*3.3. Structural Elucidation*

The strain NEAU-wh3-1 was grown preparative scale in 15.0 L of production broth for 7 days. Bioassay-guided isolation of the active components of the strain yielded eight main bioactive compounds. Compounds **2**–**8** are known compounds, which structures were elucidated as conglobatin (**2**) [75], piericidin C1 (**3**) [76], piericidin C5 (**4**) [77], piericidin A1 (**5**) [78], piericidin A3 (**6**) [76], Mer-A 2026 A (**7**) [79], and BE-52211 D (**8**) [80] by analysis of their spectroscopic data and comparison with literature values (Figure 5, Figures S12–S27). Compound **1** is a new zincophorin analogue (Figure 6, Figures S4–S11) [17].

**Figure 5.** The structures of compounds **1**–**8.** Compound **1** was isolated as white solid with [α] 25 *<sup>D</sup>* + 15 (c 0.043, EtOH) and UV (EtOH) λmax nm (log ε): 202 (4.53). Its molecular formula was established as C31H56O7 by HR-ESI-MS at *m*/*z* 539.3942 [M-H]- (calcd 539.3953 as C31H55O7). The IR spectrum revealed hydroxyl absorption at 3320 cm−<sup>1</sup> and carbonyl absorption at 1735 cm<sup>−</sup>1, as well as methyl and methylene absorptions at 2953 cm−<sup>1</sup> and 2924 cm<sup>−</sup>1.

**Figure 6.** 2D nuclear magnetic resonance (NMR) correlations of compound **1.**

Analysis of 1H NMR spectrum of **1** revealed the presence of three olefinic protons at δ<sup>H</sup> 5.52 (1H, m), 5.36 (1H, m), 5.20 (1H, d, *J* = 8.9 Hz), seven aliphatic methine protons at δ<sup>H</sup> 4.07 (1H, m), 4.06 (1H, m), 3.77 (1H, d, *J* = 8.9 Hz), 3.72 (1H, dd, *J* = 9.6, 2.1 Hz), 3.58 (1H, d, *J* = 9.2 Hz), 3.49 (1H, m), 3.28 (1H, m), seven methylene protons at δ<sup>H</sup> 2.18 (1H, m), 2.13 (1H, m), 1.77 (1H, m), 1.67 (2H, m), 1.40 (1H, m), 1.35 (1H, m), 1.28 (1H, m), one singlet methyl at δ<sup>H</sup> 1.63 s, in addition to eight doublet methyl protons at δ<sup>H</sup> 1.18 (3H, d, *J* = 7.1 Hz), 1.15 (3H, d, *J* = 7.2 Hz), 1.11 (3H, d, *J* = 7.0 Hz), 0.98 (3H, d, *J* = 6.5 Hz), 0.97 (3H, d, *J* = 6.6 Hz), 0.86 (3H, d, *J* = 6.7 Hz), 0.81 (3H, d, *J* = 6.5 Hz), 0.70 (3H, d, *J* = 6.7 Hz). The 13C NMR and DEPT135 spectra (Table 2) of 1 showed 31 resonances attributable to a carbonyl carbon at δ<sup>C</sup> 175.6, one *sp*<sup>2</sup> quaternary carbon at δ<sup>C</sup> 132.3, three *sp*<sup>2</sup> methines at δ<sup>C</sup> 136.6, 134.5, 132.4. In the sp3-carbon region, the spectrum showed six oxygenated methines at δ<sup>C</sup> 84.3, 83.3, 81.7, 76.1, 74.0, 69.4, six methines at δ<sup>C</sup> 42.2, 37.1, 36.5, 36.0, 33.0, 26.1, four methylenes at δ<sup>C</sup> 34.2, 28.6, 27.1, 24.9 and nine methyl carbons at δ<sup>C</sup> 22.9, 22.9, 17.1, 16.7, 15.4, 12.3, 11.3, 10.8, 10.1. The 1H–1H COSY correlations (Figure 6**)** of H-2/H-3/H2-4/H2-5/H-6/H-7/H-8/H-9/H-10/H-11/H-12/H-13/H2-14/H2-15/H-16/H-17/H-18/H-19 established connectivity from H-2 atom along the chain through to C-19 atom. The correlations between H-21/H-22/H3-23/H3-24, H-12/H3-27, H-10/H3-28, H-18/H3-26, H-8/H3-29 protons in the 1H–1H COSY spectrum (Figure 6**)** indicated the five structural units of C-21–C-24, C-18–C-26, C-12–C-27, C-10-C-28, C-8-C-29. The observed HMBC (heteronuclear multiple bond correlation) correlations (Figure 6**)** from H3-23, H3-24 to C-21, C-22, from H3-25 to C-19, C-21, from H3-26 to C-17, C-18, and C-19, from H3-27 to C-11, C-12, and C-13, from H3-28 to C-9, C-10, C-11, from H3-29 to C-7, C-8, C-9, from H3-30 to C-5, C-6, C-7, from H3-31 to C-2 and C-3, from H-21 to C-19, H-20 to C-18, from H-19 to C-17, from H3-23 to C-21 established the linkage of C-2–C-22. The carbonyl group was connected with C-2 by the HMBC corrections from H-2 and H3-31 to C-1 (δ<sup>C</sup> 175.6). The correlations from H-7 (δ<sup>H</sup> 3.77 d, *J* = 8.9 Hz) to C-3 (δ<sup>C</sup> 74.0) indicated the linkage of C-3 and C-7 through an oxygen atom to form a tetrahydropyran ring. Taking the molecular formula of C31H56O7 into account, four hydroxyl groups were situated at C-9, C-11, C-13, C-19, respectively, and a carboxyl group was situated at C-1. Comparison the NMR data of **1** with Zincophorin [17], a mocarboxylic acid ionophore contains one single tetrahydropyran ring, which was isolated from a strain of *Streptomyces griseus*, implied that **1** was identified to be an analogue of Zincophorin, the difference between two compounds was that the terminal ethyl group in Zincophorin was replaced by a H proton in compound **1**. On the basis of the above spectroscopic data, a gross structure of **1** was established and named Zincophorin B, and the 1H and 13C resonances in **1** were assigned (Table 2).


**Table 2.** 1H and 13C NMR data of compound 1 in CDCl3.
