Synthesis of Short and Versatile Heterobifunctional Linkers for Conjugation of Bioactive Molecules with (Radio-)Labels

Abstract

The preparation of a series of short and versatile (eleven and twelve atom length) hydrophilic heterobifunctional linkers from low-cost chemicals using simple experimental setups is described. The approach can be used to connect high molecular weight bioactive molecules with azamacrocycles to enable radiolabeling with radiometals. The ring opening reaction of three cyclic anhydrides with 2-(2-aminoethoxy)ethanol afforded precursors 4a–c, which were subsequently converted into various heterobifunctional linkers for radiofluorination, Huisgen–Click approaches, or Staudinger ligation and for solid-phase peptide synthesis. As examples for successful building block ligation using the strain-promoted Huisgen cycloaddition on the one hand and the traceless Staudinger approach on the other hand, the Cetuximab antibody was modified by using 13a in a convenient two-step procedure.

Key words

bioorthogonal conjugation - click chemistry - Staudinger ligation - solid-phase synthesis

• References and Notes

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• 15 Synthesis of O-(N-succinimidyl) 4-oxo-4-{2-[2-(tosyloxy)ethoxy]ethylamino}butanoate (11a): Compound 10a (68 mg, 0.19 mmol) was dissolved in anhydrous DMF (1.5 mL). TSTU (1.2 equiv, 68 mg) and DIEA (1.2 equiv, 39.6 μL) were added and the solution allowed to react at r.t. for 30 min. The DMF was evaporated, the residue dissolved in CH₂Cl₂ and extracted twice with 5% aqueous acetic acid. The organic phase was evaporated to give the product (81 mg, 93%) as a colorless oil; R_f = 0.25 (CH₂Cl₂–MeCN, 7:3). ¹H NMR (400 MHz, CDCl₃): δ = 7.76 (d, J = 8.4 Hz, 2 H), 7.32 (d, J = 8.0 Hz, 2 H), 6.16 (s, 1 H), 4.16 (d, J = 4.6 Hz, 2 H), 3.62 (t, J = 4.5 Hz, 2 H), 3.48 (t, J = 4.8 Hz, 2 H), 3.39 (t, J = 5.0 Hz, 2 H), 2.95 (t, J = 7.1 Hz, 2 H), 2.80 (s, 4 H), 2.58 (t, J = 7.1 Hz, 2 H), 2.42 (s, 3 H). ¹³C NMR (101 MHz, CDCl₃): δ = 170.2, 169.2, 168.4, 145.2, 133.3, 130.1, 128.1, 70.0, 69.3, 68.6, 39.4, 30.8, 27.0, 25. 8, 21.9. MS (ESI+): m/z = 457.61 [M+H]⁺, 479.61 [M+Na]⁺, 495.58 [M+K]⁺. Anal. Calcd for C₁₉H₂₄N₂O₉S: C, 49.99; H, 5.30; N, 6.14. Found: C, 50.10; H, 5.25; N, 6.40.
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• 18 Synthesis of O-(N-succinimidyl) 4-[2-(2-azidoethoxy) ethylamino]-4-oxobutanoate (13a): Compound 12a (120 mg, 0.52 mmol) was dissolved in anhydrous DMF (3 mL). TSTU (1.2 equiv, 188 mg) and DIEA (1.2 equiv, 109 μL) were added and the solution was allowed to react at r.t. for 30 min. The DMF was evaporated and the residue was dissolved in chloroform and extracted three times with brine containing 5% acetic acid. The chloroform phase was evaporated to give the product (136 mg, 80%) as a colorless oil; R_f = 0.5 (EtOAc–HOAc, 19:1). This crude product was successfully engaged in coupling reactions with the antibody Cetuximab. Additional purification by column chromatography on silica gel was achieved by using EtOAc–HOAc (19:1) as eluent. ¹H NMR (400 MHz, CDCl₃): δ = 6.11 (s, 1 H), 3.62 (t, J = 5.0 Hz, 2 H), 3.52 (t, J = 4.8 Hz, 2 H), 3.43 (q, J = 5.0 Hz, 2 H), 3.32 (t, J = 4.9 Hz, 2 H), 2.93 (t, J = 7.1 Hz, 2 H), 2.78 (s, 4 H), 2.56 (t, J = 7.1 Hz, 2 H). ¹³C NMR (101 MHz, CDCl₃): δ = 170.3, 169.2, 168.4, 70.3, 69.9, 50.8, 39.5, 30.7, 26.9, 25.8. MS (ESI+): m/z = 328.34 [M+H]⁺, 350.35 [M+Na]⁺, 366.34 [M+K]⁺. Anal. Calcd for C₁₂H₁₇N₅O₆: C, 44.04; H, 5.24; N, 21.40. Found: C, 43.96; H, 5.12; N, 21.55.
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• 21 Synthesis of 2-(diphenylphosphano)phenyl 5-(1-dansylpiperazinyl)valerate (19): Compound 18 (500 mg, 1.19 mmol) and 2-(diphenylphosphano)phenol 17 (400 mg, 1.44 mmol) were dissolved in anhydrous THF (15 mL), EDC (340 mg, 1.77 mmol) and DMAP (cat.) were added and the mixture was stirred at 50 °C overnight. Water (25 mL) was added, the layers were separated, the aqueous layer was extracted with ethyl acetate (3 × 25 mL) and the combined organic layers were dried over Na₂SO₄. After evaporation of the solvent, the crude product was purified by column chromatography (EtOAc–petroleum ether, 2:1) to give 19 (85%) as a yellow syrup. ¹H NMR (400 MHz, CDCl₃): δ = 1.27–1.47 (m, 4 H), 2.15–2.27 (m, 4 H), 2.40 (br. s, 4 H), 2.85 (s, 6 H), 3.16 (br. s, 4 H), 6.74–6.79 (m, 1 H), 7.04–7.11 (m, 2 H), 7.12 (d, J = 7.4 Hz, 1 H), 7.21–7.35 (m, 11 H), 7.50 (dd, J = 7.4, 8.6 Hz, 2 H), 8.17 (dd, J = 1.2, 7.4 Hz, 1 H), 8.42 (d, J = 8.6 Hz, 1 H), 8.52 (d, J = 8.6 Hz, 1 H). ¹³C NMR (101 MHz, CDCl₃): δ = 22.3, 33.7, 45.5, 45.6, 45.7, 52.5, 57.8, 115.4, 120.0, 122.6, 123.3, 126.2, 128.1, 128.6, 128.7, 128.8, 129.1, 130.0, 130.2, 130.7, 130.8, 131.7, 131.8, 132.6, 133.8, 133.9, 134.1, 135.8, 151.8, 152.7, 152.9, 171.3. ³¹P NMR (176 MHz, CDCl₃): δ = δ = –15.2 ppm. MS (ESI+): m/z 680.44 [M+H]⁺, 702.51 [M+Na]⁺. Anal. Calcd for C₃₉H₄₂N₃O₄PS: C, 68.90; H, 6.23; N, 6.18. Found: C, 69.00; H, 6.02; N, 6.29.