Antimicrobial activity, biocompatibility and hydrogelation ability of dipeptide-based amphiphiles

Article abstract of DOI:10.1039/b815368j

The development of new antibiotics is of increasing importance due to the growing resistance power of microbes against conventional drugs. To this end, cationic peptides are emerging as clinically potent antimicrobial agents. In the present study, we have

Full text of DOI:10.1039/b815368j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Antimicrobial activity, biocompatibility and hydrogelation ability of
dipeptide-based amphiphiles†
Rajendra Narayan Mitra, Anshupriya Shome, Pritha Paul and Prasanta Kumar Das*‡
Received 3rd September 2008, Accepted 1st October 2008
First published as an Advance Article on the web 5th November 2008
DOI: 10.1039/b815368j
The development of new antibiotics is of increasing importance due to the growing resistance power of
microbes against conventional drugs. To this end, cationic peptides are emerging as clinically potent
antimicrobial agents. In the present study, we have synthesized six dipeptide-based cationic amphiphiles
with different head group structures by varying combinations of L-amino acid residues. These
amphiphiles showed remarkable growth inhibiting activity on several Gram-positive (minimum
inhibitory concentration (MIC) = 0.1–10 mg/mL) and Gram-negative (MIC = 5–150 mg/mL) bacteria
as well as on fungus (MIC = 1–50 mg/mL). The inherent antimicrobial efficacies of these cationic
dipeptides were influenced by the head group architecture of the amphiphiles. Encouragingly, these
amphiphiles selectively attacked microbial cells, while showing biocompatibility toward mammalian
cells. The results show that the rational designing of short peptide-based cationic amphiphiles might
serve as a promising strategy in the development of antimicrobial agents with greater cell specificities.
In addition, the amphiphiles showed water gelation ability at room temperature. The formation of
non-covalent supramolecular networks in gelation was established by microscopic and spectroscopic
studies.
for the antimicrobial activity of AMPs.9 The AMPs permeate
Introduction
and disintegrate the cell membrane probably because of their
successful incorporation into the hydrophobic lipid bilayer leading
to cell death.1,8 In spite of the excellent antimicrobial activity
of AMPs there are some hurdles in their use as effective drugs.
The AMPs are not generally cell-specific i.e. they are also toxic
to mammalian cells along with the microbes.5,8 In addition,
synthetic mimics of AMPs should contain short peptide chains
to avoid low metabolic stabilitys and high production costs.10,11 To
date, there are few reports on the antimicrobial activity of short
peptides.1,10,12 The cell specificity of these AMPs is determined by
the sequence of the short peptide moiety. Most of these peptides
are comprised of amino acids like lysine and/or arginine that
get protonated easily at biological pH and thus can interact with
the negatively charged microbial cell membrane. However, if we
can design a short peptide that already contains cationic charges,
variation in the peptide structure can be substantially increased
using combinations of different categories (polar, non-polar) of
amino acids as the presence of arginine or lysine is no longer
mandatory. Thus the design of cationic amphiphiles based on short
peptide sequences with diversified cell specificities is the prime
requirement for the development of biocompatible antimicrobial
agents. In this context, cationic amphiphilic compounds are quite
well known to exhibit antimicrobial activity13 but most of them
are also cytotoxic to eukaryotic cells. In a recent report, we
showed the selective antibacterial activity of L-tryptophan based
cationic biocompatible amphiphiles.14 However for the diversified
cell specificities, biomedicinal chemistry and the allied research
front is demanding a greater number of structurally varied short
peptides as antimicrobial agents.
The design and synthesis of new antimicrobial agents is getting
exponentially important in medicinal chemistry due to the increas-
ing resistance power of microbes to conventional antibiotics.1–3
This resistance arises from newly acquired genes, which encode a
variety of resistance mediating proteins.4 However, microbes with
disrupted cell membranes are not expected to reform and hence
lose their resistance ability.1 In this regard, cationic antimicrobial
peptides (AMPs) are emerging as potent antimicrobial agents
owing to their important role in the innate defense mechanism
against microbes.5,6 The naturally derived AMPs are generally
composed of ~ 12–50 amino acid based peptides with a net
positive charge due to which it can attack the negatively charged
cell membrane.1 In addition, the AMPs also require an optimum
hydrophobicity known as ‘threshold hydrophobicity’ to exhibit
their effectiveness at killing microbes.7,8 The optimal hydropho-
bicity may be attained by the presence of a critical number of
hydrophobic amino acids in peptide chains or by the incorporation
of long alkyl chains in cationic peptide amphiphiles. Hence, a
perfect amphipathicity resulting from the optimal combination of
cationic charge and hydrophobic residue was found to be crucial
Department of Biological Chemistry, Indian Association for the Cultivation
of Science, Jadavpur, Kolkata, 700 032, India. E-mail: bcpkd@iacs.res.in;
Fax: +(91)-33-24732805
† Electronic supplementary information (ESI) available: The hydrogelation
study, related experimental details and the corresponding figures and
tables, the fluorescence micrograph of E. coli and S. aureus before and after
treating with compound 1 in the presence of LIVE/DEADꢀR BacLightTM
Bacterial Viability Kit as well as the NMR and ESIMS spectra of the
amphiphiles 1–6. See DOI: 10.1039/b815368j
In our present work, we report the synthesis of six dipeptide-
based amphiphiles with varying head group architecture (1–6,
‡ Also at the Centre for Advanced Materials, Indian Association for the
Cultivation of Science.
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Fig. 1). These amphiphiles showed inherent growth-inhibiting
activity on several Gram-positive and Gram-negative bacteria,
as well as on fungus. The antimicrobial efficiency was dependent
on the head-group structure particularly with regard to Gram-
negative bacteria and fungus. The most important feature of
these amphiphiles is their biocompatibility with different mam-
malian cell lines like HepG2, HeLa and SiHa. Thus, rational
design of small peptide-based cationic amphiphiles by head-group
modulation led to the development of inherently antimicrobial
amphiphiles with low toxicity to mammalian cells.
agents. To this end, our recent study on the alkyl chain length
dependent antimicrobial efficacy of the single amino acid-based
cationic amphiphile showed optimum antibacterial activity with
C-14 chain length.14 Thus, the development of C-14 aliphatic
chains containing short peptide-based antimicrobial cationic
amphiphiles with greater cell specificities is the key aim of the
present study. Hence, we synthesized six dipeptide-based cationic
amphiphiles that showed strong antimicrobial activity against
several Gram-positive and Gram-negative bacteria, as well as
fungus (1–6, Fig. 1) but low toxicity to mammalian cells. A very
simple synthetic route was used to prepare these amphiphiles
as discussed in our previous report.15,16 We modulated the head
group structure of the amphiphiles by changing the amino acids
of the dipeptide moiety. An attempt has been made to correlate
the antimicrobial activity with the varying head group structure
of the dipeptide amphiphiles.
The antibacterial activity of the dipeptide amphiphiles was
investigated against three strains of Gram-positive bacteria
(Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus)
and four Gram-negative bacteria (Escherichia coli, Pseudomonas
aeruginosa, Klebsiella aerogenes and Klebsiella pneumoniae). The
minimum inhibitory concentrations (MICs), the lowest concen-
tration of amphiphile at which no viable cells are present, are
reported in Table 1. The observed MIC values by both broth
dilution and the spread plate method are comparable. The killing
efficiency of amphiphiles 1–5 against Gram-positive bacteria, B.
subtilis, M. luteus, and S. aureus was very high as the observed
MIC values were very low, 0.1–1 mg/mL. The amphiphile 6
showed slightly higher MIC values (5–10 mg/mL) against three
Gram-positive bacteria. In the case of Gram-negative bacteria,
E. coli, P. aeruginosa, K. aerogenes, and K. pneumoniae, 1 showed
better killing efficiency (MIC = 5–10 mg/mL, Table 1) than
the other amphiphiles (2–6), which showed higher MIC values
(35–150 mg/mL). Again, among the others (2–6), 2 showed
comparatively better killing ability (35–75 mg/mL).
We have also tested the antifungal activity of these amphiphiles
against yeast and fungi, Candida albicans and Saccharomyces
cerevisiae, respectively. The summarized results (Table 1) revealed
that in all cases the amphiphiles showed significant antifungal
activity up to 50 mg/mL. In concurrence with the observed
antibacterial activity, amphiphile 1 showed potent antifungal
activity against both the yeast and fungus. MIC values were 1
and 5 mg/mL for C. albicans and S. cerevisiae, respectively. Am-
phiphile 2 also showed significant killing ability with C. albicans
Fig. 1 Structure of different amphiphiles 1–6.
Interestingly, these excellent antimicrobial peptide-based am-
phiphiles also exhibit water gelation ability at room tempera-
ture with varying efficiency (minimum gelation concentration;
MGC = 1.2–22%, w/v), similar to their C-16 analogues.15 The
hydrogelation ability of the amphiphiles and their supramolecular
self-assembly was investigated by microscopic and spectroscopic
techniques (ESI†).
Results and discussion
The development of new antimicrobial agents having broad-
spectrum activity against bacteria and fungi with considerable
biocompatibility is finding tremendous importance in medicinal
chemistry. AMP is an important class of antimicrobial agent
with a net positive charge and optimum hydrophobicity that
destroys the cell membrane of microbes resulting in cell-death.1,7,8
A synthetic mimic of these AMPs and/or lipopeptide, such as
peptide based cationic amphiphiles, may also act as antimicrobial
Table 1 Antimicrobial activities (MICs) of 1–6 in mg/mLa
Bacteria
Gram-positive
Gram-negative
Fungus
Amphiphile
B. subtilis
M. luteus
S. aureus
E. coli
P. aeruginosa
K. aerogenes
K. pneumoniae
C. albicans
S. cerevisiae
1
2
3
4
5
6
0.5
1
0.1
1
1
5
0.5
1
0.1
1
1
10
0.1
1
0.1
1
1
5
10
60
80
100
80
100
10
75
120
85
110
125
5
35
80
100
150
90
10
45
75
80
90
80
1
5
15
50
—
—
5
15
1
45
15
50
a The MIC values are confirmed by both broth dilution and the spread plate method.
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(MIC = 5 mg/mL) and S. cerevisiae (MIC = 15 mg/mL), although
the killing efficiency of 2 against the fungus is 3–5 times lower
than that was observed for 1. The other amphiphiles (3–6) showed
comparatively less activity towards the yeast and fungi, except for
3 against S. cerevisiae (MIC = 1 mg/mL, Table 1). However, both
amphiphiles 5 and 6 were ineffective against C. albicans even up
to a concentration of 200 mg/mL. The antimicrobial activities of
the dipeptide amphiphiles were compared with the well known
standard AMP, magainin 2, consisting of 23 amino acids.9,17,18
The dipeptide amphiphiles 1–6, showed better killing efficiency
for S. aureus and C. albicans (Table 1) compared to magainin 2
whose MIC values for the above mentioned organisms were 50 and
80 mg/mL respectively.18 Also, for E. coli, the amphiphile 1 showed
better activity than magainin 2 (MIC = 50 mg/mL) while the MIC
values of amphiphiles 2–6 are little higher (Table 1) than magainin
2.18 Interestingly it was also observed that the amphiphiles showed
better activity against Gram-positive bacteria compared to the
widely used traditional antibiotics streptomycin, gentamycin, and
kanamycin (5–10 mg/mL), tested in our recent work.14,19 How-
ever, the amphiphiles demonstrated less potency against Gram-
negative bacteria compared to these antibiotics.14,19 Interestingly,
the antifungal activity of the synthesized amphiphiles against
C. albicans was found to be better than the traditional antibiotic,
amphotericin B (25 mg/mL).19
the cationic dipeptide amphiphiles is because of their molecular
properties. 1 and 3 possess the rigid proline moiety in the interior
part of the head group. Thus, the positive charge on the quaternary
ammonium segment presumably did not delocalize on the entire
head group of 1 and 3, rather it was concentrated on a smaller
area comprised of either phenylalanine (for 1) or tryptophan (for
3) residues. While in the case of the other amphiphiles 2 and 4–6,
due to the presence of p-electron containing aromatic rings either
at the inside or both at the interior and exterior regions of the
head group, the positive charge can easily delocalize over the larger
head group area. Thus, the charge densities at the head groups of
1 and 3 were higher compared with those in other amphiphiles,
resulting in greater electrostatic interactions with the negatively
charged cytoplasmic cell membranes of microbes and enhanced
killing efficiency. Probably for a similar reason amphiphiles 5
and 6 showed comparatively lower antimicrobial activity (higher
MICs) and could not even disrupt the C. albicans cell membrane at
≤200 mg/mL.
Mechanistically, there are two major steps that contribute
to the antimicrobial activity of cationic amphiphiles. At first
the cationic head group attacks the negatively charged cell
membrane of microbes because of the electrostatic interaction.21
This electrostatic interaction is also entropically favored due
to the release of a large number of counterions.22 Next, the
hydrophobic tail of the amphiphile undergoes self-promoted
transport across the cell membrane resulting in disruption in the
bilayer and the release of the intracellular constituents leading
to cell death.23–26 In this step, an optimum hydrophobicity of the
amphiphile possibly allows it to enter and diffuse in the non-polar
environment created by the lipid bilayers of the cell membrane.
Thus the composition of the cell membrane of microbes plays
a crucial role in determining the efficiency of antimicrobial
agents. In the present study, all the amphiphiles showed higher
antimicrobial activity against Gram-positive bacteria than Gram-
negative bacteria (Table 1, Fig. 2). The cell membrane of Gram-
positive bacteria is composed of a peptidoglycan layer, a class
of polysaccharides, whereas Gram-negative bacteria contain an
outer layer consisting of lipopolysaccharide and phospholipids in
addition to the peptidoglycan layer.27,28 This additional protection
in Gram-negative bacteria was presumably the main reason behind
the higher MIC values of the amphiphiles against them (Table 1).
The better killing efficiency of the peptide amphiphiles against
Gram-positive bacteria can also be attributed to the presence of
high amounts of negatively charged lipids in their membranes
resulting in stronger electrostatic interactions.29 In the case of
fungus, the negative charge density at the cell membrane is not as
high as in a bacterial one, so most of the antibacterial compounds
do not show antifungal activity.5 However, interestingly in our
case, dipeptide-based cationic amphiphiles showed significant
antifungal activity (Table 1).
Until now we have measured the thermodynamic activity20 of
peptides in equilibrium against several microorganisms. We were
also interested in measuring the time dependent killing ability
of the cationic dipeptide amphiphiles by exposing the microor-
ganisms to the liquid medium (Luria Brutani (LB) medium for
bacteria and yeast extract, peptone, and dextrose (YEPD) medium
for fungi and yeast) for a wide range of times. The concentration
of the amphiphiles was kept at 10 mg/mL except that of 6, where
40 mg/mL was used against all the tested Gram-positive bacteria.
Fig. 2a–c shows the plot of log(survivors) versus exposure time for
all the amphiphiles against B. subtilis, M. luteus, and S. aureus,
respectively. All the Gram-positive bacteria were killed within 45–
90 min of exposure. The killing effect was more prominent in the
cases of 1 and 3, having phenylalanine-proline and tryptophan-
proline moieties at the head group. The killing kinetics of Gram-
negative bacteria, E. coli, P. aeruginosa, K. aerogenes, and K.
pneumoniae (Fig. 2d–g, respectively) were studied using all the
peptide amphiphiles at 200 mg/mL. Here also 1 and 3 showed
better killing efficiency within 120–240 min compared to the other
amphiphiles (2 and 4–6) which took 180–300 min contact time
to show the bactericidal effect. The cationic peptide amphiphiles
are more efficient at killing Gram-positive bacteria than the
Gram-negative ones. Similar to the killing kinetics trend with
Gram-negative bacteria, the amphiphiles 2 and 4–6 killed the
yeast and fungi within 300 min (Fig. 2h–i), probably with slower
permeabilization kinetics compared to 1 and 3, which took 240 min
to kill them.
To get an insight into the effect of amphiphiles on the
morphology of bacterial cells,30,31 scanning electron microscopic
(SEM) images of E. coli and S. aureus were taken before and
after treatment with amphiphile 1 at above the MIC at 50 and
1.0 mg/mL (Fig. 3a–d). The incubation of E. coli and S. aureus with
amphiphile 1 for around 30–40 min causes a significant change in
their cell morphology. The untreated bacteria E. coli (Fig. 3a) and
S. aureus (Fig. 3c) exhibited cell membrane integrity. However,
after treatment with the 1, the SEM images showed collapse in
On the whole, from the time dependent killing assay it was
observed that the amphiphiles 1 and 3 kill microbes at a faster
rate compared with the other amphiphiles. Amphiphiles 1–6 are
structurally varied at the polar head group with dipeptides of
different L-amino acids but comprising the same C-14 long chain
moiety as the hydrophobic tail. Hence, the observed antimicrobial
activity of the amphiphiles may be regulated by the molecular
architecture at the head group. So, the antimicrobial efficiency of
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Fig. 2 Log(survivors) versus exposure time (min) plots for amphiphiles 1–6 against (a) B. subtilis (b) M. luteus (c) S. aureus (d) E. coli (e) P. aeruginosa
(f) K. aerogenes (g) K. pneumoniae (h) C. albicans and (i) S. cerevisiae. Each value represents the mean SD of six determinations.
the membrane integrity of lysed E. coli (Fig. 3b) and S. aureus
(Fig. 3d).
aureus cells treated with 50 and 1 mg/mL of 1, respectively. All the
untreated bacterial cells exhibited green fluorescence (Fig. S1a
and S1c, ESI†) indicating that they were alive. After treating
with the amphiphile 1 above the corresponding MIC values, only
red fluorescence (Fig. S1b and S1d, ESI†) was observed which
confirmed the killing efficiency of the amphiphile.
The major problem with using cationic amphiphiles as antimi-
crobial agents is their potential toxicity to mammalian cells. So,
we were quite eager to know how the peptide-based amphiphiles
(1–6, Fig. 1) affect eukaryotic cells. The cytotoxicity of all the
amphiphiles in the concentration range of 10 to 200 mg/mL was
followed using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) based cell viability assay in human
hepatic cancer derived HepG2 cells, human cervical cancer derived
We have also studied the antibacterial activity of the representa-
tive amphiphile 1 by fluorescence microscopy using commercially
R
available LIVE/DEADꢀ BacLightTM bacterial viability kit.14,22,32,33
The kit is composed of two nucleic acid binding strains, known
as SYTO 9 and propidium iodide. These dyes have different cell
penetration properties as well as different spectral characteristics.
SYTO 9 can binds to the nucleic acid of both living and dead cells
and fluoresce green after excitation with BP460–495 nm filter.14
Propidium iodide can only bind to the nucleic acid of dead cells
and shows red fluorescence. Fig. S1a and S1c (ESI†) show the
control image of E. coli and S. aureus, respectively while Fig. S1b
and S1d (ESI†) show the fluorescence images of the E. coli and S.
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positively charged amphiphile preferentially binds to the more
negatively charged bacterial membrane. While in the case of
fungus, despite its eukaryotic nature, the amphiphiles selectively
kill the fungi over the mammalian cells probably due to the
presence of cholesterol in the latter membrane which is known
to prevent the membrane disruption.37 The lower membrane
potential across the mammalian cell membrane and its larger size
may also be another reason for such cell selectivity.34,35
As mentioned earlier, these antimicrobial amphiphiles (1–6,
Fig. 1) also showed water gelation capacity. The gelation behavior
of these amphiphiles was investigated by different microscopic
and spectroscopic studies and rheological studies. In this regard,
we recently reported the hydrogelation ability of the C-16 alkyl
chain analogues of these dipeptide cationic amphiphiles at room
temperature.15 The present amphiphiles with C-14 alkyl chains
exhibited notable hydrogelation efficiencies with a wide range
of minimum gelation concentrations (MGC = 1.2–22%, w/v,
Table S1, ESI†). The spectroscopic and microscopic investigations
to understand the gelation mechanism as well as its characteriza-
tion with related experimental details are discussed in the ESI†.
The dried hydrogels at their MGC showed typical entangled
fibrous networks of varying thickness in their FESEM images
(Fig. S3, ESI†). The gel-to-sol transition, Tgel was determined
by the tube inversion method (Fig. S2, ESI†), fluorescence study
(Fig. S4, ESI†), and by rheological study (Fig. S5, ESI†). The
thermoreversibility of the hydrogel was also confirmed by intrinsic
fluorescence study (Fig. S4, ESI†).
Fig. 3 Scanning electron microscopic (SEM) images of (a) control
bacterium E. coli; (b) E. coli after treatment with 50 mg/mL of amphiphile
1; (c) control bacterium S. aureus; and (d) S. aureus after treatment with
1.0 mg/mL of amphiphile 1. Scale bar = 0.5 mm.
SiHa and HeLa cells (Fig. 4).14,34 The dipeptide-based cationic
amphiphiles, irrespective of their molecular structure, showed low
toxicity to all the mammalian cells. Only a maximum of 30%
cytotoxicity was detected for amphiphile 1 at a concentration of
200 mg/mL on SiHa cells while others had a maximum toxicity up
to 26% at the same concentration in HepG2 (Fig. 4a) and SiHa
(Fig. 4b) cell lines. A maximum toxicity of 36% was observed at
a concentration of 200 mg/mL of amphiphile 3 on HeLa cell line
(Fig. 4c). At lower concentrations, all the amphiphiles were mostly
non-toxic to HepG2, SiHa and HeLa cells after an exposure of
3 h. In the same time duration most of the bacterial cells were
killed by the amphiphiles even at a much lower concentration.
Thus, all the dipeptide based cationic amphiphiles are lethal
to bacterial cells, but encouragingly safe with mammalian cells.
The differences in the lipid compositions and the membrane
potential gradient between the microbial and the mammalian
cell membranes is very crucial for such cell selectivity of the
amphiphiles.34,35 Eukaryotic cell membranes are primarily com-
prised of zwitterionic lipids such as phosphatidylcholine, sterols
and sphingomyelin.36 On the other hand, bacterial cell membranes
are made up of negatively charged phospholipids. Therefore the
Conclusion
This study demonstrates the important role of the head group
structure of the cationic dipeptide-based amphiphiles toward
influencing their antimicrobial activity as well as their physical
properties. Interestingly, all the cationic amphiphiles are lethal
to microbial cells while non-toxic to mammalian cells leading
to their greater possibility in biomedical applications. This class
of amphiphiles could be potent antimicrobial candidates in the
future since there has been a continual increase in the demand for
antimicrobial agents. Thus, we have been successful in developing
the shortest sequence of cationic peptide amphiphile that has cell
specificity toward a number of microbes over mammalian cells,
leading to its promising future in biomedicines.
Fig. 4 MTT assay based percent (a) HepG2, (b) SiHa and (c) HeLa cell viability as a function of concentration of 1–6. Each value represents the mean
SD of six determinations.
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allowed to come to room temperature and stirred for 12 h. Work
up, and extraction of this reaction were carried out similarly as
mentioned above. The residue thus obtained was then purified
by a silica gel 60–120 mesh column using ethyl acetate/toluene
as the eluent. After that the purified BOC-protected long chain
dipeptide was subjected to deprotection by trifluoroacetic acid
(TFA, 32 mmol) in dry DCM at room temperature. After 2 h
of stirring, the solvent was removed on a rotary evaporator and
the residue was taken up in ethyl acetate, which was thoroughly
washed with aqueous 10% sodium carbonate solution followed
by brine to neutrality. The organic part was dried over anhydrous
sodium sulfate and concentrated to get the corresponding amine
(7 mmol). The free amine (7 mmol) was quaternized with excess
iodomethane using anhydrous potassium carbonate (7.7 mmol for
2, 4 and 15.4 mmol for 1, 3, 5 and 6) and a catalytic amount of
18-crown-6-ether in dry DMF for 5–6 h at room temperature (25–
27 ◦C). After concentrating the reaction mixture, the residue was
taken up in ethyl acetate and washed with aqueous 5% thiosulfate
solution and brine to neutrality. The concentrated ethyl acetate
part was purified using a 60–120 mesh silica gel column using a
mixture of chloroform/methanol as the eluent and crystallized
from methanol/ether to obtain the solid quaternized iodide salt,
which was subjected to ion exchange on an Amberlite Ira-400
chloride ion exchange resin column to get the colorless chloride
salts of 1–6. The overall yields were 62, 69, 63, 70, 70, and 68% for
1–6, respectively.
Experimental
Materials
Silica gel of 60–120 mesh, L-tryptophan, L-phenylalanine,
L-proline, n-tetradecylamine, N,N-dicyclohexylcarbodiimide
(DCC), 4-N,N-(dimethylamino)pyridine (DMAP), 1-hydroxy-
benzotriazole (HOBT), iodomethane, solvents and all other
reagents were procured from SRL, India. Water used throughout
the study was Milli-Q water. Thin layer chromatography was
performed on Merck precoated silica gel 60-F254 plates. CDCl3,
Amberlite Ira-400 chloride ion exchange resins were obtained
from Aldrich Chemical Company. Ethylene diaminetetraacetic
acid (EDTA) and reagents required to prepare the Luria
Burtani (LB) and YEPD culture medium like tryptone, peptone,
yeast extract, and agar powder were purchased from Himedia
R
Chemical Company, India. The LIVE/DEADꢀ BacLightTM
bacterial viability kit was purchased from Molecular Probes,
Invitrogen Chemical Company. All the materials used in the
cell culture study such as Dulbecco’s Modified Eagles’ Medium
(DMEM), heat inactivated fetal bovine serum (FBS), trypsin
from porcine pancreas and 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) were obtained from
Sigma Aldrich Chemical Company. 1H NMR spectra were
recorded on an AVANCE 300 MHz (BRUKER) spectrometer.
Mass spectrometric (MS) data were acquired by the Electron
Spray Ionization (ESI) technique on a Q-tof-Micro Quadruple
mass spectrophotometer, Micromass.
L-Phenylalanine-L-proline-N-tetradecylamide-N¢,N¢,N¢-trimeth-
ylammonium chloride (1). [a]D +0.7 (c 1.45 in MeOH); Found:
C, 69.35; H, 10.02; N, 7.77. C31H54ClN3O2 requires C, 69.43; H,
10.15; N, 7.84%; IR (KBr): 3419 s (NH), 1666 s (CO); 1H NMR
(300 MHz, CDCl3) d 7.58–7.16 (m, 5H), 5.87–5.82 (m, 1H), 4.45–
4.42 (m, 1H), 3.55–3.43 (m, 2H), 3.36 (s, 9H), 3.19–3.15 (m, 2H),
3.05–2.93 (m, 2H), 1.96–1.44 (m, 4H), 1.18–1.16 (br, 24H), 0.86–
0.78 (t, 3H); m/z (ESI) 500.2827 (C31H54N3O2+ requires 500.4216).
Synthetic procedure and characterization of amphiphiles 1–6
All the peptide amphiphiles (1–6, Fig. 1) were synthesized
following the reaction procedure previously reported by us.15,16
Boc-protected L-amino acids (10 mmol) were coupled with the
methyl ester (11 mmol) of the corresponding L-amino acids using
dicyclohexylcarbodiimide (DCC, 11mmol)and acatalyticamount
of DMAP in the presence of HOBT (11 mmol) in dry DCM
on an ice-bath. The reaction mixture was then allowed to come
to room temperature and stirred for 12 h. Next, the resultant
mixture was filtered and the filtrate was concentrated in a rotary
evaporator. The whole residue was then extracted in ethyl acetate
washed with 2 M HCl, brine, 1 M sodium carbonate and brine
to neutrality. The organic part was dried over anhydrous sodium
sulfate and concentrated. The residue obtained was then purified
by a silica gel 60–120 mesh column using ethyl acetate/toluene as
the eluent. The concentrated column purified white solid material
(-OMe protected) was dissolved in the required amount of MeOH
and subjected to saponification by the addition of 20 mL 1 M
NaOH solution at room temperature. The stirring was carried
out for 6 h, after which the MeOH was evaporated in a rotary
evaporator. The remaining material was then added to water and
washed with diethyl ether; the pH of the aqueous part was adjusted
to 2 using 1 M HCl, and then extracted with ethyl acetate, which
was further washed with brine to remove any traces of acid. The
organic part was then dried over anhydrous sodium sulfate and
concentrated on a rotary evaporator, which yielded a solid -OMe
deprotected peptide. This free C-terminal peptide (8 mmol) was
coupled with n-tetradecylamine (8.8 mmol), DCC (8.8 mmol) and
a catalytic amount of DMAP in the presence of HOBT (8.8 mmol)
in dry DCM on an ice-bath and then the reaction mixture was
L-Proline-L-phenylalanine-N -tetradecylamide-N¢,N¢-dimethyl-
ammonium chloride (2). [a]D +3.0 (c 1.1 in MeOH); Found: C,
68.88; H, 10.11; N, 8.14. C30H52ClN3O2 requires C, 69.00; H,
10.04; N, 8.05%; IR (KBr): 3434 s (NH), 1656 s (CO); 1H NMR
(300 MHz, CDCl3) d 7.38–7.06 (m, 5H), 5.07–5.02 (m, 1H), 4.89–
4.80 (m, 1H), 3.44–3.39 (m, 2H), 3.32–3.21 (m, 4H), 2.93 (s, 3H),
2.38 (s, 3H), 2.21–2.16 (m, 2H), 1.49–1.45 (m, 2H), 1.45–1.18 (br,
24H), 0.82–0.78 (t, 3H); m/z (ESI) 486.406 (C30H52N3O2+ requires
486.4086).
L-Tryptophan-L-proline-N-tetradecylamide-N¢,N¢,N¢-trimethyl-
ammonium chloride (3). [a]D -25.0 (c 1.16 in MeOH); Found: C,
68.75; H, 9.69; N, 9.78. C33H55ClN4O2 requires C, 68.90; H, 9.64; N,
9.74%; IR (KBr): 3408 s (NH), 1648 s (CO); 1H NMR (300 MHz,
CDCl3) d 7.96 (br, 1H), 7.44–6.94 (m, 4H), 5.01–4.98 (m, 1H),
3.78 (br, 1H), 3.55–3.38 (br, 2H), 3.47 (s, 9H), 3.24–3.14 (m, 2H),
3.14–2.8 (m, 2H), 1.95 (br, 2H), 1.42–1.39 (br, 2H), 1.29–1.13
+
(br, 24H), 0.82–0.71 (t, 3H); m/z (ESI) 539.3687 (C33H55N4O2
requires 539.4325).
L-Proline-L-tryptophan-N-tetradecylamide-N¢,N¢-dimethylammo-
nium chloride (4). [a]D -19.0 (c 1.9 in MeOH); Found: C, 68.33;
H, 9.38; N, 9.77. C32H53ClN4O2 requires C, 68.48; H, 9.52; N,
9.98%; IR (KBr): 3399 s (NH), 1658 s (CO); 1H NMR (300 MHz,
CDCl3) d 7.56–7.54 (d, 1H), 7.30–6.92 (m, 4H), 4.92–4.74 (m,
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2H), 3.39–3.25 (br, 4H), 3.11 (s, 3H), 3.06–2.48 (m, 2H), 2.25 (s,
3H), 2.21–2.11 (m, 2H), 1.57–1.36 (m, 2H), 1.36–1.12 (br, 24H),
0.75–0.73 (t, 3H); m/z (ESI) 525.3685 (C32H53N4O2 requires
working concentration in the range of 106–109 and 104–105 colony
forming units (cfu)/mL for bacteria and fungus, respectively
before every experiment.
+
525.4169).
Antimicrobial studies
L-Tryptophan-L-phenylalanine-N-tetradecylamide-N¢,N¢,N¢-tri-
methylammonium chloride (5). [a]D +2.0 (c 2.05 in MeOH);
Found: C, 70.12; H, 9.23; N, 8.89. C37H57ClN4O2 requires C, 71.07;
H, 9.19; N, 8.96%; IR (KBr): 3409 s (NH), 1667 s (CO); 1H NMR
(300 MHz, CDCl3) d 7.67–7.30 (m, 5H), 7.26–6.98 (m, 5H), 5.18
(br, 1H), 4.55–4.52 (m, 1H), 3.75 (br, 2H), 3.27–3.11 (m, 2H), 2.77
(s, 9H), 1.93 (br, 2H), 1.38–1.23 (br, 24H), 0.96–0.72 (t, 3H); m/z
Minimum inhibitory concentrations (MICs) of 1–6 were estimated
by both broth dilution and the spread plate method. The MIC was
measured using a series of test tubes containing the amphiphiles
(0.05–200 mg/mL) in 5 mL liquid medium. Diluted microbial
culture was added to each test tube in identical concentration
to obtain the working concentration of B. subtilis; 7.5 ¥ 107–1 ¥
108 cfu/mL, for M. luteus; 1 ¥ 106–2.5 ¥ 106 cfu/mL, for S. aureus;
5 ¥ 106–7.5 ¥ 106 cfu/mL, for E. coli; 3.75 ¥ 107–7.5 ¥ 107 cfu/mL,
for P. aeruginosa; 9 ¥ 107–1.2 ¥ 108 cfu/mL, for K. aerogenes;
1.5 ¥ 109–6.25 ¥ 109 cfu/mL, for K. pneumoniae; 1.4 ¥ 107–
7.5 ¥ 107 cfu/mL, for C. albicans; 9.8 ¥ 104–1.3 ¥ 105 cfu/mL,
for S. cerevisiae; 1.5 ¥ 105–3 ¥ 105 cfu/mL. All the test tubes
were then incubated at 37 ◦C for 24 h. The optical density of
all the solutions was measured before and after incubation at
650 nm. Liquid medium containing microorganisms was used as a
positive control. All the experiments were performed in triplicate
and repeated twice.
+
(ESI) 589.5331 (C37H57N4O2 requires 589.4482).
L-Phenylalanine-L-tryptophan-N-tetradecylamide-N¢,N¢,N¢-tri-
methylammonium chloride (6). [a]D +6.0 (c 1.25 in MeOH);
Found: C, 70.89; H, 9.01; N, 8.89. C37H57ClN4O2 requires C,
71.07; H, 9.19; N, 8.96%; IR (KBr): 3419 s (NH), 1663 s (CO);
1H NMR (300 MHz, CDCl3) d 7.68–7.07 (m, 10H), 5.43–5.24 (m,
1H), 4.8 (br, 1H), 3.52–3.46 (m, 2H), 3.12–2.95 (br, 4H), 2.81 (s,
9H), 1.31–1.25 (br, 24H), 0.95–0.85 (t, 3H); m/z (ESI) 589.3918
+
(C37H57N4O2 requires 589.4482).
Microorganisms and culture conditions
We also tested the antimicrobial activity through the spread
plate method. For these experiments, the same amount of bacterial
culture was added to test tubes containing the desired concentra-
tion of amphiphile sol◦ution in liquid medium. The test tubes were
then incubated at 37 C for 5–7 h. Then, 50 mL from each tube
was spread onto LB agar plates inside the laminar hood. Finally,
plates were incubated for 24 h (for all the bacterial strains and for
fungi S. cerevisiae) and 48 h (for fungi C. albicans) at 37 C and
the viable cells were counted. The experiments were performed in
triplicate and were repeated twice.
The in vitro antimicrobial activity of all the cationic amphiphiles
was investigated against pathogenic representatives of Gram-
positive bacteria, Gram-negative bacteria and fungus. The Gram-
positive bacteria used in the present study were Bacillus sub-
tilis, Micrococcus luteus, and Staphylococcus aureus. The Gram-
negative strains investigated included Escherichia coli, Pseu-
domonas aeruginosa, Klebsiella aerogenes and Klebsiella pneumo-
niae. Investigations of antibacterial activities were performed by
both broth dilution and the spread plate method. The LB medium
(tryptone (10 g), yeast extract (5 g) and NaCl (10 g) in 1 L sterile
distilled water at pH 7.0) was used as a liquid medium and LB
agar (tryptone (10 g), yeast extract (5 g), NaCl (10 g) and agar
(15 g) in 1 L of sterile distilled water of at 7.0) was used as a solid
medium in all antibacterial experiments. The antifungal activity
on the two fungi Candida albicans and Saccharomyces cerevisiae
was investigated by YEPD broth dilution and the spread plate
method. The YEPD medium (yeast extract (3 g), peptone (10 g)
and dextrose (20 g) in 1 L sterile distilled water at pH 7.0) was used
as a liquid medium and YEPD agar (yeast extract (3 g), peptone
(10 g), dextrose (20 g) and agar (15 g) in 1 L sterile distilled water
at pH 7.0) was used as a solid medium for the antifungal studies.
All the microbial strains were purchased from the Institute of
Microbial Technology, Chandigarh, India. The stock solutions of
all the amphiphiles as well as the required dilutions were made in
autoclaved sterile water.
◦
Time dependent microbial killing assay
A serial dilution method was used for determining the kinetic
effect, also called Contact Germicidal Efficiency (CGE), i.e., the
time required for the antimicrobial effect. Overnight cultures on
solid agar medium were diluted with liquid medium to give a
bacterial concentration of 106–109 cfu/mL. Amphiphile (1–6)
stock solutions were added at concentrations higher than MIC
values, and were incubated at room temperature inside the laminar
hood. In the case of Gram-positive bacteria the concentrations of
1–5 were 10 mg/mL and of 6 it was 40 mg/mL while in the case
of Gram-negative bacteria it was 200 mg/mL for all amphiphiles.
At regular intervals, the same amount of aliquot was withdrawn
and kept at 4 ◦C. Immediately, the aliquots were diluted and
50 mL of diluted suspension was spread onto LB agar plates.
Next, all the plates were incubated at 37 ◦C for 24 h and after
incubation the viable cells were counted. The control experiment
was performed in the absence of amphiphile. The same technique
was followed for the two fungi. In this case 25 mg/mL (amphiphile
1) and 200 mg/mL (amphiphiles 2–6) of the amphiphiles were
utilized for the corresponding kinetic study and for C. albicans
and S. cerevisiae◦respectively. Here all the YEPD agar plates were
incubated at 37 C for 48 h (C. albicans) and 24 h (S. cerevisiae)
and after incubation the viable cells were counted. With the same
technique the control study was carried out without amphiphiles.
The freeze-dried ampules of all microbial strains were opened
and a loopful of culture was spread to give single colonies on
the respective solid agar (LB agar for bacterial strains and YEPD
agar for fungal strains) media and incubated for 24 h (for all
the bacterial strains and for fungi S. cerevisiae) and 48 h (for
fungi C. albicans) at 37 ◦C. For all the microbial strains, a
representative single colony was picked up with a wire loop and
was spread on an agar slant to give single colonies. The slants
◦
were incubated at 37 C for the respective time. These incubated
cultures of all the microorganisms were diluted as required to give a
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FESEM of dipeptide amphiphile treated E. coli and S. aureus
the number of cells alive. Reduction of the absorbance value
can be attributed to the killing of cells or inhibition of cell
proliferation by the amphiphiles. Cells were seeded at a density
15 000 cells per well in a 96-well microtiter plate for 18–24 h
before the assay. A stock solution of all the six compounds was
prepared in DMSO. Sequential dilutions of these stock solutions
were done during the experiment to vary the concentrations
of the amphiphiles (10 to 200 mg/mL) in the microtiter plate.
The amphiphiles were insoluble beyond this concentration in the
DMEM medium. The cells were incubated with 1–6 for 3 h at 37 ◦C
under 5% CO2. Then, 15 mL MTT stock solution (5 mg/mL)
in phosphate buffer saline was added to the above mixture and
the cells were further incubated for another 4 h. The precipitated
formazan was dissolved thoroughly in DMSO and the absorbance
E. coli (3.75 ¥ 107–7.5 ¥ 107 cfu/mL) and S. aureus (5 ¥ 106–7.5 ¥
106 cfu/mL) cells (1 mL) were treated with compound 1 above the
MIC at 50 and 1.0 mg/mL, respectively. The bacterial cells with
and without (untreated cells as control) amphiphile were incubated
for 30–40 min. After incubation the mixtures were centrifuged at
10 000 rpm for 10 min. The media was removed completely and
the cells were redispersed in 0.9 wt% saline. Finally, 5 mL of the
redispersed samples was mounted on a glass slide and dried under
vacuum for 4 h and the SEM images were taken on JEOL-6700F
scanning electron microscope.
Fluorescence microscopic study
R
at 570 nm was measured using BioTekꢀ Elisa Reader. The number
R
The LIVE/DEADꢀ BacLight TM bacterial kit was used to examine
of surviving cells were expressed as percent viability = (A570(treated
bacterial cell viability under a fluorescence microscope. The kit
contains a mixture of two nucleic-acid binding strains, specifically
referred to as SYTO 9 and propidium iodide. The kit was
stored at -20 ◦C in the dark, and was taken out and thawed at
room temperature just prior to assay. E. coli (3.75 ¥ 107–7.5 ¥
107 cfu/mL) and S. aureus (5 ¥ 106–7.5 ¥ 106 cfu/mL) cells
(1 mL) were treated with 1 above the MIC (50 mg/mL for E. coli
and 1 mg/mL for S. aureus, respectively) and also untreated cells
were taken in a centrifuge tube as a control. The mixtures were
centrifuged at 10 000 rpm for 10 min. Then, media was removed
completely and the cells were redispersed in 0.9 wt% saline. Finally,
BacLight dye mixture (3 mL) was added and the cells incubated
in the dark at room temperature for 15–20 min. After incubation,
5 mL of the solution mixture was mounted over microscope slides,
which were then air-dried and viewed under the light microscope
(BX61, Olympus) using an excitation filter of BP460–495 nm and
a band absorbance filter covering wavelengths below 505 nm.
cells) - background/A570(untreated cells) - background) ¥ 100.
Statistics
Significant differences between two groups of data were evaluated
by Student’s t-test.
Acknowledgements
P.K.D. is thankful to the Department of Science and Technology,
India for financial assistance through a Ramanna Fellowship
(No. SR/S1/RFPC-04/2006). A.S. acknowledges the Council of
Scientific and Industrial Research, India for Research Fellowships.
P.P. is thankful to the Indian Association for the Cultivation of
Science. We are very much thankful to Sangita Roy for helpful
discussions.
References
Cell cultures
1 A. Makovitzki, D. Avrahami and Y. Shai, Proc. Natl. Acad. Sci. U. S. A,
2006, 103, 15997–16002.
Human hepatic cancer derived HepG2 cells, human cervical cancer
derived SiHa and HeLa cells were obtained from the National
Center for Cell Science (NCCS), Pune (India), and maintained
in DMEM medium supplemented with 10% FBS, 100 mg/L
streptomycin and 100 IU/mL penicillin. Cells were grown in a
2 S. Pritz, M. Pa¨tzel, G. Szeimies, M. Dathea and M. Bienert, Org. Biomol.
Chem., 2007, 5, 1789–1794; A. Zumbuhel, L. Ferreira, D. Kuhn, A.
Astashkina, L. long, Y. Yeo, T. Iaconis, M. Ghannoum, G. R. Fink, R.
Langer and D. S. Kohane, Proc. Natl. Acad. Sci. U. S. A., 2007, 104,
12994–12998; B. Xing, C.-W. Yu, K.-H. Chow, P.-L. Ho, D. Fu and B.
Xu, J. Am. Chem. Soc., 2002, 124, 14846–14847; Z. Yang, K. Xu, Z.
Guo, Z. Guo and B. Xu, Adv. Mater., 2007, 19, 3152–3156; J. C. Tiller,
Angew. Chem., Int. Ed. Engl., 2003, 42, 3072–3075; J. C. Tiller, S. B. Lee,
K. Lewis and A. M. Klibanov, Biotechnol. Bioeng., 2002, 79, 465–471.
3 S. A. Sieber and M. A. Marahiel, Chem. Rev., 2005, 105, 715–738; E. D.
Brown and G. D. Wright, Chem. Rev., 2005, 105, 759–774.
4 J. C. B. DeNap and P. J. Hergenrother, Org. Biomol. Chem., 2005, 3,
959–966.
◦
25 mL cell culture flask and incubated at 37 C in a humidified
atmosphere of 5% CO2 to approximately 70–80% confluence. A
subculture was performed every 2–3 days. After 48–72 h, media
was removed to eliminate the dead cells. Next, the adherent
cells were detached from the surface of the culture flask by
trypsinization. Cells were now in the exponential phase of growth
for checking the viability of the cationic amphiphilic amphiphiles
(1–6).
5 A. Makovitzki and Y. Shai, Biochemistry, 2005, 44, 9775–9784.
6 M. Zasloff, Nature, 2002, 415, 389–395.
7 Y. Shai and Z. Oren, Peptides, 2001, 22, 1629–1641.
8 D. Avrahami and Y. Shai, J. Biol. Chem., 2004, 279, 12277–12285.
9 M. Dathe, M. Schu¨mann, T. Wieprecht, A. Winkler, M. Beyermann,
E. Krause, K. Matsuzaki, O. Murase and M. Bienert, Biochemistry,
1996, 35, 12612–12622; T. Wieprecht, M. Dathe, R. M. Epand, M.
Beyermann, E. Krause, W. Lee Maloy, D. L. MacDonald and M.
Bienert, Biochemistry, 1997, 36, 12869–12880; Y. Wolf, S. Pritz, Saıd
Abes, M. Bienert, B. Lebleu and J. Oehlke, Biochemistry, 2006, 45,
14944–14954; N. P. Chongsiriwatana, J. A. Patch, A. M. Czyzewski,
M. T. Dohm, A. Ivankin, D. Gidalevitz, R. N. Zuckermann and A. E.
Barron, Proc. Natl. Acad. Sci. U. S. A, 2008, 105, 2794–2799.
10 M. Dathe, H. Nikolenko, J. Klose and M. Bienert, Biochemistry, 2004,
43, 9140–9150.
Cytotoxicity assay
The cytotoxicities of amphiphiles 1–6 were assessed by the
microculture MTT reduction assay as used in the literature.
This assay is based on the reduction of a soluble tetrazolium
salt by the mitochondrial dehydrogenase of the viable cells to
form an insoluble colored product, formazan. The amount of
formazan formed can be measured spectrophotometrically after
dissolution of the dye in DMSO. The activity of the enzyme
and the amount of the formazan produced is proportional to
11 R. E. W. Hancock, Drugs, 1999, 57, 469–473.
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 94–102 | 101
©

View Article Online
12 M. B. Strom, B. E. Haug, M. L. Skar, W. Stensen, T. Stiberg and J. S.
Svendsen, J. Med. Chem., 2003, 46, 1567–1570.
13 J. Haldar, P. Kondaiah and S. Bhattacharya, J. Med. Chem., 2005, 48,
3823–3831.
14 S. Roy and P. K. Das, Biotechnol. Bioeng., 2008, 100, 756–764.
15 R. N. Mitra, D. Das, S. Roy and P. K. Das, J. Phys. Chem. B, 2007,
111, 14107–14113.
16 A. Dasgupta, R. N. Mitra, S. Roy and P. K. Das, Chem. Asian J., 2006,
1, 780–788; D. Das, A. Dasgupta, S. Roy, R. N. Mitra, S. Debnath and
P. K. Das, Chem. Eur. J., 2006, 12, 5068–5074; S. Roy, A. Dasgupta and
P. K. Das, Langmuir, 2007, 23, 11769–11776; A. Shome, S. Debnath
and P. K. Das, Langmuir, 2008, 24, 4280–4288.
17 D. Avrahami and Y. Shai, Biochemistry, 2002, 41, 2254–2263; T.
Wieprecht, M. Dathe, M. Beyermann, E. Krause, W. Lee Maloy, D. L.
MacDonald and M. Bienert, Biochemistry, 1997, 36, 6124–6132.
18 M. Zasloff, Proc. Natl. Acad. Sci. U. S. A., 1987, 84, 5449–5453.
19 C. Ramalingan, Y. T. Park and S. Kabilan, Eur. J. Med. Chem., 2006,
41, 683–696; I. Oren, O. Temiz, I. Yalcin, E. Sener and N. Altanlar,
Eur. J. Pharm. Sci., 1998, 7, 153–160.
20 N. Bodor, J. J. Kaminsky and S. Selk, J. Med. Chem., 1980, 23, 469–
474; R. M. Baird, and S. F. Bloomfield, in Microbial quality assurance in
cosmetics, toiletries and non-sterile pharmaceuticals, published by CRC
Press, 1996, pp. 258.
21 K. M. Stewart, K. L. Hortonb and S. O. Kelley, Org. Biomol. Chem.,
2008, 6, 2242–2255; D. A. Salick, J. K. Kretsinger, D. J. Pochan and
J. P. Schneider, J. Am. Chem. Soc., 2007, 129, 14793–14799.
22 R. Ku¨gler, O. Bouloussa and F. Rondelez, Microbiology, 2005, 151,
1341–1348.
23 J. A. Castillo, P. Clapes, M. R. Infante, J. Comas and A. Manresa,
J. Antimicrob. Chemother., 2006, 57, 691–698.
25 E. Jawetz, J. L. Melnick, E. A. Adelberg, G. F. Brooks, J. S. Butel, and
L. N. Ornston, in Medical microbiology, Prentice-Hall, London, 1989,
pp. 46.
26 T. J. Franklin, and G. A. Snow, in Antiseptics, antibiotics, and the cell
membrane, Biochemistry of Antimicrobial Action, Chapman and Hall
Ltd., London, 1981, pp. 58–78.
27 J. W. Costerton and K. J. Cheng, J. Antimicrob. Chemother., 1975, 1,
363–377.
28 S. P. Denyer and J. Y. Maillard, J. Appl. Micobiol., 2002, 92, 35S–
45S.
29 W. M. O’Leary and S. G. Wilkinson, in Gram-positive bacteria,
in Microbial lipids (C. Ratledge, and S. G. Wilkinson, Eds.), Aca-
demic Press Ltd., London, 1988, pp. 117–201S. G. Wilkinson,
in Gram-negative bacteria, in Microbial lipids (C. Ratledge, and
S. G. Wilkinson, Eds.); Academic Press Ltd., London, 1988, pp. 299–
488.
30 M. L. Mangoni, N. Papo, D. Barra, M. Simmaco, A. Bozzi, A. Di
Giulio and A. C. Rinaldi, Biochem. J., 2004, 380, 859–865.
31 S. Sharma, P. Sen, S. N. Mukhopadhyay and S. K. Guha, Colloids Surf.
B, 2003, 32, 43–50.
32 C. Qin, X. Zhong, X. Bu, N. L. J. Ng and Z. Guo, J. Med. Chem., 2003,
46, 4830–4833.
33 L. Boulos, M. Prevost, B. Barbeau, J. Coallier and R. Desjardins,
J. Microbiol. Methods, 1999, 37, 77–86.
34 M. B. Hansen, S. E. Nielsen and K. Berg, J. Immunol. Methods, 1989,
119, 203–210.
35 K. Matsuzaki, M. Harada, T. Handa, S. Munakoshi, N. Fujii, H.
Yajima and K. Miyajima, Biochim. Biophys. Acta, 1989, 981, 130–
134.
36 S. Munro, Cell, 2003, 115, 377–388; F. J. Alvarez, L. M. Douglas and
J. B. Konopka, Eukaryotic Cell, 2007, 6, 755–763.
37 J. Brajtburg and J. Bolard, Clin. Microbiol. Rev., 1996, 9, 512–531.
24 C. J. Ioannou, G. W. Hanlon and S. P. Denyer, Antimicob. Agents
Chemother., 2007, 51, 296–306.
102 | Org. Biomol. Chem., 2009, 7, 94–102
This journal is
The Royal Society of Chemistry 2009
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