Pyrazole derived ultra-short antimicrobial peptidomimetics with potent anti-biofilm activity

Article abstract of DOI:10.1016/j.ejmech.2016.09.071

In this study, we report on the first chemical synthesis of ultra-short pyrazole-arginine based antimicrobial peptidomimetics derived from the newly synthesized N-alkyl/aryl pyrazole amino acids. Through the systematic tuning of hydrophobicity, charge, an

Full text of DOI:10.1016/j.ejmech.2016.09.071

Accepted Manuscript
Pyrazole derived ultra-short antimicrobial peptidomimetics with potent anti-biofilm
activity
Mija Ahn, Pethaiah Gunasekaran, Ganesan Rajasekaran, Eun Young Kim, Soo-Jae
Lee, Guel Bang, Kun Cho, Jae-Kyung Hyun, Hyun-Ju Lee, Young Ho Jeon, Nam-
Hyung Kim, Eun Kyoung Ryu, Song Yub Shin, Jeong Kyu Bang
PII:
S0223-5234(16)30807-8
10.1016/j.ejmech.2016.09.071
EJMECH 8937
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 August 2016
Revised Date: 20 September 2016
Accepted Date: 21 September 2016
Please cite this article as: M. Ahn, P. Gunasekaran, G. Rajasekaran, E.Y. Kim, S.-J. Lee, G. Bang, K.
Cho, J.-K. Hyun, H.-J. Lee, Y.H. Jeon, N.-H. Kim, E.K. Ryu, S.Y. Shin, J.K. Bang, Pyrazole derived
ultra-short antimicrobial peptidomimetics with potent anti-biofilm activity, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.071.
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Pyrazole Derived Ultra-short Antimicrobial Peptidomimetics with Potent Anti-biofilm
Activity
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¶
2¶
3¶
3¶
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Mija Ahn, Pethaiah Gunasekaran, Ganesan Rajasekaran, Eun Young Kim, Soo-Jae Lee,
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5
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6,7
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Guel Bang, Kun Cho, Jae-Kyung Hyun, Hyun-Ju Lee, Young Ho Jeon, Nam-Hyung Kim,
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,9
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1,9
Eun Kyoung Ryu, Song Yub Shin, * Jeong Kyu Bang *
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2
Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chung-Buk 363-883, Republic of Korea,
Molecular Embryology Laboratory, Department of Animal Sciences, Chungbuk National University, Chung-Buk
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61-763, Republic of Korea, Department of Medical Science, Graduate School and Department of Cellular &
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Molecular Medicine, School of Medicine, Chosun University, Gwangju 501-759, Republic of Korea. College of
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Pharmacy, Chungbuk National University, Chungbuk 361-763, Republic of Korea. Biomedical Omics Group,
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Korea Basic Science Institute, Ochang, Chung-Buk 363-883, Republic of Korea, Division of Electron Microscopic
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Research, Korea Basic Science Institute, 113 Gwahakro, Daejeon 305-333, Department of Chemistry, Chungnam
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National University, Daejeon 305-764, Republic of Korea, College of Pharmacy, Korea University, 2511 Sejong-Ro,
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Sejong, 30019, Republic of Korea, Department of Bio-analytical Science,University of Science & Technology,
Daejeon 34113, Republic of Korea.
*Corresponding authors:
Jeong Kyu Bang, Ph.D.
Korea Basic Science Institute
Ochang, Chungbuk 363-883, Cheongwon
Republic of Korea
Tel: +82-43-240-5023, Fax: +82-43-240-5059
E-mail:bangjk@kbsi.re.kr
Song Yub Shin, Ph.D.
Dept. Cellular & Molecular Medicine,
School of Medicine, Chosun University
Gwangju 501-759, Republic of Korea
Tel: +82-62-230-6769, Fax: +82-62-233-6337
E-mail address: syshin@chosun.ac.kr
¶
These authors contributed equally to this work.
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Abstract
In this study, we report on the first chemical synthesis of ultra-short pyrazole-arginine based
antimicrobial peptidomimetics derived from the newly synthesized N-alkyl/aryl pyrazole amino
acids. Through the systematic tuning of hydrophobicity, charge, and peptide length, we identified
the shortest peptide Py11 with the most potent antimicrobial activity. Py11 displayed greater
antimicrobial activity against antibiotic-resistant bacteria, including MRSA, MDRPA, and VREF,
which was approximately 2-4 times higher than that of melittin. Besides its higher selectivity
(therapeutic index) toward bacterial cells than LL-37, Py11 showed highly increased proteolytic
stability against trypsin digestion and maintained its antimicrobial activity in the presence of
physiological salts. Interestingly, Py11 exhibited higher anti-biofilm activity against MDRPA
compared to LL-37. The results from fluorescence spectroscopy and transmission electron
microscopy (TEM) suggested that Py11 kills bacterial cells possibly by integrity disruption
damaging the cell membrane, leading to the cytosol leakage and eventual cell lysis. Furthermore,
Py11 displayed significant anti-inflammatory (endotoxin-neutralizing) activity by inhibiting
LPS-induced production of nitric oxide (NO) and TNF-α. Collectively, our results suggest that
Py11 may serve as a model compound for the design of antimicrobial and antisepsis agents.
Keywords: Ultra-short Peptidomimetics, Pyrazole-derived amino acids, Antimicrobial activity,
Anti-biofilm activity, Bacterial killing mechanism
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1
. Introduction
In the past few decades, the discovery of new antibiotics has gained significant importance due
to their prominent roles in treating diseases [1]. However, frequent, excessive and the widespread
use of antibiotics results in the remarkable development of drug-resistant bacteria called as
“superbugs” or “nightmare bacteria,” which are the challenging and severe threats to modern
health care [2-5]. Thus, the emergence of multidrug-resistant (MDR) microbials and the dearth
of new antibiotics led us to meet an urgent requirement for developing alternative and effective
antimicrobial agents. Antimicrobial peptides (AMPs) have gained significant importance as host
defense system in various creatures such as insects, plants, amphibians, and mammals [6-8]. In
general, AMPs are oligopeptides containing excess positively charged and hydrophobic amino
acids, which can adopt the amphipathic conformations upon contact with microbial membranes
[9-13]. Usually, AMP killing mechanism of pathogens involves either (i) disruption of the
membrane integrity by interacting with negatively charged membrane components, (ii)
association with specific intracellular targets or (iii) inhibition of proteins, DNA and RNA
synthesis [14]. Besides, AMPs have attracted significant interest as a new generation of
antibiotics because of their broad-spectrum of antimicrobial activity against fungi, bacteria, and
enveloped viruses at the physiological concentration. The distinctive mode of action and rapid
killing rate of the AMPs result in the low propensity for developing resistance and enhance the
efficacy for combating multi-drug resistance bacteria [15,16].
Although several AMPs possess promising merits as a novel tool to combat antibiotic-resistant
pathogens, their pharmaceutical advancement into therapeutic agents is restricted by the intrinsic
drawbacks such as large size, expensive production cost, susceptibility to protease degradation,
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poor activity in the presence of salts and cytotoxicity to the host cells [17]. Moreover, the clinical
use of AMPs sequences that are similar to the natural human AMPs may cause the inevitable loss
of potential in the natural human defense [18]. To circumvent the hurdles limiting their
applications, a few approaches have emerged including the de novo synthesis of peptides or
modification in sequences of peptides, and the development of new peptidomimetics resembling
the properties of AMPs. To develop new peptidomimetics, so far, several strategies have been
adapted to synthesize the antimicrobial peptidomimetics including peptoids [19−22], β-peptides
[23], aryl amide oligomers [24], oligourea [25], β-turn mimetics [26], and lipopeptides [27].
However, these moieties suffer from major disadvantageous such as poor water solubility,
complex synthetic routes, and difficulty in introducing various functional groups to achieve
activity and unique selectivity.
To overcome the above limitations and to perform the effective structure-activity
relationship (SAR), it is important to design the short and structurally simple peptidomimetics
possessing unnatural amino acids with positive charge and hydrophobicity. Taking the above
facts into the considerations, we have focused on developing the short and structurally simple
peptidomimetics having unnatural amino acids. The present study stems as a part of our research
program recently embarked on discovering the new and short peptidomimetics for antimicrobial
studies [28−31].
In this work, we have designed and synthesized the peptidomimetics consisting of only
two components, arginine (R) and N-alkyl/aryl pyrazoles (Py). The peptidomimetics were
designed in the pattern of -(RPy) - repeating units, where R is responsible for the maintenance of
n
positive charge [32], and the medicinally important pyrazole skeleton anchoring various alkyl or
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aryls at the nitrogen(-NH) maintains the hydrophobicity [33]. To synthesize ultra-short AMPs,
we have synthesized Fmoc-protected, N-substituted pyrazole amino acids for the first time,
which have been used for straight forward solid phase peptide synthesis (SPPS) method to save
the time and to maintain the structural diversity. These ultra-short peptidomimetics were
examined for their antimicrobial activities against both Gram-positive and Gram-negative
bacterial strains. The unnatural amino acid derived from pyrazole was expected to be more
resistant to protease in in vivo as well as in vitro compare to the His modified AMPs that was
reported by our group [29−31].
Among the designed peptidomimetics, Py11 displayed the most potent antimicrobial activity.
Furthermore, it was evaluated for their antimicrobial activity against several antibiotic-resistant
strains including methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant
Pseudomonas aeruginosa (MDRPA), and vancomycin-resistant Enterococcus faecium (VREF).
In addition, cytotoxicity of Py11 against sheep red blood cells (sRBCs), RAW264.7 and NIH-
3
T3 cells was also investigated. The outer membrane (OM) permeability, inner membrane (IM)
permeability, membrane depolarization, and transmission electron microscope (TEM) were
studied extensively for understanding the bacterial killing mechanism. The antimicrobial activity
in the presence of physiological salts and proteolytic stability to trypsin digestion of Py11 were
also examined. Next, the ability of Py11 to inhibit the formation of bacterial biofilm against
MDRPA was evaluated. Additionally, the anti-inflammatory (endotoxin-neutralizing) activity of
Py11 by inhibiting LPS-induced production of nitric oxide (NO) and tumor necrosis factor-α
(
TNF-α) was investigated.
2
. Results and Discussion
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2
.1 Synthesis of pyrazole-derived amino acids and peptidomimetics
In search of discovering unique scaffold for developing ultra-short antimicrobial
peptidomimetics, we have identified the N-alkyl/aryl pyrazole derivatives as a key skeleton
backbone for structural and biological properties of AMPs (Scheme 1). The commercially
available 4-nitro-3-pyrazolecarboxylic acid was used as a starting material for the synthesis of
Fmoc-protected pyrazole amino acid derivatives. Initially, the esterification of nitro-3-
pyrazolecarboxylic acid was carried out using thionyl chloride and methanol under reflux
condition to yield 1. Then, the N-alkylation of 1 was achieved using various alkyl or aryl halides
in the presence of potassium carbonate in DMF to yield 2. The Pd/C reduction of nitro group
resulted in the formation of amine, and subsequent Fmoc protection using Fmoc-Osu in the
presence of sodium bicarbonate yielded the Fmoc protected amine (3). Finally, lithium iodide
mediated methyl ester hydrolysis resulted in the formation of desired Fmoc-protected, N-
alkyl/aryl pyrazole amino acid derivatives, 4 in good yields. The synthesized Fmoc-protected
pyrazole amino acids were successfully adopted for the generation of peptide library using Rink
amide resin by the conventional solid-phase peptide synthesis (Figure 1). To initiate the sequence
extension, firstly Fmoc-Arg (pbf)-OH was added to the free amine on the resin to enable the
amide coupling in the presence of HBTU, HOBt, and DIEA in DMF. After stirring for 1 h, the
Fmoc deprotection was achieved by treating with 20% piperidine in DMF, and the next amino
acid was coupled. This procedure has been repeated for other amino acids coupling until we get
the desired peptidomimetics. Finally, the peptide cleavage from the resin was achieved by a
mixture of trifluoroacetic acid, water, and triisopropylsilane (90:5:5, v/v/v, 2 mL). The peptides
were purified using preparative RP-HPLC. The molecular weight of peptidomimetics was
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determined by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry
(MALDI-TOF MS). The cyclic peptide was synthesized from the linear side-chain protected
peptides. The Fmoc group in the linear peptides was deprotected using piperidine in DMF, and
the N-terminal amine was bromoacetylated using of BrCH COOH in the presence of DIC. Then
2
the peptide was cleaved from the resin and treated with triethylamine until the solution turns into
basic condition (pH 8-9). Under this condition, spontaneous intramolecular cyclization was taken
place through the cysteine thiol addition over N-bromoacetyl group. Further purification through
preparative RP- HPLC and MALDI-TOF-MS analysis confirmed the cyclization process.
2
.2 Structure-antimicrobial activity study
The antimicrobial activities of newly synthesized peptidomimetics were screened against
two Gram-negative bacteria (Escherichia coli [KCTC 1682] and Pseudomonas aeruginosa
KCTC 1637]) and two Gram-positive bacteria (Staphylococcus epidermidis [KCTC 1917] and
[
Staphylococcus aureus [KCTC 1621]). Human cathelicidin LL-37 and bee venom melittin are
used as positive control peptides for comparing their antimicrobial and hemolytic activities.
Initially, we started our antimicrobial screening studies for the newly synthesized dipeptide-Py1,
which attached with N-2-ethylbutyl group at pyrazole. Unfortunately, the dipeptide did not show
any considerable antimicrobial activity even at maximum concentration (64 µg/mL) (Table 1).
To increase the charge, we introduced the Arg at the N-terminal position of the dipeptide-Py1,
which resulted in the formation of Py2. This increase in charge also did not show any
considerable effect on antimicrobial activity. In view of increasing the hydrophobicity, we
synthesized the dipeptide, Py3 using propylphenyl anchoring pyrazole residue and further
increment of charge was achieved by the N-terminal Arg addition to result in the formation of
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Py4. However, Py3 and Py4 did not display any antimicrobial activity. The dipeptide Py5 was a
modification of Py3 by introducing biphenyl residue at alkyl side chain for improving the
hydrophobicity. Surprisingly, Py5 showed the appreciable antimicrobial activity towards Gram-
negative bacteria, P. aeruginosa. Even though Py5 was made up of only two residues, their
antimicrobial activity was comparable to LL-37. It is pertinent to note that the balance between
charge density and hydrophobicity is one of the crucial factors to determine the antimicrobial
activity [34]. To evaluate the effect of charge in Py5, an Arg residue was further added to the N-
terminal position to yield Py6, which exhibited strong antimicrobial activity against both P.
aeruginosa and S. aureus. Surprisingly, the activity of Py6 against S. aureus and P. aeruginosa
was about 4-8 times higher than the LL-37. Similarly, while comparing mellitin, Py6 displayed
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-4 folds of enhanced activity against S. aureus and P. aeruginosa. From the above results, it
was observed that the increase in hydrophobicity elevated the antimicrobial activity. Hence, to
further modify the structure, the dipeptide Py7 was synthesized using the naphthyl attached
pyarazole residues and the subsequent N-terminal addition of Arg to Py7 resulted in the
tripeptide, Py8. In contrast, Py7 did not show any measurable antimicrobial activity against both
the strains. However, the Py8 displayed the activity similar to LL-37 in the case of S. aureus.
Structural diversity is a key factor for studying the broad spectrum of antimicrobial activities.
Hence, to attain the structural diversity, we planned to synthesize the tetramer, hexamer, and
cyclo-peptidomimetics. Initially, tetra-peptide, Py9 with biphenyl pyrazole residue was
synthesized, which showed moderate activity against all the bacterial strains, in particular, in the
case of S. aureus, it displayed better activity than LL-37 and mellitin. This encouraging result
motivated us for the further extension of tetra peptidomimetics to hexamer Py10. However, it
displayed the reduced activity compared to the corresponding tetramer, Py9. Afterward, tetramer,
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Py11 was derived from the alternative arrangement of Arg and N-ethylnaphthyl pyrazole
residues. Interestingly, this tetramer showed the good activity against all strains range from 4~32
µg/mL. Concerning LL-37, Py11 displayed eight folds elevated activity against both P.
aeruginosa and S. aureus. Similarly, Py11 exhibited four folds of increased activity against both
P. aeruginosa and S. aureus compared to melittin. Py12 was derived from Py11 by the addition
Arg residue to increase the charge. Surprisingly there were no changes in the antimicrobial
activities compared to Py11, and it displayed the similar activity as Py11. However, a further
increment of tetramer Py11 to hexamer Py13 lost its activity completely against all the strains
except S. aureus. It was reported that the cyclization of the linear peptides enhances the activity
and protease stability [35,36]. Hence, the cyclic peptide Py14 was synthesized from the Py9
through the thioether bridging. However, Py14 displayed a poor spectrum of antimicrobial
activity compare to the linear peptide Py9. Collectively, the structure activity study infers that,
firstly, the hydrophobic units and charge find the crucial role in determining antimicrobial
activity. Secondly, the peptide length was essential in discovering efficient peptidomimetics
possessing good antimicrobial activity. For instance, even though the dipeptide Py7 and the
tripeptide Py8 did not show any antimicrobial activity, the tetrapeptides (Py9 and Py11) were
impressive in displaying antimicrobial activity against all the strains. Thus, among the all
synthesized peptidomimetics, tetrapeptide Py11 displayed the most potent antimicrobial activity
against all the tested strains. Hence, Py11 was selected as a lead compound for further studies.
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.3 Cell selectivity and cytotoxicity against mammalian cells of short AMP Py11
In terms of analyzing the cell selectivity of Py11 towards bacterial cells, the hemolytic
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activity against sRBCs of Py11 was measured in the range of 0.5~512 µg/mL (Figure 2).
Interestingly, like LL-37, Py11 produced less than 5% hemolysis even at the highest
concentration of 512 µg/mL, whereas melittin found to be relatively toxic that produced 20.9%,
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2.4%, and 74.5% hemolysis at 16, 32, and 64 µg/mL, respectively. In general, the therapeutic
potential of antimicrobial agents is mainly based on the selective killing of bacterial cells without
displaying significant cytotoxicity towards the mammalian cells. Cell selectivity of the peptides
is expressed as therapeutic index (TI) = MHC/GM, where MHC is peptide concentration needed
to reach 10% lysis of human red blood cells, while GM is expressed as the geometric mean of
MICs against four bacterial cells. The therapeutic index (TI) indicates the relative safety of the
drug and the capability of a drug candidate to differentiate any pathogen from the host cells.
Thus, larger TI values indicate greater cell selectivity. To compare the cell selectivity, we chose
LL-37 and melittin as the references. Interestingly, Py11 displayed significant cell selectivity (TI
value: 53.9) compared to melittin (TI value: 0.5) (Table 2). Besides, Py11 was about 2-folds
more selective towards bacteria cells than LL-37. To further look into the cytotoxicity of Py11
against other mammalian cells, the viability against mouse macrophage RAW264.7 and
fibroblast NIH-3T3 cells was determined using MTT assay. Py11 was found to be nontoxic to
RAW264.7 and NIH-3T3 cells up to 32 µg/mL, and 64 µg/mL, respectively (Figure 3).
2
.4 Antimicrobial activity against antibiotic-resistant bacteria of Py11
In the recent years, severe infections caused by multidrug-resistant bacteria such as
MRSA, MDRPA, and VREF are being a major challenge for conventional antibiotic treatments.
Hence, there is an urgent necessity for the evolution of new antibiotics, which can act on drug-
resistant bacteria. To probe the efficacy of short AMP Py11 against multidrug-resistant bacteria,
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we screened antimicrobial activity against three MRSA strains (CCARM 3089, CCARM 3090,
and CCARM 3095), two MDRPA strains (CCARM 2109, and CCARM 2095) and a VREF
strain (ATCC 51559). Interestingly, Py11 exhibited excellent antimicrobial activity against all
the strains compare to melittin (Table 3). For instance, Py11 displayed 2 to 4-folds of better anti-
MRSA activity than melittin. In the case of MDRPA strains, Py11 showed 4-folds of efficient
activity than melittin. Similarly, Py11 was found to disclose 2-folds of potent anti-VREF activity
than melittin. This result indicates that Py11 may serve as a new class of antimicrobial agent
against drug-resistant bacteria.
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.5 Protease stability
The poor proteolytic stability of AMPs is one of the major obstacles for their therapeutic
advancement. In particular, trypsin-like proteases that present in the body are more selective
towards the hydrolysis of C-terminal amide bonds of lysine and arginine. Since Py11 consists a
couple of arginine residues, its proteolytic stability has been investigated by tryptic degradation.
As shown in Figure 4, after the trypsin incubation for 24 h, our lead compound Py11 retained its
antimicrobial activity against E. coli and S. aureus, while melittin completely lost their
antimicrobial activity due to the proteolytic degradation by trypsin.
2
.6 Salt resistance
To examine whether the antimicrobial activity of Py11 was compromised in the presence
of salts, we treated E. coli and S. aureus with different peptide concentrations under various salt
conditions (physiological concentrations of 150 mM NaCl, 1 mM MgCl , and 2.5 mM CaCl ).
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As indicated in Table 4, Py11 maintained its strong activity against E. coli and S. aureus in the
presence of different salts. This result suggests that the activity of Py11 might be particularly
critical under physiological conditions or in the treatment of infections that disturb the normal
salt homeostasis in certain human tissues.
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.7. Antibiofilm activity
The biophysical properties of the biofilm extend extreme antibiotic resistance to
microorganisms [37]. The ability of the peptides to inhibit the formation of bacterial biofilm was
evaluated against MDRPA (CCARM 2095). The potent inhibitory activity of LL-37 on biofilm
formation of Staphylococcus epidermidis and Pseudomonas aeruginosa has been reported
[38,39]. Therefore, we used LL-37 as a reference AMP to compare the antibiofilm activity with
Py11. The IC90 (the concentration of the compounds that inhibits 90% biofilm development)
values of Py11and LL-37 against MDRPA biofilm formation was 16 ꢀg/mL, and 64 ꢀg/mL,
respectively (Figure 5). This result demonstrates that Py11 was potent enough to inhibit the
MDRPA biofilm formation. Thus, the efficient antibiofilm activity of Py11 makes it promising
anti-biofilm agents.
2
.8 Outer membrane permeability
The ability of Py11 to permeate the outer membrane of Gram-negative bacteria was
assessed by the fluorescent dye NPN uptake into the outer membrane of E. coli. The
hydrophobic fluorescent probe NPN has a tendency to quench in an aqueous environment, in
contrast, it exhibits strong fluorescence in the hydrophobic environment. Hence, there will be a
substantial increase in fluorescence, when the dye enters the damaged membrane through the
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destabilization of the bacterial outer membranes. We evaluated the NPN uptake assay using Py11
in a dose-dependent manner, and the results reveal that Py11 showed enhanced fluorescence
intensity than the standards (Figure 6(A). However, it obeys the fluorescence pattern as similar
as melittin in a concentration-dependent manner, which implies that Py11 may follow the cell
permeability mechanism similar to melittin.
2
.9 Inner membrane permeability
The inner membrane permeability of short AMP Py11 was evaluated by
spectrophotometrically at 415 nm using ONPG hydrolysis method. The inner membrane
permeability is measured by the bulk entry of nonchromogenic substrate ONPG, which
undergoes subsequent cleavage to the yellow product ONP by the cytoplasmic β-galactosidase.
To evaluate the inner membrane permeability, as shown in Figure 6(B), our lead peptide Py11
was experimented in a concentration-dependent manner. It is evident from the figure that Py11
demonstrated a significant ability to permeate the inner membrane as indicated by the greater
slope of ONPG hydrolysis. Here, buforin-2 was used as a negative control due to the weak or
poor inner membrane permeation even at higher concentrations, whereas melittin showed
enhanced permeability compare to LL-37 as illustrated in Figure 6(B). It is evident from the
above results that peptide Py11 displayed significant inner membrane permeability, and it
followed an inner membrane permeable pattern like melittin.
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.10 Cytoplasmic membrane depolarization
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To evaluate the action of Py11 on the bacterial cytoplasmic membrane depolarization, we
investigated the ability of our short peptide Py11 to depolarize the bacterial cytoplasmic
membrane using a diSC -5, which is a membrane potential-dependent probe. The dye diSC -5
3
3
was dispersed between the cells and medium, based on the cytoplasmic membrane potential, and
it gets self-quenched when concentrated inside bacterial cells. During the membrane
depolarization, there will be an increase in fluorescence intensity due to the dispersion of probe
into the medium. Here, we used melittin and LL-37 as membrane-targeting AMP, and buforin-2
was used as intracellular-target AMP. Like melittin and LL-37, the addition of Py11 triggered a
significant increase in fluorescence intensity indicating the rapid membrane depolarization
(
Figure 7). This result demonstrated that Py11 effectively induced a loss of membrane potential.
In contrast, buforin-2 did not induce membrane depolarization.
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.11 Transmission electron microscopy (TEM)
To envision the morphological changes in the E. coli and S. aureus caused by the action
of Py11, transmission electron microscopy (TEM) images were recorded for the incubated
mixtures of Py11 with E. coli and S. aureus. It was evident from Figure 8-A and 8-C that the
untreated positive controls, E. coli, and S. aureus, possessed well-defined cell morphology. In
contrast, the synthetic peptide Py11 treated cells from Figure 8-B and 8-D, showed the massive
changes in the cellular structure with disruption in the bacterial cell membrane. These damages
in cell membrane led to the formation of lysed cells with the release of cell content. Hence, it is
evident from the above images that Py11 followed the cell membrane targeting mechanism of
action against the bacterial cells.
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2
.12 Anti-inflammatory (endotoxin-neutralizing) activity
The outer membrane of Gram-negative bacteria contains lipopolysaccharide(LPS,
endotoxin) as one of the main constituents. The release of LPS from bacteria into the
bloodstream during infection may cause serious unwanted stimulation of host’s immune system,
leading to sepsis and septic shock of the patient [40,41]. In addition to their antimicrobial activity,
some AMPs have been known to show potent anti-inflammatory (endotoxin-neutralizing) ability
to suppress harmful inflammatory/septic responses [42−47]. These anti-inflammatory properties
make AMPs attractive drug candidates for Gram-negative induced endotoxic shock and sepsis
treatments. NO and TNF-α are the two important inflammatory products that are mainly
associated in promoting the inflammatory response. Therefore, we examined the anti-
inflammatory effect of Py11 using NO production and TNF-
α
release in LPS-stimulated
-
RAW264.7 cells. NO release was determined by the Griess method to detect nitrite (NO )
2
accumulation in a cultured medium. The tnf-α release was determined by the commercial mouse
ELISA kit. As shown in Figure 9, Py11 inhibited significantly NO and TNF-α release by LPS-
stimulated macrophages in a concentration-dependent manner. Py11 inhibited more than 85% of
the nitrite production at the peptide concentration of 16 ꢀg/mL. Similarly, it blocked more than
5
0% of TNF-α release at 32 ꢀg/mL. LPS induces the mRNA expression of iNOS and TNF-α in
RAW264.7 cells, which correlates with NO and TNF-α release. Therefore, the effects of the
peptides on LPS-induced the mRNA expression of inducible nitric oxide synthase (iNOS) and
TNF-α were examined by RT-PCR. Py11 efficiently suppressed the mRNA expression of iNOS
and TNF-α at 16 µg/mL (Figure 10). This result is in agreement with the observed inhibition of
NO and TNF-α release in Figure 9, confirming that Py11 has potent anti-inflammatory activity.
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3
. Conclusion
In conclusion, this article demonstrates that the first time synthesis of short
peptidomimetics comprising of Arg and N-alkyl/aryl pyrazoles (Py) residues with good
antimicrobial and anti-inflammatory activities without hemolytic activity and cytotoxicity
against mammalian cells. The easily derivable peptidomimetics led to performing the effective
SAR studies by the systematic tuning of hydrophobicity, charge, and peptide length. Also, our
peptidomimetic Py11 proved to be a very promising lead compound having anti-MRSA, anti-
VREF and anti-MDRPA activities, anti-biofilm activity and salt resistance. In addition to
antimicrobial nature, enhanced proteolytic stability also adds the additional merits for Py11 to
develop as a new class of antimicrobial agent. The mechanism of action against the pathogens
was elucidated using fluorescence spectroscopy, and TEM study. Our results revealed that our
peptidomimetic Py11 kills bacterial cells possibly by integrity disruption damaging the cell
membrane, leading to the cytosol leakage and eventual cell lysis. Collectively, our results
suggest that Py11 has tremendous potential to evolve as a model compound for antimicrobial and
antisepsis agent design.
4
. Experimental section
4
.1. Chemistry
All materials were obtained from commercial chemical suppliers and used as obtained.
Anhydrous organic solvents were purchased from Aldrich packaged under N in sure/seal bottles
2
and used directly. Thin layer chromatography (TLC) was performed on Merck TLC plates, silica
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gel 60 F254. Column chromatography was carried out on Merck silica gel 60 (70-230 mesh or
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13
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4
30-400 mesh). H and C NMR spectra were recorded on a Bruker DRX-400 spectrometer at
00 and 100 MHz, respectively. Chemical shifts (δ) 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 recorded using Shimadzu (MALDI-TOF) mass spectrometer.
Lipopolysaccharide (LPS, from Escherichia coli O111:B4), Triton X-100, N-phenyl-1-
napthylamine (NPN), colorimetric 3-(4,5-dimethyl thiozol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) dye, gramicidin D (GD), O-nitrophenyl-
sheep red blood cells (s-RBCs) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA).
,3′-Dipropylthiadicarbocyanine iodide (diSC -5) was obtained from Molecular Probes (Eugene,
β-D-galactopyranoside (ONPG), and
3
3
OR, USA). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were
supplied by HyClone (Seoul, Bioscience, Republic of Korea) and Lonza (Lonza Walkersville
Inc., MD, USA), respectively. E. coli ML-35, a lactose, permease-deficient strain with
+
constitutive cytoplasmic 3-galactosidase activity (lacI lacZ lacY), utilized for inner membrane
permeability assays, was kindly provided by Prof. J. I. Kim (Gwangju Institute of Science and
Technology, Korea). The ELISA kit for TNF-α was obtained from R&D Systems (Minneapolis,
MN, USA). All other reagents were of analytical grade. Buffers were prepared using Milli-Q
ultrapure water. All other reagents were of analytical grade.
4
.2. Synthesis of 4-Nitro-1H-pyrazole-3-carboxylic acid methyl ester (1)
To a solution of 4-nitro-3-pyrazolecarboxylic acid (3.14 g, 20.0 mmol) in MeOH (50 mL)
o
was slowly added thionyl chloride (7.3 mL, 0.1 mol) at 0 C. After the completion of the addition,
1
7

ACCEPTED MANUSCRIPT
o
the temperature of the reaction mixture increased to 70 C and stirred for 5 h. Methanol was
removed in vacuo, and the resulting residue was poured into ice water and extracted with EtOAc.
The organic layer was washed with 10 % NaHCO solution, brine and dried over Na SO . The
3
2
4
1
resulting reaction mixture concentrated in vacuo to afford 1 as a white solid (3.37 g, 99%). H
1
3
NMR (400 MHz, DMSO-d6)
δ
8.93 (s, 1H), 3.89 (s, 3H); C NMR (100 MHz, DMSO-d )
δ
6
1
61.4, 138.0, 133.7, 132.0, 53.3.
4
.3. General Procedure A for the synthesis of compound 2a-2d
4
.3.1. 1-(2-Ethylbutyl)-4-nitro-1H-pyrazole-3-carboxylic acid methyl ester (2a)
To a solution of 1 (1.0 g, 5.84 mmol) in DMF (20 mL) were added K
2
CO
3
(4.04 g, 29.2
mmol) and 1-bromo-2-ethylbutane (891 µL, 6.42 mmol). The reaction mixture was heated to 60
o
C and stirred for 15 h. The reaction mixture was diluted with water and extracted with EtOAc.
4
The organic layer was washed with water and brine, dried over MgSO , and concentrated in
vacuo. The crude product was purified by silica gel column chromatography (hexane-EtOAc,
1
3
4
:1) to afford 2a as a colorless oil (730 mg, 49%). H NMR (400 MHz, CDCl )
δ
8.12 (s, 1H),
3
.10 (d, J = 7.2 Hz, 2H), 4.00 (s, 3H), 1.93 (m, 1H), 1.40-1.28 (m, 4H), 0.93 (t, J = 7.5 Hz, 6H);
1
3
C NMR (100 MHz, CDCl3)
δ
160.5, 138.2, 134.0, 130.5, 57.1, 53.1, 41.1, 22.9, 10.3; HRMS
+
m/z calcd for C H N O (M+H) 256.1297, found 256.1298.
1
1
18
3
4
4
.3.2. 4-Nitro-1-(3-phenylpropyl)-1H-pyrazole-3-carboxylic acid methyl ester (2b)
Compound 2b was prepared from compound 1 and 3-phenylpropyl bromide following
1
the general procedure A (white solid, 66%). H NMR (400 MHz, CDCl
3
)
δ
8
.10 (s, 1H), 7.33-
7
.28 (m, 2H), 7.24-7.16 (m, 3H), 4.19 (t, J = 7.2 Hz, 2H), 3.99 (s, 3H), 2.69 (t, J = 7.4 Hz, 2H),
1
8

ACCEPTED MANUSCRIPT
1
3
2
1
2
.29 (m, 2H); C NMR (100 MHz, CDCl
3
)
δ
160.5, 139.7, 138.3, 134.1, 130.2, 128.7, 128.3,
+
26.5, 53.2, 53.1, 32.4, 30.8; HRMS m/z calcd for C14
H
16
N
3
O
4
(M+H) 290.1141, found
90.1141.
4
.3.3. 1-(3,3-Diphenyl)-4-nitro-1H-pyrazole-3-carboxylic acid methyl ester (2c)
Compound 2c was prepared from compound 1 and 3,3-diphenylpropyl bromide following
1
the general procedure A (white solid, 67%). H NMR (400 MHz, CDCl )
δ 7.94 (s, 1H), 7.35-
3
1
3
7
.22 (m, 10H), 4.16 (t, J = 7.1 Hz, 2H), 4.01 (s, 3H), 3.90 (t, J = 7.6 Hz, 1H), 2.73 (m, 2H); C
NMR (100 MHz, CDCl
3
)
δ
160.5, 142.8, 138.5, 130.4, 128.9, 127.6, 126.9, 53.2, 52.5, 48.4,
+
3
5.0; HRMS m/z calcd for C20
H
20
N
3
O
4
(M+H) 366.1454, found 366.1454.
4
.3.4. 1-[1-(2-Naphthalene-1-yl)ethyl]-4-nitro-1H-pyrazole-3-carboxylic acid methyl ester (2d)
Compound 2d was prepared from compound 1 and 1-(2-bromoethyl)naphthalene
1
following the general procedure A (white solid, 59%). H NMR (400 MHz, CDCl )
δ
7.92-7.88
3
(m, 2H), 7.79 (d, J = 8.2 Hz, 1H), 7.64 (s, 1H), 7.57-7.50 (m, 2H), 7.36 (m, 1H), 7.14 (d, J = 6.9
1
3
H, 1H), 4.52 (t, J = 7.0 Hz, 2H), 4.01 (s, 3H), 3.67 (t, J = 7.0 Hz, 2H); C NMR (100 MHz,
CDCl
3
)
δ
160.5, 138.6, 134.0, 133.9, 132.2, 131.3, 130.4, 129.2, 128.3, 127.2, 126.7, 126.0,
+
1
3
25.5, 122.6, 54.5, 53.2, 33.3; HRMS m/z calcd for C H N O (M+H) 326.1141, found
1
7
16
3
4
26.1141.
4
4
.4 General Procedure B for the synthesis of compound 3a-3d
.4.1. 4-[(9-Fluoren-9-ylmethoxy)carbonylamino]-1-(2-ethylbutyl)-1H-pyrazole-3-carboxylic
acid methyl ester (3a)
1
9

ACCEPTED MANUSCRIPT
To a solution of 2a (1.05 g, 4.11 mmol) in MeOH-CH
2 2
Cl (60 mL, 5:1) was added 5%
palladium on carbon (200 mg), and the resulting mixture was stirred under an atmosphere of H
2
at room temperature for 3 h. The reaction mixture was filtered through Celite and the filtrate was
concentrated. The crude product was used in the next reaction without further purification. To
the crude amine and NaHCO (1.03 g, 12.3 mmol) in THF-H O (50 mL, 1:1) was added Fmoc-
3
2
o
OSu (2.08 g, 6.17 mmol) at 0 C. The resulting mixture was allowed to come to room
temperature and stirred for 3 h. The reaction mixture was diluted with EtOAc and washed water
and brine, dried over Na
2
SO
4
, and concentrated in vacuo. The residue was purified by silica gel
1
column chromatography (hexane-EtOAc, 3:1) to afford 3a as a semisolid (1.33 g, 72%). H
NMR (400 MHz, CDCl
3
) δ 8.46 (s, 1H), 7.98 (s, 1H), 7.80 (d, J = 7.4 Hz, 2H), 7.65 (d, J = 7.4
Hz, 2H), 7.44 (t, J = 7.4 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H), 4.48 (d, J = 7.2 Hz, 2H), 4.31 (t, J =
7
7
1
.2 Hz, 1H), 4.06 (d, J = 7.4 Hz, 2H), 4.01 (s, 3H), 1.93 (m, 1H), 1.38-1.28 (m, 4H), 0.90 (t, J =
1
3
.4 Hz, 6H); C NMR (100 MHz, CDCl )
δ 164.2, 153.3, 143.7, 141.3, 129.1, 127.8, 127.2,
3
25.9, 125.1, 120.9, 120.1, 67.7, 57.0, 52.1, 47.0, 41.5, 23.1, 10.5; HRMS m/z calcd for
+
C H N O (M+H) 448.2236, found 448.2236.
26
30
3
4
4
.4.2. 4-[(9-Fluoren-9-ylmethoxy)carbonylamino]-1-(3-phenylpropyl)-1H-pyrazol-3-carboxylic
acid methyl ester (3b)
Compound 3b was prepared from compound 2b following the general procedure B
1
(
white solid, 67%). H NMR (400 MHz, CDCl )
δ
8.45 (s, 1H), 8.00 (s, 1H), 7.80 (d, J = 7.4 Hz,
3
2
5
H), 7.66 (d, J = 7.4 Hz, 2H), 7.44 (t, J = 7.4 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H), 7.33-7.18 (m,
H), 4.50 (d, J = 7.2 Hz, 2H), 4.31 (t, J = 7.2 Hz, 1H), 4.18 (t, J = 7.2 Hz, 2H), 4.02 (s, 3H), 2.66
1
3
(
3
t, J = 7.4 Hz, 2H), 2.26 (m, 2H); C NMR (100 MHz, CDCl ) δ 164.2, 153.3, 143.7, 141.3,
2
0

ACCEPTED MANUSCRIPT
1
3
40.4, 129.3, 128.6, 128.4, 127.9, 127.2, 126.3, 126.0, 125.1, 120.5, 120.1, 67.7, 53.0, 52.1, 47.0,
+
2.7, 31.7; HRMS m/z calcd for C29
H
28
N
3
O
4
(M+H) 482.2080, found 482.2081.
4
.4.3.4-[(9-Fluoren-9-ylmethoxy)carbonylamino]-1-(3,3-phenylpropyl)-1H-pyrazol-3-carboxylic
acid methyl ester (3c)
Compound 3c was prepared from compound 2c following the general procedure B (white
1
solid, 70%). H NMR (400 MHz, CDCl )
δ 8.40 (s, 1H), 7.89 (s, 1H), 7.78 (d, J = 7.4 Hz, 2H),
3
7
4
2
1
.63 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.35-7.17 (m, 12H), 4.47 (d, J = 7.2 Hz, 2H),
.28 (t, J = 7.2 Hz, 1H), 4.09 (t, J = 7.3 Hz, 2H), 3.98 (s, 3H), 3.90 (t, J = 7.9 Hz, 1H), 2.66 (m,
1
3
3
H); C NMR (100 MHz, CDCl ) δ 164.1, 153.3, 143.7, 143.3, 141.3, 129.4, 128.8, 127.9,
27.7, 127.2, 126.7, 125.9, 125.1, 120.7, 120.1, 67.7, 52.2, 52.1, 48.4, 47.0, 35.9; HRMS m/z
+
calcd for C H N O (M+H) 558.2393, found 558.2394.
3
5
32
3
4
4
.4.4. 4-[(9-Fluoren-9-ylmethoxy)carbonylamino]-1-[2-(naphthalen-1-yl)ethyl]-1H-pyrazole-3-
carboxylic acid methyl ester (3d)
Compound 3d was prepared from compound 2d following the general procedure B
1
(
white solid, 76%). H NMR (400 MHz, CDCl
3
)
δ
8.45 (s, 1H), 8.06 (d, J = 8.2 Hz, 1H), 7.93 (s,
1
7
4
1
1
H), 7.90 (d, J = 8.2 Hz, 1H), 7.82-7.79 (m, 3H), 7.66 (d, J = 7.2 Hz, 2H), 7.60-7.51 (m, 2H),
.47-7.35 (m, 5H), 7.30 (d, J = 7.2 Hz, 1H), 4.52 (t, J = 7.6 Hz, 2H), 4.49 (d, J = 7.1 Hz, 2H),
1
3
.31 (t, J = 7.1 Hz, 1H), 4.05 (s, 3H), 3.69 (t, J = 7.6 Hz, 2H); C NMR (100 MHz, CDCl )
δ
3
64.2, 153.2, 143.6, 141.3, 133.9, 133.2, 131.6, 129.1, 127.9, 127.87, 127.2, 127.0, 126.5, 126.0,
25.8, 125.6, 125.1, 123.1, 120.7, 120.1, 67.7, 54.1, 52.2, 47.0, 34.1; HRMS m/z calcd for
+
C
32
H
28
N
3
O
4
(M+H) 518.2080, found 518.2080.
2
1

ACCEPTED MANUSCRIPT
4
4
.5. General Procedure C for the synthesis of compound 4a-4d
.5.1.
4-[(9-Fluoren-9-ylmethoxy)carbonylamino]-1-(2-ethylbutyl)-1H-pyrazole-3-carboxylic
acid (4a)
To a solution of 3a (1.0 g, 2.23 mmol) in toluene-EtOAc (5:1, 40 mL) was added lithium
iodide (1.50 g, 11.2 mmol). The resulting mixture was refluxed for 4 h. The reaction mixture was
quenched with 10% aqueous HCl and extracted with EtOAc. The organic layer was washed with
1
0% aqueous Na
2
S
2
O
3
, brine and dried over Na
2
SO
4
, and concentrated in vacuo to afford 5a as a
8.22 (s, 1H), 7.91 (s, 1H), 7.70 (d, J =
1
white solid (950 mg, 98%). H NMR (400 MHz, CDCl
3
)
δ
7
.4 Hz, 2H), 7.55 (d, J = 7.4 Hz, 2H), 7.34 (t, J = 7.4 Hz, 2H), 7.26 (t, J = 7.4 Hz, 2H), 4.39 (d, J
=
7.2 Hz, 2H), 4.22 (t, J = 7.0 Hz, 1H), 3.99 (d, J = 7.2 Hz, 2H), 1.83 (m, 1H), 1.29-1.21 (m, 4H),
1
3
0
1
.82 (t, J = 7.4 Hz, 6H); C NMR (100 MHz, CDCl ) δ 166.7, 153.3, 143.6, 141.3, 128.6, 127.9,
3
27.2, 126.1, 125.1, 121.4, 120.1, 67.9, 57.0, 47.0, 41.6, 23.1, 10.5; HRMS m/z calcd for
+
C H N O (M+H) 434.2080, found 434.2080.
25
28
3
4
4
.5.2. 4-[(9-Fluoren-9-ylmethoxy)carbonylamino]-1-(3-phenylpropyl)-1H-pyrazol-3-carboxylic
acid (4b).
Compound 4b was prepared from compound 3b following the general procedure C
1
(
white solid, 99%). H NMR (400 MHz, CDCl )
δ
8.31 (s, 1H), 7.98 (s, 1H), 7.76 (d, J = 7.4 Hz,
3
2
2
H), 7.61 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.34-7.11 (m, 7H), 4.45 (d, J = 7.1 Hz,
1
3
H), 4.28 (t, J = 7.1 Hz, 1H), 4.15 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 2.22 (m, 2H); C
NMR (100 MHz, CDCl3)
δ
166.7, 153.3, 143.6, 141.3, 140.3, 128.9, 128.6, 128.4, 127.9, 127.2,
2
2

ACCEPTED MANUSCRIPT
1
26 3 4
26.3, 126.2, 125.1, 121.0, 120.1, 67.9, 53.0, 47.0, 32.6, 31.7; HRMS m/z calcd for C28H N O
+
(
M+H) 468.1923, found 468.1924.
4
.5.3 4-[(9-Fluoren-9-ylmethoxy)carbonylamino]-1-(3,3-phenylpropyl)-1H-pyrazol-3-carboxylic
acid (4c)
Compound 4c was prepared from compound 3c following the general procedure C (white
1
solid, 98%). H NMR (400 MHz, CDCl )
δ 8.29 (s, 1H), 7.91 (s, 1H), 7.76 (d, J = 7.4 Hz, 2H),
3
7
4
.62 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.34-7.16 (m, 12H), 4.46 (d, J = 7.2 Hz, 2H),
1
3
.28 (t, J = 7.2 Hz, 1H), 4.09 (t, J = 7.2 Hz, 2H), 3.88 (t, J = 7.8 Hz, 1H), 2.65 (m, 2H); C
166.4, 153.2, 143.6, 143.2, 141.3, 129.0, 128.8, 127.9, 127.7, 127.2,
26.7, 126.1, 125.1, 121.2, 120.1, 67.9, 52.2, 48.4, 47.0, 35.9; HRMS m/z calcd for C34
3
NMR (100 MHz, CDCl ) δ
1
30 3 4
H N O
+
(
M+H) 544.2236, found 544.2236.
4
.5.4 4-[(9-Fluoren-9-ylmethoxy)carbonylamino]-1-[2-(naphthalen-1-yl)ethyl]-1H-pyrazole-3-
carboxylic acid (4d)
Compound 4d was prepared from compound 3d following the general procedure C
1
(
3
white solid, 98%). H NMR (400 MHz, CDCl ) δ 8.20 (s, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.85 (s,
1
7
4
1
1
5
H), 7.80 (d, J = 7.9 Hz, 1H), 7.71-7.69 (m, 3H), 7.54 (d, J = 7.4 Hz, 2H), 7.50-7.41 (m, 2H),
.36-7.25 (m, 5H), 7.20 (d, J = 6.8 Hz, 1H), 4.45 (t, J = 7.7 Hz, 2H), 4.38 (d, J = 7.1 Hz, 2H),
1
3
.21 (t, J = 7.1 Hz, 1H), 3.60 (t, J = 7.7 Hz, 2H); C NMR (100 MHz, CDCl )
δ 166.7, 153.2,
3
43.6, 141.3, 133.9, 133.1, 131.6, 129.1, 127.95, 127.9, 127.2, 127.1, 126.5, 126.2, 125.9, 125.6,
+
25.1, 123.0, 121.3, 120.1, 67.9, 54.2, 47.0, 34.1; HRMS m/z calcd for C H N O (M+H)
31
26
3
4
04.1923, found 504.1923.
2
3

ACCEPTED MANUSCRIPT
4
.6. Peptidomimetic synthesis
Rink amide 4-methylbenzhydrylamine (MBHA) resin and Fmoc-amino acids were
obtained from Calbiochem-Novabiochem (La Jolla, CA). All peptides were prepared by Fmoc-
based solid-phase peptide synthesis using Rink amide resin with an initial loading of 0.61
mmol/g. Resins were swollen in DMF for 45 min before synthesis. For sequence extension, the
Fmoc-Arg (pbf)-OH (5.0 eq.) along with HBTU (5.0 eq.), HOBt (5.0 eq.) was added to the free
amine on resin in the presence of DIEA (5.0 eq.) in DMF (2 mL) and allowed to proceed for 1 h
with Vortex stirring. After washing with DMF, Fmoc deprotection was achieved with 20%
piperidine in DMF (1 x 10 min, 2 x 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,
dichloromethane, and ether, and then dried under vacuum. The peptide cleavage from the resin
and the deprotection was carried out using a mixture of trifluoroacetic acid, water, and
triisopropylsilane (90:5:5, v/v/v, 2 mL) for 2 h. The crude peptide was precipitated by the
addition of cold diethyl ether and filtered. Purification of crude peptide was carried out on the
preparative Vydac C18 column using an appropriative 10-90% water/acetonitrile gradient in the
presence of 0.05% TFA (A: water buffer, B: acetonitrile buffer). Purified peptides (> 95%) were
assessed by RP HPLC on an analytical Vydac C18 column. Mass spectra of peptides were
obtained using MALDI-TOF-MS (Shimadzu). Cyclic peptide was synthesized using Fmoc
protection strategy. The linear side-chain protected peptide was synthesized on the Rink amide
resin (0.61 mmol/g) coupled with the respective amino acids. Subsequently, Fmoc group in the
N-terminal of the resin-bound protected peptide was removed using 20% piperidine/DMF. Then,
2
the N-terminal amine was bromoacetylated using of BrCH COOH (10.0 eq.) and DIC (5.0 eq.)
2
4

ACCEPTED MANUSCRIPT
stirring for 3 h at rt. The peptide was cleaved from the resin and dissolved in 10 mL of
3
water/CH CN (1:1). Further to attain the basic solution, triethylamine was repeatedly added to
adjust the pH 8-9. Under the basic condition, cysteine thiol in the linear peptide undergoes the
nucleophilic displacement on N-bromoacetyl group to effect the spontaneous intramolecular
cyclization. This cyclization process was monitored by HPLC. After 1 h at room temperature,
3
0% aq. AcOH the solution was added and lyophilized. The crude product was purified by RP-
HPLC.
+
+
MS (MALDI-TOF): m/z Py1 367.2570 (M + H) , Py2 523.8517 (M + H) , Py3 401.2415 (M +
+
+
+
+
H) Py4 557.3426 (M + H) , Py5 477.2725 (M + H) , Py6 633.8332 (M + H) , Py7 437.2419 (M
+
+
+
+
+
H) , Py8 593.4807 (M + H) , Py9 936.9565 (M + H) , Py10 1396.4418 (M + 2H) , Py11
+
+
+
8
56.9093 (M + H) , Py12 1013.1276 (M + 2H) , Py13 1276.2438 (M + 2H) , Py14 1079.4870
+
(
M + H) .
4
.7. Bacterial strains
Two types of Gram-positive bacteria (Staphylococcus epidermidis [KCTC 1917], and
Staphylococcus aureus [KCTC 1621]) and two types of Gram-negative bacteria (Escherichia
coli [KCTC 1682] and Pseudomonas aeruginosa [KCTC 1637] were procured from the Korean
Collection for Type Cultures (KCTC) at the Korea Research Institute of Bioscience and
Biotechnology (KRIBB). Methicillin-resistant Staphylococcus aureus strains (MRSA; CCARM
3
089, CCARM 3090, and CCARM 3095) and multidrug-resistant Pseudomonas aeruginosa
strains (MDRPA; CCARM 2095, and CCARM 2109) were obtained from the Culture Collection
of Antibiotic-Resistant Microbes (CCARM) at Seoul Women’s University in Korea.
2
5

ACCEPTED MANUSCRIPT
Vancomycin-resistant Enterococcus faecium (VREF; ATCC 51559) was supplied from the
American Type Culture Collection (Manassas, VA, USA)
4
.8. Antimicrobial assay
The antimicrobial activity of the peptides against bacteria was examined using the broth
microdilution method in sterile 96-well plates. Aliquots (100 µL) of a bacterial suspension at 2 ×
6
1
0 CFU/mL in 1% peptone were added to 100 µL of the peptide solution (serial 2-fold dilutions
in 1% peptone). After incubation for 18-20 h at 37 °C, bacterial growth inhibition was
determined by measuring the absorbance at 600 nm with a microplate reader (EL 800, Bio-Tek
Instruments, VT, USA). The minimal inhibitory concentration (MIC) was defined as the lowest
peptide concentration that causes 100% inhibition of microbial growth. In addition, the MICs of
the peptides were determined in the presence of salts. 2 × 106 CFU/ml of E. coli (KCTC 1682),
and S. aureus (KCTC 1621) were treated with peptides, while different conditions were added to
LB medium, and the concentrations of physiological conditions were as follows: 150 mM NaCl,
1
mM MgCl , and 2.5mM CaCl . After these treatments, the procedures were same as MIC assay
2 2
described above.
4
.8.1. Hemolytic assay against sheep red blood cells (S-RBC)
The hemolytic activity of the peptides was measured as the amount of hemoglobin
released by the lysis of S-RBC. Fresh S-RBC were centrifuged, washed three times with PBS (35
2
6

ACCEPTED MANUSCRIPT
mM phosphate buffer, 0.15 M NaCl, pH 7.2), dispensed into 96-well plates as 100 µL of 4%
(
w/v) RBC in PBS, and 100 µL of peptide solution was added to each well. Peptide dilutions
were prepared in PBS, and the range of peptide concentrations tested went from 512 ꢀg/mL to 1
g/mL. After 1 h of incubation at 37 °C under 5% CO , cells were centrifuged at 1000 x g for 10
ꢀ
2
min and the supernatant (100 µL) was transferred to other 96 well plates. The absorbance values
of the released hemoglobin were determined at 414 nm using a microplate reader (EL 800, Bio-
Tek Instruments, VT, USA). Zero hemolysis was determined in PBS (APBS) and 100% hemolysis
was determined in 0.1% (v/v) Triton X-100 (Atriton). The hemolysis percentage was calculated as
1
00 × [(Asample – APBS) / (Atriton – APBS)].
4
.8.2. Mammalian cell cultures
RAW264.7 and NIH-3T3 cells were purchased from the American Type Culture
Collection (Manassas, VA), and cultured in DMEM supplemented with 10% fetal bovine serum
and antibiotic-antimycotic solution (100 units/mL penicillin, 100 µg/mL streptomycin and 25
µg/mL amphotericin B) in 5% CO at 37 °C. Cultures were passed every 3 to 5 days, and cells
2
were detached by brief trypsin treatment and visualized with an inverted microscope.
4
.8.3. Cytotoxicity against mammalian cells
Cytotoxicity of peptides against RAW264.7 and NIH-3T3 cells was determined using the
MTT proliferation assay as reported previously with minor modifications [48]. The cells were
4
seeded on 96-well microplates at a density of 2 × 10 cells/well in 150 µL DMEM containing
2
7

ACCEPTED MANUSCRIPT
1
2
0% fetal bovine serum. Plates were incubated for 24 h at 37 °C in 5% CO . Peptide solutions
(20 µL) (serial 2-fold dilutions in DMEM) were added, and the plates further incubated for 2
days. Wells containing cells without peptides served as controls. Subsequently, 20 µL MTT
solution (5 mg/mL) was added in each well, and the plates were incubated for a further 4 h at
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7 °C. Precipitated MTT formazan was dissolved in 40 µL of 20% (w/v) SDS containing 0.01 M
HCl for 2h. Absorbance at 570 nm was measured using a microplate ELISA reader (Molecular
Devices, Sunnyvale, CA). Cell survival was expressed as a percentage of the ratio of A570 of
cells treated with peptide to that of cells only.
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.8.4. Protease stability by radial diffusion assay
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 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 organisms. A bacteria
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suspension (2 × 10 CFU/ml in LB) was mixed with 0.7% agarose. This mixture was poured into
a 10-cm petri dish, and it dispersed rapidly. Five microliters of an aqueous peptide stock solution
(10 mg/mL) were added to 25 mL of trypsin solution in PBS (0.2 µg/mL), and incubated at 37
°C for 4 h. The reaction was stopped by freezing with liquid nitrogen, after which 10 µL aliquots
were placed in each circle paper (< 6 mm in diameter), put on the agarose plates, and then
incubated at 37 °C overnight. The diameters of the bacterial clearance zones surrounding the
circle paper were measured for the quantitation of inhibitory activities.
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