Small molecular antibacterial peptoid mimics: The simpler the better!

Article abstract of DOI:10.1021/jm401680a

The emergence of multidrug resistant bacteria compounded by the depleting arsenal of antibiotics has accelerated efforts toward development of antibiotics with novel mechanisms of action. In this report, we present a series of small molecular antibacterial peptoid mimics which exhibit high in vitro potency against a variety of Gram-positive and Gram-negative bacteria, including drug-resistant species such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. The highlight of these compounds is their superior activity against the major nosocomial pathogen Pseudomonas aeruginosa. Nontoxic toward mammalian cells, these rapidly bactericidal compounds primarily act by permeabilization and depolarization of bacterial membrane. Synthetically simple and selectively antibacterial, these compounds can be developed into a newer class of therapeutic agents against multidrug resistant bacterial species.

Full text of DOI:10.1021/jm401680a

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Article
Small Molecular Antibacterial Peptoid Mimics: the simpler the better!
Chandradhish Ghosh, Goutham B Manjunath, Padma Akkapeddi, Venkateswarlu
Yarlagadda, Jiaul Hoque, Divakara S.S.M. Uppu, Mohini Mohan Konai, and Jayanta Haldar
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm401680a • Publication Date (Web): 30 Jan 2014
Downloaded from http://pubs.acs.org on January 31, 2014
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Small Molecular Antibacterial Peptoid Mimics: the
simpler the better!
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Chandradhish Ghosh, Goutham B. Manjunath, Padma Akkapeddi, Venkateswarlu Yarlagadda,
Jiaul Hoque, Divakara S. S. M. Uppu, Mohini M. Konai and Jayanta Haldar*
Chemical Biology and Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru
Centre for Advanced Scientific Research (JNCASR), Jakkur, Bengaluru 5600064, Karnataka,
India.
ABSTRACT: The emergence of multiꢀdrug resistant bacteria compounded by depleting arsenal
of antibiotics has accelerated efforts towards development of antibiotics with novel mechanism
of action. In this report we present a series of small molecular antibacterial peptoid mimics
which exhibit high inꢀvitro potency against a variety of Gramꢀpositive and Gramꢀnegative
bacteria, including drugꢀresistant species such as methicillinꢀresistant Staphylococcus aureus and
vancomycinꢀresistant Enterococcus faecium. Highlight of these compounds is their superior
activity against the major nosocomial pathogen Pseudomonas aeruginosa. Nonꢀtoxic towards
mammalian cells, these rapidly bactericidal compounds primarily act by permeabilization and
depolarization of bacterial membrane. Synthetically simple, selectively antibacterial these
compounds can be developed into a newer class of therapeutic agents against multiꢀdrug resistant
bacterial species.
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INTRODUCTION
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Bacterial resistance to conventional antibiotics is one of the most serious problems facing world
health today. Dearth of drugs against drugꢀresistant Gram positive bacteria such as methicillinꢀ
resistant Staphylococcus aureus (MRSA) and vancomycinꢀresistant Enterococci (VRE) as well
as multiꢀdrug resistant Gramꢀnegative bacteria such as Pseudomonas aeruginosa are the most
important cause of concern.1ꢀ3 In the recent past only cationic antimicrobial peptides (AMPs)
have shown some promise as potential antibiotics and several of them are undergoing clinical
trials.4 AMPs are sentinels of innate immune system of most species and are usually the first line
of defence against any infection.5 While most of the conventional antibiotics act by targeting
intracellular organelles of bacteria, AMPs and their mimics are known to act primarily by
causing lysis of the bacterial cell membrane.6ꢀ9 Consequently, unlike in the case of conventional
antibiotics, where even point mutations can render them inactive, bacteria find it difficult to
develop resistance against antimicrobial peptides.7
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It is believed that AMPs display amphipathic conformation, i.e. facial segregation of positive
charges and hydrophobic moieties, while interacting with bacterial membranes.5, 6 AMPs act
selectively towards bacterial cells over mammalian cells, which can be attributed to the
differences of the lipid components of the cell membranes of bacterial and mammalian cells.
While bacterial membrane is largely composed of anionic lipids, the mammalian cell membrane
is mainly composed of uncharged lipids at neutral pH.10 Despite several advantages, no AMP has
yet been approved for clinical use.4 AMPs are mainly limited by their high inꢀvivo toxicity,
lability towards proteases and their high cost of manufacture. Consequently several groups
around the world have tried to develop strategies to counter the problems faced by AMPs. Most
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of these strategies, such as αꢀpeptides,11, 12 βꢀpeptides,13, 14 peptoids,15 antimicrobial polymers,16ꢀ
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oligoacyllysines,23 oligoureas,24 αꢀAA peptides,25 cationic amphiphiles26 keep the
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physiological properties of AMPs constant in their design, and try to address the problems
associated with their synthesis and degradation. Although macromolecular mimics of AMPs are
well known and abundant,27 development of small molecular mimics of AMPs has been seldom
attempted. Polymedix’s aryl amide foldamers28, 29 and Lytix biopharma’s LTX 10930, 31 are
successful examples of small molecular mimics of antimicrobial peptides. Some other examples
include the binaphthyl based dicationic peptoids,32 β2,2ꢀamino acid derivatives33 and those based
on aryl scaffolds.34, 35 Although most of these compounds involve designs that are extremely
efficient in their purpose, their major limitations lie in the complexity of their structures, the
number of steps involved in their synthesis, the cost of their manufacture and the presence of
peptide bonds which are labile to protease degradation.
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Herein we report, for the first time, the design and development of small molecular
antibacterial peptoid mimics involving only three synthetic steps. In our minimalistic design, the
two positive charges were contributed by an Lꢀlysine moiety; hydrophobicity was brought about
by an aromatic core (methyl anthracene, methyl naphthalene or methyl benzene) and an alkyl
chain (Figure 1). The hydrophobicity of the lipophilic alkyl chain and the bulky aromatic core
was varied systematically to understand the role of these parameters towards selective
antibacterial activity.
RESULTS
Synthesis. To synthesize the compounds, first aromatic aldehydes (10ꢀChloroꢀ9ꢀ
Anthracenaldehyde, naphthaldehyde and benzaldehyde) were reacted with alkyl amines to form
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Schiff’s bases, followed by reduction with sodium borohydride, to give secondary amines
(Scheme 1). Then secondary amines were coupled to BocꢀLys(Boc)ꢀOH using HBTU coupling
and finally the Boc groups were deprotected using trifluoroacetic acid to obtain the required
compounds (Figure 1); methyl anthracene core (Series I, compounds 1-5), methyl naphthalene
core (Series II, compounds 6-10) and methyl benzene core (Series III, compounds 11-16). The
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final compounds were purified by HPLC to more than 95% purity and characterized by Hꢀ
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NMR, CꢀNMR, IR and HRꢀMS. (Supporting information, Figures S1ꢀS64). Two significant
features of these compounds are that there is no imposed structural rigidity in their design and
that they include an Nꢀdisubstituted or tertiary amide bond.
Antibacterial activity. The antibacterial efficacy (Table 1) of these compounds was evaluated
against different Gramꢀpositive (Staphylococcus aureus and Enterococcus faecium) and Gramꢀ
negative (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) bacteria
including MRSA and VRE by determination of their minimum inhibitory concentrations (MICs;
the concentration of compounds required to completely inhibit the growth of bacteria).
In general all the compounds of series I (1-5) have MICs comparable to MSIꢀ78 (an AMP
currently undergoing PhaseꢀIII clinical trials as a topical antibiotic).4, 36 The shortest alkyl chain
derivative 1 (ethyl analogue) was found to be moderately active against both Gramꢀpositive and
Gramꢀnegative bacteria with MICs of 11 μg mLꢀ1 and 25 μg mLꢀ1 against S. aureus and E. coli
respectively. Increasing the long chain to butyl improves antibacterial activity by twoꢀfold as was
observed in 2. Further increase in alkyl chain yielded the hexyl analogue 3 which displayed
improved MICs of 2.4 μg mLꢀ1 and 3.5 μg mLꢀ1 against S. aureus and E. coli respectively. The
antibacterial activity was found to increase even further on increasing the long chain to octyl; 4
displayed MICs of 2.2 μg mLꢀ1 and 2.9 μg mLꢀ1 against S. aureus and E. coli respectively.
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However, further increase in alkyl chain compromised the activity of the compounds as was
observed for the decyl analogue 5. The octyl analogue 4 emerged as the most potent compound
in the series whose activity could be compared to vancomycin (MIC of 0.9 μg mLꢀ1 against S.
aureus) and colistin (MIC of 0.4 μg mLꢀ1 against E. coli). Compounds 3 and 4 were around 5
times more active against E. coli compared to MSIꢀ78 which emphasizes the effectiveness of the
design. The structureꢀactivityꢀrelationship (SAR) study indicates that if the anthracene core was
kept constant, the optimum chain length lied between butyl to octyl (Figure 2A).
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All the compounds were active against E. faecium, in fact compound 1, the least effective
compound of the series also displayed moderate activity (MIC of 13.6 μg mLꢀ1) against the
bacterium. Compound 4 with MIC of 2.5 μg mLꢀ1 was again the most potent compound while
vancomycin had MIC of 0.87 μg mLꢀ1.
The highlight of these set of compounds, however, is their superior activity towards
opportunistic human pathogen P. aeruginosa which, is a leading cause of hospitalꢀacquired
infections and is known to show resistance to almost all clinically approved antibiotics.37 All the
compounds of Series I was extremely active against P. aeruginosa. Against this bacterium, the
minimum MIC value was displayed by 3 (MIC = 1.6 μg mLꢀ1). Compound 2 also showed
excellent activity towards P. aeruginosa with MIC of 1.9 μg mLꢀ1.
In order to elucidate the role of the hydrophobic core towards antibacterial activity we
synthesized series II where the aromatic core is methyl naphthalene (6-10). It was hypothesized
that decreasing the bulkiness from anthracene to naphthalene might yield a more selective
antibacterial agent. Unlike in the case of series I, the small chain analogues of series II, e.g.
compound 6, was devoid of any activity. Compound 7, the hexyl analogue, was moderately
active and showed potency against all the drug sensitive bacteria, especially P. aeruginosa (MIC
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= 11 ꢁg mLꢀ1). The octyl analogue, 8 exhibited potent activity against all pathogens comparable
to its anthracenyl counterpart, 4, for example MICs against S. aureus and E. coli were 6.3 μg mLꢀ
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and 5 μg mLꢀ1 respectively. Its activity towards P. aeruginosa was also commendable (MIC =
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5.4 μg mLꢀ1). Compounds 9 and 10 were the most active compounds in the series (Table 1) with
superior activity against all the drug sensitive bacteria (MIC ranged from 1.6 to 4 ꢁg mLꢀ1).
Activity of 10 towards E. faecium must be mentioned here; its MIC of 1.6 ꢁg mLꢀ1 is comparable
to that of vancomycin (MIC = 0.87 ꢁg mLꢀ1). No compound in series I was active at this low
concentration.
In order to see the effect of a further decrease of aromatic core, series III (where the aromatic
core was methyl benzene) was prepared. In this series of compounds (11-16), however, no
significant activity was observed till the octyl analogue 13. The decyl analogue, 14, showed
potent activity against all the pathogens. With MICs of 5.7 ꢁg mLꢀ1 and 6.5 ꢁg mLꢀ1 against S.
aureus and E. coli, its activity was comparable to 8. Compounds 15 and 16 had comparable
antibacterial activity against all the tested pathogens (Table 1). Against S. aureus, 15 was the
most potent compound in the series with MIC of 2.7 ꢁg mLꢀ1. Against all other pathogens,
activity of 16 was slightly better, e.g. 16 displayed an MIC of 2 ꢁg mLꢀ1 compared to that of 2.6
ꢁg mLꢀ1 displayed by 15 against E. faecium.
To emphasize on the efficacy of the active compounds, their activity was tested against drug
resistant superbugs such as VRE, MRSA and βꢀlactam resistant K. pneumonia. Most of these
compounds possessed remarkable activity against VRE (Table 1). Compounds 4, 9 and 16 were
the most potent compounds with MIC values of 3 ꢁg mLꢀ1, 1.6 ꢁg mLꢀ1 and 2.5 ꢁg mLꢀ1
respectively which were far superior compared to the MIC of MSIꢀ78 (8 ꢁg mLꢀ1) while
vancomycin remained inactive till a concentration of 100 ꢁg mLꢀ1. Compounds 4, 9 and 16 were
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also potent against MRSA with MICs around 2.5 ꢁg mLꢀ1 for all. In comparison, MSIꢀ78 was
active only at 16ꢀ32 ꢁg mLꢀ1. Other compounds of series I were also active against MRSA but to
a lower extent. Compound 4 was also potent against βꢀlactam resistant K. pneumonia (MIC = 4.3
ꢁg mLꢀ1). Compounds 10 and 16 were the most active compounds in their respective series with
MICs of 4 ꢁg mLꢀ1 each whereas colistin showed MIC of 1.2 ꢁg mLꢀ1.
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Bactericidal kinetics. Inꢀvitro timeꢀkill assay with compound 3 against MRSA revealed the
rapid bactericidal properties of the compounds. Kinetics of killing increased with increase in
concentration and ~6 log10 (CFU/mL) reduction in the number of viable cells were observed in
the first hour of incubation at a concentration of 6×MIC. At 3×MIC, however, bactericidal
activity was observed in the sixth hour (Figure 2B). 7 too showed rapid bactericidal action at
2×MIC against S. aureus (Figure S65) establishing the efficacy of these compounds.
Hemolytic activity. Toxicity of these compounds towards mammalian cells was evaluated by
their ability to lyse human erythrocytes and represented as their HC50 values (i.e. the
concentration at which 50% of the red blood cells are lysed). HC50 of the compounds ranged
from 45 ꢁg mLꢀ1 to >1000 ꢁg mLꢀ1. Hemolytic toxicity studies reveal that these compounds are
selectively toxic towards bacterial cells. In general, the HC50 values of these compounds were
comparable to that of MSIꢀ78 (HC50 = 120 ꢁg mLꢀ1). Compound 7, with a HC50 value of >500
ꢁg mLꢀ1, has minimal toxicity towards human erythrocytes.
Antibacterial activity in plasma. One of the major disadvantages associated with antimicrobial
peptides is that they are prone to protease degradation. This is also reflected in the subsequent
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loss of activity in presence of blood plasma.23 Although the MIC of compound 7 (the least
hemolytic and active compound in the series II) against S. aureus in 50% blood plasma was 30
ꢁg mLꢀ1, no loss of activity was observed in physiologically relevant time frame of 3 hours
(Figure S66).
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Enzyme stability. Trypsin and chymotrypsin are well known proteases, which are omnipresent
in vertebrates. When the model compound, 7 was incubated with trypsin it was found to be
resistant to degradation up to maximum tested time 24 hours (Figure S67) confirming that this
type of antibacterial agent was not a substrate for protease.
Mechanism of action. Investigation into the mechanism of action of the most selective
compounds 3 and 7 (model compounds) using various spectroscopic techniques revealed that
these compounds primarily act by targeting the bacterial cell membrane. For example,
experiments with the membraneꢀpotential sensitive dye DiSC3(5) showed that both 3 (Figure 2C)
and 7 (Figure S68) rapidly depolarize the membrane of S. aureus cells. In fact, the membranes of
Gramꢀnegative species P. aeruginosa and E. coli were also depolarized by 3 and 7 respectively
(Figure 2D and Figure S69). Bacterial cytoplasmic membrane permeabilization was studied
using the fluorescent probe propidium iodide. Although both 3 and 7 were able to permeabilize
the S. aureus membrane (Figure 2E and Figure S70), similar effect was not observed against
Gramꢀnegative cells. However, experiments, using fluorescent probe Nꢀphenylnaphthylamine,
with 7 showed that it could cause permeabilization of Gramꢀnegative outer membrane at MIC
(Figure 2F). The same experiment could not be performed with 3 as it absorbs at the same
frequency as the dye. The effect of the compound on the bacterial membrane was also studied
using fluorescence microscopy and scanning electron microscopy. Fluorescent microscopic
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studies, using the “liveꢀdead stain”38 confirmed that on treatment with 3 and 7, bacterial cell
membranes were indeed compromised (Figure 3AꢀB and Figure S71). FESEM images (Figure
3CꢀD and Figure S72) reveal that the membranes of treated E. coli cells were disrupted and
several dead bacteria merged with each other on treatment with 3 and 7 respectively. Formation
of blebs and holes were observed on treated S. aureus cells (Figure S73 and S74). These images
are a visual proof of the membrane damaging properties of the compounds.
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DISCUSSION
In pursuit of novel antibacterial agents, much attention has been focused on developing synthetic
mimics of antimicrobial peptides. In this report we have created simple small molecular
antibacterial peptoid mimics where an aryl group, an alkyl group and a lysine moiety has been
assembled through a tertiary amide linkage. Although some of the successful designs in the field
focus on imposing rigidity into their systems,28, 29, 39 our design is devoid of any imposed rigidity.
Other than conformational rigidity, the minimum required parameters are two hydrophobic bulk
units and two cationic charges.32, 33 Although all the existing strategies mentioned above have
effectively studied the role of amphiphilicity through subtle variation of hydrophobicity and
cationic charges, we studied in our structure activity relationship (SAR), the role of both
aromatic moieties and alkyl chains together. We sought out to systematically study the effect of
aromatic core individually (keeping the alkyl chain length constant) as well as the effect of long
chains individually (keeping the aromatic core constant). Needless to say that in both the cases
the cationic charge was kept constant at two (brought about by a lysine moiety).
Keeping the aromatic ring constant, the alkyl chain has been varied in all the three series. In
case of series I a parabolic pattern of chain length dependent activity was observed. In both the
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other cases, the activity was found to increase with increase in long chain and reached a steady
state at compounds 10 and 16 respectively. Further increase in long chain would take away the
emphasis from the aromatic core and the long chain would have had more of an impact on the
activity.
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If the alkyl chain is kept constant, and the aromatic core is varied, a somewhat different SAR is
obtained. If we consider the hexyl analogues, 3 is a potent antibacterial agent. On moving to
naphthalene core, compound 7, moderate activity is retained, but the benzyl analogue 12 is
devoid of activity. From this study it is clear that the effect of the aromatic core is dominant over
the alkyl chain towards antibacterial activity. On moving to the octyl analogues, we find a
similar trend. Activity decreases as we move from anthracene to naphthalene to benzene.
However the activity of compound 8 is comparable to that of compound 4 suggesting that the
long chain has an important role to play towards activity and only aromaticity is not the
dominant factor. On moving to the decyl analogue, a somewhat different trend is observed.
While compounds 5 and 14 have comparable antibacterial activity, compound 9 is roughly twoꢀ
fold more active than both. It can be concluded that 9 has an optimum aromaticity and optimum
alkyl chain length for potent antibacterial activity. Also, it becomes clear that the decyl long
chain dominates over aromaticity towards antibacterial activity.
There is a paucity of drugs against difficult to treat Gramꢀnegative pathogens and currently
there is a huge demand for antibacterial agents towards Gramꢀnegative pathogens. Although all
the compounds here show excellent activity against drugꢀresistant Gramꢀpositive bacteria
(MRSA and VRE), the highlight of the complete set of compounds is definitely activity against
Gramꢀnegative pathogens like E. coli, P. aeruginosa and K. pneumoniae. Against P. aeruginosa,
compound 3 has a MIC which is comparable to colistin. Compounds 9 and 16 are also extremely
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potent with comparable MIC values. This superior activity may open new avenues towards
development of novel drugs against P. aeruginosa. However, selective toxicity towards Gramꢀ
negative bacteria is an important factor to consider if clinical translation is the ultimate goal.
Taking P. aeruginosa into consideration, all the compounds show selective toxicity towards
bacteria over red blood cells. In fact 3 and 7 have a good selectivity ratio of around 50 (Table
S1). At therapeutically relevant concentrations of 3×MIC and 6×MIC, these compounds show
bactericidal activity within an hour. This effect is also a consequence of the membrane damaging
ability of the compounds. The superior selectivity of the compounds is also displayed by the fact
that even at their bactericidal concentrations, they are not hemolytic. The selectivity of the
compounds has also been displayed as their therapeutic indices (HC50/MIC) against both drugꢀ
sensitive Gramꢀpositive and Gramꢀnegative bacteria (Table S1). MIC of the compounds against
S. aureus and P. aeruginosa is a good reflection of the activity of the compounds towards most
bacteria and thus these therapeutic indices are a good indicator of the preferential damage caused
by the compounds towards bacterial cells.
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The tertiary amide bond introduces an exciting new approach towards achieving plasma
stability of the compounds. This bond not only provides the point of assembly for all the three
parts of the molecule but also adds to the abiotic nature. Antibacterial activity of the compounds
in presence of plasma does not change which, proves that the proteases present in the blood are
unable to degrade the compound (also substantiated by trypsin stability experiment) and that the
bioavailability of the compound is not hindered by interaction with any other proteins.
HPLC retention time (RT values) is an effective indicator of the overall amphipathicity of the
molecules. Antibacterial activity and hemolytic activity profiles of the compounds were found to
vary with respect to the RT values as observed in the HPLC data (Table 1). As expected, in each
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series, the hemolytic activity increases with increasing RT values. The antibacterial activity was
also found to increase with increase in RT values. For series II and series III the antibacterial
activity in general increases till compounds 9 (RT = 14.2 minutes) and 15 (RT = 14.5 minutes),
then remains almost constant with further increase in RT values, whereas in series I, antibacterial
activity increases till compound 4 (RT = 14.6 minutes) and then somewhat decreases in case of
compound 5. It is also clear that in order to have some antibacterial activity the compounds must
have RT value greater than 11 minutes.
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Studies into the mechanism of action of the compounds are an indicator of the efficacy of
design of the compounds as mimics of antimicrobial peptides. The experiments performed
indicated that unlike in the case of Gramꢀpositive bacteria, where both cytoplasmic membrane
permeabilization and depolarization could be the possible mechanism of bactericidal action of
these compounds, against Gramꢀnegative bacteria, the effect was mostly on the outer membrane
permeabilization and depolarization. Like natural AMPs and known synthetic mimics of AMPs,
cytoplasmic depolarization is the dominant mechanism of action. The fact that these compounds
can permeabilize the Gramꢀpositive cell membrane opens up the question of internal targets.
Although in case of Gramꢀnegative bacteria, only outer membrane permeabilization has been
observed, other mechanisms of action cannot be ruled out. Further studies need to be performed
to draw any sort of conclusion. However, membrane lysis does seem to be the primary mode of
action, as the compounds were found to be rapidly bactericidal. Furthermore the SEM images are
visual proof of the membrane damaging properties of the molecules.
CONCLUSION
In conclusion, this report illustrates a systematic way of creating highly potent, broadꢀspectrum
smallꢀmolecular compounds which emulate the efficiency of AMPs. Prepared from inexpensive
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starting materials in only three steps, these compounds are selectively toxic towards bacterial
cells (over mammalian cells) at very low concentrations. Various spectroscopic and microscopic
studies reveal that depolarization and disruption of bacterial cell membranes are the primary
mechanisms of their bactericidal action. Therefore, these promising compounds can be
developed into a new class of antibiotics against multiꢀdrug resistant (MDR) bacteria.
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EXPERIMENTAL SECTION
Unless otherwise mentioned all commercially available chemicals were used as supplied. All the
solvents were of reagent grade and were distilled and dried prior to use wherever required. All
final compounds were purified by reverse phase HPLC using 0.1% Trifluoroacetic acid (TFA) in
water/acetonitrile (0ꢀ100%) as mobile phase to more than 95% purity.
Synthesis
Synthesis of BocꢀLys(Boc)ꢀOH
LꢀLysine hydrochloride (5 g, 27.3 mmol) was dissolved in H2O (50 ml) and to it NaHCO3 (6.9 g,
82.1 mmol) was added and stirred. To this Diꢀtꢀbutylpyrocarbonate (Boc2O) (7.16 g, 65.5 mmol)
in 50 ml Tetrahydrofuran (THF) was added at 0ºC. Then the reaction mixture was stirred at room
temperature for 12 h. After 12 h, same amount of Boc2O (7.16 g, 65.5 mmol) was added again at
0ºC and the mixture was stirred for additional 12 h at room temperature. At the end of the
reaction, THF was removed under reduced pressure and the aqueous layer was washed with
diethyl ether to remove organic impurities. Then the aqueous layer was acidified to pH 4ꢀ5 using
citric acid solution. The aqueous layer was then extracted with Dichloromethane (DCM). The
organic layer was then washed with brine, and dried over anhydrous Na2SO4. The organic layer
was removed under reduced pressure to obtain the compound in 90% yield.
General procedure for synthesis of N-alkylaminomethylarene hydrochlorides (1a-16a)
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In a typical reaction aromatic aldehydes (2.08 mmol) and alkyl amines (2.08 mmol) were
dissolved in a 1:1 mixture of dry chloroform and methanol (20 ml) and stirred at roomꢀ
temperature (under Nitrogen atmosphere) for 6 h. The resulting clear solution was then cooled to
0°C, and to it Sodium borohydride (0.142 g, 3.75 mmol) was added. This was allowed to come to
room temperature and stirred overnight. Then the solvents were evaporated under reduced
pressure (not to dryness) and diluted with diethyl ether. To this 2N NaOH (20 ml) was added and
stirred for 15 minutes. After separation from the NaOH layer, the organic layer was subsequently
washed with water twice, brine and dried over MgSO4. The organic layer was then evaporated
under reduced pressure and the residue was dissolved minimum volume of methanol. To this 4N
HCl (3 ml) was added and instantaneous formation of precipitate was observed. The solvent was
completely removed and the precipitate was dissolved in minimum volume of ethyl acetate (a
few drops of methanol was added to dissolve completely). To this hexane was added to obtain
pure crystals of the target compound (Yield: 65ꢀ90%). These crystals were filtered, dried and
subsequently characterized using 1H NMR, and Mass spectrometry.
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General procedure for amide coupling (1b-16b)
In a typical amide coupling reaction, to a stirred solution of BocꢀLys(Boc)ꢀOH ( 0.2 g, 0.58
mmol) in 5:2 DMF/CHCl3(7 ml), was added N,NꢀDiisopropylethylamine (DIPEA, 250 ꢂL, 1.44
mmol) at 0°C. To this solution was then added HBTU (0.22 g, 0.58 mmol). This mixture was
stirred for 5 minutes at 0°C and subsequently the secondary amines (1a-16a, 0.48 mmol) were
added to it. The mixture was stirred at 0°C for 30 minutes and subsequently at RT for 24 h
typically. At the end, CHCl3wasevaporated under reduced pressure and the resulting solution was
diluted to 2 times its original volume by addition of ethyl acetate. This mixture was subsequently
washed with 0.5 M KHSO4, H2O (thrice) and brine. After passage through anhydrous Na2SO4,
the organic layer was evaporated under reduced pressure and the residue was purified using
column chromatography (only CHCl3) to obtain the product in 65%ꢀ90% yield. The purified
compound was subsequently characterized using 1H NMR, IR and Mass spectrometry.
General procedure for deprotection of Boc groups (1-16):
Typically, the BocꢀLys(Boc)ꢀNꢀalkylꢀaromatic compounds (1b-16b, 0.35 mmol) were dissolved
in DCM and subsequently CF3COOH (50% by volume) was added and stirred at RT. The
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reactions were monitored by TLC until complete removal of starting material was observed. All
the volatile components were removed, and the product is purified by reverse phase HPLC using
0.1% Trifluoroacetic acid (TFA) in water/acetonitrile (0ꢀ100%) as mobile phase to more than
95% purity. C18 column (10mm diameter, 250 mm length) and UV detector (at 270 nm
wavelength) were used. After drying the compounds in freeze drier, the compounds were
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characterized by H NMR, C NMR, IR and mass spectrometry. (All spectra are furnished in
supplementary figures). Peptoids of naphthalene have been reported to show presence of
rotamers in solution.40 These compounds (Series I and Series II) too show existence of rotamers,
as is evident from their NMR spectra (furnished in Supplementary Figures).
In-vitro Biological Assays
Microorganisms and Culture conditions. The antibacterial activity of the small molecules was
evaluated against both Gramꢀpositive (S. aureus, E. faecium, methicillin resistant S. aureus and
vancomycin resistant E. faecium) and Gramꢀnegative (E. coli, P. aeruginosa and K. pneumonia)
bacteria. E. coli was cultured in Luria Bertani broth (10 g of tryptone, 5 g of yeast extract, and 10
g of NaCl in 1000 mL of sterile distilled water (pH ꢀ7) while S. aureus, P.aeruginosa (ATCC
4676) and MRSA were grown in Yeastꢀdextrose broth (1 g of beef extract, 2 g of yeast extract, 5
g of peptone and 5 g of NaCl in 1000 mL of sterile distilled water). For E. faecium and VRE
Brain Heart Infusion broth (BHI) was used. K. pneumonia was grown in nutrient media (3 g of
beef extract and 5 g of peptone in 1000 mL of sterile distilled water). For solid media 5% agar
was used along with above mentioned composition. The bacterial samples were freeze dried and
stored at ꢀ80°C. 5 ꢂL of these stocks were added to 3 mL of the nutrient broth and the culture
was grown for 6 h at 37°C prior to the experiments.
Antibacterial Activity
Antibacterial activity was reported as Minimum Inhibitory Concentration (MIC), i.e. the lowest
concentration of the antimicrobial agent that will inhibit the growth of a microorganism after
overnight incubation. MIC was determined by following previously published protocol.22 All the
final compounds were waterꢀsoluble. The compounds were assayed in a modified microꢀdilution
broth format. Stock solutions were made by serially diluting the compounds using autoclaved
Millipore water. Bacteria, to be tested, grown for 6 h in the suitable media contained ~109
cfu/mL (determined through dilution plate technique by spread plate method), which was then
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diluted to 105 cfu/mL using nutrient media. 50 ꢂL of serially diluted compound was added to a
96 well plate containing 150 ꢂL bacterial solutions. Two controls were made; one containing 150
ꢂL of media and 50 ꢂL of compound and the other containing 200 ꢂL of bacterial solution. The
plate was then incubated at 37 °C for a period of 24 h and MIC data was recorded by measuring
the O.D. value at 600 nm using a TecanInfinitePro series M200 Microplate Reader. MIC value
was determined by taking the average of triplicate O.D. values for each concentration and
plotting it against concentration using Origin Pro 8.0 software. The data was then subjected to
sigmoidal fitting. From the curve the MIC value was determined, as the point in the curve where
the O.D. was similar to that of control having no bacteria. The MIC values and errors are
reported as averages and standard errors of mean of at least two independent experiments (each
experiment was performed in triplicates) respectively. The error of the experiments is less than
7%.
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Hemolytic Activity
Hemolytic experiments were performed with slight modification of a previously reported
protocol.22 Erythrocytes were isolated from freshly drawn, heparinized human blood and reꢀ
suspended to 5 vol% in PBS (pH 7.4). In a 96ꢀwell microtiter plate, 150 ꢂL of erythrocyte
suspension was added to 50 ꢂL of serially diluted compound. Two controls were made, one
without compound and other with 50 ꢂL of 1 vol% solution of Triton Xꢀ100. The plate was
incubated for 1 h at 37°C. The plate was then centrifuged at 3,500 rpm for 5 min, 100 ꢂL of the
supernatant from each well was transferred to a fresh microtiter plate, and A540 was measured.
Percentage of hemolysis was determined as (A ꢀ A0)/(Atotal ꢀA0) x 100, where A is the absorbance
of the test well, A0 the absorbance of the negative controls (without compound), and Atotal the
absorbance of 100% hemolysis wells (with Triton Xꢀ100), all at 540 nm. The HC50 values and
errors are reported as averages and standard errors of mean of at least two independent
experiments (each experiment was performed in triplicates) respectively. The error of the
experiments is less than 10%.
Bactericidal Time-kill Kinetics
The bactericidal activity of the derivatives was assessed by the kinetics or the rate at which it
affects the killing action of the compound. Briefly, MRSA was grown in yeastꢀdextrose broth at
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37°C for 6 h. Test compound, 3 having the final concentrations of 1×MIC, 3×MIC, 6×MIC was
inoculated with the aliquots of MRSA resuspended in fresh media at approximately 1.8×106 CFU
mLꢀ1. After specified time intervals (0h, 1h, 2h, 3h, 6h and 24 h), 20 ꢂL aliquots were serially
diluted 10 fold in 0.9 % saline, plated on sterile yeastꢀdextrose agar plates and incubated at 37°C
overnight. The viable colonies were counted the next day and represented as log10(CFU/mL). A
similar protocol was followed with compound 7, but two concentrations were considered, i.e.
MIC and 2×MIC against S. aureus.
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Antibacterial activity in plasma
S. aureus was grown for 6 h in Yeast dextrose agar media and finally contained ~109 cfu/mL
(determined through dilution plate technique by spread plate method), which was then diluted to
105 cfu/mL using nutrient media. Fresh human blood cells were centrifuged at 3000 rpm for 5
minutes. The plasma, which is separated from the RBC solution, was collected carefully. The test
compound, 7 was dissolved in water at a concentration of 3200 ꢁg/mL. This was diluted twoꢀ
fold into the plasma solution so that the final concentration of the compound was 1600 ꢁg/mL.
Three such test samples were preꢀincubated in 50% plasma solution at 37°C for 0, 3 and 6 hours.
50 ꢁL of 2ꢀfold serial dilutions of this solution was added to 150 ꢁL of the bacterial solution in a
96ꢀwell plate. This was subjected to 10ꢀfold serial dilutions and incubated at 37°C for 24 hours.
Then the MIC values were determined as mentioned in the antibacterial assay.
Enzyme Stability
Compound 7 was dissolved in 0.1 M NH4HCO3 to a final concentration 1 mg mLꢀ1. 1 mg of
trypsin was dissolved in 50 mL of 0.1 M NH4HCO3 buffer to make a solution. 200 ꢁL of freshly
made trypsin solution and 200 ꢁL compound solution was incubated in 1600 ꢁL of 0.1 M
NH4HCO3 buffer at 37°C. The 200 ꢁL aliquots were taken at different time intervals like 0 min,
5 mins, 15 mins, 30 mins, 1 hr, 2hrs, 4 hrs and 24 hrs. This was diluted with 200 ꢁL of water and
analyzed using LCꢀMS. For negative control, sample without trypsin were taken at 0 min and 24
hrs. The LC chromatogram was an indicator of formation of new products (if there is
degradation) and the total ion current represented the total concentration of each chemical
species.
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Cytoplasmic Membrane Depolarization Assay
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Midꢀlog phase bacterial cells (S. aureus, P. aeruginosa or E. coli) were harvested, washed with 5
mM HEPES and 5 mM glucose and resuspended in 5 mM glucose, 5 mM HEPES buffer and 100
mM KCl solution in 1:1:1 ratio (108cfu/mL). Measurements were made in a cuvette containing 2
mL of bacterial suspension and 2 ꢁM DiSC3(5). The bacterial suspension (2 mL) was
preincubated for 10 min (20 min in case of P. aeruginosa or E. coli) with DiSC3(5) dye in a
fluorimeter cuvette (Dye uptake, and resultant selfꢀquenching). The fluorescence of the bacterial
suspension was measured and allowed to stabilize for 2 min at room temperature before the
addition of test compounds (3 and 7) of different concentrations, and the decrease in potential
was monitored by the increase in fluorescence for further 10 min. Excitation wavelength of 622
nm (slit width: 10 nm) and emission wavelength of 670 nm (slit width: 5 nm) were chosen for
the study.
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Inner Membrane Permeabilization Assay
Midꢀlog phase S. aureus cells were harvested (4000 rpm, 4°C, 10 min), washed, and resuspended
in 5 mM HEPES and 5 mM glucose pH 7.2. Then test compounds (3 and 7) of different
concentrations were added to a cuvette containing 2.0 mL of cells and 15 ꢂM propidium iodide
(PI). Excitation wavelength of 535 nm (slit width: 10 nm) and emission wavelength of 617 nm
(slit width: 5 nm) were chosen for the study. The uptake of PI was measured by the increase in
fluorescence of PI for 10 min as a measure of inner membrane permeabilization.
Outer Membrane Permeabilization Assay
Midꢀlog phase E. coli cells (grown for 6 h, 108 cells/mL) were harvested (4000 rpm, 4 °C, 10
min), washed, and resuspended in 5 mM glucose/5 mM HEPES buffer at pH 7.2. Then, 26 ꢂL of
compound 7 in water was added to a cuvette containing 2 mL of cells and 10 ꢂM Nꢀ
Phenylnaphthylamine, NPN (30 ꢂL from a 500 ꢂM stock solution in acetone). The excitation and
emission wavelengths used were 350 nm and 420 nm, respectively (slit width was 10 nm in both
cases). The uptake of NPN as a measure of outer membrane permeabilization was monitored by
the increase in fluorescence of NPN for 10 min.
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Fluorescence Microscopy26
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To 1 mL of bacterial suspension containing 109 CFU/mL, 10 times MIC of compounds 3 or 7
was added to make a final volume of 1 mL of suspension (final concentration 1 × 109 cells/mL),
and another 1 mL was left untreated as a control. The mixture was incubated for 1.5 h,
centrifuged (12,000 rpm for 1 min), and resuspended in 50 ꢂL of PBS. 5ꢂL of the bacterial
suspension was combined with 20 ꢂL of a fluorescent probe mixture containing 3.0 ꢂM green
fluorescent nucleic acid stain SYTO 9 (Invitrogen, USA) and 15.0 ꢂM red fluorescent nucleic
acid stain PI (SigmaꢀAldrich, USA). The mixture was incubated in the dark for 15 min, and a 5
ꢂL aliquot was placed on a glass slide, which was then covered with a coverslip, sealed, and
examined under a fluorescence microscope. Excitation was carried out for SYTO 9 at 450−490
nm and for PI at 515−560 nm. Emission was collected using a bandꢀpass filter for SYTO 9 at
500−550 nm and a longꢀpass filter for PI at 590− 800 nm. In all cases, a 100× objective was used
with immersion oil, giving a total magnification of 1000×. Images were captured with a Leica
DM 2500 fluorescence microscope.
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Scanning Electron Microscopy (SEM)
The bacterial cells were cultured for 6 h in suitable media (LB broth for E. coli and yeast
dextrose broth for S. aureus) at 37 °C. The cells were centrifuged and resuspended in nutrient
media at pH 7.4 (106 cells/mL). The suspension was divided into two portions. To one portion (1
mL) was added 10 × MIC of compounds 3 and 7. The other portion was left untreated as a
control. The suspension was then incubated at 37°C for 2 h (at 250 rpm shaking speed), and the
cells from both tubes were harvested by centrifugation at 12,000 rpm. After treatment, the cells
were dehydrated sequentially with 30, 50, 70, 80, 90, and 100% ethanol for 15 min. Later, 5 ꢂL
of dehydrated cells was dropped on a small piece of silicon wafer and dried at room temperature.
Before being imaged, the silicon wafer containing S. aureus was sputter coated, and E. coli was
used directly for imaging without sputtering. Images were recorded by using Quanta 3D FEG
FEI fieldꢀemission scanning electron microscopy at 20 kV for S. aureus and 10 kV for E. coli.
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ASSOCIATED CONTENT
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Supporting Information. Materials and instrumentation, Details of characterization, HPLC
data, NMR data, Mass Spectra of the final compounds and other experimental figures. This
material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: (+91) 80ꢀ2208ꢀ2565. Fax: (+91) 80ꢀ2208ꢀ2627. Eꢀmail: jayanta@jncasr.ac.in
Funding Sources
J.H. acknowledges Department of Science and Technology (DST), Govt. of India for Ramanujan
Fellowship [SR/S2/RJNꢀ43/2009] and Jawaharlal Nehru Centre for Advanced Scientific
Research for funding.
Notes
The authors declare no competing financial interest. JNCASR has filed a patent application
based on the work described in the manuscript.
ACKNOWLEDGMENT
We thank Prof. C.N.R. Rao (JNCASR) for his constant support and encouragement. J.H.
acknowledges Department of Science and Technology (DST), Govt. of India for Ramanujan
Fellowship [SR/S2/RJNꢀ43/2009].
ABBREVIATIONS
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AMP, antimicrobial peptide; MRSA, methicillinꢀresistant Staphylococcus aureus, VRE,
vancomycinꢀresistant Enterococci ; MIC, minimum inhibitory concentration; HC50, 50%
hemolytic concentration; CFU, colony forming units
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FIGURES
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Figure 1. Structures of small molecular antibacterial peptoid mimics.
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Figure 2. (A) Variation of antibacterial activity with increasing chain length in Series I; (B)
Timeꢀkill kinetics of 3 against MRSA. (Stars indicate that bacterial count is <50 CFU mLꢀ1); (C)
Cytoplasmic membrane depolarization of S. aureus by 3; (D) Cytoplasmic membrane
depolarization of P. aeruginosa by 3; (E) S. aureus membrane permeabilization by 3; (F) E. coli
outer membrane permeabilization by 7.
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Figure 3. Fluorescence microscopy images of S. aureus (A) untreated and (B) treated with 3 (10
× MIC) for 1.5 h after staining with SYTO 9 and PI. (Scale ꢀ 20 ꢂm). Scanning electron
microscopy (SEM) images of (C) untreated E. coli and (D) E. coli treated with 3 (10 × MIC).
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SCHEMES
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Scheme 1. General synthetic scheme for the preparation of the compounds.
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where, (a) RNH2 (where R is alkyl group) in MeOH, 6h; (b) NaBH4 in MeOH, 18h. (c) HCl; (d)
BocꢀLys(Boc)ꢀOH, N,NꢀDiisopropylethylamine (DIPEA), Oꢀ(Benzotriazolꢀ1ꢀyl)ꢀN,N,N’,N’ꢀ
tetramethyluroniumhexafluorophosphate (HBTU) in DMF/CHCl3 (5:2), RT, 16ꢀ24h; (e) 50%
CF3COOH in CH2Cl2, 12h.
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TABLES.
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Table 1. Inꢀvitro antibacterial and hemolytic activity of the compounds.
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Minimum Inhibitory Concentration (ꢂg mLꢀ1)
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HC50
HPLC
Retention
times
Drug sensitive bacteria
Drug resistant bacteria
Compounds
(ꢂg mLꢀ1)
S. aureus
E. faecium
E. coli
P. aeruginosa
MRSA
VRE
K. pneumoniae
(min.)
1
11
13.6
4.5
25
4
21
7.2
31
118
91
11.8
12.4
13.6
14.6
15.6
10.8
11.8
12.9
14.2
15.1
9.4
2
5.3
2.4
2.2
7.1
>100
20
4.8
3.5
2.9
26
1.9
1.6
3.8
11
6.3
5.3
17
3
3.3
2.8
5.2
16
82
4
2.5
2.3
3
4.3
64
5
4.9
4.6
5.6
7.6
71
6
>100
34
>100
25
>100
11
>100
65
>100
54
>100
100
13
>1000
508
60
7
8
6.3
2.5
3
5.5
5
5.4
3
4.4
7
9
3.5
4
2.6
1.6
5.8
54
10
1.6
3.1
>100
>100
51
3.2
>100
>100
60
2.7
3.4
4
56
11
>100
>100
46
>100
>100
60
>100
>100
>100
15.7
2.9
N.D.[a]
N.D.
>100
5.8
>100
>100
>100
31
>1000
>1000
325
95
12
10.6
12.1
13.4
14.5
15.8
N.D.
N.D.
N.D.
13
14
5.7
2.7
3.1
0.9
20
6.5
6.5
5
4
15
2.6
4
3.3
2.8
45
16
2
3.1
N.D.
0.4
2.8
N.D.
0.4
2.5
2.5
4
50
Vancomycin
Colistin
MSIꢀ78
0.87
>100
64[b]*
0.9
>100
>100
8[b]
N.D.
1.2
N.D.
N.D.
120[c]
54
8ꢀ16[b]
16ꢀ32[b] 8ꢀ16[b]
[b]
16ꢀ32[b]
8ꢀ16[b]
[a]N.D. stands for “not determined”. Literature values obtained from Ref. 36 * indicates value
for E. faecalis , Literature value obtained from Ref.28 VRE (vancomycinꢀresistant E. faecium)
[c]
and MRSA (methicillinꢀresistant S. aureus), K. pneumoniae is resistant to β–lactam antibiotics.
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SYNOPSIS.
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Table of contents graphic
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