*4.2. Biological Material*

Sponge specimens were collected on 27 May 2015 near the west shore of Ramrod Key (24◦39-38.1-- N, 81◦25-25.0-- W) in Florida. A voucher specimen is housed at the University of Wisconsin−Madison. For cultivation, a sample of sponge (1 cm3) was ground in 500 μL sterile seawater, and dilutions were made using 500 μL sterile seawater. Subsequently, 400 μL of diluted sponge sample was added to 200 μL of sterile artificial seawater and 100 μL was plated using a sterile L-shaped spreader. Diluted samples were plated on Gauze 1 media supplemented with artificial seawater. Each medium was supplemented with 50 μg/mL cycloheximide, 25 μg/mL nystatin, and 25 μg/mL nalidixic acid. Plates were incubated at 28 ◦C and colonies were isolated over the course of two months.

#### *4.3. Fermentation, Extraction and Isolation*

Two 10 mL seed cultures (25 × 150 mm tubes) in medium DSC (20 g soluble starch, 10 g glucose, 5 g peptone, 5 g yeas<sup>t</sup> extract per liter of artificial seawater) were inoculated with strain WMMC-1349 and shaken (200 RPM, 28 ◦C) for seven days. Two-liter flasks (1 × 500 mL) containing ASW-A (20 g soluble starch, 10 g D-glucose, 5 g peptone, 5 g yeas<sup>t</sup> extract, 5 g CaCO3 per liter of artificial seawater. For 13C enriched ASW-A media, 10 g D-glucose (U-13C6, 99%) was used instead of soluble starch and D-glucose) were inoculated with 20 mL seed culture and were incubated (200 RPM, 28 ◦C) for seven days. Four-liter flasks (10 × 1 L) containing medium ASW-A with Diaion HP20 (7% by weight) were inoculated with 50 mL from the 500 mL culture and shaken (200 RPM, 28 ◦C) for seven days. For producing artificial sea water, solutions I (415.2 g NaCl, 69.54 g Na2SO4, 11.74 g KCl, 3.40 g NaHCO3, 1.7 g KBr, 0.45 g H3BO3, 0.054 g NaF) and II (187.9 g MgCl2·6H2O, 22.72 g CaCl2·2H2O, 0.428 g SrCl2·6H2O) were made up separately using distilled water and combined to give a total volume of 20 L.

Filtered HP20 and cells were washed with H2O and extracted with acetone. The acetone extract was subjected to liquid-liquid partitioning using 30% aqueous MeOH and CHCl3 (1:1). The CHCl3-soluble partition (3.12 g) was fractionated by Sephadex LH20 column chromatography (column size 500 × 40 mm, CHCl3:MeOH = 1:1, 20 mL for each fraction). Fractions containing **1**–**6** (1.1 g) were subjected to RP HPLC (20%/80% to 100%/0% MeOH/H2O (with 0.1% acetic acid), 23.5 min, 20 mL/min) using a Phenomenex Gemini C18 column (250 × 30 mm). The fraction collected between 16–18 min was further fractionated by RP HPLC (22%/78% to 51%/49% MeCN/H2O (with 0.05% trifluoroacetic acid), 29.5 min, 20 mL/min) using a Phenomenex Gemini C18 column (250 × 30 mm), yielding **1** (50.2 mg, *t*R 28.2 min), **2** (35.8 mg, *t*R 27.6 min), **3** (2.2 mg, *t*R 27.3 min), **4** (3.1 mg, *t*R 28.9 min), **5** (40.5 mg, *t*R 28.6 min), **6** (32.3 mg, *t*R 29.3 min).

Bacillimidazole A (**1**): light yellow solid, UV-Vis (MeOH): λmax (log ε) 211 nm (3.83), 259 nm (2.65), 279 nm (2.55), 291 nm (2.55); IR (ATR): υmax 3385, 3144, 3033, 2935, 2873, 2834, 1781, 1679, 1563, 1498, 1456, 1399, 1357, 1199, 1129, 1083, 1029, 831, 801, 751, 720, 702 cm<sup>−</sup>1; HRMS M+ *m*/*z* = 305.2029 (calcd. for C21H25N2+ 305.2012). 1H NMR (600 MHz, MeOD) *δ*H 8.51 (s, 1H), 7.34 (t, *J* = 7.2 Hz, 4H), 7.31 (d, *J* = 7.2 Hz, 2H), 7.12 (d, *J* = 7.0 Hz, 4H), 4.35 (t, *J* = 7.0 Hz, 4H), 3.06 (t, *J* = 6.9 Hz, 4H), 2.08 (s, 6H). 13C NMR (126 MHz, MeOD) *δ*C 137.83, 135.53, 130.02, 129.97, 128.45, 128.44, 49.28, 37.07, 7.94.

Bacillimidazole B (**2**): light yellow solid, UV-Vis (MeOH): λmax (log ε) 217 nm (3.85), 274 nm (3.02), 282 nm (3.04), 290 (2.97); IR (ATR): υmax 3383, 3065, 3034, 2934, 2870, 1681, 1564, 1525, 1497, 1440, 1400, 1355, 1200, 1128, 1030, 934, 840, 801, 749, 722, 703 cm<sup>−</sup>1; HRMS M+ *m*/*z* = 319.2171 (calcd. for C22H27N2+ 319.2169). 1H NMR (600 MHz, MeOD) *δ*H 7.36–7.33 (m, 6H), 7.11 (d, *J* = 6.4 Hz, 4H), 4.31 (t, *J* = 6.7 Hz, 4H), 3.02 (t, *J* = 6.7 Hz, 4H), 2.08 (s, 6H), 2.01 (s, 3H). 13C NMR (126 MHz, MeOD) *δ*C 144.04, 138.15, 130.22, 130.08, 128.56, 127.14, 47.87, 36.33, 9.67, 8.21.

Bacillimidazole C (**3**): light yellow solid, UV-Vis (MeOH): λmax (log ε) 223 nm (4.25), 274 nm (3.67), 282 nm (3.69), 290 nm (3.61); IR (ATR): υmax 3357, 2946, 2835, 1679, 1564, 1449, 1432, 1341, 1203, 1185, 1137, 1024, 838, 802, 746, 722 cm<sup>−</sup>1; HRMS M+ *m*/*z* = 383.2228 (calcd. for C25H27N4+ 383.2230). 1H NMR (600 MHz, MeOD) *δ*H 8.12 (s, 1H), 7.39 (d, *J* = 8.1 Hz, 2H), 7.32 (d, *J* = 7.9 Hz, 2H), 7.15 (t, *J* = 7.4 Hz, 2H), 7.03 (t, *J* = 7.4 Hz, 2H), 6.96 (s, 2H), 4.25 (t, *J* = 6.7 Hz, 4H), 3.07 (t, *J* = 6.7 Hz, 4H), 2.09 (s, 9H). 13C NMR (126 MHz, MeOD) *δ*C 138.05, 135.37, 128.30, 128.19, 124.42, 122.83, 120.13, 118.43, 112.64, 110.49, 26.81, 7.94.

Bacillimidazole D (**4**): light yellow solid, UV-Vis (MeOH): λmax (log ε) 224 nm (3.98), 281 nm (3.44), 290 nm (3.38); IR (ATR): υmax 3360, 3292, 2925, 2854, 1729, 1648, 1561, 1456, 1411, 1342, 1256, 1235, 1181, 1105, 1073, 1025, 926, 744 cm<sup>−</sup>1; HRMS M+ *m*/*z* = 397.2385 (calcd. for C26H29N4+ 397.2387). 1H NMR (500 MHz, MeOD) *δ*H 7.39 (d, *J* = 8.0 Hz, 1H), 7.24 (d, *J* = 7.9 Hz, 1H), 7.14 (t, *J* = 7.5 Hz, 1H), 7.03 (t, *J* = 7.5 Hz, 1H), 6.98 (s, 1H), 4.19 (t, *J* = 5.9 Hz, 2H), 3.03 (t, *J* = 5.7 Hz, 2H), 2.18 (s, 3H), 1.64 (s, 1H). 13C NMR (126 MHz, MeOD) *δ*C 143.95, 138.00, 128.40, 126.96, 124.50, 122.88, 120.21, 118.23, 112.67, 110.76, 47.50, 25.97, 9.30, 8.29.

Bacillimidazole E (**5**): light yellow solid, UV-Vis (MeOH): λmax (log ε) 221 nm (3.87), 274 nm (3.17), 282 nm (3.18), 290 (3.11); IR (ATR): υmax 3376, 2991, 2950, 2836, 1677, 1564, 1497, 1456, 1398, 1356, 1341, 1201, 1134, 1078, 1025, 934, 834, 801, 747, 721, 703 cm<sup>−</sup>1; HRMS M+ *m*/*z* = 344.2134 (calcd. for C23H26N3+ 344.2121). 1H NMR (600 MHz, MeOD) *δ*H 8.27 (s, 1H), 7.40 (d, *J* = 8.2 Hz, 1H), 7.34 (d, *J* = 7.9 Hz, 1H), 7.32–7.26 (m, 3H), 7.15 (t, *J* = 7.4 Hz, 1H), 7.07–7.02 (m, 4H), 4.38 (t, *J* = 6.5 Hz, 2H), 4.18 (t, *J* = 7.2 Hz, 2H), 3.23 (t, *J* = 6.5 Hz, 2H), 2.84 (t, *J* = 7.2 Hz, 2H), 2.12 (s, 3H), 2.04 (s, 3H). 13C NMR (126 MHz, MeOD) *δ*C 138.08, 137.82, 135.44, 129.95, 129.87, 128.38, 128.34, 128.29, 128.27, 124.55, 122.85, 120.17, 118.37, 112.68, 110.42, 37.03, 26.76, 7.97, 7.89.

Bacillimidazole F (**6**): light yellow solid, UV-Vis (MeOH): λmax (log ε) 225 nm (3.96), 274 nm (3.55), 282 nm (3.56), 290 nm (3.51); IR (ATR): υmax 3356, 3275, 3062, 3001, 2971, 2928, 2830, 1647, 1562, 1497, 1454, 1401, 1353, 1234, 1203, 1179, 1108, 1077, 1028, 924, 745, 703 cm<sup>−</sup>1; HRMS M+ *m/z* = 358.2283 (calcd. for C24H28N3+ 358.2278). 1H NMR (500 MHz, MeOD) *δ*H 7.37 (d, *J* = 8.0 Hz, 1H), 7.28 (m, 3H), 7.23 (d, *J* = 7.9 Hz, 1H), 7.11 (t, *J* = 7.5 Hz, 1H), 7.06 – 6.97 (m, 3H), 4.31 (t, *J* = 5.5 Hz, 2H), 4.11 (t, *J* = 6.6 Hz, 2H), 3.17 (t, *J* = 5.5 Hz, 2H), 2.76 (t, *J* = 6.6 Hz, 2H), 2.15 (s, 2H), 2.07 (s, 3H), 1.77 (s, 3H). 13C NMR (126 MHz, MeOD) *δ*C 143.99, 138.09, 138.03, 130.07, 130.02, 128.47, 128.45, 127.06, 127.00, 124.64, 122.91, 120.28, 118.18, 112.72, 110.76, 47.74, 47.59, 36.27, 26.00, 9.43, 8.32, 8.18.
