Abyssomicin Monomers and Dimers from the Marine-Derived

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Cite This: J. Nat. Prod. 2018, 81, 1892−1898

Abyssomicin Monomers and Dimers from the Marine-Derived Streptomyces koyangensis SCSIO 5802 Hongbo Huang,† Yongxiang Song,† Xin Li,‡ Xin Wang,‡ Chunyao Ling,† Xiangjing Qin,† Zhenbin Zhou,† Qinglian Li,† Xiaoyi Wei,§ and Jianhua Ju*,†

J. Nat. Prod. 2018.81:1892-1898. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 08/24/18. For personal use only.

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CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China ‡ Qingdao National Laboratory for Marine Science and Technology, School of Medicine and Pharmacy, Ocean University of China, Qingdao 260000, China § Key Laboratory of Plant Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China S Supporting Information *

ABSTRACT: Three new abyssomicin monomers designated neoabyssomicins D (1), E (2), and A2 (3) and the two dimeric neoabyssomicins F (4) and G (5) were discovered from the marine-derived Streptomyces koyangensis SCSIO 5802, and their structures rigorously elucidated. Neoabyssomicin D (1) possesses an unprecedented 8/5/5/7 ring skeleton, the biosynthesis of which (as well as 2) is proposed herein. Additionally, dimeric agents 4 and 5 were found to be active against methicillin-resistant Staphylococcus aureus and vesicular stomatitis virus, respectively. Encouraged by these findings, our group has been exploring marine-derived actinomycetes as sources of structurally unique antibiotics.15,16 Recently, we reported neoabyssomicins from the marine strain Streptomyces koyangensis SCSIO 5802 isolated from the northern South China Sea.11 In reanalyzing HPLCDAD data sets for this metabolomic profile we noted several unidentified but significant signals. Large-scale 60 L fermentations of SCSIO 5802, subsequent extraction, and HPLC-based fractionations enabled the isolation of two unusual abyssomicin congeners with undescribed frameworks termed herein neoabyssomicins D (1) and E (2). Additionally, two new abyssomicin dimers, named neoabyssomicins F (4) and G (5), and one new abyssomicin monomer named neoabyssomicin A2 (3) were found. Neoabyssomicin D (1) contains a novel 8/5/5/7 ring system, whereas neoabyssomicin E (2) possesses a new furan-3(2H)-one substitution. The identifications of 4 and 5 represent only the second instance of abyssomicin dimer discovery. Herein, we report the purification, structure elucidation, and anti-MRSA and antiviral activities of new compounds 1−5.

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acterial and viral diseases continue to gain in their lethality, posing an ever-growing threat to humanity on a global scale.1 In particular, the emergence of drug-resistant disease-causing mutant microbes, so-called “superbugs”, represents a tremendous challenge to current antibiotic drugs and identification strategies leading to new antimicrobials.2 In responding to this challenge, efforts to discover potential antimicrobials with new structures/mechanisms have dramatically intensified. One consequence of this has been the discovery of the abyssomicins.3−11 This family of antimicrobials is generally composed of C19 spirotetronates often containing a four- or five-membered ring system within their architectures. The first family member reported, abyssomicin C, was obtained during the course of targeted screenings for inhibitors of p-aminobenzoate formation; abyssomicin C demonstrates activity against the Gram-positive bacteria methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) strains.3 Since 2004, more than 30 natural abyssomicins have been reported, which can be classified into either type I or type II subfamilies.12 The vast majority of abyssomicin analogues exist as monomers. To date, abyssomicin J is the only dimer.8 Interestingly, abyssomicin J and abyssomicin C and its atropisomer atrop-abyssomicin C display potent in vitro antimycobacterial activities.5,13 In 2007, Nicolaou completed total syntheses of both abyssomicin C and atrop-abyssomicin C.14 Notably, the three aforementioned abyssomicins displaying antibacterial activities, as well as anti-HIV abyssomicin 2, were identified from marine Verrucosispora/Streptomyces strains.3,8,9 © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The marine-derived S. koyangensis SCSIO 5802 was fermented on a 60 L scale. The culture was centrifuged to generate supernatant and mycelium. The supernatant was extracted Received: June 4, 2018 Published: August 2, 2018 1892

DOI: 10.1021/acs.jnatprod.8b00448 J. Nat. Prod. 2018, 81, 1892−1898

Journal of Natural Products

Article

Chart 1

Scheme 1. Proposed Biosynthetic Pathway of Neoabyssomicins D (1) and E (2)

spectrum revealed two motifs of CH3−CH−CH2−CH2− and CH3−CH−CH2−. Further HMBC associations from the protons of Me-17 and of Me-18 and Me-19 suggested the presence of typical C-7/C-6(C-17)/C-5/C-4/C-3 and C-11/ C-12(C-18)/C-13(C-19)/C-14 moieties within the abyssomicin backbone (Figure 1). However, our attempt to elucidate the intact structure of 1 was obstructed by miscellaneous HMBC correlations originating from the complicated caged skeleton. Fortunately, single crystals of 1 were obtained after very slow crystallization from MeOH. X-ray diffraction using Cu Kα radiation confirmed the framework of 1 as shown (Figure 2A).17 The novel conjugated seven- and fivemembered rings sharing the C-8/C-16/C-15 fragment are supported by noted HMBC correlations involving H-9/C-11, C-16, H-10/C-14, C-16, H-11/C-9, C-16, and H2-14/C-16. The relative configuration of 1 was elucidated on the basis of NOESY data analyses. The NOEs of H-6/H-11/H3-18/H3-19 placed H-6, H-11, Me-18, and Me-19 on the same side of 1. In addition, NOEs of H-10/H-13 and H-10/H-14a were

three times with butanone, affording, after solvent removal in vacuo, a residue that was subjected to silica gel and Sephadex LH-20 column chromatography (CC), followed by semipreparative HPLC to yield the new neoabyssomicins D, E, A2, F, and G (1−5), together with previously reported abyssomicins 2 and 4 (9 and 6, Scheme 1). The known compounds were identified on the basis of spectroscopic data comparisons to previously reported data sets.8 Neoabyssomicin D (1), as colorless crystals, displayed a molecular formula of C19H22O6 based on [M + Na]+ m/z 369.1308 in the (+)-HR-ESIMS spectrum; this inferred the presence of nine double-bond equivalents (DBEs). Inspection of the 1H and 13C NMR spectroscopic data (Table 1) revealed the presence of one ketone carbonyl at δC 213.0 (C-7), one α,β-unsaturated ester carbonyl linked with an OH moiety at δC 174.6 (C-1), 97.3 (C-2), and 179.4 (C-3), and one Z-olefin (δC 136.4, δH 5.92, J = 6.3 Hz, CH-9; δC 130.1, δH 5.78, J = 6.3 Hz, CH-10) corresponding to four DBEs. The remaining five DBEs implied the pentacyclic nature of 1. The COSY 1893



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Table 1. 1H (700 MHz) and 13C (175 MHz) NMR Data for Neoabyssomicins D (1), E (2), and A2 (3) neoabyssomicin D (1)a position

δC, type

1 2 3 4

174.6, C 97.3, C 179.4, C 30.8, CH2

5 6 7 8

−c 41.2, CH 213.0, C 40.0, C

9 10 11 12 13 14

136.4, CH 130.1, CH 82.5, CH 83.2, C 24.7, CH 34.3, CH2

15 16 17 18 19 1′ 2′ 3′ 4′ 5′ 6′

74.5, 95.3, 17.4, 22.0, 17.4,

C C CH3 CH3 CH3

δH, mult (J in Hz)

2.80, dd (16.4, 8.4); 2.46, dd (16.4, 10.9) 1.64, m 3.34, m

5.92, d (6.3) 5.78, d (6.3) 4.68, s 2.28, m 1.95, dd (12.0, 5.2); 1.50, t (12.0)

1.11, d (6.7) 1.45, s 1.02, d (7.2)

neoabyssomicin E (2)b δC, type 170.1, C 102.4, C 197.5, C 38.3, CH2 31.3, CH2 46.6, CH 213.2, C 40.8, CH2 35.4, 50.8, 73.5, 91.7, 29.8, 38.3,

CH CH CH C CH CH2

78.1, C 185.4, C 18.9, CH3 19.7, CH3 16.4, CH3 89.9, C 200.9, C 134.7, C 175.4, C 20.3, CH3 13.4, CH3

δH, mult (J in Hz)

2.96, m; 2.52, dd (12.5, 6.0) 1.92, m 2.70, m 3.00, dd (19.9, 3.7); 2.68, dd (19.9, 3.7) 2.72, dd (3.7, 3.7) 2.38, d (2.0) 3.97, d (2.0)

neoabyssomicin A2 (3)a δC, type 166.0, C 57.2, CH 206.5, C 42.7, CH2 28.6, CH2 46.8, CH 214.4, C 41.5, CH2

2.59, overlapped 2.60, overlapped; 1.20, d (9.6)

37.4, 46.0, 74.5, 85.9, 28.3, 38.0,

CH CH CH C CH CH2

1.03, d (6.9) 1.62, s 0.96, d (6.8)

79.5, C 171.4, C 18.1, CH3 19.7, CH3 16.5, CH3

δH, mult (J in Hz) 4.11, d (11.1) 2.52, overlapped; 2.37, ddd (13.4, 13.4, 3.5) 2.53, overlapped; 1.82, m 2.63, m 2.57, m; 2.08, m 2.12, overlapped 2.10, overlapped 3.62, dd (5.2, 3.2) 2.49, m 2.43, dd (12.6, 10.8); 1.50, dd (12.6, 5.0)

1.01, d (6.8) 1.30, s 0.98, d (6.9)

1.29, s 2.20, s

a

Measured in DMSO-d6. bMeasured in CD3OD. cNot observed.

Figure 1. Selected COSY (bold) and HMBC (arrows) correlations of neoabyssomicins D, E, and A2 (1−3).

observed, suggesting that H-10, H-13, and H-14a are situated opposite H-6, H-11, Me-17, and Me-18 (Figure 2A). Singlecrystal X-ray diffraction analysis confirmed the structure of 1 and revealed the absolute configurations of all stereogenic carbons to be 6S, 8S, 11S, 12S, 13S, 15S, and 16R on the basis of the refinement of the Flack parameter [x = −0.04(7)].18 Neoabyssomicin E (2) was isolated as a white powder. The (+)-HR-ESIMS spectrum was characterized by m/z 497.1776 [M + Na]+, establishing the molecular formula C25H30O9 for 2. Detailed comparisons revealed that the 1H and 13C NMR spectroscopic data (Table 1) were similar to those of abyssomicin 4,8 except for the presence of additional signals ascribed to two methyl groups (δC 20.3, δH 1.29, Me-5′; δC 13.4, δH 2.20, Me-6′) and four nonprotonated carbons (δC 89.9, C-1′; 200.9, C-2′; 134.7, C-3′; 175.4, C-4′). The 1H NMR chemical shift for Me-6′ suggested connectivity with an sp2-hybridized carbon. The HMBC correlations from Me-6′ protons to C-2′, C-3′, and C-4′ and from the Me-5′ to C-1′

Figure 2. Key NOESY correlations (blue arrows, A) and ORTEP structure (B) of neoabyssomicin D (1).

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and C-2′ established the presence of a 5-hydroxy-2,4dimethylfuran-3(2H)-one moiety. Moreover, signals attributable to one O-bearing methine at δC 64.1 and δH 4.52 (−OCH-9) in abyssomicin 4 were replaced by resonances at δC 35.4 and δH 2.72 (CH-9) in 2. The HMBC cross-peaks from H2-8, H-9, and H-10 to C-1′ and from Me-5′ to C-9 suggested that the 5-hydroxy-2,4-dimethylfuran-3(2H)-one unit is tethered to the abyssomicin framework via a C-9/C1′ linkage. In the NOESY spectrum, the correlations of H-10/H-13 and H-11/Me-18/Me-19 were observed, which indicated that H-10 and H-13 were placed in axial positions, whereas H-11, Me-18, and Me-19 were equatorial in the boat form of the substituted cyclohexane in 2 (Figure 3A). Given the likelihood of a shared

Figure 4. Key NOESY correlations (blue arrows, A) and ORTEP structure (B) of neoabyssomicin A2 (3).

2R, 6S, 9R, 10S, 11S, 12S, 13S, 15S on the basis of the Flack parameter [x = −0.01(6)].18 (+)-HR-ESIMS data for neoabyssomicin F (4) indicated a signal consistent with the Na+ salt of 4 at m/z 749.2601 [M + Na]+, corresponding to the molecular formula C38H46O12S. However, the 1H and 13C NMR spectra of 4 (Table 2), with the aid of HSQC data, disclosed the presence of only 19 carbons, including four sp2 carbons (δC 209.2 (C-7), 175.7 (C3), 173.8 (C-1), 100.2 (C-2)), three nonprotonated aliphatic carbons (δC 86.8 (C-15), 85.5 (C-16), 78.6 (C-12)), and six methine, three methylene, and three methyl groups. This implied that 4 was a symmetric structure. Further interpretation and analyses of COSY and HMBC spectra revealed a structural unit closely resembling that of abyssomicin 5,8 except that the O-bearing methine at C-9 (δH 4.77, δC 70.3) in abyssomicin 5 was replaced by a thiomethine (δH 3.89, δC 41.6) in 4. The important HMBC correlation of H-9/C-9′ indicated two monomers were connected via a C-9−S−C-9′ thioether bond to form dimeric 4, in a fashion highly similar to that in abyssomicin J.9 X-ray crystallographic analyses support the putative structure for 4 and revealed the absolute configurations for all stereogenic carbons as shown in Figure 5. The molecular formula of neoabyssomicin G (5) was determined to be C38H46O12S on the basis of (+)-HR-ESIMS data, which was the same as that of 4. Comparison of the 1H and 13C NMR spectroscopic data (Table 2) revealed that some signals for 5 were almost identical to those observed in the spectra of 4. This suggested that 5 and 4 share a monomer unit. Detailed analyses of the remaining 1H and 13C NMR signals showed the other monomer unit has similarity to the known abyssomicin 4,8 except that the O-bearing methine signals at δH 4.52 and δC 64.1 (CH-9) in abyssomicin 4 were replaced by thiomethine signals at δH 3.74 and δC 35.0 (CH9′) in 5. Meanwhile, the 13C NMR chemical resonances of the C-8′ and C-10′ in 5 shifted from δC 48.1 and 56.7 (C-8 and C10) in abyssomicin 4 to δC 45.3 and 52.7. Therefore, the structure of 5 was established as shown and confirmed by 2D NMR correlations. Neoabyssomicins D (1) and E (2) represent novel abyssomicin skeletons, which contain a unique 8/5/5/7 tetracyclic core structure and a new furan-3(2H)-one substitution, respectively. A hypothetical biosynthetic pathway for 1 and 2 is proposed in Scheme 1. As indicated, compounds 1 and 2 may be derived from abyssomicin 4 (6) and abyssomicin 2 (9), respectively. An internal Michael addition reaction at C-16 of abyssomicin 4 (6) with the enolate formed by deprotonation at C-8 would yield abyssomicin 5 (7). The

Figure 3. (A) Key NOESY correlations (blue arrows) of neoabyssomicin E (2). (B) Comparison of the experimental ECD spectrum of 2 with the calculated ECD spectra for (6S,9R,10S,11S,12S,13S,15S,1′S)-2 [(1′S)-2] and (6S,9R,10S,11S,12S,13S,15S,1′R)-2 [(1′R)-2] at the M06/TZVP level with the PCM model for MeOH [σ = 0.34, shift = +18 nm, scaling factor = 0.16 for (1′S)-2; σ = 0.34, shift = 0 nm, scaling factor = 0.21 for (1′R)-2].

biogenetic pathway for 2 and abyssomicin 4, the absolute configurations in 2 (except for C-9 and C-1′) were inferred to be 6S, 10S, 11S, 12S, 13S, and 15S. Notably, NOE cross-peaks for H-10/Me-5′ and H-11/Me-5′ were observed, indicating 9R, 1′S or 9R, 1′R configurations for 2. The measured electronic circular dichroism (ECD) spectrum of 2 was in better agreement with the calculated (theoretical) ECD spectrum of 1′S-2 than that of 1′R-2 (Figure 3B),19 supporting the assignment of the 1′S configuration for 2. On the basis of a (+)-HR-ESIMS signal at m/z 387.1422 [M + Na]+, the molecular formula of 3 was determined to be C19H24O7, one O atom less than that of neoabyssomicin A.11 The 1H and 13C NMR spectroscopic data (Table 1) revealed the presence of two ketone and two ester carbonyls, as well as two nonprotonated carbons, six methines, four methylenes, and three methyl groups. Analyses of the COSY and HMBC spectra established the planar structure of 3 (Figure 1), which was similar to that of neoabyssomicin A, except for the enol and ester groups at C-3 and C-7; in 3 these moieties are replaced by two ketone groups, respectively. The NOESY spectrum of 3 gave a correlation of H-2/H-10, demonstrating that H-2 and H-10 were orientated on the same side of 3. On the contrary, the sequential NOE correlations of H-9/H-11/ Me-18/Me-19 placed these groups on the opposite side relative to H-2 and H-10 (Figure 4A). Using X-ray crystallography, the structure of 3 was confirmed (Figure 4B), and the absolute configurations were determined to be 1895



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Table 2. 1H (700 MHz) and 13C (175 MHz) NMR Data for Neoabyssomicin F (4) in CDCl3 and Neoabyssomicin G (5) in CD3OD neoabyssomicin F (4) position

δC, type

1 2 3 4

173.8, C 100.2, C 175.7, C 29.0, CH2

5 6 7 8

23.7, CH2 47.4, CH 209.2, C 69.4, CH

9 10 11 12 13 14

41.6, 51.9, 75.2, 78.6, 27.4, 33.8,

CH CH CH C CH CH2

15 16 17 18 19

86.8, 85.5, 12.3, 20.6, 15.7,

C C CH3 CH3 CH3

δH, mult (J in Hz)

neoabyssomicin G (5) δC, type

position

δH, mult (J in Hz)

1/1′ 2/2′ 3/3′ 4/4′

173.5, C 99.9, C 176.1, C 28.9, CH2

5/5′ 6/6′ 7/7′ 8/8′

23.6, CH2 47.4, CH 209.1, C 69.7, CH

3.05, m; 1.84, m 2.60, m

43.3, 53.0, 75.2, 78.5, 27.3, 33.4,

CH CH CH C CH CH2

3.83, dd (10.2, 3.6) 2.50, d (3.6) 4.01, s

2.49, overlapped 2.49, overlapped; 1.17, dd (11.7, 7.7)

9/9′ 10/10′ 11/11′ 12/12′ 13/13′ 14/14′

1.05, d (6.8) 1.25, s 0.92, d (5.8)

15/15′ 16/16′ 17/17′ 18/18′ 19/19′

86.2, 85.7, 11.4, 19.4, 14.7,

C C CH3 CH3 CH3

2.86, ddd (16.1, 5.3, 3.4); 2.40, ddd (16.6, 12.2, 5.6) 3.08, m; 1.86, m 2.47, m 3.05, d (10.6) 3.89, dd (10.6, 3.4) 2.72, d (3.4) 4.06, s

2.82, m; 2.38, m

3.29, d (10.2)

2.39, overlapped 2.42, overlapped; 1.10, dd (8.3, 2.7)

δC, type

δH, mult (J in Hz)

169.0, C 100.9, C 196.4, C 36.5, CH2

3.20, ddd (12.6, 8.8, 6.6); 2.40, overlapped 2.03, m; 1.84, m 2.54, m

28.5, CH2 44.8, CH 208.7, C 45.3, CH2 35.0, 52.7, 71.7, 90.4, 28.6, 37.6,

2.94 dd (19.6, 6.5); 2.68, overlapped 3.74, dt (6.5, 2.1) 2.68, overlapped 3.92, d (2.8)

CH CH CH C CH CH2

2.58, d (3.0) 2.69, overlapped 1.20, dd (10.0, 3.0)

77.6, C 183.9, C 17.7, CH3 18.2, CH3 15.0, CH3

1.06, d (6.8) 1.20, s 0.91, d (6.2)

1.03, d (6.8) 1.62, s 0.98, d (7.2)

Table 3. Antibacterial Activities of Neoabyssomicins F (4) and G (5) (MICs, μg/mL) b

MRSA-shhs A1 MRSA-699b MRSA-1862b Staphylococcus aureus ATCC 29213 Enterococcus faecalis ATCC 29212

4

5

AMPa

VANa

16 16 16 32 32

16 16 16 32 32

64 4 0.125 0.25 16

0.5 0.125 0.125 0.25 0.25

a

Ampicillin (AMP) and vancomycin (VAN) were used as positive controls. bClinical isolates.

Figure 5. ORTEP structure of neoabyssomicin F (4).

vesicular stomatitis virus with IC50 values of 24 and 19 μM, respectively. Compound 1 exhibited mild antiviral activity against herpes simplex virus, presenting a low percentage (31 ± 10%) of viral replication at a concentration of 10 μM.

hydroxy at C-9 can be eliminated in tandem with the cleavage of the C-10/C-11 bond, leading to the generation of the intermediate (8) with a C-9/C-10 double bond. An aldol reaction between a C-8 enolate and C-11 yields a new sevenmembered ring to give 1. The newly installed furan-3(2H)-one moiety in 2 would be derived from the C6 precursor, 2-methyl3-oxopentanoic acid, which is formed by two propionyl-CoA units. A sequence of Michael addition between the C6 precursor and abyssomicin 2 (9), hydroxylation at C-1′ by a P450 monooxygenase and lactonization, followed by keto− enol tautomerization finally affords 2. Compounds 1−5 were evaluated for antibacterial and antiviral activities. Both dimers 4 and 5 showed growthinhibiting activities against three MRSA strains with the same MIC value of 16 μg/mL in agreement with a previously reported thioether-based prodrug approach (Table 3).9 Compounds 4 and 5 also exerted antiviral activities against

General Experimental Procedures. Optical rotations were obtained with an MCP 500 polarimeter (Anton Paar). UV spectra were recorded with a U-2600 spectrometer (Shimadzu). ECD data were recorded with a Chirascan circular dichroism spectrometer (Applied Photophysics). NMR spectra were recorded with an AVANCE-700 spectrometer (Bruker) at 700 MHz for 1H nuclei and 175 MHz for 13C nuclei. Chemical shifts (δ) are given with reference to signals of tetramethylsilane. Mass spectra were obtained using a Maxis quadrupole-time-of-flight mass spectrometer (Bruker). Semipreparative HPLC was operated with a Primaide system (Hitachi) and an ODS-A column (10 × 250 mm, 5 μm, YMC). Column chromatography was performed using silica gel (100−200 mesh, Qingdao Marine Chemical Corporation, China) and Sephadex LH-20 (Amersham Pharmacia). Single-crystal data were collected on an XtaLAB PRO MM007HF diffractometer (Rigaku) using Cu Kα radiation. All chemicals and solvents were of analytical or chromatographic grade. Stain Material and Fermentation. Streptomyces koyangensis SCSIO 5802 was isolated from a sediment sample collected from the

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final indices were R1 = 0.036, wR2 = 0.096 [I > 2σ(I)]. CCDC number: 1837968. Crystal data of 3: orthorhombic, C19H24O7, Mr = 364.38, space group P212121, a = 6.6414(1) Å, b = 14.9068(1) Å, c = 17.8924(2) Å, α = β = γ = 90°, V = 1771.38(4) Å3, Z = 4, Dcalcd = 1.366 g/cm3, μ = 0.870 mm−1, and F(000) = 776.0. Crystal size: 0.20 × 0.10 × 0.10 mm3. Independent reflections: 3472 [Rint = 0.027]. The final indices were R1 = 0.031, wR2 = 0.086 [I > 2σ(I)]. CCDC number: 1837961. Crystal data of 4: hexagonal, C38H46O12S1·2(H2O), Mr = 762.84, space group P62, a = 13.4030(8) Å, b = 13.4030(8) Å, c = 17.8604(10) Å, α = β = 90°, γ = 120°, V = 2778.6(4) Å3, Z = 3, Dcalcd = 1.368 g/cm3, μ = 1.368 mm−1, and F(000) = 1218.0. Crystal size: 0.10 × 0.05 × 0.03 mm3. Independent reflections: 3407 [Rint = 0.023]. The final indices were R1 = 0.045, wR2 = 0.123 [I > 2σ(I)]. CCDC number: 1837970. Antibacterial Assays. A preliminary screening of antibacterial activities of compounds 1−5 was conducted using a disk diffusion method. A panel of bacterial strains including Staphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus aureus shhs-A1, methicillin-resistant Staphylococcus aureus 1682, methicillin-resistant Staphylococcus aureus 669, Enterococcus faecalis ATCC 29212, Acinetobacter baumannii ATCC 19606, Klebsiella pneumoniae ATCC 13883, Vibrio alginolyticus XSBZ14, and Escherichia coli ATCC 25922 were used. The MIC values of compounds 4 and 5 were determined against MRSA-shhs A1, MRSA-1862, MRSA-699, S. aureus ATCC 29213, and E. faecalis ATCC 29212 by 2-fold serial dilutions technique using Mueller-Hinton broth in a 96-well plate according to the standard methods described by the Clinical and Laboratory Standards Institute (CLSI). The results are listed in Table 3.

South China Sea at a depth of 3536 m. The identification of SCSIO 5802 has been previously described by our group.11 Strain SCSIO 5802 was fermented on a 60 L scale, and then the fermentation broth was extracted with solvent using the previously reported method.11 Isolation and Characterization of New Compounds. The organic extract (48 g) was subjected to silica gel CC using gradient elution with a CHCl3/MeOH mixture (100/0, 98/2, 96/4, 94/6, 92/ 8, 90/10, 80/20, v/v) to give seven fractions (A1−A7), respectively. Fraction A2 was subjected to silica gel CC with petroleum ether/ EtOAc mixtures (100/0, 90/10, 80/20, 70/30, 50/50, 30/70, 0/100, v/v) to afford fractions B1−B7. Fractions B5−B7 were combined and further purified by semipreparative HPLC eluted with a linear gradient from 20% CH3CN to 100% CH3CN (CH3CN/H2O system) over the course of 0−30 min at a flow rate of 2.5 mL/min to give compounds 4 (6 mg) and 5 (18 mg). Fraction B4 was isolated by semipreparative HPLC using the same elution program to obtain compound 1 (7 mg). Fraction A3 was subjected to silica gel CC eluted with petroleum ether/EtOAc mixtures (90/10, 80/20, 70/30, 60/40, 50/50, 40/60, 30/70, 0/100, v/v) to afford fractions C1−C8. Fractions C3−C5 were isolated by semipreparative HPLC eluted with a linear gradient from 20% CH3CN to 80% CH3CN (CH3CN/H2O system) over the course of 0−35 min at a flow rate of 2.5 mL/min to afford compound 3 (5 mg). Fractions A4 was isolated by Sephadex LH-20 CC eluted with a CHCl3/MeOH (1:1, v/v) mixture to yield fractions D1−D10. Compound 2 (6 mg) was obtained from fractions D3−D5 by semipreparative HPLC purification eluted with a linear gradient from 10% CH3CN to 60% CH3CN (CH3CN/H2O system) during the course of 0−30 min at a flow rate of 2.5 mL/min. Neoabyssomicin D (1): colorless crystals (MeOH), mp 228−229 °C; [α]25D +123 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 202 (3.93), 259 (3.70) nm; 1H and 13C NMR spectroscopic data, Table 1; HRESIMS m/z 369.1308 [M + Na]+ (calcd for C19H22NaO6, 369.1308). Neoabyssomicin E (2): white powder; [α]25D +11 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 207 (3.72), 256 (3.40) nm; 1H and 13C NMR spectroscopic data, Table 1; HRESIMS m/z 497.1776 [M + Na]+ (calcd for C25H30NaO9, 497.1782). Neoabyssomicin A2 (3): colorless crystals (MeOH), mp 248−249 °C; [α]25D +47 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 203 (4.20), 269 (3.06) nm; 1H and 13C NMR spectroscopic data, Table 2; HRESIMS m/z 387.1422 [M + Na]+ (calcd for C19H24NaO7, 387.1414). Neoabyssomicin F (4): colorless crystals (MeOH), mp 210−211 °C; [α]25D −60 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 201 (3.75), 263 (4.10) nm; 1H and 13C NMR spectroscopic data, Table 2; HRESIMS m/z 749.2601 [M + Na]+ (calcd for C38H46NaO12S, 749.2602). Neoabyssomicin G (5): white powder; [α]25D −94 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 202 (3.78), 263 (4.19) nm; 1H and 13C NMR spectroscopic data, Table 2; HRESIMS m/z 749.2589 [M + Na]+ (calcd for C38H46NaO12S, 749.2602). X-ray Crystallography. Colorless crystals of 1, 3, and 4 were obtained in MeOH. The crystal data of 1, 3, and 4 were recorded with an XtaLAB PRO MM007HF diffractometer using Cu Kα radiation (λ = 1.5418 Å). The structures were solved by full-matrix least-squares calculation using Bruker SMART and SHELXL2014/7 computer programs.17 Absolute structures were determined by refinement of Flack parameters.18 Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44(0)-1233−336033 or e-mail: [email protected]]. Crystal data and structure refinements for 1, 3, and 4 are listed in Tables S3, S4, and S5 (Supporting Information), respectively. Crystal data of 1: orthorhombic, 2(C19H22O6)·H2O, Mr = 710.75, space group P212121, a = 8.32583(11) Å, b = 13.41861(19) Å, c = 29.8480(4) Å, α = β = γ = 90°, V = 3334.65(8) Å3, Z = 4, Dcalcd = 1.416 g/cm3, μ = 0.887 mm−1, and F(000) = 1512.0. Crystal size: 0.10 × 0.10 × 0.10 mm3. Independent reflections: 6537 [Rint = 0.036]. The

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00448.

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General experimental procedures, method for antiviral assays, 1D and 2D NMR spectra, HR-ESIMS spectra of compounds 1−5, and ECD calculation of 2 (PDF) X-ray crystallographic data for compound 1 (CIF) X-ray crystallographic data for compound 3 (CIF) X-ray crystallographic data for compound 4 (CIF)

AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-20-89023028. E-mail: [email protected]. ORCID

Hongbo Huang: 0000-0002-5235-739X Xiangjing Qin: 0000-0001-6708-1368 Jianhua Ju: 0000-0001-7712-8027 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by the National Natural Science Foundation of China (41476133, 41676151, 81425022, 31700755), the Chinese Academy of Sciences (XDA13020302, XDA11030403), and the Guangdong Natural Science Foundation (2016A030312014). We gratefully acknowledge support from the Guangzhou Branch of the Supercomputing Center of Chinese Academy of Sciences. Additionally, we thank Dr. Xiao and Ms. Sun, Ms. Zhang, and Ms. Ma in the analytical facility center of the SCSIO for recording NMR and MS data. 1897



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REFERENCES

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Abyssomicin Monomers and Dimers from the Marine-Derived

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