*3.1. Biological Activity-Guided Purification of Active Components from Culture Broth of S. palmae CMU-AB204T and Structure Determination of Active Components*

*S. palmae* CMU-AB204<sup>T</sup> was cultured in 40 L of ISP2 medium at 28 ◦C for seven days, and the broth and mycelia were extracted with EtOAc. The active components in culture broth extract of strain CMU-AB204<sup>T</sup> were isolated by biological activities-guided purification using paper disk assay. The extract was purified by silica gel column chromatography, Sephadex LH-20 column chromatography, preparative TLC, and preparative HPLC. The eluates were concentrated in vacuo to yield nine compounds, AB204-A (**1**, 10.2 mg), AB204-B (**2**, 8.2 mg), AB204-E (**5**, 14.2 mg), AB204-F (**6**, 9.9 mg), a mixture (6.5 mg) of AB204-C (**3**) and AB204-D (**4**), **7** (7.0 mg), **8** (1.7 mg), and **9** (17.0 mg) as depicted in Scheme 1.

AB204-A (**1**) was obtained as a pale yellow amorphous solid. It was found to be readily soluble in acetonitrile, MeOH, CHCl3, and was observed to be less soluble in water. As the HREIMS analysis showed *m*/*z* 190.1000 [M]+, the molecular formula of **1** was elucidated as C12H14O2 (calculated value of 190.0994, Figure S1). The intense band at 1706 cm−<sup>1</sup> of the IR spectrum in MeOH solution was assigned as C=O stretching frequency of dimeric carboxylic acid moiety (Figure S2). Based on 1H NMR analysis, **1** revealed four aromatic protons stacked at 7.14–7.18 ppm and a pair of olefinic protons stacked at 5.70 ppm and 6.51 ppm (Figure S3). Coupling constants (*J* = 11.5 Hz) of the two olefinic protons showed *Z*-configuration of the olefin moiety. Compound **1** had four additional methylene protons at 2.41–2.51 ppm and one methyl singlet signal at 2.24 ppm (Table 1). The 13C NMR spectrum showed 12 carbon signals: one carbonyl carbon at 177.2 ppm that indicated a carboxylic acid, eight aromatic or olefinic carbons, two methylene carbons at 23.5 and 33.7 ppm, and one methyl carbon at 19.8 ppm (Table 1, Figure S4).


**Table 1.** NMR spectroscopic data of AB204-A (**1**) and B (**2**) (δH, 500 MHz; δC, 125 MHz).

\* overlapped.


**Scheme 1.**

Purification

 procedures

 for compounds **1**–**9**.

MS, HMBC, and HMQC analyses suggested **1** contained one disubstituted aromatic ring, one methyl, and pentenoic acid moieties (Figures S5 and S6). HMBC correlations were observed from two methylene protons (2.41–2.44 ppm and 2.46–2.51 ppm) to a carboxylic carbon at 177.2 ppm, and two olefinic carbons of C-4 and C-5 (129.7 and 129.8 ppm), as are given in Table 1. A correlation between the *Z*-olefinic proton at 6.51 ppm (H-5) and one methylene carbon (C-3) at 23.5 ppm was also observed; thus **1** was believed to possess 4,5-*Z*-pentenoic acid moiety in the structure. An HMBC correlation between one methyl proton at 2.24 ppm and three aromatic carbons of C-1', C-2', and C-3' (136.2, 136.2, and 129.8 ppm, respectively), and between *Z*-olefinic protons and aromatic carbons, H-4 (5.70 ppm) and C-1' (136.2 ppm), and H-5 (6.51 ppm) and C-6' (128.8 ppm), indicated **1** was an *ortho*-methyl phenyl alkenoic acid compound, (*Z*)-5-(2-methylphenyl)-4-pentenoic acid (Figure 1). Differential NOE of **1** was observed between a methyl proton and both an aromatic 3'-proton and an olefinic proton of H-5 as well as between the two olefinic protons (Figure S7). The geometry of two substitutes of the aromatic ring was confirmed by NOE correlations, as is shown in Figure 2. From some *Streptomyces* strains, *E*-isomer of **1**, (*E*)-5-(2-methylphenyl)-4-pentenoic acid was identified [33–35]; however, there was no report on the *Z*-isomer (**1**) obtained from natural sources. Therefore, it was concluded that **1** was a novel natural product.

**Figure 1.** Antimicrobial compounds isolated from the broth extract of *Streptomyces palmae* CMU-AB204T.

**Figure 2.** Observed COSY correlation (bold lines, in **2**) and NOE correlation in AB204-A (**1**) and B (**2**).

AB204-B (**2**) was isolated as a pale yellow amorphous solid. It was readily soluble in the same solvent as **1** and less soluble in water. The molecular formula of **2** was established as C14H18O2 (calculated value of 218.1329) based on NMR data and the HREIMS ion peak of *m*/*z* 218.1301 [M] <sup>+</sup> (Figure S8), indicating compound **2** had one more C2H4 unit when compared to the molecular formula of **1**. The IR spectrum of **2** revealed the presence of C=O stretching frequency of dimeric carboxylic acid moiety at 1706 cm<sup>−</sup>1, which was similar to the spectrum of **1** (Figure S9). The 1H NMR spectrum of **2** revealed four aromatic protons at 7.14–7.16 ppm, a pair of olefinic protons at 5.69 and 6.45 ppm, eight methylene protons at 1.45, 1.63, 2.17, and 2.30 ppm and one methyl singlet signal at 2.25 ppm (Table 1, Figure S10). The coupling constant of two olefinic protons (11.5 Hz) indicated a *Z-*configuration. The 13C NMR spectrum of **2** showed 14 carbon signals: one carbonyl carbon at 177.6 ppm, eight aromatic or olefinic carbons, four methylene carbons at 24.2, 27.8, 29.1, and 33.4 ppm, and one methyl carbon at 19.9 ppm (Table 1, Figure S11). These data support the conclusion that the compound had a closely related structure to **1**. Each methylene signal was assigned by COSY, as is shown in Figure 2. Eight methylene protons constructed a C4 alkyl chain, and COSY correlation confirmed the connection between this C4 alkyl chain and *Z*-olefin (Figure S12). This connection was supported by HMBC and HMQC spectra (Figures S13 and S14). HMBC correlations were observed from one olefinic proton H-6 (5.69 ppm) to an aromatic carbon at C-1' (136.7 ppm) and from singlet methyl proton at 2.25 ppm to aromatic carbons at C-1', C-2', and C-3'. Therefore, one methyl moiety and one alkene chain were substituted for an aromatic ring in the *ortho* position. HMBC correlation from two methylene protons of the alkene chain at 1.63 and 2.30 ppm to the carbonyl carbon at 177.6 ppm, and a molecular formula of **2**, suggested that this compound had a carboxylic acid at the end of the alkene chain. The same NOE correlation was observed in **1** and **2** (Figure 2 and Figure S15). Thus, the structure of **2** was assigned as (*Z*)-7-(2-methylphenyl)-6-heptenoic acid, as is shown in Figure 1.

The structures of AB204-C (**3**) and AB204-D (**4**) were elucidated as a mixture of both compounds because of the difficulty associated with further purification. MS spectra showed the molecular formulas of **3** and **4** were C18H26O2 and C19H28O2, respectively (Figure S16). 1H NMR data suggested compounds **3** and **4** were analogs of **1** and **2**, thus **3** and **4** might be (*Z*)-11-(2-methylphenyl)-10-undecenoic acid and (*Z*)-12-(2-methylphenyl)-11-dodecenoic acid, respectively (Figure 1 and Figure S17).

AB204-E (**5**) and AB204-F (**6**) were obtained as a pale yellow amorphous solid. The accurate mass and molecular formula of compounds **5** and **6** were analyzed by both HRESIMS and HREIMS. Molecular ion peaks were exhibited at *m*/*z* 271.1705 [M + H]<sup>+</sup> and 270.1616 [M]<sup>+</sup> for **5**, and *m*/*z* 271.1689 [M + H]<sup>+</sup> and 270.1633 [M]<sup>+</sup> for **6** (Figures S18–S21). These data suggest that both compounds had the same molecular formula as C18H22O2 (calcd. 270.1620 for C18H22O2 and calcd. 271.1698 for C18H23O2). The C=O stretching frequency band at 1685 cm−<sup>1</sup> in the IR spectrum of **5** and 1684 cm−<sup>1</sup> in the spectrum of **6** was assigned as a carboxylic acid moiety (Figures S22 and S23). NMR spectra of both compounds demonstrated a structural similarity. The assignment of 1H and 13C NMR spectra of **5** and **6** are given in Table 2.


**Table 2.** NMR spectroscopic data of AB204-E (**5**) and F (**6**) (δH, 500 MHz; δC, 125 MHz).

\* overlapped.

1H NMR spectrum of **5** showed ten aromatic/olefinic protons, eight methylene protons, and one methyl triplet proton at 0.83 ppm (Figure S24). In 13C NMR spectrum of **5**, one calboxylic carbon at 170.5 ppm, twelve olefinic/aromatic carbons, four methylene carbons, and one methyl carbon were measured (Figure S25). 1H NMR spectrum of **5** suggested the existence of two pairs of *E*-olefin assigned by large coupling constants (15.5 Hz for each) and one pair of *Z*-olefin whose coupling constant was 11.5 Hz. Three partial structures were assigned by COSY correlation; one 1,2-substituted aromatic ring, one 1,2-*Z*-heptene group, and one diene group (Figure S26). HMBC correlations between diene protons of 5.99 and 7.41 ppm and a carbonyl carbon at 170.5 ppm, and between diene protons of 6.95 and 7.13 ppm and three aromatic carbons at positions C-6, C-7, and C-11 (135.6, 126.7, and 138.7 ppm, respectively) indicated that one end of the diene was connected to a carboxylic carbon and the other end of the diene was attached to an aromatic ring at position 6 (Figures S27 and S28). HMBC correlation between *Z*-olefinic protons (5.83 and 6.54 ppm) of 1,2-*Z*-heptene and aromatic ring carbons at C-10 and C-11 (131.0 and 138.7 ppm, respectively) suggested 1,2-*Z*-heptene moiety was connected to the aromatic ring at C-11. These data support the structure of **5** as (2*E*,4*E*)-5-(2-(1*Z*)-heptenylphenyl)-2,4-pentadienoic acid (Figure 1).

1H NMR and 13C NMR spectra of **6** suggested that the structure was almost the same as **5** (Figures S29 and S30). However, the 1H NMR spectrum clarified that only one pair of *E*-olefin existed, while the other two pairs of olefin were identified as *Z*-configuration by analysis of coupling constants. These data indicated that **6** was a stereoisomer of **5**. COSY correlations revealed that two partial structures of **6**, one 1,2-substituted aromatic ring and one 1,2-*Z*-heptene moiety, were identical to those of **5**; however, a diene structure was constituted of both *E* and *Z*-olefins (Figure S31). The connection of 1,2-*Z*-heptene moiety to the aromatic ring at C-11 was confirmed by the HMBC spectrum (Figure 3, Figures S32 and S33). HMBC correlations between *Z*-olefinic protons of the diene moiety and a carboxylic carbon at 169.5 ppm and between one *E*-olefinic proton (7.08 ppm) of the diene moiety and aromatic ring carbons at C-7 and C-11 (126.2 and 137.5 ppm, respectively) established the structure of **6** as (2*Z*,4*E*)-5-(2-(1*Z*)-heptenylphenyl)-2,4-pentadienoic acid, as is depicted in Figure 1.

**Figure 3.** Observed COSY (bold lines) and HMBC correlations (arrows) in AB204-F (**6**). The same correlations were observed in AB204-E (**5**).

To clarify the geometry of two substituted chains of **5** and **6**, the differential NOE experiment was conducted. NOE correlations between H-5 and H-12 were observed in both compounds (Figures S34 and S35), which supported the geometry of two side chains in **5** and **6,** as is shown in Figure 4. This is the first report of **5** and **6** obtained from natural sources. Qureshi et al. [36] found structurally related compounds, MF-EA-705a and b, along with actinopyrone A from a broth extract of *Streptomyces* MF-EA-705. The most related structures were found as cinnamoyl moieties of rare peptide compounds, pepticinnamin E, WS9326A, and RP-1776 (skyllamycin A) [37–39].

**Figure 4.** NOE correlations in AB204-E (**5**) and AB204-F (**6**).

Compounds **7** and **8** were pale-yellow amorphous solids that were determined by HRESI-MS analyses to have molecular formulas of C31H44O6 and C32H46O6, respectively (Figures S36 and S37). The planar structures of **7** and **8** (Figure 1) were confirmed by NMR spectra (Figures S38 and S39) as known compounds, anguinomycin A (**7**) [40] and leptomycin A (**8**) [41], which were known to be relative structures. Compound **9** was a colorless oil, and the molecular formula of **9** was determined to be C25H36O4 on the basis of HRESI-MS data and the signals of 1H NMR spectrum (Figures S40 and S41). These data supported the structure of **9** to be actinopyrone A (**9**) as is shown in Figure 1 [42].

Physicochemical Properties of **1**, **2**, **5**, and **6**

Compound **1** (AB204-A): pale yellow amorphous solid; [α]D –3.4 (*c* 0.1, MeOH); UV (MeOH) λmax (log ε) 204.5 (4.09), 235.5 (3.63) nm; IR (ATR) νmax 3014, 2923, 1706, 1654, 1409 cm<sup>−</sup>1; 1H and 13C NMR (chloroform-*d*) see Table 1; HREIMS *m*/*z* 190.1000 [M]<sup>+</sup> (calcd for C12H14O2, 190.0994).

Compound **2** (AB204-B): pale yellow amorphous solid; [α]D –0.3 (*c* 0.1, MeOH); UV (MeOH) λmax (log ε) 204.5 (3.98), 235.5 (3.53) nm; IR (ATR) νmax 3011, 2925, 1706, 1653, 1457 cm<sup>−</sup>1; 1H and 13C NMR (chloroform-*d*) see Table 1; HREIMS *m*/*z* 218.1301 [M]<sup>+</sup> (calcd for C14H18O2, 218.1329).

Compound **5** (AB204-E): pale yellow amorphous solid; [α]D –13.4 (*c* 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.04), 251 (3.90), 309.5 (4.26) nm; IR (ATR) νmax 2956, 2924, 2857, 1684, 1617, 1277, 1000 cm<sup>−</sup>1; 1H and 13C NMR (methanol-*d*4) see Table 2; HREIMS *m*/*z* 270.1616 [M]<sup>+</sup> (calcd for C18H22O2, 270.1620).

Compound **6** (AB204-F): pale yellow amorphous solid; [α]D –14.1 (*c* 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.76), 251 (3.51), 309.5 (3.86) nm; IR (ATR) νmax 2955, 2924, 2855, 1684, 1615, 1244, 958 cm<sup>−</sup>1; 1H and 13C NMR (chloroform-*d*) see Table 2; HREIMS *m*/*z* 270.1633 [M]<sup>+</sup> (calcd for C18H22O2, 270.1620).
