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Synlett 2003(5): 0659-0662
DOI: 10.1055/s-2003-38361
LETTER
© Georg Thieme Verlag Stuttgart ˙ New York

Synthesis of Methionine Containing Peptides Related to Native Chemical Ligation

Kandasamy Pachamuthu, Richard R. Schmidt*
Fachbereich Chemie, Universität Konstanz, Fach M 725, 78457 Konstanz, Germany
e-Mail: Richard.Schmidt@uni-konstanz.de;
Further Information

Publication History

Received 21 January 2003
Publication Date:
28 March 2003 (online)

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Abstract

Methionine chemical ligation is based on a convenient direct thioester formation at the C-terminus of the ligation site, on homocysteine reduction with dithiothreitol in order to liberate the mercapto group of the homocysteine residue at the N-terminus, and on chemoselective S-methylation with methyl iodide.

Key words

chemical ligation - methionine ligation - peptides - S-methylation

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General Procedure for the Synthesis of Peptides 8-14, 16: To a stirred solution of homocystine (200 mg, 0.75 mmol) in 0.1 M Tris buffer (10 mL, pH ca. 8.0) containing 6 M guanidinium hydrochloride was added DTT (230 mg, 1.5 mmol) and the reaction mixture was allowed to stir at r.t. After 1 h, thioester (1.5 mmol) was added to the reaction mixture. After completion of the reaction (TLC monitoring), the reaction mixture was washed with CHCl3 and the aq layer was neutralized with dilute HCl; the aq layer was then extracted with EtOAc followed by washing the EtOAc layer with brine and the organic layer was dried over anhyd Na2SO4. Evaporation of the solvent yielded the corresponding crude thiol, which was purified by recrystallization. This thiol was immediately taken up in NH3 in MeOH (3 mL) and MeI (3 equiv) was added into the reaction mixture at 0 °C. This reaction mixture was allowed to stir at 0 °C for 2.0 h and at r.t. for 30 min. Then the reaction mixture was neutralized with dilute HCl followed by extraction with EtOAc, washing the EtOAc layer with H2O and the organic layer was dried over anhyd Na2SO4. Evaporation of the organic solvent yielded the crude product which was purified by recrystallization or column chromatography. All products were characterized by NMR data.

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Compounds 8-14, 16 were characterized by [α] d , 1H NMR and 13C NMR data. 8: Mp 56-60 °C (lit. [14] : 59-62 °C); [α] d 20 -27.7 (c 1, MeOH). 9: Mp 82-88 °C; [α] d 20 +27.8 (c 1, MeOH). 10: Mp 50-54 °C; [α] d 20 -15.3 °C (c 1, MeOH). 11: Mp 70-80 °C; [α] d 20 -2.2 (c 1, MeOH). 12: Mp 145-149 °C (lit. [15] : 149-154 °C); [α] d 20 -19.0 (c 1, DMF) (lit.15 [α] d -20 (c 1, DMF). 13: Mp 83-88 °C (lit. [16] : 90 °C); [α] d -3.5 (c 1, MeOH). 14: Mp 65 °C (petroleum ether/ethyl acetate); [α] d 20 -62.0 (c 1, MeOH). 1H NMR (250 MHz, CDCl3): δ = 1.36 (d, J = 6.5 Hz, 3 H), 1.43 (s, 9 H), 1.99-2.30 (m, 6 H), 2.05 (s, 3 H), 2.46 (t, 6.4 Hz, 2 H), 2.90-3.10 (m, 2 H), 3.50-4.60 (m, 4 H), 6.20-6.40 (br s, 1 H), 7.40-7.60 (br s, 1 H). 13C NMR (62.9 MHz, CDCl3): δ = 15.4, 18.1, 25.0, 28.4, 30.8, 40.8, 47.5, 48.0, 54.4, 60.8, 77.2, 79.6, 155.5, 171.5, 173.8, 177.8; MALDI: m/z = 456 [M + K+]. 16: Mp 52-56 °C; [α] d 20 -39.5 (c 1, MeOH). 1H NMR (250 MHz, CDCl3): δ = 1.12 (d, J = 6.4 Hz, 3 H), 1.40 (s, 9 H), 1.90-2.2 (m, 2 H), 2.03 (s, 3 H), 2.50 (t, J = 7.4 Hz, 2 H), 3.33 (d, J = 3.0 Hz, 1 H), 4.06-4.09 (m, 1 H), 4.23-4.26 (m, 1 H), 4.39 (t, J = 5.4 Hz, 2 H), 4.56-4.65 (m, 1 H), 5.50 (d, J = 7.6 Hz, 1 H), 6.9 (br s, 1 H), 7.2-7.4 (m, 6 H). 13C NMR (62.9 MHz, CDCl3): δ = 15.2, 18.5, 28.2, 30.1, 31.2, 43.5, 52.6, 58.7, 67.2, 80.4, 127.4, 127.6, 128.6, 137.8, 156.2, 170.9. 171.2. MALDI: m/z = 462 [M + Na+].