Arthroamide, a Cyclic Depsipeptide with Quorum Sensing Inhibitory

Nonfilamentous actinobacteria have been less studied as secondary metabolite producers than their filamentous counterparts such as Streptomyces. From ...
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Arthroamide, a Cyclic Depsipeptide with Quorum Sensing Inhibitory Activity from Arthrobacter sp. Yasuhiro Igarashi,*,† Kazuki Yamamoto,† Takao Fukuda,† Akane Shojima,‡ Jiro Nakayama,‡ Lorena Carro,§ and Martha E. Trujillo§ †

Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan ‡ Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-14 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan § Departmento de Microbiologia y Genetica, Edificio Departamental Lab. 214, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain S Supporting Information *

ABSTRACT: Nonfilamentous actinobacteria have been less studied as secondary metabolite producers than their filamentous counterparts such as Streptomyces. From our collection of nonfilamentous actinobacteria isolated from sandstone, an Arthrobacter strain was found to produce a new cyclic peptide arthroamide (1) together with the known compound turnagainolide A (2). These compounds inhibited the quorum sensing signaling of Staphylococcus aureus in the submicromolar to micromolar range.

B

with the aid of our in-house UV database. Most strains were not productive, but Arthrobacter sp. PGVB1 produced unknown metabolites at a detectable level. Several chromatography steps of the solvent extract led to the isolation of a new depsipeptide, arthroamide (1), and its known congener turnagainolide A (2).11

iofilms on inorganic solid substrates are one of the underexplored niches for natural product screening. Some groups of rod- or coccoid-shaped actinobacteria commonly inhabit biofilms formed on the surface of inorganic material such as rock, where they colonize in association with cyanobacteria, fungi, and algae.1 Microbes residing in biofilms on rock surfaces are exposed to harsh conditions such as solar radiation, dryness, and nutritional depletion in a multispecies community. In order to survive in such a restricted space, production of inhibitory substances could be a means to overcome the activity of surrounding competitors.2 Nonfilamentous actinobacteria are regarded as less productive in secondary metabolism than filamentous actinobacteria such as Streptomyces and Micromonospora.3 Meanwhile, rapidly accumulating genomic data suggest the presence of potential secondary metabolite biosynthetic genes in previously ignored or underexplored bacterial groups including nonfilamentous actinobacteria.4,5 As part of our continuing search for new bioactive compounds from underevaluated bacteria,6−8 we isolated 66 strains from colored patinas on the outer wall surfaces of stone buildings in Salamanca, Spain, that were constructed during the 12th to 18th centuries using Villamayor sandstone.9,10 The isolates produced pigments on the isolation agar medium (Figure S1), and 16S rRNA gene analysis revealed that most of them belonged to the nonfilamentous actinobacterial group known as rock-surface species (i.e., Arthrobacter, Blastococcus, Friedmaniella, and Modestobacter) except for one Streptomyces. The strains were cultured in a set of liquid medium and extracted with an organic solvent, the extracts were analyzed by HPLC-diode array detector (DAD), and data were analyzed © XXXX American Chemical Society and American Society of Pharmacognosy

Arthroamide (1) was obtained as a white powder. HRESITOFMS analysis gave a pseudomolecular ion [M + Na]+ at m/z 565.3000 corresponding to the molecular formula C29H42N4O6 (Δ −0.3 mmu, for C29H42N4O6Na), which was corroborated by the NMR data. 13C NMR and HSQC data analysis revealed the presence of 29 carbons assignable to five carbonyl carbons, one sp2 quaternary carbon, seven sp2 methines, eight sp3 methines, one sp3 methylene, and seven methyls. In the 1H NMR spectrum of 1, resonances for a monosubstituted benzene were observed at δH 7.27, 7.34, and 7.44 (Table 1). In addition, four doublet protons (δH 7.57, 7.73, Received: June 18, 2015

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DOI: 10.1021/acs.jnatprod.5b00540 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H and 13C NMR Data for Arthroamide (1) in DMSO-d6 residue Val-1

position 1 2 3 4 5 NH

Val-2

Ala

1 2 3 4 5 NH 1 2 3 NH

Val-3

Hppa

1 2 3 4 5 NH 2 (Val-3), 1 (Hppa) 1 2

δ Ca

δH mult (J in Hz)b

HMBCb,c

d

168.67, qC 58.3, CH 28.5, CH 19.4,e CH3 19.6,e CH3

170.4, qC 57.3, CH 28.4, CH 16.6,f CH3 18.2,f CH3 173.1, qC 48.8, CH 16.3, CH3

172.3, qC 57.5, CH 29.8, CH 19.1,g CH3 18.8,g CH3

168.70,d qC 39.5, CH2

3

73.0, CH

4

126.7, CH

5 6 7, 11 8, 10 9

132.5, 135.7, 126.5, 128.7, 128.1,

CH qC CH CH CH

4.25, dd (9.7, 9.7) 2.25, m 0.90, d (6.5) 0.89, d (6.5) 7.57, d (9.6)

4.21, dd (9.7, 4.3) 2.37, m 0.83, d (7.0) 0.82, d (7.0) 8.10, d (9.7)

1, 3, 4, 5 (Val-1); 1 (Val-2) 2 (Val-1) 2, 3, 5 (Val-1) 2, 3, 4 (Val-1) 2 (Val-1); 1 (Val-2) 2, 3, 4 1, 2, 4, 5 (Val-2); 1 (Ala) 2 (Val-2) 2, 3, 5 (Val-2) 2, 3, 4 (Val-2) 2 (Val-2); 1 (Ala)

4.33, dq (6.9, 5.7) 1.19, d (6.9) 8.55, d (5.7)

1, 3 (Ala)

4.14, dd (8.3, 6.9) 1.97, m 0.88, d (6.5) 0.88, d (7.0) 7.73, d (8.6)

1, 3, 4, 5 (Val-3); 1 (Hppa) 2, 4, 5 (Val-3) 2, 3, 5 (Val-3) 2, 3, 4 (Val-3)

2.89, dd (14.3, 11.6) 2.41, dd (14.3, 2.3) 5.49, m

1, 3, 4 (Hppa)

6.28, dd (16.0, 7.0) 6.68, d (16.0) 7.44, d (7.4) 7.34, t (7.4) 7.27, t (7.4)

Figure 1. 1H−1H COSY and key HMBC correlations for 1.

sequential COSY correlations to Hppa-H4, -H3, and -H2. In addition, HMBC correlations were observed from Hppa-H3 and -H2 to the carbonyl carbon at δC 168.7. These correlation data established the presence of a 3-hydroxy-5-phenyl-4pentenoic acid (Hppa) unit. The geometry of the C-4/C-5 double bond was determined as E on the basis of a large coupling constant (3JHH = 16.0 Hz) between Hppa-H4 and -H5. HMBC correlations among these residues from Val-1-NH to Val-2-C1, Val-2-NH to Ala-C1, Ala-NH to Val-3-C1, and Val-3-NH to Hppa-C1 defined the sequence of Val-1-Val-2-AlaVal-3-Hppa. The downfield resonance of Hppa-H3 (δH 5.49) and an HMBC correlation from Hppa-H3 to Val-1-C1 connected the Hppa and Val-1 residue via an ester linkage to complete the cyclic depsipeptide structure for 1 (Figure 1). The absolute configuration of 1 was determined by applying Marfey’s method12 and chiral anisotropic analysis. The acid hydrolysate of 1 was derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA), and the HPLC retention times were compared with L-FDLA derivatives of standard amino acids (Figure S11). The derivatized D- and L-alanine standards eluted at 38.4 and 26.4 min, respectively, while the L-FDLA derivative of the acid hydrolysate eluted at 38.4 min, establishing the D-configuration for the alanine residue. The derivatized D- and L-valine standards were eluted at 64.6 and 36.9 min, respectively. The L-FDLA derivative of the acid hydrolysate gave two peaks for D-Val and L-Val with a ratio of 1:8, which was not consistent with the number of Val residues in 1, implying the partial racemization of this amino acid. Hydrolysis was then carried out in 6 M DCl−D2O, and the LFDLA derivative was subjected to LC-MS analysis (Figure S12). In the negative ion mode, only one ion peak for the nonracemized Val ([M − H]−, C17H24N5O7) was detected at m/z 410, whereas two peaks were detected for deuterized Val ([M − H]−, C17H23DN5O7) at m/z 411 (Figure S10). On the basis of this finding, the minor peak for D-Val was confirmed to be derived from racemization, thereby establishing that all three Val residues in 1 were L-enantiomers. The absolute configuration of the oxygenated methine carbon Hppa-C3 was determined by using the modified Mosher’s method.13 1 was transformed to a ring-opened methyl ester (3) by treatment with NaOMe in MeOH (Figure 2). The Δδ values of the (S)- and (R)-MTPA esters (4 and 5) prepared from 3 by the treatment with (R)- and (S)-MTPACl showed positive values for H2, H3, and NH of Val-1 and HppaH2, although one of the Hppa-H2 protons was slightly negative. Negative values were observed for H4 and H5 of Hppa (Figure 2). This distribution pattern allowed the assignment of the R-configuration at Hppa-C3, and thus the absolute configuration of 1 was established. Arthroamide (1) consists of four common amino acids and one rare structural unit, 3-hydroxy-5-phenyl-4-pentenoic acid.

1, 2 (Ala) 2, 3 (Ala); 1 (Val-3)

1 (Hppa) 1, 4, 5 (Hppa); 1 (Val-1) 2, 3, 6 (Hppa) 3, 6, 7, 11 (Hppa) 5, 9 (Hppa) 6, 8, 10 (Hppa) 7, 11 (Hppa)

a

Recorded at 100 MHz, referenced to the residual solvent signal. Recorded at 500 MHz, referenced to the residual solvent signal. c HMBC correlations are from proton(s) stated to the indicated carbon. d−gAssignment interchangeable. b

8.10, 8.55) were detected in the downfield region. These protons were assigned to the amide NH protons since they had no HSQC correlations while showing COSY correlations to the amino acid α-protons. Further COSY correlations of the isopropyl protons to the α-protons connecting to the NH protons at δH 7.57, 7.73, and 8.10 confirmed the presence of three valine residues (Figure 1; Val-1, Val-2, Val-3). The NH proton at δH 8.55 had a correlation to an α-CH at δH 4.33, which was further correlated to a doublet methyl, establishing an alanine residue (Figure 1; Ala). Doublet methine protons in the ortho-positions of the benzene ring (Hppa-H7 and -H11) had HMBC correlations to an sp2 carbon Hppa-C5, and the proton (δH 6.68) directly bonded to this carbon in turn showed B

DOI: 10.1021/acs.jnatprod.5b00540 J. Nat. Prod. XXXX, XXX, XXX−XXX

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a JASCO DIP-3000 polarimeter. The UV spectrum was recorded on a Hitachi U-3210 spectrophotometer. The IR spectrum was measured on a PerkinElmer Spectrum 100. NMR spectra were obtained on a Bruker AVANCE 400 or a Bruker AVANCE 500 spectrometer in DMSO-d6 using the signals of the residual solvent protons (δH 2.49) and carbons (δH 39.8) as internal standards. HRESITOFMS were recorded on a Bruker microTOF focus. Microorganism. Strain PGVB1 was isolated from the surface of a stone bridge (Gran Via) in Salamanca, Spain. The isolation media used was Luedemann agar22 (0.5% yeast extract, 1.5% malt extract, 1% soluble starch, 1% glucose, 0.2% CaCO3, 0.5% NaCl, 1.8% agar (pH 8.6) supplemented with 50 mg/L of cycloheximide and 10 mg/L of nalidixic acid). The strain was identified following the previously reported protocol23 as a member of the genus Arthrobacter (Figure S2) on the basis of 99.7% similarity in 16S rRNA gene sequence (1471 nucleotides; EMBL accession number HF565368) to the type strain Arthrobacter agilis DSM 20550T (accession number X80748). Fermentation. Strain PGVB1 cultured on a Bn-2 slant [soluble starch 0.5%, glucose 0.5%, meat extract (Kyokuto Pharmaceutical Industrial Co., Ltd.) 0.1%, yeast extract (Difco Laboratories) 0.1%, NZ-case (Wako Chemicals USA, Inc.) 0.2%, NaCl 0.2%, CaCO3 0.1%, agar 1.5%] was inoculated into 500 mL K-1 flasks each containing 100 mL of V-22 seed medium consisting of soluble starch 1%, glucose 0.5%, NZ-case 0.3%, yeast extract 0.2%, Tryptone (Difco Laboratories) 0.5%, K2HPO4 0.1%, MgSO4·7H2O 0.05%, and CaCO3 0.3% (pH 7.0). The flasks were placed on a rotary shaker (200 rpm) at 30 °C for 2 days. The seed culture (3 mL) was transferred into 500 mL K-1 flasks each containing 100 mL of A-3 M production medium consisting of glucose 0.5%, glycerol 2%, soluble starch 2%, Pharmamedia (Traders Protein) 1.5%, yeast extract 0.3%, and Diaion HP-20 (Mitsubishi Chemical Co.) 1%. The pH of the medium was adjusted to 7.0 before sterilization. The inoculated flasks were placed on a rotary shaker (200 rpm) at 30 °C for 6 days. Extraction and Isolation. At the end of the fermentation period, 50 mL of 1-butanol was added to each flask, and the flasks were allowed to shake for 1 h. The mixture was centrifuged at 6000 rpm for 10 min, and the organic layer was separated from the aqueous layer containing the biomass. Evaporation of the solvent gave 14.4 g of crude extract from 5 L of culture. A half-portion of the crude extract (7.2 g) was subjected to silica gel column chromatography with a step gradient of CHCl3−MeOH (1:0, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/v). Fraction 4 (4:1) was concentrated to provide 2.66 g of brown viscous oil, which was then fractionated by reversed-phase ODS column chromatography with a gradient of MeCN−0.1% HCO2H (2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2 v/v). Fractions 5 (2:1) and 6 (1:1) were combined, concentrated, and extracted with EtOAc. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated to give a pale yellow solid (270 mg). Final purification was achieved by preparative HPLC (Waters XTerra RP18 7 μm, 300 × 19 mm, 15 mL/ min, UV detection at 254 nm) with MeCN−0.1% HCO2H (42:58), followed by evaporation and extraction with EtOAc, to yield arthroamide (1, 2.8 mg, tR 22.5 min) and turnagainolide A (2, 12.8 mg, tR 17.4 min). Arthroamide (1): white powder; [α]25D +33 (c 0.050, 1:1 CH2Cl2− MeOH); UV (1:1 CH2Cl2−MeOH) λmax (log ε) 251 (4.29); IR (ATR) νmax 3283, 1727, 1638 cm−1; 1H and 13C NMR data, see Table 1; HRESITOFMS [M + Na]+ 565.3000 (calcd for C29H42N4O6Na, 565.2997). Turnagainolide A (2): white powder; [α]23D +19 (c 0.050, 1:1 CH2Cl2−MeOH); 1H and 13C NMR data were identical to the reported values;11 HRESITOFMS [M + Na]+ 579.3162 (calcd for C30H44N4O6Na, 579.3153). Marfey’s Analysis. A portion of 1 (0.5 mg) was hydrolyzed at 110 °C with 6 M HCl (200 μL) for 12 h, and the reaction mixture was evaporated to dryness. A 0.1 M NaHCO3 solution (100 μL) was added to the dried hydrolysate of 1, as well as to standards of L- and D-alanine

Figure 2. Preparation of 3 and 1H NMR ΔδS−R values for MTPA esters (4 and 5) of 3.

Hppa has been found only in turnagainolides A and B from Bacillus11 and EGM-556 from a fungus.14 No other natural products containing Hppa are known. In addition, to the best of our knowledge, 1 is the first nonribosomal peptide found from Arthrobacter sp. Arthroamide (1, 100 μg/mL) was inactive against Micrococcus luteus, Escherichia coli, and Candida albicans. However, it showed potent inhibitory effects on the agr-signaling pathway of the quorum sensing (QS) in Staphylococcus aureus. Staph. aureus is a causative agent of a wide range of infections including pneumonia, endocarditis, and septic shock. A number of genes for virulence factors such as extracellular toxins and enzymes are expressed under the control of the accessory gene regulator (agr).15,16 The expression of agr is initiated by binding of autoinducing peptide pheromone (AIP) to the cell-surface receptor. The interruption of AIP-mediated QS signaling has been proposed as a new therapeutic approach to Staph. aureus infections.17,18 A Staph. aureus strain harboring the luciferase gene under the agrP3 promoter was used to assess the QS inhibition by 1 and 2.19 Both compounds suppressed the luminescent level, namely, the agr-dependent gene expression, down to 10% of the control at 5−10 μM without showing cell toxicity (Figure 3). IC50 values were 0.3 μM for 1 and 0.8 μM for 2. This activity was more potent than WS9326A (IC50 2.5 μM)20 and avellanin C (IC50 4.4 μM),21 QS inhibitors previously found in the same assay.

Figure 3. agr-dependent quorum sensing inhibition by compounds 1 and 2. C

DOI: 10.1021/acs.jnatprod.5b00540 J. Nat. Prod. XXXX, XXX, XXX−XXX

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experimental procedure as described for (S)-MTPA ester 4: 1H NMR (500 MHz, DMSO-d6) δ 0.73 (3H, d, J = 6.9 Hz, Val-3), 0.75 (3H, d, J = 6.8 Hz, Val-3), 0.80 (3H, d, J = 6.7 Hz, Val), 0.84 (3H, d, J = 6.6 Hz, Val), 0.85 (3H, d, J = 6.5 Hz, Val), 0.87 (3H, d, J = 6.9 Hz, Val), 1.17 (3H, d, J = 7.0 Hz, Ala), 1.84 (1H, m, Val-2-H3), 1.97 (1H, m, Val), 2.01 (1H, m, Val), 2.67 (1H, dd, J = 14.7, 7.5, Hppa-H2), 2.75 (1H, dd, J = 14.7, 6.7, Hppa-H2), 3.41 (3H, s, OMe), 4.10 (1H, t, J = 6.9 Hz, Val), 4.21 (1H, dd, J = 8.5, 7.3 Hz, Val-3-H2), 4.32 (1H, dd, J = 8.5, 7.2 Hz, Val), 4.42 (1H, dq, J = 7.0, 8.0 Hz, Ala), 5.98 (1H, m, Hppa-H3), 6.38 (1H, dd, J = 16.0, 7.5 Hz, Hppa-H4), 6.72 (1H, d, J = 16.0 Hz, Hppa-H5), 7.88 (1H, d, J = 8.5 Hz, Val-NH), 8.04 (1H, d, J = 8.5 Hz, Val-3-NH), 8.17 (1H, d, J = 8.0 Hz, Ala-NH), 8.20 (1H, d, J = 8.0 Hz, Val-NH); HR-ESITOFMS m/z 789.3690 [M − H]− (calcd for C40H52F3N4O9 789.3692). Biological Assays. Antimicrobial assays were carried out according to the procedures previously reported.24 The agr inhibition assay was performed as described previously.25 In brief, the strain used was Staphylococcus aureus agr reporter strain, 8325-4, which carries plasmid pSB2035 encoding the Photorhabdus luminescens luciferase gene and gf p under control of the agrP3 promoter. An overnight culture of Staph. aureus 8325-4 (pSB2035) in Luria-Hewitt broth (LB) (Tryptone 1%, yeast extract 0.5%, NaCl 1%) supplemented with 7 μg/mL of chloramphenicol at 30 °C on a shaker was diluted in fresh LB broth (1:100), and 200 μL was dispensed into each well of a 96well microplate (96-well round bottom; Sigma-Aldrich). Sample solution was dispensed into microplate wells, and the solvent was evaporated in a SpeedVac concentrator before adding the culture. Staph. aureus 8325-4 (pSB2035) and Staph. aureus 12600 were cultured in the same way but with solvent only for the positive and negative control, respectively. The microplate was incubated at 37 °C with shaking, and the cell growth was monitored at 620 nm every hour on a plate reader (Infinite F200 Pro, Tecan) for 5 h. For the luminescence measurement, the culture was transferred to a new microplate (96F black microwell, Thermo Scientific), and luminescence was measured by a plate reader.

(Ala) and L- and D-valine (Val). A solution of 1-fluoro-2,4dinitrophenyl-5-L-leucinamide (L-FDLA) in acetone (0.05 mg in 50 μL) was added to each reaction vial. Each vial was sealed and incubated at 55 °C for 2 h. To quench reactions, 2 M HCl (50 μL) was added and then diluted with MeCN−0.2% HCO2H (100 μL, 50:50). The Marfey’s derivatives of the hydrolysate and standards were analyzed by HPLC using a Cosmosil 5C18-AR-II column (Nacalai Tesque Inc., 4.6 × 250 mm) eluted with MeCN−2% HCO2H at a flow rate of 1.0 mL/min, monitoring at 340 nm with a linear gradient of MeCN from 30% to 43% over 68 min. Retention times for the amino acid standards were 26.4 min for L-Ala-L-FDLA, 38.4 min for D-Ala-LFDLA, 36.9 min for L-Val-L-FDLA, and 64.6 min for D-Val-L-FDLA, while the L-FDLA-hydrolysate of 1 gave peaks at 36.9, 38.4, and 64.9 min (Figure S11). Hydrolysis of 1 in DCl−D2O and Marfey’s Analysis on LC-MS. A portion of 1 (1 mg) was hydrolyzed at 110 °C in 6 M DCl−D2O (200 μL) for 15 h. The hydrolysate was treated with L-FDLA in the same manner as described above. The Marfey’s derivative of the hydrolysate and standards were analyzed by LC-MS using an Imtakt Cadenza CD-C18 column (4.6 × 75 mm) with MeCN−10 mM CH3CO2NH4 (MeCN concentration: 31% for 0−23 min; 31−85% for 23−26 min; 85% for 26-31 min; 85−31% for 31−34 min) at a flow rate of 0.4 mL/min, monitoring at 340 nm. Under the negative ion mode, monitoring m/z 410 ± 0.5 gave only a L-Val peak (4.6 min), while a D-Val peak (14.9 min) was also observed together with a L-Val peak when monitoring m/z 411 ± 0.5 (Figure S12). Methanolysis of 1 to Ester 3. Compound 1 (5.0 mg, 9 μmol) was dissolved in 5% NaOMe−MeOH (3.3 mL) and stirred for 30 min at room temperature. The reaction mixture was neutralized by adding 1 M HCl. The mixture was evaporated to remove MeOH and extracted three times with EtOAc. After drying with anhydrous Na2SO4, the organic layer was concentrated to give 3 as a major product. 3 was purified by HPLC using a Cosmosil AR-II column (10 × 250 mm) with a gradient solvent system of MeCN−0.1% HCO2H (MeCN concentration: 38% for 0−26 min; 38−85% for 26−29 min; 85% for 29−34 min; 85−38% for 34−37 min) at a flow rate of 4.0 mL/min, monitoring at 254 nm, to yield 3 (2.0 mg, tR 24.1 min): 1H NMR (500 MHz, DMSO-d6) δ 0.77 (3H, d, J = 6.7 Hz, Val), 0.78 (3H, d, J = 6.5 Hz, Val), 0.79 (3H, d, J = 6.4 Hz, Val), 0.84 (3H, d, J = 6.7 Hz, Val), 0.85 (3H, d, J = 7.0 Hz, Val), 0.89 (3H, d, J = 6.8 Hz, Val), 1.19 (3H, d, J = 7.0 Hz, Ala), 1.91 (1H, m, Val), 1.98 (1H, m, Val), 2.03 (1H, m, Val), 2.43 (2H, m, Hppa-H2), 3.60 (3H, s, OMe), 4.10 (1H, t, J = 6.8 Hz, Val), 4.17 (1H, t, J = 7.5 Hz, Val), 4.29 (1H, t, J = 7.8 Hz, Val), 4.38 (1H, m, Ala-H2), 4.50 (1H, m, Hppa-H3), 5.22 (1H, d, J = 4.0 Hz, Hppa-3OH), 6.28 (1H, dd, J = 16.0, 6.0 Hz, HppaH4), 6.52 (1H, d, J = 16.0 Hz, Hppa-H5), 7.22 (1H, t, J = 7.5 Hz, Hppa-H9), 7.31 (2H, t, J = 7.0 Hz, Hppa-H8, -H10), 7.37 (2H, d, J = 7.5 Hz, Hppa-H7, -H11), 7.81 (1H, d, J = 9.0 Hz, Val), 7.92 (1H, d, J = 8.5 Hz, Val), 8.09 (1H, d, J = 7.5 Hz, Ala), 8.18 (1H, d, J = 7.5 Hz, Val); HR-ESITOFMS m/z 597.3258 [M + Na]+ (calcd for C30H46N4O7Na 597.3259). Preparation of (S)-MTPA Ester 4 and (R)-MTPA Ester 5. To a solution of 3 (0.5 mg, 0.9 μmol) in pyridine (30 μL) was added (R)MTPACl (7 μL, 37 μmol) at room temperature. The reaction mixture was stirred for 1 h at room temperature. The resulting solution was concentrated under reduced pressure to dryness to yield the (S)MTPA ester of 3 (4, 0.3 mg): 1H NMR (500 MHz, DMSO-d6) δ 0.77 (3H, d, J = 7.0 Hz, Val), 0.79 (3H, d, J = 7.4 Hz, Val), 0.81 (3H, d, J = 6.8 Hz, Val), 0.86 (3H, Val), 0.86, (3H, Val), 0.89 (3H, d, J = 6.8 Hz, Val), 1.18 (3H, d, J = 6.9 Hz, Ala), 1.86 (1H, m, Val-3-H3), 1.99 (1H, m, Val), 2.03 (1H, m, Val), 2.72 (1H, m, Hppa-H2), 2.74 (1H, m, Hppa-H2), 3.48 (3H, s, OMe), 4.11 (1H, t, J = 7.0 Hz, Val), 4.27 (1H, t, J = 7.9 Hz, Val-3-H2), 4.34 (1H, dd, J = 8.7, 7.4 Hz, Val), 4.47 (1H, dq, J = 6.9, 7.5 Hz, Ala), 5.99 (1H, m, Hppa-H3), 6.21 (1H, dd, J = 16.0, 7.0 Hz, Hppa-H4), 6.43 (1H, d, J = 16.0 Hz, Hppa-H5), 7.92 (1H, d, J = 9.0 Hz, Val-NH), 8.11 (1H, d, J = 9.0 Hz, Val-3-NH), 8.20 (1H, d, J = 7.5 Hz, Ala-NH), 8.20 (1H, d, J = 7.5 Hz, Val-NH); HRESITOFMS m/z 789.3692 [M − H]− (calcd for C40H52F3N4O9 789.3692). The (R)-MTPA ester 5 (0.2 mg) was obtained from 3 (0.5 mg, 0.9 μmol) and (S)-MTPACl (7 μL, 37 μmol) by the same

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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.5b00540. Copies of 1D and 2D NMR spectra of 1, 1H NMR spectra of 3−5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-766-56-7500. Fax: +81-766-56-2498. E-mail: yas@ pu-toyama.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Scientific Research (B) No. 24380050 from the Japan Society for the Promotion of Science to J.N.

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REFERENCES

(1) Gorbushina, A. A. Environ. Microbiol. 2007, 9, 1613−1631. (2) Elias, S.; Banin, E. FEMS Microbiol. Rev. 2012, 36, 990−1004. (3) Bérdy, J. J. Antibiot. 2012, 65, 385−395. (4) Ayuso-Sacida, A.; Genilloud, O. Microb. Ecol. 2005, 49, 10−24. (5) Gontang, E. A.; Gaudencio, S. P.; Fenical, W.; Jensen, P. R. Appl. Environ. Microbiol. 2010, 76, 2487−2499. (6) Akiyama, H.; Oku, N.; Kasai, H.; Shizuri, Y.; Matsumoto, S.; Igarashi, Y. J. Antibiot. 2014, 67, 795−798.

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DOI: 10.1021/acs.jnatprod.5b00540 J. Nat. Prod. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jnatprod.5b00540 J. Nat. Prod. XXXX, XXX, XXX−XXX