Preparative reactions
Compound 1a. 10 mg of 1 (8.02 µmol) were dissolved in 0.4 mL acetonitrile aq. 50% and mixed with 26 mL reaction buffer (100 mM NaCl, 50 mM TRIS, 10 mM MgCl2, 10 mM ATP, 5 mM DTT, pH 7.2) and 4 mL PatD-fusion enzyme solution (18.6 mg/mL in 10 mM HEPES, 50 mM NaCl, 5 mM MgCl2, 1 mM TCEP, pH 7.0). Incubation for 24 hours at 30°C and 800 rpm on an orbital shaker. The reaction was quenched with 18 mL ACN and shaken for 2 minutes then centrifuged. The quenched reaction was centrifuged (15 000 g / 10 min), filtered and then mixed with 450 mL water. This solution is pumped on a MERCK Chromolith RP18 2–10 cm column. Subsequent chromatography: phase A NH4OAc aq. 10 mM, phase B acetonitrile, gradient from 5–50% B in 30 minutes, flow 40 mL/min, UV peak detection at 222 nm. All product containing fractions were combined, frozen and dried over 3 days by lyophilization. Product 1a (7.2 mg, 6.24 µmol, purity > 95% yield = 78%) were obtained and characterized by LC-MS (TOF) and NMR. 1H-NMR, HSQC-DEPT, COSY, HMBC, and ROESY spectra can be found in Supplementary Figs. 9–13. TOF: [M + H]+ 1097.66 Da, [M + 2H]2+, 548.76 Da. 1H NMR (600MHz, DMSO-d6) δ ppm 9.18 p (br s, 1 H), δ ppm 8.6 p (m, 1 H), δ ppm 8.3 p (br d (8.6), 1 H), δ ppm 8.27 p (m, 1 H), δ ppm 8.22 p (br d (7.8), 1 H), δ ppm 8.17 p (br d (7.6), 1 H), δ ppm 7.9 p (m, 1 H), δ ppm 7.83 p (br d (8.7), 1 H), δ ppm 7.64 p (br s, 1 H), δ ppm 7.23 p (m, 2 H, J = 128.1), δ ppm 7.21 p (m, 2 H, J = 129.3), δ ppm 7.19 p (m, 1 H, J = 126.5), δ ppm 7.01 p (m, 2 H, J = 130.2), δ ppm 6.62 p (d (8.5), 2 H, J = 114.9), δ ppm 5.03 p (m, 1 H, J = 78.5), δ ppm 4.87 p (m, 1 H, J = 52.5), δ ppm 4.49 p (m, 1 H, J = 49.6), δ ppm 4.39 p (m, 1 H, J = 51.1), δ ppm 4.36 p (m, 1 H, J = 54.6), δ ppm 4.32 p (m, 2 H, J = 48.0), δ ppm 4.27 p (m, 1 H, J = 79.8), δ ppm 4.27 p (m, 1 H, J = 48.0), δ ppm 4.07 p (dd (7.5, 0.8), 1 H, J = 73.9), δ ppm 3.83 p (m, 1 H, J = 67.2), δ ppm 3.5 p (m, 1 H, J = 34.8), δ ppm 3.49 p (m, 2 H, J = 42.8), δ ppm 3.38 p (m, 1 H, J = 34.8), δ ppm 3.23 p (m, 1 H, J = 59.3), δ ppm 3.2 p (m, 1 H, J = 38.0), δ ppm 3.01 p (m, 1 H, J = 38.0), δ ppm 2.95 p (m, 1 H, J = 36.3), δ ppm 2.71 p (br dd (14.0, 9.5), 1 H, J = 36.3), δ ppm 2.56 p (m, 1 H, J = 37.1), δ ppm 2.47 p (m, 1 H, J = 37.1), δ ppm 1.73 p (m, 1 H, J = 37.1), δ ppm 1.43 p (m, 1 H, J = 24.4), δ ppm 1.28 p (d (6.3), 3 H, J = 20.9), δ ppm 1.21 p (m, 3 H, J = 18.4), δ ppm 1.19 p (m, 6 H, J = 17.9), δ ppm 1.1 p (m, 1 H, J = 24.4), δ ppm 1.07 p (d (6.3), 3 H, J = 19.5), δ ppm 0.82 p (m, 3 H, J = 11.2), δ ppm 0.81 p (m, 3 H, J = 15.1).
Compound 2a. 10 mg of 2 (9.31 µmol) were dissolved in 0.6 mL acetonitrile aq. 50% and mixed with 27 mL reaction buffer (100 mM NaCl, 50 mM TRIS, 10 mM MgCl2, 10 mM ATP, 5 mM DTT, pH 7.2) and 3 mL PatD-fusion enzyme solution (18.6 mg/mL in 10 mM HEPES, 50 mM NaCl, 5 mM MgCl2, 1 mM TCEP, pH 7.0). Incubation for 24 hours at 30°C and 800 rpm on an orbital shaker. Incubation for 24 hours at 30°C and 800 rpm on an orbital shaker. After 24 hours the reaction showed remaining 10% of the mono-heterocyclized intermediate product. Therefore, an additional amount of 13 mL reaction buffer and 1.5 mL enzyme solution were added. The reaction was incubated for additional 8 hours, 32 hours in total. Finally, the reaction was quenched with 30 mL ACN and shaken for 2 minutes. Purification and characterization in analogy to the product 1a. Product 2a (3.8 mg, 3.5 µmol, purity > 85% yield = 38%) were obtained and characterized by LC-MS (TOF) and NMR. 1H-NMR, HSQC-DEPT, HMBC, and ROESY spectra can be found in Supplementary Figs. 14–17, respectively. TOF: [M + H]+ 924.35 Da, [M + 2H]2+, 1H NMR (600MHz, DMSO-d6) 462.71 Da. 1H NMR (600MHz, DMSO-d6) δ ppm 8.59 p (br s, 1 H), δ ppm 8.31 p (br d (7.1), 1 H), δ ppm 8.28 p (br d (6.4), 1 H), δ ppm 8.04 p (br s, 1 H), δ ppm 7.76 p (br d (7.1), 1 H), δ ppm 7.54 p (br s, 1 H), δ ppm 7.23 p (m, 2 H, J = 129.0), δ ppm 7.22 p (m, 2 H, J = 128.1), δ ppm 7.17 p (m, 1 H, J = 126.4), δ ppm 7.02 p (br d (8.0), 2 H, J = 130.1), δ ppm 6.62 p (br d (8.0), 2 H, J = 114.8), δ ppm 4.97 p (br t (8.9), 1 H, J = 78.2), δ ppm 4.89 p (m, 1 H, J = 52.6), δ ppm 4.47 p (m, 1 H, J = 49.9), δ ppm 4.37 p (m, 1 H, J = 54.6), δ ppm 4.3 p (m, 1 H, J = 48.0), δ ppm 4.19 p (br d (5.7), 1 H, J = 60.3), δ ppm 3.9 p (m, 1 H, J = 57.3), δ ppm 3.9 p (m, 1 H, J = 66.5), δ ppm 3.78 p (q (7.2), 1 H, J = 53.2), δ ppm 3.46 p (m, 1 H, J = 34.8), δ ppm 3.44 p (m, 2 H, J = 43.4), δ ppm 3.35 p (m, 1 H, J = 34.8), δ ppm 3.11 p (dd (14.0, 5.6), 1 H, J = 38.5), δ ppm 2.98 p (dd (14.0, 8.3), 1 H, J = 38.3), δ ppm 2.95 p (dd (13.6, 3.6), 1 H, J = 36.4), δ ppm 2.7 p (dd (13.6, 9.8), 1 H, J = 36.4), δ ppm 2.43 p (m, 2 H, J = 38.3), δ ppm 1.71 p (m, 1 H, J = 39.7), δ ppm 1.31 p (m, 1 H, J = 23.2), δ ppm 1.2 p (m, 3 H, J = 21.2), δ ppm 1.19 p (m, 3 H, J = 18.5), δ ppm 0.99 p (br d (6.2), 3 H, J = 20.0), δ ppm 0.89 p (m, 1 H, J = 23.2), δ ppm 0.83 p (br d (6.8), 3 H, J = 15.3), δ ppm 0.74 p (t (7.2), 3 H, J = 11.4).