*2.2. Antibacterial Assay*

The antibacterial assay was used as one effective way to exclude the inactive strains and select the candidates for metabolomics analysis. Out of 521 actinomycetial strains from 13 different mangrove soil samples, 179 strains affiliated to 40 different genera with a unique colony morphology were selected to evaluate the antibacterial activities against six sets of indicator bacteria; each set contained one drug-sensitive and one drug-resistant pathogen (*Enterococcus faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, and *Enterobacter species*). Of the 179 strains, 47 exhibited antagonistic activity against at least one of the tested pathogens. These bioactive strains were distributed in 13 genera, including *Streptomyces* (29), *Micromonospora* (4), *Micrococcus* (3), *Rhodococcus* (2), *Brevibacterium* (1), *Demequina* (1), *Actinomadura* (1), *Georgenia* (1), *Gordonia* (1), *Isoptericola* (1), *Microbacterium* (1), *Streptacidiphilus* (1), and *Serinibacter* (1) (Table S4). The antibacterial spectra of the 47 isolates against indicator bacteria are shown in Figure 2. Of the 47 strains, 35 were active only against the Gram-positive bacteria, while two isolates (*Streptomyces* sp. H124 and *Streptomyces* sp. Y129) were active only against the Gram-negative bacteria. In addition, ten strains showed inhibitory activities against both the Gram-positive and Gram-negative bacteria. Interestingly, isolate M45, a rare actinomycetial strain affiliated to genus *Demequina*, showed moderate activities against all four Gram-positive bacterial strains tested. Thirty-seven isolates showed anti-MRSA activity, accounting for the highest proportion of the total active strains (78.7%). Twenty-three strains showing high anti-MRSA activity (diameter of inhibition zone ≥ 10 mm) were selected for subsequent metabolomics analyses (Table 1).

**Figure 1.** The distribution of cultivable actinomycetial strains isolated from mangrove soil in Leizhou Peninsula. (**a**) Phylogenetic tree (16S rRNA gene) obtained by neighbor-joining analysis of 40 isolates from each genus and their closely related type strains; *Escherichia coli* was used as an outgroup; (**b**) genera distribution in different sampling sites; (**c**) genera distribution according to the culture media used for the isolation.

**Figure 2.** The antibacterial spectra of 47 strains against indicator bacteria (6 mm, no inhibitory activity; *S*, sensitive; *R*, drug resistant; *E. coli*, *Escherichia coli*; *P. aeruginosa, Pseudomonas aeruginosa*; *K. pneumoniae*, *Klebsiella pneumoniae*; *A. baumannii*, *Acinetobacter baumannii*; *S. aureus*, *Staphylococcus aureus*; *E. faecalis*, *Enterococcus faecalis*; *E. faecium*, *Enterococcus faecium*).

#### *2.3. Strain Prioritization by Metabolomics*

The ethyl acetate extracts of 23 isolates shown in Table 1 were subjected to metabolomics analysis using UPLC-HRMS-PCA. PCA, an unsupervised statistical analysis method, was used to identify differing chemical features found in the outlying strains and to prioritize the strains that produced unique secondary metabolites. After UPLC-MS/MS data acquisition and processing, a total of 6544 features (RT-*m/z* pairs) and 6 principal components were generated by PCA analysis, giving the R2 (goodness of fit) and Q<sup>2</sup> (predictability) values of 0.82 and 0.70, respectively. After analyzing the scores plot (PC1 vs. PC2), the quality control (QC) samples (purple circle) clustered and were close to the center of the scores plot (Figure 3a), indicating good reproducibility and stability of the system. Four predominant outliers, *Streptomyces* sp. H7, *Streptomyces* sp. Y2, *Streptomyces* sp. H12, and *Streptomyces* sp. Y46, were observed, suggesting their chemical uniqueness from the main groups of samples in the scores plot. *Streptomyces* sp. H7 and *Streptomyces* sp. Y2 were located in the same quadrant, indicating similarities in their secondary metabolite profiles. Similarly, the metabolite profiles of *Streptomyces* sp. H12 and *Streptomyces* sp. Y46 were also alike. The loadings plot (Figure 3b) was geometrically related to the scores plot and described the variance observed in the scores plot, so it could be used to identify compounds that caused a clustered group to separate. From the loadings plot, three outlying metabolites, 12.88\_1268.6095n (n: neutral mass) (**1**), 12.69\_1254.6282n (**2**), and 11.11\_1270.6216n (**3**), were significant contributors to the group of *Streptomyces* sp. H12 and *Streptomyces* sp. Y46. They were putatively assigned as actinomycin-type antibiotics through dereplication with the Natural Products Atlas (NPAtlas) v19\_12 database [33] and StreptomeDB v3.0 database [34] and further confirmed as the

actinomycins by comparisons of their ultraviolet (UV) spectra with previously published data (Figure 3c, Table 2, and Figure S1) [35,36]. Comparison with the other extracts revealed that the outlying feature 16.71\_724.4749n (**4**) was abundant in *Streptomyces* sp. H7 and present in *Streptomyces* sp. Y2 with lower peak intensities (Figure S2). The predicted molecular formula C40H68O11 was tentatively identified as nigericin, epinigericin, or abierixin by dereplication. Abierixin was first excluded because it had UV absorptions at 200–400 nm but the feature 16.71\_724.4749n (**4**) lacked UV absorptions [37]. Meanwhile, the MS/MS fragment pattern (Figure S3) and UV characterization of 16.71\_724.4749n (**4**) strongly suggested that it was most likely to be either nigericin or epinigericin [38,39]. Another feature 18.21\_708.4796n (**6**) was putatively elucidated as 22-member macrolides (ushikulide A or B) or nigericin-type polyethers (grisorixin or epigrisorixin); all were previously isolated from *Streptomyces* spp. (Table 2, Figure 3c) [40–42].

**Table 1.** Information of 23 isolates selected for metabolomics analyses and their antimicrobial activity against drug-resistant *S. aureus* (MRSA) and drug-sensitive *A. baumannii*.


Paper disk diameter, 6 mm; -, no inhibitory zone; *A. baumannii*, *Acinetobacter baumannii*; *S. aureus*, *Staphylococcus aureus*; *S*, sensitive; *R*, drug resistant.

**Figure 3.** Discovery of natural products unique to four outlier strains (*Streptomyces* sp. Y46, *Streptomyces* sp. H12, *Streptomyces* sp. H7, and *Streptomyces* sp. Y2). (**a**) Scores plot (PC1 vs. PC2) of 23 extracts; (**b**) loadings plot (PC1 vs. PC2) of 23 extracts; the loadings plot showed compounds **1**–**3** were unique to *Streptomyces* sp. H12 and *Streptomyces* sp. Y46, and compounds **4**–**6** were special to *Streptomyces* sp. H7 and *Streptomyces* sp. Y2; 9.35\_453.2153n\* (**5**): false positive; (**c**) The structures of putative metabolites for compounds **1**–**6** from four outlier strains by database dereplication.


**Table 2.** The putative metabolites for compounds **1**–**9** against the NPAtlas (Hit 1) and StreptomeDB (Hit 2) databases in the Progenesis QI v3.0 software.

After rapid structure dereplication using high-resolution MS data of the feature 9.35\_453.2153n (**5**), it was putatively assigned to diaporisoindole D, E, aniduquinolone B, NPA001400, or NPA020460, all described as fungal metabolites by the NPAtlas database (Table 2 and Figure 3c). However, none of them matched the UV spectrum of this feature [43–45]. When searching its UV maximum (308 nm) in the UPLC-UV-HRMS chromatogram of sample *Streptomyces* sp. H7, two analogs of compound **5**, 9.53\_456.4081*m/z* and 10.90\_468.4110*m/z*, were found and they did not ge<sup>t</sup> any hits from the databases. The dereplication results of three homologs (9.35\_453.2153n (**5**), 9.53\_456.4081*m/z*, and 10.90\_468.4110*m/z*) indicated they might be the putative new metabolites. However, when we reacquired the MS/MS data in the data-dependent acquisition (DDA) method, all of them showed poor MS/MS fragmentations and high background noise (Figure S4), indicating that they probably were in-source fragments (false positives) rather than molecular ion peaks when acquiring in MSE method. As a data-independent acquisition (DIA) method, the MSE can simultaneously record exact mass precursor and fragment ion information in the full *m/z* range, but precursor and fragment spectra are aligned mainly according to retention times. Therefore, the MSE acquisition might mismatch the product ions with its parent ion in the analysis of complex samples [46–48], leading to the misidentification of compounds when processing the data in software, such as Progenesis QI. By examination of the raw MS and MS/MS data in almost the same elution time as three in-source fragments (9.35\_453.2153n (**5**), 9.53\_456.4081*m/z* and 10.90\_468.4110*m/z*), three other compounds, 9.35\_546.2571n (**7**), 9.46\_548.2717n (**8**), and 10.91\_560.2719n (**9**), had a high intensity of MS/MS fragments in the DDA acquisition, and their pseudomolecular ion peaks were also identified in MS spectra of UPLC-HRMS and HPLC-MS chromatograms (Figures 4, S5 and S6). Therefore, the corresponding compounds in the same elution time should be revised as compounds **7**–**9**, respectively. By searching databases and comparing their UV spectral data (Figures 4 and S7) with the literature data, compounds **7**–**9** were most likely to be benzoquinoid ansamycin-type compounds as shown in Figure 4 [49–51]. In summary, four predominant outliers, *Streptomyces* sp. H7, *Streptomyces* sp. Y2, *Streptomyces* sp. H12, and *Streptomyces* sp. Y46, were preliminarily excluded for prospecting new antibiotics because differing features of those strains could be matched well with known antibiotics.

Identifying the compounds responsible for groups near the center of the scores plot, such as sample *Streptomyces* sp. H37 or sample *Streptomyces* sp. M22 in the PC1 vs. PC2 scores plot (Figure 3a) was usually not straightforward. However, these groups could be separated by observing different PC planes [21]. This approach could lead to identifying more significant compounds that we might be interested in. As shown in Figure 5a, sample *Streptomyces* sp. H37 was separated in the PC1 vs. PC4 scores plot. Compounds unique to this sample were identified in the corresponding position in the loadings plot. Therefore, the major compounds unique to *Streptomyces* sp. H37 were shown as 10.64\_900.5435n (**10**) and 11.08\_928.5742n (**11**) (Figure 5b). The outlying feature (compound **10**) with neutral mass ion peak at 900.5435 Da in the predicted molecular formula C47 H80 O16 was putatively identified as either cytovaricin or W341C (Figure 5c, Table 3). The MS/MS fragment spectrum showed a series of peaks with loss of H2O (Figure S8), and a characteristic fragmentation pattern of polyether ionophores [39] representing compound **10** was likely identified as the polyether ionophore compound W341C. Database searches revealed no hit for feature 11.08\_928.5742n (**11**), suggesting that it might be a putative new compound. Two features (**10**–**11**) showed similarity in their MS/MS spectra, demonstrating they should be the structural analogs (Figure S8).

**Figure 4.** The characteristic of major benzoquinoid ansamycin-type compounds in the UPLC-UV-HRMS chromatogram of sample *Streptomyces* sp. H7 (**7**: 9.35\_546.2571n, 17-*O*-demethyl-geldanamycin; **8**: 9.46\_548.2717n, 4,5- dihydro-17-O-demethyl-geldanamycin (**8a**) or 17-O-demethylgeldanamycin hydroquinone (**8b**), or herbimycin F (**8c**); **9**: 10.91\_560.2719n, geldanamycin (**9a**) or 17-hydroxymethyl-17-demethoxygeldanamycin (**9b**), or 17-formyl-17-demethoxy18- O,21-O-dihydrogeldanamycin (**9c**)). (**a**) UV spectra at 308 nm and TIC plot. (**b**) MS spectra of three compounds, **7**–**9**. (**c**) UV spectra of three compounds, **7**–**9**. (**d**) The structures of putative metabolites for compounds **7**–**9** after comparison with the databases and literatures.

**Figure 5.** Discovery of natural products unique to strain *Streptomyces* sp. H37. (**a**) Scores plot (PC1 vs. PC4) of 23 extracts; the PC planes were adjusted to separate *Streptomyces* sp. H37; (**b**) loadings plot (PC1 vs. PC4) of 23 extracts; the loadings plot showed compounds **10**–**11** that were unique to *Streptomyces* sp. H37; \*, no hit. (**c**) The structures of putative metabolites for compound **10** from *Streptomyces* sp. H37 by database dereplication.

In the scores plot (PC1 vs. PC6) (Figure 6a), strain *Streptomyces* sp. M22 was clearly distinct from other strains. The major compounds unique to *Streptomyces* sp. M22 were 7.16\_1028.3600*m/z* (**12**), 15.40\_566.4171n (**13**), 7.47\_1028.3592*m/z* (**14**), and 9.55\_876.2968n (**15**). The dereplicated results of these outlying metabolites are shown in Table 3. The UV spectra of compounds **12**, **14**, and **15** confirmed their identities as trioxacarcin-type compounds (Figure S9), corresponding to putative gutingimycin, gutingimycin, and trioxacarcin A, respectively [52,53]. Both compounds **12** and **14** matched gutingimycin in the database searching because they had the same MS data (1028.3592*m/z*), indicating that one of them should be an isomer of gutingimycin. To the best of our knowledge, no isomers of gutingimycin have been found so far (Table S5). The presence of gutingimycin isomer in 7.16 or 7.47 min (compound **12** or **14**) indicated that one of them should be a putative new compound. After examining the UPLC-UV-HRMS profile of *Streptomyces* sp. M22, trioxacarcin-type compounds, including compounds **12**, **14**, and **15,** were presented within a retention time range of 6.5–10.0 min (Figures 7, S9 and S10). Two compounds, 6.69\_1030.3751*m/z* (**16**) and 7.94\_1013.3486*m/z* (**17**), in the chromatogram were identified as the putative new compounds after searching the databases and reverting back to the literature (Table S5). Lastly, another differing compound (**13**) was tentatively confirmed not to be 4-ketozeinoxanthin due to its lack of characteristic maximum UV absorption at 445–470 nm [54].



#### *Mar. Drugs* **2021**, *19*, 688

**Figure 6.** Discovery of natural products unique to strain *Streptomyces* sp. M22. (**a**) Scores plot (PC1 vs. PC6) of 23 extracts; the PC planes were adjusted to separate *Streptomyces* sp. M22; (**b**) loadings plot (PC1 vs. PC6) of 23 extracts; the loadings plot showed compounds **12**–**15** that were unique to *Streptomyces* sp. M22. (**c**) The structures of putative metabolites for compounds **12**–**15** from *Streptomyces* sp. M22 by database dereplication.

As shown in Table 1, among 23 strains showing anti-MRSA activity, 8 strains also displayed inhibition zones against drug-sensitive *Acinetobacter baumannii*. However, in PCA model, the groups of active or inactive strains against the *Acinetobacter baumannii* were not completely separated from each other as shown in Figure 3a. To discriminate the two classes (active vs. inactive) and identify compounds mainly contributing to the bioactivity of the eight strains, the OPLS-DA model was constructed. As shown in Figure 8a, active and inactive samples were clearly separated in the model. The R<sup>2</sup> value of 0.98 and Q<sup>2</sup> value of 0.95 suggested that the OPLS-DA model possessed reliable fitness and predictability. In the S-plot analysis, five potential marker compounds were selected to chemically distinguish the active from inactive extracts (Figure 8b). The variable importance in the projection (VIP) plot showed that all selected potential markers had high VIP values (VIP ≥ 10) (Figure S11), revealing that these marker compounds were largely responsible for the discrimination between active and inactive groups. Therefore, the main contributors to the activity were the putative three actinomycins (**1**–**3**) from the group of *Streptomyces* sp. H12 and *Streptomyces* sp. Y46, and the two compounds 10.64\_900.5435n (**10**) and 11.08\_928.5742n (**11**) from *Streptomyces* sp. H37. To the best of our knowledge, the inhibitory activity of actinomycin D against *A. baumannii* was reported [55]. Furthermore, cytovaricin and W341C, the dereplicated metabolites for compound **10**, had no antibacterial

activity against Gram-negative pathogens [56,57]. In contrast to their antibacterial spectra, compound **10** showed antibacterial activity against Gram-negative pathogens, suggesting that it might be a putative new compound.

**Figure 7.** The characteristic of major trioxacarcin-type compounds in the UPLC-UV-HRMS chromatogram of *Streptomyces* sp. M22. (**12**, 7.16\_1028.3600*m/z*; **14**, 7.47\_1028.3592*m/z*; **15**, 9.55\_876.2958n, trioxacarcin A; **16**, 6.69\_1030.3751*m/z*; **17**, 7.94\_1013.3486*m/z*; **18**, 8.43\_894.3132n, trioxacarcin B; **19**, 8.89\_878.3168n, trioxacarcin C). (**a**) UV spectra at 270 nm and TIC plot. (**b**) The structures of putative metabolites for compounds **12**, **14**–**15**, and **18**–**19** after comparison with the databases and literature.

**Figure 8.** OPLS-DA analysis of 23 extracts. (**a**) OPLS-DA scores plot against *A. baumannii*. (**b**) The OPLS-DA loadings S-plot with the selected markers (12.88\_1268.6095n (**1**), 12.69\_1254.6282n (**2**), 11.11\_1270.6216n (**3**), 10.64\_900.5435n (**10**), and 11.08\_928.5742n (**11**)).

#### *2.4. Molecular Network Analysis of Outlier Strains*

To survey the global map of the metabolites of six outliers (*Streptomyces* sp. Y46, *Streptomyces* sp. H12, *Streptomyces* sp. H7, *Streptomyces* sp. Y2, *Streptomyces* sp. H37, and *Streptomyces* sp. M22), classical molecular networking acquired by the DDA method was performed. After the removal of nodes associated with the blank medium control, the molecular network consisted of 5033 nodes connected with 5247 edges. It was noted that the number of nodes in the network did not correspond exactly to the number of metabolites, as different adducts or charges of the same compounds could generate different nodes [58]. As shown in Figure 9, six molecular families that contained spectra matching the discriminatory metabolites in the six outliers were identified. The putative actinomycin-type compounds, the trioxacarcins group, and benzoquinoid ansamycin-type compounds only existed in samples of *Streptomyces* sp. H12 and *Streptomyces* sp. Y46, *Streptomyces* sp. M22, *Streptomyces* sp. H7 and *Streptomyces* sp. Y2, respectively. They were in agreemen<sup>t</sup> with the results of the PCA analysis. In addition, the molecular network could clarify the relationship of the discriminatory metabolites in the outlier strains, assisting in the identification of the discriminatory metabolites. The two discriminatory metabolites 16.71\_724.4749n (**4**) and 18.21\_708.4796n (**6**) clustered as adjacent nodes in the nigericin family (Figure 9), indicating that compound **6** could be assigned as putative grisorixin or epigrisorixin, the analog of compound **4**. The putative metabolites (**8a** and **9a**) showed higher structural similarity with 17-*O*-demethyl-geldanamycin (**7**) than other "hit" compounds (**8b**–**8c**; **9b**–**9d**), and thus, compounds **8** and **9** in *Streptomyces* sp. H7 and *Streptomyces* sp. Y2 were further putatively deduced as 4,5-dihydro-17-*O*-demethyl-geldanamycin and geldanamycin, respectively (Figure 9). Similarly, the compounds 10.64\_900.5435n (**10**) and 11.08\_928.5742n (**11**) from *Streptomyces* sp. H37 were further confirmed as two congeners with a mass difference of 28 Da (CO or C2H4 group). In sample *Streptomyces* sp. M22, two putative new compounds, 1030.3751*m/z* (**16**) and 1013.3486*m/z* (**17**), were adjacent to gutingimycin (**12** or **14**) with mass differences of 2 and 15 Da, respectively, suggesting that the former was the putative hydrogenated gutingimycin, and the latter was the putative gutingimycin with loss of NH group (Figure 10).

**Figure 9.** Molecular network of six outliers. The different colors of the nodes represented by different outliers, but the red node represented both *Streptomyces* sp. H12 and *Streptomyces* sp. Y46. Only clusters containing at least two nodes were shown.

**Figure 10.** Molecular network analysis of putative trioxacarcins family in *Streptomyces* sp. M22 extract.

Furthermore, a large number of nodes clustered with the discriminatory metabolites suggested the presence of additional analogs in these compound classes. For example, in addition to trioxacarcin A (**15**), B (**18**), and C (**19**), the inspection of their parent mass, mass difference, and the MS/MS data implied that putative methylated trioxacarcin B (*m/z* 926.495) and its hydrogenation product (*m/z* 928.514) were present (Figure 10). Another outlier, 15.40\_566.4171n (**13**), in the *Streptomyces* sp. M22 was clustered with two other compounds, whose molecular weights were almost twice as high, indicating that its dimers might exist in the extract (Figure S12). In the cluster containing the 10.64\_900.5435n (**10**) and 11.08\_928.5742n (**11**) of *Streptomyces* sp. H37, putative dehydrogenated, methylated, and demethylated analogs were detected (Figure S13). However, most of the putative new compounds discovered from the molecular network were presented as trace components in the extracts when checking their original UPLC-HRMS/MS data.

According to the results above, actinomycin-producing strains *Streptomyces* sp. Y46 and *Streptomyces* sp. H12, and nigericin-producing strains *Streptomyces* sp. H7 and *Streptomyces* sp. Y2 were excluded since their discriminatory metabolites were well known and frequently discovered from the actinomycetial strains, even though they were chemically unique. Samples of *Streptomyces* sp. H37 and *Streptomyces* sp. M22 were prioritized for scale-up and further isolation work. The strain *Streptomyces* sp. H37 was selected because the PCA analysis revealed that it contained the putative novel compounds. Further analysis using the OPLS-DA model identified these putative novel compounds contributing to significant inhibitory activity against drug-sensitive *A. baumannii*. Lastly, a variety of their potential new analogs were found through molecular network analysis. The purification and structural elucidation of new putative compounds in *Streptomyces* sp. H37 is still underway. The strain *Streptomyces* sp. M22 was selected because the putative novel trioxacarcin-type compounds were identified by the metabolomics-based dereplication approach (PCA, metabolic profile, molecular network, and database searching). Furthermore, the structures of some putative novel compounds were deduced from molecular networking. As a result, two novel trioxacarcin analogs, gutingimycin B (**16**) and trioxacarcin G (**20**) (Figure 11), along with gutingimycin (**12**) from the strain *Streptomyces* sp. M22, were isolated and structurally identified after large-scale fermentation.

**Figure 11.** Chemical structures of gutingimycin B (**16**) and trioxacarcin G (**20**).

#### *2.5. Structure Elucidation of Trioxacarcin Compounds from Strain Streptomyces sp. M22*

Gutingimycin B (**16**) was obtained as a yellow powder. It fluoresced green in solution upon irradiation with 365 nm light and showed the UV absorption maxima at 274 nm and 408 nm (Figure S14), typical for trioxacarcin-type compounds. Its molecular formula was assigned as C47H59N5O21 on the basis of HRESIMS peak at *m/z* 1030.3778 [M+H]+ (calcd for C47H60N5O21, 1030.3781) (Figure S15), 2 mass units more than gutingimycin. The 1H NMR spectra (Table 4 and Figure S17) showed characteristic resonances for an exchangeable OH proton (*δ*H 13.84 (s)), two aromatic protons (*δ*H 8.22 (s) and 7.48 (s)), two anomeric protons (*δ*H 5.64 (d, 3.6) and 5.51 (d, 4.2)), a pair of coupling oxygenated methines (*δ*H 5.22 (d, 4.2) and 5.10 (d, 4.2)), a pair of geminal coupling methylenes (*δ*H 5.02 (d, 15.6) and 4.33 (d,15.6)), three methoxyl singlets (*δ*H 3.95 (s), 3.66 (s) and 3.53 (s)), two methyls attached to olefinic or carbonyl carbon (*δ*H 2.62 (s) and 2.25 (s)), three methyl doublets (*δ*H 1.37 (d, 6.6), 1.26 (d, 6.6) and 1.26 (d, 6.6)), and one methyl singlet (*δ*H 1.18 (s)). In accordance with the molecular formula, the 13C (Table 4 and Figure S18) and DEPT NMR spectra (Figure S19) showed the 47 carbon resonances assigned to 1 keto carbonyl (*δ*C 207.6), 1 ester carbonyl (*δ*C 173.3), a characteristic of the guanine group (*δ*C 157.8, 153.8, 151.8, 140.6, and 108.1), 3 naphthalene carbons bearing to oxygen (*δ*C 162.9, 152.7, and 144.6), 6 acetal carbons or electron-rich *sp2* atoms (*δ*C 108.8, 108.3, 103.3, 100.9, 99.6, and 92.5), 15 oxygenated methines (*δ*C 56.1–84.4), 4 methylenes (*δ*C 46.2, 37.7, 37.1, and 33.8), and 6 methyls (*δ*C 15.7–27.3). The aforementioned NMR data of compound **16** showed very close similarities with those of gutingimycin [52]. The differences were an additional methine quartet at *δ*H 3.99 (q, 6.6) and a methyl doublet at *δ*H 1.37 (d, 6.6) in compound **16** instead of the methyl singlet at *δ*H 2.50 (s) in gutingimycin. In the 13C NMR spectra, the carbonyl at *δ*C 211.1 in gutingimycin was missing, and additional oxygenated methine at *δ*C 70.3 in compound **16** was detected. The missing ketone signal, together with the additional two mass units compared to gutingimycin, indicated that the ketone group in the sugar moiety of gutingimycin was reduced to an alcohol in compound **16**.


**Table 4.** 1H (600 MHz) and 13C (150 MHz) NMR data for compounds **16** and **20** in CDCl3.

The abovementioned structural features in compound **16** were confirmed by the 2D NMR spectra (Figures S20–S22). The crucial 1H-1H COSY correlation of H-6 (δH 3.99, q, 6.6)/H3-7 (*δ*H 1.37, d, 6.6) and the key HMBC correlation from H3-7 to C-6" (*δ*C 70.3), C-4 (72.1) (Figure 12) indicated that the hydroxyl group was linked to the C-6" position as the keto-reduced trioxacarcinose B.

**Figure 12.** The key 1H-1H COSY and HMBC correlations of **16** and **20**.

Trioxacarcin G (**20**) was obtained as a yellow powder. The UV absorption maxima at 232, 271, and 400 nm (Figure S23) and fluorescence under 365 nm light indicated that compound **20** is a trioxacarcin-type compound. The HRESIMS suggested that its molecular formula was determined to be C42H56O21 (*m/z* 914.3644 [M+NH4]+, calcd C42H60NO21, 914.3658) (Figure S24), 2 mass units more than trioxacarcin B. 1H and 13C NMR of compound **20** (Table 4, Figures S26–S28) were nearly identical to those of trioxacarcin B [59,60], except for an additional methine quartet at *δ*H/*δ*C 3.91 (q, 6.6)/70.7 and a methyl doublet at *δ*H/*δ*C 1.34 (d, 6.6)/18.2 in replacement of the carbonyl at *δ*C 210.9 and the methyl singlet at *δ*H /*δ*C 2.46 (s)/28.1. These were similar to the difference between compound **16** and gutingimycin, indicating that the ketone group in the L-trioxacarcinoses B of trioxacarcin B was also reduced to an alcohol in the keto-reduced trioxacarcinose B of compound **20**. The location of hydroxyl group was assigned at C-6 based on the 1H-1H COSY correlation of H-6 (*δ*H 3.91, q, 6.6)/H3-7 (*δ*H 1.34, d, 6.6) and the HMBC correlations from H3-7 to C-6" (*δ*C 70.7), C-4 (*δ*C 72.6) and from H-6 to C-3" (*δ*C 68.4), C-5 (*δ*C 66.3) (Figures 12 and S29–S31).

The NMR data of the keto-reduced trioxacarcinose B in **16** and **20** were closely similar with those of trioxacarcin C rather than epi-6"-trioxacarcin C, indicating that the absolute configuration at C-6"of **16** and **20** was determined as 6"S, the same as trioxacarcin C [61]. According to the literature [60], the X-ray structure of gutingimycin delivered the stereochemistry of the trioxacarcin skeleton, and the sugar moieties of the trioxacarcins A–B were identified previously as L-trioxacarcinoses A and B. In addition, compounds **16** and **20** had the same specific rotation sign as the known trioxacarcin, also isolated in the present study, gutingimycin (**12**) ([*α*]25D −60.0◦ (c 0.02, ACN)). Therefore, the absolute configuration of compounds **16** and **20** was established, as shown in Figure 11. In addition, the structures of known compounds were identified as gutingimycin (**12**) by comparison of their physio-chemical and spectroscopic data (Figures S32–S37) with those of the literature [52].
