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J. Makker et al. / Journal of Molecular Structure 654 (2003) 119–124
2.1. Synthesis of Z-DVal-OH (1)
was stirred for 80 h. The solvent was
evaporated and the residue was dissolved in ethyl
acetate. It was washed with 10% sodium bicarbon-
ate, 5% citric acid and water, respectively, and
dried over anhydrous sodium sulphate. The
solvent was removed under reduced pressure and
the solid product (4) was obtained at a yield of
83%.
The compound (1) was synthesized by the
condensation of 2-oxo-3-methyl-butanoic acid
(0.9 g, 7.8 mmol) with benzyl carbamate (1.4 g,
9.4 mmol) and p-toluene sulfonic acid (0.27 g,
9.4 mmol) in dry benzene. The reaction mixture was
refluxed at 100 8C using Dean and Stark water
remover for 8 h. Then the solution was extracted
with saturated sodium bicarbonate. The extracts were
neutralized by adding concentrated hydrochloric acid
drop wise to yield a white solid, which was filtered
and recrystallized from benzene. The solid product of
Z-DVal-OH was obtained with a yield of 67%.
2.5. Removal of Boc from compound (4) to get
TFA-Val-DPhe-Ile-Ome (5)
Compound (4) (2.16 g, 4.7 mmol) was dissolved in
0.5 ml of trifluoroacetic acid (TFA) and 0.5 ml of
DCM and stirred for 1 h. The solvent was removed in
vacuo to yield compound (5) on addition of dry ether
with a yield of 78%.
2.2. Synthesis of Boc-Val-(b-OH)-Phe-OH (2)
To a precooled solution of (1) (1 g, 4.6 mmol) in
tetrahydrofuran (THF), N-methylmorpholine (NMM)
(0.5 ml, 4.6 mmol) and isobutylchloroformate (IBCF)
(0.61 ml, 4.6 mmol) were added and stirred for 20 min
at 210 8C. To this, a precooled solution of Phe-(b-
OH) (1 g, 5.5 mmol) in 1N NaOH (5.5 ml) was added
and the mixture was stirred for 3 h at 0 8C and then at
room temperature overnight. The organic solvent was
removed in vacuo and the aqueous phase was acidified
with citric acid to pH 3 and extracted with ethyl
acetate. The organic layer was washed with water and
dried over anhydrous sodium sulphate and evaporated
to yield 80% of compound (2).
2.6. Synthesis of Z-DVal-Val-DPhe-Ile-Ome (6)
To a solution of the compound (1) (0.18 g,
0.68 mmol) in DCM (10 ml), compound (5) was
added along with TEA (0.4 ml, 2.71 mmol). The
coupling was carried out as mentioned for (4) to
obtain 74% of the final compound.
2.7. 1H NMR of Z-DVal-Val-DPhe-Ile-Ome
In order to confirm the correctness of the final
synthesis of the peptide, 1H NMR spectra were
recorded in CDCl3 with 400 MHz Bruker DRX 400
instrument and the following results were obtained:
d 0.88–0.95 (m, 6H, Cg2, Cd Ile); d 1.04–1.06 (m,
2H, Cg1 Ile); d 1.52 (m, 12H, Cg1, Cg2, DVal, Val);
d 1.77 (m, 3H, Cg1, Cb Ile); d 1.98 (m, 1H, Cb
Val); d 3.74 (s, 3H, Ome); d 4.46 (m, 1H, Ca Ile);
d 4.63–4.67 (m, 2H, –CH2, Z); d 4.82–4.85 (d,
1H, Ca Val); d 6.12 (bs, 1H, NH DVal); d 6.31–
6.33 (bd, 1H, NH Val); d 7.26–7.28 (m, 11H, Ar,
Z; DPhe, Cb DPhe); d 7.52 (s, 1H, NH DPhe), d
8.71 (s, 1H, NH Ile). The observed 1H NMR
spectra clearly indicated the synthesis of the correct
peptide.
2.3. Synthesis of Boc-Val-DPhe-azlactone (3)
Compound (2) (1.78 g, 4.7 mmol) was reacted with
anhydrous sodium acetate (0.46 g, 5.6 mmol) and
freshly distilled acetic anhydride (10 ml) for 24 h at
room temperature. The reaction mixture was then
poured over crushed ice, the resultant product was
washed with 5% sodium bicarbonate and water and
finally recrystallized from acetone–water mixture to
yield 82% of compound (3).
2.4. Synthesis of Boc-Val-DPhe-Ile-Ome (4)
To a solution of compound (3) (1 g, 2.9 mmol)
in dichloromethane (DCM) (10 ml), Ile-Ome·HCl
(0.63 g, 3.4 mmol) was added followed by triethy-
lamine (TEA) (0.47 ml, 3.4 mmol). This mixture
2.8. Structure determination
The peptide was crystallized from its solution in
acetone–water mixture at room temperature
Makker
