Hydrogelation through self-assembly of fmoc-peptide functionalized cationic amphiphiles: Potent antibacterial agent

Article abstract of DOI:10.1021/jp909520w

The present work reports a new class of antibacterial hydrogelators based on anti-inflammatory N-fluorenyl-9-methoxycarbonyl (Fmoc) amino acid/peptides functionalized cationic amphiphiles. These positively charged hydrogelators were rationally designed and developed by the incorporation of a pyridinium moiety at the C-terminal of Fmoc amino acid/peptides, because the pyridinium-based amphiphiles are a known antibacterial agent due to their cell membrane penetration properties. The Fmoc amino acid/peptide-based cationic amphiphiles efficiently gelate (minimum gelation concentration ～0.6-2.2%, w/v) water at room temperature. Judicious variation of amino acid and their sequences revealed the architectural dependence of the molecules on their gelation ability. Several microscopic techniques like field-emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) were used to obtain the visual insight of the morphology of the gel network. A number of spectroscopic techniques like circular dichroism, FTIR, photoluminescence, and XRD were utilized to know the involvement of several noncovalent interactions and participation of the different segments of the molecules during gelation. Spectroscopic results showed that the - Interaction and intermolecular hydrogen bonding are the major responsible factors for the self-assembled gelation process that are oriented through an antiparallel β-sheet arrangement of the peptide backbone. These Fmoc-based cationic molecules exhibited efficient antibacterial activity against both Gram-positive and Gram-negative bacteria.

Full text of DOI:10.1021/jp909520w

J. Phys. Chem. B 2010, 114, 4407–4415
4407
Hydrogelation Through Self-Assembly of Fmoc-Peptide Functionalized Cationic
Amphiphiles: Potent Antibacterial Agent
Sisir Debnath, Anshupriya Shome, Dibyendu Das, and Prasanta Kumar Das*
Department of Biological Chemistry, Indian Association for the CultiVation of Science,
JadaVpur, Kolkata - 700 032, India
ReceiVed: October 5, 2009; ReVised Manuscript ReceiVed: December 22, 2009
The present work reports a new class of antibacterial hydrogelators based on anti-inflammatory N-fluorenyl-
9-methoxycarbonyl (Fmoc) amino acid/peptides functionalized cationic amphiphiles. These positively charged
hydrogelators were rationally designed and developed by the incorporation of a pyridinium moiety at the
C-terminal of Fmoc amino acid/peptides, because the pyridinium-based amphiphiles are a known antibacterial
agent due to their cell membrane penetration properties. The Fmoc amino acid/peptide-based cationic
amphiphiles efficiently gelate (minimum gelation concentration ∼0.6-2.2%, w/v) water at room temperature.
Judicious variation of amino acid and their sequences revealed the architectural dependence of the molecules
on their gelation ability. Several microscopic techniques like field-emission scanning electron microscopy
(FESEM) and atomic force microscopy (AFM) were used to obtain the visual insight of the morphology of
the gel network. A number of spectroscopic techniques like circular dichroism, FTIR, photoluminescence,
and XRD were utilized to know the involvement of several noncovalent interactions and participation of the
different segments of the molecules during gelation. Spectroscopic results showed that the π-π interaction
and intermolecular hydrogen bonding are the major responsible factors for the self-assembled gelation process
that are oriented through an antiparallel ꢀ-sheet arrangement of the peptide backbone. These Fmoc-based
cationic molecules exhibited efficient antibacterial activity against both Gram-positive and Gram-negative
bacteria.
Introduction
as well as bacterial infections. Also for the biomedicinal use of
hydrogels, it is preferable the gelator to be antibacterial to avoid
Low molecular weight hydrogels (LMWHs), a novel class
of soft materials, have attracted large interest for the last two
decades due to their wide-ranging applications from biomedi-
cines to advanced materials.1-5 Noncovalent interactions like
electrostatic, dipole-dipole, van der Waals, π-π stacking, and
hydrogen bonding between the small molecules leads to the
formation of a self-assembled three-dimensional (3D) network
that can immobilize water.6 Among several LMWHs, peptide-
based gelators are finding great importance because of their
potential biocompatibility and applications in drug delivery,
tissue engineering, cell culture, and so on.7 These self-assembled
peptide hydrogels are known to form through varied supramo-
lecular structural motifs like R-helix, ꢀ-sheet, ꢀ-hairpin, coiled-
coil, and so on.8 Often it is seen that a small peptide or even an
amino acid with modified C- and N-termini either by aromatic
ring or a long alkyl chain can self-assemble to form supramo-
lecular gel.9
To this end, peptides containing aromatic moieties like
N-fluorenyl-9-methoxycarbonyl (Fmoc) are known to induce
excellent self-assembling properties resulting in hydrogela-
tion.7a-c,10 Interestingly, the Fmoc amino acid/dipeptides possess
intrinsic anti-inflammatory properties.11 In this context, li-
popolysaccharide (LPS), the key constituents of outer layer
membrane of Gram-negative bacteria, is also known to be one
of the major causes for stimulating proinflammatory responses
that may lead to sepsis.12 Therefore, the design of anti-
inflammatory agent having strong antibacterial activity will
surely eliminate the possibility of LPS-induced inflammation
the material associated nosocomial infection. Thus, to incor-
porate the antibacterial property, judicious functionalization (for
example, inclusion of cationic charge) in the molecular archi-
tecture of the Fmoc-based peptides is necessary. These cationic
peptides would exterminate the microbial cell membranes
resulting in the failure of the innate defense mechanism of
microorganisms.13 In this regard, pyridinium group is known
for its proven antimicrobial efficacies.13c Additionally, the planar
aromatic moiety can also induce π-π stacking interactions, a
key factor for the formation of supramolecular gels. Hence, the
self-assembly of a rationally designed molecule comprising of
a pyridium group, along with the intrinsic properties of Fmoc
amino acids/peptides will lead to the formation of a multifaceted
antimicrobial gel.
In the present work, we have designed and developed a class
of Fmoc amino acid/peptide functionalized cationic amphiphiles
(Figure 1) having pyridinium moiety at the C-terminal. These
amphiphiles exhibited excellent gelation ability (minimum
gelation concentration (MGC) ∼0.6-2.2%, w/v) in plain water
over a wide range of pH at room temperature. The aliphatic/
aromatic residues of the amino acids and their sequences were
altered to find out the respective roles on the gelation efficiency
of these Fmoc-based amphiphiles. These cationic amphiphilic
peptides were arranged as antiparallel ꢀ-sheets as confirmed
from XRD, FTIR, and fluorescence studies. The changes in the
supramolecular arrangement of the hydrogels with structural
variation at the molecular level were investigated using several
spectroscopic and microscopic techniques. Intriguingly, these
molecules showed excellent bactericidal activities having similar
* To whom correspondence should be addressed. Fax: +(91)-33-
24732805. E-mail: bcpkd@iacs.res.in.
10.1021/jp909520w  2010 American Chemical Society
Published on Web 03/18/2010

4408 J. Phys. Chem. B, Vol. 114, No. 13, 2010
Debnath et al.
protection was done by the reaction of this amine with Fmoc
chloride in dry DCM in presence of 1.1 equiv dry triethylamine
for 30 min. After the reaction the DCM solution was washed
with brine for several times and the pure Fmoc-protected product
was obtained by column chromatography using 100-200 mesh
silica gel and MeOH/CHCl3 as eluent. Next, the quaternization
at the pyridine “N” was done by the stirring with excess
iodomethane in dry DCM for 6 h. The reaction mixture was
taken in CHCl3 and washed with aqueous thiosulphate solution
and water, respectively. The pure quaternized iodide product
was obtained by column chromatography using 60-120 mesh
silica gel with MeOH/CHCl3 as solvent. The iodide salt thus
obtained was subjected to ion exchange on Amberlite Ira-400
chloride resin column to get the pure chlorides (1 and 2). Overall
yield was ∼50-60%.
Synthesis of Amphiphile 3-6. The amino acid coupled with
m-amino pyridine at “C” terminal end with free N-terminal after
deprotection (Boc) was obtained as described earlier. This amine
was further coupled with another Boc-protected amino acid
using DCC and HOBt in dry DCM at room temperature for
12 h. The product was purified in a similar way as mentioned
earlier and also Boc group was removed using TFA. Then the
Fmoc protection was done at the free N-terminal of the dipeptide
in a similar ways as described above. After completion of the
reaction, the product was precipitated out from the solvent DCM
and it was obtained by simple filtration and several time washing
with DCM. The product was further purified by column
chromatography using 100-200 mesh silica gel with MeOH/
CHCl3 as eluent. Then the quaternization at the pyridine “N”
was done by stirring with excess iodomethane in dry CHCl3/
MeOH mixture for 6 h and purified in a similar way as
mentioned above. The iodide salt thus obtained was subjected
to ion exchange on Amberlite Ira-400 chloride resin column to
get the pure chlorides (3-6). Overall yield was ∼40-50%.
Synthesis of Amphiphile 7. m-Amino pyridine is coupled
with Fmoc chloride in dry DCM in presence of 1.1 equiv dry
triethylamine for 1 h. The reaction mixture was worked up and
purified in a similar way as described above. Also, the
quaternization at the pyridine “N” was done exactly following
the same protocol as mentioned above. The pure quaternized
iodide product was obtained by column chromatography using
60-120 mesh silica gel and MeOH/CHCl3 as solvent. The
iodide salt thus obtained was subjected to ion exchange on
Amberlite Ira-400 chloride resin column to get the pure chloride
(7). Overall yield was ∼70-80%.
Figure 1. Structure of amphiphiles 1-13.
efficacies against both Gram-negative and Gram-positive bac-
teria. The antibacterial property strongly depends on the
structures of the gelators reaching up to a minimum inhibitory
concentration (MIC) of 10 µg/mL.
Experimental Section
Materials. All amino acids, di-tert-butyl dicarbonate (Boc
anhydride), N,N′-dicyclohexylcarbodiimide (DCC), 1-hydroxy-
benzotriazole (HOBT), Fmoc chloride, and solvents were
procured from SRL (India). Trifluoro acetic acid (TFA) was
purchased from Spectrochem, India. CDCl3 was obtained from
Aldrich Chemical Co. Thin layer chromatography were per-
1
formed on Merck precoated silica gel 60-F254 plates. H NMR
spectra were recorded in AVANCE 300 MHz (BRUKER)
spectrometer. Mass spectrometric data were acquired by the
electron spray ionization (ESI) technique on a Q-tof-micro
quadruple mass spectrometer (Micromass). The specific rotations
of the synthesized compounds were measured in Perkin-Elmer
(model 341LC) polarimeter. Fluorescence and FT-IR experi-
ments were done on Varian Cary Eclipse luminescence spec-
trometer and Perkin-Elmer Spectrum 100 FT-IR spectrometer,
respectively.
Synthesis of Amphiphile 8-13. The pure Boc-protected
amino acid/peptide coupled with m-amino pyridine was prepared
as described above. Then the quaternization at the pyridine “N”
was done exactly following the same protocol as mentioned
above. The pure quaternized iodide product was obtained by
column chromatography using 60-120 mesh silica gel and
MeOH/CHCl3 as solvent. The iodide salt thus obtained was
subjected to ion exchange on Amberlite Ira-400 chloride resin
column to get the pure chloride (8-13). Overall yield was
∼70-80. Characterization data for all the amphiphiles are given
in Supporting Information.
Preparation of Hydrogel. Required amount of the com-
pounds were added in 1 mL water in a screw-capped vial with
internal diameter (i.d.) of 10 mm and slowly heated until the
solid was completely dissolved. Then the solutions were cooled
to room temperature with out any disturbance. After 1 h,
formation of gel was confirmed by stable to inversion of the
glass vial. Only compound 2 gave gelation after keeping the
solution for 12 h at room temperature.
Synthetic Procedure. Synthetic scheme for all the am-
phiphiles (1-13) are given in Figure S1 (Supporting Informa-
tion).
Synthesis of Amphiphile 1 and 2. Required amino acid was
protected with tert-butyloxycarbonyl (Boc) group. Free COOH
group of this Boc protected L-amino acid was coupled with
m-amino pyridine using DCC and HOBt in DCM at room
temperature for 12 h. The reaction mixture was washed with
alkali followed by brine solution. Then DCM was evaporated
and the pure product was isolated by column chromatography
using 100-200 mesh silica gel with 1% MeOH/CHCl3 as eluent.
The product was subjected to deprotection by TFA (2 equiv)
in dry DCM. After 2 h of stirring, solvents were removed on a
rotary evaporator and the mixture was taken in ethyl acetate.
The EtOAc part was thoroughly washed with aqueous 10%
sodium carbonate solution followed by brine to neutrality. The
organic part was dried over anhydrous sodium sulfate and
concentrated to get the corresponding amine. Then Fmoc

Fmoc-Peptide Functionalized Cationic Amphiphiles
J. Phys. Chem. B, Vol. 114, No. 13, 2010 4409
Microscopic Studies. FESEM was performed on JEOL-
6700F microscope. A piece of hydrogel was mounted on glass
slide and dried for few hours under vacuum before imaging.
The morphology of the dried gels of 2 and 4 was studied using
atomic force microscopy (Veeco, modelAP0100) in noncontact
mode. A piece of gel was mounted on a silicon wafer and dried
for a few hours under vacuum before imaging.
Circular Dichroism (CD). CD spectra of the aqueous
solutions of 2, 4, and 11 at varying concentration were recorded
by using a quartz cuvette of 1 mm path length in a Jasco J-815
spectropolarimeter. Varying temperature CD spectra were also
recorded for compound 2 and 4 at concentration 0.025%, w/v
from 20 to 70 °C.
FTIR Measurements. FTIR measurements of the gelators
2 and 4 in CHCl3 solution and in gel state in D2O were carried
out in a Perkin-Elmer Spectrum 100 FT-IR spectrometer using
KBr and CaF2 windows, respectively, with 1 mm Teflon spacers
at their MGC.
Fluorescence Spectroscopy. The emission spectra of the
compounds 2 and 4 were recorded on Varian Cary Eclipse
luminescence spectrometer in a concentration range from
0.0025%, w/v, to above MGC. A super stock solution of 2 and
4 was prepared, which was diluted as required. Solutions were
excited at λex ) 280 nm. The excitation and emission slit width
were 5 and 5 nm, respectively.
aureus, 5 × 106-7.5 × 106 cfu/mL; for E. coli, 3.75 × 107-7.5
× 107 cfu/mL; and for P. aeruginosa, 9 × 107-1.2 × 108 cfu/
mL. All the test tubes were then incubated at 37 °C for 24 h.
The optical density of all the solutions was measured at 650
nm before and after incubation. Liquid medium containing
microorganisms was used as a positive control. All the experi-
ments were performed in triplicate and repeated twice.
FESEM of Amphiphile 4 Treated E. coli. E. coli (3.75 ×
107-7.5 × 107 cfu/mL) cells (1 mL) were treated with
compound 4 above MIC at 200 µg/mL. The bacterial cells with
and without (untreated cells as control) amphiphile were
incubated for 30-40 min. After incubation, the mixtures were
centrifuged at 5000 rpm for 5 min at 4 °C. The media was
removed completely and the cells were redispersed in 0.9 wt
% saline. Finally, 5 µL of the redispersed samples were mounted
on a glass slide and dried under vacuum for 4 h, and the SEM
images were taken on JEOL-6700F scanning electron microscope.
Fluorescence Microscopic Study. The Live/Dead BacLight
TM Bacterial Viability Kit was used to examine bacterial cell
viability under a fluorescence microscope. The kit contains a
mixture of two nucleic acid binding stains, specifically referred
to as SYTO 9 and propidium iodide. The kit was stored at -20
°C in dark, which is taken out and thawed at room temperature
just prior to assay. E. coli (3.75 × 107-7.5 × 107 cfu/mL) and
S. aureus (5 × 106-7.5 × 106 cfu/mL) cells (1 mL) were treated
with four above MIC and also untreated cells were taken in
centrifuge tube. The mixtures were centrifuged at 5000 rpm for
5 min at 4 °C. Then, media was removed completely and the
cells were redispersed in 0.9 wt % saline. Finally, 3 µL of the
BacLight dye mixture was added and incubated in dark at room
temperature for 15-20 min. After incubation, 5 µL of the
solution mixture was mounted over microscope slides, which
then air-dried and viewed under the light microscope (BX61,
Olympus) using an excitation filter of BP460-495 nm and a
band absorbance filter covering wavelength below 505 nm.
X-ray Diffraction (XRD). XRD measurements were taken
in Seifert XRD 3000P diffractometer and the source was Cu
KR radiation (R ) 0.15406 nm) with a voltage and current of
40 kV and 30 mA, respectively. Gel of 4 was mounted on the
glass slide and dried under vacuum. The xerogel was scanned
from 1-40°.
Microorganisms and Culture Conditions. The in vitro
antimicrobial activity of all the cationic amphiphiles was
investigated against representative Gram-positive and Gram-
negative bacteria. Gram-positive bacteria used in the present
study were Bacillus subtilis and Staphylococcus aureus. Gram-
negative bacteria investigated include Escherichia coli and
Pseudomonas aeruginosa. Investigations of antibacterial activi-
ties were performed by both broth dilution and spread plate
method. The LB medium (tryptone (10 g), yeast extract (5 g),
and NaCl (10 g) in 1 L sterile distilled water at pH 7.0) was
used as a liquid medium and LB agar (tryptone (10 g), yeast
extract (5 g), NaCl (10 g), and agar (15 g) in 1 L of sterile
distilled water at pH ) 7.0) was used as a solid medium in all
antibacterial experiments. All the microbial strains were pur-
chased from Institute of Microbial Technology, Chandigarh,
India. The stock solutions of all the amphiphiles as well as the
required dilutions were made in autoclaved sterile water.
Results and Discussion
Rational design and synthesis of low molecular weight
gelators (LMWGs) with a correlation between the structure and
gelation property are intensified in recent years due to their
potential applications in diversified scientific arena. To develop
functional hydrogels, herein we have synthesized a series of
N-terminal protected Fmoc amino acids/peptides that have
pyridinium moiety at the C-terminal (1-6, Figure 1). Cationic
charge within these compounds were introduced through the
coupling of m-amino pyridine at the C-terminal of Fmoc-
protected amino acids or peptides followed by methylation at
the heterocyclic nitrogen of the pyridine ring (Figure S1,
Supporting Information). The gelation ability of the amphiphiles
was tested by stable to inversion of glass vial at room
temperature (Table 1).
Gelation Studies. To check the gelation efficiency we started
with Fmoc-protected aliphatic (L-isoleucine, 1) and aromatic (L-
phenylalanine, 2) amino acid compounds functionalized at
C-terminal. Amphiphile 1 with aliphatic side chain substituted
amino acid was found to be a nongelator. Amphiphile 2
comprising of an aromatic moiety at the side chain exhibited
gelation ability with a MGC value of 1%, w/v (Table 1), while
12 h was required for this gelation. This result indicates that,
in addition to the two aromatic segments, Fmoc and the
pyridinium (Py+) moiety, the presence of another π-π stacking
unit (phenyl ring of 2) is important to induce gelation for single
amino acid based amphiphiles. At this point, we were interested
to know whether the presence of a dipeptide unit instead of the
The freeze-dried ampules of all bacterial strains were opened
and a loopful of culture was spread to give single colonies on
the respective solid LB agar media and incubated for 24 h at
37 °C. A representative single colony was picked up with a
wire loop and was spread on an agar slant to give single
colonies. The slants were incubated at 37 °C for the respective
time. These incubated cultures of all the bacteria were diluted
as required to give a working concentration in the range of
106-109 colony forming units (cfu)/mL before every experiment.
Antimicrobial Studies. Minimum inhibitory concentrations
(MICs) of 1-6 were estimated by both broth dilution and spread
plate method. MIC was measured using a series of test tubes
containing the amphiphiles (0.05-200 µg/mL) in 5 mL of liquid
medium. Diluted microbial culture was added to each test tube
in identical concentration to obtain the working concentration
of bacteria as for B. subtilis, 7.5 × 107-1 × 108 cfu/mL; for S.

4410 J. Phys. Chem. B, Vol. 114, No. 13, 2010
TABLE 1: Gelation Results of 1-13 in Plain Water
Debnath et al.
were swapped in these amphiphiles (Figure 1). It would be
intriguing to know which position is preferred by the aromatic
moiety in the molecular architecture of the gelator for displaying
better gelation efficiency. In the case of 5, where the aromatic
amino acids was closer to the N-terminal Fmoc segment and
the aliphatic amino acid was attached to the Py+ unit, MGC
value was 1.5%, w/v (Table 1), which is slightly lower than
that found in case of the 3 (MGC ) 1.7%, w/v). However, as
the dipeptide sequence in the gelator 5 was reversed to form
compound 6 having aliphatic amino acid at the N-terminal
attached to Fmoc moiety, gelation efficiency decreased (MGC
) 2.2%, w/v) with the formation of an opaque gel. This result
indicates that the presence of a nonplanar aliphatic substitution
closer to the Fmoc unit possibly hinders the π-π stacking of
the extended aromatic rings resulting decline in the gelation
efficiency of 6. The importance of the amino acid/peptide moiety
in the hydrogelation by Fmoc functionalized compounds was
further established when compound 7 (Figure 1), having no
amino acid unit between Fmoc and Py+ unit, was found to be
a nongelator.
compound
statea
MGC (%, w/v)
1
2
3
4
5
6
7
S
TG
TG
TG
TG
OG
S
1
1.7
0.6
1.5
2.2
8
9
S
S
10
11
12
13
S
TG
S
3.0
S
a S: solution (soluble up to 10%, w/v); TG: transparent gel; OG:
opaque gel.
single amino acid between the Fmoc and the Py+ moieties can
improve the gelation. To this end, we have synthesized different
dipeptide compounds (3-6, Figure 1) comprising both aromatic
(L-phenylalanine) and aliphatic (L-isoleucine) amino acids in
varying combinations. Fascinatingly, all these compounds
showed excellent hydrogelation ability (Table 1), and the
gelation occurs within a few minutes of dissolving the com-
pounds in water. Interestingly, the dipeptide gelator 3 with two
aliphatic isoleucine amino acids showed MGC at 1.7%, w/v
(Table 1). This result indicates the importance of the additional
hydrogen bonding peptide linkage within the dipeptide moiety
in hydrogelation. Even the absence of an aromatic moiety in
the amino acid unit can be compensated with the presence of
an extra hydrogen-bonding moiety between two aliphatic amino
acids that converted the nongelator (1) to an efficient gelator
(3). So the presence of an aromatic side chain for single amino
acid (2) or at least a peptide bond (3) is crucial toward
hydrogelation of the Fmoc amino acid/peptide functionalized
amphiphiles. On this basis, we have synthesized Fmoc dipeptide
functionalized cationic amphiphile, 4, having a diphenylalanine
unit with the expectation of better gelation efficiency. Indeed,
the amphiphilic dipeptide 4 showed superior gelation efficiency
with a MGC value of 0.6%, w/v.
Furthermore, the importance of the planar aromatic ring of
Fmoc on the gelation efficiency of these cationic amphiphiles
(1-6) was also investigated. For this purpose, tert-butyloxy-
carbonyl (Boc) unit, which neither had the aromaticity nor the
planarity, was utilized as the protecting agent at the N-terminal
of amino acid/peptide. Keeping all other structural motif
identical as in amphiphiles 1-6, only the Fmoc group was
replaced by the Boc moiety in 8-13 (Figure 1), respectively.
All the Boc containing cationic amino acids or dipeptides were
found to be nongelators except 11 that showed the ability to
form a transparent gel having MGC ) 3.0%, w/v. The loss of
ordered supramolecular arrangement in presence of nonplanar
Boc moiety is the most obvious reason for the nongelation of
all the Boc-protected cationic amphiphiles. However, the two
aromatic rings of the diphenylalanine peptide in compound 11
might have helped in the restoration of the ordered supramo-
lecular arrangement through its π-π interaction during gelation.
This observation indicates the important participation of π-π
stacking of diphenylalanine units in the self-assembling process
that resulted in the hydrogelation of 11. However, the importance
of the extended aromatic ring of Fmoc was stressed once again
as the gelation efficiency of 11 (MGC ) 3.0%, w/v) is five
times lower than the corresponding Fmoc analogue, 4 (MGC
To investigate the effect of dipeptide moiety comprising both
aliphatic and aromatic side chains substituted amino acids in
the gelation efficiency, compounds 5 and 6 were prepared. The
positions of L-isoleucine and L-phenylalanine at N- and C-termini
Figure 2. (a-f) FESEM images of the dried gels of 2-6 and 11, respectively at MGC.

Fmoc-Peptide Functionalized Cationic Amphiphiles
J. Phys. Chem. B, Vol. 114, No. 13, 2010 4411
Figure 3. (a,b) AFM images of 2 and 4 at their respective MGC (scale
bars ) 500 nm).
) 0.6%, w/v). All the hydrogels were thermoreversible and the
gel-to-sol transition temperature was ∼50-60 °C at their MGC.
Microscopy. Supramolecular morphologies of the gelators
2-6 and 11 at their MGC was investigated by field emission
scanning electron microscopy (FESEM). All the dried gel
showed fibrous networks (Figure 2a-f), although their nature
and size were different due to the variation in the supramolecular
arrangement, depending on the structure of LMWGs. Gelator
2, having one amino acid (L-phenylalanine) unit, showed
entangled fibrous morphology with an individual fiber diameter
of ∼60 nm. Some of the fibers were coiled together to form
thicker fibers of ∼300 nm diameter (Figure 2a). Xerogels of
dipeptides 3 (aliphatic-aliphatic) and 4 (aromatic-aromatic)
also gave an intertwined fibrous network having a fiber thickness
of ∼80 and 150 nm, respectively (Figure 2b,c). The length of
the fibers of gelator 4 (Figure 2c) was found to be several
micrometers with uniform diameter. This intertwined fibrous
network of 4 with a large length and relatively thicker diameter
possibly helps to entrap more water molecules within its
supramolecular structure leading to the formation of most
efficient hydrogel among all the amphiphiles. Compound 5 also
showed similar intertwined fibrous morphology having fiber
thickness ∼100 nm (Figure 2d). However, amphiphile 6
exhibited very thin fibrous network having fiber thickness
∼40 nm, while the only Boc-protected gelator 11 showed a
similar thin scattered fibril structure (Figure 2e,f). The thin
fibrous morphology of compounds 6 and 11 made them
relatively weak gelators with a MGC value of 2.2 and 3%, w/v,
respectively. The fibrous morphology of xerogels of 2 and the
best gelator 4 has been further confirmed from their atomic force
microscopy (AFM) images (Figure 3a,b). In concurrence with
the observed FESEM images, xerogel of 2 showed intertwined
fibrous network having fiber diameter ∼300 nm (Figure 3a) and
the dipeptide 4 gave several micrometer length fiber of 140 nm
diameter (Figure 3b).
Circular Dichroism (CD) Spectroscopy. The supramolecular
arrangement in the organized aggregates was further confirmed
from circular dichroism (CD) spectroscopy of the gelators 2, 4,
and 11 (Figure 4a-c) in water. Compound 2 showed negative
cotton effects in the CD spectra having double minima at 190
and 210 nm (Figure 4a). The nature of the spectra is analogous
to that was generally observed for the helical structure of protein
by its pattern14 but not by the positions of the double minima.
However, the supramolecular arrangements of dipeptide gelators
(4, 11) are different (Figure 4b,c) from that of the single amino
acid containing 2. The CD spectrum of gelator 4 showed a
negative peak at 218 nm and a positive peak at 197 nm (Figure
4b). This particular pattern of CD spectra confirmed that the
self-assembled structure of compound 4 is mostly dominated
with ꢀ-sheet arrangement of the peptide backbone.15 Gelator
11 having same dipeptide moiety with Boc protection also
Figure 4. CD spectra of 2, 4, and 11 with varying concentration (%,
w/v) in plain water at 25 °C.
showed similar CD spectra to that of 4, which proves that the
dipeptide unit is the key factor for this ꢀ-sheet dominated
supramolecular structure. In all the cases, the formation of a
higher ordered structure was originated from the supramolecular
association of individual molecules that showed a sharp increase
in the peak intensity with the increasing gelator concentration.
The noncovalent packing of the molecules was further confirmed
from the temperature dependent CD experiment of 2 and 4 at
0.025%, w/v (Figure S2, Supporting Information) in water. In
both cases, intensity of the CD peak gradually decreased with
an increase in temperature from 20 to 70 °C due to the
destruction of the 3D-aggregated structure.
FT-IR Spectroscopy. To investigate the involvement of
hydrogen bonding of the peptide (amide) moiety during the self-
assembly of gelators, FTIR spectra of 2 and 4 were taken at
the self-assembled state in D2O and at non-self-assembled state
in chloroform solution. Compound 2 in chloroform gives
carbonyl-stretching frequency (amide-I) at 1708 cm-1, which

4412 J. Phys. Chem. B, Vol. 114, No. 13, 2010
Debnath et al.
Figure 6. (a) Luminescence spectra of compound 2 at varying
concentrations in water at room temperature at λex 280 nm: [2] (%,
w/v) a ) 0.0025; b ) 0.005; c ) 0.01; d ) 0.05; e ) 0.1; f ) 0.5; g
) 1; h ) 1.5; Inset is the normalized spectra of 0.0025 and 1%, w/v,
of 2. (b) Luminescence spectra of compound 4 at varying concentrations
in water at room temperature at λex 280 nm: [4] (%, w/v) a ) 0.0025;
b ) 0.005; c ) 0.01; d ) 0.05; e ) 0.1; f ) 0.5; g ) 1; Inset is the
normalized spectra of 0.0025 and 0.5%, w/v, of 4.
Figure 5. (a,b) FT-IR spectra of 2 and 4 in CHCl3 solution (---) and
in D2O at the gel state (s).
at the gel state in D2O decreased to 1655 cm-1 (Figure 5a).
This shifting indicates the formation of intermolecular H-
bonding of carbonyl group of one molecule with the amide-
NH of another molecule at the gel state. Existence of the
intermolecular interactions was also seen for 4 as the two
amide-I peaks at 1708 and 1678 cm-1 in CHCl3 solution shifted
to 1684 and 1636 cm-1, respectively, at the gel state in D2O
(Figure 5b). The presence of this two characteristic peaks
designate the ꢀ-sheet structure of the supramolecular aggregates
with antiparallel arrangement of the peptide backbone.16
Luminescence Studies. Participation of the fluorenyl moiety
of the Fmoc group in the gelation was investigated by taking
the luminescence spectra of gelators 2 and 4 with varying
concentrations in water (Figure 6). The molecules were excited
at 280 nm. At very dilute concentration (0.0025%, w/v),
compounds 2 and 4 were found to remain at the molecular state,
as it showed emission at λem ) 307 nm. With the increase of
concentration, the intensity of the peaks initially increases
followed by decrease with continuous red shift of the λem of
fluorenyl group. At a concentration of 1%, w/v of both the
gelators, the emission was observed at λem ) 323 nm, which
confirms the self-assembly of the fluorenyl moiety where this
extended aromatic rings were overlapped in an antiparallel
orientation.17 The red shift of the short wavelength emission
(normalized intensity) with increase in concentration of gelator
is shown in the inset of the corresponding figure. Also, at higher
concentration of the amphiphiles, a broad phosphorescence peak
appeared at 425 nm. This indicates the more efficient overlap-
ping of fluorenyl as well as the phenyl ring at the self-assembled
state.17c At 1%, w/v, of compound 2, a small peak at 361 nm is
generated, which is the characteristic peak for parallel orientation
of fluorenyl group.17b Thus, in the gel state, these Fmoc
functionalized cationic amphiphiles self-assemble mostly by the
antiparallel overlapping through π-π stacking of the aromatic
rings.
Wide Angle X-ray Scattering (WAXS). To establish further
the molecular packing and the possible orientation of the gelator
in the supramolecular fibrous networks, we have taken the wide-
angle X-ray diffraction (WXRD) spectra of the dried gels of 4.
Xerogel obtained from gel 4 gave a peak at 2θ ) 20.05°
corresponding to d-spacing 4.6 Å, which is accompanied by
another peak at 2θ ) 9.4° with a d-spacing of 9.5 Å (Figure
7). These two peaks indicate the presence of the cross ꢀ-structure
between the dipeptide moieties with an antiparallel alignment
in the peptide strand.18 This observation is also supported from
the results obtained from the CD and FTIR spectroscopy where

Fmoc-Peptide Functionalized Cationic Amphiphiles
J. Phys. Chem. B, Vol. 114, No. 13, 2010 4413
TABLE 2: Antibacterial Activity (MICs) of the Amphiphiles
1-6 in µg/mL
Gram-positive
Gram-negative
E. coli P. aerugenosa
amphiphile
B. subtilis
S. aureus
1
2
3
4
5
6
150
30
30
25
30
50
200
20
30
10
20
150
75
20
40
40
20
50
a
150
100
200
200
200
a No killing up to 200 µg/mL.
activity.13 As the Fmoc-based compounds possess positively
charged pyridinium moiety, they also could be potential small
molecule antibacterial agents. Antibacterial activities of all the
Fmoc-based peptides (1-6) were checked against both types
of Gram-positive (Bacillus subtilis and Staphylococcus aureus)
and Gram-negative (Escherichia coli and Pseudomonas aerugi-
nosa) bacteria. Minimum inhibitory concentrations (MIC), the
lowest amphiphile concentration at which no viable bacterial
cell is present, are presented in Table 2. Interestingly, almost
all the Fmoc-based cationic amphiphiles showed killing efficacy
against both Gram-positive and Gram-negative bacteria. Com-
pound 1 having nonaromatic L-isoleucine showed relatively low
antibacterial activity against Gram-positive bacteria (MIC 150
and 200 µg/mL for B. subtilis and S. aureus, respectively). MICs
of 1 against Gram-negative bacteria E. coli and P. aerugenosa
were 150 and 75 µg/mL, respectively. However, dipeptide of
L-isoleucine (3) showed better activity than 1 (Table 1), but it
cannot kill E. coli up to 200 µg/mL. Among all the Fmoc-based
amphiphiles, 2, 4, and 5 are most effective in killing both types
of bacteria. Compound 2 had the lowest MIC values within
20-50 µg/mL, irrespective of the nature of bacteria. Also,
amphiphile 4 exhibited the lowest MIC (10 µg/mL) against
Gram-positive S. aureus. Interestingly, in these three am-
phiphiles, L-phenylalanine was attached to the Fmoc moiety.
The close proximity of fluorenyl and phenyl moieties is
presumably helping to achieve an optimum hydrophobicity
known as “threshold hydrophobicity” that is required for the
Figure 7. XRD diagrams of the dried gels of 4.
the presence of the ꢀ-structure between the dipeptide moieties
was indicated. The periodicity of 4.6 Å arises from spacing
between the peptide chains with in a ꢀ-sheet where as the other
peak of periodicity 9.4 Å was due to the spacing between two
stacked ꢀ-sheet layers. Another peak at d-spacing 3.5 Å (2θ )
23.01°) further established the presence of π-π stacking
between two antiparallel Fmoc group in the self-assembled
ꢀ-sheet structure.19 The observed spectroscopic and microscopic
studies suggest an ordered arrangement of the Fmoc-peptide
functionalized cationic amphiphile involving the H-bonding,
π-π stacking interaction, and the higher order packing in the
3D network of the hydrogel (representative example is given
for 4 in Figure 8).
Antibacterial Activity. As mentioned earlier that biome-
dicinal applications of these anti-inflammatory Fmoc-peptide-
based cationic amphiphiles can be dramatically enhanced if they
also become simultaneously antibacterial. To this end, “cationic
peptide antibiotics”, as well as quaternary ammonium and
pyridinium amphiphiles, are known to exhibit antibacterial
Figure 8. Probable arrangement of 4 in the gel state.

4414 J. Phys. Chem. B, Vol. 114, No. 13, 2010
Debnath et al.
Conclusion
Herein, we have developed Fmoc-peptide functionalized
cationic hydrogel. Systematic analysis with special emphasis
on the structural aspects as well as the mechanism of the gelation
processes reveals that a minute architectural change at the
molecular level influences the self-assembling mechanism.
Noncovalent interactions including π-π stacking and intermo-
lecular hydrogen bonding were found to be the major responsible
factors for the gelation process. Several spectroscopic and
microscopic studies confirm the antiparallel ꢀ-sheet arrangement
of the self-assembled peptides in the gel phase. These Fmoc-
based cationic peptide exhibited efficient antibacterial activity
against both Gram-positive and Gram-negative bacteria. De-
velopment of such antibacterial soft matters (biomaterials) from
Fmoc-protected amino acid/peptide-based cationic gelators
comprising efficient antibacterial activity will have immense
importance in the material science.
Figure 9. Scanning electron microscopic (SEM) image of (a) control
bacterium E. coli; (b) E. coli after treatment with 200 µg/mL of
amphiphile 4.
effective killing of bacteria.20 Again, the swapping of L-
phenylalanine from N-terminal Fmoc unit to C-terminal pyri-
dinium group in compound 6 resulted in comparatively poor
antibacterial efficacy, possibly because of the loss of said
“threshold hydrophobicity”. Thus, antibacterial activity of these
Fmoc-peptide-based cationic amphiphiles is dependent on each
structural motif as well as on their sequence in the molecule.
Although exact mechanism of bactericidal effect remains to
be better understood, but in general it seems that the antimi-
crobial effect is originated from the ability of the cationic
compounds to penetrate the cell membrane of the microorgan-
isms.21 Positively charged peptide/amphiphiles are believed to
attack negatively charged cell membrane of microbes, which is
also entropically favorable as huge numbers of counterions are
released.22 Next, the hydrophobic moiety helps in the diffusion
of the small molecules by “self-promoted” transport across the
bacterial membrane resulting in release of cytoplasmic con-
stituents leading to the death of bacteria.13b,23 In general, it has
been observed that it is relatively easier to kill Gram-positive
bacteria rather than Gram-negative because the former have a
simple cell wall whose major constituent is peptidoglycan, a
polysaccharide. In contrast, Gram-negative has an additional
outer bilayer membrane composed of lipopolysaccharides and
phospholipids.13c However, the Fmoc-based cationic amphiphiles
used in the present study are almost equally effective in killing
both bacteria. To unravel the killing mechanism further in depth
investigation is required.
Acknowledgment. P.K.D. is thankful to Department of
Science and Technology, India for financial assistance through
Ramanna Fellowship (No. SR/S1/RFPC-04/2006). S.D., A.S.,
and D.D. acknowledge Council of Scientific and Industrial
Research, India for their Research Fellowships.
Supporting Information Available: Generalized synthetic
schemes, characterization for all the amphiphiles, temperature
dependence CD spectra and Live/Dead fluorescence micrograph
of E. coli and S. aureus before and after treating with 4, are
provided in the Supporting Information. This material is
available free of charge via the Internet at http://pubs.acs.org.
References and Notes
(1) (a) Terech, P.; Weiss, R. G. Chem. ReV. 1997, 97, 3133. (b) Estroff,
L. A.; Hamilton, A. D. Chem. ReV. 2004, 104, 1201. (c) Tiller, J. C. Angew.
Chem., Int. Ed. 2003, 42, 3072. (d) Sangeetha, N. M.; Maitra, U. Chem.
Soc. ReV. 2005, 34, 821. (e) Guan, J.; He, H.; Hansford, D. J.; Lee, L. J. J.
Phys. Chem. B 2005, 109, 23134.
(2) (a) George, M.; Weiss, R. G. Acc. Chem. Res. 2006, 39, 489. (b)
Bhattacharya, S.; Srivastava, A.; Pal, A. Angew. Chem., Int. Ed. 2006, 45,
2934. (c) Vemula, P. K.; John, G. Chem. Commun. 2006, 2218. (d) Vemula,
P. K.; Aslam, U.; Mallia, V. A.; John, G. Chem. Mater. 2007, 19, 138. (e)
Song, F.; Zhang, L. J. Phys. Chem. B 2008, 112, 13749.
To get the insight into the effect of amphiphiles on the
morphology of bacteria,13b,24 FESEM images of E. coli were
taken before and after the treatment with amphiphile 4 above
MIC at 200 µg/mL (Figure 9). The untreated bacteria E. coli
(Figure 9a) exhibited integrity of cell membrane. The incubation
of E. coli with amphiphile 4 for around 30-40 min causes a
significant change in their cell morphology where the SEM
image showed deformation in the membrane integrity of dead
E. coli (Figure 9b). The antibacterial activity of the representa-
tive amphiphile 4 was also studied by fluorescence microscopy
using commercially available Live/Dead BacLight bacterial
viability kit.13a,b The kit is composed of two nucleic acid binding
stains, known as SYTO 9 and propidium iodide. These dyes
have different cell penetration properties as well as different
spectral characteristics. SYTO 9 binds to the nucleic acid of
both living and dead cells and fluoresce green after excitation
with BP460-495 nm filter. Propidium iodide can only bind to
the nucleic acid of the dead cell and shows red fluorescence.
The untreated bacterial cells of E. coli and S. aureus exhibited
green fluorescence (Figures S3a and S3c, Supporting Informa-
tion), indicating that they were alive. After treating with the
200 and 50 µg/mL of 4 for E. coli and S. aureus cells,
respectively, only red fluorescence (Figures S3b and S3d,
Supporting Information) was observed that confirmed the killing
efficiency of the amphiphile.
(3) (a) Ko¨hler, K.; Fo¨rster, G.; Hauser, A.; Dobner, B.; Heiser, U. F.;
Ziethe, F.; Richter, W.; Steiniger, F.; Drechsler, M.; Stettin, H.; Blume, A.
J. Am. Chem. Soc. 2004, 126, 16804. (b) Herres, A.; van der Pol, C.; Stuart,
M.; Friggeri, A.; Feringa, B. L.; van Esch, J. J. Am. Chem. Soc. 2003, 125,
14252. (c) Nakashima, T.; Kimizuka, N. AdV. Mater. 2002, 14, 1113. (d)
Bieser, A. M.; Tiller, J. C. Chem. Commun. 2005, 3942.
(4) (a) van Bommel, K. J. C.; van der Pol, C.; Muizebelt, I.; Friggeri,
A.; Meetsma, A.; Feringa, B. L.; van Esch, J. Angew. Chem., Int. Ed. 2004,
43, 1663. (b) Estroff, L. A.; Hamilton, A. D. Angew. Chem., Int. Ed. 2000,
39, 3447.
(5) (a) Yang, Z.; Xu, B. Chem. Commun. 2004, 2424. (b) Kohler, K.;
Forster, G.; Hauser, A.; Dobner, B.; Heiser, U. F.; Ziethe, F.; Richter, W.;
Steiniger, F.; Drechsler, M.; Stettin, H.; Blume, A. Angew. Chem., Int. Ed.
2004, 43, 245. (c) Friggeri, A.; Feringa, B. L.; van Esch, J. J. Controlled
Release 2004, 97, 241. (d) John, G.; Vemula, P. K. Soft Matter 2006, 2,
909. (e) Kar, T.; Debnath, S.; Das, D.; Shome, A.; Das, P. K. Langmuir
2009, 25, 8639.
(6) (a) Suzuki, M.; Nanbu, M.; Yumoto, M.; Shiraib, H.; Hanabusa,
K. New. J. Chem. 2005, 29, 1439. (b) Suzuki, M.; Sato, T.; Shiraib;
Hanabusa, K. New. J. Chem 2006, 30, 1184.
(7) (a) Jayawarna, V.; Ali, M.; Jowitt, T. A.; Miller, A. E.; Saiani, A.;
Gough, J. E.; Ulijn, R. V. AdV. Mater. 2006, 18, 611. (b) Zhang, Y.; Gu,
H. W.; Yang, Z. M.; Xu, B. J. Am. Chem. Soc. 2003, 125, 13680. (c) Mahler,
A.; Reches, M.; Rechter, M.; Cohen, S.; Gazit, E. AdV. Mater. 2006, 18,
1365. (d) Fung, S. Y.; Yang, H.; Bhola, P. T.; Sadatmousavi, P.; Muzar,
E.; Liu, M.; Chen, P. AdV. Funct. Mater. 2009, 19, 74.
(8) (a) Smith, A. M.; Acquah, S. F. A.; Bone, N.; Kroto, H. W.;
Ryadnov, M. G.; Stevens, M. S. P.; Walton, D. R. M.; Woolfson, D. N.
Angew. Chem., Int. Ed. 2005, 44, 325. (b) Zhang, S. G.; Holmes, T.;
Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3334. (c)

Fmoc-Peptide Functionalized Cationic Amphiphiles
J. Phys. Chem. B, Vol. 114, No. 13, 2010 4415
Haines, L. A.; Rajagopal, K.; Ozbas, B.; Salick, D. A.; Pochan, D. J.;
Schneider, J. P. J. Am. Chem. Soc. 2005, 127, 17025.
(15) (a) Mayo, K. H.; Ilyina, E.; Park, H. Protein Sci. 1996, 5, 1301.
(b) Laczko, I.; Vass, E.; Soos, K.; Fulop, L.; Zarandi, M.; Penke, B. J.
Pept. Sci. 2008, 14, 731. (c) Escuder, B.; Miravet, J. F. Langmuir 2006,
22, 7793.
(16) (a) Matsuzawa, Y.; Ueki, K.; Yoshida, M.; Tamaoki, N.; Nakamura,
T.; Sakai, H.; Abe, M. AdV. Funct. Mater 2007, 17, 1507. (b) Rapaport,
H.; Kjaer, K.; Jensen, T. R.; Leiserowitz, L.; Tirrell, D. A. J. Am. Chem.
Soc. 2000, 122, 12523. (c) Rapaport, H.; Grisaru, H.; Silberstein, T. AdV.
Funct. Mater. 2008, 18, 2889.
(17) (a) Channon, K. J.; Devlin, G. L.; Magennis, S. W.; Finlayson,
C. E.; Tickler, A. K.; Silva, C.; MacPhee, C. E. J. Am. Chem. Soc. 2008,
130, 5487. (b) Gao, J.; Wang, H.; Wang, L.; Wang, J.; Kong, D.; Yang, Z.
J. Am. Chem. Soc. 2009, 131, 11286. (c) Yang, Z.; Gu, H.; Zhang, Y.;
Wanga, L.; Xu, B. Chem. Commun. 2004, 208.
(18) Balbach, J. J.; Ishii, Y.; Antzutkin, O. N.; Leapman, R. D.; Rizzo,
N. W.; Dyda, F.; Reed, J.; Tycko, R. Biochemistry 2000, 39, 13748.
(19) George, S. J.; Ajayaghosh, A. Chem.sEur. J. 2005, 11, 3217.
(20) (a) Shai, Y.; Oren, Z. Peptides 2001, 22, 1629. (b) Avrahami, D.;
Shai, Y. J. Biol. Chem. 2004, 279, 12277.
(21) Salick, D. A.; Kretsinger, J. K.; Pochan, D. J.; Schneider, J. P.
J. Am. Chem. Soc. 2007, 129, 14793.
(22) Ku¨gler, R.; Bouloussa, O.; Rondelez, F. Microbiology 2005, 151,
1341.
(23) (a) Castillo, J. A.; Clapes, P.; Infante, M. R.; Comas, J.; Manresa,
A. J. Antimicrob. Chemother. 2006, 57, 691. (b) Ioannou, C. J.; Hanlon,
G. W.; Denyer, S. P. Antimicrob. Agents Chemother. 2007, 51, 296.
(24) Mangoni, M. L.; Papo, N.; Barra, D.; Simmaco, M.; Bozzi, A.; Di
Giulio, A.; Rinaldi, A. C. Biochem. J. 2004, 380, 859.
(9) (a) Roy, S.; Dasgupta, A.; Das, P. K. Langmuir 2007, 23, 11769.
(b) Shome, A.; Debnath, S.; Das, P. K. Langmuir 2008, 24, 4280. (c) Das,
D.; Dasgupta, A.; Roy, S.; Mitra, R. N.; Debnath, S.; Das, P. K. Chem.sEur.
J. 2006, 12, 5068. (d) Bhattacharya, S.; Ghosh, Y. K. Chem. Commun.
2001, 185. (e) Mitra, R. N.; Das, D.; Roy, S.; Das, P. K. J. Phys. Chem. B
2007, 111, 14107. (f) Gao, P.; Zhan, C.; Liu, L.; Zhoua, Y.; Liu, M. Chem.
Commun. 2004, 1174. (g) Dutta, S.; Shome, A.; Debnath, S.; Das, P. K.
Soft Matter 2009, 5, 1607. (h) Debnath, S.; Shome, A.; Dutta, S.; Das,
P. K. Chem.sEur. J. 2008, 14, 6828.
(10) (a) Smith, A. M.; Williams, R. J.; Tang, C.; Coppo, P.; Collins,
R. F.; Turner, M. L.; Saiani, A.; Ulijn, R. V. AdV. Mater. 2008, 20, 37. (b)
Yang, Z.; Xu, K.; Wang, L.; Gu, H.; Wei, H.; Zhangc, M.; Xu, B. Chem.
Commun. 2005, 4414. (c) Todd, S. J.; Scurr, D. J.; Gough, J. E.; Alexander,
M. R.; Ulijn, R. V. Langmuir 2009, 25, 7533.
(11) Burch, R. M.; Weitzberg, M.; Blok, N.; Muhlhauser, R.; Martin,
D.; Farmer, S. G.; Bator, J. M.; Connor, J. R.; Ko, C.; Kuhn, W.; Mcmillan,
B. A.; Raynor, M.; Shearer, B. G.; Tiffany, C.; Wilkins, D. Proc. Natl.
Acad. Sci. U.S.A. 1991, 88, 355.
(12) Muhle, S. A.; Tam, J. P. Biochemistry 2001, 40, 5777.
(13) (a) Roy, S.; Das, P. K. Biotechnol. Bioeng. 2008, 100, 756. (b)
Mitra, R. N.; Shome, A.; Paul, P.; Das, P. K. Org. Biomol. Chem. 2009, 7,
94. (c) Haldar, J.; Kondaiah, P.; Bhattacharya, S. J. Med. Chem. 2005, 48,
3823. (d) Zasloff, M. Nature 2002, 415, 389. (e) Makovitzki, A.; Avrahami,
D.; Shai, Y. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15997. (f) Liu, L.;
Xu, K.; Wang, H.; Tan, J.; Fan, P. K. W.; Venkatraman, S. S.; Li, L.; Yang,
Y. Nat. Nanotechnol. 2009, 4, 457.
(14) (a) Kuroda, Y.; Maeda, Y.; kagawa, T. J. Am. Chem. Soc. 2000,
122, 12596. (b) Kunjithapatham, R.; Oliva, F. Y.; Doshi, U.; Perez, M.;
Vila, J. A.; Munoz, V. Biochemistry 2005, 44, 149.
JP909520W