Tetrocarcins N and O, glycosidic spirotetronates from a marine-derived Micromonospora sp. identified by PCR-based screening

Abstract

Two new glycosidic spirotetronate antibiotics, tetrocarcins N (1) and O (2), were isolated and identified from the marine-derived Micromonospora sp. 5-297 using a PCR-based genetic screening method targeting the dTDP-glucose-4,6-dehydratase gene. Their structures were determined by extensive IR, NMR, and MS spectroscopic analyses. Tetrocarcin O (2) is a derivative of 1 that lacks the terminal L-amicetose moiety at C-9. Compound 1 and 2 exhibited antibacterial activity against Bacillus subtilis with MIC values of 2 and 64 μg mL⁻¹, respectivly. It seems that the sugar moieties at C-9 and the formyl group at C-32 play important roles in the antibacterial activity of the tetrocarcins.

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Introduction

A large number of the antibiotics produced by microorganisms, such as aminocyclitols, anthracyclines, glycopeptides, enediynes, and macrolides, are glycosides. A recent analysis revealed that among the 15 940 bacterial natural products reported in the literature through early 2013, over one-fifth (3426 compounds) were glycosides, among which glycosylated macrolides and macrolactams represented the largest allocation (738 compounds, 21.5% of all bacterial glycosides).¹ The glycosidic residues are essential for their activity.² Most of the sugars appended in bacterial natural products are 6-deoxyhexoses.³ dTDP-glucose-4,6-dehydratase (dTGD) is a key enzyme that catalyzes the formation of the important intermediate, dTDP-4-keto-6-deoxy-D-glucose, in the early stage of most deoxysugars biosynthesis.⁴ Therefore, the dTGD gene can serve as a tool during genetic screening for diverse 6-deoxyglycosidic antibiotics.⁵ During a search for new antibiotics from marine-derived microorganisms, we used a molecular polymerase chain reaction (PCR)-based approach targeting the dTGD gene to identify potential glycosidic-antibiotic-producing strains.⁵ Guided by the PCR results, we previously isolated 6-deoxyhexose-containing elaiophylins from Streptomyces sp. 7-145.⁶ In the present study, the PCR amplification of the genomic DNA from marine-derived Micromonospora sp. 5-297 using the 4,6-dehydratase degenerate primers yielded two gene fragments, both of which showed high homology to the dTGDs of several types of antibiotics (Fig. S1, ESI†). Further chemical investigation of the cultures of strain 5-297 led to the isolation and characterization of two new glycosidic macrolides, tetrocarcins N (1) and O (2), together with three known analogues, tetrocarcins A (3), G (4), and H (5, Fig. 1). Tetrocarcins are a family of spirotetronate antibiotics consisting of an unusual polycyclic aglycone (tetronolide), a nitro sugar (tetronitrose) and four deoxysugars (two L-amicetose and two L-digitoxose residues).^7–9 Herein, we report the isolation, structure elucidation, and biological activities of these compounds.

Fig. 1 Structures of compounds 1–5.

Results and discussion

The strain 5-297 was isolated from a sediment sample collected from the Bohai Bay, Dalian, China. The strain was cultured for five days and then extracted using Amberlite XAD-7HP resin. The resulting extract was purified by a combination of silica gel, reversed-phase (RP) flash chromatography, Toyopearl gel, and preparative RP-HPLC to afford pure samples of compounds 1–5.

Tetrocarcin N (1) was isolated as a white powder with the molecular formula C₆₇H₁₀₀N₂O₂₄ as determined by HRESIMS. The IR spectrum showed absorption bands at 3434, 1736, and 1632 cm⁻¹, suggesting the presence of hydroxyl, ester, and amide carbonyl functionalities, respectively. The ¹H NMR spectrum of 1 in CDCl₃ (Table 1) displayed characteristic resonances ascribed to four olefinic protons [δ_H 5.74 (d, H-11), 5.40 (ddd, H-12), 5.16 (m, H-15), and 5.18 (d, H-19)], five glycosyl anomeric protons [δ_H 4.44 (dd, H-1_A), 4.83 (d, H-1_B), 4.88 (brd, H-1_C), 4.90 (dd, H-1_D), 4.92 (brs, H-1_E)], and 13 methyl protons. Analysis of the ¹³C NMR and DEPT spectra revealed the presence of 11 nonprotonated carbons (including one ketone carbonyl at δ_C 206.6, and three ester or amide carbonyls at δ_C 170.6, 167.2, and 157.4); four olefinic methine carbons at δ_C 126.3, 126.2, 123.2, and 118.5; 19 oxygenated methine carbons (including five anomeric carbons at δ_C 99.5, 98.6, 96.8, 92.7, and 92.0, representing five sugar moieties); eight aliphatic methine carbons; 12 methylene carbons (one oxygenated carbon at δ_C 67.1); and 13 methyl carbons. From the 19 degrees of unsaturation deduced from the molecular formula, five were assigned to glycosyl units, four to carbonyls, four to olefinic double bonds, and one to a nitro group. This indicates that 1 contains five ring systems. The NMR data of 1 showed good similarity with those of co-isolated tetrocarcin A (3),^8–11 suggesting that 1 is a tetrocarcin A analogue. The structure of 1 was further elucidated by 2D NMR data analysis.

Table 1 ¹H and ¹³C NMR data for compounds 1 and 2^a

No.	1 (CDCl3)		2 (CD3COCD3)
	δC, type	δH mult. (J in Hz)	δC, type	δH mult. (J in Hz)
a The assignments were based on ^1H–^1H COSY, TOCSY, HSQC and HMBC experiments.b nd: not detected.
1	167.2, C		167.3, C
2	100.9, C		100.1, C
3	206.6, C		203.4, C
4	51.1, C		51.9, C
5	43.2, CH	1.99, m	44.6, CH	2.16, m
6	31.3, CH	1.57, m	30.3, CH	1.60, m
7	41.7, CH2	1.57, m; 1.50, m	42.5, CH2	1.71, m; 1.50, m
8	34.7, CH	2.21, m	35.7, CH	2.22, m
9	84.5, CH	3.43, dd (10.8, 5.4)	85.5, CH	3.46, dd (10.8, 5.4)
10	38.6, CH	2.07, m	40.5, CH	2.11, m
11	126.3, CH	5.74, d (10.2)	126.4, CH	5.79, d (10.2)
12	126.2, CH	5.40, ddd (10.2, 4.8, 2.4)	128.4, CH	5.36, m
13	53.8, CH	3.35, d (4.8)	52.3, CH	3.67, m
14	136.0, C		136.3, C
15	123.2, CH	5.16, m	124.2, CH	5.33, m
16	31.0, CH2	2.29, 2H, m	31.1, CH2	2.50, m; 2.13, m
17	78.4, CH	4.25, m	79.0, CH	4.26, m
18	140.5, C		140.6, C
19	118.5, CH	5.18, d (10.2)	119.8, CH	5.21, d (10.2)
20	48.1, CH	2.76, t (10.2)	49.6, CH	2.66, t (10.2)
21	70.3, CH	4.05, td (10.2, 3.6)	71.1, CH	3.77, td (10.2, 4.2)
22	36.2, CH2	2.20, m; 1.32, m	37.6, CH2	2.10, m; 1.06, m
23	34.6, CH	2.18, m	35.7, CH	1.97, m
24	35.2, CH2	1.92, m; 1.73, m	37.1, CH2	2.02, m; 1.64, m
25	85.8, C		86.0, C
26	201.7, C		nd^b
27	15.1, CH3	1.61, s	16.0, CH3	1.70, s
28	22.2, CH3	0.61, d (5.4)	24.0, CH3	1.13, d (6.0)
29	14.1, CH3	1.08, d (7.2)	14.6, CH3	1.16, d (7.2)
30	14.2, CH3	1.34, s	13.8, CH3	1.31, s
31	16.0, CH3	1.48, s	16.5, CH3	1.65, s
32	67.1, CH2	3.66, dd (10.8, 6.6); 3.56, dd (10.8, 6.0)	67.4, CH2	3.42, m; 3.36, m
A-1	96.8, CH	4.44, dd (9.6, 1.8)	97.2, CH	4.62, brd (9.6)
A-2	36.0, CH2	2.72, brd (9.6); 1.64, m	36.4, CH2	2.60, brd (9.6); 1.80, m
A-3	91.4, C		92.2, C
A-4	53.9, CH	4.35, dd (10.2, 2.4)	54.8, CH	4.39, brd (10.2)
A-4-NH		5.11, d (10.2)		6.47, d (10.2)
A-5	69.2, CH	3.48, m	69.5, CH	3.54, m
A-6	17.1, CH3	1.15, d (6.0)	17.3, CH3	1.10, d (6.0)
A3-CH3	25.4, CH3	1.58, s	25.8, CH3	1.59, s
A4-NHCOOC̲H̲3̲	52.9, CH3	3.71, s	52.5, CH3	3.66, s
A4-NHC̲O̲OCH3	157.4, C		158.6, C
B-1	98.6, CH	4.83, d (4.8)	99.2, CH	4.83, d (4.8)
B-2	31.3, CH2	2.24, dd (14.4, 3.0); 1.79, m	31.9, CH2	2.33, m; 1.83, m
B-3	66.8, CH	4.16, m	67.3, CH	4.15, m
B-4	74.5, CH	4.58, dd (9.6, 3.0)	75.4, CH	4.51, dd (9.6, 3.6)
B-5	62.2, CH	4.36, m	62.8, CH	4.37, m
B-6	17.6, CH3	1.14, d (6.0)	17.9, CH3	1.10, d (6.0)
B4-OCOC̲H̲3̲	21.2, CH3	2.08, s	21.0, CH3	2.08, s
B4-OC̲O̲CH3	170.6, C		170.4, C
C-1	92.7, CH	4.88, brd (3.0)	93.0, CH	4.93, brd (2.4)
C-2	29.7, CH2	1.88, m; 1.75, m	30.2, CH2	1.86, m; 1.70, m
C-3	26.4, CH2	2.03, m; 1.97, m	27.4, CH2	1.99, m; 1.86, m
C-4	81.4, CH	3.21, td (9.6, 4.8)	81.2, CH	3.18, m
C-5	68.0, CH	3.73, m	68.7, CH	3.68, m
C-6	18.2, CH3	1.15, d (6.0)	18.5, CH3	1.11, d (6.0)
D-1	99.5, CH	4.90, dd (9.6, 1.8)	100.0, CH	4.89, brd (9.6)
D-2	37.1, CH2	2.15, dt (14.4, 1.8); 1.67, m	39.1, CH2	1.99, m; 1.60, m
D-3	64.0, CH	4.25, m	68.7, CH	4.03, m
D-4	75.3, CH	3.46, dd (9.6, 3.0)	73.7, CH	3.16, dd (9.6, 3.0)
D-5	67.9, CH	3.85, dq (9.6, 6.0)	70.1, CH	3.69, m
D-6	19.0, CH3	1.32, d (6.0)	18.8, CH3	1.20, d (6.0)
E-1	92.0, CH	4.92, brs
E-2	29.8, CH2	1.83, 2H, m
E-3	27.5, CH2	1.90, m; 1.74, m
E-4	71.9, CH	3.30, td (9.6, 4.8)
E-5	70.4, CH	3.63, dq (9.6, 6.0)
E-6	17.9, CH3	1.24, d (6.0)

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The ¹H–¹H COSY spectrum of 1 displayed a series of correlations establishing structure fragments consisting of H-5–H-13, H-15–H-17, and H-19–H-24 spin systems (Fig. 2). The HMBC correlations of H₃-27 (δ_H 1.61)/C-3, C-4, C-5, and C-13 and H-5/C-9, C-10, and C-11 further corroborated the structure of C-5–C-13 as an octahydronaphthalene ring. Furthermore, HMBC correlations of H-13/C-14, H-16/C-14 and C-18, and H-19/C-17 and C-18 allowed the construction of the skeleton of C-3–C-24. The HMBC correlations of H-24a and 24b/C-20, C-25, and C-26 indicated the presence of a cyclohexane ring between C-20 and C-25 and extended the polyketide chain from C-24 to C-26. Comparison of the NMR data of 1 and tetrocarcin A indicated that the double bond between C-22 and C-23 of the tetronolide⁷ aglycone in tetrocarcin A was replaced by a saturated single bond [C-22 (δ_C 36.2) and C-23 (δ_C 34.6)] in 1. Additionally, HMBC correlations from H-22a, 22b, 24a, and 24b to C-32 (δ_C 67.1) revealed that the formyl group (C-32) in tetrocarcin A was replaced by a hydroxymethyl group in 1. The characteristic chemical shifts of C-1 (δ_C 167.2), C-2 (δ_C 100.9), C-3 (δ_C 206.6), and C-26 (δ_C 201.7) suggested that the spiro γ-lactone moiety was retained in 1, akin to that of tetrocarcin A.¹¹ Finally, the five glycosyl moieties and their linkage positions were deduced to be the same as those of tetrocarcin A on the basis of NMR data and HMBC correlations.

Fig. 2 Key ¹H–¹H COSY (thick lines), HMBC (red arrows) and ROESY (blue arrows) correlations of 1.

The relative configuration of the cyclohexane ring moiety (C-20–C-25) was assigned by analysis of the ¹H NMR and ROESY data. The large vicinal coupling constant (10.2 Hz) between H-20 and H-21 established their anti relationship and, therefore, axial positions. The 1,3-ROESY correlation between H-21 and H-23 suggested their cis relationship and consequently an equatorial arrangement for the hydroxymethyl (C-32) group. In addition, the ROE correlations of H₃-27/H-6, H-10, and H-13; and H₃-29/H-10 revealed that these protons were oriented on the same face of the octahydronaphthalene ring, whereas the ROE correlations of H-9/H-5 and H-8; and H₃-28/H-5 indicated that they were on the opposite face of the ring. On the basis of this evidence, the relative configuration of the octahydronaphthalene ring in 1 could be assigned as the same as that of tetrocarcin A. Therefore, the relative structure of 1 was determined as shown in Fig. 1 and named as tetrocarcin N.

Tetrocarcin O (2) was isolated as a white solid. The (+)-HRESIMS-derived molecular formula, C₆₁H₉₀N₂O₂₂, 114 amu less than 1, indicates one missing sugar residue. The UV data and the NMR spectra of 2 in acetone-d₆ revealed its similarity to 1, except that the proton and carbon signals ascribed to the terminal L-amicetose (sugar E) were absent. The chemical shift of C-4_D was significantly shielded (δ_C 73.7), whereas the resonances of its neighbouring carbon C-3_D (δ_C 68.7) and C-5_D (δ_C 70.1) were deshielded, due to the attachment of the free hydroxy group at C-4_D of the sugar D moiety. The structure of 2 was confirmed by ¹H–¹H COSY, HSQC, HMBC, and ROESY experiments (Fig. S16–S20, ESI†), which exhibited the same aglycone moiety as 1 and the same sugar types and connections as found in other tetrocarcins. Therefore, the new compound 2 was named tetrocarcin O.

Compounds 1–3 showed very similar Cotton effects (CE) in their spectra, which all displayed a negative CE at 216 nm and a positive CE near 300 nm (Fig. S21†). However, the observed positive CE around 240 nm in the CD spectrum of 3, ascribed to the π → π* transition of the α,β-unsaturated aldehyde group,¹² was obscured in 1 and 2 due to lack of the double bond between C-22 and C-23. Comparison of the CD spectra of 1–3 indicated that they possess the same absolute configuration in the aglycone moiety. In addition, the configurations of the sugar units of 1 and 2 were presumed to be the same as those of 3 on the basis of biosynthetic considerations.

Tetrocarcins have demonstrated antibiotic activity against Gram-positive bacteria, as well as antitumor activity against sarcoma 180, P388 leukemia, and B16 melanoma.^8,9 Recent studies have suggested that tetrocarcins can induce the apoptosis of various tumour cells in a cell-type-dependent manner.^13–15 In this study, antibacterial activities were tested against Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 29213. Compounds 1–5 exhibited antibacterial activity against B. subtilis with minimum inhibitory concentrations (MICs) of 0.125–64 μg mL⁻¹ (Table 2). Remarkably, the new compound 1, which contains four sugar moieties at C-9, showed good activity against B. subtilis with an MIC of 2 μg mL⁻¹, whereas compound 2, which contains three sugar moieties at C-9, displayed weak activity with an MIC of 64 μg mL⁻¹. This result is consistent with the previously reported structure–activity relationship of tetrocarcins in which the antibacterial activity was proportional to the numbers of deoxy sugars.¹⁶ In comparison to tetrocarcin A (3, 0.125 μg mL⁻¹), the 32-hydroxymethyl (4, 2 μg mL⁻¹), and 32-carboxy (5, 64 μg mL⁻¹) derivatives suffered from dramatic decreases in activity, suggesting that the aldehyde group at C-32 is also essential to exert antibacterial properties. None of compounds isolated in this study except tetrocarcin A showed any significant inhibitory activity against S. aureus at 256 μg mL⁻¹. The cytotoxicities of 1–5 were evaluated against three human cancer cell lines, HepG2 (hepatocellular carcinoma), MCF-7 (breast adenocarcinoma), and K562 (leukemia), using the sulforhodamine B (SRB) assay method.¹⁷ None of the compounds was considered active (IC₅₀ > 10 μM).

Table 2 Antimicrobial bioassay results (MIC, μg ml⁻¹) of compounds 1–5

Compound	Bacillus subtilis	Staphylococcus aureus
1	2	>256
2	64	>256
3	0.125	64
4	2	>256
5	64	>256
Erythromycin	0.25	0.5

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Experimental section

General experimental procedures

Optical rotations were determined using a Perkin-Elmer model 343 polarimeter. UV spectra were obtained on a Shimadzu UV-2550 UV-vis spectrometer. CD spectra were recorded on an Applied Photophysics Chirascan spectropolarimeter. IR spectra were recorded on a Nicolet 5700 FT-IR microscope spectrometer (FT-IR microscope transmission). 1D and 2D-NMR spectra were obtained at 600 MHz for ¹H and 150 MHz for ¹³C, respectively, on a Bruker Avance III HD 600 MHz spectrometer in acetone-d₆ (δ_H 2.050 and δ_C 29.840) and CDCl₃ (δ_H 7.260 and δ_C 77.160) with solvent peaks used as references. HRESIMS data were obtained using a Thermo LTQ Orbitrap XL mass spectrometer. Preparative HPLC was conducted on an Agilent 1200 series (quaternary pump, autosampler, diode array detector, Agilent Technologies Ltd.). TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 7% H₂SO₄ in 95% aqueous EtOH followed by heating.

Strain isolation and characterization

A marine sediment sample was collected at Bohai Bay, Dalian, China. The wet sediment was dried overnight in a laminar. About 1 g of the dried ground-up sediment was added to 10 mL 0.1% sodium cholate solution in seawater and treated by vigorous vortexing for 20 min, thermal shock at 50 °C for 20 min and centrifugation at 500 g for 1 min. The resulting supernatant was streaked on an isolation medium (casein 1.0 g, starch 1.0 g, K₂HPO₄ 0.2 g, MgSO₄ 0.2 g, artificial sea salt 30.0 g, cycloheximide 100 mg, nalidixic acid 50 mg, agar 20 g, deionized water 1 L), giving rise to individual colonies of strain 5-297 after 4 weeks. On the basis of the 16S rDNA gene sequence (GenBank accession no. HM467159) analysis, strain 5-297 is most closely related to Micromonospora haikouensis (GenBank accession no. GU130129, 99.85% identity), identifying the strain as a Micromonospora sp.

PCR amplification of dTGD gene

Genomic DNA was extracted using a microwave-based method.¹⁸ PCR amplification of dTGD gene from genomic DNA was performed using the degenerated primers: forward primer (5′-GSGGSGSSGCSGGSTTCATSGG-3′) and reverse primer (5′-GGGWRCTGGYRSGGSCCGTAGTTG-3′) (R = A/G, W = A/T, Y = C/T, S = C/G).¹⁹ The PCR reaction mixture contained 20–50 ng of DNA, 400 pM of each primer, 10 μL 2 × PCR mixture (Dongsheng Biotech Co., Ltd.), and 8 μL ddH₂O in a total volume of 20 μL. Reactions were carried out at 96 °C for 1 min; 31 cycles of 96 °C for 30 s, 65 °C for 30 s, and 72 °C for 1 min; 72 °C for 5 min. The purified dTGD-like gene fragments were then sequenced and deposited in GenBank under accession no. HQ241421 and HQ241422.

Fermentation and isolation

Strain 5-297 was grown on ISP4 medium prepared with 3.0% artificial sea salt for about 10 days at 28 °C and then inoculated into 600 replicate 500 mL Fernbach flasks each containing 100 mL of sterile fermentation medium (composed of 10 g of starch, 25 g of glucose, 10 g of cottonseed flour, 3 g of peptone, 5 g of CaCO₃, 0.1 g of KH₂PO₄, 0.1 g of MgSO₄, and 30 g of artificial sea salt in 1 L of H₂O) and cultured on rotary shakers (200 rpm) at 28 °C for 5 days. The culture broth was centrifuged, and the resulted supernatant was subjected to an Amberlite XAD-7HP macroporous adsorbent resin column (6 L) and the column was washed with H₂O and then successively eluted with 50% and 100% aqueous acetone. The two latter fractions were combined and then concentrated under reduced pressure to afford a crude extract (21 g). The extract was applied to silica gel column chromatography eluting with a step-gradient of CH₂Cl₂–MeOH (100 : 0–0 : 100, v/v) on the basis of TLC results. The CH₂Cl₂–MeOH (20 : 1) eluting fraction (10.3 g) was further chromatographed on a reversed-phase C₁₈ flash column eluting with a linear gradient of MeOH–H₂O (50% to 80%). Fractions (0.97 g) from the 75–80% MeOH–H₂O elutions were combined and further separated by Toyopearl gel HW-40F (100% MeOH). The eluate was purified by a preparative C₁₈ HPLC (Agilent SB-C18 5 μm, 21.2 mm × 150 mm, CH₃CN–H₂O 53 : 47, 10 mL min⁻¹, 272 nm detection) to yield 1 (t_R: 53 min, 11.0 mg) and 2 (t_R: 36 min, 3.7 mg).

Tetrocarcin N (1). White amorphous powder; [α]²⁰_D −62.1 (c 0.80, Me₂CO); UV (MeOH) λ_max (log ε) 240 (3.12), 266 (3.07) nm; CD (c 7.6 × 10⁻⁴ M, MeOH) 216 (Δε −56.0), 297 (Δε +3.4) nm; IR ν_max 3434, 2934, 1736, 1632, 1545, 1453, 1374, 1240, 1052 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) and ¹³C NMR (CDCl₃, 150 MHz) data, Table 1; HRESIMS m/z 1339.6581 [M + Na]⁺ (calcd for C₆₇H₁₀₀N₂O₂₄Na, 1339.6558).

Tetrocarcin O (2). White amorphous powder; [α]²⁰_D −50.8 (c 0.25, Me₂CO); UV (MeOH) λ_max (log ε) 240 (3.09), 266 (3.02) nm; CD (c 4.2 × 10⁻⁴ M, MeOH) 216 (Δε −54.6), 297 (Δε +3.0) nm; IR ν_max 3421, 2935, 1712, 1680, 1624, 1544, 1421, 1381, 1204, 1052 cm⁻¹; ¹H NMR (acetone-d₆, 600 MHz) and ¹³C NMR (acetone-d₆, 150 MHz) data, Table 1; HRESIMS m/z 1225.5894 [M + Na]⁺ (calcd for C₆₁H₉₀N₂O₂₂Na, 1225.5877).

Antibacterial assay

Antibacterial activities against B. subtilis ATCC 6633 and S. aureus ATCC 29213 were evaluated using the broth microdilution method described by the Clinical and Laboratory Standards Institute (CLSI).²⁰ The test bacteria were grown on Mueller-Hinton (MH) agar at 37 °C for 24 h. Bacterial inocula were prepared in MH broth to achieve a turbidity equivalent to the 0.5 McFarland turbidity standard (about 1.5 × 10⁸ cfu per mL) for each strain and then diluted to a final density of 5 × 10⁵ cfu per mL. Bacterial cultures (99 μL) were inoculated into each well of 96-wells plates containing DMSO solutions (1 μL) of the tested compounds in a series of concentrations ranging from 256 to 0.125 μg mL⁻¹. The plates were incubated at 37 °C for 18 h. DMSO was used as a negative control, whereas erythromycin was used as a positive control. All experiments were performed in triplicate. The lowest concentration at which no bacterial growth was observed was recorded as the MIC.

Cytotoxicity assay

The cytotoxicity assay of 1–5 against the human cancer cells HepG2 (hepatocellular carcinoma cell), MCF-7 (breast adenocarcinoma cell), K562 (leukemia cell) was performed by using the SRB method as described previously.²¹ HepG2 and K562 cells were maintained in RPMI 1640 medium (Hyclone), and MCF7 cells were cultured in Eagle's minimum essential medium (MEM) with Earle's balanced salts solution (Hyclone). All media contained 100 units per mL of penicillin, 100 mg mL⁻¹ of streptomycin and 10% fetal bovine serum. For the cytotoxicity assays, cells were inoculated into 96-well plates at a concentration of 4000 cells per well. After incubation at 37 °C under a humidified atmosphere containing 5% CO₂ for 24 h, cells were treated with test compounds at eight different concentrations ranging from 300 to 0.1 μM in triplicate and further incubated for 48 h. Cell proliferation was determined by the SRB assay. The IC₅₀ value was defined as the compound concentration which produces 50% inhibition of cell growth during 2 days of compound treatment and calculated using SigmaPlot 10.0 software. Doxorubicin hydrochloride was used as a positive control.

Conclusions

In conclusion, we isolated and identified two new tetrocarcin analogues from marine-derived Micromonospora sp. 5-297 using a PCR-based genetic screening method targeting the dTDP-glucose-4,6-dehydratase gene. This genetic screening strategy was useful in identifying glycosidic antibiotics from microorganisms, and would be applicable for other microbial secondary metabolites with characteristic structures. Tetrocarcin N (1) showed potent antibacterial activity against B. subtilis. The antibacterial assay results revealed that the sugar moiety at C-9 and the formyl group at C-32 are essential structural features for the antibacterial activity of tetrocarcins.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Grant No. 81273414) and the National Science and Technology Project of China (Grant No. 2014ZX09507009-008 and 2012ZX09301002-003).

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Footnote

† Electronic supplementary information (ESI) available: MS, IR, and NMR spectra of compounds 1 and 2. See DOI: 10.1039/c6ra17026a

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