Synthesis of ²H-Labelled α-Azidoisobutyryl Chloride (D₆-Azib-Cl) as ²H₆-Aib Equivalent Building Block in Peptide Synthesis

Abstract

A new synthesis of ²H-labelled α-azidoisobutyryl ­chloride for application as a building block in peptide synthesis is presented. Starting from readily available acetone cyanohydrin-D₆, an efficient synthetic protocol provides the deuterated target molecule α-azidoisobutyryl chloride (D₆-Azib-Cl) in good yield with very high deuterium incorporation. This compound represents an activated, N-protected equivalent of ²H-labelled α-aminoisobutyric acid (D₆-Aib), which will be especially useful for NMR investigations on peptides with a high Aib content.

Key words

isotopic labelling - azido acids - peptides - bioorganic chemistry - deuterium

References

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12

Typical procedure: 4-Toluene sulfonyl chloride (17.2 g, 90.0 mmol) is added at 0 °C to a solution of acetone cyanohydrin-D₆ (D ₆ -1) (5.47 g, 60.0 mmol) and DABCO (11.2 g, 100 mmol) in dry CH₂Cl₂ (100 mL). The reaction mixture is stirred at 0 °C for 5 min and then for an additional 1.5 h at r.t. EtOAc (500 mL) is added, the solid is filtered off and washed with EtOAc. The filtrate is washed with water and sat. NaHCO₃, dried over Na₂SO₄ and concentrated to 50 mL. The pure product is obtained by crystallization from EtOAc. Further concentration and crystallization gives a mixture of the product, 4-toluene sulfonyl chloride and DABCO hydrochloride, which can be separated by flash-chromatography (silica gel, petroleum ether-EtOAc, 12:1); D ₆ -2 is obtained as a colorless solid (10.2 g, 70%). Mp: 98 °C; ¹H NMR (250.1 MHz, CDCl₃): δ = 2.46 (s, 3 H), 7.36 (m, 2 H), 7.85 (m, 2 H); ¹³C NMR (62.9 MHz, CDCl₃): δ = 21.7, 28.4 (m), 74.6, 118.2, 127.9, 129.9, 134.3, 145.5; IR (KBr): ν [cm^-1] = 3061, 2360, 1594, 1359 (st), 1181 (st), 1089, 885 (sst), 715, 676; MS (EI, 70 eV): m/z = 245 (29%), 173 (99%), 155 (25%), 91 (100%); degree of deuteration (¹H NMR): 92%.

13

Typical procedure: 2-(4′-Toluene sulfonyloxy)-2-methyl propionitrile-D₆ (D ₆ -2) (7.19 g, 29.3 mmol) is added to a stirred solution of NaN₃ (2.86 g, 44.0 mmol) in DMSO (60 mL) at 40 °C. After stirring overnight at 40 °C, aqueous work up (300 mL of water) and extraction with Et₂O, the organic layers are washed with water and brine, dried over Na₂SO₄ and evaporated. The pure product D ₆ -3 is obtained as a colorless liquid (2.54 g, 75%). ¹³C NMR (62.9 MHz, CDCl₃): δ = 25.7 (sept, J = 20 Hz), 54.5, 119.4; IR (Film): ν [cm^-1] = 2953, 2458, 2244, 2111 (sst), 1456, 1239 (st), 1160, 1101, 1032 (st), 810, 775, 672; GC-MS: t _R = 3.4 min (100%); MS: m/z = 116 (11%), 74 (87%), 46 (100%).

17

Typical procedure: 2-Azido-2-methyl propionitrile-D₆
D ₆ -3 (6.39 g, 55.0 mmol) is stirred in dry THF (400 mL) at -15 °C under argon. DIBALH (110 mL, 110 mmol, 1.0 M in THF) is added and the mixture is stirred at -15 °C for 30 min and then at r.t. for 2 h. Water (100 mL) is added carefully at 0 °C and the THF is evaporated in vacuo. After acidic workup (500 mL of water, 150 mL of H₂SO₄) and extraction with CH₂Cl₂, the organic layers are dried over Na₂SO₄ and concentrated to 50-100 mL at ambient temperature and pressure. The crude azido aldehyde D ₆ -4 and sodium chlorite (67.8 g, 600 mmol, 80%) are dissolved in a mixture of tert-butanol (60 mL) and water (500 mL). Amido sulfuric acid (58.3 g, 600 mmol) is added carefully and the reaction mixture is stirred at r.t. for 1 h. The aqueous mixture is extracted with Et₂O, the organic layers are washed with brine, dried over Na₂SO₄ and the solvent is evaporated. Dicyclohexyl amine (16 mL, 80.0 mmol) in dry Et₂O (200 mL) is added to the crude azido acid D ₆ -5 and the solvent is evaporated. The residue is recrystallized from Et₂O, CH₂Cl₂-n-hexane (1:1). The isolated colorless solid is dissolved in a minimum of water and the solution is acidified to pH 1-2 with concentrated HCl. The precipitate is filtered off and the aqueous solution is extracted with Et₂O. The organic layers are washed with 1 M HCl, dried over Na₂SO₄ and the solvent is evaporated to give an oily colourless substance, which crystallizes at reduced temperature to give colorless needles (0.76 g, 10%). Mp: 31-32 °C; ¹H NMR (250.1 MHz, CDCl₃): δ = 9.02 (s); ¹³C NMR (62.9 MHz, CDCl₃): δ = 23.4 (sept, J = 20 Hz), 62.6, 178.5; IR (Film): ν [cm^-1] = 2934 (sst), 2858 (st), 2361, 2111 (st), 1627 (st), 1451, 1383, 1267; MS (EI, 70 eV): m/z = 89 (13%), 76 (32%).