3.1. General Experimental Procedures
Optical rotations were measured at 589 nm on a 343 polarimeter (Perkin-Elmer, Waltham, MA, USA) at 20 °C. CD spectra were recorded on a J-810 spectropolarimeter (Jasco Corporation, Cremella, Italy) [λ([θ]) in nm]. H
1 and C
13 NMR spectra were recorded on a DRX-400 spectrometer (Bruker, Billerica, MA, USA) at 298 K.
1H and
13C chemical shifts are reported relative to CHCl
3 (δ
H 7.26 ppm) or CDCl
3 (δ
C 77.0 ppm) as internal reference.
13C chemical shifts for compounds
1a,
1b and
3 is described with two decimals for comparison with previously published data containing two decimals. High resolution mass spectra (HRMS) data was recorded with micrOTOF II (Bruker, Billerica, MA, USA). TLC was performed on silica gel 60 F
254 (Sigma-Aldrich, St. Louis, MO, USA) with detection of UV light and anisaldehyde-sulfuric acid as spray reagent. Flash Column chromatography [eluent for flash chromatography is given between brackets in the
Experimental Section] was carried out on silica gel (particle size, 60 Å, 230–400 mesh) Sigma-Aldrich. Preparative HPLC was performed using VP 150/10 Nucleodur C-18, HTEC, 5 µm column (Macherey-Nagel, Düren, Germany) on a Gilson 333/334 Prep-Scale system with a flow rate of 7 mL/min, detection at 210 nm Gilson 151 (Gilson, Middleton, WI, USA), and an CH
3CN (0.005% HCO
2H)/H
2O (0.005% HCO
2H) eluent system. Analytical HPLC was performed using EC 150/4.6 Nucleodur C18 HTec, 5 µm column (Macherey-Nagel, Düren, Germany) on a Gilson 322 with a diode array detector Gilson 172 and evaporated light scattering detector Sedex 85 (Sedere, Oilvet, France). Reductions were performed using a H-Cube (ThalesNano, Budapest, Hungary) continuous-flow hydrogenation system utilizing a 10% Pd/C CatCart catalyst at 1 bar pressure with a flow rate of 1 mL/min at 20 °C. Cultivation of bacteria was carried out in 500 mL Erlenmeyer flasks with one baffle (Glasgerätebau OCHS, Bovenden, Germany).
3.7. Extraction and Isolation of Secondary Metabolites from Marine Actinobacteria
The pH of the supernatant from 5 L broth (PM2 with 4% sea salt) was adjusted to pH 8 using 1 M NaOH (aq) and extracted twice with EtOAc, 1:1 v:v. After centrifugation, the organic layer was concentrated under vacuum that gave a residue of 1.8 and 2.2 g for the 48 and 96 h culture, respectively. The bacterial pellet was dissolved in deionized water and extracted with EtOAc, 1:1 v:v. After centrifugation, the organic phase was concentrated which gave an oily residue of 8.5 g. The EtOAc extract of 48 h culture was mixed with hexane and filtrated through glass-wool to remove an orange precipitate. After concentration, the resulting residue was pre-purified with flash chromatography over silica gel [CHCl3:MeOH, 95:5]. Fractions 9–11 (197 mg) contained compounds 3–4, fractions 12–14 (61 mg) contained compounds 1a, 1b, 2 and 3 and fractions 15–19 (36 mg) contained compounds 1a, 1b, and 2. Fractions 9–11 was mixed with 450 μL DMSO and purified with preparatory HPLC (C18, Nucleodur HTEC, 110 Å/5 µm, 10 × 160 mm) using a gradient from 30% to 100% CH3-CN/H2O (0.005% HCO2H) during 10 min. Fractions containing compounds 3 and 4 were neutralized with NaHCO3 (aq) and extracted with CHCl3. Organic phases were concentrated in vacuum which gave 1.5 mg of compound 3 and 1 mg of compound 4 as colorless oils. In the same way, fractions 12–14 and 15–19 were purified using a gradient from 10% to 100% CH3-CN/H2O (0.005% HCO2H) during 40 min which gave 1.3 mg of compound 2, 6 mg of compounds 1a and 1b (12:1) and 0.8 mg of compound 3. The workup of EtOAc extract from 96 h culture was performed in the same way as the 48 h culture that gave 2.1, 5.8 and 8 mg of compounds 1a, 1b, 2 and 3, respectively. The oily residue of the bacterial pellet was mixed with hexane and filtrated through a plug of glass wool to remove an orange precipitate. After concentration the resulting residue was pre-purified with flash chromatography over silica gel [Pentane:Et2O, 3:1]. Fractions containing compound 4 (34–59) was concentrated and mixed with 450 μL DMSO. The resulting mixture was purified using preparatory HPLC, seven injections (Nucleodur C18 HTec, 110 Å/5 µm, 10 × 160 mm) using a gradient from 50% to 100% CH3-CN/H2O (0.005% HCO2H) during 10 min. Fractions containing compound 4 were neutralized with NaHCO3 (aq) and extracted twice with CHCl3. The combined organic phases were concentrated in vacuum that gave 11.4 mg of compound 4 as a colorless oil.
1a: Isolated after 48h, [α]
22D +74.4° (
c 0.93, MeOH); CD (
c 0.110, MeOH) ∆
ε208 +15;
1H-NMR (400 MHz CDCl
3) δ 7.44 (
J1 = 5.7,
J2 = 1.4 Hz, 1 H), 6.11 (dd,
J1 = 5.7,
J2 = 1.9 Hz, 1 H), 5.06–5.01 (m, 1H), 3.68–3.61 (m, 1H), 1.83–1.72 (m, 1H), 1.72–1.61 (m, 1H) 1.59–1.17 (m, 9H), 1.13 (d,
J = 6.34 Hz, 3H), 0.86 (d,
J = 6.69 Hz, 3H);
13C-NMR data of
1a are listed in
Table 1. HRMS (M + Na
+) calculated for C
13H
22O
3Na: 249.1467; found, 249.1466.
1a and 1b (1:1 mixture): Isolated after 96 h, [α]
22D +80.8° (
c 1.15, MeOH), CD (
c 0.115, MeOH) ∆
ε208 +19;
1H-NMR (400 MHz CDCl
3) δ 7.44 (
J1 = 5.7,
J2 = 1.4 Hz, 1 H), 6.10 (dd,
J1 = 5.7,
J2 = 1.9 Hz, 1 H), 5.06–5.01 (m, 1H), 3.74–3.67, 3.67–3.60 (each m, 1H), 1.83–1.72 (m, 1H), 1.72–1.61 (m, 1H) 1.59–1.17 (m, 9H), 1.15 (d,
J = 6.34 Hz, 3H), 1.12 (d,
J = 6.34 Hz, 3H), 0.88 (d,
J = 6.69 Hz, 3H), 0.86 (d,
J = 6.69 Hz, 3H);
13C-NMR (100 MHz CDCl
3) δ 173.3, 156.4, 121.7, 83.5, 71.9, 71.5, 40.1, 39.9, 33.3, 32.6, 32.5, 29.8, 27.3, 27.1, 25.12, 25.10, 20.4, 19.7, 14.7, 14.3; Subtracted
13C-NMR data of
1b are listed in
Table 1 as
1a + (
1b); HRMS (M + Na
+) calculated for C
13H
22O
3Na: 249.1467; found, 249.1474.
2: Isolated after 48h, [α]
22D +66.5° (
c 0.927, MeOH); CD (
c 0.093, MeOH) ∆
ε205 +16;
1H-NMR (400 MHz CDCl
3) δ 7.44 (dd,
J1 = 5.8,
J2 = 1.5 Hz, 1 H), 6.11 (dd,
J1 = 5.8,
J2 = 2.0 Hz, 1 H), 5.05–5.00 (m, 1H), 1.84–1.72 (m, 1H), 1.72–1.61 (m, 1H), 1.53–1.40 (m, 6H), 1.40–1.31 (m, 4H), 1.14 (s, 3H), 1.11 (-OH broad singlett), 0.89 (t,
J = 7.5 Hz, 3H);
13C-NMR data of
2 are listed in
Table 1; HRMS (M + Na
+) calculated for C
13H
22O
3Na: 249.1467; found, 249.1470.
3: Isolated after 48h, [α]
22D +48.8° (
c 1.475, MeOH); CD (
c 0.147, MeOH) ∆
ε204 +26;
1H-NMR (400 MHz CDCl
3) δ 7.44 (dd,
J1 = 5.7,
J2 = 1.4 Hz, 1 H), 6.11 (dd,
J1 = 5.7,
J2 = 2.0 Hz, 1 H), 5.05–5.00 (m, 1H), 2.53–2.44 (m, 1H), 2.13 (s, 3H), 1.82–1.72 (m, 1H), 1.70–1.58 (m, 2H), 1.51–1.38 (m, 2H), 1.39–1.21 (m, 5 H), 1.08 (d,
J = 7.4 Hz, 3 H);
13C-NMR data of
3 are listed in
Table 1; HRMS (M + Na
+) calculated for C
13H
20O
3Na: 247.1310; found, 247.1310.
4: Isolated after 48h, [α]
22D +58° (
c 1.033, MeOH); CD (
c 0.103, MeOH) ∆
ε204 +17;
1H-NMR (400 MHz CDCl
3) δ 7.45 (dd,
J1 = 5.7,
J2 = 1.5 Hz, 1 H), 6.11 (dd,
J1 = 5.7,
J2 = 1.9 Hz, 1 H), 5.06–5.01 (m, 1H), 1.82-–1.62 (m, 2H), 1.51–1.38 (m, 2H), 1.38–1.21(m, 7H), 1.18–1.03 (m, 2H), 0.87–0.82 (m, 6H);
13C-NMR data of
4 are listed in
Table 1; HRMS (M + Na
+) calculated for C
13H
22O
2Na: 233.1518; found, 233.1517.
5: Compound 3 (4.3 mg, 0.019 mmol) was dissolved in absolute EtOH and hydrogenated utilizing H-cube. The eluate was concentrated and co-concentrated with CHCl3 three times which gave 3.77 mg (0.017 mmol) as a colorless oil in 89% yield. [α]22D −54° (c 0.76, MeOH); 1H-NMR (400 MHz CDCl3) δ 4.53–4.42 (m, 1H), 2.56–2.45 (m, 3H), 2.37–2.27 (m, 1H), 2.13 (s, 3H), 1.90–1.79 (m, 1H), 1.75–1.68 (m, 1H), 1.67–1.57 (m, 2H), 1.51–1.21 (m, 7H), 1.08 (d, J = 7 Hz, 3H); 13C-NMR (100 MHz CDCl3) δ 212.8, 177.2, 80.9, 47.1, 35.5, 32.7, 29.3, 28.8, 28.0, 28.0, 27.0, 25.1, 16.2; HRMS (M + Na+) calculated for C13H22NaO3: 249.1467; found, 249.1473.
6: Compound 4 (3.5 mg, 0.017 mmol) was dissolved in absolute EtOH and hydrogenated utilizing H-cube. The eluate was concentrated followed by Flash chromatography [3:1 pentane:Et2O] over silica gel to give 2.2 mg (0.010 mmol) as a colorless oil in 62% yield. [α]22D −38° (c 0.79, MeOH); 1H-NMR (400 MHz CDCl3) δ 4.52–4.44 (m, 2H), 2.56–2.49 (m, 2H), 2.37–2.26 (m, 1H), 1.92–1.80 (m, 1H), 1.79–1.69 (m, 1H), 1.65–1.54 (m, 1H), 1.50–1.42 (m, 1H), 1.41–1.21 (m, 8H), 1.18–1.03 (m, 2H), 0.91–0.78 (m, 6H); 13C-NMR (100 MHz CDCl3) δ 177.3, 81.1, 36.5, 35.6, 34.3, 29.7, 29.4, 28.9, 28.0, 26.9, 25.2, 19.2, 11.4; HRMS (M+ Na+) calculated for C13H24NaO2: 235.1674; found, 235.1679.