Angucycline Glycosides from Mangrove-Derived Streptomyces diastaticus subsp. SCSIO GJ056

Nine new angucycline glycosides designated urdamycins N1–N9 (1–9), together with two known congener urdamycins A (10) and B (11), were obtained from a mangrove-derived Streptomyces diastaticus subsp. SCSIO GJ056. The structures of new compounds were elucidated on the basis of extensive spectroscopic data analysis. The absolute configurations of 6–9 were assigned by electronic circular dichroism calculation method. Urdamycins N7 (7) and N8 (8) represent the first naturally occurring (5R, 6R)-angucycline glycosides, which are diastereomers of urdamycins N6 (6) and N9 (9), respectively.

During the course of searching for novel anti-infective and antitumor agents from the marine environment, we found that the chemical profile of strain SCSIO GJ056 cultivated in AM2 medium revealed an array of secondary metabolites showing typical UV/VIS absorptions, which were similar to those of angucyclines/anthracyclines. Subsequent solvent extraction and isolation procedures led to the purification and structure elucidation of nine new angucycline glycosides, named urdamycins N1-N9 (1-9), together with two known urdamycins A (10) and B (11). Urdamycins N7 (7) and N8 (8) represent the first naturally occurring (5R, 6R)-angucycline glycosides. Herein, we report the fermentation, isolation, and structure elucidation of these compounds.

Results and Discussion
The strain SCSIO GJ056 was fermented (15 L) and the fermentation broth was extracted with butanone. The extract was subjected to repetitive silica gel column chromatography, followed by preparative HPLC purification to yield compounds 1-11 ( Figure 1). The known urdamycins A (10) Mar. Drugs 2018, 16 and B (11) were identified by comparisons of MS, 1 H, and 13 C NMR spectroscopic data with those previously reported [8]. Compound 1 was obtained as a yellowish powder. Its molecular formula was determined to be C 38 H 46 O 14 on the basis of HRESIMS peak at m/z 725.2834 [M − H] − , indicating 16 degrees of unsaturation. The 13 C and DEPT NMR spectra of 1 displayed 38 carbon resonances, including five methyls, six methylenes, 14 methines, and 13 nonprotonated carbons. The 1 H NMR spectrum showed one chelated hydroxy group signal at δ H 12.55 (1H, br s, 8-OH), a pair of ortho-coupled aromatic proton signals at δ H 7.81 (d, 7.8 Hz, H-10) and 7.59 (d,7.8 Hz,, and a singlet aromatic proton signal at δ H 7.65 (s, H-6). The HMBC correlations ( Figure 2) from H-6 to C-4a, C-5, C-7, and C-12a, from H-11 to C-7a, C-9, and C-12, and from H-10 to C-8, C-9, and C-11a confirmed the existence of the anthraquinone skeleton (rings B, C, and D). Further HMBC correlations of H 2 -2/C-1, C-12b, C-4; H 2 -4/C-2, C-4a, C-12b; and H 3 -13/C-2, C-3, C-4 allowed the assignment of the angular ring (ring A) with a methyl group (CH 3 -13) substitution at C-3. A methoxy group (OCH 3 -14) attached at C-5 in ring B was deduced by the HMBC correlation of H 3 -14/C-5. A hydroxy group linked at C-3 in ring A was inferred based on the 13 C NMR chemical shift at δ C 71.8. The absolute configuration of C-3 was tentatively deduced to be R, which was identical with that of urdamycinone B and N05WA963D in light of the similar 13 C NMR resonances of C-3 and CH 3 -13, as well as the similar biosynthetic pathway [8,9].
Compound 2, isolated as a dark red powder, has the molecular formula of C 38 H 44 O 13 on the basis of HRESIMS peak at m/z 707.2708 [M − H] − , showing 17 degrees of unsaturation and an 18 amu less than that of compound 1. An obvious red shift on the UV-VIS spectrum of 2 relative to that of 1 indicated an additional conjugated system in 2. The 13 C and DEPT NMR data of 2 displayed 38 carbon signals attributable to five methyls, four methylenes, 16 methines, and 13 nonprotonated carbons. The 1 H and HSQC NMR spectra suggested three singlet olefnic proton signals at δ H 7.58 (H-4), δ H 7.54 (H-6), and δ H 6.99 (H-2), and a pair of ortho-coupled aromatic proton signals at δ H 7.82 (d, 7.6 Hz, H-10) and 7.68 (d, 7.6 Hz, H-11). Comparing the 1 H and 13 C NMR spectroscopic data to those of 1 revealed that 2 possessed a similar core structure with that of 1. The difference between 2 and 1 was the aromatization of ring A in 2, which supported by the HMBC correlations from CH 3 -13 to C-2, C-3, and C-4, from H-2 to C-1, C-4, and C-12b, and from H-4 to C-2, C-4a, and C-12b. Compound 2 possessed the same trisaccharide moiety with 1 according to similar 1 H and 13 C NMR signals in aliphatic area. The structure of 2 was elucidated as shown in Figure 1 by detailed analysis of 2D NMR spectra data.
Compound 3, a dark green powder, was isolated as minor component from the extract. Its molecular formula of C 26 H 24 O 8 was determined by the HRESIMS peak at m/z 463.1409 [M − H] − , indicating 15 degrees of unsaturation. Comprehensive analysis of its 1 H and 13 C NMR spectroscopic data revealed that 3 had the same aglycone with that of 2. However, a set of 1 H and 13 C resonances ascribed to β-olivose-(1→4)-α-rhodinosyl moiety disappeared, indicating the absence of two sugar units in 3. This is consistent with the HRESIMS data, which showing a C 12 H 20 O 5 fragment loss relative to 2. Therefore, the structure of 3 was established and named urdamycin N3.  Compound 4 was obtained as a dark green powder. Its molecular formula was determined to be C 37 H 42 O 13 by the HRESIMS peak at m/z 693.2554 [M − H] − . The 1 H and 13 C NMR data of 4 were closely similar to those of 2, except that the methoxy signals at δ H 4.14, δ C 56.6 in 2 were absent. The 13 C NMR signal of C-5 shifted from δ C 160.3 in 2 to δ C 163.6 in 4, indicating the OMe-5 in 2 was replaced by OH-5 in 4. Compound 4 was named urdamycin N4.
Compound 5 was obtained as a red powder. The molecular formula of C 37 H 42 O 12 , as determined by HRESIMS, which was one oxygen atom less than that of 4. The 1 H and 13 C NMR spectroscopic data were similar with those of 4, except that two pairs of ortho-coupled aromatic signals were observed. Additionally, the 13 C NMR signal at δ C 163.6 for the oxygen-bearing aromatic C-5 in 4 was replaced by an aromatic methine signal at δ C 135.4. Thus, the structure of 5 was determined as 5-demethoxy-urdamycin N2, designated as urdamycin N5.
The molecular formulae of compounds 6 and 7 were determined both to be C 27 H 28 O 9 by HRESIMS, indicating 14 degrees of unsaturation. The 1 H and 13 C NMR spectroscopic data of 6 were similar with those of 3, except that two aromatic carbon signals at δ C 160.5 (C-5) and 100.0 (C-6) in 3 were replaced by two oxygen-bearing methine carbon signals at δ C 78.1 (C-5) and 70.2 (C-6). Furthermore, two methoxys were attached at C-5 and C-6 based on the HMBC correlations of H 3 -14/C-5 and H 3 -15/C-6, respectively. Small coupling constants (2.8 Hz) between H-5 and H-6 revealed a trans configuration of H-5 and H-6, indicating an (5R, 6R) or (5S, 6S) configuration of 6.
To determine the absolute configurations of 6, comparisons of the experimental and ECD spectra using a time-dependent density functional theory (TDDFT) were employed. Comparison of the experimental and calculated CD spectra ( Figure 4) established the absolute configuration as (5S, 6S) for 6, which were the same as those of PMO70747, PD116740, and TAN-1085 [12][13][14][15][16][17]. Compound 7 possessed the same planar structure with that of 6, as deduced by the COSY and HMBC spectra ( Figure 2). However, the experimental and calculated CD spectra of 7 showed cotton effects totally opposite to those of 6, respectively, inferring the (5R, 6R) configuration for 7 ( Figure 4). Compounds 6 and 7 were named urdamycins N6 and N7, respectively. Compound 4 was obtained as a dark green powder. Its molecular formula was determined to be C37H42O13 by the HRESIMS peak at m/z 693.2554 [M − H] − . The 1 H and 13 C NMR data of 4 were closely similar to those of 2, except that the methoxy signals at δH 4.14, δC 56.6 in 2 were absent. The 13 C NMR signal of C-5 shifted from δC 160.3 in 2 to δC 163.6 in 4, indicating the OMe-5 in 2 was replaced by OH-5 in 4. Compound 4 was named urdamycin N4.
Compound 5 was obtained as a red powder. The molecular formula of C37H42O12, as determined by HRESIMS, which was one oxygen atom less than that of 4. The 1 H and 13 C NMR spectroscopic data were similar with those of 4, except that two pairs of ortho-coupled aromatic signals were observed. Additionally, the 13 C NMR signal at δC 163.6 for the oxygen-bearing aromatic C-5 in 4 was replaced by an aromatic methine signal at δC 135.5. Thus, the structure of 5 was determined as 5-demethoxy-urdamycin N2, designated as urdamycin N5.

Bacterial Materials
Strain SCSIO GJ056 was isolated from a mangrove-derived sediment sample collected in Yalong bay, China. It was identified as Streptomyces diastaticus subsp. on the basis of morphological characteristics and 16S rRNA sequence analysis by comparisons with other sequences in the GenBank database. The DNA sequence has been deposited in GenBank (accession no. MH368281). The strain was preserved at the RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences and also at the China General Microbiological Culture Collection Center (CGMCC, Beijing, China), CGMCC No. 13648.