Antimicrobial Spirotetronate Metabolites from Marine-Derived

Article pubs.acs.org/jnp

Antimicrobial Spirotetronate Metabolites from Marine-Derived Micromonospora harpali SCSIO GJ089 Chun Gui,†,‡ Shanwen Zhang,†,‡ Xiangcheng Zhu,§ Wenjuan Ding,†,‡ Hongbo Huang,† Yu-Cheng Gu,⊥ Yanwen Duan,§ and Jianhua Ju*,†,‡ †

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, 164 West Xingang Road, Guangzhou 510301, People’s Republic of China ‡ University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 110039, People’s Republic of China § Xiangya International Academy of Translational Medicine, Central South University, National Engineering Research Center of Combinatorial Biosynthesis for Drug Discovery, 172 Tongzipo Road, Changsha 410013, People’s Republic of China ⊥ Syngenta Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, U.K. S Supporting Information *

ABSTRACT: Two new spirotetronate aglycones, 22-dehydroxymethyl-kijanolide (1) and 8-hydroxy-22-dehydroxymethyl-kijanolide (2), along with seven new spirotetronate glycosides, microsporanates A−F (3−8) and tetrocarcin P (9), together with three known tetrocarcins [tetrocarcins A (10), B (11), and AC6H (12)], were isolated from fermentation broths of the marine-derived Micromonospora harpali SCSIO GJ089. The structures of 1−9 were elucidated on the basis of 1D and 2D NMR and MS spectroscopic data. Compounds 3− 8 feature an α,β-unsaturated carbonyl moiety within their spirotetronate skeletons. Moreover, compounds 3−12 displayed strong to moderate antibacterial activities against Gram positive bacteria Bacillus thuringiensis BT01 and B. subtilis BS01 with MIC values ranging from 0.016 to 8.0 μg/mL.

S

Careful dissection and component analyses of these extracts led to the isolation and characterization of nine new spirotetronate metabolites termed here 22-dehydroxymethyl-kijanolide (1), 8hydroxy-22-dehydroxymethyl-kijanolide (2), microsporanates A−F (3−8), and tetrocarcin P (9), as well as three known compounds, tetrocarcins A (10), B (11), and AC6H (12). Herein we report the isolation, structure elucidation, and antibacterial activities of these spirotetronate compounds.

pirotetronate-containing natural products mainly originate from microorganisms.1,2 To date, more than 60 spirotetronates from Actinomycetes have been reported. These spirotetronates belong to an important family of polyketide compounds characterized by a spirocyclic tetronate moiety linked to a cyclohexene ring or conjugated with a trans-decalin moiety by a carbonyl or ester carbonyl unit. Typical structures of the spirotetronate group include tetrocarcin A, chlorothricin, and kijanimicin.1,2 Among them, tetrocarcin A and several spirotetronates have been isolated from Micromonospora species, and the biosynthetic gene cluster for tetrocarcin A from Micromonospora chalcea has been studied previously.3 Spirotetronate compounds often exert a broad range of biological activities including antibacterial, antitumor, and anti-inflammatory activities.4−7 In the past two decades, marine-derived microorganisms have proven themselves as rich sources of novel bioactive natural products. Efforts in our laboratory have enabled us to identify new marine-derived metabolites with anti-infective and antitumor activities from the Actinomycetes isolated from sediments collected in the South China Sea.8−13 We recently identified, on the basis of bioassays, extracts of the marinederived strain SCSIO GJ089 possessing strong activity against Gram-positive Bacillus thuringiensis BT01 and B. subtilis BS01. © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Strain SCSIO GJ089 was isolated from a sediment sample collected in the northern South China Sea. Phylogenetic 16S rRNA sequence analysis (GenBank accession no. KX470413) revealed a similarity of 99% to Micromonospora harpali NEAUJC6. A 15 L scale cultivation of this strain was carried out, and a subsequent battery of purification procedures including silica gel chromatography, Sephadex LH-20 chromatography, and semipreparative HPLC rendered compounds 1−12. Notably, compounds 10−12 were identified as tetrocarcin A (10),14,15 tetrocarcin B (11),16,17 and AC6H (12),18 respectively, on the Received: March 1, 2017 Published: May 10, 2017 1594

DOI: 10.1021/acs.jnatprod.7b00176 J. Nat. Prod. 2017, 80, 1594−1603

Journal of Natural Products

Article

Chart 1

basis of 1H and 13C NMR spectroscopic data comparisons with previously reported data sets. Compound 1 was obtained as a white, amorphous powder. The HRESIMS data gave ion peaks at m/z 523.3060 [M + H]+ and m/z 545.2887 [M + Na]+. Its molecular formula of C32H42O6 revealed the presence of 12 degrees of unsaturation. The IR spectrum of 1 showed characteristic absorptions of hydroxy (3335 cm−1), carboxyl (1740 cm−1), and olefinic groups (1616, 1550, and 1435 cm−1). The 13C and DEPT NMR spectra (Table 1, Figures S3 and S4, Supporting Information) displayed the presence of 32 carbon signals ascribed to 12 sp2 carbons including two carbonyl carbons (C-1 and C-3) and one oxygen-bearing nonprotonated sp2 carbon (C-26) and 20 sp3 carbons including two nonprotonated carbons, nine methine, three methylene, and six methyl groups. The COSY spectrum together with the analysis of HMBC correlations revealed three spin systems (Figure 1), which led to the assignment of fragments C-5/C-6(C-28)/C-7/C-8(C29)/C-9/C-10/C-11/C-12/C-13, C-15/C-16/C-17, and C-19/ C-20/C-21/C-22/C-23(C-32)/C-24. In its HMBC spectrum, the correlations of H3-27/C-4, C-5, and C-13 revealed a substituted decalin moiety (rings A and B). The key correlations from methyl protons H3-30 to C-13, C-14, C-15 and from H3-31 to C-17, C-18, and C-19 established a long fragment of C-14−C-24 linked with the decalin at C-13. Additionally, four carbon signals at δC 167.5 (C-1), 102.1 (C2), 201.2 (C-26), and 83.1 (C-25) indicated the existence of a tetronic acid moiety (ring E) in 1.7,18 The HMBC correlations from H-20 and H2-24 to C-25 and C-26 indicate fusion of the tetronic acid with a cyclohexene to form a [5,6] spiro-ring system (rings D and E). The linkage of the carbonyl C-3 with C-2 and C-4 to form ring C was deduced on the basis of the HMBC correlation of H 3 -27/C-3 and the remaining unoccupied one degree of unsaturation. Three hydroxy groups were assigned to the oxygen-bearing carbons C-9, C-17, and C26, consistent with the molecular formula. The Z-configurations of the Δ11,12 and Δ21,22 double bonds were assigned on the basis of the coupling constants JH‑11/H‑12 (10.2 Hz) and JH‑21/H‑22 (10.2 Hz). The NOE correlations of H13/H-15 and H3-31/H-20 indicate that both double bonds Δ14,15 and Δ18,19 are in the respective E-configurations. The

relative configuration of 1 was established on the basis of NOESY experiments (Figure 2). The NOE correlations of H13/H3-27/H-10/H-6 and H-10/H3-29/H-7α placed these protons and methyl groups on the same side of the decalin ring. Correspondingly, the cross-peaks of H-5/H-9/H-7β in the NOESY spectrum suggested H-5, H-9, and H-7β were on the same side. The NOE associations of H-20/H-24β/H-23 and H24α/H3-32 suggested placement of H-20, H-24β, and H-23 on the same side, while H-24α and 32-Me on the other side of the cyclohexene ring. Due to the conserved configurations displayed in the spirotetronate backbone and also from the perspective of biosynthesis,19,20 the absolute configuration of 1 is suggested as shown. The structure of 1 is closely related to kijanolide,21 with the exception of the absence of hydroxymethyl at C-22. Thus, compound 1 was named 22dehydroxymethyl-kijanolide. Compound 2 was obtained as a white, amorphous powder. Its molecular formula was established as C32H42O7 by HRESIMS, which had one more oxygen atom than 1. Comparison of its 1H, 13C, and 2D NMR spectroscopic data with those of 1 (Table 1) revealed that 2 possesses an additional C-8 OH substitution compared to 1; the methine at C-8 (δH 2.21, δC 34.6) in 1 is replaced by an oxygen-bearing nonprotonated carbon (δC 74.1) in structure 2. Consistently, the HMBC correlations from H3-29, H-7, and H-9 to C-8 confirmed this substitution. The NOE correlations of H-13/H327/H-10/H-6 and H-10/H3-29/H-7α revealed the protons H13, H-10, H-6, and H-7α and the methyl groups H3-27 and H329 were on the same side of the decalin ring and thus established a β OH at C-8. The absolute configurations of 2 were established to be the same as those of 1 since 2 and 1 displayed similar circular dichroism (CD) curves, which showed a negative Cotton effect at 217 nm and a positive Cotton effect at about 300 nm (Figure S64). Hence, the structure of 2 was established as 8-hydroxy-22-dehydroxymethyl-kijanolide. Microsporanate A (3) had a molecular formula of C70H100N2O24 on the basis of a deprotonated molecule peak at m/z 1351.6646 [M − H]−. The UV spectrum of 3 showed characteristic absorption bands at 203, 242, and 274 nm, which was slightly different from those of 1 (204, 241, 267 nm) and 2 (203, 240, 267 nm), suggesting 3 has a similar core structure, 1595



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Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1, 2, and 9 1a position 1 2 3 4 5 6 7α 7β 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24α 24β 25 26 27 28 29 30 31 32 NS1 NS2 NS3 NS4 NS5 NS6 NS3-CH3 NS4-NHCO2− NS4-NHCO2CH3 DG1 DG2 DG3 DG4 DG5 DG6 DG4-O2CDG4-O2CCH3 AM/DG′1 AM/DG′2 AM/DG′3 AM/DG′4 AM/DG′5 AM/DG′6 DG″1 DG″2

δC, type 167.5, 102.1, 206.4, 50.9, 42.7, 31.1, 41.7,

C C C C CH CH CH2

34.6, 76.0, 39.0, 125.5, 126.4, 53.2, 135.6, 123.1, 31.9, 72.8, 141.0, 117.8, 39.1, 125.0, 131.9, 27.7, 34.1,

CH CH CH CH CH CH C CH CH2 CH C CH CH CH CH CH CH2

83.1, 201.2, 15.1, 22.3, 13.0, 13.7, 14.8, 21.5,

C C CH3 CH3 CH3 CH3 CH3 CH3

2b

δH mult. (J in Hz)

1.96, 1.56, 1.49, 1.56, 2.21, 3.61, 2.02, 5.99, 5.37, 3.44,

m overlapped m overlapped m dd (5.2, 10.4) m d (10.2) d (10.2) d (3.7)

5.17, d (10.0) 2.17, m; 2.33, m 4.14, br s 5.22, d (10.4) 3.50,dd (10.4, 1.6) 5.39, d (10.2) 5.77, d (10.2) 2.51, m 1.72, d (14.3) 2.31, dd (14.4, 7.7)

1.56, 0.60, 0.99, 1.34, 1.34, 1.18,

s d (4.9) d (7.0) s s d (7.3)

δC, type 174.2, C 103.3,d C 204.6, C 51.1, C 42.8, C 34.2, CH 48.7, CH2 74.1, 80.4, 41.2, 125.0, 126.6, 52.9, 135.8, 122.9, 32.0, 72.7, 140.7, 118.0, 39.2, 125.3, 131.9, 27.6, 34.0,

C CH CH CH CH CH C CH CH2 CH C CH CH CH CH CH CH2

83.3, 200.7, 14.7, 22.0, 20.8, 13.5, 14.8, 21.4,

C C CH3 CH3 CH3 CH3 CH3 CH3

1596

9c

δH mult. (J in Hz)

2.05 1.39, m 1.45, overlapped 1.62, d (11.8) 3.28, 1.79, 5.95, 5.31, 3.47,

d (10.8) m d (10.1) overlapped overlapped

5.11, d (9.4) 2.1, d (16.3); 2.31, m 4.05, br s 5.14, 3.42, 5.34, 5.72, 2.45, 1.66, 2.25,

1.47, 0.59, 1.18, 1.29, 1.28, 1.14,

d (9.8) d (10.1) overlapped d (10.1) m d (14.3) dd (14.1, 7.6)

s d (6.0) s s s d (7.3)

δC, type 177.4, 98.7, 199.4, 52.9, 45.5, 32.7, 43.3,

C C C C C CH CH2

36.2, 86.6, 40.4, 127.0, 128.9, 53.0, 138.5, 122.9, 32.1, 80.1, 139.7, 122.0, 46.7, 71.9, 151.1, 139.7, 30.8,

CH CH CH CH CH CH C CH CH2 CH C CH CH CH CH C CH2

85.4, 200.4, 15.5, 23.1, 14.7, 15.1, 16.4, 195.0, 96.8, 37.9, 57.9, 55.5, 68.9, 17.4, 24.6, 160.0, 52.8, 99.9, 32.1, 67.9, 76.2, 63.5, 18.4, 172.1, 21.0, 93.7, 27.5, 30.5, 82.1, 69.3, 17.9, 100.6, 39.6,

C C CH3 CH3 CH3 CH3 CH3 C CH CH2 C CH CH CH3 CH3 C CH3 CH CH2 CH CH CH CH3 C CH3 CH CH2 CH2 CH CH CH3 CH CH2

δH mult. (J in Hz)

2.11, 1.51, 1.49, 1.59, 2.21, 3.43, 2.04, 5.75, 5.39, 3.62,

m overlapped overlapped; m m dd (10.7, 5.2) m d (10.2) ddd (9.8, 4.9, 2.2) m

5.21, m 2.22, m; 2.32, m 4.26, br s 5.16, 2.83, 4.46, 6.86,

m t (9.5) dm (9.5) s

2.25, d (19.1) 2.76, dt (18.8, 2.7)

1.49, 0.66, 1.12, 1.38, 1.51, 9.52, 4.77, 1.79,

s d (5.9) d (7.1) s s s dd (10.1, 2.1) m, 1.90, m

3.59, 3.94, 1.18, 1.34,

br s m d (7.1) s

3.68, 4.82, 1.81, 4.16, 4.54, 4.39, 1.14,

s d (4.0) m; 2.23, m dt (2.9, 3.0) dd (9.7, 3.0) dq (9.8, 6.5) d (6.7)

2.09, 4.93, 1.87, 1.75, 3.21, 3.66, 1.13, 4.90, 1.65,

s d (2.9) m; 2.00, m m; 1.90, m ddd (10.0, 6.7, 4.5) m d (6.4) dd (9.9, 1.7) m; 2.00, m



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Table 1. continued 1a position

δC, type

δH mult. (J in Hz)

2b δC, type

DG″3 DG″4 DG″5 DG″6

δH mult. (J in Hz)

9c δC, type 69.2, 74.3, 70.7, 18.7,

CH CH CH CH3

δH mult. (J in Hz) 4.00, 3.14, 3.76, 1.24,

dt (3.0, 2.9) dd (9.5, 3.0) dq (9.5, 6.3) d (6.2)

a

Recorded in CDCl3 at 500 MHz for 1H NMR, 125 MHz for 13C NMR. bRecorded in CDCl3/CD3OD at 500 MHz for 1H NMR, 125 MHz for 13C NMR. cRecorded in CD3OD at 700 MHz for 1H NMR, 176 MHz for 13C NMR. dDeduced from the HMBC spectrum.

tetronitrose (NS) unit at C-17 were identical to those in tetrocarcin A on the basis of 1D and 2D NMR data (Figure 3). The acetyl group in the DG unit and the methyl carbamate at C-4 (NS) were confirmed by the HMBC correlations from protons of H-4 (NS) (δH 4.58) and −CH3 (δH 2.08, δC 21.2) to the carbonyl at δC 170.6, and of H-4 (NS) (δH 4.36) and −OCH3 (δH 3.70, δC 52.9) to the carbonyl at δC 157.4, respectively. Therefore, the structure of 3 was assigned as shown and designated as microsporanate A. Microsporanate B (4) was isolated as a white, amorphous powder. Its molecular formula was established as C64H90N2O22 by HRESIMS, which is one C6H10O2 fragment less than that of 3. Subsequent analysis of the 1H and 13C NMR spectra revealed that only four anomeric methines were observed at δH 4.44 and δC 96.6, δH 4.83 and δC 98.6, δH 4.88 and δC 92.6, and δH 4.87 and δC 99.4, indicating the absence of one sugar unit in 4 compared to those of 3. In addition, the 13C NMR resonance of C-3 (DG″) was shifted from δC 64.0 in 3 to 68.3 in 4, and C-4 (DG″) was shifted from δC 75.3 in 3 to 73.1 in 4, suggesting that the C-4 (DG″) was not O-glycosylated. These data showed that the AM′ in 3 was missing in compound 4. Thus, the structure of 4 was elucidated as shown. The 1H and 13C NMR data were assigned on the basis of detailed COSY, HSQC, and HMBC analyses. Microsporanate C (5) was obtained as a white, amorphous powder. HRESIMS established the molecular formula of C64H90N2O23 based on the deprotonated molecule peak at m/z 1253.5921 [M − H]−, which revealed that 5 has one more oxygen atom than 4. The 1H and 13C NMR spectroscopic data of 5 were similar to those of 4, except that the methylene signals at δH 1.94 and 2.00, δC 26.4 (CH2-3 (DG′)) in 4 were absent, while signals consistent with an oxygen-bearing methine were observed at δH 4.20, δC 67.4 in 5, suggesting the presence of a hydroxy group at C-3 (DG′). COSY and HMBC correlations (Figure 3) further validated the assertion that the AM moiety in 4 was replaced by a DG′ moiety in 5; the presence of a trisaccharide comprising 4-acetyl-DG/DG′/DG″ is apparent in compound 5. A similar linkage consisting of a DG/DG′/DG″ chain has been previously noted in lobophorin B.5 Microsporanate D (6) was obtained as a white, amorphous powder. Its molecular formula was determined to be C70H102N2O22 on the basis of HRESIMS analysis, which gave a deprotonated molecular peak at m/z 1321.6856 [M − H]−. The 1H and 13C NMR spectroscopic data of 6 were almost identical to those of 3 except that the 13C NMR chemical shift for C-3 (NS) changed from δC 91.6 in 3 to δC 58.0 in 6, suggesting that the nitro group at C-3 (NS) in 3 was replaced by an amino group in 6. This change is consistent with a molecular formula (and subsequent mass) of 6 requiring 30 amu less than that of 3. In the spirotetronate family, the same −NO2/−NH2 substitution has been observed in TCA/

Figure 1. COSY (bold) and selected HMBC (arrow) correlations for 22-dehydroxymethyl-kijanolide (1).

Figure 2. NOESY correlations for 22-dehydroxymethyl-kijanolide (1).

but different chromophores than 1 and 2. The 13C NMR and HSQC spectra revealed five carbonyls, 12 olefinic carbons in the deshielded sp2-region, and 53 aliphatic carbons in the shielded area. On comparing the 1H and 13C spectroscopic data of 3 with those for previously established tetrocarcin A (10)14,15,22 (Table S1, Supporting Information), it was revealed that a close structural relationship exists between the two compounds. The most distinct difference is that the formyl group at C-23 in tetrocarcin A is replaced by an α,β-unsaturated butanone in 3; this was confirmed on the basis of the HMBC correlations from H-32 to C-22, C-23, C-24, and C-34 and from H-33 to C-23, C-34, and C-35. The double bond of C-32/ C-33 was assigned the E-configuration based on the coupling constant JH‑32/H‑33 = 16.2 Hz. The s-trans conformer of the two conjugated double bonds Δ22,23 and Δ32,33 was assigned on the basis of the NOE correlation of H-22/H-32. The α-amicetosyl (AM′)-(1→4)-β-digitoxosyl (DG″)-(1→4)-α-amicetosyl (AM)-(1→3)-α-digitoxosyl (DG) chain at the C-9 and the β1597



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Figure 3. COSY (bold) and selected HMBC (arrow) correlations for microsporanates A, C, and D (3, 5, and 6, respectively).

AC6H,18 lobophorins A and B,4,5 and arisostatins A and B.22 Thus, the structure of 6 was determined as shown. Compounds 7−9 were also obtained as a white, amorphous powders. The molecular formulas C64H92N2O20, C64H92N2O21, and C61H88N2O20 for 7, 8, and 9 were determined by HRESIMS; all corresponding molecular weights were found to be 30 amu less than those of 4, 5, and the known compound 11, respectively. The 1H and 13C NMR spectroscopic data of 7, 8, and 9 closely resembled those of 4, 5, and 11, respectively. The slight differences were that the 13C NMR chemical resonances of C-3 (NS) changed from δC 91.6 in 4, 5, and 11 to δC 57.7 in 7, δC 56.8 in 8, and δC 57.9 in 9. In a fashion consistent with that observed in 3 and 6, the nitro groups at C3 (NS) in 4, 5, and 11 were substituted by amino groups in 7, 8, and 9. Accordingly, compounds 7 and 8 were designated as microsporanates E and F, respectively. Compound 9 was closely related to tetrocarcins and thus designated as tetrocarcin P. All natural spirotetronates described in the literature to date have been obtained from actinobacteria. Biosynthetic investigations3,23−28 have revealed that the assembly of the spirotetronate starts with a linear carbon skeleton catalyzed by a PKS-I module, followed by a [4 + 2] cycloaddition (postulated Diels−Alder reaction) to effect the requisite macrocylization. Subsequent hydroxylations and glycosylations at different aglycone positions lead to significant structural

diversity across members of the spirotetronate group. On the basis of carbon number (Cn) for the central ring system (the macrocycle framework), spirotetronates have been further classified into small (C11), medium (C13), or large (>C13) sized family members. Compounds 1−12 belong to the medium-sized spirotetronates. Only three decalin-containing aglycones, nominicin, maklamicin, and 29-deoxymaklamicin, have been previously reported.7,29,30 22-Dehydroxymethylkijanolide (1) and 8-hydroxy-22-dehydroxymethyl-kijanolide (2) represent additional examples of nonglycosylated spirotetronates beyond the established agents nominicin, maklamicin, and 29-deoxymaklamicin. Microsporanates A−F (3−8) feature an α,β-unsaturated carbonyl chain at C-23; these compounds represent the first group to contain this functionality within the spirotetronate family. Structurally speaking, nonenzymatic aldol condensation of compounds 9−12 with acetone would yield compounds 7, 3, 4, and 6, respectively. To test if the nonenzymatic aldol condensation process could occur, compound 12 (1.0 mg in total) was selected and suspended in (i) 250 μL of acetone, (ii) 250 μL of acetone supplemented with 0.1% acetic acid, (iii) 250 μL of acetone supplemented with 0.1% triethylamine, (iv) 250 μL of acetone supplemented with 0.1% acetic acid and 3% XAD-16 resin, and (v) 250 μL of acetone supplemented with 0.1% triethylamine and 3% XAD-16 resin. The reaction mixtures were stirred at 28 °C for 24 h and then subjected 1598



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Table 2. 1H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data for Microsporanates A−C (3−5) in CDCl3 3 position 1 2 3 4 5 6 7α 7β 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24α 24β 25 26 27 28 29 30 31 32 33 34 35 NS1 NS2 NS3 NS4 NS5 NS6 NS3-CH3 NS4-NHCO2− NS4-NHCO2CH3 DG1 DG2 DG3 DG4 DG5 DG6 DG4-O2C− DG4-O2CCH3 AM/DG′1 AM/DG′2 AM/DG′3 AM/DG′4 AM/DG′5

δC, type 166.8, 100.9, 206.6, 51.3, 43.3, 31.3, 41.7,

C C C C CH CH CH2

34.7, 84.4, 38.6, 126.5, 126.1, 54.3, 136.1, 123.1, 30.8, 78.0, 141.3, 118.7, 44.9, 69.7, 139.6, 130.9, 32.3,


84.5, 201.9, 15.6, 22.2, 14.1, 14.5, 16.2, 143.8, 127.2, 198.4, 27.7, 96.6, 36.1, 91.6, 53.8, 69.4, 17.9, 25.4, 157.4, 52.9, 98.6, 31.6, 66.8, 74.5, 62.2, 18.2, 170.6, 21.2, 92.7, 29.7, 26.4, 81.3, 68.0,

C C CH3 CH3 CH3 CH3 CH3 CH CH C CH3 CH CH2 C CH CH CH3 CH3 C CH3 CH CH2 CH CH CH CH3 C CH3 CH CH2 CH2 CH CH

4

δH mult. (J in Hz)

2.0, m 1.58, overlapped 1.52, overlapped 1.56, overlapped 2.20, m 3.45, m 2.08, overlapped 5.75, d (10.5) 5.41, ddd (9.8, 4.8, 2.1) 3.25, m 5.15, d (9.1) 2.22, m; 2.31, m 4.27, br s 5.21, d (10.0) 3.0, t (9.7) 4.72, dm (8.7) 6.35, s 2.43, d (17.7) 2.88, d (17.7)

1.63, 0.65, 1.08, 1.35, 1.52, 7.18, 6.07,

s d d s s d d

(4.5) (7.0)

(16.2) (16.2)

2.29, s 4.44, dd (9.8, 1.6) 1.64, m; 2.69, d (14.7) 4.36, 3.48, 1.16, 1.58,

d (9.8) m d (6.1) s

3.70, 4.83, 1.77, 4.15, 4.58, 4.35, 1.13,

s d (4.1) m; 2.23, m dt (3.0, 2.9) dd (9.8, 2.8) dd (9.8, 1.4) d (6.3)

2.08, 4.88, 1.75, 1.95, 3.20, 3.70,

s d (2.8) m; 1.87, m m; 2.0, m ddd (10.4, 4.7) m

δC, type 166.8, 100.9, 206.6, 51.3, 43.3, 31.3, 41.7,

C C C C CH CH CH2

34.7, 84.4, 38.6, 126.4, 126.1, 54.3, 136.1, 123.1, 30.8, 78.0, 141.3, 118.7, 44.9, 69.7, 139.6, 130.9, 32.3,


84.5, 201.9, 15.6, 22.2, 14.1, 14.5, 16.2, 143.8, 127.2, 198.4, 27.7, 96.6, 36.1, 91.6, 53.8, 69.4, 17.9, 25.4, 157.4, 53.0, 98.6, 31.3, 66.8, 74.5, 62.2, 18.2, 170.6, 21.2, 92.6, 29.7, 26.4, 81.4, 68.0,


1599

5

δH mult. (J in Hz)

2.0, m 1.58, overlapped 1.52, overlapped 1.56, overlapped 2.20, m 3.44, m 2.08, overlapped 5.75, d (10.2) 5.41, ddd (9.8, 4.7, 2.0) 3.26, d (3.3) 5.15, d (9.4) 2.22, m; 2.31, m 4.27, br s 5.21, d (10.0) 3.0, t (9.7) 4.72,dm (8.7) 6.35, s 2.43, d (17.7) 2.88, d (17.7)

1.63, 0.65, 1.08, 1.35, 1.52, 7.18, 6.07,

s d d s s d d

(4.0) (7.0)

(16.2) (16.2)

2.29, s 4.44, dd (9.7) 1.64, m; 2.69, d (14.9) 4.36, 3.48, 1.16, 1.58,

d (9.8) m d (6.6) s

3.71, 4.83, 1.77, 4.15, 4.58, 4.35, 1.13,

s d (3.9) m; 2.23, m dt (2.9, 2.2) dd (9.8, 2.4) dd (9.7) d (6.4)

2.08, 4.88, 1.74, 1.94, 3.20, 3.72,

s m m; 1.87, m m; 2.0, m ddd (10.2, 4.6) m

δC, type 166.8, 101.0, 206.5, 51.3, 43.4, 31.3, 41.7,

C C C C CH CH CH2

34.6, 84.7, 38.6, 126.2, 126.3, 54.3, 136.0, 123.3, 30.8, 78.0, 141.3, 118.7, 44.9, 69.7, 139.6, 130.9, 32.4,


84.5, 201.8, 15.1, 22.2, 14.4, 14.5, 16.2, 143.7, 127.2, 198.3, 27.7, 96.6, 36.1, 91.6, 53.8, 69.4, 17.9, 25.4, 157.4, 53.0, 98.3, 30.0, 66.2, 73.6, 62.0, 17.0, 170.6, 21.0, 92.8, 35.3, 67.4, 82.2, 62.9,

C C CH3 CH3 CH3 CH3 CH3 CH CH C CH3 CH CH2 C CH CH CH3 CH3 C CH3 CH CH2 CH CH CH CH3 C CH3 CH CH2 CH CH CH

δH mult. (J in Hz)

2.0, m 1.59, overlapped 1.52, overlapped 1.56, overlapped 2.21, m 3.46, m 2.11, overlapped 5.73, d (10.0) 5.42, ddd (9.8, 4.7, 2.0) 3.27, d (3.9) 5.16, overlapped 2.22, m; 2.31, m 4.27, br s 5.21, d (9.9) 3.0, t (9.7) 4.72, dm (8.4) 6.35, s 2.43, d (17.7) 2.89, d (17.7)

1.64, 0.65, 1.11, 1.36, 1.52, 7.19, 6.07,

s d d s s d d

(3.9) (7.0)

(16.2) (16.2)

2.29, s 4.44, dd (9.8) 1.64, m; 2.69, d (14.9) 4.36, 3.48, 1.16, 1.58,

d (9.8) m d (6.3) s

3.71, 4.83, 1.80, 4.20, 4.64, 4.30, 1.14,

s d (3.6) m; 2.35, m overlapped dd (9.8, 2.6) dd (9.9, 3.6) d (6.3)

2.13, 5.15, 1.95, 4.20, 3.20, 3.80,

s overlapped m; 2.14, m overlapped dd (9.3, 6.6) m



Article

Table 2. continued 3 position AM/DG′6 DG″1 DG″2 DG″3 DG″4 DG″5 DG″6 AM′1 AM′2 AM′3 AM′4 AM′5 AM′6

δC, type 17.6, 99.5, 37.1, 64.0, 75.3, 67.9, 19.0, 92.0, 27.5, 29.8, 71.8, 70.4, 17.8,

CH3 CH CH2 CH CH CH CH3 CH CH2 CH2 CH CH CH3

4

δH mult. (J in Hz) 1.13, 4.89, 1.67, 4.25, 3.44, 3.85, 1.31, 4.92, 1.72, 1.80, 3.28, 3.62, 1.23,

d (6.3) dd (9.8, 1.8) m; 2.14, m dt (3.1, 2.7) m dq (9.5, 6.3) d (6.2) br s m; 1.88, m m; 1.87, m dd (4.3, 10.5) dq (9.1, 6.3) d (6.2)

δC, type 18.2, 99.4, 38.1, 68.3, 73.1, 68.3, 18.3,

δH mult. (J in Hz)

CH3 CH CH2 CH CH CH CH3

1.15, 4.87, 1.67, 4.10, 3.31, 3.73, 1.29,

■

to HPLC analyses. However, no desired product 6 was observed in all of the above-mentioned conditions. Compounds 9, 10, and 11 were subsequently processed under the aforementioned same conditions; similarly, the desired products of 3, 4, and 7 were not observed. The results of these experiments indicate that compounds 3, 4, 6, and 7 could not be formed via simple nonenzymatic chemical processes. On the other hand, enzymatic catalyzed aldol condensations to yield an α,β-unsaturated keto group have been reported. For instance, RedH catalyzes the condensation of 4-methoxy-2,2′bipyrrole-5-carboxaldehyde and 2-undecylpyrrole to yield undecylprodiginine.31 Thus, we conclude that the α,βunsaturated carbonyl containing compounds 3, 4, 6, and 7 are naturally occurring metabolites. All of these compounds were evaluated for their antibacterial activities against B. subtilis BS01, B. thuringiensis BT01, methicillin-resistant Staphylococcus aureus shhs-A1 (MRSA, a clinical isolate), S. aureus ATCC 29213, and Enterococcus faecalis ATCC 29212. Compounds 3−5, 10, and 11 showed significant growth-inhibiting activities against B. subtilis BS01 and B. thuringiensis BT01 with MIC values ranging from 0.016 to 0.5 μg/mL. Notably, these MICs indicate compounds with activities superior to those of the vancomycin, kanamycin, and ampicillin positive controls. Compounds 6−9 and 12 exhibited moderate activities against B. subtilis BS01 and B. thuringiensis BT01 with MIC values between 1.0 and 8.0 μg/mL. Aglycones 1 and 2 exhibited no activity against B. subtilis BS01 or B. thuringiensis BT01, nor did they display any activity against methicillin-resistant S. aureus shhs-A1, S. aureus ATCC 29213, or E. faecalis ATCC 29212. On the basis of these structure− activity relationship findings we postulate the following: (i) the sugar moieties are indispensable for antimicrobial activity in agreement with previous studies showing that deglycosylated kijanimicin is devoid of antibacterial activity, unlike its glycosylated counterpart;32 (ii) nitro-sugars as seen in compounds 3−5, 10, and 11 impart greater antibacterial activity to their scaffolds than do their amino-sugar counterparts (as in 6−9 and 12); and (iii) the apparent interchangeability of the C-23 α,β-unsaturated carbonyl moiety with the −CHO group suggests that substitution at this position either has little overall influence upon activity or that some flexibility exists with respect to the nature of the electrondeficient and electrophilic element placed at C-23.

5 d (6.6) overlapped m; 2.14, m dt (3.1, 2.5) dd (9.4, 2.7) m d (6.1)

δC, type 17.7, 99.7, 37.8, 68.3, 72.7, 69.2, 18.1,


δH mult. (J in Hz) 1.20, 4.89, 1.81, 4.10, 3.36, 3.72, 1.27,

d (6.1) d (9.2) m; 2.11, m m dd (8.0) overlapped d (5.9)

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained with an MCP-500 polarimeter (Anton Paar). UV spectra were measured with a UV-2600 spectrometer (Shimadzu). IR spectra were obtained using an IRAffinity-1 spectrophotometer (Shimadzu). NMR spectra were acquired with a Bruker Avance 500 or a Bruker Avance 700 spectrometer. Carbon signals and the residual proton signals of CD3OD (δC 49.0 and δH 4.87) and CDCl3 (δC 77.16 and δH 7.26) were used for calibration. Low-resolution and high-resolution mass spectrometric data were acquired using an amaZon SL ion trap mass spectrometer (Bruker) and MaXis quadrupole-time-of-flight mass spectrometer (Bruker), respectively. Column chromatography (CC) was carried out on silica gel (100−200 mesh, Yantai Jiangyou Silica Gel Development Co., Ltd.). Reversed-phase HPLC was performed using LC-3000 solvent delivery modules equipped with a UV detector 2550 (Knauer) and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Bacterial Materials. Strain SCSIO GJ089 was isolated from a marine sediment at a depth of −1565 m collected in the northern South China Sea. This strain was identified as Micromonospora harpali on the basis of morphological characteristics and 16S rRNA gene sequence (GenBank accession no. KX470413) analysis by comparisons with other sequences in the GenBank database. The strain was preserved at the RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, and was also preserved at the China Center for Type Culture Collection (CCTCC) with the access number CCTCC M 2016409. Fermentation and Extraction. The fermentation was carried out using A1 medium (soluble starch 10 g/L, yeast extract 4 g/L, bacterial peptone 2 g/L, sea salt (Guangdong Province Salt Industry Group, Guangzhou, China) 10 g/L, pH 7.2−7.4) as seed medium. The seed medium was inoculated at 28 °C on rotary shakers (200 rpm) for 24 to 36 h, then transferred into 1 L flasks each containing 250 mL of N4 medium (soluble starch 15 g/L, fish peptone 8 g/L, bacterial peptone 5 g/L, glycerol 6 g/L, KBr 0.2 g/L, CaCO3 2 g/L, sea salt 10 g/L, pH 7.2−7.4) with 3% XAD-16 resin. The culture was grown at 28 °C for 7 days on rotary shakers at 200 rpm. The XAD-16 resin along with the mycelium was separated by filtration through a metal sieve (40 mesh) and then eluted three times with 1.5 L of acetone. The acetone fraction was concentrated under reduced pressure to afford the extract. Isolation. The extract (15.2 g) was subjected to a silica gel column using gradient elution with a CHCl3/MeOH mixture (100/0, 99/1, 98/2, 96/4, 94/6, 92/8, 90/10, 80/20, 0/100 v/v) to give nine fractions (A1−A9), respectively. Fractions A1−A5 were combined and further purified by Sephadex LH-20 chromatography and semipreparative HPLC to get compounds 1 (30.0 mg, tR 24.3 min), 3 (12.0 mg, tR 24.1 min), 4 (10.2 mg, tR 23.5 min), 5 (10.0 mg, tR 21.9 min), 10 (7.6 mg, tR 24.0 min), and 11 (8.0 mg, tR 23.0 min). Fractions A6 and A7 were combined and subjected to Sephadex LH-20 1600



Article

Table 3. 1H and 13C NMR Spectroscopic Data for Microsporanates D−F (6−8) 6a position 1 2 3 4 5 6 7α 7β 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24α 24β 25 26 27 28 29 30 31 32 33 34 35 NS1 NS2 NS3 NS4 NS5 NS6 NS3-CH3 NS4-NHCO2− NS4-NHCO2CH3 DG1 DG2 DG3 DG4 DG5 DG6 DG4-O2C− DG4-O2CCH3 AM/DG′1 AM/DG′2 AM/DG′3 AM/DG′4 AM/DG′5

δC, type 177.2, 98.9, 199.5, 52.8, 45.5, 32.6, 43.3,

C C C C CH CH CH2

36.2, 86.6, 40.4, 127.0, 128.9, 53.0, 138.4, 123.0, 32.1, 80.0, 139.4, 122.3, 46.6, 72.2, 140.8, 134.3, 33.4,


85.8, 200.5, 15.5, 23.2, 14.7, 15.2, 16.4, 146.3, 127.6, 201.2, 27.9, 96.6, 37.8, 58.0, 55.4, 68.9, 17.3, 24.7, 160.0, 52.9, 99.9, 32.1, 67.8, 76.1, 63.5, 18.3, 172.1, 21.1, 93.7, 27.5, 30.4, 82.1, 69.3,


7b

δH mult. (J in Hz)

2.10, 1.49, 1.49, 1.59, 2.20, 3.43, 2.04, 5.76, 5.40, 3.64,

m overlapped overlapped m m dd (10.1, 4.4) m d (10.1) m overlapped

5.21, d (5.3) 2.23, m; 2.32, m 4.27, br s 5.15, d (9.3) 2.80, t (9.5) 4.38, d (9.3) 6.3, s 2.26, overlapped 2.87, d (18.0)

1.49, 0.66, 1.11, 1.38, 1.50, 7.30, 6.22,

s d d s s d d

(3.9) (8.4)

(16.0) (16.1)

2.29, s 4.79, d (10.0) 1.79, m; 1.92, m 3.62, 3.93, 1.17, 1.35,

br s m d (6.0) s

3.68, 4.83, 1.75, 4.16, 4.54, 4.39, 1.13,

s d (3.7) m; 2.30, m m dd (9.8, 2.3) m overlapped

2.09, 4.93, 1.87, 1.86, 3.21, 3.66,

s overlapped m; 1.99, m m; 1.89, m ddd (9.8, 5.4) overlapped

δC, type

8b

δH mult. (J in Hz)

177.2, 98.9, 199.5, 52.9, 45.5, 32.7, 43.3,

C C C C CH CH CH2

36.2, 86.6, 40.4, 127.0, 128.9, 53.0, 138.4, 122.9, 32.1, 80.0, 139.4, 122.3, 46.6, 72.2, 140.9, 134.3, 33.4,


85.8, 200.5, 15.5, 23.2, 14.7, 15.2, 16.4, 146.3, 127.5, 201.3, 27.9, 96.7, 37.9, 57.7, 55.6, 68.9, 17.3, 24.7, 160.0, 52.8, 99.9, 32.1, 67.9, 76.1, 63.5, 18.4, 172.1, 21.1, 93.7, 27.5, 30.4, 82.1, 69.3,


1601

2.10, 1.49, 1.49, 1.59, 2.20, 3.43, 2.04, 5.75, 5.40, 3.63,

m overlapped overlapped m m dd (10.5, 5.0) m d (10.1) ddd (9.7, 4.8, 2.1) d (4.2)

5.21, d (5.6) 2.23, m; 2.32, m 4.26, br s 5.15, 2.79, 4.35, 6.30,

d (9.5) t (9.4) d (9.2) s

2.26, overlapped 2.87, d (17.9)

1.49, 0.66, 1.12, 1.38, 1.50, 7.30, 6.21,

s d d s s d d

(4.0) (8.0)

(16.1) (16.1)

2.29, s 4.78, dd (9.9, 1.5) 1.76, m; 1.88, m 3.57, 3.94, 1.17, 1.33,

br s m d (6.1) s

3.68, 4.83, 1.75, 4.16, 4.54, 4.39, 1.14,

s d (3.3) m; 2.3, m m dd (9.7, 2.6) m d (5.5)

2.09, 4.93, 1.87, 1.86, 3.20, 3.66,

s overlapped m; 1.99, m m; 1.89, m ddd (9.9, 4.8) m

δC, type 177.2, 98.9, 199.3, 52.9, 45.6, 32.6, 43.3,

C C C C CH CH CH2

36.1, 87.1, 40.3, 127, CH 129.1, 52.8, 138.5, 123.0, 32.2, 80.0, 139.4, 122.4, 46.6, 72.2, 140.9, 134.3, 33.4,

CH CH CH CH CH C CH CH2 CH C CH CH CH CH CH CH2

85.8, 200.6, 15.5, 23.1, 14.9, 15.2, 16.4, 146.4, 127.4, 201.5, 27.9, 97.0, 38.2, 56.8, 56.0, 68.9, 17.4, 25.1, 160.0, 53.0, 99.7, 30.7, 67.6, 75.3, 63.4, 17.9, 172.1, 21.1, 94.2, 35.9, 69.1, 83.0, 64.2,

C C CH3 CH3 CH3 CH3 CH3 CH CH C CH3 CH CH2 C CH CH CH3 CH3 C CH3 CH CH2 CH CH CH CH3 C CH3 CH CH2 CH CH CH

δH mult. (J in Hz)

2.10, 1.51, 1.49, 1.59, 2.20, 3.45, 2.06, 5.75, 5.41, 3.63,

m overlapped overlapped m m dd (10.6, 5.1) m d (10.1) ddd (9.9, 5.1, 2.3) d (4.6)

5.21, overlapped 2.23, m; 2.30, m 4.26, br s 5.15, 2.79, 4.37, 6.30,

d (9.6) t (9.3) d (8.9) s

2.25, overlapped 2.87, d (17.8)

1.48, 0.65, 1.13, 1.38, 1.51, 7.31, 6.22,

s d (5.2) overlapped s s d (16.1) d (16.1)

2.29, s 4.79, d (10.1) 1.72, m; 1.87, m 3.52, 3.96, 1.16, 1.30,

br s m d (6.2) s

3.68, 4.83, 1.85, 4.23, 4.62, 4.32, 1.14,

s d (4.3) m; 2.39, m m overlapped m overlapped

2.12, s 5.2, d (2.4) 2.02, m; 2.11, m 4.17, m 3.28, dd (9.7, 2.6) 3.80, dq (9.9, 6.3)



Article

Table 3. continued 6a δC, type

position AM/DG′6 DG″1 DG″2 DG″3 DG″4 DG″5 DG″6 AM′1 AM′2 AM′3 AM′4 AM′5 AM′6 a

17.4, 100.6, 39.2, 64.0, 76.4, 69.5, 19.3, 92.9, 28.3, 30.4, 72.6, 71.4, 18.4,

CH3 CH CH2 CH CH CH CH3 CH CH2 CH2 CH CH CH2

7b

δH mult. (J in Hz) 1.13, 4.93, 1.62, 4.26, 3.28, 3.91, 1.29, 4.92, 1.73, 1.72, 3.13, 3.59, 1.20,

overlapped overlapped m; 2.01, m overlapped dd (9.5, 2.0) m d (6.2) br s m; 1.80, m m; 1.75, m dd (9.7, 4.9) m d (6.1)

δC, type

8b

δH mult. (J in Hz)

17.86, CH3 100.6, CH 39.6, CH2 69.2, CH 74.3, CH 70.7, CH 18.7, CH3

1.13, 4.90, 1.65, 4.00, 3.14, 3.76, 1.24,

d (6.0) overlapped m; 2.01, m dt (3.1, 2.8) dd (9.5, 2.8) dq (9.5, 6.2) d (6.2)

δC, type 17.9, 101.1, 39.3, 69.2, 74.3, 70.7, 18.7,


δH mult. (J in Hz) 1.20, d (6.3) 4.93, overlapped 1.75, m; 2.02, m 4.0, dt (3.0, 2.9) 3.16, dd (9.5, 2.9) 3.75, dq (9.6, 6.2) 1.24, d (6.2)

Recorded in CD3OD at 500 MHz for 1H NMR, 125 MHz for 13C NMR. bRecorded in CD3OD at 700 MHz for 1H NMR, 176 MHz for 13C NMR.

Table 4. Antimicrobial Activitiesa of Compounds 1−12 (MIC, μg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 vancomycin kanamycin ampicillin

B. subtilis BS01

B. thuringiensis BT01

MRSA (clinical isolate shhs-A1)

S. aureus ATCC 29213

E. faecalis ATCC 29212

64 >128 0.03 0.13 0.5 2.0 2.0 8.0 2.0 0.03 0.063 2.0 1.0 8.0 0.25

64 >128 0.016 0.063 0.25 1.0 1.0 4.0 1.0 0.016 0.063 1.0 2.0 8.0 32

128 >128 32 32 >128 >128 >128 >128 >128 32 32 >128 2.0 64 1.0

128 >128 32 32 >128 >128 128 >128 >128 32 32 >128 1.0 16 2.0

128 >128 64 64 128 >128 >128 >128 >128 64 64 >128 4.0 32 2.0

(3.80) nm; 1H and 13C NMR spectroscopic data, Table 2; (+)-HRESIMS m/z 1375.6580 [M + Na] + (calcd for C70H100N2O24Na, 1375.6558); (−)-HRESIMS m/z 1351.6646 [M − H]− (calcd for C70H99N2O24, 1351.6646). Microsporanate B (4): amorphous, white powder; [α]25D −49 (c 0.41, MeOH); UV (MeOH) λmax (log ε) 204 (3.88), 244 (3.56), 274 (3.79) nm; 1H and 13C NMR spectroscopic data, Table 2; (+)-HRESIMS m/z 1261.5876 [M + Na]+ (calcd for C64H90N2O22 Na, 1261.5877); (−)-HRESIMS m/z 1237.5909 [M − H]− (calcd for C64H89N2O22, 1237.5912). Microsporanate C (5): amorphous, white powder; [α]25D −33 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 203 (3.89), 242 (3.48), 274 (3.69) nm; 1H and 13C NMR spectroscopic data, Table 2; (+)-HRESIMS m/z 1277.5807 [M + Na] + (calcd for C64H90N2O23Na, 1277.5827); (−)-HRESIMS m/z 1253.5921 [M − H]− (calcd for C64H89N2O23, 1253.5862). Microsporanate D (6): amorphous, white powder; [α]25D −120 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.34), 243 (4.10), 274 (4.26) nm; 1H and 13C NMR spectroscopic data, Table 3; (−)-HRESIMS m/z 1321.6856 [M − H]− (calcd for C70H101N2O22, 1321.6851). Microsporanate E (7): amorphous, white powder; [α]25D −84 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 203 (4.30), 245 (4.08), 274 (4.30) nm; 1H and 13C NMR spectroscopic data, Table 3; (−)-HRESIMS m/z 1207.6190 [M − H]− (calcd for C64H91N2O20, 1207.6171). Microsporanate F (8): amorphous, white powder; [α]25D −51 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 203 (4.18), 244 (3.88), 275 (4.07) nm; 1H and 13C NMR spectroscopic data, Table 3; (−)-HRESIMS m/z 1223.6132 [M − H]− (calcd for C64H91N2O21, 1223.6120). Tetrocarcin P (9): amorphous, white powder; [α]25D −48 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 202 (4.08), 234 (3.83), 274 (3.62) nm; 1H and 13C NMR spectroscopic data, Table 1; (−)-HRESIMS m/ z 1167.5866 [M − H]− (calcd for C61H87N2O20, 1167.5858). Tetrocarcin A (10): amorphous, white powder; [α]25D −58 (c 0.20, MeOH); [α]21D −70 (c 0.10, acetone); the specific rotation data in acetone were consistent with those in the literature;16 1H and 13C NMR spectroscopic data, Table S1. Tetrocarcin B (11): amorphous, white powder; [α]25D −23 (c 0.18, MeOH), [α]19D −59 (c 0.13, acetone); the specific rotation data in acetone were consistent with those in the literature.16 AC6H (12): amorphous, white powder; [α]25D −85 (c 0.2, MeOH); the specific rotation data were consistent with those in the literature.18 Antibacterial Activity Assay. The antibacterial activities of the 12 compounds were evaluated using sequential 2-fold serial dilutions of each agent in Mueller-Hinton broth according to the previously reported protocol and standard methods provided by Clinical and Laboratory Standards Institute (CLSI).33 Five pathogenic bacteria

a

Vancomycin, kanamycin, and ampicillin served as positive controls. The tests were performed in triplicate.

chromatography to get seven subfractions (C1−C7). Fractions C2 and C3 were combined and further purified by semipreparative HPLC to give compounds 2 (5.0 mg, tR 22.4 min), 9 (7.0 mg, tR 14.0 min), and 12 (15.3 mg, tR 15.1 min). Fractions C4−C9 were further purified by semipreparative HPLC to yield compounds 6 (6.4 mg, tR 17.9 min), 7 (5.0 mg, tR 17.2 min), and 8 (4.5 mg, tR 15.9 min). 22-Dehydroxymethyl-kijanolide (1): amorphous, white powder; [α]25D +16 (c 0.24, MeOH); UV (MeOH) λmax (log ε) 204 (4.17), 241 (3.47), 267 (3.82) nm; 1H and 13C NMR spectroscopic data, Table 1; (+)-HRESIMS m/z 523.3060 [M + H]+ (calcd for C32H43O6, 523.3054); (+)-HRESIMS m/z 545.2887 [M + Na]+ (calcd for C32H42O6Na, 545.2874); (+)-HRESIMS m/z 1067.5871 [2M + Na]+ (calcd for C64H84O12Na, 1067.5855). 8-Hydroxy-22-dehydroxymethyl-kijanolide (2): amorphous, white powder; [α]25D −31 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.95), 240 (3.47), 267 (3.39) nm; 1H and 13C NMR spectroscopic data, Table 1; (+)-HRESIMS m/z 539.2995 [M + H]+ (calcd for C32H43O7, 539.3003); (+)-HRESIMS m/z 561.2820 [M + Na]+ (calcd for C32H42O7Na, 561.2823); (−)-HRESIMS m/z 537.2852 [M − H]− (calcd for C32H41O7, 537.2858). Microsporanate A (3): amorphous, white powder; [α]25D −60 (c 0.48, MeOH); UV (MeOH) λmax (log ε) 205 (3.91), 242 (3.57), 274 1602



Article

including B. subtilis BS01, B. thuringiensis BT01, methicillin-resistant S. aureus shhs-A1, S. aureus ATCC 29213, and E. faecalis ATCC 29212 were used in this study.

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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.7b00176. Spectra of 1D and 2D NMR for compounds 1−9 (PDF)

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AUTHOR INFORMATION

Corresponding Author

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

Hongbo Huang: 0000-0002-5235-739X 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 (81425022, U1501223, and 41476133), the Program of Chinese Academy of Sciences (XDA11030403), Natural Science Foundation of Guangdong Province (2016A030312014), and the Syngenta Ph.D. Fellowship awarded to C.G. We are grateful to Ms. Xiao, Ms. Sun, Ms. Zhang, and Mr. Li in the analytical facility at SCSIO for recording spectroscopic data.

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