Compound 1-A2

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Synthetic procedure: See article for the definitive version of this procedure and for full experimental details.

Salinospora arenicola CNR107 was isolated off the California coast and was most kindly provided by Paul R. Jensens Laboratory at the Scripps Institution of Oceanography. It was routinely grown in A1M1 medium (5 g/L soluble starch, 2 g/L peptone, 2 g/l yeast, 22 g/L instant ocean®, pH= 6.8). Salinospora arenicola CNR107 was grown in liquid A1M1 medium. We observed that the concentration of bacteria must be kept relatively high to achieve significant growth. Therefore, dilutions of the culture were kept to a factor of 4-5 fold. From a glycerol stock was taken 50 µL and added into a culture tube (5 mL medium). The bacteria were grown at 28°C, and shaken at 220 r.p.m. After 4 days, the 5 mL were transferred to an Erlenmeyer flask (100 mL) with fresh medium (15 mL) and left at 28 °C and 170 r.p.m. After three days, this was transferred to an indented Erlenmeyer flask (500 mL) with fresh media (80 mL) and left at 28 °C and 140 r.p.m.. After six days, the culture was divided in two and each transferred to an Erlenmeyer flask (1 L) with fresh media (200 mL), and left for four days at 28 °C and 170 r.p.m.. Finally the culture was used to start a total of 14 liters of A1M1 media in indented Erlenmeyer flasks (5 L) with a total of two liters per flask. These were grown at 28 °C and 100 r.p.m. for three days while monitoring the BE-43547 content in 5 mL aliquots. Every day from this point a 5 mL aliquot of culture was taken, extracted with EtOAc, dried over Na₂SO₄, filtered, and concentrated. The concentrated aliquot was dissolved in 250 µL MeOH, filtered and analyzed by LC-MS to follow the production of BE-43547 molecules. The 14 L culture was extracted with EtOAc, filtered, and concentrated to a final volume of approximately 100 mL EtOAc. MeOH was added and the volume was reduced to approximately 50 mL. This was repeated three times to remove all the EtOAc. This mixture was diluted with water to yield a final solution of the bacterial extract in 20% MeOH in water. By avoiding full concentration we were able to obtain better results from the isolation, we speculate that the BE-43547 molecules show some instability when concentrated.

The extract was loaded onto a C18 column (LiChroprep RP-18, 40-63 µm, 25 g, 3.5 cm diameter, 4 cm high). Fractions of 250 mL were run through the column as follows: Flowthrough, 20% MeOH, 40% MeOH, 60% MeOH, 70% MeOH, 2 x 100%. LCMS analysis show that the BE-43547 molecules were found in the 100% MeOH, mainly in the first 100% MeOH fraction. The two fractions were concentrated to approximately 25 mL, water was added (approx. 225 mL) and the mixture was frozen in an acetone/dry ice bath before concentrated to dryness by lyophilization. The BE-43547 molecules were separated using semi-prep LC-MS (Phenomenex, Luna, 5 µ, C18(2), 100 Å, 250 x 10 mm). Method: 7 mL/min, H₂O/MeCN (no acid). The method went from 25% to 80% MeCN over 15 min followed by an isocratic plateau for 10 min before increasing to 100% MeCN over 1 min and holding for 4 min. This method effectively separated the molecules into A_1-2 (21 min), B_1-3 (23 min), and C_1-2 (27 min). The A_1-2 fraction was lyophilized and a final purification was performed on an analytical HPLC (Phenomenex, Luna, 5 µ, C8(2), 100 Å, 150 x 4.60 mm). Method: 1 mL/min, H₂O/MeCN (no acid). The method started at 25% MeCN for 2 min, then ramped to 62% over 1 min, from 62% to 65% over 27 min. Before washing at 95% for 5 min. BE-43547A₁ eluted at approx. 28 min. and BE-43547A₂ at approx. 30 min. The relevant fractions were combined and lyophilized. From this we isolated BE-43547A₁ (<1 mg), BE-43547A₂ (1 mg). The relative amount of the natural products seen by LC-MS in the bacteria culture aliquot appears to match the isolated amount. (14S,16S,17S,E)-17-hexyl-14-hydroxy-4,14,16-trimethyl-8-methylene-1-oxa-4,9,12-triazacycloheptadec-6-ene-2,5,10,13,15-pentaone (BE-43547A₂): ¹H NMR (950 MHz, DMSO-d₆) δ 8.79 (brs, 1H), 8.69 (s, 1H), 6.98 (d, J = 15.2 Hz, 1H), 6.34 (d, J = 15.2 Hz, 1H), 5.77 (s, 1H), 5.40 (s, 1H), 4.96 (td, J = 9.4, 2.6 Hz, 1H), 4.41 (d, J = 19.2 Hz, 1H), 4.15 (d, J = 19.3 Hz, 1H), 3.96 (dd, J = 16.1, 4.2 Hz, 1H), 3.68 (dd, J = 16.1, 5.1 Hz, 1H), 3.39 (dt, J = 9.6, 6.9 Hz, 1H), 2.87 (s, 3H), 1.68 (ddq, J = 12.3, 7.6, 2.7 Hz, 1H), 1.60 (s, 3H), 1.54 – 1.49 (m, 1H), 1.48 – 1.43 (m, 1H), 1.23 (s, 21H), 1.08 (d, J = 6.8 Hz, 3H), 0.85 (t, J = 7.2 Hz, 3H). ¹³C NMR (239 MHz, DMSO-d₆) δ 211.9, 173.8, 168.8, 167.7, 166.4, 139.4, 137.3, 116.8, 114.2, 80.6, 76.5, 50.2, 44.3, 43.5, 35.0, 31.3, 29.0, 29.0, 29.0, 29.0, 28.9, 28.9, 28.7, 28.7, 28.6, 28.5, 26.6, 26.5, 25.1, 24.5, 22.1, 20.9, 18.5, 15.7, 13.9.