Catenulobactins A and B, Heterocyclic Peptides from Culturing

Aug 21, 2018 - The production of two new heterocyclic peptide isomers, catenulobactins A (1) and B (2), in cultures of Catenuloplanes sp. RD067331 was...
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Catenulobactins A and B, Heterocyclic Peptides from Culturing Catenuloplanes sp. with a Mycolic Acid-Containing Bacterium Shotaro Hoshino,† Masahiro Ozeki,† Takayoshi Awakawa,†,‡ Hiroyuki Morita,§ Hiroyasu Onaka,‡,⊥ and Ikuro Abe*,†,‡ †

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan § Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan ⊥ Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

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‡

S Supporting Information *

ABSTRACT: The production of two new heterocyclic peptide isomers, catenulobactins A (1) and B (2), in cultures of Catenuloplanes sp. RD067331 was significantly increased when it was cocultured with a mycolic acid-containing bacterium. The planar structures and absolute configurations of the catenulobactins were determined based on NMR/MS and chiral-phase GC-MS analyses. Catenulobactin B (2) displayed Fe(III)-chelating activity and moderate cytotoxicity against P388 murine leukemia cells.

A

RD067331 was significantly enhanced in the presence of the mycolic acid-containing bacterium Tsukamurella pulmonis TPB0596 (Figure 1A). Herein, we report the isolation and structural elucidation of two novel heterocyclic peptides, named catenulobactins A and B (1 and 2, Figure 1B), as well as the cytotoxicity and Fe(III)-chelating property of 2. Initially, we cultured the Catenuloplanes sp. RD067331 in the presence or absence of T. pulmonis TP-B0596 (a mycolic acidcontaining bacterium) and analyzed each metabolic profile by HPLC. Intriguingly, the production of two metabolites was significantly enhanced in the presence of the mycolic acidcontaining bacterium (1 and 2, Figure 1A). To determine their chemical structures, the combined-culture was scaled up (2.0 L), and the extract from the culture was subjected to a series of chromatographic purifications, yielding the catenulobactins A (1, 3.1 mg) and B (2, 2.4 mg). The molecular formula of catenulobactin A (1) was established as C27H30N4O9 based on HR-ESIMS analysis, which indicated 14 degrees of unsaturation. The 1D NMR (1H, 13C) and HMQC data revealed the presence of 27 carbons (Table 1), consisting of five carbonyls/imidate (δC 173.5−165.9), two aromatic carbons adjacent to an oxygen atom (δC 160.2 and 159.7), two sp2 nonprotonated carbons (δC 115.7 and 110.6), eight aromatic methines (δC 134.7− 117.1), six aliphatic methines adjacent to oxygen/nitrogen atoms (δC 79.5−52.5), three aliphatic methylenes (δC 45.0, 28.8, and 23.6), and two methyl groups (δC 21.1 and 17.2). The 1H NMR spectrum also displayed three exchangeable proton resonances, including typical amide NHs (δH 9.21 and

ctinomycetes are Gram-positive bacteria and prolific sources of bioactive compounds. Phylogenetically, actinomycetes are often divided into Streptomyces and nonStreptomyces (rare actinomycetes). In contrast to Streptomyces, which is the largest genus of actinobacteria and whose secondary metabolites have been extensively investigated,1,2 those from rare actinomycetes have been less exploited, except as described in some reports.3−5 Recent genome sequencing projects have revealed that rare actinomycetes are a rich source of cryptic natural product biosynthetic gene clusters,6,7 whereas the number of identified secondary metabolites is much less than that of natural product biosynthetic gene clusters. Considering that most of the gene clusters are likely not expressed in the traditional laboratory conditions, activation of these cryptic natural product biosynthetic gene clusters would lead to isolation of various novel compounds. Coculture is a simple but potent approach to activate the cryptic natural product biosynthetic gene clusters in certain microorganisms.8−11 In nature, microorganisms often interact via signaling molecules and/or physical contacts, which may trigger the production of secondary metabolites. Coculture is thought to mimic such an interaction factor and induce the expression of silent natural product biosynthetic gene clusters. Indeed, we previously demonstrated that the cryptic natural product biosynthetic gene clusters in several Streptomyces species were efficiently activated by the “combined-culture” with a mycolic acid-containing bacterium,8,9 which resulted in the isolation of 17 novel secondary metabolites.8 Recently, we also demonstrated that this method can be applied to rare actinomycetes.12,13 During screening of metabolites from the combined-culture with rare actinomycetes, we found that the production of secondary metabolites in Catenuloplanes sp. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 27, 2018

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DOI: 10.1021/acs.jnatprod.8b00261 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. NMR Data for Catenulobactins A (1) and B (2) Recorded in d6-DMSO (1H: 500 MHz, 13C: 125 MHz) catenulobactin A (1) position 1 1-OH 2 3 4 5 6 7 8 9 10 11 12(NH) 13 14

159.7, C

15 16

23.6, CH2 45.0, CH2

17 18 19

173.5, C 167.6, C 57.7, CH

20(NH) 21

78.6, CH

22 23 24 25 26 27 28 29 29-OH

Figure 1. (A) HPLC chromatograms of crude extracts from Catenuloplanes sp. cocultured with a mycolic acid-containing bacterium (top), the pure culture of Catenuloplanes sp. (bottom), and the pure culture of the mycolic acid-containing bacterium (bottom). (B) Chemical structures of 1 and 2 (Sal: salicylic acid, MeOzn: methyloxazoline, OHOrn: N-hydroxyornithine).

δC, type

a−g

117.1, CH 134.7,a CH 119.6, CH 128.6, CH 110.6, C 165.9, C 73.9, CH 169.9, C 79.5, CH 21.1, CH3 52.5, CH 28.8, CH2

17.2, CH3 169.4, C 115.7, C 128.8, CH 119.3, CH 134.6,a CH 118.0, CH 160.2, C

catenulobactin B (2)

δH (J in Hz)

δC, type

δH (J in Hz)

159.8,c C (11.79, s)h 6.96, d (7.5) 7.43,b dd (7.5) 6.90, m 7.59, d (7.5)

117.1, CH 134.6,d CH 119.6,e CH 128.6,f CH 110.6, C 165.9, C 4.53, d (7.5) 73.9, CH 169.8, C 4.84, dq (7.5, 6.5) 79.5, CH 1.43, d (6.5) 21.0, CH3 8.42, brd (7.5) 4.18, m 52.6, CH 1.79, m 29.0, CH2 1.69, m 1.61−1.57, m 23.2, CH2 3.56, m 47.8, CH2 3.45, m 173.7, C 169.5, C 4.70, dd (10.5, 70.8, CH 7.5) 9.21, d (7.5) 4.42, dq (10.5, 79.3, CH 6.0) 1.35, d (6.0) 20.9, CH3 165.9, C 110.6, C 7.84, d (8.0) 128.4,f CH 6.89, m 119.5,e CH b 7.40, dd (7.5) 134.5,d CH 6.91, m 117.1, CH 158.7,c C h (11.79, s)

12.02,g s 6.97, m 7.43, m 6.91, m 7.59, m

4.52, d (7.5) 4.81, m 1.42, d (6.5) 8.39, brd (7.0) 4.18, m 1.75, m 1.60, m 1.63−1.58, m 3.65, m 3.45, m

5.02, d (5.5)

4.91, m 1.42, d (6.5)

7.59, 6.91, 7.43, 6.97,

m m m m

11.79,g s

h

Assignments are interchangeable. The signal at 11.79 ppm is assigned to either 1-OH or 29-OH.

8.42) and a phenolic OH (δH 11.79). The presence of two sets of o-substituted benzene rings was inferred from the COSY correlations (Figure 2, H-2/H-3/H-4/H-5 and H-25/H-26/H27/H-28), and both were confirmed as salicylic acids (Sal1 and Sal2 in Figure 1B) based on the HMBC correlations from aromatic protons (Figure 2). Further NMR analyses unveiled the presence of three amino acid residues (Thr1, Thr2, and Orn, Figure 1B). The Thr1 residue was clearly indicated by the COSY correlations of H-8/H-10/H3-11 and the HMBC correlations of H-8/C-9 and H-10/C-9. Considering the downfield-shifted 13C NMR resonance at C-8 (δC 73.9, Figure S15)14,15 and the absence of an NH signal in the 1H NMR, the C-8 position is thought to be adjacent to an imine nitrogen atom (−CN−C) rather than to an amide nitrogen (C( O)−NH−C) (Figure S15),14,15 and the Thr1 residue was assumed to form a methyloxazoline (MeOzn) ring. Likewise, the Thr2 residue was identified by the COSY correlations of NH-20/H-19/H-21/H3-22 and the HMBC correlation of H19/C-18 (Figure 2). The Orn residue was inferred from the COSY correlations of NH-12/H-13/H-14/H-15/H-16 and the HMBC correlations of H-13/C-17 and H-14/C-17 (Figure 2). The connection of each substructure was completely determined based on further HMBC analyses. The HMBC

Figure 2. Key HMBC and COSY correlations in 1 and 2.

correlations from two NH protons (NH-12/C-9 and NH-20/ C-23) clearly indicated the connection of Thr1/Orn and Thr2/Sal2 through amide bonds (Figure 2). In addition, the HMBC correlations of H-8/C-7 and H-10/C-7 established the connection of Sal1/Thr1 through methyloxazoline ring B

DOI: 10.1021/acs.jnatprod.8b00261 J. Nat. Prod. XXXX, XXX, XXX−XXX

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formation. Finally, the HMBC correlation of H-16/C-18 revealed the amide bond formation between the Orn and Thr2 residues. The above observations could explain 13 out of the 14 degrees of unsaturation in 1, indicating that 1 has one more ring system. Based on the molecular formula and the absence of an NH signal at the side chain of the Orn residue, the final ring system was proposed to be an isoxazolidinone ring between the Orn and Thr2 residues, which contains an N−O linkage (Figure 1B). The assignment of the isoxazolidinone structure was further confirmed by the 13C NMR resonances of the Thr2 residue, which includes characteristic upfield-shifted α-carbon (C-19, δC 57.7) adjacent to an amide-type nitrogen (C(O)−NH−C) (Figure S15).14,15 Finally, we analyzed the fragment pattern in the ESIMS/MS spectrum of 1, which supported the proposed structure of 1 (Figure S16). The molecular formula of catenulobactin B (2) was also established as C27H30N4O9, based on the HR-ESIMS analysis. The NMR spectra and the fragment pattern of the ESIMS/MS spectrum of 2 were similar to those of 1, and the detailed spectroscopic analyses indicated the presence of two salicylic acids, two Thr, and Orn substructures with the same sequences as in 1 (Figures 1B, 2, and S17). In contrast, the 13C NMR resonances of the Thr2 residue were quite different from those of 1 (Figure S15), and the 1H NMR signal corresponding to NH-20 was not observed. Therefore, the Thr2 and Sal2 residues were expected to be connected through the methyloxazoline ring in 2, and this was further supported by the HMBC correlation of H-21/C-23 (Figure 2). Moreover, to satisfy the molecular formula of 2, a hydroxy group should be attached to the nitrogen atom in the side chain of the Orn residue (OHOrn, Figure 1B). The relationship between 1 and 2 resembles that of acinetobactin and preacinetobactin, which are bacterial siderophores produced by Acinetobacter baumannii (Figure 3A).16−18 Walsh and co-workers reported that acinetobactin was initially biosynthesized and released from a nonribosomal peptide synthase (NRPS) assembly line, as the preacinetobactin containing an N-OH oxazoline structure.17 However, the N-OH oxazoline structure was unstable under physiological conditions (aqueous, pH > 7) and rapidly isomerized to acinetobactin with an isoxazolidinone structure by the attack of a hydroxamate oxygen to the oxazoline ring in an SN2 manner (Figure 3A).17,18 Therefore, the isoxazolidinone ring of 1 was likely derived from the N-OH oxazoline structure of 2 by nonenzymatic rearrangement (Figure 3B). To test our hypothesis, we incubated the purified compound 2 in phosphate buffer (pH = 7.0). As expected, 2 was indeed rapidly isomerized to 1 (Figure 3C), suggesting that 1 is likely an artifact created from 2. To determine their absolute configurations, 1 and 2 were subjected to total acid hydrolysis and subsequent chiral-phase GC-MS analysis. Under the hydrolysis conditions, the MeOzn/ OHOrn moieties were converted to Thr/Orn with retention of the stereochemistry (Figure S18). The amino acids in the hydrolysates were then derivatized to N-trifluoroacetyl methyl esters (Figure S18). Each hydrolysate was subsequently subjected to chiral-phase GC-MS analysis, and the retention time was compared with those of amino acid standards derivatized in the same manner. As a result, L-Thr and D-Orn were detected in both hydrolysates (Figure S19), indicating that all of the MeOzn units are derived from L-Thr, and the Orn residues have the D-configuration in 1 and 2. Considering the mechanism of the isoxazolidinone ring formation in 1

Figure 3. (A) Proposed mechanism of preacinetobactin isomerization to acinetobactin. The hydroxamate oxygen attacks the adjacent oxazoline ring in an SN2 manner. (B) Proposed mechanism of the isomerization of 2 to 1 in 0.1 M phosphate buffer (pH = 7.0). (C) HPLC chromatograms of 2 incubated in 0.1 M phosphate buffer for 5, 40, 80, and 150 min. (D) Key NOESY correlations and vicinal coupling constants in the isoxazolidinone moiety of 1.

(Figure 3B), the absolute configuration of C-19 should be retained between 1 and 2, while that of C-21 was expected to be inverted. Consistent with this expectation, the large coupling constant between H-19 and H-21 and the NOESY correlation of H-19/H3-22 indicated the 1,2-trans relationship between H-19 and H-21 (Figure 3D). Therefore, the absolute configuration of the isoxazolidinone ring in 1 was determined to be (19S, 21S). Since the chemical structures of catenulobactins are closely related to those of known bacterial siderophores,16,19,20 we expected that the catenulobactins would also act as Fe(III) chelators. To test the Fe(III)-binding activities, 1 and 2 were treated with an excess amount of tris(2,4-pentanedionato)iron(III) (Fe(acac)3) and analyzed by LC-MS. As expected, 2 formed a 1:1 Fe(III) complex, which displayed the characteristic 56Fe- and 54Fe-containing positive ion species (Figures 4 and S20). In contrast, 1 did not show a noticeable m/z change upon treatment with Fe(acac)3 (Figure S21), indicating that the N-OH ornithine and oxazoline moieties of 2 are essential for the Fe(III)-binding activity, as described in the previous structure−function studies of acinetobactin analogues.14 We also evaluated the cytotoxicities of 1 and 2 against P388 murine leukemia cells by the methylthiazole tetrazolium (MTT) assay.21 2 showed moderate cytotoxicity against P388 murine leukemia cells, with an IC50 of 22.4 μM, while 1 did not exhibit any cytotoxicity up to a 100 μM concentration. In conclusion, we identified two novel heterocyclic peptides, catenulobactins A (1) and B (2), by combined-culture. To the best of our knowledge, the catenulobactins are the first secondary metabolites identified from the genus CatenuloC

DOI: 10.1021/acs.jnatprod.8b00261 J. Nat. Prod. XXXX, XXX, XXX−XXX

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1.0% HP-20 resin, pH = 7.2) and shaken under the same conditions for 5 days. After the fermentation, a 10 mL portion of the culture broth was lyophilized, extracted with 10 mL of an equal mixture of methanol−chloroform, and concentrated in vacuo. The residual material was dissolved in 0.5 mL of extractant, and an aliquot (10 μL) was analyzed by HPLC with a Cosmosil 5C18-MS-II column (4.6 × 250 mm, Nacalai Tesque, Japan) in a CH3CN (solvent A)−0.05% formic acid (solvent B) gradient system (solvent A: 20% (5 min), 70% (12 min), 100% (15−25 min), 1.0 mL min−1). In parallel, we also prepared the pure cultures of Catenuloplanes/Tsukamurella, and their metabolic profiles were analyzed in the same manner. The production levels of 1 and 2 were evaluated based on the peak areas of HPLC chromatograms detected at 300 nm. Under the combined-culture conditions, the production of 1 increased 12.9-fold and the production of 2 increased 19.4-fold, as compared with the pureculture conditions. Examination of the temporal production of 1 and 2 during a 5-day cultivation revealed that the formation of 1 and 2 was not observed at 48, 72, and 96 h. Isolation of Catenulobactins. Catenuloplanes sp. RD067331 was combined-cultured with a mycolic acid-containing bacterium on a large scale (100 mL × 20), as described above. The mixture of the cell pellet and HP-20 resin was collected by centrifugation (6000 rpm), lyophilized, and extracted with a 1:1 mixture of methanol−chloroform (1.0 L). Evaporation of the organic solvent gave a crude extract (3.6 g), which was subjected to flash silica gel column chromatography (19 × 320 mm, methanol−chloroform, 0/100, 10/90, 20/80, 50/50, and 100/0, 50 mL each). Both 1 and 2 were found in the 50/50 methanol−chloroform fraction, and they were further purified by semipreparative reverse-phase HPLC with a Cosmosil C18-ARII column (10 × 250 mm, Nacalai Tesque, Japan) in a CH3CN (solvent A)−0.05% formic acid (solvent B) gradient system (solvent A: 55− 65% (12 min), 100% (13−16 min), 3.0 mL min−1). The final yields of 1 (tR = 8.8 min) and 2 (tR = 10.2 min) were 3.1 and 2.4 mg, respectively. Catenulobactin A (1): white powder; [α]25D −19.9 (c 0.033, MeOH); UV (MeOH) λmax (log ε) 206, 238, 303 nm; IR (KBr) νmax 3394, 2980, 2362, 2339, 1683, 1640, 1451, 1309, 1256, 1229, 1074, 1028, 828, 758 cm−1; 1H and 13C NMR data, Table 1; HR-ESIMS m/ z 555.2075 [M + H]+ (calcd. for C27H31N4O9, 555.2092). Catenulobactin B (2): white powder; [α]25D +55.7 (c 0.023, MeOH); UV (MeOH) λmax (log ε) 206, 244, 306 nm; IR (KBr) νmax 3393, 2925, 2361, 2334, 1721, 1641, 1493, 1454, 1258, 1231, 1074, 1036, 825, 758 cm−1; 1H and 13C NMR data, Table 1; HRESIMS 555.2071 [M + H]+ (calcd for C27H31N4O9, 555.2092). Isomerization of 2 to 1 in Phosphate Buffer. Compound 2 (0.1 mg) was placed into a microtube containing 0.1 M phosphate buffer (0.1 mL, pH = 7.0) and incubated at 30 °C. After 5, 40, 80, and 150 min of incubation, 10 μL portions of the solution were subjected to an HPLC analysis with a Cosmosil 5C18-MS-II column (4.6 × 250 mm, Nacalai Tesque, Japan) in a CH3CN (solvent A)−0.05% formic acid (solvent B) gradient system (solvent A: 20% (5 min), 70% (12 min), 100% (15−25 min), 1.0 mL min−1), to monitor the conversion from 2 to 1. Chiral-Phase GC-MS Analysis. In a sealed tube, each catenulobactin (0.1 mg) was completely hydrolyzed by heating in 6 M HCl (0.4 mL) at 110 °C for 12 h, and the resulting hydrolysate was lyophilized to dryness. The hydrolysate was then derivatized with a methyl ester group by heating in 5−10% HCl−MeOH reagent (0.4 mL, Tokyo Chemical Industry Co., Ltd.) at 110 °C for 30 min. After the solvent was removed by evaporation, the hydrolysate was further derivatized with an N-trifluoroacetyl (N-TFA) group, by boiling in an equal mixture of trifluoroacetic acid anhydride and CH2Cl2 (total 0.4 mL) at 110 °C for 5 min. The reaction mixture was dried under an argon atmosphere, and the residue was dissolved in acetone (0.3 mL) and used as the sample for the chiral-phase GC-MS analysis. All of the amino acid standards (DL-Thr, DL-allo-Thr, and DL-Orn) were derivatized with a methyl ester−N-TFA in the same manner. Chiral-phase GC-MS analyses were performed with a CP-ChirasilDEX column (Alltech, 0.25 mm × 25 m; He as the carrier gas;

Figure 4. LC-MS charts of (i) total ion chromatogram (TIC) of 2 treated with Fe(acac)3, (ii) extracted ion chromatogram (EIC) of 2 treated with Fe(acac)3 at m/z 555.2 ([M + H]+), (iii) EIC of 2 treated with Fe(acac)3 at m/z 608.1 ([M + 56Fe − 2H]+), (iv) TIC of 2 (untreated), (v) EIC of 2 (untreated) at m/z 555.2, and (vi) EIC of 2 (untreated) at m/z 608.1.

planes, illustrating the broad applicability of our combinedculture strategy. Intriguingly, 2 formed the Fe(III) complex, suggesting its functional role as a bacterial siderophore. From the perspective of competition for environmental Fe(III) species, it is reasonable that Catenuloplanes sp. increased the production of 2 in response to a mycolic acid-containing bacterium. Although the detailed mechanism of the formation of the siderophore still remains to be elucidated, we propose that physical interactions with a mycolic acid-containing bacterium is crucial, and this triggers the catenulobactin biosynthesis as in the case of previously reported cryptic natural product biosynthetic gene clusters.8,9

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained with a JASCO DIP-1000 digital polarimeter. The UV spectra were measured by a Shimadzu UV-1280. Infrared spectra were recorded with KBr pellets on a Jasco FT/IR-460 Plus spectrometer. All NMR analyses were performed on a JEOL ECX-500 spectrometer (1H NMR, 500 MHz; 13C NMR, 125 MHz). The HR-ESIMS and ESIMS/MS spectra were obtained with a Bruker Compact QqTOF mass spectrometer. Bacterial Strains. Catenuloplanes sp. RD067331 was purchased from the National Institute of Technology and Evaluation (NITE). The characterization of Tsukamurella pulmonis TP-B0596 was previously reported.11 Monitoring the Production of Catenulobactins. Initially, Catenuloplanes sp. RD067331 and T. pulmonis TP-B0596 were separately precultured in 500 mL flasks containing 100 mL of V-22 medium11 up to full growth (Catenuloplanes: 3 days, Tsukamurella: 2 days) on a rotary shaker at 160 rpm and 30 °C. Then, aliquots of each preculture (Catenuloplanes: 3 mL, Tsukamurella: 0.3 mL) were combined in a 500 mL baffled flask containing production medium (2.5% starch, 1.5% soy flour, 0.2% yeast extract, 0.4% CaCO3, and D

DOI: 10.1021/acs.jnatprod.8b00261 J. Nat. Prod. XXXX, XXX, XXX−XXX

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program rate: 50−200 °C at 4 °C/min). As a result, L-Thr and D-Orn were confirmed to be present in both hydrolysates of 1 and 2. Monitoring the Fe(III)-Binding Activity of Catenulobactins. Methanol solutions of compounds 1 and 2 (50 μg dissolved in 50 μL of methanol) were treated with 2.0 equiv of tris(2,4-pentanedionato)iron(III) (Fe(acac)3, 64 μg dissolved in 50 μL of methanol). Each mixture was incubated at room temperature for 1 h and analyzed by LC-MS with a Cosmosil 2.5C18-MS-II column (2.0 × 75 mm, Nacalai Tesque, Japan) in a CH3CN (solvent A)−20 mM formic acid (solvent B) gradient system (solvent A: 20% (3 min), 100% (15−20 min), 0.2 mL min−1). As references, methanol solutions of 1 and 2 (untreated with Fe(acac)3) were also analyzed by LC-MS in the same manner. Cytotoxicity Assay. The cytotoxicities of 1 and 2 against P388 murine leukemia cells were evaluated using the MTT assay.21 P388 murine leukemia cells were cultured in RPMI 1640 (Wako Chemicals) medium, containing 10 μg/mL of penicillin−streptomycin and 10% fetal bovine serum (MP Biomedicals), at 37 °C under a 5% CO2 atmosphere. The methanol solutions of 1 and 2 (100 μL each; 100, 50, 25, 12.5, 6.3, 3.1, 1.6, and 0.78 μM) were added to the wells of a 96-well microplate containing 100 μL of 1 × 105 cells/mL tumor cell suspension. The plates were incubated for 4 days at 37 °C under a 5% CO2 atmosphere, and then 50 μL of MTT solution (1 mg/mL in DMSO) was added. The plates were further incubated for 4 h under the same conditions. The plates were centrifuged, and the precipitates were dissolved in 50 μL of DMSO. Finally, the absorbance at 570 nm was measured with a microplate reader. As a positive control, we employed doxorubicin, and its IC50 value was determined to be 5.2 μM.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00261.

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Note

Copies of NMR and ESIMS/MS spectra of catenulobactins A and B (1 and 2), 13C NMR resonances of isoxazolidinone/oxazoline structures in 1 and 2 compared with those of reference compounds, graphical outline of acid hydrolysis and chiral-phase GC-MS analysis, GC-MS chromatograms, HR-ESIMS spectrum of 2-Fe(III) complex, and LC-MS chromatograms of 2 treated with Fe(acac)3 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +81-3-5841-4740. Fax: +81-3-5841-4744. E-mail: abei@ mol.f.u-tokyo.ac.jp. ORCID

Ikuro Abe: 0000-0002-3640-888X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (JSPS KAKENHI Grant Nos. JP16H06443 and JP18H02120), JST/NSFC Strategic International Collaborative Research Program, and JSPS Research Fellowships for Young Scientists (to S.H.). We thank Dr. S. Asamizu (The University of Tokyo) for helpful advice and the maintenance of Tsukamurella pulmonis TP-B0596. E

DOI: 10.1021/acs.jnatprod.8b00261 J. Nat. Prod. XXXX, XXX, XXX−XXX