allowed to come to room temperature and stirred for 12 h. Work
up, and extraction of this reaction were carried out similarly as
mentioned above. The residue thus obtained was then purified
by a silica gel 60–120 mesh column using ethyl acetate/toluene
as the eluent. After that the purified BOC-protected long chain
dipeptide was subjected to deprotection by trifluoroacetic acid
(TFA, 32 mmol) in dry DCM at room temperature. After 2 h
of stirring, the solvent was removed on a rotary evaporator and
the residue was taken up in ethyl acetate, which was thoroughly
washed with aqueous 10% sodium carbonate solution followed
by brine to neutrality. The organic part was dried over anhydrous
sodium sulfate and concentrated to get the corresponding amine
(7 mmol). The free amine (7 mmol) was quaternized with excess
iodomethane using anhydrous potassium carbonate (7.7 mmol for
2, 4 and 15.4 mmol for 1, 3, 5 and 6) and a catalytic amount of
18-crown-6-ether in dry DMF for 5–6 h at room temperature (25–
27 ◦C). After concentrating the reaction mixture, the residue was
taken up in ethyl acetate and washed with aqueous 5% thiosulfate
solution and brine to neutrality. The concentrated ethyl acetate
part was purified using a 60–120 mesh silica gel column using a
mixture of chloroform/methanol as the eluent and crystallized
from methanol/ether to obtain the solid quaternized iodide salt,
which was subjected to ion exchange on an Amberlite Ira-400
chloride ion exchange resin column to get the colorless chloride
salts of 1–6. The overall yields were 62, 69, 63, 70, 70, and 68% for
1–6, respectively.
Experimental
Materials
Silica gel of 60–120 mesh, L-tryptophan, L-phenylalanine,
L-proline, n-tetradecylamine, N,N-dicyclohexylcarbodiimide
(DCC), 4-N,N-(dimethylamino)pyridine (DMAP), 1-hydroxy-
benzotriazole (HOBT), iodomethane, solvents and all other
reagents were procured from SRL, India. Water used throughout
the study was Milli-Q water. Thin layer chromatography was
performed on Merck precoated silica gel 60-F254 plates. CDCl3,
Amberlite Ira-400 chloride ion exchange resins were obtained
from Aldrich Chemical Company. Ethylene diaminetetraacetic
acid (EDTA) and reagents required to prepare the Luria
Burtani (LB) and YEPD culture medium like tryptone, peptone,
yeast extract, and agar powder were purchased from Himedia
R
Chemical Company, India. The LIVE/DEADꢀ BacLightTM
bacterial viability kit was purchased from Molecular Probes,
Invitrogen Chemical Company. All the materials used in the
cell culture study such as Dulbecco’s Modified Eagles’ Medium
(DMEM), heat inactivated fetal bovine serum (FBS), trypsin
from porcine pancreas and 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) were obtained from
Sigma Aldrich Chemical Company. 1H NMR spectra were
recorded on an AVANCE 300 MHz (BRUKER) spectrometer.
Mass spectrometric (MS) data were acquired by the Electron
Spray Ionization (ESI) technique on a Q-tof-Micro Quadruple
mass spectrophotometer, Micromass.
L-Phenylalanine-L-proline-N-tetradecylamide-N¢,N¢,N¢-trimeth-
ylammonium chloride (1). [a]D +0.7 (c 1.45 in MeOH); Found:
C, 69.35; H, 10.02; N, 7.77. C31H54ClN3O2 requires C, 69.43; H,
10.15; N, 7.84%; IR (KBr): 3419 s (NH), 1666 s (CO); 1H NMR
(300 MHz, CDCl3) d 7.58–7.16 (m, 5H), 5.87–5.82 (m, 1H), 4.45–
4.42 (m, 1H), 3.55–3.43 (m, 2H), 3.36 (s, 9H), 3.19–3.15 (m, 2H),
3.05–2.93 (m, 2H), 1.96–1.44 (m, 4H), 1.18–1.16 (br, 24H), 0.86–
0.78 (t, 3H); m/z (ESI) 500.2827 (C31H54N3O2+ requires 500.4216).
Synthetic procedure and characterization of amphiphiles 1–6
All the peptide amphiphiles (1–6, Fig. 1) were synthesized
following the reaction procedure previously reported by us.15,16
Boc-protected L-amino acids (10 mmol) were coupled with the
methyl ester (11 mmol) of the corresponding L-amino acids using
dicyclohexylcarbodiimide (DCC, 11mmol)and acatalyticamount
of DMAP in the presence of HOBT (11 mmol) in dry DCM
on an ice-bath. The reaction mixture was then allowed to come
to room temperature and stirred for 12 h. Next, the resultant
mixture was filtered and the filtrate was concentrated in a rotary
evaporator. The whole residue was then extracted in ethyl acetate
washed with 2 M HCl, brine, 1 M sodium carbonate and brine
to neutrality. The organic part was dried over anhydrous sodium
sulfate and concentrated. The residue obtained was then purified
by a silica gel 60–120 mesh column using ethyl acetate/toluene as
the eluent. The concentrated column purified white solid material
(-OMe protected) was dissolved in the required amount of MeOH
and subjected to saponification by the addition of 20 mL 1 M
NaOH solution at room temperature. The stirring was carried
out for 6 h, after which the MeOH was evaporated in a rotary
evaporator. The remaining material was then added to water and
washed with diethyl ether; the pH of the aqueous part was adjusted
to 2 using 1 M HCl, and then extracted with ethyl acetate, which
was further washed with brine to remove any traces of acid. The
organic part was then dried over anhydrous sodium sulfate and
concentrated on a rotary evaporator, which yielded a solid -OMe
deprotected peptide. This free C-terminal peptide (8 mmol) was
coupled with n-tetradecylamine (8.8 mmol), DCC (8.8 mmol) and
a catalytic amount of DMAP in the presence of HOBT (8.8 mmol)
in dry DCM on an ice-bath and then the reaction mixture was
L-Proline-L-phenylalanine-N -tetradecylamide-N¢,N¢-dimethyl-
ammonium chloride (2). [a]D +3.0 (c 1.1 in MeOH); Found: C,
68.88; H, 10.11; N, 8.14. C30H52ClN3O2 requires C, 69.00; H,
10.04; N, 8.05%; IR (KBr): 3434 s (NH), 1656 s (CO); 1H NMR
(300 MHz, CDCl3) d 7.38–7.06 (m, 5H), 5.07–5.02 (m, 1H), 4.89–
4.80 (m, 1H), 3.44–3.39 (m, 2H), 3.32–3.21 (m, 4H), 2.93 (s, 3H),
2.38 (s, 3H), 2.21–2.16 (m, 2H), 1.49–1.45 (m, 2H), 1.45–1.18 (br,
24H), 0.82–0.78 (t, 3H); m/z (ESI) 486.406 (C30H52N3O2+ requires
486.4086).
L-Tryptophan-L-proline-N-tetradecylamide-N¢,N¢,N¢-trimethyl-
ammonium chloride (3). [a]D -25.0 (c 1.16 in MeOH); Found: C,
68.75; H, 9.69; N, 9.78. C33H55ClN4O2 requires C, 68.90; H, 9.64; N,
9.74%; IR (KBr): 3408 s (NH), 1648 s (CO); 1H NMR (300 MHz,
CDCl3) d 7.96 (br, 1H), 7.44–6.94 (m, 4H), 5.01–4.98 (m, 1H),
3.78 (br, 1H), 3.55–3.38 (br, 2H), 3.47 (s, 9H), 3.24–3.14 (m, 2H),
3.14–2.8 (m, 2H), 1.95 (br, 2H), 1.42–1.39 (br, 2H), 1.29–1.13
+
(br, 24H), 0.82–0.71 (t, 3H); m/z (ESI) 539.3687 (C33H55N4O2
requires 539.4325).
L-Proline-L-tryptophan-N-tetradecylamide-N¢,N¢-dimethylammo-
nium chloride (4). [a]D -19.0 (c 1.9 in MeOH); Found: C, 68.33;
H, 9.38; N, 9.77. C32H53ClN4O2 requires C, 68.48; H, 9.52; N,
9.98%; IR (KBr): 3399 s (NH), 1658 s (CO); 1H NMR (300 MHz,
CDCl3) d 7.56–7.54 (d, 1H), 7.30–6.92 (m, 4H), 4.92–4.74 (m,
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The Royal Society of Chemistry 2009
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