Bioactive Polycyclic Tetramate Macrolactams from Lysobacter

Article pubs.acs.org/jnp

Bioactive Polycyclic Tetramate Macrolactams from Lysobacter enzymogenes and Their Absolute Configurations by Theoretical ECD Calculations Liangxiong Xu,†,‡ Ping Wu,† Stephen J. Wright,‡ Liangcheng Du,*,‡ and Xiaoyi Wei*,† †

Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China ‡ Department of Chemistry, University of Nebraska−Lincoln, Lincoln, Nebraska 68588, United States S Supporting Information *

ABSTRACT: Two new polycyclic tetramate macrolactams, lysobacteramides A (1) and B (2), together with HSAF (heat-stable antifungal factor, 3), 3-dehydroxy HSAF (4), and alteramide A (5) were isolated from a culture of Lysobacter enzymogenes C3 in nutrient yeast glycerol medium. Their structures were determined by MS and extensive NMR analysis. The absolute configurations of 1−5 were assigned by theoretical calculations of their ECD spectra. Although HSAF and analogues were reported from several microorganisms, their absolute configurations had not been established. The isolation and the absolute configurations of these compounds revealed new insights into the biosynthetic mechanism for formation of the polycycles. Compounds 1−4 exhibited cytotoxic activity against human carcinoma A549, HepG2, and MCF-7 cells with IC50 values ranging from 0.26 to 10.3 μM. Compounds 2 and 3 showed antifungal activity against Fusarium verticillioides with IC50 value of 47.9 and 6.90 μg/mL, respectively.

H

polyene-containing, partially cyclized intermediates due to their limited stability and yield. Information on the structure of these potential intermediates is valuable in understanding the biosynthetic mechanism for this distinct group of natural products. Moreover, L. enzymogenes produces HSAF and related analogues only in nutrient-depleted media, such as 1/ 10 TSB, but not in rich media, such as the full-strength TSB.1 HSAF is the end product of the biosynthetic pathway, and we hypothesized that some intermediates might be isolable under certain growth conditions. We thus tested various culture conditions to see if new metabolites would be accumulated in the culture. Configuration is often crucial to the biological activity of chiral molecules and hence needed to be elucidated before further biological and chemical studies. However, the absolute configuration of HSAF and related analogues has not been assigned, except carbon-2, which was assigned the S configuration on the basis of the biosynthetic origin of the tetramic acid moiety.1,9,13 In this work, we report the isolation, structure determination, and bioactivity of two new (1, 2) and three known (3−5) HSAF analogues and the absolute configuration of 1−5 by quantum chemical calculation of ECD spectra. The new insights into the biosynthetic mechanism revealed by these metabolites are also discussed.

SAF (heat-stable antifungal factor) was isolated as a major metabolite from Lysobacter enzymogenes C3, a Gram-negative bacterium used in the biological control of fungal diseases in crop plants.1 It is a polycyclic tetramate macrolactam (PTM),2 which is distinct from any existing antifungal drug or fungicide. HSAF acts by inhibiting the polarized growth of filamentous fungi by disrupting the biosynthesis of sphingolipids.3 The structure of sphingolipids in fungal cells is distinct from that in mammalian cells, and none of the existing antifungal drugs and fungicides target sphingolipids.4 The distinct structure and novel mode of action of HSAF and related analogues have made these compounds attractive subjects of current research.1,2,5−8 We have been studying the biosynthesis of HSAF and have proposed a biosynthetic pathway.1,9−13 However, not all details of the biosynthesis are clear, particularly the mechanism for formation of the polycycles from the polyene intermediates that are biosynthesized by HSAF-PKS/NRPS, a hybrid polyketide synthase/nonribosomal peptide synthetase.12,13 HSAF-PKS/ NRPS is a highly unusual enzyme in that it contains only a single set of functional domains of the typical bacterial modular PKS and NRPS and yet is able to synthesize two separate polyene chains and to assemble a hexaene-ornithine-hexaene intermediate.13 The intermediates are covalently linked to the PKS/NRPS and eventually lead to the polycyclic structure through the action of a series of yet-to-be characterized tailoring enzymes.12 It has been challenging to isolate the © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 30, 2015

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DOI: 10.1021/acs.jnatprod.5b00099 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Article

RESULTS AND DISCUSSION

Table 1. NMR Spectroscopic Data (400 MHz) for 1 and 2 in DMSO-d6

In the course of analyzing cultures of L. enzymogenes C3 by HPLC-MS, we observed cultures in media rich in glycerol such as NYG medium (2% glycerol, 0.5% peptone, 0.3% yeast extract) generating several new peaks. These new peaks contained metabolites with a molecular mass around 478− 510 Da, which is close to but differs from HSAF (3),1 3-deOHHSAF (4),10 and alteramide A (5).14 EtOAc extracts of NYG cultures were fractionated by ODS column chromatography and HPLC to furnish two new PTMs, lysobacteramides A (1) and B (2), together with HSAF (3), 3-deOH-HSAF (4), and alteramide A (5). The structures of 3−5 were confirmed by analyzing their spectroscopic data and comparing these with the reported spectroscopic data and HPLC retention time of reference compounds.

1 position

δC, type

1 2 3

195.3, C 61.8, CH 28.8, CH2

4

25.0, CH2

5

38.0, CH2

6

Lysobacteramide A (1), obtained as colorless needles, had a molecular formula of C 29 H 38 N 2 O 4 , as determined by HRESIMS. The 1H and 13C NMR data (Table 1), assigned by HSQC, 1H−1H COSY, and HMBC spectra (Figure 1), showed the presence of two active protons [δH 8.92; 8.09 (each 1H, br s)], four carbonyl groups [δC 195.3, 185.4, 175.6, 165.7], four double bonds [δC 138.8, 137.0, 135.1, 134.5, 130.0, 127.8, 125.2, 123.8; δH7.08 (dd, J = 14.8, 12.0 Hz, 1H), 6.20 (dd, J = 12.0, 10.5 Hz, 1H), 6.13−6.00 (m, 2H), 5.92 (d, J = 14.8 Hz, 1H), 5.66 (t, J = 10.5 Hz, 1H), 5.47 (ddd, J = 11.2, 10.5, 4.8 Hz, 1H), 5.29 (dd, J = 13.9, 9.8 Hz, 1H)], and two methyl groups [δH 1.07 (d, J = 6.0 Hz, 3H); 0.86 (t, J = 7.2 Hz, 3H)]. The 1 H−1H COSY correlations, especially those between H-12/19, H-14/18, and H-16/H-17, and the HMBC correlations from H-5 to C-7 and from H-2 to both C-1 and C-27 (Figure 1) indicated that 1 also contains a 5/5-bicyclic unit and a tetramic acid ring system similar to those in alteramide A (5), the main PTM obtained from this strain. The configurations of double bonds were assigned as 8E, 10Z, 20E, and 22Z from the proton coupling constants, J8,9 = 14.8 Hz, J10,11 = 10.5 Hz, J20,21 = 13.9 Hz, and J22,23 = 11.2 Hz, respectively. Although 1 shares the same carbon skeleton with 5, it does not have hydroxy groups at C-3 and C-20 as 5. Furthermore, besides the difference in configurations of double bonds (two E and two Z in 1 vs all E in 5), the 21,23-diene in 5 is shifted to a 20,22-diene in 1, disconnecting the diene from the conjugation system of the tetramate ring, which is unusual in PTM compounds.

7 8 9

165.7, C 125.2, CH 134.5, CH

10

127.8, CH

11 12

138.8, CH 48.1, CH

13

39.3, CH2

14 15

41.4, CH 40.5, CH2

16 17 18

54.0, CH 46.9, CH 57.3, CH

19

56.5, CH

20

137.0, CH

21

130.0, CH

22 23

135.1, CH 123.8, CH

24

25 26 27 28 29 30 31 28-Me

37.2, CH2

2 δH, mult. (J in Hz)

3.83 d (4.6) 1.63 m a 1.42 m b 1.03 m a 3.60 m b 2.51 m 8.09 dd (7.6, 4.2) 5.92 d (14.8) 7.08 dd (14.8, 12.0) 6.20 dd (12.0, 10.5)

12.9, CH3

194.7, C 66.3, CH 24.5, CH2 20.3, CH2 38.5, CH2

166.0, C 124.6, CH 139.5, CH

δH, mult. (J in Hz) 3.82 d (5.0) a 2.06 m b 1.72 m a 1.27 m b 1.02 m a 3.26 m b 2.36 m 7.89 t (5.2)

5.66 t (10.5) 3.26 dt (10.5, 5.3) α 1.80 dd (12.4, 8.0) β 1.53 m 2.67 m α 2.06 ddd (12.5, 7.3, 5.8) β 0.90 m 1.38 m 1.19 m 1.75 dd (10.4, 8.0) 2.43 ddd (10.4, 9.8, 6.0) 5.29 dd (13.9, 9.8) 6.06 m

43.9, CH 48.0, CH

5.73 d (11.5) 5.91 dd (11.5, 2.0) α 1.93 br d (15.8) β 3.50 ddd (15.8, 10.8, 2.0) ax 1.28 m ax 1.65 m

40.9, CH2

α 0.81 m

41.9, CH 37.7, CH2

β 2.02 m 2.35 m α 1.98 m

54.0, CH 46.9, CH 58.5, CH

β 0.87 m 1.33 m 1.28 m 1.77 m

59.6, CH

ax 1.10 m

73.1, CH

ax 3.26 m

42.3, CH2

6.07 m 5.47 ddd (11.2, 10.5, 4.8) α 2.88 d (9.8) β 3.90 dd (11.2, 9.8)

46.1, CH 150.2, CH

ax 1.77 m eq1.26 m ax 2.09 m 6.56 dd (15.6, 10.5) 6.95 d (15.6)

185.4, C 103.6, C 174.9, C 19.0, CH3 26.0, CH2

δC, type

28.5, CH2

121.8, CH

170.6, C 101.3, C 173.0, C 8.97 br s 0.84 d (6.4) a 1.53 m b 1.06 m 0.86 t (7.3)

18.9, CH3 26.5, CH2 13.1, CH3 26.3, CH3

1.07 d (6.0) a 1.57 m b 1.06 m 0.86 t (7.2) 2.87 s

In the ROESY spectrum, the presence of correlations for H11/H-14, H-11/H-18, H-18/H-20, and H-18/H-29 (Figure 2) indicated H-14, H-18, and 17-CH3, together with two side chains at C-12 and C-19, are all β-orientated. Key ROE correlations among the protons on the other side, such as H-9/ B



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C-27 were observed. In the ROESY spectrum, the presence of ROE correlations of H-9/H-12, H-9/H-22, H-12/H-20, H-12/ H-22, H-14/H-18, H-18/H-20, H-20/H-22, and H-22/H-24 (Figure 3) suggested that the six-membered ring is in a chairlike conformation with H-12, H-20, and H-22 at axial positions. Key ROE correlations of H-11/H-13β, H-11/H-19, H-11/H-21, H13β/H-15β, H-17/H-19, and H-21/H-23 for protons on the opposite face of the 5/5/6-tricyclic system were also present in the ROESY spectrum (Figure 3). These findings showed that 2 has the same relative configuration as that of 4. Therefore, 2 was deduced to be 28-methyl-3-deOH-HSAF. For the configuration of previously reported PTMs, the S configuration of C-2 in the ornithine unit is assignable on the basis of the biosynthetic mechanism, which demonstrates that this unit in 3 and 4 is generated from L-ornithine,9,13 and the relative configuration of the 5/5/6 or 5/5 ring-containing lipophilic unit has been solved from NOE/ROESY correlations. However, the stereochemical correlation between L-ornithine and the lipophilic polycyclic units has so far not been clarified in PTMs except aburatubolactam A, cylindramide, and ikarugamycin.15−22 The great challenge to establish the correlation between the two units lies in the uncertainty in predicting stable conformations of the macrolactam rings due to their flexible nature. Furthermore, because of the keto−enol tautomerism between C-25 and C-1,23,24 1 and 2 may exist as an equilibrium of three structures, as shown in Figure 4, which gives increased complexity in conformational analysis of the rings. In order to assign the absolute configurations, we carried out thereotical calculations of ECD spectra of 1 and 2. Two possible stereoisomers of (2S)-1 and (2S)-2 were both calculated for reliable comparative analysis. In order to save computational cost, the truncated structures (Figure 4) were used in the detailed calculations based on our preliminary tests. Three tautomeric structures of each stereoisomer were separately subjected to MMFF conformational searches and the subsequent DFT geometry optimization. The low-energy conformers were obtained from the entirety of energy minima of three tautomeric structures. As a result (Tables S1 and S2 in the Supporting Information), the dominant tautomers were found to be (25Z)-1-keto-25-enol in (12R,19S)-1 (1c), (12S,19R)-1 (1C), and (11S,22S)-2 (2C), and (25E)-1-keto25-enol in (11R,22R)-2 (2b), whereas 25-keto-1-enol structures (1a, 1A, 2a, and 2A) were the least dominant tautomers in all stereoisomers of 1 and 2. The low-energy conformers accounting for more than 2% equilibrium population were applied to ECD/TDDFT calculations. The calculated ECD spectra for the two possible stereoisomers of 1 and 2 are shown in Figures 5 and 6,

Figure 1. 1H−1H COSY (bold lines) and HMBC correlations (arrows) of 1.

Figure 2. Key ROESY correlations (curves) and the relative configuration of 1.

H-12, H-12/H-19, H-13α/H-19, H-16/H-19, H-17/H-19, and H-19/H-21, were also observed in the spectrum (Figure 2), suggesting the relative configuration of the 5/5-bicyclic unit in 1 is identical to that of alteramide A (5). Lysobacteramide B (2) was isolated as a white powder. Its molecular formula was determined as C 30H42N2O5 by HRESIMS. The 1H and 13C NMR data (Table 1) in combination with 1H−1H COSY and HSQC correlations suggested 2 is structurally similar to 3-deOH-HSAF (4),10 a known PTM also obtained in the present study, except for the presence of an additional methyl group (δH 2.87; δC 26.3) and the absence of the proton for 28-NH. The location of the additional methyl group at N-28 was determined by HMBC, in which the key correlations from the methyl protons to C-2 and

Figure 3. Key ROESY correlations (curves) and the relative configuration of 2. C



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Figure 4. Truncated structures and three tautomers of each stereoisomer of 1 and 2 for configuration of the relevant carbons, 1a, 1b, and 1c with (12R, 19S), 1A, 1B, and 1C with (12S, 19R), 2a, 2b, and 2c with (11R, 22R), and 2A, 2B, and 2C with (11S, 22S).

of 1, while the calculated spectrum for (12S,19R)-1 is similar to the mirror image of the experimental spectrum. Therefore, the 2S,12R,19S configuration can be assigned for the macrolactam ring of 1. The absolute configurations of other chiral centers in 1 can be assigned as 14S,16R,17R,18R according to their relative configuration. For compound 2, as shown in Figure 6, the simulated ECD spectrum of (11R,22R)-2 provides an excellent fit with the measured spectrum, whereas the theoretical spectrum of (11S,22S)-2 is obviously different from the experimental spectrum in the range 250−350 nm. Thus, the absolute configuration of 2 was assigned as 2S,11R,12S,14S,16R,17R,18R,19S,20R,22R. HSAF (3) and 3-deOH-HSAF (4) are similar to 2 in both skeletons and relative configurations. In order to assign their absolute configurations, we measured their ECD spectra. As expected, both provided an ECD spectrum very similar to that of lysobacteramide B (2) (Figure 7). Therefore, their absolute configurations could be assigned to be the same as that of 2. On the basis of the chiroptical analysis of ECD data, we found that compounds 1−4 share the same absolute configurations at the first cyclopentyl ring. Then we considered that this might be a common feature in the configuration of this group of compounds and predicted that alteramide A (5) may have the same absolute configuration in the 5/5-bicyclic unit as

Figure 5. Comparison of B3LYP/TZVP-calculated ECD spectra of (12R,19S)- and (12S,19R)-isomers with the experimental spectra of 1 in MeOH. σ = 0.30 eV; shift = +5 nm; scaled by 0.30 and 0.46, respectively.

Figure 6. Comparison of B3LYP/TZVP-calculated ECD spectra of (11R,22R)- and (11S,22S)-isomers with the experimental spectrum of 2 in MeOH. σ = 0.35 eV; shift = −2 nm; scaled by 0.65 and 0.35, respectively.

respectively. It is clear that the simulated ECD spectrum of (12R,19S)-1 is in good agreement with the measured spectrum

Figure 7. Comparison among the experimental ECD spectra of 2−4. D



Article

lysobacteramide A (1). In order to investigate this, we performed ECD calculations of 5 using the above-described method. As can be seen in Figure 8, the calculated ECD

Scheme 1. Proposed Biosynthetic Pathway of 1−5

Figure 8. Comparison of CAM-B3LYP/TZVP-calculated ECD spectra of (12S,19R)- and (12R,19S)-isomers with the experimental spectra of 5 in MeOH. σ = 0.37 eV; shift = −9; scaled by 0.55 and 0.27, respectively.

spectrum of the (12S,19R)-isomer matched the measured spectrum of 5 rather than that of the predicted (12R,19S)isomer, indicating that the 5/5-bicylic unit in 5 is enantiomeric to that in 1. This result was consistent with our findings that HSAF-PKS/NRPS was unable to convert alteramide A (5) to any HSAF analogues (3 or 4) in feeding experiments, for which we postulated that alteramide A might be produced in an opposite cyclization on the second ring, which resulted in a dead-end shunt product.13 The present study further clarified that, although both HSAF (3) and alteramide A (5) contain a fused 5/5-bicyclic unit, the absolute configuration of the first cyclopentyl ring in alteramide A is enantiomeric to the first cyclopentyl ring in HSAF. The opposite cyclization in the first cyclopentyl ring of alteramide A may contribute to the configuration of the second cyclopentyl ring of alteramide A, which is also different from the second cyclopentyl ring of HSAF. The difference in the second cycopentyl ring may prevent alteramide A from forming the third ring (cyclohexyl ring) in HSAF. Further, compound 1 is probably also a deadend shunt product because the absolute configuration of C-12 is different from that of HSAF. In light of these findings, we propose that the structural diversity in HSAF and related analogues arises from multiple cyclization/isomerization pathways of the polyene intermediates in the biosynthesis, as shown in Scheme 1. We postulate that both alteramide A and lysobacteramide A are byproducts of the main biosynthetic pathway, which probably result from a less than absolute stereospecificity of the cyclization and/or alkene isomerization. This less than absolute stereospecificity is probably also related to the reactive nature of the polyene intermediates synthesized by HSAF-PKS/NRPS and might also depend on the cooperation between HSAF-PKS/NRPS and the tailoring enzymes that are responsible for the cyclization and isomerization reactions.12,13 Compounds 1−4 were evaluated for the growth inhibitory activity against human carcinoma A549, HepG2, and MCF-7 cells by the MTT method.25 Compounds 2−4 showed activity against these three cell lines with half inhibitory concentration

(IC50) values of 0.26−4.1 μM, and 1 showed activity with IC50 values ranging from 7.6 to 10.3 μM as shown in Table 2. The antifungal activities of 1−5 were also tested. Compounds 2−5 were active, while 1 was inactive against Fusarium verticillioides at a dose of 50 μg per paper disk. The IC50 values against spore germination of 2−5 were determined to be 47.9, 6.90, 71.2, and 76.3 μg/mL, respectively (Table 2). Among these PTM analogues, HSAF (3) exhibited the strongest activity, with an IC50 close to thiram, a fungicide widely used in agriculture to control plant pathogenic fungi. The results indicate that the 5−5−6 tricyclic system and 3hydroxy group are critical for antifungal activity against F. verticillioides, an important mycotoxin-producing pathogen of crop plants.26 E



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Antifungal Activity Assay. The antifungal activities of compounds 1−5 were tested by an agar diffusion method using paper disks. Five microliters of conidia of F. verticillioides was inoculated in the middle of a Petri dish containing PDA medium; autoclaved paper disks (6 mm diameter) were placed around the fungal inoculant on the same Petri dish, and each of the paper disks contained 50 μg of testing samples or an equivalent volume of methanol. The inhibitory zones were observed after growing for 2 days at 30 °C in the dark. The IC50 of compounds 1−5 against F. verticillioides spore germination was also evaluated using 48-well culture plates (see Supporting Information for details). Cytotoxic Activity Assay. The cytotoxic activity assay was performed by the MTT colorimetric method as previously described.25 The test compounds 1−5 in DMSO were serially diluted with culture medium. The final concentrations of each compound in wells were 50, 10, 2, 0.4, 0.08, and 0.016 μM, and the experiments were performed in quadruplicate. Adriamycin (ADM) was used as a positive control. The inhibitory rate of cell growth was calculated according to the following formula: Inhibition rate (%) = {1 − (ODtreated − ODcontrol)/(ODcontrol − ODblank)} × 100%. IC50 values were determined by nonlinear regression analysis of logistic dose−response curves (SPSS 16.0 statistic software). Computational Methods. Molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with the Spartan’14 software package (Wavefunction Inc., Irvine, CA, USA) and Gaussian 09 program package, 27 respectively, using default grids and convergence criteria. A MMFF conformational search generated lowenergy conformers within a 10 kcal/mol energy window, which were subjected to geometry optimization using the DFT method at the B3LYP/6-31G(d,p) level. Frequency calculations were run at the same level to verify that each optimized conformer was a true minimum and to estimate their relative thermal free energies (ΔG) at 298.15 K. Energies of the low-energy conformers in MeOH were calculated at the B3LYP/6-311+G (2d,p) level. Solvent effects were taken into account by using the polarizable continuum model (PCM). The TDDFT calculations were performed using the long-range-corrected hybrid CAM-B3LYP and the hybrid B3LYP functionals and Ahlrichs’ basis sets SVP (split valence plus polarization)28 and TZVP (triple-ζ valence plus polarization).29 The number of excited states per molecule was 40 (for 1) or 48 (for 2 and 5). The ECD spectra were generated by the program SpecDis30 using a Gaussian band shape with a 0.30 (for 1), 0.35 (for 2), or 0.37 (for 5) eV exponential halfwidth from dipole-length dipolar and rotational strengths. The equilibrium population of each conformer at 298.15 K was calculated from its relative free energy using Boltzmann statistics. The calculated spectra of compounds 1, 2, and 5 were generated from the low-energy conformers according to the Boltzmann weighting of each conformer in MeOH solution. In the preliminary tests with tautomers (11R,22R)1a and (12R,19S)-2a, the truncated structures, in which the 16-ethyl and 17-methyl groups in 1 and the outer five-membered ring in 2 were omitted, provided simulated ECD spectra identical to those by the intact structures. Therefore, the subsequent theoretical calculations were carried out with the truncated structures (Figure S1) in order to save computational cost.

Table 2. Cytotoxicity and Antifungal Activity of 1−5 tumor cell growth inhibition (IC50, μM)a

compound 1 2 3 4 5 ADM thiram

A549 10.3 0.80 0.26 0.95 ND 0.69

± ± ± ±

0.9 0.02 0.003 0.09

± 0.06

HepG2 9.5 1.7 2.1 3.5 ND 1.22

± ± ± ±

0.6 0.1 0.1 0.02

± 0.02

MCF-7 7.6 4.1 2.2 2.6 ND 0.17

± ± ± ±

0.4 0.4 0.2 0.2

spore germination inhibition against F. verticillioides (IC50, μg/ mL)a >160 47.9 6.90 71.2 76.3

± ± ± ±

6.8 0.5 8.3 8.2

± 0.03 0.55 ± 0.2

Values represent means ± SD based on three individual experiments. ND means not detected. ADM is adriamycin. a

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a PerkinElmer 343 spectropolarimeter. Melting points were measured with a Vernier Melt Station. ESIMS data were obtained on a Finnigan LCQ mass spectrometer. Experimental CD spectra in MeOH were recorded in a quartz cuvette of 1 mm optical path length on a JASCO J-815 CD spectrometer (Tokyo, Japan). The CD spectrum of MeOH was used as a baseline and subtracted from the experimental spectra. 1H NMR, 13C NMR, and 2D NMR spectra were recorded on a Bruker Avance 400 instrument with residual solvent peaks as references. HRESIMS data were collected on a Q-TOF Synapt G2 HDMS system (Waters, Pittsburgh, PA, USA) in positiveion mode. Analysis and preparative HPLC were performed with a Phenomenex column (150 × 4.6 mm) and an Agilent ZORBAX SBC18 column (250 × 10 mm). The cytotoxic assays were determined with a Genois microplate reader (Tecan Group, Männedorf, Zürich, Switzerland). The spore germination percentages were counted with a B203LED optical microscope (Chongqing Optec Instrument Co., Ltd., Chongqing, China). Fermentation of Lysobacter enzymogenes C3. The HSAFproducing bacterium L. enzymogenes C3 was from Professor Gary Yuen’s laboratory at University of Nebraska−Lincoln. The strain was grown in a 150 × 250 mL Erlenmeyer flask containing 100 mL of NYG medium (0.5% peptone; 0.3% yeast extract; 2% glycerol; pH 6.4) on a rotary shaker for 4 days at 30 °C. Extraction and Isolation. The liquid cultures of L. enzymogenes C3 were extracted two times with an equal volume of EtOAc at room temperature. The organic layers were combined and concentrated in vacuo to obtain the brown crude extracts (3.40 g). The extract was fractionated by ODS column chromatography and eluted with aqueous acetonitrile (40−100%) with 0.1% formic acid to afford six fractions (Fr-1−6). Fr-2 was further separated with HPLC using 65% acetonitrile to provide compounds 3 (4 mg) and 5 (10 mg). Fr-3 was purified by HPLC using 70% acetonitrile to obtain 4 (10 mg). Fr4 was separated with HPLC using 85% acetonitrile to yield compounds 1 (18 mg) and 2 (15 mg). Lysobacteramide A (1): colorless needles (MeOH); it began to decompose into a dark brown char above 160 °C; [α]25D +30 (c 0.01, MeOH); UV(MeOH) λmax (log ε) 229 (4.12), 266 (4.01) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) see Table 1; positive ESIMS m/z 479 [M + H]+, 957 [2 M + H]+, 979 [2 M + Na]+; HRESIMS m/z 523.2535 [M − H + 2Na] + (calcd for C29H38N2O4Na2, 523.2549). Lysobacteramide B (2): white powder; it decomposed into a dark brown liquid above 260 °C; [α]25D +250 (c 0.01, MeOH); UV(MeOH) λmax (log ε) 320 (3.90) nm; 1H NMR (400 MHz) and 13 C NMR (100 MHz) see Table 1; positive ESIMS m/z 511 [M + H]+, 1021 [2 M + H]+, 1043 [2 M + Na]+; HRESIMS m/z: 511.3160 [M + H]+ (calcd for C30H43N2O5, 511.3166).

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ASSOCIATED CONTENT

S Supporting Information *

1

H, 13C, and 2D NMR spectra, together with HRESIMS of 1 and 2. Measured CD spectra of 1−5. Relative and free energies and calculated ECD spectra of the low-energy conformers of 1 and 2. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jnatprod.5b00099.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +1-402-472-2998. Fax: +1-402-472-9402. E-mail: ldu3@ unl.edu (L. Du). F



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*Tel: +86-20-37252538. Fax: +86-20-37252537. E-mail: wxy@ scbg.ac.cn (X. Wei).

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank R. Cerny, M. Morton, and K. Wulser for technical assistance. This work was supported in part by NSFC (31329005 and 81172942) and the NIH (R01AI097260).

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