56
A. Dutta et al. / Journal of Molecular Structure 930 (2009) 55–59
was stirred at room temperature for 2 days. The progress of the
reaction was monitored by TLC. After completion of the reaction
the methanol was evaporated. The residue obtained was diluted
with water and washed with diethylether. The aqueous layer was
cooled in an ice-bath and neutralized with 2 M HCl and then ex-
tracted with ethyl acetate. The solvent was evaporated in vacuo
to give a white solid. Yield: 1.1 g (94.1%).
O
O
O
O
H
H
H
H
H
N
H
N
H
N
OMe
N
N
N
O
N
H
H
H
H
O
O
O
O
Fig. 1. Schematic diagram of peptide I.
egy [24]. The t-butyloxycarbonyl and methyl ester group were
used for amino and carboxyl protections, respectively, and N,N0-
dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBT)
as coupling agents. Deprotections were performed using trifluoro-
acetic acid or saponification, respectively. Methyl ester hydrochlo-
rides of Aib, Leu and Val were prepared by the thionyl chloride–
methanol procedure. All the intermediates obtained were checked
for purity by thin layer chromatography (TLC) on silica gel and
used without further purification. The final peptide was purified
by column chromatography using silica gel (100–200 mesh) as
the stationary phase and ethyl acetate and petroleum ether mix-
ture as the eluent. The reported peptide was fully characterized
by NMR studies and X-ray crystallography.
2.1.5. Boc-Ile-Aib-Leu-OMe (5)
This peptide was prepared following the literature method
[25].
Boc-Ile-Aib-Val-m-ABA-Ile-Aib-Leu-OMe (Peptide I): To Boc-Ile-
Aib-Leu-OMe (5), (0.47 g, 1.07 mmol) trifluoroacetic acid (3 ml)
was added at 0 °C and stirred at room temperature. The removal
of the Boc-group was monitored by TLC. After 3 h the trifluoroace-
tic acid was removed under reduced pressure to afford the crude
trifluoroacetate salt. The residue was taken up in water and
washed with diethyl ether. The pH of the aqueous solution was ad-
justed to eight with sodium bicarbonate and extracted with ethyl
acetate. The extracts were pooled, washed with saturated brine,
dried over sodium sulfate, and concentrated to a highly viscous li-
quid that gave a positive ninhydrin test. This free base of the tri-
peptide was added to a well ice-cooled solution of compound 4
(0.57 g, 1.07 mmol) in DMF (6 ml) followed by DCC (0.33 g,
1.60 mmol) and HOBt (0.17 g, 1.28 mmol). The reaction mixture
was stirred at room temperature for 4 days. The residue was taken
up in ethyl acetate and DCU was filtered off and to the filtrate
20 ml of ethyl acetate was added. The organic layer was washed
with 2 M HCl (3 ꢂ 50 ml), 1 M Na2CO3 solution (3 ꢂ 50 ml) and
brine, dried over anhydrous Na2SO4 and evaporated in vacuo, to
yield a white solid. Purification was done using silica gel as station-
ary phase and ethyl acetate–petroleum ether mixture as the elu-
ent. Single crystals were grown from methanol and ethyl acetate
mixture by slow evaporation and were stable at room temperature.
Yield: 0.81 g (88.3%). Mp = 150–152 °C; IR (KBr): 3313, 1717,
2.1.1. Boc-Ile-Aib-Val-OMe (1)
The peptide was prepared using the literature method [25].
2.1.2. Boc-Ile-Aib-Val-OH (2)
Compound 1 (1.4 g, 3.16 mmol) was dissolved in methanol
(15 ml) and 2 M NaOH (5 ml) was added. The reaction mixture
was stirred at room temperature for 2 days. The progress of the
reaction was monitored by TLC. After completion of the reaction
the methanol was evaporated. The residue obtained was diluted
with water and washed with diethylether. The aqueous layer was
cooled in an ice-bath and neutralized with 2 M HCl and then ex-
tracted with ethyl acetate. The solvent was evaporated in vacuo
to give a white solid. Yield: 1.3 g (95.8%).
1674, 1526 cmꢁ1 1H NMR 300 MHz (DMSO-d6, d ppm): 9.96 (m-
;
2.1.3. Boc-Ile-Aib-Val-m-ABA-OMe (3)
Compound 2 (1.2 g, 2.79 mmol) was dissolved in DMF (5 ml). m-
ABA-OMe obtained from its hydrochloride (1.05 g, 5.6 mmol) was
added, followed by DCC (0.86 g, 4.2 mmol) and HOBT (0.38 g,
2.79 mmol). The reaction mixture was stirred at room temp for
3 days. The precipitated N,N0-dicyclohexylurea (DCU) was filtered
and to the filtrate 20 ml of ethyl acetate was added. The organic
layer was washed with 1 N HCl (3 ꢂ 30 mL), 1 M Na2CO3 solution
(3 ꢂ 30 mL) and water. The solvent was then dried over anhydrous
Na2SO4 and evaporated in vacuo, giving a light yellow gum. Yield:
1.4 g (91.6%). Purification was carried out using silica gel as the sta-
tionary phase and ethyl acetate–petroleum ether mixture as the
eluent. Mp = 178–180 °C; IR (KBr): 3420, 3302, 1724, 1665, 1604,
ABA(4) NH, 1H, s); 8.37 (m-ABA(4) Hd, 1H, d, J = 6.6 Hz); 8.34 (m-
ABA(4) Ha, 1H, s); 8.08 (m-ABA(4) Hb and Aib (6) NH, 2H, bs);
7.74 (Leu(7) NH, 1H, d, J = 7.8 Hz); 7.57 (Ile(5) NH, 1H, d,
J = 7.8 Hz); 7.33 (m-ABA(4) Hc, Aib (2) NH, 2H, m); 7.07 (Val(3)
NH, 1H, d, J = 8.4 Hz); 6.76 (Ile(1) NH, 1H, d, J = 7.5 Hz); 4.26–4.23
a
a
(C Hs of Ile(5) and Leu (7), 2H, m); 4.12–4.07 (C H of Val (3),1H,
a
m); 3.80–3.76 (C H of Ile(1),1H, m); 3.57 (–OCH3, 3H, s); 1.59–
2.15 (CbHs of Ile(1), Val(3), Ile(4) and Leu(6), 5H, m); 1.56 (CbHs
of Aib(6), 6H, s); 1.53 (Boc-CH3s, 9H, s); 1.45 (CbHs of Aib(2), 6H,
c
s); 1.30–1.25 (C Hs of Ile(1), Ile(5) and Leu(6), 11H, m); 1.0–0.87
c
(C Hs of Val(3), CdHs of Ile(1), Ile(5) and Leu(7), 18H, m); 13C
NMR 75 MHz (DMSO-d6, d ppm): 173.93, 173.83, 173.02, 171.70,
171.27, 170.08, 167.03, 155.71, 138.80, 134.62, 128.47, 122.47,
122.19, 119.01, 78.19, 56.28, 56.09, 51.79, 50.24, 36.23, 34.97,
30.62, 28.19, 26.94, 25.73, 25.31, 24.69, 24.25, 23.72, 23.08,
22.41, 21.19, 19.21, 17.97, 15.40, 15.12, 11.10, 10.57; HR–MS
(M+Na+) = 882.03, Mcalcd = 860.11.
1547, 1511 cmꢁ1 1H NMR 300 MHz (CDCl3, d ppm): 9.12 (m-ABA
;
NH, 1H, s); 8.49 (m-ABA (4) Ha, 1H, s); 8.17 (m-ABA (4) Hd, 1H, d,
J = 6.9 Hz); 7.75(m-ABA (4) Hb, 1H, d, J = 7.8 Hz); 7.36 (m-ABA (4)
Hc, 1H, t, J = 8.1 Hz); 6.95 (Val (1) NH, 1H, d, J = 8.4 Hz); 6.68
Aib(2) NH, 1H, s); 5.04 (Ile (3) NH, 1H, d, J = 3.9 Hz); 4.58–4.54
a
a
(C H of Val (3), 1H, m); 3.91 (–OCH3, 3H, s); 3.89–3.84 (C H of
Ile(1), 1H, m); 2.70–2.65 (CbHs of Val (3), 1H, m); 1.95-1-93 (CbHs
of Ile (1), 1H, m);1.55 (CbHs of Aib, 6H, s);1.44 (Boc–CH3s, 9H, s);
2.2. FTIR spectroscopy
1.29–1.27 (C Hs of Ile (1) , 5H, m); 1.01–0.92 (CdHs of Ile (1) and
c
IR spectrum of peptide I was examined using Perkin Elmer-782
model spectrophotometer. The solid state FTIR measurements
were performed using the KBr disk technique.
c
C Hs of Val (3), 9H, m); 13C NMR (75 MHz, CDCl3, d ppm): 173.9,
171.9, 170.1, 167.1, 156.5, 138.9, 130.5, 128.6, 124.9, 124.5,
121.1, 81.2, 60.7, 59.3, 57.2, 51.9, 36.4, 29.1, 28.1, 27.5, 25.3,
24.0, 19.5, 16.9, 15.74, 11.52; HR–MS (M+Na+) = 571.31,
Mcalcd = 548.67.
2.3. NMR experiments
All 1H and 13C NMR spectra of peptide I were recorded on Bru-
ker Avance 300 model spectrometer operating at 300 and 75 MHz,
respectively. The peptide concentrations were 10 mM in CDCl3 for
1H NMR and 40 mM in CDCl3 for 13C NMR.
2.1.4. Boc-Ile-Aib-Val-mABA-OH (4)
Compound 3 (1.2 g, 2.19 mmol) was dissolved in methanol
(15 ml) and 2 M NaOH (5 ml) was added. The reaction mixture