Pyridinium based amphiphilic hydrogelators as potential antibacterial agents

Article abstract of DOI:10.3762/bjoc.6.101

The numerous applications of hydrogelators have led to rapid expansion of this field. In the present work we report the facile synthesis of amphiphilic hydrogelators having a quaternary pyridinium unit coupled to a hydrophobic long alkyl chain through an amide bond. Different amphiphiles with various hydrophobic chain length and polar head groups were rationally designed and synthesized to develop a structure-property relation. A judicious combination of hydrophilic and hydrophobic segments led to the development of pyridinium based amphiphilic hydrogelators having a minimum gelation concentration of 1.7%, w/v. Field emission scanning electronic microscopy (FESEM), atomic force microscopy (AFM), photoluminescence, FTIR studies, X-ray diffraction (XRD) and 2D NOESY experiments were carried out to elucidate the different non-covalent interactions responsible for the self-assembled gelation. The formation of three-dimensional supramolecular aggregates originates from the interdigitated bilayer packing of the amphiphile leading to the development of an efficient hydrogel. Interestingly, the presence of the pyridinium scaffold along with the long alkyl chain render these amphiphiles inherently antibacterial. The amphiphilic hydrogelators exhibited high antibacterial activity against both Gram-positive and Gram-negative bacteria with minimum inhibitory concentration (MIC) values as low as 0.4 μg/mL. Cytotoxicity tests using MTT assay showed 50% NIH3T3 cell viability with hydrogelating amphiphile 2 up to 100 μg/mL.

Full text of DOI:10.3762/bjoc.6.101

Pyridinium based amphiphilic hydrogelators as
potential antibacterial agents
Sayanti Brahmachari, Sisir Debnath, Sounak Dutta
and Prasanta Kumar Das*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2010, 6, 859–868.
Department of Biological Chemistry, Indian Association for the
Cultivation of Science, Jadavpur, Kolkata – 700 032, India. Fax:
+(91)-33-24732805
doi:10.3762/bjoc.6.101
Received: 05 May 2010
Accepted: 15 July 2010
Email:
Published: 21 September 2010
Prasanta Kumar Das* - bcpkd@iacs.res.in
Guest Editor: J.-P. Desvergne
* Corresponding author
© 2010 Brahmachari et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
antibacterial; bilayer structure; hydrogel; pyridinium; self-assembly
Abstract
The numerous applications of hydrogelators have led to rapid expansion of this field. In the present work we report the facile syn-
thesis of amphiphilic hydrogelators having a quaternary pyridinium unit coupled to a hydrophobic long alkyl chain through an
amide bond. Different amphiphiles with various hydrophobic chain length and polar head groups were rationally designed and
synthesized to develop a structure-property relation. A judicious combination of hydrophilic and hydrophobic segments led to the
development of pyridinium based amphiphilic hydrogelators having a minimum gelation concentration of 1.7%, w/v. Field emis-
sion scanning electronic microscopy (FESEM), atomic force microscopy (AFM), photoluminescence, FTIR studies, X-ray diffrac-
tion (XRD) and 2D NOESY experiments were carried out to elucidate the different non-covalent interactions responsible for the
self-assembled gelation. The formation of three-dimensional supramolecular aggregates originates from the interdigitated bilayer
packing of the amphiphile leading to the development of an efficient hydrogel. Interestingly, the presence of the pyridinium scaf-
fold along with the long alkyl chain render these amphiphiles inherently antibacterial. The amphiphilic hydrogelators exhibited high
antibacterial activity against both Gram-positive and Gram-negative bacteria with minimum inhibitory concentration (MIC) values
as low as 0.4 μg/mL. Cytotoxicity tests using MTT assay showed 50% NIH3T3 cell viability with hydrogelating amphiphile 2 up to
100 μg/mL.
Introduction
Gels are an outstanding group of soft materials lying at the entrap water) are of special importance owing to their tremen-
interface of solid and liquid, and find numerous applications in dous potential in biomedicine [12-16]. These hydrogels can be
various fields including tissue engineering, biosensors, food of natural origin [17] (collagens, polysaccharides) as well as of
processing, cosmetics, photography, controlled drug delivery synthetic origin [18] (poly(acrylic acid) and derivatives,
etc. [1-11]. Amongst the variety of gels, hydrogels (those that polypeptides and small molecules). This fascinating class of
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materials results from the spontaneous self-assembly of poly- features of the amphiphiles such as the aliphatic chain length
meric/non-polymeric molecules that lead to the formation of and the polar head group was systematically carried out to
supramolecular three dimensional (3D) networks with inter- understand their influence on the self-assembled hydrogelation.
stitial space for the immobilization of solvents. Low-molecular- The various factors involved in the formation of supra-
weight-gelators (LMWG) have received more attention than molecular aggregates leading to hydrogelation were studied
their polymeric analogues for a number of scientific applica- using FTIR, XRD and fluorescence spectroscopy. The topo-
tions due to their thermo-reversible nature and their prompt graphical features of the soft matter were visualized using
response to external stimuli [19]. A critical balance between different microscopic techniques (scanning electron microscopy
hydrophilic and hydrophobic interactions is mandatory for any (SEM), atomic force microscopy (AFM)). Interestingly, these
gelation process. Non-covalent interactions such as hydrogen compounds were found to show excellent antibacterial activity
bonding, ionic interactions, π–π stacking or van der Waals against Gram-positive and Gram-negative bacteria with
forces play a pivotal role in self-assembled gelation [12]. minimum inhibitory concentration (MIC) values as low as 0.4
Tuning the structure of gelator molecules leads to a better µg/mL for Micrococcus luteus. In addition, amphiphile 2 was
understanding of the contribution of the different interactive investigated for cytotoxicity with mammalian cells (NIH3T3)
forces and an insight into the 3D-morphology of supra- and showed sufficient viability throughout a range of concentra-
molecular aggregates [20-22]. In this context, low molecular tions.
weight hydrogels (LMWH) are of greater importance compared
to polymeric ones as the former (i) can have diversified supra-
molecular morphology by varying the structure of its precursor
molecules, (ii) have the ability for quick response to external
stimuli, and (iii) are potentially biocompatible [12,23,24]. Thus,
the huge range of applications of the hydrogels and the benefi-
cial aspects of small molecule gelators including ease of prepar-
ation have synergistically led to a surge in the development of
tailor-made LMWHs.
The presence of an aromatic ring (for example, phenyl, naph-
thalene, N-fluorenyl-9-methoxycarbonyl (Fmoc), indole, pyri-
dine) in small molecule gelator is known to have crucial influ-
ence in inducing self-aggregation towards gelation. The planar
aromatic moiety favors π–π stacking interactions between the
molecules and leads to the formation of 3D networks of
viscoelastic gels [25-27]. Interestingly, among all these
aromatic rings, the positively charged pyridine (pyridinium)
unit is well known to impart antibacterial properties to
Figure 1: Structure of amphiphiles 1–5.
amphiphilic molecules [28-31]. The cationic charge of the
amphiphile plays an instrumental role in disrupting the innate
defense mechanism of microorganisms by disrupting the micro- Results and Discussion
bial cell membrane [32,33]. Hence, it would be interesting to Gelation is simply a macroscopic manifestation of the self-
develop amphiphilic hydrogelators that have pyridinium assembled aggregation at the molecular level due to the
moieties in order to exploit its favorable π–π stacking inter- optimum combination of hydrophilic and hydrophobic interac-
action towards self-assembled gelation as well as an ability to tions between molecules [12]. The formation, nature and
kill bacteria. Furthermore, a very simple method of synthe- morphology of these supramolecular 3D-networks are primarily
sizing such amphiphilic antibacterial hydrogelators with pyri- dictated by the architecture of the gelating molecules. To estab-
dinium units would definitely boost its importance and utility lish the different nature of interactions taking place within the
for a wide spectrum of applications.
supramolecular assemblies, a structure-property correlation for
the gelators is necessary. In the present work we have synthe-
In the present work, we report the facile synthesis of pyri- sized a series of amphiphilic compounds containing a quater-
dinium based amphiphiles (1–5, Figure 1) of which amphiphiles nary pyridinium unit as the polar head group and varied the
1 and 2 were efficient hydrogelators with minimum gelation length of alkyl chain of the hydrophobic part (1–5, Figure 1)
concentrations (MGC) ≈1.7–2.0%, w/v. Modification of certain using very simple methodology (Scheme 1). Variation at the
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Scheme 1: Synthetic procedure of the amphiphiles.
hydrophilic as well as the hydrophobic segment was performed (inner diameter = 10 mm) in a thermostated oil bath and raising
to understand the critical balance imperative for hydrogelation. the temperature slowly at a rate of 2 °C/min. Tgel is defined as
the temperature (±0.5 °C) at which the gel melts and starts to
Amphiphile 1, with a N-methylated pyridinium moiety as the flow from an inverted glass vial. Both hydrogels of 1 and 2 at
polar head linked by a C-18 alkyl chain through an amide bond, their MGC showed Tgel at 35 and 36 °C, respectively. In agree-
exhibited efficient water gelation ability (MGC of 2.0%, w/v). ment with the previous reports it was found that the Tgel of the
The stable to inversion of container method confirmed the for- hydrogels steadily increased with increase in gelator concentra-
mation of gel. However, the hydrogel was not stable as the tion (Figure 2) [34,35]. This clearly indicates the enhancement
amphiphile precipitated from the gel after 4–5 h. It is possible in the strength of the non-covalent intermolecular interaction in
that the C-18 alkyl chain is too hydrophobic to maintain the the aggregated state with increasing gelator concentration. Also
optimum hydrophilic–hydrophobic balance necessary for effi- the thermo-reversible nature of hydrogelation was established
cient gelation. Consequently, keeping all other segments iden- as the sol formed on heating returned to the gel state upon
tical as in 1, the C-18 alkyl chain was replaced by a shorter lowering the temperature. Interestingly, the Tgel curve of
alkyl chain, C-16 in case of amphiphile 2. In accord with our amphiphile 2 maintained a slightly higher profile throughout the
expectations, amphiphile 2 exhibited better water gelation range of the concentrations suggesting the comparatively better
ability with a MGC of 1.7%, w/v. The transparent hydrogel of 2 hydrogelation efficiency of 2 over that of 1.
was stable for several months. At this point, we were curious to
know how the gelation efficiency would be affected by further
lowering the alkyl chain length to C-14 (amphiphile 3).
However, compound 3 was found to be a non-gelator. A
decrease in the hydrophobicity in amphiphile 3 possibly
destroyed the hydrophilic–hydrophobic balance required for
water gelation. Following the importance of the hydrophobic
segment of the amphiphile in gelation, we were also interested
to investigate the influence of the polar head group of the pyri-
dinium-based amphiphiles in hydrogelation. To this end we
made very minor modifications to the quaternized nitrogen of
the pyridine moiety keeping other segments unaltered as in the
efficient gelator 2. N-methylated pyridinium of 2 was changed
first of all to a simple protonated pyridinium moiety in
amphiphile 4 and then to N-ethylated pyridinium group in the
case of 5 (Figure 1). Neither of these amphiphiles exhibited any
water gelation ability, which reiterates the importance of the
Figure 2: Variation of the Tgel with concentration of amphiphiles 1 and
2.
optimum balance between hydrophilic and hydrophobic char-
acter within a molecule for gelation.
The gel-to-sol transition temperature (Tgel) for both the hydro- The formation of three dimensional higher ordered structures
gels was determined by placing the gel-containing glass vial during self-assembled hydrogelation was investigated by field
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emission scanning electron microscopy (FESEM). Morphology
of the dried xerogels showed the formation of different supra-
molecular structures that are involved in the gelation process of
1 and 2 (Figure 3). SEM image of hydrogel 1 showed an aggre-
gated form of porous networks (Figure 3a), which were respon-
sible for the entrapment of the solvent. In case of hydrogel 2,
formation of thin intertwined fibrillar networks of 200–300 nm
dimensions (Figure 3b) was observed. The fibrillar architecture
of 2 at the aggregated state was further confirmed by Atomic
Force Microscopic (AFM) images. Two and three dimensional
AFM images of xerogel 2 (Figure 3c, d) showed the involve-
ment of fibrillar networks in self-assembled hydrogelation. The
dimension of the fibril network observed in the AFM image was
also in accord with the FESEM images.
Figure 4: Luminescence spectra of 2 in water (λex = 330 nm) at
various concentrations and room temperature.
corresponding MGC which is also almost three times higher
than its critical micellar concentration (0.011%, w/v). Hence,
the amphiphile 2 began to self-assemble towards hydrogelation
above 0.035%, w/v. Moreover, the red shifted emission peak up
to MGC and above indicates that the intermolecular π–π inter-
actions between the pyridine moieties plays an important role in
gelation [28]. Consequently, the fluorescence quenching of
pyridine by the cationic charge was due to the close proximity
of the head groups during gelation.
To determine the involvement of intermolecular hydrogen
bonding between the amide N–H and carbonyl oxygen we
investigated both hydrogels by FTIR spectroscopy. As the pres-
ence of H2O in FTIR spectroscopy may create difficulties in
extracting information on intermolecular interactions, we
measured the FTIR spectra of gelators 1 and 2 in D2O (self-
aggregated state) and in CHCl3 (non-aggregated state). The
absorption frequency for the C=O stretching band (amide I) in
gels is always lower compared to that in CHCl3. The transmis-
sion bands of C=O stretching for the gel 1 and 2 in D2O
appeared at 1660 and 1655 cm−1, respectively, which is charac-
teristic of hydrogen bonded amide groups (Figure 5). Whereas
the corresponding amide I stretching frequency at 1700 and
1703 cm−1 for 1 and 2 in CHCl3 demonstrates the existence of a
non-hydrogen bonded amide group. Hydrogen bond formation
is accompanied by a decrease in the bond order and hence the
observed shift in the carbonyl stretching frequency to a lower
value underlines the participation of intermolecular H-bonding
in the gel state [37]. In addition, the N–H stretching frequency
of amide for both gelators appeared at ≈3400 cm−1 in the gel
state which was shifted to 3430 cm−1 in the non-aggregated
form in CHCl3. This shift in the N–H stretching confirms the
Figure 3: (a, b) FESEM images of the dried gels of 1 and 2, respect-
ively at their MGC. (c, d) Two- and three-dimensional AFM image of
xerogel 2.
The participation of the pyridinium ring in hydrogelation was
investigated by taking the luminescence spectra of the efficient
gelator 2 at various concentrations (0.01–3.0%, w/v) in water
(Figure 4) at room temperature. The amphiphile 2 was excited
at λ = 330 nm and the emission spectra were recorded between
of 340–550 nm. At a very low concentrations (0.01%, w/v), 2
showed an emission peak at λem = 402 nm. With a gradual
increase in the concentration of 2, a steady increase in the fluo-
rescence intensity was observed up to 0.035%, w/v. With
further increase in the concentration of 2, the fluorescence
intensity decreased with a continuous red shift of the λem from
402 nm to 416 nm. The observed increase in the fluorescence
intensity as well as continuous quenching of the signal after
0.035%, w/v to MGC and above is probably due to the inter-
action between the pyridinium ring and the cationic charge
(Figure 4) [36]. Notably, the quenching in the emission of pyri-
dine started at a concentration that is ≈50 times lower than the
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Figure 5: FTIR spectra of (a) 1 and (b) 2 in CHCl3 solution (dashed line) and in D2O at the gel state (solid line).
participation of the amide N-H in the intermolecular hydrogen
bonding. Furthermore, the increase in intensity of the meth-
ylene scissoring vibration δ(CH2) band at ≈1460 cm−1 for both
the gelators (Figure 5) in D2O indicates the high trans con-
formational packing of alkyl chain [38].
To establish further the intermolecular interaction between the
gelators as noted above, we carried out 2D NOESY experi-
ments for gelator 2 (2.0%, w/v) in the aggregated state in
DMSO-d6 in the presence of 70% water and also in the non
self-assembled state of the amphiphile in neat DMSO-d6. At
70% water content in DMSO-d6, off-diagonal cross peaks were
observed between the aromatic rings and the methyl group on
the quaternized nitrogen of pyridine (Figure 6). The presence of
off-diagonal peaks in the aggregated form clearly indicates the
existence of through space interaction between the neighboring
gelator molecules which plays a crucial role in gelation. No
such off-diagonal peak was observed for 2 in neat DMSO-d6
which is in accord with the absence of any kind of
Figure 6: 2D-NOESY spectra of 2 (2%, w/v) in DMSO-d6 with 70%
water.
intermolecular interaction in the non-gelated state of the
amphiphile.
To investigate the molecular packing and orientation of the Thus, on the basis of the aforementioned spectroscopic, micro-
gelator molecules in the supramolecular self-assembled state, scopic studies as well as from the XRD results, it can be
the xerogel of 2 was examined by X-ray diffraction (XRD). A concluded that in the gelation process the amphiphiles are
sharp diffraction peak was obtained in the small angle region at possibly forming repeating bilayers in which the molecules are
2θ = 2.37° which corresponds to a d-spacing of 3.71 nm indi- connected by intermolecular hydrogen bonding and
cating an ordered arrangement of the molecules in the gel state hydrophobic interaction. The probable interdigitated bilayer
(Figure 7). The observed d-spacing was greater than the length packing of the amphiphile 2 is represented in Figure 8 [39].
of a single surfactant molecule, 2.6 nm (calculated using
MOPAC AMI method, CS Chem Office) but smaller than twice As noted earlier the pyridinium component is well known to
the fully extended molecular length of gelator 2 (5.2 nm).
impart antibacterial properties to a molecule [28-31]. Thus, we
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Table 1: Antibacterial activities (MICs) of 1 and 2 in μg/mL.
Amphiphile
Gram-positive
Gram-negative
B. subtilis M. luteus E. coli K. aerogenes
1
2
2.0
1.0
0.6
0.4
20.0
10.0
5.0
5.0
effective in killing bacteria with MIC values of 0.4–2.0 μg/mL
for Gram-positive bacteria and 5.0–20.0 μg/mL for Gram-nega-
tive bacteria. However, 2 was found to have slightly better
antibacterial activity than 1 with MIC values of only 0.4 μg/mL
for Gram-positive Micrococcus luteus and 10 μg/mL for Gram-
negative Escherichia coli. Interestingly, the pyridinium based
amphiphilic hydrogelators showed antibacterial activity against
both type of bacteria which is in contrast to the antibacterial
Figure 7: XRD diagram of the dried gel of 2.
envisaged that it might be possible to develop inherently activity of conventional quaternary cationic amphiphiles which
antibacterial soft matter based on amphiphilic pyridinium com- are, in general, ineffective against Gram-negative bacteria. The
pounds. The antibacterial activities of both hydrogelating positively charged amphiphiles are presumably adsorbed on the
amphiphiles (1 and 2) were tested against two types of Gram- negatively charged cell membrane of microbes due to electro-
positive (Bacillus subtilis and Micrococcus luteus) and Gram- static interaction. This interaction is also entropically favorable
negative (Escherichia coli and Klebsiella aerogenes) bacteria. as huge numbers of counterions are released. Next, the
Minimum inhibitory concentrations (MIC), the lowest hydrophobic chain penetrates the hydrophobic cell membrane
amphiphile concentration at which no viable bacterial cell is by ‘self-promoted’ transport resulting in release of the cyto-
present, are presented in Table 1. Both 1 and 2 were found to be plasmic constituents thus leading to the death of bacteria
Figure 8: Schematic representation of the possible arrangement of molecules during hydrogelation of 2.
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[29,40]. The pyridinium-based amphiphiles 1 and 2 are struc- gelation process were found to be non-covalent interactions
turally different from those studied earlier [29] and their such as π–π stacking and intermolecular hydrogen bonding.
antibacterial activity is similar to that of the reported pyri- These cationic amphiphilic molecules exhibited antibacterial
dinium compounds. Most importantly, the hydrogelation ability activity against both Gram-positive and Gram-negative bacteria
along with the inherent antibacterial properties of the present and were found to be viable towards mammalian cells. The
amphiphiles make them interesting scaffolds for biomedicinal antibacterial activity conjugated with low cytotoxicity and water
applications.
gelation ability makes this class of compound an attractive
target for the development of antibacterial biomaterials.
Application of antibacterial biomaterials becomes more versa-
tile and significant only when they are also non-toxic to living
Experimental
cells. Consequently, the cytotoxicity of amphiphile 2 (as a Materials
representative example) in NIH3T3 cells was investigated using Myristic acid, palmitic acid, stearic acid and ethyl bromide were
MTT based assay. Encouragingly, the molecule showed more purchased from SRL, India. Thionyl chloride, 3-aminopyridine,
than 96% viability up to a concentration of 20 μg/mL. However, methyl iodide were purchased from Spectrochem, India. D2O,
as the concentration of the amphiphile increased, viability DMSO-d6 and CDCl3 were obtained from Aldrich Chemical
towards the cell decreased. Nevertheless, even up to a concen- Co. Thin layer chromatography was performed on Merck pre-
tration of 100 μg/mL, greater than 50% viability was noted coated silica gel 60-F254 plates. All the material used in the cell
(Figure 9). Thus, the cationic amphiphiles are potentially lethal culture study, such as Dulbecco’s Modified Eagles’ Medium
to bacteria, but encouragingly viable to mammalian cells. Such (DMEM), heat inactivated fetal bovine serum (FBS), trypsin
cell selectivity may originate from the difference in the lipid from porcine pancreas and MTT, were obtained from Sigma
composition as well as in the membrane potential gradient Aldrich Chemical Company. 1H NMR spectra were recorded on
between the target prokaryotic and the non-target eukaryotic an AVANCE 300 MHz (BRUKER) spectrometer. Mass spec-
cell membranes [41,42].
trometric data were acquired by the electron spray ionization
(ESI) technique on a Q-Tof-micro Quadruple mass spectrom-
eter (Micromass). Fluorescence and FTIR spectra were
measured on a Varian Cary Eclipse luminescence spectrometer
and a Perkin Elmer Spectrum 100 FTIR spectrometer, respect-
ively.
General synthetic procedure
Synthesis of amphiphiles 1–5
The acid (3 g) was refluxed with thionyl chloride (1 mL) for 4 h
in an oil bath at 70 °C. The unreacted thionyl chloride was
removed with a rotary-evaporator. The resulting compound
(90% yield) was dissolved in dry dichloromethane (DCM) and
then 3-aminopyridine (1.5 equiv dissolved in minimum quan-
tity of dry DCM) added dropwise with stirring and ice cooling.
The solution was stirred for 3–4 h, the DCM removed and the
residue dissolved in ethyl acetate. The solution was washed
with NaOH to remove excess acid and to convert the pyri-
dinium salt to the free pyridine base. The organic layer was
washed with brine until neutral. The ethyl acetate was then
removed and the alkylated compound coupled through the
amide linkage purified by column chromatography on
Figure 9: MTT assay based percent NIH3T3 cell viability as a function
of concentration of amphiphile 2.
Conclusion
We have utilized a combination of a quaternary pyridinium unit 60–120 mesh silica gel with 1% methanol/chloroform mixture
and hydrophobic long chain to build a scaffold, which can as eluent (75% yield). The compound thus obtained was stirred
gelate water. The routes adopted for the synthesis of such mole- with methyl iodide (1.1 equiv) in dry DCM for 4–5 hours. After
cules were extremely simple. Systematic variations of the struc- the reaction, the DCM was removed and the compound
ture of the amphiphile reveal that minute architectural changes dissolved in ethyl acetate. The product was purified by column
at molecular level influences the self-assembling mechanism of chromatography on 60–120 mesh silica gel with methanol/chlo-
the gelation process. The major responsible factors for the roform as eluent. The resulting iodide salt was subjected to ion
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Beilstein J. Org. Chem. 2010, 6, 859–868.
exchange on Amberlite IRA-400 chloride resin to produce the (t, 2H), 4.61–4.64 (q, 2H), 7.85 (s, 1H), 8.38 (s, 1H), 9.18–9.20
pure chloride salt. The overall yield was ≈50–60%. Amphiphile (d, 1H), 9.8 (s, 1H), 11.28 (s, 1H) ppm; ESI-MS: m/z calcd for
5 was quaternized with ethyl bromide (2 equiv) and stirred for C23H41N2O (the quaternary ammonium ion, 100%): 361.3213;
36 h. The reaction mixture was taken in chloroform and washed found 361.1093 [M]+; Elemental analysis calcd (%) for
with aqueous sodium thiosulphate and brine solutions. The C23H41N2OCl: C, 69.58; H, 10.41; N, 7.06; found: C, 69.36; H,
organic layer was evaporated (rotary evaporator) and finally 10.26; N, 7.12.
purified by column chromatography on 60–120 mesh silica gel
with methanol/chloroform as the eluent. In order to synthesize Preparation of hydrogel
compound 4 the corresponding coupled compound was The required amount of the amphiphile was added in 1 mL
dissolved in methanol (minimum quantity) and HCl gas was water at pH = 7.0 to a screw-capped vial with an internal diam-
passed through it. The precipitate formed was filtered and eter of 10 mm and heated slowly until the solid had completely
collected. General synthetic scheme for the preparation of all dissolved. The solution was then cooled to room temperature
the amphiphiles (1–5) is shown in Scheme 1.
without any disturbance. After 1 h, formation of gel was
confirmed by stable to inversion of the glass vial.
Data for 1: 1H NMR (300 MHz, CDCl3, 25 °C): δ = 0.88 (t,
3H), 1.26 (br, 28H), 1.66–1.75 (m, 2H), 2.65 (t, 2H), 4.45 (s, Microscopic studies
3H), 7.88–7.93 (m, 1H), 8.44–8.46 (d, 1H), 9.24–9.27 (d, 1H), FESEM was performed on JEOL-6700F microscope. A piece of
9.85 (s, 1H), 10.79 (br, 1H) ppm; ESI-MS: m/z calcd for hydrogel was mounted on a glass slide and dried for few hours
C24H43N2O (the quaternary ammonium ion, 100%) 375.3370; under vacuum before imaging. The morphology of the dried gel
found 375.3335 [M]+; Elemental analysis calcd (%) for of compound 2 was also studied using AFM (Veeco, model
C24H43N2OCl: C, 70.12 H, 10.54; N, 6.81; found: C, 69.86; H, AP0100) in the non-contact mode. A piece of gel was mounted
10.31; N, 6.53.
on a silicon wafer and dried for a few hours under vacuum
before imaging.
Data for 2: 1H NMR (300 MHz, CDCl3, 25 °C): δ = 0.87 (t,
3H), 1.25 (br, 24H), 1.65–1.74 (m, 2H), 2.67 (t, 2H), 4.44 (s, Fluorescence spectroscopy
3H), 7.88–7.93 (m, 1H), 8.44–8.46 (d, 1H), 9.24–9.27 (d, 1H), The emission spectra of the compound 2 were recorded on
9.85 (s, 1H), 10.80 (br, 1H) ppm; ESI-MS: m/z calcd for Varian Cary Eclipse luminescence spectrometer in the concen-
C22H39N2O (the quaternary ammonium ion, 100%) 347.3057, tration range from 0.01%, w/v to above MGC (3%, w/v). A
found 347.2011 [M]+; Elemental analysis calcd (%) for super stock solution of 2 was prepared and diluted as required.
C22H39N2OCl: C, 68.99; H, 10.26; N, 7.31; found: C, 69.21; H, The compound was excited at λex = 330 nm and emission
10.15; N, 7.23.
recorded between 340–550 nm. The excitation and emission slit
widths were 5 nm and 5 nm, respectively.
Data for 3: 1H NMR (300 MHz, CDCl3, 25 °C): δ = 0.87 (t,
3H), 1.24 (br, 20H), 1.66–1.70 (m, 2H), 2.68 (t, 2H), 4.56 (s, FTIR measurements
3H), 7.80–7.82 (m, 1H), 8.11 (br, 1H), 9.42–9.43 (d, 1H), 10.05 FTIR measurements of the gelators 1 and 2 in CHCl3 solution
(s, 1H), 12.55 (br, 1H) ppm; ESI-MS: m/z calcd for C20H35N2O and in D2O (gel state) were taken in a Perkin Elmer Spectrum
(the quaternary ammonium ion, 100%) 319.2744; found 100 FTIR spectrometer using KBr and CaF2 windows, respect-
319.1093 [M]+; Elemental analysis calcd (%) for ively with 1 mm Teflon spacers at their MGC.
C20H35N2OCl: C, 67.67; H, 9.94; N, 7.89; found: C, 67.49; H,
10.02; N, 8.07.
NMR measurements
1H NMR and 2D-NOESY spectra were recorded on AVANCE
Data for 4: 1H NMR (300 MHz, CDCl3, 25 °C): δ = 0.87 (t, 300MHz (BRUKER) spectrometer at 2%, w/v for 2 in DMSO-
3H), 1.26 (br, 24H), 1.59 (br, 2H), 2.29 (t, 2H), 7.53 (m, 1H), d6 and in water (70%) and DMSO-d6 (30%).
7.69–7.73 (m, 2H), 7.81 (s, 1H), 8.19 (br, 1H) ppm; ESI-MS:
m/z calcd for C21H37N2O (the quaternary ammonium ion, X-ray diffraction (XRD)
100%) 333.2900; found 333.1956 [M]+; Elemental analysis XRD measurements were taken with Seifert XRD 3000P
calcd (%) for C21H37N2OCl: C, 68.36; H, 10.11; N, 7.59; diffractometer. The source was Cu Kα radiation (λ =
found: C, 68.43; H, 9.98; N, 7.37.
0.15406 nm) with a voltage and current of 40 kV and 30 mA,
respectively. The gel was mounted on a glass slide and
Data for 5: 1H NMR (300 MHz, CDCl3, 25 °C): δ = 0.87 (t, dried under vaccum. The xerogel was scanned from 2Θ =
3H), 1.25 (br, 24H), 1.41–1.45 (m, 2H), 1.70–1.75 (t, 3H), 2.61 1–40°.
866

Beilstein J. Org. Chem. 2010, 6, 859–868.
Microorganisms and culture conditions
now in the exponential phase of growth for checking the
The in vitro antimicrobial activity of the cationic amphiphiles viability of amphiphile 2.
was investigated against representative Gram-positive and
Gram-negative bacteria. Gram-positive bacteria used in the Cytotoxicity assay
present study were Bacillus subtilis and Micrococcus luteus. The cytotoxicity of amphiphile 2 was assessed by the microcul-
Gram-negative bacteria investigated include Escherichia coli ture MTT reduction assay as described in the literature. This
and Klebsiella aerogenes. Investigations of antibacterial activi- assay is based on the reduction of a soluble tetrazolium salt by
ties were performed by the broth dilution method. The LB me- mitochondrial dehydrogenase of the viable cells to form an
dium (tryptone (10 g), yeast extract (5 g) and NaCl (10 g) in 1 L insoluble colored formazan product. The amount of formazan
sterile distilled water at pH 7.0) was used as the liquid medium product formed can be measured spectrophotometrically after
in all antibacterial experiments. All the microbial strains were dissolution of the dye in DMSO. The activity of the enzyme and
purchased from Institute of Microbial Technology, Chandigarh, the amount of the formazan produced is proportional to the
India. The stock solutions of all the amphiphiles as well as the number of live cells. Reduction of the absorbance value can be
required dilutions were made in autoclaved sterile water. attributed to the killing of the cells or inhibition of the cell
Freeze-dried ampoules of all bacterial strains were opened and a proliferation by the molecule. 150 μL of cell solution were
loopful of culture was spread to give single colonies on the seeded (20,000 cells per well) in a 96-well microtiter plate for
respective solid LB agar media and incubated for 24 h at 37 °C. 18–24 h before the assay. A stock solution of the amphiphile 2
A representative single colony was picked up with a wire loop was prepared. Sequential dilution of this stock solution was
and was spread on an agar slant to give single colonies. The carried out during the experiment to vary the concentrations of
slants were incubated at 37 °C for the respective time. These in- the amphiphile in the microtiter plate. The cells were incubated
cubated cultures of all the bacteria were diluted as required to with the amphiphile solutions at different concentrations for 4 h
give a working concentration in the range of 106–109 colony at 37 °C under 5% CO2. Then, 15μL of MTT stock solution
forming units (cfu)/mL before every experiment.
(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 in DMSO and
Antimicrobial studies
Minimum inhibitory concentrations (MICs) of hydrogelating absorbance at 570 nm was measured using BioTek® Elisa
amphiphiles 1 and 2 were estimated by both the broth dilution Reader. The number of surviving cells were expressed as
and the spread plate method. MIC was measured using a series percent viability = [A570 (treated cells)−background/
of test tubes containing the amphiphiles (0.05–200 μg/mL) in A570(untreated cells)−background] × 100.
5 mL liquid medium. Diluted microbial culture was added to
each test tube at identical concentrations to obtain the working Acknowledgements
concentration of bacteria: for B. subtilis 7.5 × 107–1 × 108 cfu/ P.K.D. is thankful to the Department of Science and Tech-
mL, for M. luteus 5 × 106–7.5 × 106 cfu/mL, for E. coli 3.75 × nology (DST), India for financial assistance through the
107–7.5 × 107 cfu/mL, for K. aerogenes 9 × 107–1.2 × 108 cfu/ Ramanna Fellowship (No. SR/S1/RFPC-04/2006). S.B., S.D.
mL. All the test tubes were then incubated at 37 °C for 24 h. and S.D. acknowledge Council of Scientific and Industrial
The optical density of all the solutions was measured at 650 nm Research, India for Research Fellowships. We thank
before and after incubation. Liquid medium containing micro- Anshupriya Shome for her help in antibacterial and cytotoxic
organisms was used as a positive control. All the experiments studies.
were performed in triplicate and repeated twice.
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