Self-assembly to function: Design, synthesis, and broad spectrum antimicrobial properties of short hybrid e -vinylogous lipopeptides

Article abstract of DOI:10.1021/jm400884w

Nonribosomal E-vinylogous γ-amino acids are widely present in many peptide natural products and have been exploited as inhibitors for serine and cysteine proteases. Here, we are reporting the broad spectrum antimicrobial properties and self-assembled nanostructures of various hybrid lipopeptides composed of 1:1 alternating α- and E-vinylogous residues. Analysis of the results revealed that self-assembled nanostructures also play a significant role in the antimicrobial and hemolytic activities. In contrast to the α-peptide counterparts, vinylogous hybrid peptides displayed excellent antimicrobial properties against various bacterial and fungal strains. Peptides that adopted nanofiber structures displayed less hemolytic activity, while peptides that adopted nanoneedle structures displayed the highest hemolytic activity.

Full text of DOI:10.1021/jm400884w

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Article
Self-Assembly to Function: Design, Synthesis and Broad Spectrum
Antimicrobial Properties of Short Hybrid E-Vinylogous Lipopeptides
Hosahudya N Gopi, Sushil N Benke, Narem Nagendra, Prabhakar
Lal Srivastava, Hirekodathakallu V Thulasiram, and S Shiva Shankar
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 13 Oct 2013
Downloaded from http://pubs.acs.org on October 21, 2013
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Self-Assembly to Function: Design, Synthesis and Broad Spectrum
Antimicrobial Properties of Short Hybrid E-Vinylogous
Lipopeptides
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Shiva Shankar S.,1 Sushil N. Benke, 1 Narem Nagendra, 1 Prabhakar Lal Srivastava, 2
Hirekodathakallu V. Thulasiram,*2 and Hosahudya N. Gopi*1
1Department of Chemistry, Indian Institute of Science Education and Research, Dr. Homi
Bhabha Road, Pune-41108, 2 National Chemical Laboratory, Dr. Homi Bhabha Road, Pune-
411008, INDIA
E-mail: hv.thulasiram@ncl.res.in; hn.gopi@iiserpune.ac.in
Abstract: Non-ribosomal E-vinylogous γ-amino acids are widely present in many peptide
natural products and have been exploited as inhibitors for serine and cysteine proteases. Here,
we are reporting the broad spectrum antimicrobial properties and self-assembled nanostructures
of various hybrid lipopeptides composed of 1:1 alternating α-and E-vinylogous residues.
Analysis of the results revealed that self-assembled nanostructures also play a significant role in
the antimicrobial and hemolytic activities. In contrast to the α-peptide counterparts, vinylogous
hybrid peptides displayed excellent antimicrobial properties against various bacterial and fungal
strains. Peptides that adopted nanofiber structures displayed less hemolytic activity, while
peptides that adopted nanoneedle structures displayed highest hemolytic activity.
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Introduction
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As a consequence of evolution, a large number of bacterial and fungal strains have
developed resistance towards the conventional antibiotics, creating an urgent need for the design
of new antibiotics with different mode of action. Indeed an overwhelming body of literature
over the past several years demonstrated that naturally occurring antimicrobial peptides (AMPs)
are evolutionary conserved components of the innate immune response capable of targeting wide
range of “multidrug resistant” strains.1 However, potential therapeutic applications of AMPs
have been suffering with several inherent problems including selectivity, toxicity and
bioavailability. In this regard, various approaches have been adopted to mimic the cationic and
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amphiphilic nature of natural AMPs including synthetic peptides,2 β-peptides, peptoids,4, 5
polypyridines,6 polynorboranes,7 polysaccharides,8
peptidopolysaccharides,9 and so on.
Lipopeptides are a recently emerged class of AMPs and the broad spectrum of natural
lipopeptides has paved way for the development of new class of synthetic analogues.10 Most of
the native lipopeptides isolated from bacteria and fungi are non-specific and highly toxic.
However, after the approval of drug daptomycin and the re-emergence of Polymyxin B as
potential chemotherapeutic agents for the treatment of microbial infections, these lipopeptides
have attracted considerable attention of medicinal chemists.11 Previous studies of many natural
and synthetic hybrid lipopeptides revealed that the antimicrobial activity of lipopeptides mainly
depends on the sequence of peptides, length of fatty acid chain and overall hydrophobicity of the
peptides.12 These lipopeptides permeate rapidly and disintegrate the membrane morphology of
pathogens.13 In addition to this, many N-terminal fatty acid acylated amphiphilic peptides are
self-assembled into cylindrical nanofibers with diameters ranging from nanometers to
micrometers, lead to the formation of hydrogels, nanotubes, nanobelts and nanoribbons.14, 15 The
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applications of self-assembled nanostructures of amphiphilic peptide based materials have been
found in various fields like bone regeneration and enamel formation.16
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α, β-Unsaturated γ-amino acids (E-vinylogous amino acids) are important components of
many biologically active peptides 17 and also have been used as potential candidates for design of
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inhibitors of serine and cysteine protease.18 We have been interested in the synthesis and
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conformational analysis of hybrid peptides containing E-vinylogous amino acids.19,
The
structural analysis of the E-vinylogous amino acids in hybrid peptides reveal that due to the
geometrical constraints of the double bonds they prefer to adopt extended β-sheet type of
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structures.20,
Here, we sought to explore the antimicrobial properties of short hybrid
lipopeptides containing geometrically constrained E-vinylogous amino acids. Herein, we are
reporting the design, antimicrobial activities, self-assembling properties and the mode of action
of short 1:1 alternating alpha and E-vinyologus hybrid lipopeptides. Systematic investigation of
the mode of action reveals that the nature of the self-assemblies of these hybrid lipopeptides play
a crucial role in their antimicrobial activities and the mechanism of action. The self-assembly
properties associated with the mode of action can be controlled by tuning the hydrophobic
threshold of lipopeptides.
Results and Discussion
In order to understand their antimicrobial activity, we designed two series of short hybrid
E-vinylogous lipopeptides containing C12 and C8 fatty acids. The schematic representation of the
short hybrid lipopeptides is shown in Scheme 1. The design is based on the analysis of the
natural lipopeptides which generally constitute equal proportions of hydrophobic and hydrophilic
residues. The α-amino acids were chosen to be hydrophilic residues while E-vinylogous amino
acids as hydrophobic residues in short tetrapeptide sequences. All E-vinylogous amino acids
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Peptide
S1
X
AA1
C11H23 dgAla
C7H15 dgAla
C11H23 dgTyr
C7H15 dgTyr
C11H23 dgAla
C7H15 dgAla
C11H23 dgPhe
AA2
Lys
Lys
Lys
Lys
Arg
Arg
Lys
Lys
Lys
Lys
Lys
AA3
AA4
Lys
Lys
Lys
Lys
Arg
Arg
Lys
Lys
Lys
Lys
Lys
tR
dgAla
dgAla
dgTyr
dgTyr
dgAla
dgAla
dgPhe
dgPhe
dgVal
dgPhe
Val
41.14
31.96
38.61
30.65
33.30
31.63
48.19
35.60
37.50
25.25
39.22
S2
S3
S4
S5
S6
S7
S8
C7H15
C7H15
CH3
dgPhe
dgLeu
dgPhe
Leu
S9
S10
S11
C7H15
Scheme 1. Schematic representation of the hybrid E-vinylogous lipopeptides (dg stands for α, β-
dehydro γ-amino acids).
were synthesized using Wittig reaction as reported earlier.19, 20 All peptides were synthesized by
solid phase method on Knorr amide resin and purified through reverse phase HPLC on C18
column. Based on the reverse phase HPLC retention time, the order of hydrophobicity of the E-
vinylogous hybrid lipopeptides was found to be S7> S1> S3> S9>S8> S5> S2> S6> S4. The
lipopeptide with α-amino acids (S11) and E-vinylogous hybrid peptide without fatty acid (S10)
displayed the retention times 39.22 and 25.25, respectively.
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All E-vinylogous hybrid peptides with N-terminal C12 fatty acid (S1, S3, S5, and S7) and
C8 fatty acid (S2, S4, S6, S8 and S9) along with α-peptide control S11 and the hybrid peptide
with N-acetyl group (S10) were subjected to antimicrobial studies. Instructively, except S7, all
peptides with C12 fatty acids, displayed potent antimicrobial activity with various Gram-negative
Table 1: MICs of vinylogous hybrid lipopeptides (µg/ml)
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Microorganisms
Escherichia coli
Escherichia coli K12
Klebsiella
S1
S2
S3
S4
S5
S6
S7
25
25
S8
S9
S10
S11
6.25 >100 6.25 25
6.25 >100 6.25 25
12.5 >100
12.5 >100
1.56 12.5 >100 >100
1.56
3.12
-
>100 >100
>100 >100
3.12 >100 3.12 50
6.25 >100 3.12 50
6.25 >100 3.12 50
6.25 >100 3.12 50
6.25 >100
3.12 >100
6.25 >100
3.12 >100
25
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25
pneumoniae
Pseudomonas
aeruginosa
3.12
3.12
3.12
50
50
50
>100 >100
>100 >100
>100 >100
Staphylococcus
aureus
Salmonella
typhimurium
Candida albicans
Candida glabrata
Cryptococcus
neoformans
3.12 >100 1.56 12.5 3.12 >100 3.12 3.12
6.25 50 3.12 25 6.25 >100 6.25 3.12
25
50
>100 >100
>100 >100
6.25 >100 6.25 25
12.5 >100 6.25 3.12 6.25 >100 >100
Saccharomyces
cerevisiae
3.12 >100 3.12 12.5 3.12 >100 6.25 3.12
48 58 2.5 54 22 52 <1.56 16
50
>100 >100
Hemolytic HD10
-Nil- -Nil- -Nil-
and Gram-positive bacterial strains as well as various fungal strains. Among all lipopeptides in
the series of C8 fatty acid containing hybrid peptides, S8 displayed potent activity against all
bacterial and fungal strains while moderate activities were observed for S4, S7 and S9. The MIC
values of all the peptides are given in the Table 1. Similar MIC values of the individual peptides
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against Gram-positive, Gram-negative and fungal strains suggest the unique mode of action of
these lipopeptides. Peptides which showed strong inhibitory action against bacteria also
displayed the strong inhibitory action against fungi (S1, S3, S5 and S8). In comparison with S7,
S3 containing phenolic –OH groups displayed excellent antimicrobial activity. Interestingly, S8
showed more potent activity than S7, while S4 showed lower potency than that of the C12
analogue S3. In order to understand the role of basic residues, we replaced Lys with Arg in the
peptides S5 and S6. Results reveal that the peptides containing Arg also displayed similar
antimicrobial activities. In comparison with S8, S9 showed moderate activity against both
bacteria and fungi. Further the control peptides, hybrid peptide with N-acetyl group (S10) and
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i)
ii)
iii)
iv)
(A)
(B)
Figure 1: A) Atomic Force Microscopy images of Escherichia coli K12 (i) and Candida
albicans (ii) before the treatment of lipopeptides, iii and iv after the treatment lipopeptide S1,
respectively. B) Fluorescence images (FITC) of E.coli without peptides (a) and (b). Fluorescence
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images (FITC) of the E. coli after the treatment of S3 (c and d) and S8 (e and f) lipopeptides
(Scale bar 10 µm).
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the α-peptide with C8 fatty acid (S11) showed no activity even at higher concentration. These
results suggest that E-vinylogous hybrid lipopeptides can be used as potential antimicrobial
candidates. In addition, the antimicrobial activity further depends on the side-chains of amino
acids as well as length of the fatty acids. We find it difficult to correlate the disparity in the
antimicrobial activity of these hybrid lipopeptides with their hydrophobicity. However, the
hybrid lipopeptide with higher hydrophobicity S7 or lower hydrophobicity S2 and S6 peptides
displayed moderate or no activity against the microorganisms. All active peptides showing
similar MIC values promoted us to probe the plausible mechanism of action of these
lipopeptides.
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The atomic force microscopy images of Escherichia coli (E. coli) and fungus Candida
albicans (C.albicans) before and after the treatment of S1 lipopeptide are shown in Figure 1. The
change in the morphology of both the organisms clearly indicates that the possible action of
these peptides is through the disruption of cell membrane. Similar results were also observed for
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Figure 2: A) Peptide induced leakage of β-galactosidase from Escherichia coli Top10
(Invitrogen) cells. The graph represents relative amount of β-galactosidase present in the medium
after treating with the peptides (20 ꢀg/ml). Error bars are based on triplicate experiments for each
peptide. B) Hemolytic activity of hybrid lipopeptides with 4% hRBCs. Curves were fitted based
on the average of two independent replicates.
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other peptides (data not shown). Though the cell wall components are different for bacteria and
fungi, the mode of action of these peptides indeed appears to be the perturbation of the cell
membrane. We further validate the ability of cell membrane disruption by the lipopeptides using
FITC (MW 389.4 Da) uptake assay using E.coli.22 Since the entry of FITC requires significant
membrane damage, the cell membrane disruption was studied using S3 and S8. The fluorescent
image of E.coli before and after the treatment of the lipopeptides is shown in Figure 1. No
fluorescence was observed in E.coli cells in the absence of hybrid peptides. The observed
fluorescence behavior of the cells suggests that lipopeptides render E. coli cells permeable to
FITC.
Further, to understand the mechanism of the action of these peptides on the inner cell
membrane, we studied the enzyme β-galactosidase leakage assay 23 using peptides S1, S3 and S8
on E.coli (pUC19 plasmid transformed Top10 (Invitrogen) competent cells). The leakage of β-
galactosidase (MW≈116kDa) from E.coli was used as an indicator of the disruption of the
cytoplasmic membrane. β-Galactosidase release was measured using the initial velocity of the
reaction by standard fluorogenic substrate. Results reveal very intriguing information regarding
the action of lipopeptides (Figure 2A). No reaction of β-galactosidase with the substrate
observed in the case S1 suggests that the peptide probably disrupts the outer cell membrane by
binding through the negatively charged lipopolysaccharides. In contrast, S3 and S8 displayed
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equal amount of fluorescence intensity indicating the release of β-galactosidase upon the
treatment of lipopeptides. This experiment suggests that these lipopeptides act on both outer and
inner membrane and facilitate the release of β-galactosidase. Though the peptides S1, S3 and S8
display nearly similar MIC values, these results suggest their different mode of action.
The encouraging results of the antimicrobial activities of these peptides inspired us to
subject them for hemolytic activity. Results of the hemolytic activities are shown in Figure 2B.
Out of all the peptides tested, S7 and S3 (both highly hydrophobic) showed the highest hemolytic
activity followed by S8. In contrast the highly potent antimicrobial peptide S1 displayed
comparatively lesser hemolytic activity. Other peptides which show poor activity against the
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Figure 3: FE-SEM images of S1 (A), S5 (B), S3 (C, D) and S8 (E, F) hybrid lipopeptides.
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bacteria and fungi have shown lesser hemolytic activity. Analysis of these results reveals that
peptides containing aromatic side chains have shown greater hemolytic activity than those
containing aliphatic side chain amino acids. Even though S1 is more hydrophobic than S8, it
displayed lower hemolytic activity. Except S3 and S7 all other peptides which showed
antimicrobial activities are non-hemolytic at their MICs. The control peptides S10 and S11
which are inactive in the antimicrobial studies displayed no hemolytic activities.
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The disparity in the antimicrobial activities upon addition or truncation of N-terminal
fatty acid between S8 and S7, also the different mode of action between S1, S3 and S8 promoted
us to speculate the role of self-organization of these lipopeptides. To address this possibility and
gain further knowledge regarding their mode of action we analyzed the self-assembly patterns of
lipopeptides in both solution and on the surface.
Dynamic light scattering (DLS) experiments confirmed that all lipopeptides undergo a
spontaneous self association to give nano-assemblies in solution. Mono dispersed self-assemblies
of peptides in DLS are shown in supporting information. Intriguingly, the size of mono dispersed
self-assemblies are ranging from 124 to 950 nm. Lipopeptides S1, S3, S7 and S8 displayed
average size of the nano-assemblies 164, 220, 615 and 950 nm, respectively. Interestingly, the
zeta-potential values of these self-assembled lipopeptides were found to be 54 3, 55 1, 47
3
and 43 4 respectively. Irrespective to the size of assembly, these peptides showed excellent
surface potential charges. In order to understand the pattern of self-association, we subjected
them to FE-SEM (Field Emission Scanning Electron Microscope) analysis. The FE-SEM images
of S1, S3, S5, and S8 are shown in Figure 3. Peptides S1 and S5 containing dehydro alanine
(dgA) adopted nanofibers type of morphology similar to the amyloid fibrils.24 Peptide S3 and S8
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with aromatic amino acid side chains underwent diffusion limited aggregation, however, with a
different morphology. Peptide S3 was found to be composed of fractals with a needle shaped
nano-structures. Ironically, S8 displayed spectacular fractal trees similar to the polyelectrolyte
complexes and peptide containing aromatic amino acids.25-27 S7 display fractals different than
that of S8. Though the higher concentrations are required for the SEM analysis, however, the
different morphologies observed in the self-assembly of these hybrid lipopeptides partially
explains the disparity in their biological activity. Even though electric potential of self-
assemblies are very similar, substantial change in the self-assembly pattern is observed between
the peptides S1 and S3. The robust growth of fractals and needle shaped nano-structures from S3
and S8 may be influencing their antimicrobial and high hemolytic activities. As S7 showed poor
antimicrobial activity, we anticipate that this peptide might have adopted different mechanism
from that of S3 and S8. The low hydrophobicity and low surface charges of S2, S4 and S6 is
reflected in their poor antimicrobial and lower hemolytic activity values.
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Conclusion
In conclusion, we demonstrated the broad spectrum antimicrobial properties of short hybrid
lipopeptides composed α- and E-vinylogous amino acids. Due to their geometrical restriction of
the backbone double bonds, these short hybrid peptides with C12 and C8 N-terminal fatty acids
displayed better activity than the α- as well as β-lipopeptides containing C16 fatty acid at the N-
terminal.12c, 12d The different nano-structures and assemblies of these lipopeptides may be
influencing the disparity in their biological activities. The hybrid peptides with moderate
hydrophobicity and high electrical potential of self-assemblies showed better antimicrobial and
low hemolytic activities (S1). These peptides may possibly bind to the negatively charged outer
membrane of the microorganisms and affecting the transmembrane electrical potential. The
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robust growth of nano needles from the hybrid peptides containing aromatic amino acids may be
responsible for the disruption of both outer and inner membranes. The antimicrobial activity, the
mode of action and the self-assembly patterns of these short E-vinylogous hybrid lipopeptides,
particularly S1 and S8 provide a unique opportunity to further design potent antimicrobial
candidates as well as to explore their utility as smart biomaterials.
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Experimental section
General. All amino acids, DCC, PPh3 were purchased from Aldrich. The solvents THF, DCM,
Toluene were purchased from Merck. Ethyl bromoacetate, di-tert-butyl dicarbonate , Fmoc-OSu
were purchased from Spectrochem and used without further purification. Column
chromatography was performed on Merck silica gel (100-200 mesh). Peptides were filtered and
purified through reverse phase HPLC on C18 column using MeOH/H2O gradient. 1H NMR
13
spectra were recorded on Jeol 400 MHz and C NMR on100 MHz spectrometer using residual
solvent as internal standard (DMSO-d6 δH, 2.5 ppm, δc 39.51 ppm) The chemical shifts (δ) were
reported in ppm and coupling constant (Ј) in Hz. Mass spectra were obtained from the MALDI-
TOF/TOF. All HPLC purified peptides with the purity > 95% were used for the biological
activities.
Synthesis of N-Fmoc-protected α, β-unsaturated γ-amino acids: N-Fmoc-protected α,β-
unsaturated γ-amino acids were synthesized using reported procedure.19, 20 Briefly, the Boc-
amino aldehyde (10 mmol) and ylide (15 mmol, 5.22g) were dissolved in 30 mL of dry THF at
room temperature and stirred for about 5hrs. Completion of reaction was monitored by TLC.
The reaction mixture was concentrated under reduced pressure to give crude product, which was
further purified on silica gel column chromatography using EtOAc/Pet-ether (1:5) to get pure
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ethyl ester of Boc-protected α, β-unsaturated γ-amino acid. To a solution of ethyl ester of Boc-α,
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β-unsaturated γ- amino acid (2 mmol) in 5 mL of ethanol, mL of 1N NaOH was added dropwise.
After completion of the reaction (~30 min), ethanol was evaporated and the residue was acidified
using 10 mL of 5% HCl (5% volume in water) at cold conditions. The product was extracted
with ethyl acetate (3× 40 mL). Combined organic layer was washed with brine (30 mL) and
dried over anhydrous Na2SO4. The solvent was concentrated under reduced pressure to give Boc-
α, β-unsaturated γ-amino acid as gummy product in a quantitative yield.
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The Boc-α, β-unsaturated γ-amino acid (1 mmol) was dissolved in 5 mL of DCM and
cooled to 0 °C in ice bath followed by addition of 5 mL neat TFA to the reaction mixture. After
30 min, TFA was removed from reaction mixture under vacuum. Residue was dissolved in 15
mL of water (15 mL) and the pH was adjusted to ~9-10 by the slow addition of solid Na2CO3.
The solution of Fmoc-OSu (1 mmol) in 10 mL of THF was added slowly to the reaction mixture
and stirred overnight at room temperature. After completion of the reaction, the reaction mixture
was acidified with 20 mL of 20% HCl (20% volume in water) in cold condition. Product was
extracted with ethyl acetate (3× 50 mL). Combined organic layer was washed with brine (30 mL)
and dried over anhydrous Na2SO4. The solvent was concentrated under reduced pressure to give
gummy product, which was recrystallized using EtOAc/Pet-ether. The pure solid Fmoc- α, β-
unsaturated γ-amino acid was subjected for SPPS.
SPPS peptide synthesis and Purification
Peptides were synthesized at 0.25 mmol scales on Knorr amide resin using standard Fmoc-
chemistry. HBTU/HOBt was used as coupling reagents. The coupling reactions were monitored
by Kaiser Test. After completion of the synthesis, peptides were cleaved from the resin using
cocktail mixture of TFA: water: thioanisole (98:1:1). After cleavage, the resin was filtered and
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washed with TFA. The cleavage mixture was then evaporated under reduced pressure to give
gummy product and purified through reverse phase HPLC on C18 column using MeOH/H2O
gradient. The purity of the peptides was further confirmed by analytical C18 column with the
same gradient system.
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Antibacterial activity:
The bacteria used in these experiments were collected from National Collection of Industrial
Microorganisms (NCIM) Escherichia coli (NCIM 2065), Escherichia coli K12 (NCIM2563),
Klebsiella pneumoniae (NCIM 2957), Pseudomonas aeruginosa (NCIM 5029), Salmonella
typhimurium (NCIM 2501), and Staphylococcus aureus (NCIM 5021).
The antibacterial activities of lipopeptides were carried out in 96-well microtiter plate
using broth dilution method. The bacterial cultures were grown over night at 37 °C and serially
diluted to a concentration of 106 colony forming units/mL with sterile MHB (Mueller-Hinton
broth) medium. Two-fold serial dilutions with MHB medium were performed triplicate for each
lipopeptide in a sterile 96-well plate to a final volume of 50 ꢀl in each well. The final volume
was made to 100 ꢀl by adding an aliquot of 50 ꢀl bacterial suspension to each well, and the plate
was incubated at 37 °C for 18-20 h. Controls were done without peptide and the MIC of peptide
was defined as the lowest concentration of the peptide required for the complete inhibition or
killing the bacterial inoculum. The growth inhibition was monitored by measuring the
absorbance at 492 nm.12b The MIC values reported were reproducible between three
independent triplicates.
Antifungal activity:
The antifungal activity of lipopeptides was performed using 96 well plate methods (TPP brand)
as follows: The 50 ꢀl of water containing peptide was serially two fold diluted in PDB (Potato
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dextrose broth, Hi-Media) medium. The final volume was adjusted to 100 ꢀl by adding 50 ꢀl of
yeast suspension containing a concentration of 2×103 colony forming units/ml. A culture without
peptide was also grown parallel as a control. Inoculated plates were incubated at 25 °C for 48-72
h and the growth inhibition was determined by measuring the absorbance at 620 nm. The yeasts
used were Candida albicans (NCIM 3471), Candida glabrata (NCIM 3237), Cryptococcus
neoformans (NCIM 3542), and Saccharomyces cerevisiae (NCIM 3454). Experiments were
carried out in triplicate. The MIC was determined as the lowest concentration of peptide required
for the complete growth inhibition of each organism after 48-72 h incubation.
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Hemolysis assay:
Fresh hRBCs (Human Red Blood Cells) were collected with EDTA. The hRBC were washed
with Tris buffered saline (10 mM Tris, 150 mM NaCl, and pH 7.2) four times and diluted to a
final concentration of 4% v/v. The assay was performed in sterile 96-well plate in a final volume
of 100 µl as follows: aliquot of 50 µl of hRBC suspension were added to 50 µl (100 µg/ml) of
two-fold serial diluted lipopeptide in Tris buffer. The plate was incubated at 37 ºC for 1 h and
then centrifuged for 15 min at 3000 rpm. The supernatant (50 µl) from each well was transferred
to a fresh 96-well plate containing 50 µl of water and release of hemoglobin was monitored by
measuring the absorbance at 540 nm. The experiments were performed in duplicate and the
hRBCs suspensions with Tris buffer and 1% Triton-X was consisted as negative and positive
controls. Percentage of hemolysis was defined as (A-AN)/ (AP-AN) × 100, where A is the
absorbance of test well, AN is the absorbance of the negative control and AP is the absorbance of
the positive control (100% hemolysis).
Atomic Force Microscopy Imaging:
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A culture of E. coli K12 (NCIM 2563) were grown in MHB medium and Candida albicans
(NCIM 3471) were grown in potato dextrose broth. The cells were centrifuged and resuspended
in deionized distilled water. The bacteria were incubated with 50 ꢀM of S1 and S8 lipopeptide at
37 °C for 1 h. After incubation, 50 ꢀl droplets of each sample were applied onto Poly-L-Lysine
(PLL) pretreated glass slides and allowed to dry for 30 min at 25 °C. For control, cells without
peptide were similarly prepared and imaged within 2 h of being resuspended in distilled water.
For AFM analysis of peptides the samples were applied on the freshly cleaved mica surface and
dried for 10 min at 25 °C. The AFM images were acquired using a JPK Nano Wizard (Berlin,
Germany) mounted on a Zeiss Axiovert 200 inverted microscope. Measurements were carried
out in Intermittent contact (IC) mode using Tap 300 E-G (Cr/Pt Conductive coating) cantilevers
from silicon AFM probes (Spring constant, k = 40 N/m, Resonance Frequency around 300 KHz,
Radius <10 nm). Height and size information were acquired using JPK data processing software.
FITC uptake assay:
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To analyze the membrane permeabilization of lipopeptides impermeant fluorescent probe
Fluorescein isothiocyanate (FITC) was used. The assay was done as previously described (2)
Culture of E.coli K12 (NCIM 2563) were grown to absorbance OD 0.6 in MHB medium. The
bacteria were washed and suspended (2×107 cfu /ml) in 10 mM sodium phosphate buffer (pH
7.4). The bacterial suspension was incubated with 50 ꢀM of S1, S3 and S8 lipopeptides at 37 °C
for 60 min. The microorganisms were immobilized on Poly-L-Lysine (PLL) coated glass slide
for 50 min at 30 °C, followed by addition of 100 µl FITC (10 µg/ml) suspended in the same
buffer. The slides were washed with 10 mM sodium phosphate buffer (pH 7.4) and viewed under
Zeiss Axiovert apotome microscope equipped with an AxioCam camera. The images were
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processed with Axiovision 4.7 software. The samples with peptide solvents were taken as a
control.
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Beta-galactosidase leakage from E.coli cells:
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The PUC19 plasmid containing LacZ reporter gene was transformed in to the chemically
competent Escherichia coli (TOP10, Invitrogen) cells. E.coli cells were grown in Luria Bertani
broth with 100 µg/ml of ampicillin at 37 °C to an absorbance 0.6 at 660 nm. The cells were
centrifuged and washed with fresh medium for three times to remove extracellular β-
galactosidase. The 10 µl of water containing peptide of stock solution (200 µg/ml) were added in
a sterile 96-well plate. The final volume was adjusted to 100 µl by adding an aliquot of 90 µl
bacterial suspension to each well, and the plate was incubated at 37 °C for 1 h. After 1 h, the
plate was centrifuged at 4000 rpm for 10 min to remove cell debris and 80 µl of supernatant was
removed and separated in a fresh well. An aliquot 20 µl (0.4 mg/ml) of fluorescent indicator 4-
Methylumbelliferyl-β-galactosidase (MUG, Sigma) was added to the well and β-galactosidase
release was monitored for 1 h. Initial velocities of enzyme reaction were taken from the linear
plot of fluorescence versus time. The samples with water were taken as a blank.
Scanning Electron Microscopy imaging:
The peptides were dissolved in water of concentration (50 µg/ml) was spotted on silicon wafers
and air dried for 45 min. The samples were sputter coated with gold and observed in Zeiss FE-
SEM. The sample was spotted on silicon wafers and recorded on FESEM. The images were
converted to binary file and fractal dimension were analyzed by using fractal box count of Image
J software.
Dynamic light scattering:
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For DLS measurements, lipopeptides of concentration 1mg/ml were dissolved in water and by
using poly propylene glass cuvette measurements were performed in Zeta sizer (Malvern
Instrument). The average count rate at each time point was recorded.
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Author Information
* To whom correspondence should be addressed. Tel: +91-20-2590 8075; Fax: +91-20-
25908186; E-mail: hn.gopi@iiserpune.ac.in; hv.thulasiram@ncl.res.in
Abbreviations Used
AMP’s, Antimicrobial peptides; MIC, Minimum inhibitory concentration; FITC, Fluorescein
isothiocyanate; hRBC’s, Human red blood cells; dgX, α, β- dehydro γ-amino acid (ex. dgA, α,β-
dehydro γ-alanine); FESEM, Field emission scanning electron microscopy; AFM, Atomic force
microscopy; DLS, Dynamic light scattering, DCC, N, N’-dicyclohexylcarbodiimide, PPh3,
Triphenylphosphine; Fmoc-OSu, 9-fluorenylmethylsuccinimidyl carbonate; HBTU, N,N,N’,N’-
Tetramethyl-O -(1H-benzotriazol-1-yl)-uronium hexafluorophoshate; NCIM, Nation collection
of industrial microorganisms; MHB, Mueller Hinton broth; MALDI-TOF, Matrix assisted laser
desorption ionisation-time of flight; HOBt, N-Hydroxybenzotriazole.
Acknowledgements
This work was supported by Department of Science and Technology, Govt. of India and IISER-
Pune. SNB and PLS are thankful to UGC and CSIR, respectively for research fellowship. HVT
acknowledges CSIR network projects (BSC0124 and CSC0106) for financial support.
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Supporting Information Available: 1H NMR and 13C NMR for all E-vinylogous amino acids,
mass spectra for all compounds, images of AFM and FE-SEM, DLS histograms and HPLC
profiles. This material is available free of charge via the Internet at htpp:// pubs.acs.org.
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