*4.2. Strain Maintenance*

*Streptomonospora* sp. DSM 106425T was isolated in 2017 by the serial dilution method from a sand sample that had been collected from a beach of the North Sea at Cuxhaven, Germany. The strain grew well in the presence of 7% NaCl. This percentage of sodium chloride was added to all media that we used for culturing and production media. A section of the agar containing bacterial colonies and aerial mycelium was stored in glycerol 20% at −80 ◦C. The strain was transferred to 100 mL of liquid GYM medium (0.4% glucose, 0.4% yeas<sup>t</sup> extract, 1% malt extract, 0.2% CaCO3; pH 7.2). The inoculated flask was incubated on a rotary shaker (160 rpm) for 5 days at 30 ◦C.

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

The 5-day-old preculture was transferred to production medium 1:10 in eight 1000-mL flasks, filled with 800 mL of medium 5294 (1% soluble starch, 0.2% yeas<sup>t</sup> extract, 1% glucose, 1% glycerol, 0.25% corn steep liquor, 0.2% peptone, 0.1% NaCl, 0.3% CaCO3; pH 7.2) incubated at 30 ◦C for 8 days on rotary shaker (160 rpm). The 8-day-old culture medium was centrifuged using a Sorvall RC-5 refrigerated superspeed centrifuge for 30 min, at 8500 rpm. The supernatant was discarded and 311 g cell mass was extracted with two liters of acetone three time in an ultrasonic bath (3 × 30 min). The solution obtained was evaporated to yield an aqueous phase, which was further extracted with ethyl acetate (3 × 500 mL) and the ethyl acetate portion was dried out with evaporator to yield 575.2 mg of crude cell mass extract. An initial pre-separation of 575.2 mg crude extract was applied with a Strata TM-X 33 UM Polymeric reversed phase 1 g/<sup>12</sup> mL Giga tube (Phenomenex) and washed three times with methanol. Subsequently, fractionation of 265.2 mg crude extract was completed by preparative HPLC (Gilson) (1 run using a linear gradient of solvent B from 20% to 50% solvent B in 30 min, 50% to 100% B in 20 min followed by isocratic conditions for 10 min at a flow rate of 50 mL/min). Fractions were collected and combined according to UV absorption at 220, 280 and 350 nm and yielded 5.6 mg of **1** at a retention time of 37.5–38.5 min and 0.5 mg of **2** at 36.5–37 min, respectively.

Litoralimycin A (**1**): light-yellow oil; [ α]D = +251 (*c* = 1 mg/mL in acetone); 1H NMR (700 MHz, DMSO-*d*6): see Table 1; 13C NMR (175 MHz, DMSO-*d*6): see Table 1; ESI-MS: *m*/*z* 1120.38 [M + H]<sup>+</sup>, 1118.43 [M + H]<sup>+</sup>; HRESIMS: *m*/*z* 1120.2429 [M + H]+ (calcd. for C48 H46 N15 O10S4 1120.2429), 1142.2246 [M + Na]+ (calcd. for C48 H45 N15 O10S4Na 1142.2249).

Litoralimycin B (**2**): light-yellow oil; [ α]D = +399 (*c* = 1 mg/mL in acetone); 1H NMR (700 MHz, DMSO-*d*6): δH 10.67 (br s, 43–NH), 9.81 (br s, 21–NH), 9.74 (br s, 31–NH), 9.69 (br s, 28–NH), 8.95 (dd, *J* = 6.2, 5.2 Hz, 10–NH), 8.75 (br d, *J* = 8.2 Hz, 15–NH), 8.52 (d, *J* = 8.2 Hz, 39–H), 8.45 (s, 2–H), 8.31 (s, 23–H), 8.29 (s, 17–H), 8.25 (s, 12–H), 8.22 (d, *J* = 8.2 Hz, 40–H), 8.17 (br s, 44–NHa), 8.07 (br d, *J* = 9.2 Hz, 5–NH), 7.66 (br s, 44–NHb), 6.58 (s, 45–Ha), 6.52 (q, *J* = 6.9 Hz, 26–H), 6.48 (br s, 30–Ha), 5.81 (br s, 45–Hb), 5.72 (br s, 30–Hb), 5.63 (br s, 35–Ha), 5.44 (m, 15–H), 5.42 (br s, 35–Hb), 4.70 (dd, *J* = 15.9, 6.2 Hz, 10–Ha), 4.54 (dd, *J* = 15.9, 5.2 Hz, 10–Hb), 4.37 (dd, *J* = 9.2, 7.3 Hz, 5–H), 2.64 (s, 36–H3), 2.10 (dspt, *J* = 7.2, 6.9 Hz, 7–H), 1.79 (br d, *J* = 6.9 Hz, 27–H3), 1.55 (br d, *J* = 6.9 Hz, 20–H3), 0.90 (d, *J* = 6.9 Hz, 9–H3), 0.87 (d, *J* = 6.9 Hz, 8–H3) ppm; 13C NMR (175 MHz, DMSO-*d*6): δC 173.6 (C, C–16), 170.9 (C, C–6), 168.6 (C, C–11), 167.3 (C, C–22), 164.9 (C, C–44), 163.8 (C, C–1), 161.2 (C, C–42), 159.98 (C, C–4), 159.94 (C, C–14), 159.2 (C, C–19), 158.9 (C, C–25), 155.6 (C, C–32), 150.2 (C, C–33), 149.22 (C, C–3), 149.18 (C, C–41), 148.7 (C, C–18), 148.6 (2xC, C–13, C–24), 147.7 (C, C–37), 141.2 (CH, C–39), 133.8 (C, C–28), 133.6 (C, C–43), 133.1 (C, C–34), 130.6 (C, C–38), 129.2 (C, C–21), 129.0 (C, C–31), 128.3 (CH, C–26), 125.3 (CH, C–12), 125.1 (CH, C–17), 125.0 (CH, C–23), 104.5 (CH2, C–30), 102.5 (CH2, C–45), 58.2 (CH, C–5), 46.9 (CH, C–15), 40.0 (CH2, C–10), 30.7 (CH, C–7), 20.6 (CH3, C–20), 19.3 (CH3, C–8), 18.4 (CH3, C–9), 14.2 (CH3, C–27), 11.6 (CH3, C–26); ESI-MS: *m*/*z* 1051.32 [M + H]<sup>+</sup>, 1049.34 [M + H]<sup>+</sup>; HRESIMS: *m*/*z* 1051.2219 [M + H]+ (calcd. for C45 H43 N14 O9S4 1051.2215), 1073.2031 [M + Na]+ (calcd. for C45 H42 N14 O9S4Na 1073.2034).

### *4.4. Ozonolysis, Hydrolysis and Marfey's Derivatization with* l*-FDAA*

For the ozonolysis reaction a stream of O3 was bubbled through a solution of **1** (2.3 mg) dissolved in methanol (6 mL) at −78 ◦C until the solution obtained a characteristic blue color and stirred for 30 min. Subsequently, the solvent was removed in vacuo and the resulting oxidized material was subjected to hydrolysis in 3 mL of 6 n HCl at 110 ◦C for 24 h as described in [6]. Afterwards, the solvent was removed under a stream of nitrogen for 3 h and the remainder dissolved in H2O (200 μL), of which 100 μL were proceeded further. A total of 1 n NaHCO3 (20 μL) and 1% 1-fluoro-2,4-dinitrophenyl-5-Lalaninamide (100 μL in acetone) were added, and the mixture was heated at 40 ◦C for 40 min [17]. After being cooled to room temperature, the solutions were neutralized with 2 n HCl (20 μL) and evaporated to dryness. The residues were dissolved in MeOH and analyzed by HPLC−MS. Retention times in minutes of FDAA-derivatized amino acids were 6.5 for Val and 5.0 for Ala. Retention times of the authentic amino acid standards were l-Val 6.5, dl-Val 6.5/7.5, l-Ala 5.0, dl-Ala 5.0/6.0.
