also
disrupted. As a class of natural products, however, their
mechanisms of antimicrobial action are quite complex.
Some target membrane receptors such as lipopolysac-
charide (e.g., polymyxin), whereas others clearly exhibit
carbonyl;
HATU,
2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate; HBTU, 2-[1H-benzotriazole-
1-yl]-1,1,3,3-tetramethyluronium hexafluorophosphate; HOAt, N-hy-
droxy-9-azabenzotriazole; HOBt, N-hydroxybenzotriazole; Pbf, 2,2,4,
6,7-pentamethyldihydrobenzofuran-5-sulfonyl; TIPS, triisopropyl-si-
lane; TFA, trifluoroacetic acid.
Keywords: Antibiotic; Secondary structure; Haemolysis; Protein epi-
tope mimetic.
*
Corresponding author. Tel.: +41 1635 4242; fax: +41 1635 6833;
e-mail: robinson@oci.unizh.ch
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.009

2056
J. A. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 2055–2064
other biological activities (e.g., members of the cathelic-
idin family12). It seems that cationic peptides (and pep-
tidomimetics) may have multiple actions on cells,
ranging from cell permeabilization to targeting intracel-
lular receptors, and that these actions may vary with
changes in sequence and stereochemistry.
other cationic peptides targeting the cell membrane.41–43
This behaviour is consistent with the peptides interact-
ing mainly with the fatty acyl chains in the cell mem-
brane. However, PG-I is also able to bind the
lipopolysaccharide and lipid-A in the membranes of cer-
tain Gram-negative bacteria.44
The starting point for our peptidomimetic design was
the naturally occurring antimicrobial peptide protegrin
I (PG-I) (1) (Fig. 1), one of a family of five homologous
peptides first isolated from porcine leucocytes.13,14 PG-I
is an 18-amino acid peptide containing two disulfide
bridges (Cys6–15 and Cys8–13). These disulfides con-
strain the peptide backbone into a b-hairpin conforma-
tion, with a b-turn formed by residues 9–12, as detected
by NMR in water and DMSO solution, and in DPC mi-
celles.15–17 Other naturally occurring disulfide bridged
antimicrobial b-hairpin peptides of similar size and
shape include tachyplesins18–21 and polyphemusins22
from horseshoe crabs, androctonin from scorpions,23,24
thanatin from insects,25 lactoferricin B from cows,26 hu-
man hepcidin,27 an antimicrobial protein from plants
(Ib-AMp1),28 and gomesin from spiders.29,30 Related
backbone cyclic antimicrobial peptides that also adopt
b-hairpin structures include gramicidin S31 and tyroci-
dine A,32 both of bacterial origin, and the backbone cyc-
lic and disulfide cross-linked peptide RTD-I (also called
theta-defensin) from primate leukocytes.33,34
Biophysical studies of the interaction of PG-I with
model membrane bilayers confirm that the peptide
inserts into membranes, forms oligomeric aggregates
that contact both the polar head groups and the hydro-
phobic core, and causes thinning and disruption of lipid
packing. The average orientation, mobility and depth of
insertion seems to depend upon the constitution (e.g.,
charge, lipid chain length, degree of unsaturation) of
the membrane lipids used and the presence of choles-
terol.45–50 The degree of insertion into anionic contain-
ing monolayers is significantly larger than that of
zwitterionic bilayers. Lipid A films are completely desta-
bilized,47 consistent with the preferential damage caused
to bacterial over mammalian cell membranes.
Detailed structure–activity relationship (SAR) studies
on PG-I revealed that linear analogues, lacking the
disulfide bridges, have no regular secondary structure
and exhibit reduced antimicrobial activity.51–55
Although a large number of amino acid substitutions
in the PG-I structure are tolerated, the amphiphilicity
of the b-hairpin, and total number of positively charged
residues impact significantly on the activity. SAR stud-
ies52 culminated in the discovery of analogue IB367 (2)
with Cys5–14 and Cys7–12 disulfide bridges, which has
now been tested in the clinic to treat ulcerative oral
mucositis, ventilator associated pneumonia, and respira-
tory infections associated with cystic fibrosis. However,
a property limiting the wider application of PG-I as an
antibiotic is its mammalian toxicity. One key indicator
of this toxicity is its high haemolytic action on human
red blood cells, causing ca. 37% lysis (see below) at a
PG-I concentration of 100 lg/mL.
PG-I typically kills susceptible microorganisms within
minutes,35–37 consistent with a bactericidal effect caused
by disruption of the cell membrane.13,38–40 Both enan-
tiomeric forms of PG-I show essentially the same anti-
microbial activity,37,39 a characteristic shared with
F12
V14
V16
R18
NH2
R1
R11
R9
C13
Y7
C15
L5
G17
G3
Protegrin-I
1
R10
C8
C6
R4
G2
In earlier work,56,57 we reported an approach to PG-I
peptidomimetics of type 3 (Fig. 1) based on the use of
b-hairpin-stabilizing organic templates. The template is
chosen for its ability to promote a b-hairpin loop struc-
ture, as depicted in 3, thereby dispensing with the need
for disulfide bridges to constrain the loop into a b-hair-
pin geometry. We have used this design principal to
prepare b-hairpin peptidomimetics of antibody hyper-
variable loops,58 protease inhibitors based on the
Bowman–Birk fold,59 RNA-binders that inhibit the
interaction of Tat with TAR-RNA,60 and in the design
of novel p53-HDM2 inhibitors.61
F11
V13
V15
R17
NH2
R10
C12
C14
G16
IB-367
2
R8
Y6
L4
G2
G9
C7
C5
G3
R1
Temp.
D-Pro
R8
R10
R12
O
H
H
N
O
H
N
O
R7
N
N
H
N
H
N
H
O
H
O
R9
R4
O
R11
R2
N
3
O
O
H
N
H
N
H
N
O
O
N
O
N
H
N
H
N
H
R6
R5
O
R3
O
R1
L-Pro
O
The PG-I mimetic 4 (Fig. 1), described earlier, adopts a
b-hairpin structure in dodecylphosphocholine micelles,
and exhibits good antimicrobial activity (MICs 6–
12 lg/mL) against Gram-positive and Gram-negative
bacteria. However, compared to PG-I, 4 shows a much
reduced haemolytic activity on human erythrocytes; ca.
1% lysis at a peptide concentration of 100 lg/mL. In this
work, we report new data on 4 and an extensive SAR
study of this mimetic.
Temp.
D-Pro13
W8
Y10
V12
R7
K9
L3
R11
L1
4
K5
R6
K4
R2
L-Pro14
Figure 1. Structures 1–4.

J. A. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 2055–2064
2057
2. Results and discussion
2.1. Enantioselectivity
It was shown earlier56 that 4 has a rapid bactericidal
action upon both Gram-negative and Gram-positive
bacteria, effectively reducing the number of viable bacte-
rial cells by three orders of magnitude within a few min-
utes. The enantiomer of 4 was prepared here, and shown
to have essentially similar or even slightly higher antimi-
crobial activity (data not shown). The enhancement in
activity of ent-4 is small (less than or equal to a factor
of two in MIC compared to 4), which possibly reflects
a higher proteolytic stability in bacterial cultures. As
with PG-I, however, the comparable activity of the
enantiomer suggests that the mechanism of action does
not involve binding to a chiral receptor, but rather
reflects a nonenantiospecific interaction with membrane
lipids.
2.2. Activity on model lipid membranes
The ability of 4 to bind and cause leakage through lipid
bilayers was assessed by measuring dequenching of cal-
cein fluorescence upon release from a concentrated solu-
tion within liposomes, which are used as model cell
membranes. Large unilamellar liposomes containing cal-
cein were prepared from various binary ratios of zwitter-
ionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) and negatively charged 1-palmitoyl-2-oleoyl-
sn-glycero-3-[phospho-(1-glycerol)] (POPG). As a con-
trol, the dye release assays were also performed with
PG-I.
Figure 2. Concentration dependence of calcein release from liposomes
induced by A, peptide mimetic 4; and B, by PG-1. The liposomes were
composed of pure POPG (d); pure POPC (h); POPC/POPG (80:20)
(j) and POPC/POPG (70:30) (s). See Experimental.
PG-I induced substantial leakage of calcein from vesi-
cles composed of POPC and POPG (80:20 and 70:30),
as well as from pure POPC and POPG liposomes (Fig.
2B). Similar experiments have been described already
for PG-I62,63 and the related cationic peptide antibiotics
polyphemusin and gramicidin S.64 Mimetic 4 showed
comparable leakage activity only toward the highly nega-
tively charged, and hence more susceptible POPG lipo-
somes (Fig. 2A). Liposomes containing 20% or 30%
zwitterionic POPC, or POPC alone, experienced much
reduced dye release with 4 compared to that caused by
PG-I under comparable conditions. These data show
that mimetic 4 possesses membranolytic activity in this
model system, but at a level significantly reduced com-
pared to that for PG-I. This parallels the reduced hae-
molytic behaviour on human red blood cells observed
for 4, compared to that seen for PG-I.
was performed in dilute DMF solution with HATU/
HOAt, and a final deprotection step was performed with
a mixture of TFA/TIPS/H2O (95/2.5/2.5). The desired
macrocycle was typically the main component in the
product. This was purified by preparative HPLC and
characterized by electrospray ionization MS. The purity
of the peptides used for biological assays was P95%.
The sequences of the analogues, their antimicrobial
activities against three test organisms, and their haemo-
lytic activity on fresh human red blood cells, are re-
ported in Tables 1–3. We focus first on analogues
arising through substitutions in the hydrophobic resi-
dues in 4, namely Leu-1, Leu-3, Trp-8, Tyr-10 and
Val-12. The results (Table 1) show that good antimicro-
bial activities were retained in many of the analogues,
although large improvements were not observed. It has
been noted often, that antimicrobial peptides acting on
bacterial cell membranes typically have MICs at best
in the low lM range, but not significantly below this
(e.g., into the low nM range). Many of the analogues
tested contained hydrophobic residues of differing size,
yet showed antimicrobial activities close to those of 4.
Substituting a polar residue for a nonpolar residue,
however, often had deleterious effects on MICs.
2.3. Structure–activity relationships
In order to probe how the antimicrobial activity of 4 is
related to its sequence, libraries of analogues were syn-
thesized, by substituting a variety of different amino
acids at each of the 12 positions around the b-hairpin.
For the synthesis of these analogues, a linear precursor
was first assembled on 2-chlorotrityl chloride resin using
Fmoc-chemistry, as described earlier.56,57 The linear,
side chain protected precursors were then cleaved from
the resin with 1% TFA in CH2Cl2, macrocyclization
The haemolytic activity on red blood cells was also inves-
tigated (see Tables 1–3). It is apparent that substitutions
at position-10 can cause large increases in haemolytic

2058
J. A. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 2055–2064
Table 1. Minimal inhibitory concentrations (MICs) in lg/mL against the test organisms shown, and the percentage haemolysis of human red blood
cells caused at a peptide concentration of 100 lg/mL
Position-1 (Leu)
Leu
Phe
Cha
Tyr
Trp
Hfe
1-Nal
2-Nal
ClF
His
Bip
Orn
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
18
25
25
12.5
25
25
50
25
2.2
12.5
12.5
25
12.5
12.5
12.5
13.2
25
6.2
3.1
25
100
100
12.5
6.2
50
100
100
9.4
9.4
1.1
12.5
6.2
7.7
12.5
1.4
6.2
7.1
25
6.2
11.5
6.2
17.6
0.6
6.8
0.2
0.7
Position-3 (Leu)
Leu
Phe
Cha
Trp
Hfe
1-Nal
2-Nal
Val
ClF
His
Bip
Orn
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
25
25
50
2.6
25
12.5
12.5
12.5
4.5
12.5
6.2
12.5
12.5
12.5
5.2
6.2
25
6.2
6.2
25
50
50
25
100
100
12.5
12.5
25
50
100
100
12.5
12.5
3.1
12.5
1.4
12.5
3.8
12.5
5.8
50
13.1
1.1
1.2
7.7
1.5
Position-8 (Trp)
Trp
Phe
Tyr
Y(B)
Hfe
1-Nal
2-Nal
Val
ClF
Leu
25
Ile
Orn
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
25
25
25
25
1.7
25
25
12.5
6.2
25
25
12.5
25
2
12.5
6.2
25
50
50
25
12.5
50
6.2
6.2
4.5
9.4
25
6.2
9.4
3.5
12.5
50
1.3
12.5
25
12.5
1.4
12.5
2.7
12.5
3.3
2.1
3.1
2.3
3.1
Position-10 (Tyr)
Tyr
Phe
Cha
Trp
Hfe
1-Nal
2-Nal
Val
ClF
Leu
6.2
Bip
Orn
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
12.5
12.5
12.5
30.1
25
50
25
25
25
18
50
50
50
50
4.9
12.5
9.4
6.2
6.2
6.2
33
50
50
50
12.5
9.4
3.9
12.5
9.4
12.5
12.5
29.3
9.4
12.5
26.6
12.5
1.4
12.5
57.6
12.5
39.6
12.5
34.8
31.8
0.1
Position-12 (Val)
Val
Phe
Cha
Trp
Hfe
1-Nal
2-Nal
Tyr
ClF
Leu
Bip
Orn
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
6.2
12.5
12.5
10.1
50
12.5
6.2
12.5
12.5
12.5
21.7
25
25
25
25
50
6
12.5
6.2
25
12.5
6.2
50
25
12.5
6.2
12.5
6.2
3.7
12.5
18
12.5
25
12.5
1.4
12.5
10.5
12.5
6.4
6.2
18.4
100
35.2
23.3
4.5
0.3
Shown are the amino acid substitutions made at the positions Leu-1, Leu-3, Trp-8, Tyr-10 and Val-12 in 4. The first entry in each row shows the
activities of 4 without substitution. The normal three letter code is used for proteinogenic amino acids, and Cha = L-cyclohexylalanine; Hfe = L-
homophenylalanine; 1-Nal = L-1-naphthylalanine; 2-Nal = L-2-naphthylalanine; ClF = L-4-chlorophenylalanine; Bip = L-4-biphenylalanine;
Orn = L-ornithine; Y(B) = L-(O-benzyl)tyrosine.
activity. Substituting Tyr-10 to Phe, for example, or
introducing larger aromatic groups enhances haemolytic
activity dramatically. Also, the biphenyl side chain at po-
sition-10 improves slightly the MICs, but induces much
higher haemolysis than seen with 4. A significant worsen-
ing of the haemolytic activity is seen with several other
substitutions made at position-12. On the other hand,
similar substitutions at positions-1 and -3, on the other
side of the hairpin, or at position-8, were much less del-
eterious. An explanation for these selectivity effects is not
yet available, since the precise molecular mechanism(s)
of haemolysis is(are) unknown.
(4-N-acylamino)proline analogue (14A–S), or a (4-O-
alkoxy)proline derivative (14T and 14U) (Fig. 3). The
functionalized proline derivatives 7 and 9 required for
the synthesis of these peptides were prepared from N-
Boc-(2S,4S)-4-azidoprolinemethyl ester65 (5) or N-Boc-
(2S,4R)-4-hydroxyproline (8), as shown in Scheme 1.
Most of the analogues 14A–U contain additional hydro-
phobic groups, which should facilitate interactions with
a lipid bilayer. However, any beneficial effects on the
MIC of a new substituent were typically small and usu-
ally accompanied by an increase in the haemolytic activ-
ity (see Table 3). Only 14A has an improved
antimicrobial profile, without a significant increase in
haemolytic activity.
The results of substitutions of cationic residues in 4 are
summarized in Table 2. Of the many substitutions
tested, none gave rise to significant improvements in
MICs, although in some cases a significant increase in
haemolytic activity was observed.
In summary, the SAR studies of 4 have involved the syn-
thesis and biological profiling of over 100 single site
substituted analogues. These studies revealed that the
antimicrobial activity of 4 was tolerant to a large num-
ber of the substitutions tested. Some analogues show
slightly (2–4-fold) improved MIC values against the pa-
nel of test organisms. The SAR studies also revealed
that the haemolytic activity of 4 on human red blood
cells, was significantly increased following substitutions,
in particular, at positions-10 and -12 in the putative
hairpin. Finally, several new derivatives have been iden-
tified, which show an improved antimicrobial profile,
In a final round of screening, substitutions were also
made in the D-Pro-L-Pro template (Table 3), by substi-
tuting the L-Pro-14 by a different L-amino acid. Some
analogues showed good antimicrobial activity (e.g.,
Pro-14 to L-Phe) without an increased haemolytic activ-
ity, whereas in other cases the haemolytic activity was
also increased significantly. A further library of ana-
logues was prepared by substituting L-Pro-14 for an

J. A. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 2055–2064
2059
Table 2. Minimal inhibitory concentrations (MICs) in lg/mL against the test organisms shown, and the percentage haemolysis of human red blood
cells at a peptide concentration of 100 lg/mL
Position-2 (Arg)
Arg
Tyr
Trp
Leu
His
Thr
Gln
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
6.2
12.5
25
6.2
12.5
25
25
12.5
18
12.5
25
12.5
50
12.5
25
1.3
12.5
25
12.5
1.4
12.5
0.2
1.4
1.6
1.8
1.1
Position-4 (Lys)
Lys
Arg
Trp
Leu
His
Thr
Gln
6.2
25
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
12.5
6.2
50
100
100
4
25
25
25
12.5
12.5
25
25
25
25
12.5
1.4
12.5
1.2
25
1
0.5
0.4
0.7
Position-5 (Lys)
Lys
Arg
Trp
Leu
12.5
His
Thr
25
Gln
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
12.5
6.2
25
100
100
6
18
25
50
4.1
12.5
25
12.5
25
1.3
12.5
25
12.5
1.4
12.5
2.4
12.5
27.4
1.1
Position-6 (Arg)
Arg
Tyr
Trp
Leu
His
Thr
Gln
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
12.5
25
25
25
50
25
12.5
12.5
50
25
25
25
25
12.5
25
12.5
1.4
50
100
100
1.8
9.4
2.6
0.3
2.5
0.6
Position-7 (Arg)
Arg
Tyr
Trp
12.5
6.2
25
1.3
Leu
His
Gln
12.5
Thr
25
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
12.5
12.5
50
25
25
6.2
12.5
50
12.5
50
6.2
12.5
1.4
100
12.5
2.7
4.6
2.5
6.7
2.5
Position-9 (Lys)
Lys
Tyr
Trp
Leu
6.2
Thr
Gln
His
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
12.5
18.8
25
12.5
18.8
25
6.2
12.5
25
6.2
12.5
25
12.5
25
12.5
12.5
3.5
12.5
1.4
12.5
1.4
2.3
1.6
2.7
3.7
Position-11 (Arg)
Arg
Tyr
Trp
Leu
Orn
Cit
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
6.2
6.2
12.5
25
6.2
12.5
12.5
9.9
12.5
25
25
25
25
50
6.2
25
12.5
1.4
50
4.4
5.9
2.2
1.4
The amino acid substitutions made at the positions Arg-2, Lys-4, Lys-5, Arg-6, Arg-7, Lys-9 and Arg-11 in 4 are shown. The first entry in each row
shows the activities of 4 without substitution. The normal three letter code is used for proteinogenic amino acids, and Cit = L-citrulline; Orn = L-
ornithine.
compared to 4, whilst maintaining a much lower haemo-
lytic activity in comparison to PG-I.
resin was then washed extensively (with CH2Cl2/
MeOH/DIEA, 17/2/1; then CH2Cl2; then DMF; then
CH2Cl2; and Et2O, three times each) and dried under
vacuum. Following removal of the Fmoc-group using
40% piperidine/DMF, chain elongation was performed
sequentially with 5 equiv of each protected amino acid
and HOBt/HBTU (each 5 equiv) for activation, diiso-
propylethylamine (5 equiv) as base and DMF as solvent.
After completion of the synthesis, the resin was washed
with CH2Cl2 and MeOH, and then treated with 1% TFA
in CH2Cl2 (three times). The filtrate was neutralized
with DIEA in CH2Cl2 and evaporated to give the linear
protected peptide. The product (100 mg) was then cyc-
lized overnight at rt in DMF (9 mL) with HOAt and
HATU (each 3 equiv) and 1% v/v DIEA. After evapora-
tion of the DMF, the product was dissolved in CH2Cl2,
3. Experimental
3.1. Peptide synthesis
The peptidomimetic libraries were made by parallel syn-
thesis on a Syro-II (Microsyntec, Germany) peptide syn-
thesizer typically using 12–24 reaction vessels, and a
method described earlier.56 A typical procedure is de-
scribed. In one reaction vessel, 2-chlorotrityl chloride
resin (0.5 g, 0.83 mmol/g) was suspended in CH2Cl2
and then treated with the first amino acid (0.415 mmol)
and DIEA (4 equiv) with shaking at rt for 18 h. The

2060
J. A. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 2055–2064
Table 3. Amino acid substitutions made at position-14 in 4
Position-14 (Pro)
Pro
12.5
Gly
Arg
Tyr
Phe
6.2
Trp
6.2
Leu
12.5
Ile
6.2
Val
12.5
Gln
Cha
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
12.5
25
6.2
12.5
25
12.5
25
25
1
25
50
12.5
6.2
6.2
12.5
1.4
6.2
12.5
0.7
12.5
12.5
4.1
12.5
50
25
25
2
12.5
50
1
50
100
12.5
11.7
1.1
1.7
2.7
1.2
Position-14 (Pro)
Ybzl
Hfe
2-Nal
1-Nal
Nle
Bip
ClF
S(B)
Orn
hCha
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
25
12.5
12.5
12.5
5.9
12.5
12.5
12.5
18.2
25
12.5
6.2
50
12.5
6.2
12.5
6.2
25
25
25
6.2
6.2
6.2
12.5
12.5
13.1
6.2
12.5
14.7
12.5
13.1
12.5
4.2
12.5
19.7
100
6.2
13.8
1.6
0.8
Position-14 (Pro)
14A
14B
14C
14D
14E
14F
14G
14H
14I
9.4
14J
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
3.1
3.1
6.2
2.7
6.2
6.2
6.2
6.2
6.2
6.2
6.2
18
12.5
25
50
100
100
25
50
12.5
25
12.5
6.2
25
12.5
25
6.2
19.6
6.2
23.6
100
0.5
100
50
1.9
0.6
0.5
1.7
2.7
Position-14 (Pro)
14K
14L
14M
14N
14O
14P
12.5
14Q
12.5
9.4
25
0.2
14R
14S
25
6.2
14T
14U
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
Haemolysis (%)
50
25
12.5
6.2
12.5
12.5
12.5
3.9
12.5
12.5
25
12.5
6.2
25
25
25
50
0.7
12.5
6.2
18
12.5
25
1
12.5
12.5
11
100
0.9
12.5
3.4
25
3
12.5
3.4
0.8
2.2
MICs in lg/mL against the test organisms are shown, and the percentage haemolysis of human red blood cells caused at a peptide concentration of
100 lg/mL. The first entry shows the activity of 4 without substitution. The normal three letter code is used for proteinogenic amino acids, and
Nle = L-norleucine; S(B) = L-(O-benzyl)serine; hCha = L-homocyclohexylalanine; see also Tables 1 and 2 and Figure 3 for 14A–U.
washed with water and dried. The cyclic protected pep-
tide was treated with TFA/H2O/TIPS (95/2.5/2.5) at rt
for 2.5 h. After evaporation, the residue was dissolved
in H2O/acetic acid (3/1) and extracted with diisopropyl
ether. The aqueous phase was evaporated and the prod-
uct purified by reverse phase HPLC-MS (C18 column,
gradient from 5% to 95% MeCN/H2O + 0.1% TFA).
For analysis of purified products (C18 column, flow
1 mL/min, gradient from 5–95% MeCN/H2O + 0.1%
TFA over 20 min) the HPLC instrument was interfaced
with a Finnigan AQA electrospray ionization (ESI)
mass spectrometer (MS). The ESI-MS of all mimetics
was measured to an accuracy of 1 in 1000, and the spec-
tra confirmed in every case the expected molecular
weight (see Tables 4A and 4B).
3.2. Antimicrobial assays
The antimicrobial activities were determined by the
standard NCCLS broth microdilution method. Innocula
of the microorganisms were diluted into Mueller–Hin-
ton (MH) broth to give approximately 106 colony form-
ing units (CFU) per mL. Aliquots (50 lL) of the
innocula were added to MH broth (50 lL) containing
the peptide in serial twofold dilutions. Antimicrobial
activities are expressed as the minimal inhibitory con-
centration (MIC) in lg/mL at which 100% inhibition
of growth was observed after 18–20 h incubation at
37 ꢁC.
3.3. Haemolytic activity
Fresh human red blood cells were washed three times
with phosphate buffered saline (PBS), and then incu-
bated with peptide at a concentration of 100 lg/mL
for 1 h at 37 ꢁC. The final erythrocyte concentration
was ca. 0.9 · 109/mL. The values of 0% and 100% lysis
Figure 3. Structures of the analogues 14A–U. Compare structure 4.

J. A. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 2055–2064
2061
Scheme 1.
Table 4A. Sequences, retention times (tR, min) on HPLC, synthesis yield (%), and the mass confirmed by ESI-MS for the mimetics described in
Tables 1 and 2
Mimetic
1
2
3
4
5
6
7
8
9
10
11
12
tR
%
Mass
1
Leu
Phe
Cha
Tyr
Trp
Hfe
1Nal
2Nal
ClF
His
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
10.6
10.5
10.6
10.4
10.5
9.6
51
52
36
38
40
95
47
69
53
63
39
84
1878.4
1912.4
1918.4
1928.4
1951.4
1926.4
1962.4
1962.4
1946.8
1902.3
1988.5
1879.3
2
3
4
5
6
7
11.8
9.6
8
9
11.1
10.3
11.4
8.4
10
11
12
Bip
Orn
13
14
15
16
17
18
19
20
21
22
23
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Phe
Cha
Trp
Hfe
1Nal
2Nal
Val
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
10.6
10.8
10.5
10.6
10.9
11.0
10.0
10.9
10.3
11.3
9.2
44
41
41
32
43
42
47
50
40
39
48
1912.4
1918.4
1951.4
1926.4
1962.4
1962.4
1864.3
1946.8
1902.4
1988.5
1879.3
ClF
His
Bip
Orn
24
25
26
27
28
29
30
31
32
33
34
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Phe
Tyr
Y(B)
Hfe
1Nal
2Nal
Val
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
10.0
9.9
54
48
33
52
34
53
49
49
46
57
31
1839.3
1855.3
1945.4
1853.3
1889.4
1889.4
1791.2
1873.8
1805.3
1805.3
1806.3
11.0
10.3
10.5
10.5
9.9
ClF
Leu
Ile
10.4
10.1
10.1
9.7
Orn
35
36
37
38
39
40
41
42
43
44
45
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Phe
Cha
Trp
Hfe
1Nal
2Nal
Val
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
10.9
11.1
10.8
11.3
11.6
11.7
10.6
11.2
10.7
11.6
9.4
45
42
56
32
24
30
48
48
18
46
49
1862.3
1868.4
1901.4
1876.4
1912.4
1912.4
1814.3
1896.8
1828.3
1938.5
1829.3
ClF
Leu
Bip
Orn
(continued on next page)

2062
J. A. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 2055–2064
Table 4A (continued)
Mimetic
1
2
3
4
5
6
7
8
9
10
11
12
tR
%
Mass
46
47
48
49
50
51
52
53
54
55
56
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Phe
Cha
Trp
Hfe
1Nal
2Nal
Tyr
10.6
11.2
10.4
10.8
11.3
11.2
10.2
11.0
10.5
11.7
8.3
35
60
69
95
89
30
42
32
56
37
75
1926.4
1932.5
1965.4
1940.4
1976.5
1976.5
1942.2
1960.8
1892.4
2002.5
1893.4
ClF
Leu
Bip
Orn
57
58
59
60
61
62
Leu
Leu
Leu
Leu
Leu
Leu
Tyr
Trp
Leu
His
Thr
Gln
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Trp
Trp
Trp
Trp
Trp
Trp
Lys
Lys
Lys
Lys
Lys
Lys
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Arg
Arg
Arg
Arg
Arg
Arg
Val
Val
Val
Val
Val
Val
10.3
10.4
10.6
10.3
10.4
10.4
42
55
88
40
94
55
1885.4
1908.4
1835.3
1859.3
1823.3
1850.3
63
64
65
66
67
68
Leu
Leu
Leu
Leu
Leu
Leu
Arg
Arg
Arg
Arg
Arg
Arg
Leu
Leu
Leu
Leu
Leu
Leu
Arg
Trp
Leu
His
Thr
Gln
Lys
Lys
Lys
Lys
Lys
Lys
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Trp
Trp
Trp
Trp
Trp
Trp
Lys
Lys
Lys
Lys
Lys
Lys
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Arg
Arg
Arg
Arg
Arg
Arg
Val
Val
Val
Val
Val
Val
10.3
10.5
10.9
10.2
10.4
10.3
45
35
62
30
94
76
1906.4
1936.4
1863.3
1887.3
1851.3
1878.3
69
70
71
72
73
74
Leu
Leu
Leu
Leu
Leu
Leu
Arg
Arg
Arg
Arg
Arg
Arg
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Lys
Lys
Lys
Lys
Arg
Trp
Leu
His
Thr
Gln
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Trp
Trp
Trp
Trp
Trp
Trp
Lys
Lys
Lys
Lys
Lys
Lys
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Arg
Arg
Arg
Arg
Arg
Arg
Val
Val
Val
Val
Val
Val
10.4
10.3
11.9
10.5
11.4
10.4
37
45
43
53
45
50
1906.4
1936.4
1863.3
1887.3
1851.3
1878.3
75
76
77
78
79
80
Leu
Leu
Leu
Leu
Leu
Leu
Arg
Arg
Arg
Arg
Arg
Arg
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Tyr
Trp
Leu
His
Thr
Gln
Arg
Arg
Arg
Arg
Arg
Arg
Trp
Trp
Trp
Trp
Trp
Trp
Lys
Lys
Lys
Lys
Lys
Lys
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Arg
Arg
Arg
Arg
Arg
Arg
Val
Val
Val
Val
Val
Val
10.5
10.8
10.8
10.2
10.4
10.4
65
35
58
46
82
36
1885.4
1908.4
1835.3
1859.3
1823.3
1850.3
81
82
83
84
85
86
Leu
Leu
Leu
Leu
Leu
Leu
Arg
Arg
Arg
Arg
Arg
Arg
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Arg
Arg
Arg
Arg
Arg
Arg
Tyr
Trp
Leu
His
Gln
Thr
Trp
Trp
Trp
Trp
Trp
Trp
Lys
Lys
Lys
Lys
Lys
Lys
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Arg
Arg
Arg
Arg
Arg
Arg
Val
Val
Val
Val
Val
Val
10.6
10.8
10.7
10.3
10.4
10.3
60
70
50
74
40
45
1885.4
1908.4
1835.3
1859.3
1850.3
1823.3
87
88
89
90
91
92
Leu
Leu
Leu
Leu
Leu
Leu
Arg
Arg
Arg
Arg
Arg
Arg
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Trp
Trp
Trp
Trp
Trp
Trp
Tyr
Trp
Leu
Thr
Gln
His
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Arg
Arg
Arg
Arg
Arg
Arg
Val
Val
Val
Val
Val
Val
10.5
10.7
10.6
10.5
10.5
10.4
88
65
44
84
52
49
1913.4
1936.4
1863.3
1851.3
1878.3
1887.3
93
94
95
96
97
Leu
Leu
Leu
Leu
Leu
Arg
Arg
Arg
Arg
Arg
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Trp
Trp
Trp
Trp
Trp
Lys
Lys
Lys
Lys
Lys
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Trp
Leu
Orn
Cit
Val
Val
Val
Val
Val
10.6
10.9
10.7
10.2
10.5
54
47
65
92
54
1885.3
1908.4
1835.3
1836.3
1880.7
were determined by incubation of cells with PBS or 0.1%
Triton X-100 in water, respectively. The samples were
centrifuged, the supernatant diluted 20-fold in PBS,
and the optical density was measured at 540 nm. The
reproducibility in determinations of haemolytic activity
was typically 1%.
4. Dye release from liposomes
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
(POPG) were purchased from Genzyme (Liestal, Swit-
zerland). Large unilamellar vesicles (LUVs) of approxi-

J. A. Robinson et al. / Bioorg. Med. Chem. 13 (2005) 2055–2064
2063
Table 4B. Retention times (tR) on HPLC, synthesis yield (%), and the
mass confirmed by ESI-MS for the mimetics described in Table 3; also
shown is the residue used to replace L-Pro-14 (see also Fig. 3)
The apparent percent calcein leakage was calculated
according to:
ðF ꢀ F 0Þ
Mimetic
tR
%
Av.
mass
Mimic tR
%
Av.
mass
% Dye leakage ¼
ꢁ 100
ðF max ꢀ F 0Þ
(min)
(min)
1
2
3
4
5
6
7
8
9
Pro
Gly
Arg
Tyr
Trp
Leu
Ile
10.6
9.5
51 1878.4 14A
58 1838.3 14B
57 1937.4 14C
29 1944.4 14D
25 1967.5 14E
21 1894.4 14F
42 1894.4 14G
50 1880.4 14H
33 1909.4 14I
36 1934.5 14J
51 2034.6 14K
76 1942.5 14L
27 1978.5 14M
46 1978.5 14N
53 1894.4 14O
43 2004.5 14P
67 1962.9 14Q
51 1958.5 14R
76 1895.4 14S
64 1948.5 14T
14U
11.1
11.8
11.5
11.8
9.4
51 1991.5
43 2019.6
37 2087.6
23 2087.6
27 1978.5
30 2006.5
48 1935.4
42 1949.4
55 1963.4
44 1977.5
23 1963.4
20 2003.5
18 2017.5
21 1977.5
30 1997.4
32 2011.5
28 2050.5
24 2064.5
22 2073.5
20 2034.6
21 2018.3
where F is the fluorescence intensity induced by the pep-
tide, and F0 and Fmax denote the fluorescence intensities
without peptide or detergent and with Triton X-100,
respectively.
9.3
9.9
10.7
10.5
10.4
10.5
9.8
9.4
10.0
10.0
10.1
10.3
10.4
10.3
10.9
10.5
10.5
10.6
10.9
9.9
References and notes
Val
Gln
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