V.V. Ryakhovsky, A.S. Ivanov / Tetrahedron 68 (2012) 7070e7076
7075
was stirred at 0 ꢀC for 6 h. The solvents were removed under vacuum
at 10e15 ꢀC, the residue dissolved in solution A, and filtered through
a plug of Al2O3 (4 g). The filtrate was evaporated under vacuum and
Na-Boc-protected dipeptide alkyl ester (2.5 mmol) in methanol
(5 mL). The mixture was stirred at rt for 4 h. The solvents were
evaporated to dryness, the residue dissolved in water (20 mL), and
washed with ether. The aqueous solution was then acidified with
citric acid to pH 3, saturated with NaCl, and extracted with ethyl
acetate. The organic phase was washed with saturated aqueous NaCl,
dried over anhydrous MgSO4, and evaporated under vacuum. The
the residue dried in a vacuum desiccator over P4O10
.
4.3.4. Preparation ofalkyl esters from Na-BoceNa-alkylamino acids. A
suspension of NaH in mineral oil (40 mmol) was added to a stirred
solution of Na-BoceNa-alkylamino acid (10 mmol) in anhydrous THF
(30 mL), and the mixture was stirred at 20 ꢀC for 10 min. Then alkyl
iodide (100 mmol) was added, and the mixture was stirred at 60 ꢀC
for 10 h, and maintained at rt overnight. The solvent was removed
under vacuum, the residue partitioned in a mixture of ethyl acetate
(100 mL) and water (25 mL), and acidified with citric acid to pH 3.
The organic phase was separated, washed with saturated aqueous
NaCl, containing 1% of Na2S2O3, and dried over anhydrous MgSO4.
After evaporation of ethyl acetate, the residue was loaded on Al2O3
(30 g), and eluted consecutively with heptane (to remove mineral
oil), eluent A (for BoceXaaeOAlk), and finally, with eluent L (for
BoceXaaeOH). The fractions were evaporated under vacuum. The
traces of acetic acid from eluent L were removed by the azeotrope
distillationwith toluene under reduced pressure. The products were
dried in a desiccator under the residual pressure of 0.13e0.26 kPa.
residual product was dried in a vacuum desiccator over P4O10
.
4.3.9. Amidation of peptide methyl esters. A solution of Na-Boc-
peptide methyl ester (0.5 mmol) in methanol (5 mL) was cooled to
0 ꢀC, saturated with ammonia, passed through the KOH-filled
drying column, and maintained at rt for 16e18 h (for regular
amino acids) or for 48e72 h (for Na-alkylamino acids). The solution
was evaporated to dryness, and the residual product was dried in
vacuum desiccator over P4O10
.
4.3.10. Oxidation of methionine to methionine sulfoxide in
peptides. 38% Hydrogen peroxide (0.094 mL,1.2 mmol) was added to
a solution of BoceMet or a methionine-containing peptide in acetic
acid (7 mL). The solution was maintained at 20 ꢀC for 30 min. After
that 5% Pd/C (0.2 g) was charged, and the mixture stirred for another
30 min. The charcoalwasfiltered offand the filtrate evaporatedunder
vacuum. In the case of BoceMet, the residue was dissolved in toluene,
evaporated under vacuum, and the product dried in a vacuum des-
iccatorover P4O10. In thecase of a methionine-containingpeptide, the
residue was dissolved in water and lyophilized.
4.3.5. Peptide coupling using mixed anhydrides with pivalic
acid. NMM (15e20 mmol) was added to a solution of Na-Boc amino
acid (10 mmol) in THF (20 mL). The mixture was cooled to ꢂ15 ꢀC,
then pivaloyl chloride (10 mmol) was added, and the mixture
stirred for 10 min. After that, a solution of an amino component
(9 mmol) and NMM (9 mmol) in 20 mL of a proper solvent (DCM,
chloroform, or DMF) was added, and the resulting solution was
stirred at 20 ꢀC for 1 h (for Pro, Sar, EtGly) or for 24 h (for MeAla,
MeLeu, MePhe). The reaction mixture was then diluted with ethyl
acetate (150 mL), washed with 10% solution of KHSO4 and with
demineralized water, and dried over anhydrous MgSO4. The sol-
vents were evaporated under vacuum to yield the solid material. In
some cases the product was purified by column chromatography on
silica gel.
4.3.11. Reduction of sulfoxide group in peptides, containing
BoceMeMet(O). A solution of BoceMeMet(O)eR (2.8 mmol) and
dithioerythritol (14 mmol) in water (23 mL) was maintained at
50e55 ꢀS for 6 h under the argon atmosphere, and then overnight
at rt. The reaction mixture was extracted with ethyl acetate, the
extract washed with water repeatedly, dried over anhydrous
MgSO4, and the solvent evaporated under vacuum. The residue was
loaded on Al2O3 (20 g), the excess of dithioerythritol was eluted
with eluent A, and a peptide productdwith eluent L. The traces of
acetic acid were removed from the eluate, containing a peptide, by
azeotropic distillation with toluene. The product was dried in
a vacuum desiccator over P4O10 and KOH.
4.3.6. Peptide coupling using mixed anhydrides with isobutylcarbonic
acid. NMM (10 mmol) was added to a stirred solution of Na-Boc
amino acid (10 mmol) in THF (20 mL). The mixture was cooled
to ꢂ20 ꢀC, then isobutylchloroformate (10 mmol) was added, and the
mixture was stirred for 5 min. After that, a solution of an amino
component (9 mmol) and NMM (9 mmol) in 20 mL of a proper sol-
vent (DCM, chloroform, or DMF) was added dropwise at ꢂ15 ꢀC. The
resulting solutionwas stirred for 1e2 h at ꢂ15 ꢀC and1 h at 20 ꢀC. The
work-up was similar to that described in the previous procedure.
Supplementary data
Synthesis and properties of intermediate compounds are pre-
sented. Supplementary data associated with this article can be
4.3.7. Peptide coupling using pentafluorophenyl esters. DCC
(1.05 mmol) was added to a solution of Na-Boc-protected tripeptide
(1 mmol) and PfpOH (1 mmol) in methylene dichloride (6 mL) at0 ꢀC.
The mixture was slowly heated to 20 ꢀC and maintained for 16 h. The
precipitate of N,N0-dicyclohexylurea was filtered off, the filtrate
evaporated to dryness, and the residue re-dissolved in DMF (6 mL). To
References and notes
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this solution a mixture of
D-phenylalanine (2 mmol) and KOH
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solution was stirred for 1 h, the solvents evaporated under vacuum,
the residue treated with water (30 mL), and the mixture filtered. The
filtrate was acidified with citric acid to pH 3, saturated with NaCl, and
extracted with ethyl acetate. The extract was washed with saturated
aqueous solution of NaCl, dried over anhydrous MgSO4, and evapo-
rated to dryness under vacuum. The residue was purified by column
chromatography on silica gel using eluent systems A and G.
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2011, 76, 6686e6693.
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Kessler, H. J. Am. Chem. Soc. 2010, 132, 8115e8128.
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7. Scanlon, D.; Harris, K. S.; Coley, A. M.; Karas, J. A.; Casey, J. L.; Hughes, A. B.;
Foley, M. Int. J. Pept. Res. Ther. 2008, 14, 381e386.
8. Veber, D. F.; Saperstein, R.; Nutt, R. F.; Freidinger, R. M.; Brady, S. F.; Curley, P.;
Perlow, D. S.; Paleveda, W. J.; Colton, C. D.; Zacchei, A. G.; Tocco, D. J.; Hoff, D. R.;
Vandlen, R. L.; Gerich, J. E.; Hall, L.; Mandarino, L.; Cordes, E. H.; Anderson, P. S.;
Hirschmann, R. Life Sci. 1984, 34, 1371e1378.
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Mandl, J.; Reubi, J. C.; Keri, G.; Kessler, H. ACS Med. Chem. Lett. 2011, 2, 509e514.
4.3.8. Saponification of peptide alkyl esters. Water (0.15 mL) and 2 M
methanolic solution of NaOH (3.75 mL) were added to a solution of
Ryakhovsky, Vladimir V.
