Reaction Steps
tert-butyl 3-({3-[(3-{(3β)-cholest-5-en-3-yl[(2-nitrophenyl)sulfonyl]amino}propyl)amino]-3-oxopropyl}amino)-3-oxopropylcarbamate (18, 52 mg, 0.060 mmol) was treated with trifluoroacetic acid in CH2Cl2 (2:25, 10 mL). After 1 h at 23° C., TLC analysis (MeOH/CH2Cl2, 1:25) revealed conversion to the more polar primary amine. Aqueous NaOH (1 M, 40 mL) was added, the mixture was extracted with CH2Cl2 (2×10 mL), the organic layers were dried over anhydrous Na2SO4, and the solvent was removed in vacuo. The remaining residue was dissolved in CH2Cl2 (10 mL) and 6-(2,4-dinitrophenyl)aminohexanoic acid succinimidyl ester (71 mg, 0.180 mmol) and diisopropylethylamine (50 μL, 0.287 mmol) were added. After 8 h at 23° C., ethylenediamine (50 μL) was added with stirring for 30 min to consume the excess active ester. CH2Cl2 (5 mL) and aqueous citric acid (10%, 25 mL) were added followed by extraction with CH2Cl2 (2×5 mL). The organic layer was dried over anhydrous Na2SO4 and removed in vacuo. Flash column chromatography (MeOH/CH2Cl2, 1:25) afforded 21 (60 mg, 96%), mp 106-108° C.; 1H NMR (500 MHz, CDCl3) δ 9.10 (d, J=2.59 Hz, 1H), 8.57 (t, J=4.98 Hz, 1H), 8.25 (dd, J=2.6 Hz, J=9.5 Hz, 1H), 8.01-7.97 (m, 1H), 7.74-7.68 (m, 2H), 7.64-7.59 (m, 1H), 6.93 (d, J=9.6 Hz, 1H), 6.70 (t, J=5.5 Hz, 1H), 6.65 (t, J=5.7 Hz, 1H), 6.58 (d, J=5.1 Hz, 1H), 5.19 (d, J=4.9 Hz, 1H), 3.57-3.51 (m, 5H), 3.44-3.34 (m, 6H), 2.43 (t, J=5.9 Hz, 2H), 2.38 (t, J=5.9 Hz, 2H), 2.21 (d, J=7.4 Hz, 2H), 2.01-1.67 (m, 13H), 1.58-1.22 (m, 14H), 1.18-0.97 (m, 9H), 0.94 (s, 3H), 0.90 (d, J=6.5 Hz, 3H), 0.87 (d, J=2.4 Hz, 3H), 0.86 (d, J=2.2 Hz, 3H), 0.66 (s, 3H); 13C NMR (125.8 MHz, CDCl3) δ 172.9 172.1 (×2), 148.6, 148.1, 140.0, 136.1, 133.9, 133.8, 131.9, 130.8, 130.5, 130.4, 124.5, 124.3, 122.7, 114.2, 59.3, 56.8, 56.3, 50.2, 43.5, 42.5, 41.8, 39.8, 39.7, 38.4, 37.8, 36.7, 36.4, 36.4, 36.3, 35.9, 35.9, 35.8, 34.1, 32.0, 31.9, 28.6, 28.4, 28.2, 27.7, 26.6, 25.8, 25.2, 25.1, 24.4, 24.0, 23.0, 22.7, 21.1, 19.6, 18.9, 12.0; IR (film) νmax 3378, 3287, 2937, 2867, 1634, 1622, 1548, 1337 cm−1; CI m/z 1049.5764 (MH+, C54H81N8O11S requires 1049.5745).
