An Operationally Simple and Efficient Synthesis of Orthogonally Protected l-threo-β-Hydroxyasparagine

Abstract

A synthesis of orthogonally protected l-threo-β-hy­droxyasparagine from l-aspartic acid is reported. Iodocyclization of 3-benzoylaminoaspartic acid provided an intermediate oxazoline dicarboxylate that was efficiently hydrolyzed to l-threo-β-hy­droxyaspartic acid. The synthetic route for conversion of the free β-hydroxy-α-amino acid into the target compound is highly efficient and amenable to the preparation of various orthogonally protected asparagine derivatives, on a multigram scale.

asymmetric synthesis - amino acids - amides - peptides - protecting groups

References and Notes

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l- threo -BocNH-β-OH-Asp(OMe)-OH ( 4): l-threo-β-Hydroxyaspartic acid (1.00 g, 5.41 mmol) was dissolved in a solution of concentrated HCl (0.89 mL, 10.8 mmol) and MeOH (18.0 mL) at 0 °C. The reaction was heated at reflux for 3 h, then cooled to r.t., and concentrated in vacuo. The crude l-threo-β-OH-Asp(OMe)-OH was triturated with Et₂O, collected by filtration, and used without further purification in the following reaction. An aqueous solution of 10% Na₂CO₃ (18.0 mL) was added to l-threo-β-OH-Asp(OMe)-OH and the resulting mixture was cooled in an iced bath. A solution of Boc₂O (3.65 g, 16.2 mmol) in dioxane (18.0 mL) was added dropwise to the reaction mixture and the resulting solution was stirred overnight at r.t. After concentration by rotary evaporation, the residue was dissolved in EtOAc (150 mL) and washed with 1 N HCl solution (3 × 100 mL). The organic layer was separated, dried over Na₂SO₄, filtered, and concentrated in vacuo. Flash chromatography (SiO₂, 35% EtOAc-hexanes followed by 10-25% MeOH-CHCl₃) provided 4 (0.88 g, 62%) as a foamy white solid; mp 109-110 °C; [α]²³ _D +22 (c = 1.00, MeOH). ¹H NMR (500 MHz, CD₃OD): δ = 4.79 (s, 1 H), 4.50 (s, 1 H), 3.77 (s, 3 H), 1.46 (s, 9 H). ¹³C NMR (125 MHz, CD₃OD): δ = 174.2, 170.2, 155.1, 80.4, 73.0, 59.0, 52.8, 28.7. IR (film): 3388, 2981, 2934, 2858, 1739, 1699, 1608, 1512, 1393, 1367, 1251, 1167, 1107, 1061 cm^-1. HR-EI-MS: m/z [M⁺] calcd for C₁₀H₁₇O₇N: 263.1000; found: 263.1001.
l- threo -BocNH-β-OH-Asn ( 5): A sample of l-threo-BocNH-β-OH-Asp(OMe)-OH (4; 1.30 g, 4.94 mmol) was dissolved in MeOH (16.5 mL) and treated with NH₃ gas until the solution became saturated. The resulting mixture was stirred at r.t. for 3 d. The reaction was concentrated by rotary evaporation and the crude material was purified by flash chromatography (SiO₂, 25% → 50% MeOH-CHCl₃), which provided 5 (1.10 g, 90%) as a crystalline solid; mp 165-166 °C; [α]²³ _D -11 (c = 1.0, MeOH). ¹H NMR (400 MHz, CD₃OD): δ = 4.63 (s, 1 H), 4.48 (s, 1 H), 1.40 (s, 9 H). ¹³C NMR (125 MHz, CD₃OD): δ = 177.8, 177.7, 157.6, 80.4, 73.6, 59.4, 28.8. IR (KBr): 3369, 2980, 2933, 1694, 1602, 1510, 1395, 1251, 1168, 1109, 1102, 1059, 1029 cm^-1. HR-EI-MS: m/z [M⁺] calcd for C₉H₁₆O₆N₂: 249.1003; found: 249.1002.
l- threo -BocNH-β-OH-Asn-OBn ( 6): A solution of l-threo-BocNH-β-OH-Asn (5; 0.910 g, 3.67 mmol) in DMF (18.3 mL) at 0 °C was treated with NaHCO₃ (0.616 g, 7.33 mmol) and benzyl bromide (1.74 mL, 14.3 mmol). The resulting solution was stirred at 0 °C for 2 h and then for 24 h at r.t. The solution was cooled to 0 °C and H₂O (100 mL) was slowly added. The resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic extracts were washed with H₂O (100 mL). The organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by flash chromatography (SiO₂, 0% → 5% MeOH-CHCl₃), which provided 6 (1.06 g, 86%) as a crystalline solid; mp 135-136 °C; [α]²³ _D -17 (c = 1.0, CHCl₃). ¹H NMR (400 MHz, CD₃OD): δ = 7.30-7.38 (m, 5 H), 5.19 (s, 2 H), 4.70 (d, J = 2.0 Hz, 1 H), 4.59 (d, J = 2.5 Hz, 1 H), 1.40 (s, 9 H). ¹³C NMR (125 MHz, CD₃OD): δ = 176.2, 171.7, 157.8, 136.9, 129.3, 129.2, 129.0, 128.9, 80.8, 72.7, 68.2, 58.1, 28.7. IR (film): 3358, 2979, 1685, 1653, 1559 cm^-1. HR-EI-MS: m/z [M⁺] calcd for C₁₆H₂₂O₆N₂: 338.1472; found: 338.1471.
l- threo -FmocNH-β-OH-Asn-OBn ( 7): l-threo-BocNH-β-OH-Asn-OBn (6; 0.740 g, 2.19 mmol) was dissolved in 4 N HCl-dioxane (16.4 mL) and stirred at r.t. for 2 h. The solution was concentrated by rotary evaporation and dried under high vacuum. The residue obtained was dissolved in 50% dioxane-H₂O (32.4 mL, 1:1) and the resulting solution was cooled to 0 °C. Fmoc-OSuc (1.14 g, 3.28 mmol) and NaHCO₃ (0.368 g, 4.37 mmol) were added to the solution and the resulting mixture was stirred at r.t. for 12 h. The reaction solution was diluted with EtOAc (100 mL) and sat. aq NaHCO₃ (150 mL). The aqueous layer was separated and extracted with EtOAc (2 × 75 mL). The combined organic extracts were dried over Na₂SO₄ and concentrated by rotary evaporation. The crude product was dissolved in the minimal amount of 20% MeOH-CHCl₃ solution. Precipitation with hexanes and collection of the solid by filtration provided 7 as a white solid; mp 173-174 °C; [α]²³ _D -18 (c = 0.80, CHCl₃). ¹H NMR (400 MHz, CD₃OD): δ = 7.78 (d, J = 7.2 Hz, 2 H), 7.64 (d, J = 6.0 Hz, 2 H), 7.26-7.38 (m, 9 H), 5.21 (d, J = 3.2 Hz, 2 H), 4.64 (d, J = 2.4 Hz, 1 H), 4.35 (dd, J = 3.6, 6.0 Hz, 1 H), 4.22 (m, 2 H). ¹³C NMR (125 MHz, CD₃OD): δ = 176.4, 171.7, 158.8, 145.2, 145.1, 142.5, 142.4, 137.0, 129.5, 129.2, 129.1, 128.8, 128.7, 128.2, 128.1, 126.3, 126.2, 120.1, 72.8, 68.4, 68.3, 58.6, 48.2. IR (film): 3328, 1724, 1695, 1655, 1539, 1451, 1299, 1255, 1109 cm^-1. HR-EI-MS: m/z [M + H⁺] calcd for C₂₆H₂₄O₆N₂: 460.1629; found: 460.1638.
l- threo -FmocNH-β-OH-Asn(Tr)-OBn ( 8): A solution of l-threo-FmocNH-β-OH-Asn-OBn (7; 0.863 g, 1.87 mmol) and Tr-OH (5.05 g, 18.7 mmol) in HOAc (6.52 mL) was heated to 50 °C and treated with concd H₂SO₄ (65 µL, 1.12 mmol) and Ac₂O (0.443 mL, 4.69 mmol). The resulting mixture was stirred at 50 °C for 2.5 h. After cooling to r.t., the reaction solution was diluted with EtOAc (100 mL) and sat. aq NaHCO₃ (150 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic layers were dried over Na₂SO₄ and concentrated in vacuo. Purification by flash chromatography (SiO₂, 15% → 30% EtOAc-hexanes) provided 8 (1.03g, 1.46 mmol, 78%) as a white solid; mp 74-75 °C; [α]²³ _D -14 (c = 0.93, CHCl₃). ¹H NMR (400 MHz, CD₃OD): δ = 7.79 (d, J = 3.2 Hz, 1 H), 7.77 (d, J = 3.2 Hz, 1 H), 7.67 (d, J = 7.6 Hz, 1 H), 7.62 (d, J = 7.2 Hz, 1 H), 7.17-7.38 (m, 24 H), 5.20 (s, 2 H), 4.81 (d, J = 2.0 Hz, 1 H), 4.65 (d, J = 2..0 Hz, 1 H), 4.51 (dd, J = 4.0, 9.6 Hz, 1 H), 4.17 (m, 2 H), ¹³C NMR (125 MHz, CHCl₃): δ = 172.2, 171.8, 158.9, 145.7, 145.3, 145.0, 142.5, 142.4, 137.1, 129.9, 129.7, 129.5, 129.2, 129.0, 128.9, 128.8, 128.7, 128.2, 128.1, 126.5, 126.2, 120.9, 73.4, 71.5, 68.6, 68.3, 58.6, 48.2. IR (film): 3347, 3061, 3022, 2915, 2522, 1723, 1709, 1670, 1493, 1451, 1333, 1218 cm^-1. HR-EI-MS: m/z [M⁺] calcd for C₄₅H₃₈O₆N₂: 702.2724; found: 702.2721.
l- threo -FmocNH-β-OH-Asn(Tr)-OH ( 2): Pd/C (0.086 g) was cautiously added to a solution of l-threo-FmocNH-β-OH-Asn(Tr)-OBn (8; 0.860 g 1.22 mmol) in EtOH (12.2 mL). The solution was placed under an atmosphere of hydrogen gas for 1 h. The catalyst was removed by filtration through Celite and the filter cake was washed with EtOH. The combined filtrates were concentrated in vacuo and purified by flash chromatography (SiO₂, 25% → 50% MeOH-CHCl₃), which provided 2 (0.674g, 90%) as a white solid; mp 189-190 °C (dec.); [α]²³ _D -17 [c = 1.00, MeOH-CHCl₃ (1:1)]. ¹H NMR (400 MHz, DMSO-d ₆): δ = 8.29 (s, 1 H), 7.88 (d, J = 7.5 Hz, 2 H), 7.76 (d, J = 6.5 Hz, 1 H), 7.65 (d, J = 7.0 Hz, 1 H), 7.38 (q, J = 5.0, 7.0 Hz, 2 H), 7.08-7.27 (m, 18 H), 4.52 (d, J = 12 Hz, 1 H), 4.35 (m, 3 H), 4.17 (t, J = 6.5 Hz, 1 H). ¹³C NMR (DMSO, 125 MHz): δ = 173.8, 171.0, 156.1, 144.7, 143.8, 143.7, 140.7, 128.4, 127.7, 127.1, 126.7, 125.3, 120.1, 72.8, 69.1, 65.8, 57.7, 46.7. IR (KBr): 3387, 3062, 2926, 2857, 1954, 1695, 1601, 1510, 1448, 1404, 1328, 1248, 1106, 1058 cm^-1. HR-EI-MS: m/z [M⁺] calcd for C₃₈H₃₂O₆N₂: 612.2255; found: 612.2256.