Gram-Scale Solution-Phase Synthesis of Heptapeptide Side Chain of Teixobactin^[1]

^◊ These authors contributed equally to this work

Abstract

We report herein a scalable synthesis of linear heptapeptide side chain of the depsipeptide natural product teixobactin through solution phase. The synthesis of heptapeptide was achieved through an efficient coupling of suitably protected tripeptide and tetrapeptide comprising of three d-amino acids and four usual l-amino acid subunits.

Keywords

peptides - amino acids - antibiotics - solution-phase synthesis - heptapeptide - depsipeptide

• References and Notes

• 1 CSIR-IICT communication number: IICT/Pubs./2019/316.
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• 26 Synthetic Procedure for Key Dipeptide Intermediate, Methyl O-Benzyl-N-[(tert-butoxycarbonyl)-l-isoleucyl]-l-serinate (7) To a mixture of commercially available Boc-l-Ser(OBn)-OH (11, 5 g, 16.9 mmol, 1.0 equiv) in MeOH (50 mL) under nitrogen atmosphere, was added MeCOCl (1.8 mL, 25.3 mmol, 1.5 equiv) slowly at 0 °C. The reaction mixture was stirred at reflux for 3 h, and the solvent was distilled off in vacuo to get l-Ser(OBn)-OMe as a hydrochloride (4.1 g, 16.9 mmol). In another round-bottomed flask, Boc-l-isoleucine (12, 3.9 g, 16.9 mmol, 1.0 equiv) was dissolved in CH₂Cl₂ (50 mL) under nitrogen atmosphere, and HOBt (2.5 g, 18.5 mmol, 1.1 equiv) and EDC·HCl (4.85 g, 25.3 mmol, 1.5 equiv) were added sequentially at 0 °C and stirred for 15 min. To this reaction mixture was added dropwise a solution of the above-obtained l-Ser(OBn)-OMe hydrochloride salt (4.1 g, 16.9 mmol, 1.0 equiv) and DIPEA (11.5 mL, 67.6 mmol, 4.0 equiv) in dry CH₂Cl₂ (20 mL) at 0 °C and stirred for 1 h. Then reaction mixture was maintained at room temperature for 12 h. The reaction mixture was diluted with CH₂Cl₂ (100 mL) and washed with saturated aqueous NH₄Cl solution (2 × 75 mL). The organic layer was separated and washed with saturated aqueous NaHCO₃ solution (2 × 75 mL) followed by brine solution (75 mL). The organic layer was separated, dried over anhydrous Na₂SO₄, and concentrated under vacuum. The residue was purified by silica gel column chromatography (15% EtOAc in hexanes) to give key dipeptide 7 (6.39 g, 90%) as white solid. R_f = 0.5 (20% EtOAc in hexanes); mp 160–162 °C; [α]_D ²⁰ +26.30 (c 1, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 7.36–7.31 (m, 2 H), 7.31–7.23 (m, 3 H), 6.67 (d, J = 6.9 Hz, 1 H), 5.10 (d, J = 7.4 Hz, 1 H), 4.73 (dt, J = 7.9, 3.1 Hz, 1 H), 4.50 (q, J = 12.2 Hz, 2 H), 4.08–3.98 (m, 1 H), 3.89 (dd, J = 9.5, 3.2 Hz, 1 H), 3.72 (s, 3 H), 3.66 (dd, J = 9.5, 3.2 Hz, 1 H), 1.95–1.79 (m, 1 H), 1.51–1.45 (m, 1 H), 1.44 (s, 9 H), 1.20–1.09 (m, 1 H), 0.95 (d, J = 6.8 Hz, 3 H), 0.90 (t, J = 7.4 Hz, 3 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 171.4, 170.5, 155.7, 137.5, 128.6, 128.0, 127.8, 79.9, 73.4, 69.6, 59.2, 52.6, 37.8, 28.4, 24.8, 15.6, 11.7 ppm. IR (thin film): ν_max = 3285, 2964, 2928, 1707, 1642, 1512, 1367, 1165, 863, 749, 666 cm^–1. HRMS (ESI): m/z calcd for [M + Na]⁺ C₂₂H₃₄N₂O₆Na: 445.2315; found: 445.2313.
• 27 Synthetic Procedure for Tripeptide, Methyl O-Benzyl-N-(tert-butoxycarbonyl)-d-alloisoleucyl-l-isoleucyl-l-serinate (14) To a solution of Boc-Ile-Ser(OBn)-OMe (7, 5.0 g, 11.83 mmol, 1.0 equiv) in dry CH₂Cl₂ (5.0 mL) under nitrogen atmosphere was added TFA (5.0 mL, 65.2 mmol, 5.53 equiv) at 0 °C and stirred for 30 min at this temperature. Then reaction was maintained at room temperature for 2 h. After completion, the volatiles were removed in vacuo. The residue was dried under high vacuum for 1 h to get the corresponding amine salt (5.2 g, crude) as a pale brown thick liquid which was used for the next step directly. Boc-d-allo-isoleucine (10, 2.73 g, 11.80 mmol, 1.0 equiv) was dissolved in CH₂Cl₂ (30 mL) under nitrogen atmosphere. HOBt (1.75 g, 12.98 mmol, 1.1 equiv) and EDC·HCl (3.4 g, 17.7 mmol, 1.5 equiv) were added sequentially at 0 °C. A mixture of amine salt (5.2 g, crude, ca. 11.9 mmol, 1.0 equiv) and DIPEA (8.08 mL, 47.32 mmol, 4.0 equiv) in dry CH₂Cl₂ (50 mL) was added dropwise to the above reaction mixture at 0 °C and stirred for 30 min. Then reaction mixture was maintained at room temperature for 18 h. After completion of reaction, the reaction mixture was diluted with CH₂Cl₂ (100 mL) and washed with saturated aqueous NH₄Cl solution (2 × 75 mL). The organic layer was separated and washed with saturated aqueous NaHCO₃ solution (2 × 75 mL) followed by brine solution (75 mL). The organic layer was separated, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (25% EtOAc in hexanes) to give tripeptide 14 (5.16 g, 82%, over 2 steps) as white solid. R_f  = 0.5 (30% EtOAc in hexanes); mp 134–136 °C; [α]_D ²⁰ +13.80 (c 1, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ = 7.37–7.24 (m, 5 H), 6.75–6.64 (m, 2 H), 5.02 (d, J = 6.6 Hz, 1 H), 4.73 (dt, J = 8.0, 3.2 Hz, 1 H), 4.57–4.43 (m, 2 H), 4.41 (dd, J = 8.6, 6.3 Hz, 1 H), 4.24–4.10 (m, 1 H), 3.89 (dd, J = 9.5, 3.3 Hz, 1 H), 3.73 (s, 3 H), 3.64 (dd, J = 9.5, 3.0 Hz, 1 H), 2.07–1.95 (m, 1 H), 1.95–1.85 (m, 1 H), 1.58–1.48 (m, 1 H), 1.44 (s, 9 H), 1.42–1.34 (m, 1 H), 1.24–1.12 (m, 2 H), 0.98–0.87 (m, 9 H), 0.82 (d, J = 6.8 Hz, 3 H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ = 171.8, 170.8, 170.4, 155.8, 137.5, 128.6, 128.1, 127.8, 80.1, 73.4, 69.5, 58.3, 57.5, 52.7, 37.7, 37.3, 28.4, 26.6, 24.9, 15.4, 14.2, 11.8, 11.5 ppm. IR (thin film): ν_max = 3323, 2966, 2930, 1747, 1658, 1501, 1365, 1211, 1163, 1020, 867, 749, 665 cm^–1. HRMS (ESI): m/z calcd for [M + Na]⁺ C₂₈H₄₅N₃O₇Na: 558.3155; found: 558.3156.