Expedient Synthesis of Fmoc-(S)-γ-Fluoroleucine and Late-Stage Fluorination of Peptides

Abstract

A concise synthesis of (S)-γ-fluoroleucine is described in five steps from commercially available compounds, with an overall yield of 57%. The Markovnikov hydrofluorination reaction of the unsaturated amino acid precursor as last step of the synthesis proved to be effective. This fluorination can also be performed directly on a short peptide model with the same efficiency. This reaction could be in principle applicable for the preparation of radiolabeled amino acids and peptides.

Key words

(S)-γ-fluoroleucine - peptides - stereoselective synthesis - Selectfluor - late-stage fluorination

• References and Notes

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• 20 Fmoc-(S)-γ-fluoroleucine (5) Iron(III) oxalate hexahydrate (319 mg, 0.33 mmol) was stirred in H₂O (13.8 mL) until complete dissolution (ca. 2 h). The solution was cooled to 0 °C and degassed for 10 min. Selectfluor (234 mg, 0.66 mmol) and MeCN (6.6 mL) were added to the reaction mixture. A solution of 4 (135 mg, 0.33 mmol) in MeCN (6.6 mL) was transferred into the reaction mixture, and NaBH₄ (37.5 mg, 0.99 mmol) was added at 0 °C. After 2 min, the reaction was treated with an additional portion of NaBH₄ (37.5 mg, 0.99 mmol). The resulting mixture was stirred for 30 min at 0 °C before being quenched by addition of 28–30% aq NH₄OH (6.6 mL). The mixture was extracted with 10% MeOH in CH₂Cl₂, the organic layer was dried over MgSO₄, and concentrated under reduced pressure. Flash chromatography (SiO₂, cyclohexane–EtOAc = 95:5) provided 5 as a colorless oil (105 mg, 76%). ¹H NMR (300 MHz, CDCl₃): δ = 1.32 (d, J = 5.2 Hz, 3 H), 1.35 (s, 3 H), 1,38 (s, 9 H), 1.90–2.08 (m, 2 H), 4.15 (t, J = 7.1 Hz, 1 H), 4.25–4.35 (m, 2 H), 5.35 (br d, J = 7.3 Hz, 1 H), 7.22 (dt, J ₁ = 1.2, J ₂ = 7.4 Hz, 2 H), 7.31 (t, J = 7.4 Hz, 2 H), 7.52 (d, J = 7.3 Hz, 2 H), 7.70 (d, J = 7.4 Hz, 2 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 171.4, 155.7, 143.9, 143.8, 141.3, 127.7, 127.0, 125.1, 119.9, 95.0 (J = 166.1 Hz), 82.2, 67.0, 52.0, 47.2, 42.7 (J = 22.1 Hz), 27.9, 27.4 (J = 24.6 Hz), 26.6 (J = 24.6 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ = –135.7 ppm. ESI-HRMS: m/z C₂₅H₃₀NO₄NaF calculated [M + Na]: 450.2053; found: 450.2057. Compound 6 The dipeptide Boc-Ala-Gly-OH was directly coupled to the free amino ester extracted after acidic cleavage of benzophenon­imine derivative 3, in DMF, in the presence of BOP (1 equiv) and DIPEA (3 equiv). The mixture was stirred for 24 h at room temperature. The solvent was evaporated, and the residue was diluted with EtOAc and consecutively extracted with 1 M KHSO₄ (2×), aq NaHCO₃ (2×) and brine (2×), dried over Na₂SO₄, and the solvent was evaporated under reduced pressure to afford the crude product. Flash chromatography (SiO₂, cyclohexane–EtOAc = 20:80) provided 6 as a colorless oil (210 mg, 85%). ¹H NMR (300 MHz, CDCl₃): δ = 1.35 (d, J = 7.1 Hz, 2 H), 1.41 (s, 9 H), 1.43 (s, 9 H), 1.43 (s, 3 H), 2.36 (dd J ₁ = 7.9 Hz, J ₂ = 13.9 Hz, 1 H), 2.48 (dd J ₁ = 6.1 Hz, J ₂ = 13.9 Hz, 1 H), 3.94 (ddd, J ₁ = 5.5 Hz, J ₂ = 16.6 Hz, J ₃ = 34.0 Hz, 2 H), 4.17–4.22 (m, 1 H), 4.55 (q, J = 6.2 Hz, 1 H), 4.72 (s, 1 H), 4.80 (t, J = 1.5 Hz, 2 H), 5.34 (br d, J = 6.9 Hz, 1 H), 6.87 (br d, J = 7.7 Hz, 1 H), 7.16 (br t, J = 5.2 Hz, 1 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 173.4, 171.0, 168.5, 155.5, 140.5, 114.3, 103.3, 82.2, 80.0, 51. 2, 50.2, 42.9, 40.6, 28.3, 27.9, 21.8, 18.4 ppm. ESI-HRMS: m/z calcd for C₂₀H₃₅N₃O₆Na [M + Na]: 436.2421; found: 436.2424. Compound 7 Following the procedure described for compound 5, peptide 7 was prepared starting from precursor 6 (37 mg, 0.089 mmol) and purified by flash chromatography (SiO₂, n-hexane–EtOAc = 30:70) providing the desired compound as a colorless oil (30 mg, 77%). ¹H NMR (300 MHz, CDCl₃): δ = 1.29 (d, J = 8.9 Hz, 3 H), 1.31 (d, J = 7.0 Hz, 3 H), 1.37 (s, 9 H), 1.37 (s, 9 H), 1.93–2.08 (m, 2 H), 3.90 (ddd, J ₁ = 5.6 Hz, J ₂ = 16.8 Hz, J ₃ = 37.4 Hz, 2 H), 4.06–4.16 (m, 1 H), 4.44–4.51 (m, 1 H), 5.01 (br d, J = 6.8 Hz, 1 H), 6.71 (br d, J = 6.0 Hz, 1 H), 6.82 (br t, J = 5.0 Hz, 1 H). ¹³C NMR (75 MHz, CDCl₃): δ = 173.1, 170.8, 168.3, 155.5, 95.1 (J = 165.9 Hz), 82.2, 80.3, 50.4, 43.0, 42.1 (J = 22.0 Hz), 28.3, 27.8, 27.3 (J = 25.6 Hz), 26.5 (J = 24.5 Hz), 18.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ = –135.6 ppm. ESI-HRMS: m/z calcd for C₂₀H₃₇N₃O₆F [M + H]: 434.2666; found: 434.2666. Compound 8 Cbz-l-Asp(OBzl)-OH was directly coupled to the free amine extracted after acid cleavage of benzophenonimine derivative 3 in a solution of the free amino ester in DMF in the presence of HATU (1 equiv) and DIPEA (3 equiv). The mixture was stirred for 24 h at room temperature. The solvent was evaporated, and the residue was diluted with EtOAc and consecutively extracted with 1 M KHSO₄ (2×), aq NaHCO₃ (2×) and brine (2×), dried over Na₂SO₄, and the solvent was evaporated under reduced pressure to afford the crude product. Flash chromatography (SiO₂, cyclohexane–EtOAc = 80:20) provided 8 as colorless oil (590 mg, 75%). ¹H NMR (300 MHz, CDCl₃): δ = 1.47 (s, 9 H), 1.76 (s, 3 H), 2.43 (ddd, J ₁ = 6.0 Hz, J ₂ = 14.0 Hz, J ₃ = 52.4 Hz, 2 H), 2.76 (dd, J ₁ = 6.4 Hz, J ₂ = 17.2 Hz, 1 H), 3.08 (dd, J ₁ = 4.1 Hz, J ₂ = 17.2 Hz, 1 H), 4.51–4.55 (m, 1 H), 4.63–4.67 (m, 1 H), 4.74 (s, 1 H), 4.83 (s, 1 H), 5.11–5.19 (m, 4 H), 5.95 (br d, J = 8.4 Hz, 1 H), 6.88 (br d, J = 6.7 Hz, 1 H), 7.36–7.39 (m, 10 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 171.7, 170.6, 169.8, 155.9, 140.6 136.0, 135.3, 128.6, 128.4, 128.3, 128.1, 114.5, 82.1, 67.2, 66.9, 51.3, 50.8, 40.5, 36.2, 27.9, 21.8 ppm. ESI-HRMS: m/z calcd for C₂₉H₃₇N₂O₇ [M + H]: 525.2603; found: 525.2601. Compound 9 Following the same procedure described for compound 5, peptide 9 was prepared starting from precursor 8 (170 mg, 0.324 mmol) and purified by flash chromatography (SiO₂, cyclohexane–EtOAc = 80:20) providing the desired compound as a colorless oil (133 mg, 75%). ¹H NMR (300 MHz, CDCl₃): δ = 1.36 (d, J = 11.2 Hz, 3 H), 1.41 (d, J = 11.2 Hz, 3 H), 1.44 (s, 9 H), 1.93–2.15 (m, 2 H), 2.74 (dd, J ₁ = 6.17 Hz, J ₂ = 17.3 Hz, 1 H), 3.10 (dd, J ₁ = 3.9, J ₂ = 17.1 Hz, 1 H), 4.46–4.51 (m, 1 H), 4.61–4.66 (m, 1 H), 5.08–5.16 (m, 4 H), 5.93 (d, J = 8.4 Hz, 1 H), 7.04 (d, J = 6.0 Hz, 1 H), 7.34–7.36 (m, 10 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 171.7, 170.5, 169.9, 156.0, 136.0, 135.3, 128.6, 128.5, 128.3, 128.3, 128.2, 128.1, 95.5 (J = 165.8 Hz), 82.1, 67.2, 66.9, 50.8, 50.7, 42.0 (J = 21.8 Hz), 36.0, 27.8, 27.4 (J = 24.5 Hz), 26.3 (J = 25.5 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ = –135.4 ppm. ESI-HRMS: m/z calcd for C₂₉H₃₈N₂O₇F [M + H]: 545.2659; found: 545.2663. Compound 10 Boc-l-Lys(Cbz)-OH was directly coupled to the free amine extracted after acid cleavage of benzophenonimine derivative 3 in a solution of the free amino ester in DMF in the presence of HATU (1 equiv) and DIPEA (3 equiv). The mixture was stirred for 24 h at room temperature. The solvent was evaporated, and the residue was diluted with EtOAc and consecutively extracted with 1 M KHSO₄ (2×), aqueous NaHCO₃ (2×) and brine (2×), dried over Na₂SO₄, and the solvent was evaporated under reduced pressure to afford the crude product. Flash chromatography (SiO₂, cyclohexane–EtOAc = 70:30) provided 10 as colorless oil (600 mg, 73%). ¹H NMR (300 MHz, CDCl₃): δ = 1.30–1.59 (m, 25 H), 1.67 (s, 3 H), 1.71–1.80 (m, 2 H), 2.36 (ddd, J ₁ = 6.0 Hz, J ₂ = 14.0 Hz, J ₃ = 52.4 Hz, 2 H), 3.10–3.15 (m, 2 H), 4.01 (m, 1 H), 4.46 (m, 1 H), 4.67 (s, 1 H), 4.75 (t, J = 1.5 Hz, 1 H), 4.95 (br s, 1 H), 5.02 (s, 2 H), 5.06 (br s, 1 H), 6.33 (d, J = 7.1 Hz, 1 H), 7.22–7.28 (m, 5 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 171.6, 171.0, 156.5, 155.6, 140.6, 136.6, 128.5, 128.1, 128.0, 114.4, 82.2, 79.9, 66.6, 54.1, 51.0, 40.6, 40.4, 32.0, 29.3, 28.3, 27.9, 22.3, 21.9 ppm. ESI-HRMS: m/z calcd for C₂₉H₄₆N₃O₇ [M + H]: 548.3336; found: 548.3336. Compound 11 Following the same procedure described for compound 5, peptide 11 was prepared starting from precursor 10 (170 mg, 0.310 mmol) and purified by flash chromatography (SiO₂, cyclohexane–EtOAc = 80:20) providing the desired compound as a colorless oil (130 mg, 74%) ¹H NMR (300 MHz, CDCl₃): δ = 1.36–1.64 (m, 30 H), 1.79–1.85 (m, 1 H), 1.97–2.14 (m, 2 H), 3.17–3.22 (m, 2 H), 4.09 (m, 1 H), 4.50–4.52 (m, 2 H), 5.01 (br s, 1 H), 5.09 (s, 2 H), 5.11 (br s, 1 H), 6.57 (d, J = 5.1 Hz), 7.29–7.35 (m, 5 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 171.5, 170.8, 156.5, 155.5, 136.6, 128.4, 128.1, 128.0, 95.2 (J = 165.5 Hz), 82.2, 66.6, 54.1, 50.4, 42.2 (J = 21.9 Hz), 40.4, 32.0, 29.3, 28.3, 27.8, 27.4 (J = 24.3 Hz), 26.9, 26.4 (J = 24.5 Hz), 22.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ = –135.75 ppm. ESI-HRMS: m/z calcd for C₂₉H₄₇N₃O₇F [M + H]: 568.3399; found: 568.3398.
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