Discovery of novel histidine-derived lipo-amino acids: Applied in the synthesis of ultra-short antimicrobial peptidomimetics having potent antimicrobial activity, salt resistance and protease stability

Article abstract of DOI:10.1016/j.ejmech.2013.07.008

Here we report for the first time the synthesis of Histidine (His) derived lipo-amino acids having pendant lipid tails at N(τ)- and N(π)-positions on imidazole group of His and applied it into synthesis of lipo-peptides. The attachment of His-derived lipo-amino acid into the very short inactive cationic peptides endows potent antimicrobial activity against Gram-positive and Gram-negative bacteria without hemolytic activity. Furthermore, our designed His-derived lipo-peptidomimetics (HDLPs) consisting of two or three residues displayed strong anti-MRSA activity and protease stability as well as retained potent antimicrobial activity under high salt concentration. Our results demonstrate that the novel lipo-amino acid is highly flexible to synthesize and carry out the extensive structure-activity relationship (SAR) on lipo-antimicrobial peptidomimetics and represents a unique amenable platform for modifying parameters important for antimicrobial activity. Through this study, we proved that the discovery of His-derived lipo-amino acid and the corresponding HDLPs are an excellent candidate as a lead compound for the development of novel antimicrobial agents.

Full text of DOI:10.1016/j.ejmech.2013.07.008

European Journal of Medicinal Chemistry 68 (2013) 10e18
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of novel histidine-derived lipo-amino acids: Applied in the
synthesis of ultra-short antimicrobial peptidomimetics having potent
antimicrobial activity, salt resistance and protease stability
Mija Ahn a , Ravichandran N. Murugan , Binu Jacob , Jae-Kyung Hyun
,
1
a
,
1
d
,
1
b,1
,
a
a
b
a
a
Chaejoon Cheong , Eunha Hwang , Hyo-Nam Park , Ji-Hyung Seo , G. Srinivasrao ,
Kyung S. Lee , Song Yub Shin
Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Cheongwon, Chung-Buk 363-883, Republic of Korea
Division of Electron Microscopic Research, Korea Basic Science Institute, 113 Gwahanno, Daejeon 305-333, Republic of Korea
Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
Department of Bio-Materials, Graduate School and Department of Cellular & Molecular Medicine, School of Medicine, Chosun University, Gwangju 501-
c
d,
a,
*
*
*
, Jeong Kyu Bang
a
b
c
d
7
59, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 May 2013
Received in revised form
Here we report for the first time the synthesis of Histidine (His) derived lipo-amino acids having pendant
lipid tails at N(s)- and N(p)-positions on imidazole group of His and applied it into synthesis of lipo-
peptides. The attachment of His-derived lipo-amino acid into the very short inactive cationic peptides
endows potent antimicrobial activity against Gram-positive and Gram-negative bacteria without he-
molytic activity. Furthermore, our designed His-derived lipo-peptidomimetics (HDLPs) consisting of two
or three residues displayed strong anti-MRSA activity and protease stability as well as retained potent
antimicrobial activity under high salt concentration. Our results demonstrate that the novel lipo-amino
acid is highly flexible to synthesize and carry out the extensive structureeactivity relationship (SAR) on
lipo-antimicrobial peptidomimetics and represents a unique amenable platform for modifying param-
eters important for antimicrobial activity. Through this study, we proved that the discovery of His-
derived lipo-amino acid and the corresponding HDLPs are an excellent candidate as a lead compound
for the development of novel antimicrobial agents.
16 July 2013
Accepted 18 July 2013
Available online 26 July 2013
Keywords:
Histidine-derived amino acid
Antimicrobial peptidomimetics
Salt resistance
Anti-MRSA activity
Protease stability
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
peptide scaffolds and fatty acid residues of C14eC18 length that are
known to be integral to their activities [4,5]. In recent years, several
Most of all organisms have antimicrobial peptides (AMPs) as a
defense system against invading pathogens. The AMPs have consid-
erable attention as new antibiotics because of their potent broad-
spectrum antimicrobial activity and rapid action. It makes bacteria
more difficult to acquire resistance to AMPs compared to conven-
tional antibiotic therapies [1e3]. Among them, lipo-peptides are an
important class of AMPs because daptomycin and polymyxin have
been already successfully evaluated in the clinics. Both daptomycin
and polymyxin are formed by dual composition as such the cyclic
groups reported that fatty acid attachment of cationic linear peptides
endows inactive cationic peptides with antimicrobial activity. These
lipo-peptides are generally composed of aliphatic acids attached to
the N-terminus of cationic linear or cyclic peptides which displayed
broad activity against both Gram-positive and Gram-negative bac-
teria [6e15]. In addition, the higher hydrophobicity gained through
fatty acid attachment can be considered as a useful remedy for the
salt sensitive peptide binding that limits the bacterial potency at
physiological ionic strength [16]. However, a potential downside of
these lipo-peptides is their high production cost due to large size
which doesn’t allow large scale synthesis and the decrease in water
solubility due to lipidation that can have severe pharmacokinetic
consequences. In addition, there are limitations on the bioavailability
of lipo-peptides since they can be easily degraded by the peptidase
like trypsin owing to multiple Arg or Lys residues.
*
Corresponding author. Tel.: þ82 43 240 5023; fax: þ82 43 240 5059.
*
* Corresponding author. Tel.: þ82 62 230 6769; fax: þ82 62 233 6337.
E-mail addresses: syshin@chosun.ac.kr (S.Y. Shin), bangjk@kbsi.re.kr (J.K. Bang).
These authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.008

M. Ahn et al. / European Journal of Medicinal Chemistry 68 (2013) 10e18
11
Herein, we focused on developing the short Histidine-derived
lipo-peptidomimetics (HDLPs) as a new class of lipidated peptido-
mimetics having ultra-short length (2 or 3 residues) by modulating
the charge, hydrophobicity and amphipathicity. This is the critical
issue for the therapeutic development of AMPs, since the long
peptide sequence of AMPs, typically having 10e50 amino acids,
limits their use in systematic administration and biological uptake
N-terminal (HDLP6) and C-terminal (HDLP7), respectively. Finally,
to understand the role of charge on HDLP2 in the antimicrobial
activity, we derived dimeric peptide HDLP8 by the N-terminal
deletion of Arg and analyzed the antimicrobial activity (Fig. 1).
2.2. Antimicrobial and hemolytic activities
[
17]. Thus, if AMPs potency and selectivity can be retained for short
All of the peptides were assayed for their antimicrobial activities
towards three Gram-negative bacteria (Escherichia coli, Pseudo-
monas aeruginosa and Salmonella typhimurim) and three Gram-
positive bacteria (Staphylococcus epidermidis, Bacillus subtilis and
Staphylococcus aureus). The cytotoxicity of the peptides was eval-
uated by testing hemolysis with human red blood cells (hRBCs). For
the purpose of comparison, the previously reported active AMPs,
human cathelicidin LL-37 and bee venom melittin, were served as a
positive control for antimicrobial and hemolytic activities.
HDLPs, it would open up new avenues for the wider pharmaceutical
applicability of antimicrobial peptidomimetics. To achieve this goal,
we have developed a pendant alkyl-tagging on Histidine (His)
amino acid with hydrophobic groups stretches as a key residue for
the facile and readily tunable approach to increase the lipophilicity
[18,19]. The solid-phase synthesis of HDLPs is straightforward, and
their potential diversification is large by the introduction of a va-
riety of functional groups.
In this study we modified the short HDLPs by positioning from C-
to N-terminus with His residue that was lipidated on the side chain of
imidazole group. Until now, this strategy has been rarely employed:
usually N-terminal lipidation is performed to investigate structuree
activity study on antimicrobial peptides. In order to identify the most
promising, cost-effective ultra-short AMPs as a drug candidate, our
designed HDLPs were compared in terms of (i) broad-spectrum of
antimicrobial activity, (ii) prokaryotic selectivity, (iii) anti-MRSA ac-
tivity, (iv) proteolytic stability and (v) salt resistance.
Our assay results demonstrated that ultra-short HDLPs appear
to be very effective and a novel class of antimicrobial agents. As
shown in Table 1, almost all the HDLPs except HDLP3 displayed
potent antimicrobial activity (average MIC range: 3.7e24.0
against Gram-positive and Gram-negative bacteria. The HDLP1
bearing two Arg residues and one His derivative which has C alkyl
tail at N( )-, N( )-position, displayed one of the most potent anti-
microbial activity with the MIC value 2e8 g/ml against all bacteria
mg/ml)
7
s
p
m
tested. Particularly, the activity of HDLP1 displayed the same or
little higher activity compared with melittin which was known to
possess the strong antimicrobial activity. The therapeutic potential
of peptide antimicrobial drugs lies in their prokaryotic selectivity to
effectively kill bacterial cells without exhibiting significant cyto-
toxicity toward mammalian cells such as hRBCs. To determine
prokaryotic selectivity of the peptides, we calculated their thera-
peutic index (TI) (Table 1). The TI is a widely employed parameter to
represent the prokaryotic selectivity of antimicrobial agents. The TI
of the peptides is calculated as the ratio of the HC50 to GM (geo-
metric mean of MICs against six bacterial strains); thus, larger
values of TI indicate greater prokaryotic selectivity. When there was
2
. Results and discussion
2.1. Design and synthesis
The design of the 8 HDLPs is based on the fact that a global
distribution of cationic and hydrophobic side chains is a key to
antimicrobial activity, which can be easily constructed by attaching
aliphatic alkyl tail to cationic peptide. The synthesis of HDLPs was
carried out on solid phase and characterized by HPLC and MALDI-
TOF Mass. Fmoc-His(Trt)-OH was used as a starting material to
synthesize the lapidated His derivatives as a building blocks. As a
first step, Fmoc-His(Trt)-OH was coupled with the excess of
no hemolysis at the highest concentration tested (256
mg/ml),
512 g/ml was used for the TI calculation, since the test was carried
m
0
methanol in the presence of N-(3-dimethylaminopropyl)-N -eth-
out by two-fold serial dilution. HDLP1showed the highest TI value
among our designed HDLPs. HDLP1 displayed almost 70 times
higher TI value in comparison to melittin, which augments its po-
tential development as antimicrobial agents to treat both Gram-
negative and Gram-positive bacterial infections (Table 1 and Fig. 2).
Next, to investigate theeffectoflength of alkyl tailonantimicrobial
activity, we designed and synthesized HDLP2 and HDLP3 having
either C10 or C14 as pendant alkyl tail, respectively (Fig. 1). Since anti-
microbial activity is positively correlated with hydrophobicity, we
expected that HDLP2 and HDLP3 display more potent antimicrobial
activity than HDLP1 because of increasing the hydrophobicity by
extending the alkyl tail length. As shown inTable 1, changing the alkyl
ylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole
(
HOBt) to carry out the esterification reaction. Then, the N(
s)- and
N( )-positions on Fmoc-His(Trt)-OMe were dialkylated using alkyl
p
or alkenyl alcohols in the presence of triflic anhydride and diiso-
propylethylamine (DIEA), followed by the treatment of trifluoro-
acetic acid (TFA) and triisopropylsilane (TIS) for 2 h at room
temperature. Finally, lapidated Fmoc-protected His derivatives
were obtained in good yield through hydrolysis of ester (Scheme 1).
Next, the above synthesized N(s)-alkyl, N(p)-alkyl bis-adducted His
monomers were successfully used to generate our focused pepti-
domimetic derivatives using Rink amide resin by the conventional
solid phase peptide synthesis (Scheme 1 and Fig. 1).
7
tail length from C (HDLP1) to C10 (HDLP2) did not alter the antimi-
Our focused HDLP derivatives contain variety of lipophilic acids
including saturated C -, C10-, and C14-, and an unsaturated lipid
7
moiety in order to study how chain length and aggregation (satu-
ration vs unsaturation) of the lipophilic moiety affects the antimi-
crobial activity. For example, HDLP1e3 are cationic lipo-
crobial activity even though hemolytic activity was increased (Fig. 2).
Against expectations, HDLP3 having the highest hydrophobic prop-
erties among the HDLPs series due to the presence of C14 as pendant
alkyl tail completely lost its antimicrobial activity and reduced its
hemolytic activity (Table 1). It is possible that there is an optimum
length of lipophilic tail for HDLP activity or lipophilicity of the HDLP is
self-limiting due to aggregation or an unknown mechanism.
peptidomimetics having either C
7
, C10, or C14 as pendant alkyl at
) mono-
N( )-, N( )-position, respectively. HDLP4 containing N(p
s
p
alkylated tail was generated to see the effect of hydrophobicity
on antimicrobial activity compared to HDLP2. HDLP5 having same
sequence with HDLP4 but with unsaturated lipid tail was derived to
see the effect of unsaturation on increasing the potency of anti-
microbial activity by limiting the aggregation of peptides. HDLP6
and HDLP7 were generated to investigate the positional effect on
antimicrobial activity by moving alkyl tail from center (HDLP2) to
On the basis of above results, further evaluation was carried out
to see the effect of hydrophobicity by replacing dialkylated HDLP2
to N(p)-mono-alkylated peptide, HDLP4 (Fig. 1). The assay results
showed that the hydrophobicity played an important role in anti-
microbial activity against Gram-negative bacteria because all the
strains showed 2e4 fold decreased activity compared to HDLP2. On
the other hand, the Gram-positive bacteria were insensitive to

12
M. Ahn et al. / European Journal of Medicinal Chemistry 68 (2013) 10e18
Scheme 1. Synthetic protocol of Fmoc-His-OH derivatives and trimeric HDLP1 using solid phase peptide synthesis (SPPS): (a) R-OH (2.2 eq.), Tf
2
O (2.2 eq.), DIEA (2.2 eq.),
ꢁ
0
ꢁ
CH
2
Cl
2
, ꢀ78 Cert, 15 h. (a ) trans-2-decen-1-ol (1.1 eq.), Tf
2
O (1.1 eq.), DIEA (1.1 eq.), CH
2
Cl
2
, ꢀ78 Cert, 15 h. (b) TFA (10 eq.), TIS (1.1 eq.), CH
2 2
Cl , rt, 2 h. (c) 2 N HCl-1,4-dioxane
(
1:1), reflux, 10 h. (d) i. 20% piperidine in DMF, 15 min. ii. Fmoc-Arg(pbf)-OH, HBTU, HOBt, DIEA, DMF, 1 h. (e) i. 20% piperidine in DMF, 15 min. ii. 7, HBTU, HOBt, DIEA, DMF,
2
overnight. (f) i. 20% piperidine in DMF, 15 min. ii. TFA/TIS/H O (90:5:5), 2 h.
change in hydrophobicity (Table 1). We also designed and syn-
thesized the HDLP5, having identical sequence to HDLP4 but con-
tains unsaturated lipophilic tail at N(p) position and assayed its
showed that there was no appreciable change on the antimicrobial
activity by both N-terminal and C-terminal segregation of hydro-
phobic alkyl tail when compared to the HDLP2 (Fig. 1 and Table 1).
This result demonstrated that antimicrobial activity was less sen-
sitive to the spatial positioning of the lipophilic tail.
Finally, to understand the role of charge on HDLPs in the anti-
microbial activity, we synthesized dimeric peptide HDLP8 by the N-
terminal deletion of Arg and analyzed the antimicrobial activity.
Being very short lipidated HDLP, HDLP8 surprisingly displayed
similar activity against Gram-negative bacteria and even more active
against Gram-positive bacteria compared to trimeric HDLP2 (Fig. 1
and Table 1). This indicates that lipophilicity of the pendant alkyl
tail on HDLPs was critical and the decreased cationic property by the
deletion of Arg at the N-terminus did not affect the antimicrobial
activity, but induced almost 2-fold decrease in the TI value due to
antimicrobial activity. But, the 2-fold decreased in antimicrobial
activity of HDLP5 against HDLP4 suggests that the unsaturated
lipophilic tail on HDLPs may not enhance the potency of antimi-
crobial activity (Fig. 1 and Table 1).
An important, yet rarely explored in various studies, question is
how the activity of the amphiphilic HDLPs varies as a function of
spatial positioning of the positive charge and lipophilic pendant
alkyl tail [20]. Since our HDLPs are very unique by having lipophilic
tail at the middle of the sequence, we focused to study the influence
of spatial positioning of the hydrophobic alkyl tail on antimicrobial
activity by incorporating either at the N-terminus (HDLP6) or C-
terminus (HDLP7) from the middle (HDLP2). The assay result

M. Ahn et al. / European Journal of Medicinal Chemistry 68 (2013) 10e18
13
Fig. 1. The structures of HDLPs tested for their antimicrobial activity.
increase in hemolytic activity. To the best of our knowledge, HDLP8 is
one of most short lipo-peptidomimetics having potent broad-
spectrum antimicrobial activity.
frequency of deaths from MRSA, a major cause of skin and soft-
tissue infections in the hospital, is rapidly increasing and has
greater than those caused by human immunodeficiency virus/ac-
quired immune deficiency syndrome (HIV/AIDS) [21]. Hence, in
addition to the above broad-spectrum activity study, MRSA strains
2.3. Anti-MRSA activity
(MRSA 1, 2, and 3) were employed as a multi-drug resistant
Next, we tested the antimicrobial activity of our designed
HDLPs against methicillin-resistant S. aureus (MRSA). The annual
bacteria. Surprisingly, most of our HDLPs (1, 2, 4, 5, 6, 7 & 8) were
found to retain the activity against the methicillin-resistant strains
whereas HDLP3 didn’t showed the any activity against MRSA
strains tested (Table 2).
100
2
.4. Salt resistance
8
0
0
0
0
The salt sensitivity of AMPs is a major obstacle in their devel-
6
4
2
opment as novel therapeutic agents. It was well known that the
electrostatic attractions between AMPs and bacterial membranes
at high ionic strength showed reduced driving force for AMP
adsorption and in partial or complete loss in antimicrobial activity
at physiological conditions. Salt sensitivity has been observed in
several AMPs including
a-defensins, and cecropins [22]. For
effective use in clinical pharmacotherapy, AMPs need to remain
active in the presence of physiological levels of salt (150 mM
NaCl). For this reason, we tested the effects of NaCl on antimi-
crobial activity of our designed HDLPs against six bacterial strains.
As shown in Table 3, HDLPs 2, 6, 7 and 8 appeared to be relatively
resistant to NaCl concentration of 150 mM. In contrast, HDLPs 1, 3,
0
0
50
100
150
200
250
Peptide [ g/ml]
µ
Fig. 2. Concentration-response curves of percent hemolysis of the peptides against human
red blood cells. Peptides are indicated as follows: HDLP1 (C), HDLP2 (B), HDLP3 (;),
4
and 5 showed a drastic increase in their MIC values in the
HDLP4 (7), HDLP5 (-), HDLP6 (,), HDLP7 (A), HDLP8 (>), LL-37 (:), melittin (6).
presence of 150 mM NaCl.

14
M. Ahn et al. / European Journal of Medicinal Chemistry 68 (2013) 10e18
Table 1
Antimicrobial and hemolytic activities and therapeutic index of the designed peptides.
Peptides
Minimal inhibitory concentration (MIC) (
m
g/ml)
GMa
HC50b
g/ml)
TIc
(m
g/ml)
(
m
(HC50/GM)
E. coli
P. aeruginosa
S. typhimurium
S. epidermidis
B. subtilis
S. aureus
[
KCTC 1682]
[KCTC 1637]
[KCTC 1926]
[KCTC 1917]
[KCTC 3068]
[KCTC 1621]
HDLP1
HDLP2
HDLP3
HDLP4
HDLP5
HDLP6
HDLP7
HDLP8
LL-37
4
8
>64
16
32
8
16
4
32
8
8
8
>64
16
32
8
8
8
64
8
8
8
>64
32
64
8
8
4
16
8
2
4
>64
2
4
4
8
2
16
8
2
4
>64
2
4
4
8
2
32
4
4
4
>64
4
8
4
8
2
32
4
4.7
5.3
128.0
12.0
24.0
6.0
9.3
3.7
32.0
6.7
>256
121
>256
>256
>256
110
116
49
>256
10.5
108.9
22.8
4.0
42.7
21.3
18.3
12.5
13.2
16.0
1.6
Melittin
a
The geometric mean (GM) of the MIC values for four bacterial strains. When no detectable antimicrobial activity was observed at the maximum concentration (64
g/ml) was used for calculation of the therapeutic index.
HC50 is the peptide concentration that produces 50% hemolysis against human red blood cells. When no detectable hemolytic activity was observed at the maximum
mg/ml),
the value of twice maximum concentration (128
m
b
concentration (256
m
g/ml), the value of twice maximum concentration (512
m
g/ml) was used for calculation of the therapeutic index.
c
Therapeutic index (TI) is the ratio of the HC50 value to the GM.
2.5. Protease stability
enhancing antimicrobial potency of ultra-short, highly cationic
AMPs against Gram-positive and Gram-negative bacteria including
Another main drawback of AMPs into development of therapeutic
MRSA strains. Second, the results indicate that the proteolytic sta-
bility is achieved by the precise addition of lipophilic tail on His
imidazole ring, the latter being particularly sensitive to proteolytic
inactivation. Third, the optimized HDLPs retained high antimicro-
bial potency at high NaCl ionic strength. Collectively, our findings
demonstrated that the small amphipathic HDLPs may be a prom-
ising class of antimicrobial agents by means of having a similar
range of antimicrobial potency and prokaryotic selectivity as larger
cationic AMPs in addition to improved proteolytic stability, salt
resistance and lower costs of production.
applications is intrinsic instability by protease. Of great concern are
trypsin-like proteases that abound in the body and are selective for
basic residues. Trypsin specifically catalyzes the hydrolysis of C-ter-
minal amide bonds of lysine and arginine [23]. Since our HDLPs
possess one or two arginine residues, their proteolytic stability was
examined by tryptic degradation. The HDLPs 1, 2, 4, 6, 7 and 8 having
relatively high antimicrobial activity were pretreated with trypsin
and their residual antimicrobial activity was assayed by the broth
microdilution assay. All of our HDLPs tested preserved completely
their antimicrobial activity against E. coli and S. aureus after trypsin
treatment, while melittin and LL-37 partially or completely abolished
their antimicrobial activity (Fig. 3).
4. Experimental
4.1. General experimental
2.6. Mechanistic studies using TEM
Reagents were obtained commercially (SigmaeAldrich, TCI) and
As a brief study of the mechanism of action, the effects of
used without further purification. All reactions were carried out
under nitrogen atmosphere in oven-dried glassware. CH Cl was
distilled under N from CaH immediately before use. Analytical
treating E. coli and S. aureus with HDLPs 1, 2, 6 and 7 were studied
by transmission electron microscopy (TEM). In comparison to un-
treated control, obvious disruption of cellular structure was
observed when HDLPs 1, 2, 6 and 7 were added (Fig. 4) in which
clear rupturing of bacterial cells and release of internal materials
were clearly visible.
2
2
2
2
TLC was performed on silica gel plate using UV light and/or
ninhydrin stain followed by heating. Column chromatography was
1
13
performed on silica gel 60 (230e400 mesh). H and C NMR
spectra were obtained on a Bruker DRX 400 spectrometer at 400
and 100 MHz, respectively. Chemical shifts (d) are reported in parts
per million (ppm) measured relative to an internal standard, and
coupling constants (J) are expressed in hertz (Hz). Mass spectra
were obtained on a Waters SYNAPT G2 (ESI) mass spectrometer.
3
. Conclusion
In conclusion, three main findings are presented in this report.
First, it shows the applicability of lipophilic tagging as a means for
4
.2. Synthesis of N(
a)-[(9H-fluoren-9-ylmethoxy)carbonyl]-N(s)-
Table 2
(triphenylmethyl)- -histidine methyl ester (1)
L
Antimicrobial activity of the peptides against methicillin-resistant Staphylococcus
aureus (MRSA) strains.
To a solution of Fmoc-His(Trt)-OH (3.10 g, 5.00 mmol) in CH
2 2
Cl
Peptide
Minimal inhibitory concentration (MIC) (mg/ml)
(30 mL) were added EDCI (1.05 g, 5.50 mmol), HOBt (743 mg,
MRSA 1
MRSA 2
(CCARM 3090)
MRSA 3
(CCARM 3095)
5.50 mmol), and MeOH (1 mL, excess) at room temperature and
stirred for 15 h. The mixture was diluted with CH Cl and washed
with H O. The organic layer was dried (Na SO ) and evaporated in
vacuo. The residue was purified by silica gel column chromatog-
raphy (CH Cl /MeOH, 20:1) to provide 1 (3.0 g, 95%) as a white foam.
H NMR (400 MHz, CDCl ):
(
CCARM 3089)
2
2
HDLP1
HDLP2
HDLP3
HDLP4
HDLP5
HDLP6
HDLP7
HDLP8
LL-37
4
8
>64
8
16
4
8
8
4
>64
8
16
4
8
4
4
>64
8
16
4
8
2
2
4
2
2
1
3
d
7.75 (d, 2H, J ¼ 7.5 Hz), 7.62 (t, 2H,
J ¼ 7.5 Hz), 7.53 (s, 1H), 7.42e7.26 (m, 13H), 7.13e7.10 (m, 6H), 6.57
(s, 1H), 6.53 (d, 1H, J ¼ 8.2 Hz), 4.63 (m, 1H), 4.38e4.22 (m, 3H), 3.63
2
>32
4
>32
2
>32
13
(
s, 3H), 3.13e3.03 (m, 2H). C NMR (100 MHz, CDCl
3
):
d
172.0,
156.2, 144.1, 143.9, 142.2, 141.2, 138.8, 136.2, 129.7, 128.12, 128.09,

M. Ahn et al. / European Journal of Medicinal Chemistry 68 (2013) 10e18
15
Table 3
Antimicrobial activity of the designed peptides in the presence of 150 mM NaCl.
Peptides
Minimal inhibitory concentration (MIC) (mg/ml)
E. coli
KCTC 1682]
P. aeruginosa
[KCTC 1637]
S. typhimurium
[KCTC 1926]
S. epidermidis
[KCTC 1917]
B. subtilis
[KCTC 3068]
S. aureus
[KCTC 1621]
[
HDLP1
HDLP2
HDLP3
HDLP4
HDLP5
HDLP6
HDLP7
HDLP8
LL-37
>32
16
>64
>64
>64
16
16
8
16
8
8
8
>64
16
64
8
8
8
64
8
32
16
>64
>64
64
16
16
8
32
16
4
>64
32
64
4
4
2
>64
4
8
4
>64
8
16
4
4
2
16
8
8
4
>64
32
32
8
4
2
>64
2
Melittin
16
1
3
27.6,127.1, 125.4,125.3,119.9, 119.7, 75.4, 67.2, 54.3, 52.2, 47.2, 33.9,
0.1.
All the values are in agreement with the reported values [24].
CH
2
Cl
2
(45 mL) dropwise over a 30 min and stirred for an additional
Cl
2
30 min. Subsequently, a solution of 1 (1.50 g, 2.37 mmol) in CH
(20 mL) was added dropwise. After 10 min, the reaction mixture
2
was slowly warmed to room temperature and stirred for 15 h. At
ꢁ
0
3
C, satd. NaHCO solution was slowly added to the reaction
4.3. General procedure I for alkylation of histidine methyl ester 2e
2 2
mixture and stirred for 30 min, and extracted with CH Cl . The
6
. The synthesis was performed in accordance to Qian et al. [24]
2 4
organic extract was washed with brine and dried (Na SO ), and
evaporated in vacuo. To a solution of resulting product in CH
(20 mL) were added TFA (1.81 mL, 23.70 mmol) and triisopro-
pylsilane (535 L, 2.61 mmol) and stirred at room temperature for
2 2
Cl
4
.3.1. N(
a
)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(
p), N(s)-
bis(heptyl)-
L
-histidine methyl ester (2)
m
To a solution of triflic anhydride (877
m
L, 5.21 mmol) in CH
2
Cl
2
2 h. The solvent was removed in vacuo, and the residue was purified
ꢁ
(
35 mL) under nitrogen at ꢀ78 C were added a solution of 1-
by silica gel column chromatography (CH Cl /MeOH, from 100:1 to
2
2
heptanol (736
mL, 5.21 mmol) and DIEA (907
mL, 5.21 mmol) in
20:1) to provide 2 (1.08 g, 77%) as a colorless gum.
1
H NMR (400 MHz, CDCl
3
):
d
8.96 (s, 1H), 7.73 (d, 2H, J ¼ 7.5 Hz),
7
.66e7.59 (m, 2H), 7.37 (t, 2H, J ¼ 7.4 Hz) 7.33e7.28 (m, 3H), 6.73 (d,
0.20
0.15
0.10
0.05
Control
2H, J ¼ 8.0 Hz), 4.55 (m, 1H), 4.35e4.20 (m, 2H), 4.17 (m, 1H), 4.13e
E. coli
Trypsin +
4
2
0
1
1
.04 (m, 2H), 4.4.0e3.99 (t, 2H, J ¼ 7.4 Hz), 3.77 (s, 3H), 3.33e3.17 (m,
Trypsin
-
H), 1.86e1.75 (m, 2H), 1.74e1.64 (m, 2H) 1.33e1.09 (m, 16H), 0.89e
13
.76 (m, 6H). C NMR (100 MHz, CDCl
41.2, 135.7, 131.4, 127.8, 127.2, 127.1, 125.4, 125.3,122.3,120.4, 120.0,
19.1, 67.4, 53.0, 52.5, 50.0, 47.4, 46.9, 31.5, 31.45, 30.0, 29.9, 28.6,
3
): d 170.6,156.2, 143.8, 143.5,
28.5, 26.2, 26.0, 25.9, 22.5, 22.4, 14.0, 13.9. MS (ESI) m/z 588.3 (M).
4
.3.2. N(
a
)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(
-histidine methyl ester (3) and N( )-[(9H-fluoren-9-
ylmethoxy)carbonyl]-N( )-(decyl)- -histidine methyl ester (4)
p), N(s)-
bis(decyl)-
L
a
p
L
Compound 3 and 4 were synthesized using general procedure I,
Tf
2
O (1.75 mL, 10.41 mmol), 1-decanol (1.99 mL, 10.41 mmol), his-
tidine methyl ester 1 (3.00 g, 4.73 mmol), DIEA (1.81 mL,
0.41 mmol), TFA (3.63 mL, 47.34 mmol), and TIS (1.07 mL,
.20 mmol). The crude product was purified by silica gel column
chromatography (CH Cl /MeOH, from 100:1 to 20:1) to provide 3
(2.6 g, 82%) as a colorless gum and 4 (0.6 g) as a white foam.
0
.00
1
5
HDLP1 HDLP2 HDLP4 HDLP6 HLDP7 HDLP8 LL-37 Melittin
2
2
0.20
Control
S. aureus
Trypsin +
1
Trypsin
-
Compound 3: H NMR (400 MHz, CDCl
3
): d 8.88 (s, 1H), 7.74 (d,
2
H, J ¼ 7.4 Hz), 7.62 (m, 2H), 7.39 (t, 2H, J ¼ 7.4 Hz), 7.31 (t, 2H,
0
0
0
0
.15
.10
.05
.00
J ¼ 7.4 Hz), 7.13 (s, 1H), 6.50 (d, 1H, J ¼ 7.7 Hz), 4.58 (m, 1H), 4.36 (m,
1
3
H), 4.28 (m, 1H), 4.20 (t, 1H, J ¼ 7.2 Hz), 4.13e4.02 (m, 4H), 3.79 (s,
H), 3.25 (m, 2H), 1.80e1.71 (m, 4H),1.27e1.22 (m, 28H), 0.87 (t, 6H,
13
J ¼ 6.7 Hz). C NMR (100 MHz, CDCl
41.2, 135.7, 131.4, 127.8, 127.17, 127.15, 125.5, 125.3, 122.3, 120.3,
20.0,119.1, 67.5, 53.0, 52.5, 50.0, 47.4, 46.9, 31.8, 29.97, 29.91, 29.44,
9.43, 29.4, 29.36, 29.25, 29.23, 29.0, 28.9, 26.3, 26.1, 25.8, 22.6,14.1.
3
): d 170.6, 156.4, 143.8, 143.5,
1
1
2
MS (ESI) m/z 672.5 (M).
1
Compound 4: H NMR (400 MHz, CDCl
3
):
d
8.56 (s, 1H), 7.77 (d,
2
H, J ¼ 7.4 Hz), 7.57 (m, 2H), 7.41 (t, 2H, J ¼ 7.4 Hz), 7.32 (t, 2H,
J ¼ 7.4 Hz), 7.15 (s, 1H), 5.90 (d, 1H, J ¼ 7.3 Hz), 4.56 (m, 1H), 4.41 (m,
HDLP1 HDLP2 HDLP4 HDLP6 HLDP7 HDLP8 LL-37 Melittin
2
2
H), 4.19 (t, 1H, J ¼ 6.8 Hz), 4.01 (m, 2H), 3.79 (s, 3H), 3.13e3.25 (m,
13
H), 1.79 (br s, 2H), 1.24e1.29 (m, 14H), 0.88 (t, 3H, J ¼ 6.5 Hz).
C
Fig. 3. Inhibition of antimicrobial activity of the peptides by trypsin assessed using the
broth microdilution assay.
NMR (100 MHz, CDCl
3
): d 170.5, 156.0, 143.6, 143.5, 141.3, 134.7,

16
M. Ahn et al. / European Journal of Medicinal Chemistry 68 (2013) 10e18
Fig. 4. Electron micrographs of negatively stained E. coli and S. aureus untreated or treated with HDLPs and melittin. All images are scaled, and the scale bar represents 1 mm.
1
3
29.4, 127.8, 127.1, 125.0, 120.0, 119.0, 67.3, 53.1, 52.9, 47.0, 46.9, 31.8,
1H NMR (400 MHz, CDCl
3
):
d
8.13 (s, 1H), 7.75 (d, 2H, J ¼ 7.4 Hz),
0.1, 29.4, 29.3, 29.2, 28.9, 26.3, 26.1, 22.6, 14.1. MS (ESI) m/z 532.3
7.56 (d, 2H, J ¼ 7.4 Hz), 7.39 (t, 2H, J ¼ 7.4 Hz), 7.30 (t, 2H, J ¼ 7.4 Hz),
þ
(
M þ H) .
7.05 (s, 1H), 5.97 (m, 1H), 5.67 (m, 1H), 5.47 (m, 1H), 4.57e4.52 (m,
3
H), 4.37 (m, 2H), 4.18 (t, 1H, J ¼ 6.6 Hz), 3.75 (s, 3H), 3.18e3.13 (m,
13
4
(
.3.3. N(
tetradecyl)-
Compound 5 was synthesized using general procedure I, Tf
1.76 mL, 10.49 mmol), 1-tetradecanol (2.25 g, 10.49 mmol), histidine
a
)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(
p
), N(
s)-bis
2H), 2.02 (m, 2H),1.34e1.24 (m,10H), 0.87 (t, 3H, J ¼ 6.6 Hz). C NMR
L
-histidine methyl ester (5)
(100 MHz, CDCl ): 170.9, 155.9, 143.6, 143.58, 141.3, 138.3, 135.7,
3
d
2
O
128.3,127.8, 127.1, 125.0,122.5, 122.0, 120.0, 67.2, 53.0, 52.9, 48.1, 47.0,
32.1, 31.7, 29.1, 29.0, 28.7, 26.4, 22.6,14.1. MS (ESI) m/z 530.3 (M þ H) .
þ
(
methyl ester 1 (3.02 g, 4.77 mmol), DIEA (1.83 mL, 10.49 mmol), TFA
3.65 mL, 47.66 mmol), and TIS (1.08 mL, 5.25 mmol). The crude
product was purified by silica gel column chromatography (CH Cl
MeOH, from 100:1 to 20:1) to provide 5 (1.45 g, 39%) as a colorless
gum and N( )-[(9H-fluoren-9-ylmethoxy)carbonyl]-N( )-(tetra-
decyl)- -histidine methyl ester (0.69 g).
H NMR (400 MHz, CDCl ):
8.92 (s, 1H), 7.74 (d, 2H, J ¼ 7.4 Hz),
(
4.4. General procedure II for the hydrolysis of esters 7e11
2
2
/
4.4.1. N(
a
)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(p), N(s)-bis
a
p
(heptyl)-L
-histidine (7)
L
To a solution of compound 2 (1.08 g, 1.83 mmol) in 1,4-dioxane
(35 mL) was added 2 N HCl (35 mL) and refluxed for 10 h. The
solvent was removed in vacuo, and the residue was partitioned
1
3
d
7
.62 (m, 2H), 7.38 (t, 2H, J ¼ 7.4 Hz), 7.30 (t, 2H, J ¼ 7.4 Hz), 7.17 (s,1H),
6
4
2
CDCl
1
.59 (br s, 1H), 4.57 (m, 1H), 4.34 (m, 1H), 4.26 (m, 1H), 4.18 (m, 1H),
between CH
evaporated in vacuo. The residue was purified by silica gel column
chromatography (CH Cl /MeOH, from 20:1 to 5:1) to provide 7
(563 mg, 54%) as a colorless gum.
2 2 2 2 4
Cl and H O. The organic layer was dried (Na SO ) and
.13e4.00 (m, 4H), 3.78 (s, 3H), 3.25 (m, 2H), 1.80 (br s, 2H), 1.71 (br s,
13
H), 1.25e1.18 (m, 44H), 0.88 (t, 6H, J ¼ 6.5 Hz). C NMR (100 MHz,
): 170.6, 156.4, 143.7, 143.5, 141.2, 135.7, 131.3, 127.8, 127.17,
27.15, 125.4, 125.2, 122.3, 120.2, 120.0, 119.1, 67.5, 53.1, 52.5, 50.0,
7.4, 46.9, 31.9, 29.98, 29.9, 29.7, 29.67, 29.62, 29.61, 29.5, 29.4, 29.39,
2
2
3
d
1
H NMR (400 MHz, CDCl
3
):
d
8.86 (s, 1H), 7.73 (d, 2H, J ¼ 7.1 Hz),
4
7.66e7.48 (m, 2H), 7.43e7.23 (m, 4H), 6.66 (s,1H), 4.60 (m,1H), 4.35e
4.19 (m, 2H), 4.18e3.92 (m, 5H), 3.34e3.16 (m, 2H),1.84e1.64 (m, 4H),
2
9.37, 29.0, 28.9, 26.3, 26.2, 25.9, 22.7, 14.1. MS (ESI) m/z 784.6 (M).
13
1
.31e1.08 (m,16H), 0.82 (t, 6H, J ¼ 6.2 Hz). C NMR (100 MHz, CDCl
3
)
4
2
.3.4. N(
-en-1-yl)-
Compound 6 was synthesized using general procedure I, Tf
293 L,1.74 mmol), trans-2-decen-1-ol (324 L,1.74 mmol), histidine
methyl ester 1 (1.00 g, 1.58 mmol), DIEA (303 L, 1.74 mmol), TFA
1.21 mL,15.78 mmol), and TIS (357 L,1.74 mmol). The crude product
was purified by silica gel column chromatography (CH Cl /MeOH,
from 100:1 to 20:1) to provide 6 (0.29 g, 35%) as a colorless gum.
a
)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(
p
)-(trans-dec-
d 171.5, 156.2, 143.8, 143.3, 141.3, 141.2, 135.7, 130.9, 127.9, 127.24,
L
-histidine methyl ester (6)
127.18,125.3, 125.1, 122.0,121.1, 120.0,118.8, 67.1, 52.9, 50.1, 47.5, 46.9,
31.5, 30.1, 30.0, 28.6, 28.5, 26.2, 26.1, 22.4,14.0. MS (ESI) m/z 574.3 (M).
2
O
(
m
m
m
4.4.2. N(
a)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(p), N(s)-bis
(
m
(decyl)- -histidine (8)
L
2
2
Compound 8 was synthesized using general procedure II, com-
pound 3 (500 mg, 0.74 mmol), 2 N HCl (10 mL), and 1,4-dioxane

M. Ahn et al. / European Journal of Medicinal Chemistry 68 (2013) 10e18
17
(
10 mL). The crude product was purified by silica gel column
chromatography (CH Cl /MeOH, from 10:1 to 5:1) to provide 8
242 mg, 50%) as a colorless gum.
Calbiochem-Novabiochem (La Jolla, CA). Other reagents used for
2
2
peptide synthesis included trifluoroacetic acid (TFA; SigmaeAldrich),
0
0
(
(
piperidine (Merck), O-benzotriazole-1-yl-N,N,N ,N -tetramethylu-
ronium hexafluorophosphate (HBTU; SigmaeAldrich), 1-hydrox-
ybenzotriazole (HOBt; SigmaeAldrich) and N,N-dimethylformamide
(DMF, peptide synthesis grade; Biolab). All other reagents were of
analytical grade. The buffers were prepared in double glass-distilled
water. All the HDLPs were prepared by Fmoc SPPS methods using
Rink amide resin with an initial loading of 0.61 mmol/g, unless
otherwise noted. Resins were swollen in DMF for 45 min prior to
synthesis. For sequence extension, the Fmoc-Arg(Pbf)-OH (5.0 eq.)
along with HBTU (5.0 eq.) and HOBt (5.0 eq.) was added to the free
amine on resin in the presence of N,N-diisopropylethylamine (DIEA)
(5.0 eq.) in 2 mL DMF and allowed to proceed for 1 h with Vortex
stirring. After washing with DMF, Fmoc deprotection was achieved
with 20% piperidine in DMF (1 ꢂ 10 min, 2 ꢂ 3 min). The resin was
washed once again, and the process was repeated for the next amino
acid and finally the resin was washed with DMF, methanol, di-
chloromethane and ether, and then dried under vacuum. Linear
peptides were cleaved from the resin with 5% triisopropylsilane (TIS)
1
HNMR(400MHz, CDCl
3
):
d
8.71(s,1H), 7.74(d, 2H,J¼ 7.4 Hz), 7.58
m, 2H), 7.38 (t, 2H, J ¼ 7.4 Hz), 7.32e7.27 (m, 4H), 6.58 (d, 1H,
J ¼ 6.4 Hz), 4.53 (m, 1H), 4.34e4.24 (m, 2H), 4.13 (t, 1H, J ¼ 7.3 Hz),
4
0
.09e3.97 (m, 4H), 3.25 (m, 2H), 1.73 (br s, 4H), 1.26e1.19 (m, 28H),
1
3
.86 (t, 6H, J ¼ 6.5 Hz). C NMR (100 MHz, CDCl
3
): d 172.1,156.2,143.8,
1
1
2
43.4, 141.3, 141.2, 135.0, 131.2, 127.8, 127.2, 125.3, 125.0, 122.1, 121.1,
20.0, 118.9, 67.3, 53.4, 49.9, 47.4, 46.9, 31.84, 31.82, 30.0, 29.95, 29.4,
9.38, 29.2, 29.0, 28.9, 26.3, 26.2, 22.6, 14.1. MS (ESI) m/z 658.5 (M).
4
.4.3. N( )-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N( )-(decyl)-
a
p
L-
histidine (9)
Compound 9 was synthesized using general procedure II, com-
pound 4 (440 mg, 0.83 mmol), 2 N HCl (5 mL), and 1,4-dioxane
(
5 mL). The crude product was purified by silica gel column chro-
matography (CH Cl /MeOH, from 10:1 to 5:1) to provide 9 (270 mg,
3%) as a white solid.
2
2
6
1
H NMR (400 MHz, CDCl
.57 (t, 2H, J ¼ 7.4 Hz), 7.38 (t, 2H, J ¼ 7.4 Hz), 7.28 (t, 2H, J ¼ 7.4 Hz),
.16 (s, 1H), 6.28 (br s, 1H), 4.44 (br s, 1H), 4.36 (m, 2H), 4.18 (t, 1H,
J ¼ 6.8 Hz), 3.98 (m, 2H), 3.27 (m, 2H),1.69 (br s, 2H),1.24 (br s,14H),
3
):
d
8.26 (s, 1H), 7.74 (d, 2H, J ¼ 7.4 Hz),
7
7
2
and 5% H O inTFA (approximately 2 mL of TFA per 100 mg of resin) for
2 h. The cleavage mixture was mixed with cold ether to precipitate the
peptide and then filtered. Purificationofcrudepeptidewas carried out
1
3
0
1
1
2
.86 (t, 3H, J ¼ 6.7 Hz). C NMR (100 MHz, CDCl
3
):
d
173.6, 156.0,
on thepreparative Vydac C18 column (15
an appropriate 10e90% water/acetonitrile gradient in the presence of
0.05% TFA (A: water buffer, B: acetonitrile buffer). The final purity of
m
m, 20 mm ꢂ 250 mm) using
43.9, 143.6, 141.3, 141.28, 133.5, 130.5, 127.8, 127.1, 125.1, 125.0,
20.0, 119.5, 67.0, 54.4, 47.1, 46.7, 31.8, 30.2, 29.4, 29.36, 29.2, 29.0,
þ
6.4, 26.3, 22.6, 14.1. MS (ESI) m/z 518.3 (M þ H) .
the HDLPs(>98%)wasassessedby RP-HPLCon an analytical Vydac C18
ꢀ
column (4.6 mm ꢂ 250 mm, 300 A, 5
mm particle size). The molecular
4
.4.4. N(
a
)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(
p
), N(
s
)-
masses of HDLPs were determined using matrix-assisted laser-
desorption ionization-time-of-flight mass spectrometry (MALDI-TOF
MS, Voyager-DE STR). MS (MALDI-TOF) m/z: HDLP 1; 663.6 (M), HDLP
bis(tetradecyl)-
L
-histidine (10)
Compound 10 was synthesized using general procedure II,
compound 5 (1.34 g, 1.71 mmol), 2 N HCl (20 mL), and 1,4-dioxane
þ
2;747.7(M), HDLP3;859.8(M),HDLP4;607.6(Mþ H) , HDLP5;605.6
þ
(
20 mL). The crude product was purified by silica gel column
chromatography (CH Cl /MeOH, from 10:1 to 5:1) to provide 10
610 mg, 46%) as a colorless gum.
(M þ H) , HDLP 6; 747.7 (M), HDLP 7; 747.8 (M), HDLP 8; 591.6 (M).
2
2
(
4.6. Antimicrobial activity (MIC)
1
H NMR (400 MHz, CDCl
3
):
d
8.67 (s, 1H), 7.73 (d, 2H, J ¼ 7.4 Hz),
7
1
4
.58 (m, 2H), 7.38 (t, 2H, J ¼ 7.4 Hz), 7.30e7.28 (m, 3H), 6.63 (br s,
The antimicrobial activity of the peptides against three Gram-
positive bacterial strains and three Gram-negative bacterial strains
H), 4.55 (br s, 1H), 4.33e4.23 (m, 2H), 4.12 (t, 1H, J ¼ 7.0 Hz), 4.09e
.00 (m, 4H), 3.25 (m, 2H), 1.71 (br s, 4H), 1.29e1.18 (m, 44H), 0.88
was examined by using the broth microdilution method in sterile 96-
13
6
(
1
1
2
t, 6H, J ¼ 6.7 Hz). C NMR (100 MHz, CDCl
43.4, 141.3, 141.2, 135.0, 131.1, 127.9, 127.8, 127.2, 125.3, 125.1, 122.0,
21.1, 120.0, 118.9, 67.3, 53.4, 49.9, 47.4, 46.9, 31.9, 30.0, 29.9, 29.7,
9.67, 29.63, 29.5, 29.4, 29.37, 29.0, 28.9, 26.3, 26.2, 22.7, 14.1. MS
ESI) m/z 770.6 (M).
3
)
d
172.3, 156.2, 143.9,
well plates. Aliquots (100
m
L) of a bacterial suspension at 2 ꢂ 10
colony-forming units (CFU)/mL in 1% peptone with 0 or 150 mM NaCl
were added to 100
in 1% peptone). After incubation for 18e20 h at 37 C, bacterial
m
L of the peptide solution (serial 2-fold dilutions
ꢁ
(
growth inhibition was determined by measuring the absorbance at
600 nm with a Microplate Autoreader EL 800 (Bio-Tek Instruments,
4
2
.4.5. N(
-en-1-yl)-
Compound 11 was synthesized using general procedure II,
compound 6 (170 mg, 0.32 mmol), 2 N HCl (3 mL), and 1,4-dioxane
3 mL). The crude product (155 mg, 94%) was obtained as a white
solid and used without further purification in the following step.
a
)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(
p
)-(trans-dec-
VT). The minimal inhibitory concentration (MIC) was defined as the
minimum peptide concentration inhibited bacteria growth. Three
types of Gram-positive bacteria (B. subtilis [KCTC 3068], S. epidermidis
[KCTC 1917] and S. aureus [KCTC 1621]) and three types of Gram-
negative bacteria (E. coli [KCTC 1682], P. aeruginosa [KCTC 1637]
and S. typhimurium [KCTC 1926]) were procured from the Korean
Collection for Type Cultures (KCTC) at the Korea Research Institute of
Bioscience and Biotechnology (KRIBB). Methicillin-resistant S. aureus
(MRSA) (CCARM 3089, CCARM 3090 and CCARM 3095) were ob-
tained from the Culture Collection of Antibiotic-Resistant Microbes
(CCARM) at Seoul Women’s University (Seoul, Korea).
L
-histidine (11)
(
1
H NMR (400 MHz, CDCl
d, 2H, J ¼ 7.4 Hz), 7.57 (m, 2H), 7.37 (t, 2H, J ¼ 7.4 Hz), 7.28 (t,
H, J ¼ 7.4 Hz), 7.19 (s, 1H), 6.30 (br s, 1H), 5.69 (m, 1H), 5.42
m, 1H), 4.57e4.47 (m, 3H), 4.37 (m, 2H), 4.16 (m, 1H), 3.26 (m,
3
): d 11.9 (br s, 1H), 8.28 (s, 1H), 7.74
(
2
(
13
2
H), 1.97 (m, 2H), 1.26e1.21 (m, 10H), 0.86 (t, 3H, J ¼ 6.6 Hz).
): 173.4, 156.1, 143.8, 143.6, 141.3, 141.2,
39.4, 133.8, 130.5, 127.8, 127.1, 125.1, 121.2, 120.0, 119.4, 67.0,
C
NMR (100 MHz, CDCl
1
5
3
d
4.7. Hemolytic activity
4.2, 48.7, 47.1, 32.2, 31.7, 29.1, 29.0, 28.6, 26.3, 22.6, 14.1. MS
þ
(
ESI) m/z 516.39 (M þ H) .
Fresh human red blood cells (hRBCs) were centrifuged, washed
three times with PBS, dispensed into 96-well plates as 100 mL of 4%
(v/v) hRBC in PBS, and 100 mL of peptide solution was added to
4
.5. General procedures for peptide synthesis
ꢁ
each well. Plates were incubated for 1 h at 37 C, then centrifuged at
Rink amide 4-methylbenzhydrylamine (MBHA) resin and 9-
fluorenylmethoxycarbonyl (Fmoc) amino acids were obtained from
1000ꢂ g for 5 min. Samples (100 mL) of supernatant were trans-
ferred to 96-well plates and hemoglobin release was monitored by

18
M. Ahn et al. / European Journal of Medicinal Chemistry 68 (2013) 10e18
measuring absorbance at 414 nm. Zero hemolysis was determined
in PBS (APBS) and 100% hemolysis was determined in 0.1% (v/v)
Triton X-100 (Atriton). The percentage hemolysis was calculated as:
References
[
1] B. Findlay, G.G. Zhanel, F. Schweizer, Cationic amphiphiles, a new generation
of antimicrobials inspired by the natural antimicrobial peptide scaffold,
Antimicrob. Agents Chemother. 54 (2010) 4049e4058.
%
hemolysis ¼ 100 ꢂ [(Asample ꢀ APBS)/(Atriton ꢀ APBS)].
[
[
[
2] G.G. Perron, M. Zasloff, G. Bell, Experimental evolution of resistance to an
antimicrobial peptide, Proc. Biol. Sci. 273 (2006) 251e256.
3] A.T. Yeung, S.L. Gellatly, R.E. Hancock, Multifunctional cationic host defence
peptides and their clinical applications, Cell. Mol. Life Sci. 68 (2011) 2161e2176.
4] H. Tsubery, I. Ofek, S. Cohen, M. Fridkin, N-Terminal modifications of Poly-
myxin B nonapeptide and their effect on antibacterial activity, Peptides 22
(2001) 1675e1681.
4.8. Proteolytic stability
E. coli (KCTC 1682) and S. aureus (KCTC 1621) were grown
ꢁ
overnight for 18 h at 37 C in 10 mL of LB broth and then 10
mL of
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this culture was inoculated into 10 mL of fresh LB and incubated for
ꢁ
an additional 3 h at 37 C to obtain mid-logarithmic phase organ-
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5
0
m
g/ml peptide and 0.2
m
g/ml trypsin in 50 mM TriseHCl buffer,
L) was added to
ꢁ
pH 7.5 at 37 C for 2 h. The reaction solution (50
m
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ꢁ
After incubation at 37 C for 18e20 h, the bacterial growth inhi-
bition was determined by measuring absorbance at 600 nm with a
Microplate Autoreader EL 800 (Bio-Tek Instruments).
[
[
4.9. Morphological changes of bacteria upon HDLPs addition
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Morphologicalchanges ofa Gram-negative bacterial strain(E. coli
[
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KCTC 1682]) and a Gram-positive bacterial strain (S. aureus [KCTC
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transmission electron microscopy (TEM). Bacterial culture at 2 ꢂ10
colony-forming units (CFU)/mL in LB media was washed 3 times in
phosphate buffered saline (PBS) via a series of centrifugation at
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0,000ꢂ g, for 5 min, and re-suspension.100
mL of HDLPs in PBS was
added to an equal volume of bacterial suspension to a final con-
centration at ꢂ3 MIC. Following the addition of HDLPs, the samples
ꢁ
were incubated for 1 h at 37 C. Bacterial cell pellet after centrifu-
(2010) 563e569.
gation was re-suspended in 20
tion. 5 L of sample solution was loaded onto a carbon film-coated
TEM grid that was rendered hydrophilic by glow discharge. After
0 s, excess sample solutionwas washed off with distilled water. 5 mL
mL PBS for TEM specimen prepara-
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peptides, Biopolymers 55 (2000) 4e30.
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for 1 min, and excess stain solution was blotted using a piece of filter
2
paper. Samples were imaged using a Tecnai G Spirit electron mi-
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6
croscope (FEI) equipped with lanthanum hexaboride (Lab ) gun,
operating at 120 kV. Images were recorded using Ultrascan 4000
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Acknowledgments
1250e1254.
This work was supported in part by the Korea Basic Science
Institute’s research grant T33418 (J.K.B), T33518 (J-K Hyun), the
National Cancer Institute’s Intramural Research grant (K.S.L) and
the Korea Research Foundation funded by the Korean Government
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L. Temime, Impact of antibiotic exposure patterns on selection of community-
associated methicillin-resistant Staphylococcus aureus in hospital settings,
Antimicrob. Agents Chemother. 55 (2011) 4888e4895.
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(
KRF-2011-0009039 to S.Y. Shin).
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Appendix A. Supplementary data
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8890.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.07.008.