PLoS One 8; DOI: 10

PLoS One 8; DOI: 10.1371/journal.pone.0084864. 7.36 (m, 1H), 4.97C4.08 (m, 4H), 4.08C3.00 (m, 16H), 2.84C2.59 (m, 2H), 2.31C2.56 (m, 2H). 13C NMR (100 MHz, CDCl3): = 174.1, 173.2, 166.7, 165.9, 157.7, 143.9, 130.33, 128.2, 70.4, 70.2, 70.0, 69.9, 66.9, 66.4, 65.4, 42.9, 36.3, 34.6, 31.9. MS (MALDI) calcd. for C23H33N5O8: 507.2; discovered: 530.7 ([M + Na]+). Substance Tz(NHS). To a remedy of PEG-4 tetrazine derivative Tz(OH) (0.08 g, 0.16 mmol) in DMF (1.0 mL) was added = 10.17 (s, 1H), 8.50 (m, 2H), 7.50 (m, 2H), 4.54 (m, 2H), 4.98C3.37 (m, 20H), 2.88C2.73 (m, 2H), 2.62 (s, 4H), 2.60C2.43 (m, 2H). 13C NMR (100 MHz, CDCl3): = 172.6, 172.2, 169.1, 166.2, 157.7, 144.3, 130.33, 128.4, 128.2, 70.4, 70.4, 70.1, 67.2, 66.5, 65.6, 51.7, 42.9, 36.7, 34.7, 32.06, 25.4, MS (MALDI) calcd. for C27H36N6O10: 604.2; discovered: 627.4 ([M + Na]+). Substance 5. To a remedy of 5-norbornene-2-carboxylic acidity (4, 0.10 g, 0.72 mmol) in DMF (2.0 mL) was added the N-Boc-2,2-(ethylenedioxy)diethylamine (0.23 g, 0.92 mmol), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (0.14 g, 0.72 mmol), and triethylamine (0.07 g, 0.72 mmol). The resultant option was stirred for 12 h, as well as the solvent evaporated. The crude item was purified by adobe flash chromatography (ethyl acetate) to provide a triethylene glycol (PEG-3) customized norbornene derivative 5 (0.19 g, 0.50 mmol, 70%) like a colorless oil. 1H NMR (400 MHz, CDCl3): = 6.37 (s, 1H), 5.93 (m, 2H), 5.13 (s, 1H), 3.43 (m, 2H), 3.36 (m, 2H), 3.27 (m, 2H), 3.11 (m, 2H), 2.72 (m, 2H), 1.85 (m, 1H), 1.73 (m, 1H), 1.56 (m, 1H), 1.25 (s, 9H), 1.11 (m, 2H). 13C NMR (100 MHz, CDCl3): = 175.7, 155.9, 137.9, 135.9, 78.9, 70.1, 70.0, 69.8, 47.1, 46.1, 41.4, 40.1, 39.1, 28.26. MS (MALDI) calcd. for C19H32N2O5: 368.2; discovered: 369.3 ([M + H]+). Substance EN-H. The perfect solution is of PEG-3 norbornene derivative (5, 0.19 g, 0.50 mmol) was stirred with TFA (2.0 mL) for 4 h. After deprotection, the solvent was evaporated and the merchandise was purified by adobe flash chromatography (ethyl acetate) to provide EN-H (0.12 g, 0.44 mmol, 88%) like a colorless vicious water. 1H NMR (400 MHz, CDCl3): = 6.95 (s, 1H), 6.08 (m, 1H), 6.04 (m, 1H), 3.82C3.48 (m, 7H), 3.41 (m, 2H), 3.20 (m, 2H), 2.86 (m, 2H), 2.43 (m, 1H), 2.11 (m, 1H), 1.73 (m, 1H), 1.50 (m, 1H), 1.32 (m, 1H). 13C NMR (100 MHz, CDCl3): = 178.8, 137.9, 135.6, 69.9, 69.8, 66.1, 46.7, 44.4, 41.4, 39.9, 39.6, 30.5. MS (MALDI) calcd. for C14H24N2O3: 268.2; discovered: 269.3 ([M + H]+). Substance CB-EN. To a remedy of PEG-3 norbornene derivative (EN-H, 0.12 g, 0.43 mmol) in DMF (2.0 mL) was added protected cross-bridged chelator (CB-TE2A(tBu)-COOH, 0.19 g, 0.36 mmol), 1-ethyl-3-(3-(dimethylamino)-propyl) carbodiimide hydrochloride (0.07 g, 0.36 mmol), and Rabbit polyclonal to IL27RA triethylamine (0.04 g, 0.36 mmol). The resultant option was stirred for 12 h, filtered, as well as the solvent evaporated. The BMS-962212 crude secured item was purified by HPLC (0.11 g, 0.14 mmol, 40%) like a white good. 1H NMR (400 MHz, CDCl3): = 9.09 (m, 2H), 6.08 (m, 2H), 3.95C3.45 (m, 10H), 3.48C3.00 (m, 12H), 2.97C2.54 (m, 10H), 2.52C2.19 (m, 4H), 2.15C1.65 (m, 10H), 1.58C1.33 (m, 15H), 1.33C1.05 (m, 4H). 13C NMR (100 MHz, CDCl3): 176.1, 175.9, 138.2, 136.1, 82.5, 81.9, 70.3, 70.1, 70.0, 68.7, 62.4, 56.2, 55.9, 51.1, 47.9, 47.2, 46.3, 44.6, 44.4, 41.5, 39.6, 39.2, 32.9, 30.4, 28.1, 25.2. MS (MALDI) calcd. for C41H72N6O8: 776.5; discovered: 799.5 ([M + Na]+). The shielded item was blended with TFA (2.0 mL) and the perfect BMS-962212 solution is was stirred for 12 h. Following the response, the solvent was evaporated and the merchandise was purified by HPLC to provide CB-EN (0.08 g, 0.13 mmol, 90%) like a red viscous water. 1H NMR (400 MHz, CDCl3): = 6.13 (m, 2H), 3.96C3.49 (m, 12H), 3.49C2.97 BMS-962212 (m, 13H), 2.97C2.70 (m, 5H), 2.70C2.31 (m, 3H), 2.31C1.91 (m, 5H), 1.91C1.61 (m, 3H), 1.61C1.43 (m, 2H), 1.43C1.25 (m, 2H),1.03 (s 1H). 13C NMR (100 MHz, CDCl3): 179.9, 175.4, 141.9, 139.9, 73.9, 73.6, 72.7, 61.3, 54.1, 51.0, 50.0, 48.1, 46.6, 45.4, 43.4, 42.9, 36.3, 34.2, 31.7, 31.2, 26.9. MS (MALDI).