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FONINA et al.
(200 ml), washed with 5% NaHCO3 (2 × 50 ml), water (100 ml), treated with 1 N NaOH (9 ml), and hydroge-
nated over 10% Pd/C until the disappearance of starting
substance (TLC monitoring). The catalyst was filtered
off, and 1 N H2SO4 (9 ml) was added to the filtrate. The
precipitate was filtered, washed with water, and dried in
a vacuum desiccator. The amorphous powder contain-
ing the target product was dissolved in 0.01 N
CH3COONH4 (10 ml) and subjected to HPLC on a Dia-
sorb C 16-T column. The fractions containing the target
product were combined and concentrated on a rotary
evaporator to final volume of 10 ml. This solution was
diluted with water to the volume of 50 ml and
lyophilized; yield 3.2 g (46%); Rf 0.61 (A), 0.48 (B),
(1 × 50 ml), 5% H2SO4 (2 × 50 ml), and, finally, with
water (2 × 50 ml). The ethyl acetate solution was dried
with MgSO4 and evaporated in a vacuum. The oily res-
idue was dissolved in 3 : 7 ethyl acetate–hexane mix-
ture (20 ml) and applied onto a column with silica gel
60 (90–130 µm) equilibrated with the same mixture.
The target substance was eluted with the same solvent
mixture (500 ml). The fractions containing the target
product were combined and evaporated in a vacuum.
The oily residue was dissolved in ethanol (100 ml) and
hydrogenated over10% Pd/C (1 g) at 18°C until the dis-
appearance of starting substance (TLC monitoring in
9 : 1 chloroform–methanol mixture). The catalyst was 0.64 (D).
filtered off, and the filtrate was evaporated on a rotary
Synthesis of retro-MP-2 was carried out by the
evaporator at the temperature no higher than 30°C. The
oily residue was triturated with anhydrous ether (2 ×
30 ml). The solvent was filtered off, and the resulting
amorphous powder was washed with anhydrous ether
and dried in a vacuum desiccator; yield 7.62 g (73%);
Rf 0.61 (C), 0.32 (D).
solid phase method using the chloromethylated copoly-
mer of styrene and 1% divinylbenzene with the chlorine
content of 1 mmol per g. Boc-amino acids were
attached by the carbodiimide method with the addition
of equimolar amounts of N-hydroxybenzotriazole for
the inhibition of the racemization. The resulting peptide
was cleaved from the polymer by the treatment with
70% trifluoroacetic acid.
Z-Leu-Val-Val-Tyr-Pro. Z-Leu-ONSu (5.8 g,
16 mmol) was added to a stirred solution of H-Val-Val-
Tyr(But)-Pro-OBut (7.62 g) in DMF (150 ml). The
reaction mixture was stirred for 12 h at 18°ë and evap-
orated. The oily residue was dissolved in ethyl acetate
(200 ml); successively washed with water (1 × 50 ml),
saturated solution of NaHCO3 (2 × 50 ml), water, 2%
H2SO4 (2 × 50 ml), water (2 × 50 ml); dried with
MgSO4; and evaporated. The oily residue was dissolved
in TFA (70 ml) and stirred for 1 h at 18°ë. TFA was
removed on a rotary evaporator at 18°ë. The oily resi-
due was triturated with anhydrous ether (2 × 50 ml),
and the amorphous powder was filtered and dried in a
vacuum desiccator over NaOH. The product was dis-
solved in a 9 : 1 chloroform–methanol mixture and
applied onto a column (30 × 200 mm) filled with silica
gel 60 (90–130 µm) and equilibrated with the same sol-
vent mixture. The column was successively eluted with
the following chloroform–methanol mixtures: 9 : 1, 9 :
2, and 9 : 3 (200 ml of the each mixture). The fractions
containing the target Z-Leu-Val-Val-Tyr-Pro were com-
bined and evaporated; yield 6.55 g (70%); Rf 0.45 (A),
0.31 (B).
Amino acid sequences of the MP-2 and retro-MP-2
peptides were determined on a gas phase sequencer
(Applied Biosystem 477A, United States) connected
with an automatic analyzer of phenylthiohydantoins of
amino acids.
Molecular masses of the hexapeptides determined
by mass spectrometry on a Thermo Bioanalysis Vision
2000 device (England) were equal to 776.
CD spectra were registered on a Jasco 500 C
dichrograph (Japan) at the temperature of 20°ë in
demountable quartz cuvettes (Hellma) 10–2 cm in thick-
ness. Concentration of the peptide solution was
1 mg/ml. The presented spectra are averaged after three
scans.
IR spectra were measured on a Perkin-Elmer 1725
X spectrometer at a 4 cm–1 resolution and 20°ë. The
spectrometer was preliminary blown off with dry nitro-
gen for the removal of water vapour. The measurements
were done in cuvettes of 0.2 mm in thickness made
from CaF2. The number of scans was 300.
The proliferative response of T-lymphocytes to the
action of the phytohemagglutinin mitogen was esti-
mated according to the technique described in [8].
Leu-Val-Val-Tyr-Pro-Trp-OH.
H-Trp-OBzl
hydrochloride (3.63 g, 11 mmol), N-hydroxybenzotria-
zole (1.35 g, 10 mmol), and N-methylmorpholine
(1.70 ml) were added to a solution of Z-Leu-Val-Val-
Tyr-Pro-OH (6.55 g, 9 mmol) in DMF (80 ml). The
reaction mixture was cooled to –20°ë, and dicyclohex-
ylcarbodiimide (2.06 g, 10 mmol) was added at stirring.
The reaction mixture was stirred for 1 h at −20°ë and
for 12 h at 20°ë and evaporated in a vacuum. The resi-
due was dissolved in 1 : 1 butanol–ethyl acetate mix-
ture; washed with water (1 × 30 ml), saturated solution
of NaHCO3 (2 × 50 ml), water, 2% H2SO4 (2 × 50 ml),
Antitumor activities of the peptides were deter-
mined on various types of mouse tumors (Lewis lung
carcinoma, Ca 755 adenocarcinoma of mammal gland,
and S 180 sarcoma). The tumors were obtained from
the Tumor Strain Bank of the Blokhin Cancer Research
Center and passed on linear mice two times. The linear
and hybrid female mice (18–20 g of the body mass)
were purchased from the Stolbovaya breeding nursery
of the RussianAcademy of Medical Sciences. The solid
and, finally, with water to pH 7. The organic layer was tumors were transplanted by the introduction of the cell
evaporated. The residue was dissolved in methanol suspension in the 199 medium at a dose of 50 mg per
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 3 2005
Fonina
