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the solution was acidified with 3 N HCl to pH 3.5, and the
product was extracted with ethyl acetate (5×30 mL). The
organic layer was washed with 20 mL of water, 20 mL of
saturated NaCl, and dried over Na2SO4. The solvent was
removed in a vacuum, and the residue was triturated with
hexane. Yield 3.02 g (49%), white crystals.
6.4 Hz), 7.23–7.33 m (15Н, Ar), 7.79 d (1H, NH, J =
8.8 Hz), 8.23 d (1Н, NH, J = 7,1 Hz), 9.15 s (1Н, ОН).
HRMS, m/z: 825.9661 [M + H].
RP(O)(OPr-i)–Boc–Tyr–Thr(OBn)–Lys(Z)OBn
(16a, 16b). Sodium hydride, 0.17 g (0.0007 mol), pre-
washed with hexane to remove mineral oil, was added
with stirring to a coled (5°С) suspension of 0.412 g
(0.0005 mol) of compound 10 in 40 mLof anhydrous ace-
tonitrile. The mixture was stirred for 30 min at room tem-
perature, after which 0.0006 mol phosphonoyl chloride
15a or 15b in 10 mLof anhydrous acetonitrile was added,
and stirring was continued for an additional 2 h. The
reaction progress was monitored by TLC. The reaction
mixture was reduced by half in a vacuum and poured into
40 mL of water. The reaction product was extracted with
chloroform (3×30 mL), and the extract was washed with
20 mLof water, dried over MgSO4, and concentrated in a
vacuum to leave a colorless viscous liquid. The reaction
products were isolated by chromatography on a column
(150×30 mm) of silica gel (GeduranSi 60, 40–63 μm,
Merck), eluent ethyl acetate‒methylene chloride (3 : 1),
elution rate 1 mL/min. The purity of the products after
column chromatography was 85–87 % (by 31P NMR).
Boc–Thr(OBn)–Lys(Z)OBn (7). Asolution of 2.06 g
(10 mmol) of dicyclohexylcarbodiimide in 10 mLof DMF
was added dropwise to a cold (0°С) solution of 3.72 g
(9.2 mmol) of compound 4, 2.84 g (9.2 mmol) of com-
pound 6, 1.53 g (10 mmol) of N-hydroxybenzothriazole,
and 1.0 g (10 mmol) of N-methylmorpholine in 50 mL
of DMF. The reaction mixture was stirred for 1 h at 0°С
and then for 12 h at room temperature. A 5% solution of
citric acid, 100 mL , was added, and the reaction product
was extracted with ethyl acetate (5×30 mL), washed with
5% NaHCO3 (2×20 mL), 20 mL of water, and 10 mL of
saturated NaCl and dried over Na2SO4. The solvent was
removed in a vacuum, and the residue was triturated with
20 mL of hexane. Yield 5.86 g (96.2%), light-colored
crystals.
HCl·H–Thr(OBn)–Lys(Z)OBn (8). To 5.86 g
(8.86 mmol) of compound 7, a 15% solution of HCl in
dioxane, 25 mL, was added. The mixture was stirred
for 2 h at room temperature, the solvent was removed
in a vacuum, and the residue was triturated with 20 mL
of ether and dried in air to constant weight. Yield 4.9 g
(99%), white crystals.
CH3P(O)(OPr-i)–Boc–Tyr–Thr(OBn)–Lys(Z)–
OBn (16a). Yield 0.372 g (76 %), semicrystalline mate-
rial. 31P NMR spectrum (CDCl3): δP 26.70 ppm. HRMS,
m/z: 946.0530 [M + H].
CD3P(O)(OPr-i)–Boc–Tyr–Thr(OBn)–Lys(Z)–
OBn (16b). Yield 0.399 g (81%), pale yellow semi-
crystalline material. 31P NMR spectrum (CDCl3): δP
26.79 ppm. HRMS, m/z: 949.0714 [M + H].
Boc–Tyr–Thr(OBn)–Lys(Z)OBn (10). A solution
of 2.06 g (10 mmol) of dicyclohexylcarbodiimide in 10
mL of DM was added dropwise to a solution of 4.9 g
(8.8 mmol) of compound 8, 2.75 g (9.8 mmol) of Boc–
Tyr–OH 9, 1.53 g (10 mmol) of N-hydroxybenzothriazole,
and 1.1 g (11 mmol) of N-methylmorpholine in 80 mL
of DMF. The reaction mixture was stirred for 1 h at 0°С
and then for 12 h at room temperature. A 5% solution of
citric acid, 100 mL, was added, and the reaction product
was extracted with ethyl acetate (5×30 mL), washed
with 20 mL of water, 20 mL of 5% NaHCO3, and 10 mL
of saturated NaCl and dried over Na2SO4. The solvent
was removed in a vacuum, and the residue was triturated
RP(O)(OPr-i)–Tyr–Thr(OBn)–Lys(Z)–OBn (17a,
17b). A 15% solution of anhydrous HCl in dioxane,
10 mL, was added to phosphorylated peptide 16a or 16b.
The mixture was stirred for 1 h at room temperature,
and the solvent was removed in a vacuum. The residue
was dissolved in 40 mL of ethyl acetate, the solution
was washed with 5% NaHCO3 (2×20 mL) and 10 mL of
saturated NaCl, and dried over Na2SO4. The solvent was
removed to leave an amorphous white material, which
purification was subjected to hydrogenolysis to remove
benzyl protective groups.
1
with 20 mL of hexane. Yield 4.1 g (49.8%). Н NMR
CH3P(O)(OPr-i)-Tyr-Thr(OBn)-Lys(Z)OBn (17a).
31P NMR spectrum (CDCl3): δP 26.76 ppm. HRMS, m/z:
845.9371 [M + H].
spectrum (DMSO-d6), δ, ppm: 1.09 d (3Н, CH3CH, J =
6.1 Hz), 1.34 s [9Н, (CH3)3C], 1.20–1.39 m (4Н,
СH2CH2), 2.85 t, 2.91 d (2Н, СН2), 2.92 m (2Н, СН2N),
4.15 m, 4.29 m, 4.32 m, 4.41 m (4Н, СН), 4.44 s, 4.99
s, 5.12 s (6Н, СН2Аr), 6.63 d (2Н, ArOH, J = 8.3 Hz),
7.03 d (2Н, ArCH2, J = 8.3 Hz), 7.04 d (1Н, NH, J =
CD3P(O)(OPr-i)–Tyr–Thr(OBn)–Lys(Z)OBn
(17b). 31P NMR spectrum: δP 26.92 ppm. HRMS, m/z:
848.9556 [M + H].
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 89 No. 10 2019