New antimicrobial hexapeptides: Synthesis, antimicrobial activities, cytotoxicity, and mechanistic studies

Article abstract of DOI:10.1002/cmdc.200900330

Synthetic antimicrobial peptides have recently emerged as promising candidates against drug-resistant pathogens. We identified a novel hexapeptide, Orn-D-Trp-D-Phe-Ile-D-Phe-His(1-Bzl)-NH₂, which exhibits broad-spectrum antifungal and antibacterial activity. A lead optimization was undertaken by conducting a full amino acid scan with various proteinogenic and non-proteinogenic amino acids depending on the hydrophobic or positive-charge character of residues at various positions along the sequence. The hexapeptide was also cyclized to study the correlation between the linear and cyclic structures and their respective antimicrobial activities. The synthesized peptides were found to be active against the fungus Candida albicans and Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis, as well as the Gram-negative bacterium Escherichia coli; MIC values for the most potent structures were in the range of 1-5 μg mL^-1 (IC₅₀ values in the range of 0.02-2 μg mL^-1). Most of the synthesized peptides showed no cytotoxic effects in an MTT assay up to the highest test concentration of 200 μg mL^-1. A tryptophan fluorescence quenching study was performed in the presence of negatively charged and zwitterionic model membranes, mimicking bacterial and mammalian membranes, respectively. The results of the fluorescence study demonstrate that the tested peptides are selective toward bacterial over mammalian cells; this is associated with a preferential interaction between the peptides and the negatively charged phospholipids of bacterial cells.

Full text of DOI:10.1002/cmdc.200900330

MED
DOI: 10.1002/cmdc.200900330
New Antimicrobial Hexapeptides: Synthesis, Antimicrobial
Activities, Cytotoxicity, and Mechanistic Studies
Rohit K. Sharma,[a] Sandeep Sundriyal,[a] Nishima Wangoo,[b] Werner Tegge,*[c] and
Rahul Jain*[a]
Synthetic antimicrobial peptides have recently emerged as
promising candidates against drug-resistant pathogens. We
Staphylococcus epidermidis, as well as the Gram-negative bacte-
rium Escherichia coli; MIC values for the most potent structures
were in the range of 1–5 mgmLꢀ1 (IC50 values in the range of
0.02–2 mgmLꢀ1). Most of the synthesized peptides showed no
cytotoxic effects in an MTT assay up to the highest test con-
centration of 200 mgmLꢀ1. A tryptophan fluorescence quench-
ing study was performed in the presence of negatively
charged and zwitterionic model membranes, mimicking bacte-
rial and mammalian membranes, respectively. The results of
the fluorescence study demonstrate that the tested peptides
are selective toward bacterial over mammalian cells; this is as-
sociated with a preferential interaction between the peptides
and the negatively charged phospholipids of bacterial cells.
identified
a novel hexapeptide, Orn-d-Trp-d-Phe-Ile-d-Phe-
His(1-Bzl)-NH2, which exhibits broad-spectrum antifungal and
antibacterial activity. A lead optimization was undertaken by
conducting a full amino acid scan with various proteinogenic
and non-proteinogenic amino acids depending on the hydro-
phobic or positive-charge character of residues at various posi-
tions along the sequence. The hexapeptide was also cyclized
to study the correlation between the linear and cyclic struc-
tures and their respective antimicrobial activities. The synthe-
sized peptides were found to be active against the fungus
Candida albicans and Gram-positive bacteria such as methicil-
lin-resistant Staphylococcus aureus and methicillin-resistant
Introduction
The widespread and irrational use of antibiotics has led to the
emergence of resistant strains of pathogenic microorganisms.
Therefore, the search for novel antimicrobial agents has gained
importance over the past several years. Nature has provided a
solution to this problem in the form of antimicrobial peptides
(AMPs) that are not only lethal to a broad spectrum of patho-
gens but also have a unique low tendency to cause develop-
ment of resistance. A variety of AMPs and proteins have been
isolated from virtually all kingdoms and phyla including plants,
microbes, insects, larger animals, and humans.[1–4] Hundreds of
such AMPs have been listed in the antimicrobial peptide data-
base (APD).[5] These naturally occurring peptides are an integral
part of the host defense system and provide an immediate re-
sponse to invading microorganisms; they display a broad spec-
trum of bactericidal and fungicidal actions.[6] Consequently,
AMPs are specifically toxic toward pathogens and are able to
differentiate between host and microbial cells. This specific rec-
ognition may be governed by differences in cell membrane
composition and hence charge, membrane asymmetry, trans-
membrane potential, and specific receptor binding.[7] Such se-
lective action is also demonstrated by preferential interaction
with the anionic lipopolysaccharide (LPS) of the bacterial cell
membrane rather than with the net neutral mammalian cell
membrane, which is composed of zwitterionic phospholipids
and cholesterol.[8–10] The therapeutic potential of AMPs is at-
tributed mainly to their membrane lytic properties. This in-
cludes initial binding of the peptide monomer to the microbial
membrane followed by peptide aggregation and insertion,
which generally results in the formation of pores or disruption
of the cell membrane. The peptides have demonstrated their
ability to rapidly kill a broad spectrum of microorganisms in-
cluding multi-drug-resistant bacteria, fungi, and viruses. Devel-
opment of resistance by sensitive microbial strains against
these AMPs is less probable, because AMPs exert their action
by forming multimeric pores in the cell membranes, leading to
cell lysis,[11] or interaction with the RNA or DNA after penetra-
tion into the cell.[12] Models proposed to explain the mecha-
nism of microbial cytoplasmic membrane disruption by AMPs
include the ’barrel-stave’ model, the ’toroid pore’ or ’worm-
hole’ model, and the ’carpet’ model.[7,13–15] In general, the initial
association of AMPs with the microbial membrane occurs
[a] Dr. R. K. Sharma, Dr. S. Sundriyal, Prof. R. Jain
Department of Medicinal Chemistry
National Institute of Pharmaceutical Education and Research
Sector 67, S.A.S. Nagar – 160 062, Punjab (India)
Fax: (+91)172-2214692
E-mail: rahuljain@niper.ac.in
[b] N. Wangoo
Institute of Microbial Technology
Sector 39, Chandigarh – 160 036, Punjab (India)
[c] Dr. W. Tegge
Department of Chemical Biology
Helmholtz Centre for Infection Research
Inhoffenstrasse 7, 38124 Braunschweig (Germany)
E-mail: werner.tegge@helmholtz-hzi.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.200900330.
86
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ChemMedChem 2010, 5, 86 – 95

Antimicrobial Hexapeptides
through electrostatic interactions between the cationic peptide
and the anionic LPS or other negatively charged components
of the membrane bilayer, leading to membrane perturbation.
Most of these peptides are cationic in nature with a significant
number of hydrophobic residues, and their antimicrobial po-
tency has been correlated with positive charge, although not
always linearly.[16]
intact in all designed peptides. Modulation of histidine resi-
due(s), either by substitution on the imidazole ring or by re-
placement with other amino acids, could profoundly influence
a peptide’s biological activities.[22] Moreover, it has been report-
ed that the replacement of basic amino acids, such as arginine
and lysine, with histidine increases the antimicrobial activity of
peptides, particularly under acidic conditions.[23] Therefore, we
first investigated the role of His6 and synthesized peptides in
which it is replaced by d-His, 1-Bzl-His, and 2-pyridylalanine (2-
Pal) (Table 1). In one particular case (peptide 3), in addition to
replacement of His6 with 1-Bzl-His, the role of Arg1 was also
investigated by its replacement with the less basic non-protei-
nogenic ornithine (Orn) residue.
Although naturally occurring AMPs appear to be promising
candidates against resistant pathogens owing to their specifici-
ty and infrequent problems of resistance development, their
clinical utility is limited by problems of enzymatic degradation,
bioavailability, and cost. Thus, synthetic congeners that possess
similar structural features but which contain unnatural amino
acids may provide the solution.
Short cationic AMPs have
Table 1. In vitro antifungal and antibacterial activities of representative hexapeptides 3–6.
reached the realm of classical
drug molecules in terms of mo-
lecular size and complexity. They
are attracting considerable at-
tention and are the focus of
more research in their develop-
ment as future antibiotics to
treat multi-drug-resistant infec-
tions.[17–19] Thus, our aim is to
identify potential AMPs and to
manipulate their structure to
create synthetic analogues to
successfully treat fungal and
bacterial infections.
Peptide
Sequence[a]
C. neoformans
MRSA
MSSA
[c]
[c]
[c]
MIC[b]
IC50
MIC[b]
IC50
MIC[b]
IC50
3
4
5
6
O-W-F-I-F-H(1-Bzl)-NH2
R-W-F-I-F-H-NH2
R-W-F-I-F-H(1-Bzl)-NH2
R-W-F-I-F-Pal-NH2
5.0
10.0
5.0
1.5
6.0
3.5
3.5
10.0
NT
10.0
5.0
3.5
>20
10.0
NT
10.0
10.0
3.5
>20
6.5
3.5
6.0
3.5
5.0
[a] O=ornithine; amino acids in italics represent the d-isomers. [b] MIC [mgmLꢀ1] (minimum inhibitory concen-
tration): the lowest test concentration that completely inhibits microorganism growth; NT: not tested.
[c] IC50 [mgmLꢀ1]: the concentration that affords 50% inhibition of bacterial growth; standards used: C. neofor-
mans (amphotericin B: IC50 =0.34 mgmLꢀ1), MRSA (vancomycin: IC50 =0.14 mgmLꢀ1), MSSA (ciprofloxacin: IC50
0.05 mgmLꢀ1).
=
Peptides 3–6 displayed promising antifungal activity against
C. neoformans (Table 1), but they were not active against Asper-
gillus fumigatus and showed moderate inhibition (IC50 =
20 mgmLꢀ1) against C. albicans (data not shown). Peptide 3
was the most promising antifungal compound of the series,
displaying pronounced antifungal activity with an MIC value of
5.0 mgmLꢀ1 (IC50 =1.5 mgmLꢀ1) against C. neoformans (Table 1),
whereas peptides 4–6 displayed MIC values in the range be-
tween 5.0 and 10.0 mgmLꢀ1. Peptides 3, 5, and 6 displayed
promising antibacterial activities against methicillin-sensitive
Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus
(MRSA). Peptide 3 was the most potent and exhibited antibac-
Results and Discussion
We came across an earlier study in which the hexapeptide His-
d-Trp-d-Phe-Phe-d-Phe-Lys-NH2 (1), structurally similar to
growth hormone releasing hexapeptide (GHRP-6, His-d-Trp-
Ala-Trp-d-Phe-Lys-NH2),[20] was found to display antifungal ac-
tivity against Candida albicans (IC50 =28.6 mm) and Cryptococ-
cous neoformans (MIC=6.8 mm). Further optimization of 1 was
done by using the combinatorial approach in which d-amino
acids at positions 2, 3, and 5, which were found to be essential
for activity, were retained. This led to the identification of pep-
tide 2, Arg-d-Trp-d-Phe-Ile-d-Phe-His-NH2, which is more
potent against C. albicans (IC50 =6.8 mm) but displays similar
potency against C. neoformans (MIC=6.8 mm). To examine the
effect of increased peptide chain length on biological activity,
peptide 2 was chosen as a motif upon which to develop a
nonapeptide library. This approach led to the peptide Arg-d-
Trp-d-Phe-Ile-d-Phe-His-Lys-Arg-Lys-NH2, which is more potent
than 2 against both C. albicans (IC50 =3.3 mm) and C. neofor-
mans (MIC=2.4 mm).[21]
terial activity with MIC values of 10 mgmLꢀ1 (IC50 =3.5 mgmLꢀ1
)
against MRSA and MSSA. The in vitro cytotoxicities of peptides
3–6 were tested against four human cancer cell lines and two
noncancerous mammalian kidney cell lines up to a concentra-
tion of 23.8 mgmLꢀ1 using a neutral red assay procedure, as de-
scribed earlier.[24,25] None of the peptides showed any cytotoxic
effects up to the highest test concentration. The antimicrobial
activities discussed above led to the identification of the hexa-
peptide Orn-d-Trp-d-Phe-Ile-d-Phe-His(1-Bzl)-NH2 (3) as a lead
compound, which provides an interesting pharmacophore for
the design of more potent AMPs. Interestingly, upon careful
examination the chemical structure of peptide 3, the clear-cut
demarcation between the positively charged termini (residues
X1 and X6) and the inner hydrophobic core sequence (residues
X2–X5) is readily apparent (Figure 1). This segregation of posi-
tively charged and hydrophobic features correlates directly
with the amphiphilic structure of AMPs, and this has been
In our preliminary study, we set out to optimize hexapeptide
2 in terms of structure and biological activity, without increas-
ing the peptide backbone length, by replacing its key constitu-
ent amino acids with selected unnatural amino acids. We con-
centrated our efforts on a systematic amino acid replacement
strategy with peptide 2. Keeping in mind the observations
made earlier,[20] the residues at positions 2–5 in 2 were kept
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87

R. Jain, W. Tegge, et al.
MED
(Table 2). These peptides were synthesized using the Boc strat-
egy for solid-phase peptide synthesis (SPPS).
Modifications at positions 2–5
Keeping the hydrophobic character intact, the d-Trp2, d-Phe3,
Ile4 and d-Phe5 groups of peptide 3 were systematically re-
placed with various proteinogenic and non-proteinogenic
amino acids, including aminobutyric acid (Abu), 4,4’-biphenyla-
lanine (Bip), 4-fluoro-l-phenylalanine [Phe(4-F)], 4-methyl-l-
phenylalanine [Phe(4-Me)], cyclohexylalanine (Cha), phenylgly-
cine (Phg), 3-benzothienylalanine (Bal), 4-tert-butyl-l-phenylala-
nine [Phe(4-tBu)], 1-napthylalanine (Nal), 4-trifluoromethyl-l-
phenylalanine [Phe(4-CF3)], 3-(9-anthryl)-l-alanine [Ala(9-anth)],
dibutylglycine (Dbg), 3,3-diphenylalanine (Dip), norleucine
(Nle), pentafluoro-l-phenylalanine (Pfp), l/d-Trp, l/d-Phe, l/d-
Leu, l/d-Ile, l/d-Val, l/d-Tyr, l/d-Cys, l/d-Met, l/d-Ala, and l/d-
Pro. These replacements resulted in peptides 16–47 (substi-
tuted at X2), 48–80 (substituted at X3), 81–112 (substituted at
X4) and 113–145 (substituted at X5) (Table 2), which were syn-
thesized using the Fmoc strategy for SPPS.
Figure 1. Lead hexapeptide 3 with cationic termini and inner hydrophobic
amino acids.
widely reported to be essential for the antimicrobial activity of
this peptide class. Encouraged by these observations, we de-
cided to undertake lead optimization in the form of a full
amino acid scan with various proteinogenic and non-proteino-
genic (natural/unnatural) amino acids on lead hexapeptide 3.
Our aim was to increase antimicrobial activity and to identify
residues that increase activity at various sequence positions in
the lead compound. In particular, the positively charged termi-
nal amino acids were replaced with similar cationic residues at
positions 1 and 6 of lead peptide 3. The selection of replace-
ment residues was made on the assumption that they partially
or fully retain the cationic character of amino acids already
present. Similarly, the amino acids at positions 2–5 of lead pep-
tide 3 were replaced with residues in a manner that conserves
the hydrophobic character at this position.
Cyclization
To investigate the antimicrobial activities of linear versus cyclic
structures, we also synthesized an N-to-C-terminus-cyclized
version of peptide 3. The linear hexapeptide, which was syn-
thesized by Fmoc SPPS, was cyclized in the presence of PyBop,
HOAt, and DIEA in DMF to afford the cyclic peptide 155
(Scheme 1).
Antimicrobial activity
The synthesized peptides were evaluated for antibacterial and
antifungal activities against three Gram-negative bacteria [Es-
cherichia coli (ATCC 35218), Pseudomonas aeruginosa
(ATCC 9027), and Klebsiella pneumoniae (ATCC 700603)], two
Gram-positive bacteria [MRSA (DSM 50128509) and methicillin-
resistant Staphylococcus epidermidis (MRSE; DSM 50160384)],
and one fungal strain [C. albicans (ATCC 10231)], which were
obtained from the German Collection of Microorganisms and
Cell Cultures (DSMZ) and cultured using procedures described
earlier.[26–28]
We also synthesized a specific peptide in which the lead
hexapeptide 3 is constrained by cyclization. This was undertak-
en to correlate the linear and cyclic structures of the peptides
with their respective antimicrobial activities. The synthesized
peptides were evaluated for antimicrobial activity against two
Gram-positive bacteria, three Gram-negative bacteria, and one
fungal strain. To further assess their therapeutic potential, cyto-
toxicity studies were performed with the MTT test on mouse fi-
broblasts. To investigate the relative extent of peptide burial in
the model membranes, we performed a fluorescence quench-
ing experiment in small unilamellar vesicles (SUVs) using the
water-soluble neutral fluorescence quencher acrylamide.
The antimicrobial activities of peptides 7–155 are listed in
Table 2. Among peptides 7–15, which represent replacements
at position X1 of peptide 3, peptides 10 (X1 =d-Lys) and 11
(X1 =Lys) are the most potent against C. albicans (MIC=
20 mgmLꢀ1; IC50 =9.9–11.7 mgmLꢀ1). These observations clearly
indicate that the presence of Lys, either as the l or d isomer,
increases the potency of X1-position-substituted peptides
toward the fungus to the greatest extent. They also reflect that
the presence of an amino group (as a positively charged spe-
cies) on the side chain at position X1, consistent with peptide
3 (X1 =Orn), is essential for activity against C. albicans. Regard-
ing Gram-positive bacterial strains, analogue 8 displayed
potent activity against MRSA, with an excellent MIC value of
2 mgmLꢀ1 (IC50 =0.75 mgmLꢀ1). Analogues 7 and 11 (X1 =d-
Modifications at positions 1 and 6
The Orn1 and His(1-Bzl)6 residues at the respective N- and C-
termini of peptide 3 were selectively replaced with cationic
amino acids based on the assumption that they partially or
fully retain the cationic character at positions X1 and X6. The
substitute cationic residues chosen were Lys, Arg, His, d-Lys, d-
Arg, d-His, 2,4-diaminobutyric acid (Dab), 4-amino-l-phenylala-
nine [Phe(4-NH2)], His(1-Bzl), and Orn; this resulted in peptides
7–15 (substituted at X1) and 146–154 (substituted at X6)
88
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Antimicrobial Hexapeptides
Table 2. Antifungal and antimicrobial activities of hexapeptides 7–155.
[b]
Peptide
Sequence[a]
MIC (IC50) [mgmLꢀ1
]
C. albicans
MRSA
MRSE
E. coli
K. pneumoniae
P. aeruginosa
7
8
9
K-W-F-I-F-H(1-Bzl)-NH2
R-W-F-I-F-H(1-Bzl)-NH2
H-W-F-I-F-H(1-Bzl)-NH2
K-W-F-I-F-H(1-Bzl)-NH2
R-W-F-I-F-H(1-Bzl)-NH2
H-W-F-I-F-H(1-Bzl)-NH2
Dab-W-F-I-F-H(1-Bzl)-NH2
F(4-NH2)-W-F-I-F-H(1-Bzl)-NH2
H(1-Bzl)-W-F-I-F-H(1-Bzl)-NH2
O-Abu-F-I-F-H(1-Bzl)-NH2
O-Bip-F-I-F-H(1-Bzl)-NH2
O-F(4-F)-F-I-F-H(1-Bzl)-NH2
O-F(4-Me)Abu-F-I-F-H(1-Bzl)-NH2
O-Cha-F-I-F-H(1-Bzl)-NH2
O-Phg-F-I-F-H(1-Bzl)-NH2
O-Bal-F-I-F-H(1-Bzl)-NH2
O-F(4-tBu)-F-I-F-H(1-Bzl)-NH2
O-Nal-F-I-F-H(1-Bzl)-NH2
O-F(4-CF3)-F-I-F-H(1-Bzl)-NH2
O-A(9-anth)-F-I-F-H(1-Bzl)-NH2
O-Dbg-F-I-F-H(1-Bzl)-NH2
O-W-F-I-F-H(1-Bzl)-NH2
O-F-F-I-F-H(1-Bzl)-NH2
20 (11.7)
50 (18.6)
100 (20.9)
20 (9.9)
100 (27.4)
100 (37.2)
100 (22.8)
100 (47.3)
50 (18.0)
200
10 (4.8)
2 (0.75)
>200
100 (17.2)
5 (2.6)
>200
>200
>200
>200
>200
50 (21.4)
100 (3.4)
100 (26.9)
10 (7.3)
>200
5 (3.7)
10 (6.7)
>200
20 (8.0)
5 (1.9)
>200
50 (14.3)
>200
50 (36.1)
20 (13.3)
>200
100 (43.1)
100 (56.8)
>200
>200
>200
200
>200
200
200
100 (32.4)
200
>200
>200
200
>200
>200
>200
200
100 (23.6)
10 (13.5)
>200
>200
>200
>200
200
>200
100 (36.0)
>200
2 (1.4)
>200
>200
>200
200
>200
>200
>200
>200
>200
>200
>200
>200
200
100 (52.1)
100 (66.9)
200
200
200
>200
>200
200
100 (51.8)
200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
200
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
100 (37.6)
>200
>200
>200
>200
>200
>200
>200
>200
100 (56.1)
>200
>200
>200
>200
>200
>200
>200
>200
>200
200
50 (36.7)
5 (4.0)
2 (1.5)
50 (16.5)
50 (22.0)
100 (32.6)
5 (3.6)
50 (28.4)
200
50 (22.1)
10 (3.8)
50 (33.6)
>200
5 (2.9)
20 (11.2)
10 (9.4)
50 (27.1)
2 (1.1)
10 (7.2)
200
>200
50 (19.0)
10 (3.7)
50 (16.2)
>200
200
>200
>200
10 (7.9)
50 (35.0)
10 (14.9)
100 (39.0)
200
100 (59.1)
50 (20.7)
100 (43.0)
100 (27.0)
100 (64.3)
5 (4.5)
50 (40.3)
100 (64.1)
10 (6.1)
100 (57.3)
200
5 (1.1)
50 (38.0)
>200
>200
>200
>200
50 (23.1)
>200
100 (31.5)
100 (42.1)
50 (8.6)
>200
>200
>200
200
>200
>200
100 (49.7)
>200
200
>200
5 (3.9)
2 (0.25)
5 (1.8)
2 (0.11)
10 (6.8)
20 (8.8)
2 (1.8)
2 (1.1)
2 (1.7)
2 (0.94)
NT
10 (6.1)
20 (12.4)
200
>200
>200
>200
50 (11.6)
>200
>200
>200
5 (2.0)
>200
>200
>200
200
>200
100 (36.5)
>200
200
>200
O-F-F-I-F-H(1-Bzl)-NH2
O-l-F-I-F-H(1-Bzl)-NH2
O-l-F-I-F-H(1-Bzl)-NH2
O-I-F-I-F-H(1-Bzl)-NH2
O-I-F-I-F-H(1-Bzl)-NH2
O-V-F-I-F-H(1-Bzl)-NH2
O-V-F-I-F-H(1-Bzl)-NH2
O-Y-F-I-F-H(1-Bzl)-NH2
>200
200
>200
>200
>200
>200
200
200
O-Y-F-I-F-H(1-Bzl)-NH2
O-C-F-I-F-H(1-Bzl)-NH2
>200
>200
100 (72.3)
>200
200
>200
>200
>200
>200
100 (65.8)
200
>200
200
200
200
200
200
>200
200
200
200
NT
>200
200
200
200
O-C-F-I-F-H(1-Bzl)-NH2
O-M-F-I-F-H(1-Bzl)-NH2
O-M-F-I-F-H(1-Bzl)-NH2
O-A-F-I-F-H(1-Bzl)-NH2
O-A-F-I-F-H(1-Bzl)-NH2
O-P-F-I-F-H(1-Bzl)-NH2
>200
>200
200
100 (72.2)
200
50 (23.1)
200
O-P-F-I-F-H(1-Bzl)-NH2
>200
>200
>200
>200
50 (28.7)
50 (16.2)
100 (18.9)
>200
20 (5.9)
100 (52.6)
100 (79.0)
200
50 (21.5)
NT
100 (32.5)
>200
200
O-Dip-F-I-F-H(1-Bzl)-NH2
O-W-Abu-I-F-H(1-Bzl)-NH2
O-W-Bip-I-F-H(1-Bzl)-NH2
O-W-F(4-F)-I-F-H(1-Bzl)-NH2
O-W-F(4-Me)-I-F-H(1-Bzl)-NH2
O-W-Cha-I-F-H(1-Bzl)-NH2
O-W-Nle-I-F-H(1-Bzl)-NH2
O-W-Phg-I-F-H(1-Bzl)-NH2
O-W-Bal-I-F-H(1-Bzl)-NH2
O-W-F(4-tBu)-I-F-H(1-Bzl)-NH2
O-W-Nal-I-F-H(1-Bzl)-NH2
O-W-F(4-CF3)-I-F-H(1-Bzl)-NH2
O-W-A(9-anth)-I-F-H(1-Bzl)-NH2
O-W-Dbg-I-F-H(1-Bzl)-NH2
O-W-F-I-F-H(1-Bzl)-NH2
O-W-W-I-F-H(1-Bzl)-NH2
O-W-W-I-F-H(1-Bzl)-NH2
O-W-l-I-F-H(1-Bzl)-NH2
O-W-l-I-F-H(1-Bzl)-NH2
O-W-I-I-F-H(1-Bzl)-NH2
O-W-I-I-F-H(1-Bzl)-NH2
O-W-V-I-F-H(1-Bzl)-NH2
O-W-V-I-F-H(1-Bzl)-NH2
O-W-Y-I-F-H(1-Bzl)-NH2
O-W-Y-I-F-H(1-Bzl)-NH2
100 (78.7)
100 (60.3)
20 (13.0)
10 (6.6)
2 (1.0)
50 (14.8)
100 (28.1)
50 (17.7)
10 (7.2)
50 (27.7)
10 (4.0)
NT
100 (63.9)
20 (13.5)
20 (11.2)
10 (5.1)
10 (8.1)
50 (12.2)
20 (9.6)
50 (20.9)
100 (31.1)
50 (7.9)
100 (53.8)
50 (22.6)
>200
5 (2.7)
5 (2.4)
1 (0.2)
2 (0.86)
5 (1.6)
20 (6.9)
1 (1.0)
1 (0.56)
1 (0.02)
1 (0.81)
NT
200
>200
200
>200
200
100 (79.9)
200
>200
200
200
NT
200
200
>200
>200
100 (58.8)
>200
>200
>200
>200
>200
>200
>200
>200
>200
100 (22.7)
10 (9.0)
50 (18.3)
100 (61.7)
100 (21.1)
50 (17.1)
200
20 (4.0)
>200
>200
10 (3.8)
1 (0.18)
2 (1.1)
10 (6.3)
20 (11.5)
10 (8.6)
100 (76.8)
100 (36.4)
100 (54.1)
100 (26.4)
100 (46.9)
>200
200
200
>200
200
200
200
200
>200
200
>200
>200
>200
>200
>200
>200
>200
200
ChemMedChem 2010, 5, 86 – 95
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
89

R. Jain, W. Tegge, et al.
MED
Table 2. (Continued)
[b]
Peptide
Sequence[a]
MIC (IC50) [mgmLꢀ1
]
C. albicans
MRSA
MRSE
E. coli
K. pneumoniae
P. aeruginosa
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
O-W-C-I-F-H(1-Bzl)-NH2
O-W-C-I-F-H(1-Bzl)-NH2
O-W-M-I-F-H(1-Bzl)-NH2
O-W-M-I-F-H(1-Bzl)-NH2
O-W-A-I-F-H(1-Bzl)-NH2
O-W-A-I-F-H(1-Bzl)-NH2
O-W-P-I-F-H(1-Bzl)-NH2
O-W-P-I-F-H(1-Bzl)-NH2
O-W-Dip-I-F-H(1-Bzl)-NH2
O-W-F-Abu-F-H(1-Bzl)-NH2
O-W-F-Bip-F-H(1-Bzl)-NH2
O-W-F-F(4-F)-F-H(1-Bzl)-NH2
O-W-F-F(4-Me)-F-H(1-Bzl)-NH2
O-W-F-Cha-F-H(1-Bzl)-NH2
O-W-F-Nle-F-H(1-Bzl)-NH2
O-W-F-Phg-F-H(1-Bzl)-NH2
O-W-F-Bal-F-H(1-Bzl)-NH2
O-W-F-F(4-tBu)-F-H(1-Bzl)-NH2
O-W-F-Nal-F-H(1-Bzl)-NH2
O-W-F-Pfp-F-H(1-Bzl)-NH2
O-W-F-F(4-CF3)-F-H(1-Bzl)-NH2
O-W-F-A(9-anth)-F-H(1-Bzl)-NH2
O-W-F-Dbg-F-H(1-Bzl)-NH2
O-W-F-F-F-H(1-Bzl)-NH2
O-W-F-F-F-H(1-Bzl)-NH2
O-W-F-W-F-H(1-Bzl)-NH2
O-W-F-W-F-H(1-Bzl)-NH2
O-W-F-l-F-H(1-Bzl)-NH2
O-W-F-l-F-H(1-Bzl)-NH2
O-W-F-I-F-H(1-Bzl)-NH2
O-W-F-V-F-H(1-Bzl)-NH2
O-W-F-V-F-H(1-Bzl)-NH2
O-W-F-Y-F-H(1-Bzl)-NH2
O-W-F-C-F-H(1-Bzl)-NH2
O-W-F-C-F-H(1-Bzl)-NH2
O-W-F-M-F-H(1-Bzl)-NH2
O-W-F-A-F-H(1-Bzl)-NH2
O-W-F-A-F-H(1-Bzl)-NH2
O-W-F-P-F-H(1-Bzl)-NH2
O-W-F-P-F-H(1-Bzl)-NH2
O-W-F-Dip-F-H(1-Bzl)-NH2
O-W-F-I-Abu-H(1-Bzl)-NH2
O-W-F-I-Bip-H(1-Bzl)-NH2
O-W-F-I-F(4-F)-H(1-Bzl)-NH2
O-W-F-I-F(4-Me)-H(1-Bzl)-NH2
O-W-F-I-Cha-H(1-Bzl)-NH2
O-W-F-I-Nle-H(1-Bzl)-NH2
O-W-F-I-Phg-H(1-Bzl)-NH2
O-W-F-I-Bal-H(1-Bzl)-NH2
O-W-F-I-F(4-tBu)-H(1-Bzl)-NH2
O-W-F-I-Nal-H(1-Bzl)-NH2
O-W-F-I-F(4-CF3)-H(1-Bzl)-NH2
O-W-F-I-A(9-anth)-H(1-Bzl)-NH2
O-W-F-I-Dbg-H(1-Bzl)-NH2
O-W-F-I-F-H(1-Bzl)-NH2
O-W-F-I-I-H(1-Bzl)-NH2
100 (28.4)
200
100 (39.2)
100 (66.9)
200
100 (38.5)
100 (51.1)
200
10 (6.4)
200
10 (5.6)
5 (2.1)
200
100 (26.6)
>200
200
2 (1.4)
100 (40.6)
50 (20.9)
>200
>200
>200
>200
>200
>200
100 (35.6)
200
100 (52.5)
10 (8.2)
200
50 (34.0)
>200
100 (36.2)
100 (60.6)
10 (6.5)
>200
>200
50 (32.0)
>200
>200
100 (68.63)
>200
>200
200
>200
200
>200
>200
50 (22.2)
>200
>200
>200
NT
>200
>200
200
200
>200
20 (6.5)
>200
>200
200
100 (34.2)
>200
>200
>200
50 (27.1)
>200
>200
>200
>200
>200
>200
>200
>200
100 (42.7)
>200
>200
20 (8.6)
>200
>200
100 (62.5)
>200
>200
>200
>200
>200
>200
>200
>200
>200
100 (65.6)
200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
100 (75.6)
>200
>200
>200
>200
>200
>200
>200
200
>200
>200
>200
NT
>200
>200
>200
>200
>200
>200
>200
200
>200
>200
>200
>200
>200
>200
100 (56.0)
>200
>200
200
>200
>200
>200
>200
>200
100 (37.5)
100 (32.2)
>200
>200
>200
>200
200
200
200
>200
200
200
200
>200
200
>200
200
>200
200
>200
>200
100 (46.6)
>200
100 (38.7)
>200
200
10 (4.8)
20 (11.1)
100 (33.9)
200
200
100 (17.7)
>200
1 (0.58)
50 (12.8)
2 (1.2)
1 (0.78)
1 (0.07)
>200
>200
>200
2 (1.2)
>200
2 (1.4)
2 (0.95)
1 (0.42)
>200
>200
20 (9.4)
1 (0.58)
20 (12.8)
>200
1 (0.63)
200
200
100 (63.4)
20 (15.3)
20 (11.3)
20 (7.0)
100 (26.8)
200
100 (48.7)
100 (38.9)
50 (23.8)
100 (49.7)
50 (17.7)
100 (31.5)
20 (4.4)
200
100 (63.6)
>200
200
50 (29.3)
100 (31.7)
50 (12.5)
100 (54.1)
50 (26.2)
200
>200
NT
200
200
200
200
50 (22.6)
200
200
50 (30.2)
100 (44.7)
50 (16.1)
>200
200
200
20 (5.8)
200
100 (65.9)
200
>200
1 (1.2)
1 (0.23)
1 (0.63)
>200
20 (19.5)
5 (2.5)
100 (38.9)
100 (42.0)
5 (4.0)
20 (10.3)
20 (16.3)
20 (9.5)
200
200
>200
50 (20.3)
50 (25.8)
100 (56.8)
>200
>200
NT
>200
>200
100 (57.4)
>200
1 (0.70)
>200
200
200
200
200
200
100 (32.6)
5 (2.1)
50 (18.4)
20 (10.5)
10 (3.6)
100 (37.9)
200
>200
100 (26.9)
50 (7.8)
50 (20.4)
>200
>200
NT
>200
200
>200
>200
200
97
98
99
>200
200
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
>200
>200
100 (58.5)
>200
>200
>200
NT
>200
200
>200
200
>200
>200
>200
>200
>200
>200
>200
200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
200
>200
200
>200
>200
50 (12.4)
100 (20.1)
200
200
>200
50 (14.6)
200
>200
>200
>200
200
>200
>200
200
200
200
200
>200
>200
200
200
200
20 (8.0)
200
50 (19.0)
100 (29.7)
100 (43.1)
100 (27.4)
200
100 (58.8)
>200
200
>200
>200
200
>200
>200
200
200
>200
200
>200
100 (56.5)
200
50 (20.5)
50 (26.4)
100 (41.5)
>200
100 (34.0)
200
100 (50.8)
200
100 (27.6)
200
O-W-F-I-W-H(1-Bzl)-NH2
O-W-F-I-l-H(1-Bzl)-NH2
O-W-F-I-l-H(1-Bzl)-NH2
O-W-F-I-I-H(1-Bzl)-NH2
O-W-F-I-V-H(1-Bzl)-NH2
O-W-F-I-V-H(1-Bzl)-NH2
O-W-F-I-Y-H(1-Bzl)-NH2
O-W-F-I-Y-H(1-Bzl)-NH2
O-W-F-I-C-H(1-Bzl)-NH2
>200
>200
200
>200
100 (40.1)
>200
>200
100 (39.4)
90
www.chemmedchem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 86 – 95

Antimicrobial Hexapeptides
Table 2. (Continued)
[b]
Peptide
Sequence[a]
MIC (IC50) [mgmLꢀ1
]
C. albicans
MRSA
MRSE
E. coli
K. pneumoniae
P. aeruginosa
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
O-W-F-I-C-H(1-Bzl)-NH2
O-W-F-I-M-H(1-Bzl)-NH2
O-W-F-I-M-H(1-Bzl)-NH2
O-W-F-I-A-H(1-Bzl)-NH2
O-W-F-I-A-H(1-Bzl)-NH2
O-W-F-I-P-H(1-Bzl)-NH2
O-W-F-I-P-H(1-Bzl)-NH2
O-W-F-I-W-H(1-Bzl)-NH2
O-W-F-I-Dip-H(1-Bzl)-NH2
O-W-F-I-F-K-NH2
200
>200
200
200
>200
200
>200
>200
50 (17.9)
>200
>200
100 (35.5)
200
5 (1.6)
200
>200
>200
>200
>200
>200
20 (14.7)
20 (10.5)
20 (14.7)
20 (9.5)
20 (11.0)
1 (0.12)
10 (4.7)
20 (9.5)
10 (8.4)
10 (6.2)
10 (3.9)
5 (1.2)
>200
>200
>200
>200
>200
>200
>200
100 (59.9)
>200
200
50 (16.5)
50 (13.2)
5 (1.5)
20 (5.6)
20 (8.2)
20 (10.4)
50 (12.3)
20 (6.4)
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
50 (25.1)
200
>200
>200
200
>200
>200
>200
>200
>200
200
>200
>200
>200
>200
>200
>200
200
>200
>200
200
100 (32.6)
50 (19.2)
100 (48.2)
100 (30.3)
20 (11.3)
50 (17.3)
50 (15.1)
100 (28.6)
20 (12.7)
50 (11.8)
100 (39.4)
50 (18.6)
10 (2.6)
O-W-F-I-F-R-NH2
O-W-F-I-F-H-NH2
O-W-F-I-F-K-NH2
O-W-F-I-F-R-NH2
>200
50 (20.0)
20 (11.9)
50 (15.1)
20 (6.7)
10 (4.1)
>200
O-W-F-I-F-H-NH2
O-W-F-I-F-Dab-NH2
O-W-F-I-F-O-NH2
O-W-F-I-F-F(4-NH2)-NH2
cyclo-[O-W-F-I-F-H(1-Bzl)]
200
>200
200
>200
>200
20 (10.8)
50 (15.3)
>200
5 (3.3)
200
[a] O=ornithine; amino acids in italics represent the d-isomers. [b] Standards used: C. albicans (amphotericin B: IC50 =0.34 mgmLꢀ1), MRSA (vancomycin:
IC50 =0.14 mgmLꢀ1), MRSE (vancomycin: IC50 =0.14 mgmLꢀ1), E. coli (streptomycin: IC50 =0.73 mgmLꢀ1), K. pneumoniae (neomycin: IC50 =0.6 mgmLꢀ1), P. aeru-
ginosa (ciprofloxacin: IC50 =1.18 mgmLꢀ1).
Arg) were also promising, with MIC values of 10 and 5 mgmLꢀ1,
respectively. On the other hand, analogue 11 was active
against MRSE, with an excellent MIC value of 5 mgmLꢀ1 (IC50 =
1.9 mgmLꢀ1). As in the case of MRSA, it appears that d-Arg at
X1 is also best suited among the tested peptides for activity
against MRSE. In the case of Gram-negative bacterial strains,
analogue 8 was found to be most potent against E. coli, with
an MIC value of 20 mgmLꢀ1 (IC50 =13.3 mgmLꢀ1). However,
none of the peptides was active against K. pneumoniae, where-
as peptides 7, 8, and 15 were moderately active against P. aer-
uginosa. Therefore, in general, out of all the peptides in which
Orn1 of hexapeptide 3 is replaced, peptides 7 and 8 (X1 =Lys
and Arg, respectively) were active against four tested strains,
exhibiting a broad spectrum of antimicrobial activities.
Among
peptides
16–47,
which are substituted at posi-
tion X2 of the lead peptide 3,
analogues 24 (X2 =Nal), 19 [X2 =
Phe(4-Me)] and 20 (X2 =Cha)
were observed to be the most
potent against C. albicans, with
MIC values of 2, 2, and
5 mgmLꢀ1 (IC50 =1.1, 1.5, and
2.9 mgmLꢀ1), respectively. Inter-
estingly, most of the peptides
that are active against C. albi-
cans have unnatural amino acids
as the substituent at position X2.
In the case of MRSA, analogue
28 exhibited highly potent activ-
ity
(MIC=5 mgmLꢀ1
;
IC50 =
1.1 mgmLꢀ1). Peptide 38 was the
most potent analogue against
MRSE, with an MIC value of
5 mgmLꢀ1
(IC50 =2.0 mgmLꢀ1).
Scheme 1. Reagents and conditions: a) Fmoc-His(Bzl)-OH, DIEA, CH2Cl2, 2 h; b) Fmoc-d-Phe-OH, TBTU, DIEA, DMF,
3 h, then 20% piperidine in DMF, 15 min; c) Fmoc-Ile-OH, TBTU, DIEA, DMF, 3 h, then 20% piperidine in DMF,
15 min; d) Fmoc-d-Phe-OH, TBTU, DIEA, DMF, 3 h, then 20% piperidine in DMF, 15 min; e) Fmoc-d-Trp(Boc)-OH,
TBTU, DIEA, DMF, 3 h, then 20% piperidine in DMF, 15 min; f) Fmoc-Orn(Boc)-OH, TBTU, DIEA, DMF, 3 h, then 20%
piperidine in DMF, 15 min; g) 1% TFA in CH2Cl2, 10 min, then pyridine, 5 min; h) PyBop, HOAt, DMF, 24 h, room
temperature; i) 20% TFA in CH2Cl2, 30 min, room temperature.
This observation clearly indi-
cates that the presence of hy-
drophobic amino acids with aro-
matic rings on the side chain at
position X2 increases the poten-
ChemMedChem 2010, 5, 86 – 95
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
91

R. Jain, W. Tegge, et al.
MED
cy against the tested Gram-positive bacterial stains. Of all the
tested analogues, 38 was the most active, with an MIC value
of 2 mgmLꢀ1 (IC50 =1.4 mgmLꢀ1) against E. coli. Most of the
peptides were inactive against K. pneumoniae and P. aerugi-
nosa, with the exception of analogues 29 (X2 =d-Phe), 38, 40,
and 47, which displayed modest activities. Broadly, out of all
peptides evaluated with substitutions at position X2, 38 and 28
(substituted with Tyr and Trp, respectively) were found to be
active against five and four tested microbial strains exhibiting
a broad spectrum of activities.
logues 83, 87, 89, 95, and 103, which displayed modest activi-
ties. Broadly speaking, analogue 95 displayed activity against
five tested microbial strains, thereby exhibiting a broad spec-
trum of activities, whereas analogues 83, 89, and 103 dis-
played activity against four tested microbial strains.
The antimicrobial data for peptides 113–145, with substitu-
tions at position X5 of the lead peptide 3, show that 121 [X5 =
Phe(4-tBu)] is the most potent against C. albicans (MIC=
20 mgmLꢀ1; IC50 =5.8 mgmLꢀ1). In the case of MRSA, peptides
117, 113 (X5 =Abu), and 142 (X5 =Pro) exhibited moderate ac-
tivity, with MIC values of 50, 100, and 50 mgmLꢀ1 (IC50 =14.6,
26.1 and 17.9 mgmLꢀ1), respectively. On the other hand, ana-
logues 116 [X5 =Phe(4-Me)], 142, 143 (X5 =d-Pro), 144, and
145 were active against MRSE, with MIC values of 20 mgmLꢀ1
(IC50 =8.0, 14.7, 10.5, 14.7, and 9.5 mgmLꢀ1, respectively). In
general, the range of activities for peptides 113–145 is smaller
than that of the preceding series of peptides toward Gram-
positive bacterial strains. Analogue 133 (X5 =Val) was the most
active peptide, with an MIC value of 20 mgmLꢀ1 (IC50 =
8.6 mgmLꢀ1) against E. coli. In parallel with the observation
made earlier, most of the peptides were inactive against
K. pneumoniae and P. aeruginosa. In summary, analogues 117,
121, and 145, respectively containing Cha, Phe(4-tBu), and Dip
residues, were active against three tested microbial strains, ex-
hibiting a broad spectrum of activities.
In the case of peptides 48–80, in which d-Phe3 of peptide 3
is replaced, analogue 52 (X3 =Cha) is the most potent against
C. albicans, with an MIC value of 2 mgmLꢀ1 (IC50 =1.0 mgmLꢀ1).
In fact, 52 was the most active analogue against C. albicans
among all the tested peptides. Analogues 58 [X3 =Phe(4-CF3)],
63 (X3 =Trp), and 80 (X3 =Dip) also exhibited promising activi-
ties against C. albicans. The presence of a bulky ring substitu-
tion at position X3 seems to be critical for potency against the
fungal strain. In the same manner, peptides 52, 50 [X3 =Phe(4-
F)], and 58 exhibited potent activity, with MIC values of
2 mgmLꢀ1 (IC50 =0.11, 0.25 and 0.94 mgmLꢀ1
against MRSA. Analogue 57, on the other hand, displayed
potent activity against MRSE (MIC=1 mgmLꢀ1
IC50 =
, respectively)
;
0.02 mgmLꢀ1). Other peptide analogues such as 51 [X3 =Phe(4-
Me)], 52, 56, 58, 62 (X3 =d-Trp), and 80 also displayed high ac-
tivities against MRSE. In general, most of the peptides exhibit-
ed good activities against both the Gram-positive bacterial
strains. Comparatively fewer peptides were active against
E. coli, with analogue 54 (X3 =Phg) displaying the greatest po-
tency, with an MIC value of 20 mgmLꢀ1 (IC50 =5.9 mgmLꢀ1). In
parallel with the observation made earlier, most peptides were
found inactive against K. pneumoniae and P. aeruginosa. To con-
clude, peptide 54, containing a Phg residue, was found to be
active against five tested microbial strains exhibiting a broad
spectrum of activities, whereas analogues 51, 52, 58, 73 (X3 =
Cys), and 80 displayed promising activity against four tested
microbial strains.
For peptides 146–154, in which His(1-Bzl)6 in peptide 3 is
replaced, peptide analogue 152 (X6 =Dab) was the most
potent against C. albicans, with an MIC value of 10 mgmLꢀ1
(IC50 =2.6 mgmLꢀ1). Analogue 147 (X6 =Arg) exhibited the
highest potency, with an MIC value of 5 mgmLꢀ1 (IC50 =
1.6 mgmLꢀ1) against MRSA. Similarly, for MRSE, peptide 147 ex-
hibited the most promising activity, with an excellent MIC
value of 1 mgmLꢀ1 (IC50 =0.12 mgmLꢀ1). Some other analogues
such as 153 and 154 also displayed high activities, with MIC
values of 5 mgmLꢀ1 (IC50 =1.2 and 3.3 mgmLꢀ1, respectively). In
the case of E. coli, analogue 149 (X6 =d-Lys) was the most
active, with an MIC value of 5 mgmLꢀ1 (IC50 =1.5 mgmLꢀ1).
Except 149 (MIC=50 mgmLꢀ1), no compound was found to be
active against Gram-negative bacterial strains of K. pneumoniae
and P. aeruginosa. Generally speaking, analogues 149, 151,
152, 153, and 154, containing d-Lys, d-His, Dab, Orn, and
Phe(4-NH2) residues, respectively, at position X6, were active
against four tested microbial strains, exhibiting a broad spec-
trum of activities.
For peptides 81–112, in which substitutions at position X4 of
peptide 3 were carried out, analogues 95 (X4 =d-Phe) and 86
(X4 =Nle) displayed moderate activity against C. albicans, with
MIC values of 20 mgmLꢀ1 (IC50 =4.4 and 7.0 mgmLꢀ1, respec-
tively). In the case of MRSA, analogues 84, 88 (X4 =Bal), 91
(X4 =Pfp), and 83 [X4 =Phe(4-F)] were highly potent, with ex-
cellent MIC values of 1, 1, 1, and 2 mgmLꢀ1 (IC50 =0.42, 0.58,
0.63, and 0.95 mgmLꢀ1), respectively. Analogue 84 was highly
potent against MRSE, with an excellent MIC value of 1 mgmLꢀ1
(IC50 =0.07 mgmLꢀ1). Some other analogues such as 83 [X4 =
Phe(4-F)], 88, 89 [X4 =Phe(4-tBu)], and 112 (X4 =Dip) also dis-
played high activities, with MIC values of 1 mgmLꢀ1 (IC50 =0.78,
0.23, 0.63, and 0.70 mgmLꢀ1, respectively) against MRSE. Inter-
estingly, most of the active peptides against MRSA and MRSE
contained an unnatural amino acid with a bulky residue as the
side chain at position X4. In the case of Gram-negative bacterial
strains, analogue 89 was most active against E. coli, with an
MIC value of 10 mgmLꢀ1 (IC50 =6.5 mgmLꢀ1). However, consis-
tent with earlier results, these peptides were inactive against
K. pneumoniae and P. aeruginosa, with the exception of ana-
The cyclic peptide 155 was inactive against all the tested
strains (MICꢁ200 mgmLꢀ1). This observation clearly indicates
that linearity is essential for activity against microbial strains in
this particular series of peptides. The constraint induced by
cyclization possibly limits the flexibility of the peptide to a
large extent, thereby preventing the cationic termini from in-
teracting with the anionic phospholipids of the microbial cell
membrane for disruption.
Cytotoxicity experiments
All the synthesized peptides were screened for cytotoxicity
using an MTT assay with mouse fibroblasts using a procedure
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Antimicrobial Hexapeptides
described earlier.[27] Most of the tested linear synthetic hexa-
peptides showed no cytotoxic effects up to the highest test
concentration used (200 mgmLꢀ1). Peptides 29 and 55, howev-
er, displayed some cytotoxic behavior. Peptide 29 exhibited
high cytotoxicity, with an observed IC50 value of 10 mgmLꢀ1,
whereas 55 was 50% toxic at 100 mgmLꢀ1
.
Tryptophan fluorescence quenching studies
The selectivity of active peptides toward bacterial cells may be
related to differences in their interaction with the outer mem-
brane monolayers of bacterial versus mammalian cells. Such
differences can be elucidated by the relative extent of peptide
burial in model membranes; to measure this, we performed a
tryptophan fluorescence quenching study with both negatively
charged and zwitterionic model membranes, which mimic bac-
terial and mammalian membranes, respectively. For this pur-
pose, we selected the twelve hexapeptides 11, 15, 70, 75, 87,
113, 118, 134, 146, 147, 149, and 152 that exhibited modest
to promising activity against the microorganism strains tested.
All the selected peptides contain d- or l-tryptophan in their se-
quence, along with phenylalanine and tyrosine in some of the
hexapeptides.
The Stern–Volmer plots of acrylamide-mediated quenching
of tryptophan in the absence and presence of lipid vesicles are
shown in Figure 2. Tryptophan fluorescence in the peptides
decreased in a concentration-dependent manner through the
addition of acrylamide to the peptide solution, both in the ab-
sence and presence of liposomes. However, relative to the
measurements in the absence of liposomes, the slopes were
decreased in the presence of small unilamellar vesicles (SUVs)
of egg yolk l-a-phosphatidylcholine (EYPC)/egg yolk l-a-phos-
phatidyl-d,l-glycerol (EYPG) (7:3 w/w) or SUVs of EYPC/choles-
terol (10:1 w/w), suggesting that the indole moiety of trypto-
phan is buried in the bilayers, where it is inaccessible for
quenching by acrylamide. In the presence of EYPC/EYPG (7:3
w/w) SUVs, which mimic the negatively charged bacterial
membrane, all of the peptides exhibited similar extents of
quenching, suggesting that their indole moieties are buried ef-
fectively in the negatively charged phospholipid membranes.
Likewise, the order for the relative extent of tryptophan fluo-
rescence quenching in the presence of EYPC/cholesterol (10:1
w/w) SUVs, which mimic the mammalian membrane, also ex-
hibited similar extents of quenching. In particular, the slope for
the tested peptides in the presence of EYPC/cholesterol (10:1
w/w) SUVs was much higher than in the presence of EYPC/
EYPG (7:3 w/w) SUVs, suggesting that the peptides have a pref-
erential interaction with negatively charged phospholipids.
This clearly indicates that the peptides effectively embed into
the negatively charged membrane but not into the zwitterion-
ic membrane, suggesting that the selectivity of these peptides
toward bacterial cells is associated with preferential interaction
with negatively charged phospholipids. Thus, it can be safely
stated that the observations made from the tryptophan fluo-
rescence quenching studies with the selected most active hex-
apeptides are in line with the differences between bacterial
(negatively charged phospholipids) and mammalian (zwitter-
Figure 2. Stern–Volmer plots for the quenching of Trp fluorescence in the
peptides by the aqueous quenching agent, acrylamide.
ionic phospholipids and cholesterol) cell membrane composi-
tion.
Conclusions
Solid-phase peptide synthesis (SPPS) protocols were used with
peptide 3 as a starting point in the design and synthesis of
small synthetic peptides containing natural and unnatural
amino acids, which were successfully used to identify leads
against various microbial pathogens. The synthesized peptides
displayed a broad spectrum of activities, and were particularly
more active against the Gram-positive bacterial strains. These
peptides also exhibited potent activities against the fungus
C. albicans. Of all the peptides tested against C. albicans and
MRSA, Orn-d-Trp-Cha-Ile-d-Phe-His(1-Bzl)-NH2 (52) was the
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93

R. Jain, W. Tegge, et al.
MED
most active. Peptide 52 also displayed an excellent IC50 value
against MRSE and moderate activity against E. coli. Notably, 52
exhibited better activities than peptide 3 against all the tested
fungal, Gram-positive, and Gram-negative bacterial strains.
Thus, peptide 52 has been identified as a new lead that dis-
plays broad-spectrum activities against a panel of pathogenic
microorganisms. On the other hand, Orn-d-Trp-d-Phe-Ile-d-
Phe-d-Lys-NH2 (149) was most active against both E. coli and
K. pneumoniae, whereas Orn-d-Trp-Nal-Ile-d-Phe-His(1-Bzl)-NH2
(57) and Orn-d-Trp-d-Phe-Phe(4-tBu)-d-Phe-His(1-Bzl)-NH2 (89)
exhibited the best potency against MRSE and P. aeruginosa, re-
spectively. It seems that an N-to-C-terminus cyclization restricts
the flexibility of the peptide and leads to blockage of terminal
functional groups, resulting in the loss of antimicrobial activity
in peptide 155. The fluorescence studies not only indicate that
the active hexapeptides interact preferentially with the nega-
tively charged bacterial membrane over the neutral mammali-
an membrane, but also that these hexapeptides exhibit their
activity in a well-proven manner. Therefore, it can be safely
stated that the synthesized active peptides might employ any
of the traditional mechanistic models (barrel-stave, carpet, or
toroid pore) in disruption of the cell membrane that ultimately
results in killing of the microbial cell.
General procedure for Fmoc SPPS
TentaGel S Ram resin was used as the solid support. The Fmoc
group in TentaGel S Ram resin was first removed using 20% piperi-
dine in DMF for 15 min to yield free resin. The free resin was cou-
pled with the first Fmoc-protected amino acid in the presence of
TBTU or benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexa-
fluorophosphate (PyBop) as coupling agent (depending on the
amino acid to be coupled) and DIEA as activating reagent in DMF
for 3 h. The Fmoc group was removed using 20% piperidine in
DMF for 15 min to afford the resin-coupled amino acid. This cycle
of coupling and deprotection was repeated with subsequent
Fmoc-protected amino acids to afford the resin-coupled hexapepti-
des. Finally, the resin and orthogonal protecting groups were
cleaved using TFA in the presence of 3% triisopropylsilane as scav-
enger and 2% H2O for 2.5 h. After filtration of the resin and subse-
quent TFA evaporation, crude peptides were precipitated and
washed with tert-butylmethyl ether before being dried under
vacuum.
Synthesis of cyclic peptide 155
Peptide 155 was synthesized as shown in Scheme 1. 2-Chlorotrityl
chloride polystyrene resin, which was used as the solid support,
and Fmoc-His(1-Bzl)-OH were coupled by adding DIEA and dry
CH2Cl2, and shaking the reaction mixture for 2 h at ambient tem-
perature. The optical density of the Fmoc group was measured by
UV/Vis spectroscopy of the cleaved Fmoc group. Depending on
the loading of amino acid on the resin, the resin-coupled His(1-Bzl)
was coupled with Fmoc-d-Phe-OH using TBTU and DIEA for 3 h to
afford the Fmoc-protected resin-coupled dipeptide. The Fmoc
group was cleaved by using 20% piperidine in DMF for 15 min.
This cycle of coupling and deprotection was repeated with Fmoc-
Ile-OH, Fmoc-d-Phe-OH, Fmoc-d-Trp(Boc)-OH, and Fmoc-Orn(Boc)-
OH to afford the resin-coupled orthogonally protected hexapep-
tide. Treatment with three cycles of 1% TFA in CH2Cl2 cleaved the
protected hexapeptide from the resin. The filtrate was treated with
pyridine to neutralize the TFA, and the solvent was removed under
vacuum. The resulting residue was dissolved in 1,4-dioxane and
lyophilized to yield the linear hexapeptide Orn(Boc)-d-Trp(Boc)-d-
Phe-Ile-d-Phe-His(1-Bzl). This linear hexapeptide was allowed to cy-
clize in the presence of PyBop, 1-hydroxy-7-azabenzotriazole
(HOAt), and DIEA in DMF as solvent for 24 h at ambient tempera-
ture. Finally, the Boc groups were removed using 20% TFA in
CH2Cl2 for 30 min to yield the required cyclic hexapeptide 155.
Experimental Section
Materials
Amino acids, coupling reagents, N,N-diisopropylethylamine (DIEA),
and trifluoroacetic acid (TFA) were purchased from either Chem-
Impex International or NovaBiochem (Merck Ltd.). Egg yolk l-a-
phosphatidylcholine (EYPC), egg yolk l-a-phosphatidyl-d,l-glycerol
(EYPG), acrylamide, and cholesterol were supplied by Sigma Chemi-
cal Co. The buffers were prepared in double glass-distilled water.
All solvents used for synthesis were of analytical grade and were
used without further purification unless otherwise stated. All other
reagents were of analytical grade. We used both Fmoc and Boc
methods of peptide synthesis to optimize various sets of condi-
tions for the series of hexapeptides reported herein.
General procedure for Boc SPPS
MBHA·2HCl resin was used as the solid support, and was swelled
in CH2Cl2 and neutralized with 10% DIEA in CH2Cl2. Free resin was
coupled with the Boc-protected first amino acid using O-(benzo-
triazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU)
as coupling agent in the presence of DIEA as activating base in
DMF for 90–120 min. The completion of coupling reactions was
routinely monitored by Kaiser’s test. The Boc group was then re-
moved using 20% TFA for 20 min in CH2Cl2. The peptide was neu-
tralized using 10% DIEA in CH2Cl2 to afford the resin-coupled
amino acid. This cycle of coupling, deprotection, and neutralization
was repeated with subsequent Boc-protected amino acids to
afford the resin-coupled hexapeptides. The resin and orthogonal
protecting groups were cleaved using 30% HBr solution in acetic
acid, in the presence of pentamethylbenzene and thioanisole as
scavengers in TFA for 1 h at ambient temperature to yield the
linear hexapeptides. After filtration of the resin and subsequent
TFA evaporation, crude peptides were precipitated and washed
with Et2O before being dried under vacuum.
Peptide purification and characterization
The initial mass spectra were recorded with a Finnigan Mat LCQ
spectrometer (APCI) and a Kratos III MALDI mass spectrometer.
Preparative HPLC was done using a Merck Hitachi HPLC system
equipped with a Nucleosil 100 C18, 250ꢁ40 mm column. All final
peptides were checked for homogeneity on a Shimadzu SPD-M20A
HPLC system using a SupelcosilTM LC-8 (5 mm, 250ꢁ4.6 mm i.d.)
column by employing a solvent system of CH3CN/H2O (0.1% TFA)
with a 14 min gradient of 0!50% CH3CN over 12 min, 50!60%
CH3CN over 1 min, and 60!100% CH3CN over 1 min. Final confir-
mation of peptide purity was done on an Agilent 1100 series LCMS
with an Applied Biosystems API100 (MS) using a Polymer Labs
PLRP-S 100A (3 mm, 50ꢁ2.1 mm) column by employing a solvent
system of CH3CN/H2O (0.1% TFA) with a 15 min gradient of 5!
95% CH3CN over 10 min and 100% CH3CN over 5 min. In general,
the percentage purity obtained for the synthesized compounds
94
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ChemMedChem 2010, 5, 86 – 95

Antimicrobial Hexapeptides
was found to be >90%. However, peptides 24, 26, and 93 showed
a purity of 86, 76, and 86%, respectively, mainly because of the
presence of inseparable racemic sequence isomers.
Centre for Infection Research for his support in carrying out the
cytotoxicity experiments, and Dr. C. Raman Suri at the Institute of
Microbial Technology, for the fluorescence studies.
Preparation of small unilamellar vesicles (SUVs)
Keywords: antimicrobial
peptides
·
cytotoxicity
·
fluorescence · peptides · solid-phase synthesis
SUVs were prepared by a standard procedure with required
amounts of either EYPC/EYPG (7:3 w/w) or EYPC/cholesterol (10:1
w/w) for tryptophan fluorescence. Dry lipids were dissolved in
CHCl3 in a small glass vessel. Solvents were removed by rotary
evaporation to form a thin film on the wall of a glass vessel. The
dried thin film was resuspended in Tris-HCl buffer by vortex
mixing. The lipid dispersions were then sonicated in an ice/H2O
mixture for 10–20 min with an ultrasonicator until the solution
became transparent.
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AMPs=antimicrobial peptides, APD=antimicrobial peptide data-
base, Boc=tert-butyloxycarbonyl, DIEA=N,N-diisopropylethyla-
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a-phosphatidyl-d,l-glycerol, Fmoc=9-fluorenylmethyloxycarbonyl,
HPLC=high-pressure liquid chromatography, IC50 =inhibitory con-
centration that affords 50% inhibition of microbial growth, LPS=
lipopolysaccharide, MBHA=4-methylbenzhydrylamine, MIC=mini-
mum inhibitory concentration, MRSA=methicillin-resistant Staphy-
lococcus aureus, MRSE=methicillin-resistant Staphylococcus epider-
midis, MSSA=methicillin-sensitive Staphylococcus aureus, MTT=3-
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(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
PyBop=benzotriazol-1-yloxytris(pyrrolidino)phosphonium
bromide,
hexa-
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TFA=trifluoroacetic acid, YPD=yeast extract/peptone/dextrose.
Acknowledgements
[31] H. Zhao, P. K. Kinnunen, J. Biol. Chem. 2002, 277, 25170–25177.
Rohit K. Sharma thanks the Council of Scientific and Industrial
Research (CSIR) New Delhi for the award of Senior Research Fel-
lowship. The authors thank Dr. Florenz Sasse at the Helmholtz
Received: August 10, 2009
Revised: November 4, 2009
Published online on November 26, 2009
ChemMedChem 2010, 5, 86 – 95
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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95