Synthesis of Methionine Containing Peptides Related to Native Chemical Ligation

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.

chemical ligation - methionine ligation - peptides - S-methylation

References

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10

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.

11

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⁺].

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