Niizalactams A–C, Multicyclic Macrolactams Isolated from Combined

Dec 1, 2015 - RIKEN Center for Life Science Technologies, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. §Graduate School of Agricultural...
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Niizalactams A−C, Multicyclic Macrolactams Isolated from Combined Culture of Streptomyces with Mycolic Acid-Containing Bacterium Shotaro Hoshino,† Masahiro Okada,† Toshiyuki Wakimoto,† Huiping Zhang,‡ Fumiaki Hayashi,‡ Hiroyasu Onaka,§ and Ikuro Abe*,† †

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan RIKEN Center for Life Science Technologies, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan § Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan ‡

S Supporting Information *

ABSTRACT: A terrestrial bacterium, Streptomyces sp. NZ-6, produced niizalactams A−C (1−3), unprecedented di- and tricyclic macrolactams, by coculturing with the mycolic acidcontaining bacterium Tsukamurella pulmonis TP-B0596. Their complete structures, including absolute configurations, were elucidated on the basis of spectroscopic data and chemical derivatization. Their unique skeletons are proposed to be biosynthesized from a common 26-membered macrolactam intermediate by SN2 cyclization or an intramolecular Diels−Alder reaction.

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the previous studies.6,8−10 As a result, one of the strains, Streptomyces sp. NZ-6, produced or significantly enhanced the production of three novel macrolactams with unprecedented skeletons, subsequently named niizalactams A−C (1−3), when cocultured with T. pulmonis TP-B0596. Herein, we report the isolation and the complete structural elucidation of compounds 1−3 obtained by this coculture strategy based on detailed spectroscopic analysis and chemical derivatizations.

ctinomycetes are a phylum of Gram-positive bacteria characterized by high productivity of secondary metabolites with unique structures, providing large numbers of druglike compounds. In this respect, the genus Streptomyces occupies a quite important position, as almost all of the actinomycetes-derived secondary metabolites have been isolated from this extraordinary genus.1,2 Recent studies revealed that the genomes of Streptomyces bacteria include far more biosynthetic gene clusters responsible for the production of secondary metabolites than previously expected.3 Therefore, we are able to access only a fraction of their secondary metabolites, while the remaining potential from cryptic genes is unexpressed under classical culture conditions. In order to discover the unique secondary metabolites, cryptic genes must be effectively and conveniently activated.4,5 Recently, several kinds of mycolic acid-containing bacteria were revealed to have the potential to activate the expression of genes responsible for cryptic secondary metabolite production in the genus Streptomyces. Indeed, 41 out of 112 Streptomyces bacteria produced new secondary metabolites when they were cocultured with the mycolic acid-containing bacterium Tsukamurella pulmonis.6 As a result, alchivemycin A,7 the novel polyketide antibiotic with a unique chemical structure, was isolated from Streptomyces endus S-522. The feasibility of this fermentation method, named combined culture, was demonstrated by further applications, leading to the isolation of novel secondary metabolites with various biosynthetic backgrounds including alkaloids,8,9 butanolides,10 and polyketides.11 To discover new cryptic secondary metabolites in Streptomyces bacteria, we applied the combined-culture method to our terrestrial Streptomyces collections, while employing the mycolic acid-containing bacterium T. pulmonis TP-B0596, as in © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

To investigate the alteration of metabolic profiles, Streptomyces sp. NZ-6 and T. pulmonis TP-B0596 were separately or simultaneously cultured in A-3M medium.6 These culture broths were directly extracted with ethyl acetate, and the organic layers were subjected to HPLC analysis. As a result, Streptomyces sp. NZ-6 produced two new compounds (1, 2), and enhanced the production of compound 3 more than 5 times (Figure 1). To determine the structures of 1−3, 10 L of coculture broth was separated into cell pellet and supernatant by centrifugation. The lyophilized cell pellet was extracted with acetone, and the supernatant was extracted with ethyl acetate. Both of the crude extracts were purified by flash silica gel column chromatography and subsequent HPLC. We performed all processes from cultivation to purification under dark condition and with ice cooling because compounds 1−3 were highly unstable and gradually degraded when they were exposed to light or left at room temperature. We obtained a significant amount of 1 (60 Received: September 7, 2015

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remaining two trisubstituted alkenes were revealed to be 6E and 18E based on the NOESY correlations from the corresponding allyric methyl protons. Therefore, all of the atom connections in 1 were established as shown in Figure 1. The molecular formula of niizalactam B (2) was established as C28H35NO6, suggesting that 2 lacks one hydroxy group as compared to 1. The NMR data of 2 were very similar to those of 1, except for the absence of signals corresponding to the oxygenated methine at C-10 in 1. Alternatively, the NMR spectroscopy of 2 showed new signals corresponding to the aliphatic methylene group (δH 2.08 and 2.65, δC 36.0), and its position was determined to be at C-10 based on the 1H−1H COSY analysis (Figure S8). Therefore, the chemical structure of niizalactam B (2) was determined to be the C-10 deoxy form of 1. The molecular formula of niizalactam C (3) was established as C28H35NO6, indicating 12 degrees of unsaturation and an identical molecular formula to that of 2. The 1H NMR, 13C NMR, and HSQC spectra in CD3OD revealed the presence of two ketones (C-3 and C-13), one amide carbonyl (C-1), two trisubstituted olefinic carbons (C-6 and C-18), and 10 olefinic methines (C-4, C-5, C-7, C-15, C-16, C-19, to C-23), satisfying 9 out of 12 degrees of unsaturation. Therefore, 3 should have a tricyclic skeleton to explain the remaining three degrees of unsaturation. These NMR data also indicated the presence of three oxygenated methines (C-10 to C-12), five aliphatic methines (C-8, C-9, C-14, C-17, and C-24), two aliphatic methylenes (C-2 and C-25), and three methyl groups (C-26, C27, and C-28). The 1H NMR spectrum in d6-DMSO showed the presence of an additional four D2O exchangeable signals (δH 4.63, 4.94, 5.29, and 7.99), corresponding to the three hydroxy groups at the C-10, C-11, and C-12 methines and the amide proton adjacent to the C-25 methylene. The DQFCOSY (in CD3OD) and 1H−1H COSY (in d6-DMSO) analyses provided three substructures (C-7 to C-9 and C-14 to C-17, C-10 to C-12, and C-19 to C-25 including the C-28 methyl group), in addition to an isolated methylene group (C2) and a stand-alone double bond (C-4 and C-5). DQF-COSY analysis also indicated the connection between C-9 and C-10 based on the weak correlation signals from H-9 to H-10 and H11 (Figure 2 and Figure S14). The connection between C-9 and C-10 was also supported by the NOESY correlations of H9/H-10 and the HMBC correlations from H-9 to C-10 and C11 (Figure 2). The 1H homonuclear decoupling experiment irradiating H-15 and H-17 signals (δH 5.86 and 2.50) suggested the presence of allylic coupling between H-15 and H-17 (4JH15H17 = 2.5 Hz, Figure S15) via the C-16/H-16 position, although the DQF-COSY correlation between H-16 and H-17 was completely absent in 3 probably because of its dihedral angle of nearly 90°. The connection between C-16 and C-17 was further supported by the NOESY correlation of H-16/H17 and the HMBC correlations of H-16/C-8 and H-17/C-15 (Figure 2). The linkages across the quaternary carbons (C-1 to C-4, C-5 to C-7, C-12 to C-14, and C-17 to C-19) were also confirmed by the HMBC analysis (Figure 2). Finally, the amide bond formation was inferred from the molecular formula and the chemical shift of the C-1 carbonyl signal (δC 166.2). Except for the Δ15,16-alkene group constructing the cyclohexene moiety, all of the C−C double-bond geometries were assigned as E, on the basis of the 3JHH coupling constants (3JH4H5 = 15.0 Hz, 3JH20H21 = 14.5 Hz, 3JH22H23 = 15.0 Hz) and the NOESY correlations from allylic methyl groups (Figure 2). Therefore, all of the atom connections of 3 were determined as shown in

Figure 1. Chemical structures of 1−3 and HPLC profiles of ethyl acetate extracts of (i) Streptomyces sp. NZ-6 cultured with T. pulmonis, (ii) Streptomyces sp. NZ-6 pure culture, and (iii) T. pulmonis pure culture. All peaks were detected by UV absorption at 300 nm, and a few minor peaks observed in the extract of T. pulmonis pure culture were derived from A-3M medium.

mg) and enough 2 (5.8 mg) and 3 (1.3 mg) for structural elucidation. The molecular formula of niizalactam A (1) was determined to be C28H35NO7 based on a MALDI-TOF2-HRMS analysis and NMR data (Table 1), indicating 12 degrees of unsaturation. The 1H NMR, 13C NMR, and HMQC spectra indicated the presence of two ketones (C-3 and C-13), one amide carbonyl (C-1), two trisubstituted olefinic carbons (C-6 and C-18), and 12 olefinic methines (C-4, C-5, C-7 to C-9, C-14 to C-17, and C-19 to C-21), explaining 10 out of 12 degrees of unsaturation. Therefore, 1 should be a bicyclic structure, to satisfy the remaining two degrees of unsaturation. In addition, these NMR spectroscopic data revealed the presence of four oxygenated aliphatic methines (C-10 to C-12 and C-23), two aliphatic methines (C-21 and C-24), two aliphatic methylenes (C-2 and C-25), three methyl groups (C-26 to C-28), and four D2O exchangeable signals (δ H 4.64, 4.80. 4.81, and 5.44) corresponding to the four hydroxy groups. Interpretation of the 1H−1H COSY spectrum showed the presence of three substructures (C-7 to C-12, C-14 to C-17, and C-19 to C-25, including the C-28 methyl group, Figure 2), in addition to a discrete methylene group (C-2) and an isolated double bond (C-4 and C-5). Additionally, the HMBC analysis completed all of the carbon−carbon bonds in 1 (Figure 2). The presence of a pyrrolidinol ring (C-22 to C-25) was indicated by the HMBC correlation of H-25a/C-22, along with the moderately downfield shifted signals at C-22 and C-25 (δC 67.5 and 50.1). Finally, the amide bond formation between the C-1 carbonyl and the pyrrolidinol moiety was constructed to satisfy the molecular formula and the upfield shift of the carbonyl signal (δC 165.9). All of the C−C double-bond geometries were elucidated based on the vicinal coupling constants and the NOESY correlations. The large 3JHH coupling constants (3JH4H5 = 15.5 Hz, 3JH8H9 = 15.0 Hz, 3JH16H17 = 15.5 Hz, 3JH20H21 = 15.5 Hz) indicated the 4E, 8E, 16E, and 20E configurations in 1. The 14Z configuration was established by the NOESY correlation (Figure 2) and the relatively small 3JHH coupling constant (3JH14H15 = 11.0 Hz). The geometries of the B

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Table 1. 1H NMR (500 MHz for 1 and 2, 900 MHz for 3) and 13C NMR (125 MHz for 1 and 2, 225 MHz for 3) Data of 1−3 (in d6-DMSO for 1 and 2, in CD3OD for 3) 1 no. 1 2a

165.9, C 50.0, CH2

3 4 5 6 7 8 9 10 10-OH 11 11-OH 12 12-OH 13 14 15 16 17 18 19 20 21 22 23 23-OH 24 25

192.6, 122.6, 145.7, 132.5, 140.4, 129.0, 138.9, 69.6,

26 27 28 −NH a

δC, type

C CH CH CH CH CH CH CH

73.1, CH 79.3, CH 198.4, 120.6, 143.1, 126.1, 146.3, 135.6, 134.6, 128.6, 137.5, 67.5, 82.1,

C CH CH CH CH C CH CH CH CH CH

38.1, CH 50.1, CH2 12.0, CH3 12.2, CH3 14.5, CH3

2 δH

3.90 d (17.5) 3.44 d (17.5) 6.10 d (15.5) 6.77 d (15.5) 6.07 6.34 5.47 4.02 4.80 3.82 4.64 4.34 4.81

d (11.5) dd (11.5, 15.0) dd (9.0, 15.5) t (9.0) brs dd (2.0, 9.0) brs brs brs

6.17 6.67 7.42 6.62

d (11.0) dd (11.0, 11.0) dd (11.0, 15.5) d (15.5)

6.66 6.40 5.50 3.93 3.47 5.44 1.84 3.85 2.79 1.79 1.63 1.01

d (11.5) dd (11.5, 15.5) dd (9.0, 15.5) dd (7.0, 9.0) m m m dd (8.0, 12.0) dd (12.0, 12.0) s s d (6.5)

3

δC, type 165.9, C 49.8, CH2 192.7, 122.3, 146.0, 130.6, 141.0, 128.0, 137.0, 36.0,

C CH CH C CH CH CH CH2

69.8, CH 80.2, CH 198.3, 120.7, 143.1, 126.0, 146.6, 135.5, 134.9, 128.6, 137.6, 67.5, 82.0,

C CH CH CH CH C CH CH CH CH CH

38.1, CH 50.1, CH2 12.0, CH3 12.0, CH3 14.5, CH3

δH

δC, type 166.2, C (50.2, CH2)b

3.90 d (18.0) 3.45 d (18.0) 6.04 d (15.5) 6.73 d (15.5) 6.01 6.21 5.56 2.65

d (12.0) ddd (1.5, 12.0, 15.0) ddd (4.0, 11.0, 15.0) ddd (11.0, 11.0, 15.0) 2.08 brd (15.0)

4.10 4.88 4.30 4.76

brd (11.0) brs brs brd (4.0)

6.17 6.65 7.34 6.61

d (11.5) dd (11.5, 11.5) dd (11.5, 15.5) d (15.5)

6.14 6.39 5.46 3.90 3.45 5.44 1.83 3.84 2.77 1.75 1.60 1.00

d (12.0) dd (12.0, 15.5) dd (10.5, 15.5) dd (8.0, 10.5) m brs m dd (8.5, 11.5) dd (11.5, 11.5) s s d (6.5)

192.5, 121.7, 148.9, 136.6, 146.2, 38.4, 49.2, 70.6,

C CH CH C CH CH CH CH

78.5, CH 74.3, CH 210.4, 44.6, 123.8, 130.8, 53.3, 134.4, 130.6, 126.1, 132.6, 131.3, 137.0,

C CH CH CH CH C CH CH CH CH CH

38.8, CH 44.5, CH2 12.5, CH3 12.0, CH3 17.9, CH3

δH 3.53 s

6.13 d (15.0) 7.30 d (15.0) 5.74 d (10.0) 2.77 ddd (10.0, 10.0, 12.0) 2.55 dd (6.5, 12.0) 4.05 brs (5.29 s)a 4.23 m (4.94 s)a 4.70 d (3.5) (4.63 s)a 3.66 5.86 5.83 2.50

m ddd (2.5, 5.0, 10.0) d (10.0) dd (10.0, 2.5)

5.44 6.16 5.82 6.08 5.29

d (11.0) dd (11.0, 14.5) dd (10.5, 14.5) dd (10.5, 15.0) dd (9.5, 15.0)

2.42 m 3.45 dd (11.0, 13.5) 2.95 dd (5.0, 13.5) 1.42 s 1.59 s 1.01, d (6.5) (7.99 m)a

Observed in d6-DMSO. bFrom HSQC correlation.

dimethoxypropane (DMP) and pyridinium p-toluenesulfonate (PPTS), both 1 and 2 were converted into the corresponding acetonide-protected forms at room temperature (4 and 5, Scheme 1). The anti-configuration of the 10,11-diol in 1 was determined based on the similar magnetic environments of the isopropylidene methyl groups (ΔδH = 0.01) and the relatively large coupling constant between H-10 and H-11 (3JH10H11 = 9.0 Hz) in 4, while the syn-configuration of the 11,12-diol in 2 was established based on the dissimilar proton chemical shifts of the isopropylidene methyl groups (ΔδH = 0.16) and the relatively small coupling constant between H-11 and H-12 (3JH11H12 = 4.5 Hz) in 5.12−14 Considering the NMR analysis and their structural relationships, the stereochemistries of 1 and 2 should be mutually applicable; therefore, the relative configurations of the 1,2,3-triol moiety in 1 were determined to be (10R*, 11S*, 12S*). In the same manner, the (22S*, 23R*, 24S*) configurations of 2 were also inferred from the structural comparison with 1. The absolute configurations of 1 were determined by combining the modified Mosher’s method15 and Trost’s method.16 The modified Mosher’s method was applied to 4

Figure 2. 2D NMR correlations of niizalactams A (1) and C (3).

Figure 1, which is structurally related to 1 and 2 but contains an unprecedented [18,6,6]-ring system. The relative stereochemistries of the pyrrolidinol ring (C-22 to C-25 in 1 and 2) were established as (22S*, 23R*, 24S*) based on NOESY correlations around the pyrrolidinol moiety of 1 (Figure 2). On the other hand, the NOESY and the other NMR spectroscopic data did not give beneficial information for stereochemistries of the 1,2,3-triol moiety in 1 (1,2-diol moiety in 2). To determine their relative stereochemistries, acetonides 4 and 5 were prepared (Scheme 1). In the presence of 2,2C

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Scheme 1. Chemical Derivatization of Niizalactams A (1) and B (2) and NMR Analyses of the Derivatives

Scheme 2. Proposed Biosynthetic Pathway of Niizalactams 1−3

D

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using α-methoxy(trifluoromethyl)phenylacetic acid chloride (MTPA-Cl),15 and the mono-(S)- and (R)-MTPA esters 6a and 6b were obtained (Scheme 1). The 1H NMR and 1H−1H COSY analysis of both forms of the MTPA esters allowed us to conclude that the absolute stereochemistries of the pyrrolidinol moiety were (22S, 23R, 24S). In spite of significant efforts, we could not obtain C-12 MTPA esters of 4. Alternatively, the absolute stereochemistries of the 1,2,3-triol moiety in 1 were determined based on Trost’s method, using α-methoxyphenylacetic acid (MPA).16 In the presence of N,N-dimethylaminopyridine (DMAP) and N,N′-diisopropyl carbodiimide (DIC), 4 was converted into the desired bis-MPA derivatives 7a and 7b at room temperature. The detailed NMR analysis of these MPA esters led us to establish the absolute stereochemistries for the 1,2,3-triol moiety as (10R, 11S, 12S). On the basis of the structural comparison, the absolute configurations of 2 were established as (11S, 12S, 22S, 23R, 24S). The stereochemistries of 3 were determined based on structural comparison and NMR analysis. The partial stereochemistries for the 1,2,3-triol moiety and the C-24 chiral center of 3 were determined as (10R, 11S, 12S, 24R) from comparison to the stereochemistries of 1. The remaining stereochemistries in the cyclohexene moiety (C-8, C-9, C-14, and C-17) were fully elucidated based on NMR data. The combination of the 3 JHH coupling constants (3JH8H9 = 12.0 Hz, 3JH8H17 = 10.0 Hz, 3 JH9H14 = 6.5 Hz) established their relative configurations as (8R*, 9S*, 14R*, 17S*), and the NOESY correlations of H-7/ H-9, H-7/H-17, H-9/H-17, and H-9/H-14 also supported these assignments. Finally, the absolute stereochemistries of the cyclohexene ring in 3 were determined to be (8R, 9S, 14R, 17S) based on the NOESY correlation between H-12 and H-14 (Figure 2). The plausible biosynthetic relationship among the niizalactams is depicted in Scheme 2. First, as in the biosyntheses of vicenistatin17 and lobosamides,18 the type I polyketide synthase (type I PKS) assembly lines construct a core 26-membered macrolactam structure employing aminoacyl-ACP as a starter unit, which is then converted into a common intermediate 8 by subsequent enzymatic oxidation. The pyrrolidinol moiety of 2 is assumed to be constructed through the epoxidation of the Δ22,23-alkene and the spontaneous SN2 cyclization of 8. If hydroxylation of the C-10 methylene group precedes the pyrrolidinol ring formation, then 1 would be biosynthesized via 9, in the same manner. In addition, 3 would also be biosynthesized from 9 via intermediate 10 through doublebond isomerization of the Δ14,15-alkene moiety and subsequent intramolecular Diels−Alder cyclization (Scheme 2). Although we could not prove the presence of the common precursor 8, the chemical structure of 9 is identical to that of the previously reported sceliphrolactam,19 a 26-membered antimicrobial macrolactam isolated from a wasp-associated Streptomyces strain, whose stereochemistries were not determined in the original research. Notably, the NMR data, the optical rotation, and UV absorption of 3 were highly similar to those of the previously reported tripartilactam (11)20 isolated from a dung beetlerelated Streptomyces bacterium (Figure 3A and Figure S35). The tripartilactam (11) possesses the same 18-membered polyene macrolactam ring moiety as 3 and an unusual [4,8]ring moiety (Figure 3A). As noted above, niizalactam C (3) probably shares the same biosynthetic background as 1 and 2. Furthermore, the direct bond formation between C9 and C10 was definitely confirmed on the basis of the 1H−1H COSY

Figure 3. Chemical structures of tripartilactam and structurally related compounds to niizalactams.

analysis (Figures S13 and S14), corresponding to C9 and C11 in tripartilactam 11.20 Considering these results, the structure of the tripartilactam 11 should be revised to 3. To our best knowledge, this is the first report of macrolactams containing a [5,23]-ring system (1 and 2) or an [18,6,6]-ring system (3). Furthermore, pyrrolidinol-bearing macrolactams are relatively rare in nature; only ciromicins11 and heronamide A21 have been reported as pyrrolidinolcontaining macrolactams (Figure 3B). Unfortunately, 1−3 did not show any antimicrobial activities or cytotoxicities. The results of bioactive assays for 3 do not conflict with the relationship between 3 and 11.20 Our study strongly supports the efficacy of the combinedculture strategy to discover structurally unique cryptic secondary metabolites from Streptomyces bacteria. Further applications will lead to the isolation of secondary metabolites with attractive structures and bioactivities.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO DIP-1000 digital polarimeter. The UV spectra of 1−3 were measured with a JASCO MD-2010 Plus multiwavelength detector. Except for the NMR spectra of 3 in CD3OD, all of the NMR spectra were recorded on a JEOL ECX-500 or ECA-500 spectrometer (1H NMR, 500 MHz; 13C NMR, 125 MHz). The NMR spectra of 3 in CD3OD were obtained from a Bruker AVANCE III HD 900 spectrometer (900 MHz). The MALDI-TOF-TOF HRMS data of 1−3 were recorded with an AXIMA-TOF2 mass spectrometer (Shimadzu), and the ESI-LRMS data of chemical derivatives 4−7b were obtained from an Esquire 4000 (Bruker). Isolation and Identification of Streptomyces sp. NZ-6. The soil sample was obtained at Niiza city, Saitama, Japan, in November 2014, and the producing microorganism Streptomyces sp. NZ-6 was isolated from this sample according to Hayakawa and Nonomura’s protocol.22 To identify the 16S rRNA gene sequence, Streptomyces sp. NZ-6 was cultured in tryptosoya broth (TSB, 100 mL) for 3 days (160 rpm, 30 °C) and its genomic DNA was extracted as a template. The 16S rRNA gene region was amplified by PCR reaction using universal primers, and the reaction product was inserted into the pT7Blue vector and sequenced. The BLAST analysis revealed that the partial E

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Compound 4: 1H NMR (d6-DMSO, 500 MHz) δ 7.17 (1H, dd, J = 12.0, 15.0 Hz, H-16), 6.77 (1H, d, J = 16.0 Hz, H-5), 6.60 (1H, dd, J = 11.0, 12.0 Hz, H-15), 6.59 (1H, d, J = 15.0 Hz, H-17), 6.48 (1H, dd, J = 11.5, 15.0 Hz, H-20), 6.41 (1H, dd, J = 11.5, 15.0 Hz, H-8), 6.20 (1H, d, J = 11.0 Hz, H-14), 6.13 (1H, d, J = 16.0 Hz, H-4), 6.13 (1H, m, H-19), 6.12 (1H, m, H-7), 5.51 (1H, dd, J = 10.5, 15.0 Hz, H-9), 5.46 (1H, dd, J = 8.5, 15.0 Hz, H-21), 5.45 (1H, m, 23-OH), 5.05 (1H, d, J = 5.5 Hz, 10-OH), 4.64 (1H, dd, J = 2.0, 5.5 Hz, H-12), 4.37 (1H, dd, J = 9.0, 10.0 Hz, H-10), 4.14 (1H, dd, J = 2.0, 9.0 Hz, H-11), 3.92 (1H, d, J = 18.0 Hz, H-2b), 3.89 (1H, dd, J = 6.5, 9.0 Hz, H-22), 3.85 (1H, dd, J = 7.0, 11.5 Hz, H-25b), 3.47 (1H, d, J = 18.0 Hz, H-2a), 3.41 (1H, m, H-23), 2.77 (1H, t, J = 11.5 Hz, H-25a), 1.83 (1H, m, H24), 1.81 (3H, s, H-26), 1.61 (3H, s, H-27), 1.34 (3H, s, CH3 in acetonide), 1.33 (3H, s, CH3 in acetonide), 1.00 (3H, d, J = 6.5 Hz, H28); ESI-LRMS m/z 538.4 [M + H]+ (calcd for C31H39NO7, 538.3). Acetonide Protection of Niizalactam B. A 1.0 mg (2.1 μmol) amount of 2 and 0.6 mg (2.4 μmol) of PPTS were dissolved in 1.0 mL of 1:4 DMP−acetone and stirred for 3 h at room temperature without light exposure. The reaction mixture was concentrated in vacuo and separated by flash silica gel column chromatography (methanol− chloroform = 0:100−10:90). Finally, 0.8 mg of 5 was collected from a 4:96 methanol−chloroform fraction (1.5 μmol, 71%). Compound 5: 1H NMR (d6-DMSO, 500 MHz) δ 7.21 (1H, dd, J = 12.0, 15.0 Hz, H-16), 6.75 (1H, d, J = 16.0 Hz, H-5), 6.62 (1H, t, J = 11.0 Hz, H-15), 6.62 (1H, d, J = 15.0 Hz, H-17), 6.40 (1H, dd, J = 11.5, 15.0 Hz, H-20), 6.32 (1H, dd, J = 11.5, 15.0 Hz, H-8), 6.15 (1H, d, J = 11.0 Hz, H-14), 6.11 (1H, d, J = 11.5 Hz, H-19), 6.06 (1H, d, J = 16.0 Hz, H-4), 6.04 (1H, d, J = 11.5 Hz, H-7), 5.51 (1H, m, H-9), 5.44−5.47 (2H, m, H-21 and 23-OH), 4.91 (1H, d, J = 4.5 Hz, H-12), 4.55 (1H, m, H-11), 3.95−4.00 (2H, m, H-2b and H-22), 3.84 (1H, dd, J = 8.0, 11.5 Hz, H-25b), 3.44−3.48 (2H, m, H-2a and H-23), 2.78 (1H, t, J = 11.5 Hz, 25a), 2.58 (1H, m, H-10b), 2.33 (1H, m, H-10a), 1.84 (1H, m, H-24), 1.76 (3H, s, H-26), 1.61 (3H, s, H-27), 1.45 (s, 3H, CH3 in acetonide), 1.29 (3H, s, CH3 in acetonide), 1.00 (3H, d, J = 6.5 Hz, H-28); ESI-LRMS m/z 522.5 [M + H]+ (calcd for C31H39NO6, 522.3). Preparing the MTPA Esters of 4. A 2.8 mg (5.2 μmol) amount of 4 and 10 mg (40 μmol) of (R)-(−)-MTPA chloride were dissolved in 1.0 mL of anhydrous CH2Cl2 and stirred for 5 min at room temperature. To this solution was added a catalytic amount of DMAP, and the mixture stirred for 2 h at room temperature (all processes conducted without light). The reaction mixture was concentrated in vacuo, the resulting residue was separated by silica-gel flash column chromatography, and 0.4 mg of 6a was obtained from the 4:96 methanol−chloroform fraction (0.5 μmol, 10%). A 0.7 mg amount of 6b was also obtained from 1.9 mg of 4 (0.9 μmol, 26%) using (S)(−)-MTPA chloride. Compound 6a: key 1H NMR data, see Figure S34; ESI-LRMS m/z 754.6 [M + H]+ (calcd for C41H46F3NO9, 754.3). Compound 6b: key 1H NMR data, see Figure S34; ESI-LRMS m/z 754.7 [M + H]+ (calcd for C41H46F3NO9, 754.3). Preparing the Bis-MPA Esters of 4. A 2.0 mg (3.7 μmol) amount of 4 and few crystals of DMAP were suspended in 0.5 mL of anhydrous CH2Cl2 and stirred for 5 min at room temperature. To this solution were added 4.8 μL of DIC (31 μmol) and 2.0 mg of (R)(−)-MPA (12 μmol), and the mixture was stirred at room temperature. After stirring for 30 min, the same amount of DIC and (R)-(−)MPA were added to the solution again and stirred for additional 1.5 h (all processes were performed in the dark). The reaction mixture was purified by HPLC equipped with a Cosmosil cholester column (10 × 250 mm, Nacalai Tesque, Japan) with the isocratic solvent system (65% aqueous CH3CN, 7a was eluted at 8.0 min), and 0.4 mg of 7a (0.5 μmol, 14%) was obtained as a yellow solid. Likewise, 0.4 mg of 7b was obtained from 2.0 mg of 4 (0.5 μmol, 14%). Compound 7a: key 1H NMR data, see Figure S34; ESI-LRMS m/z 834.7 [M + H]+ (calcd for C49H55NO11, 834.4). Compound 7b: key 1H NMR data, see Figure S34; ESI-LRMS m/z 834.8 [M + H]+ (calcd for C49H55NO11, 834.4).

16S rRNA gene sequence of Streptomyces sp. NZ-6 was completely included in that of Streptomyces sp. Ahbb2 (see Supporting Information). Analysis of the Metabolic Profiles of Streptomyces sp. NZ-6. Streptomyces sp. NZ-6 and Tsukamurella pulmonis TP-B0596 were independently inoculated into V-22 medium6 (100 mL, in 500 mL Erlenmeyer flasks); Streptomyces sp. NZ-6 was cultured for 3 days, and T. pulmonis TP-B0596 was cultured for 2 days (160 rpm, 30 °C). The precultures (Streptomyces sp. NZ-6, 3 mL. T. pulmonis, 1 mL) were simultaneously put into A-3M medium (100 mL, in a 500 mL baffled Erlenmeyer flask) and cultured for 6 days at 160 rpm and 30 °C. As controls, each of the strains was individually cultured under the same conditions, respectively. After cultivation, the whole culture broths were extracted with 100 mL of ethyl acetate and the organic layers were collected by centrifugation (10 000 rpm, 5 min) and concentrated under reduced pressure. The resulting residues were dissolved in 50:50 methanol− chloroform (1 mL), and 10 μL of the samples was analyzed by HPLC with a Cosmosil 5C18-MS-II column (4.6 × 250 mm, Nacalai Tesque, Japan) in a CH3CN (solvent A)−H2O with 0.1% trifluoroacetic acid (solvent B) gradient system (solvent A: 10−60% (15 min) to 100% (20−30 min)), at 35 °C and a flow rate of 1.0 mL min−1. 1−3 were detected at 15.9, 17.3, and 20.3 min, respectively, based on the UV absorption at 300 nm. The productions of 3 in combined culture and pure culture were evaluated by integrating the corresponding peaks in an HPLC chart at 279 nm. Isolation of Niizalactams A−C (1−3). The combined-culture broth (2.0 L, 20 × 100 mL) of Streptomyces sp. NZ-6 was centrifuged (6000 rpm, 10 min) to obtain cell pellet and supernatant. The cell pellet (containing HP-20 resin in A-3M medium) was extracted with 500 mL of acetone twice after lyhophilization, and the supernatant was extracted with 700 mL of ethyl acetate three times, respectively. HPLC analysis revealed that 1 and 2 were mainly present in the cell extract and 3 was mainly present in the supernatant extract. The cell extract (2.6 g) was separated by flash silica gel column chromatography (methanol−chloroform = 0:100−20:80). A 10:90 methanol−chloroform fraction (kept on ice) contained 2, and a 20:80 methanol−chloroform fraction contained 1. The supernatant extract (560 mg) was also separated by flash silica gel column chromatography (methanol−chloroform = 0:100−10:90), and 3 was present in a 10:90 methanol−chloroform fraction. The final purification was performed by semipreparative reversed-phase HPLC equipped with a Cosmosil cholester column (10 × 250 mm, Nacalai Tesque, Japan) with an isocratic solvent system (1: 9.9 min in 23% aqueous CH3CN; 2: 8.0 min in 30% aqueous CH3CN; 3: 9.5 min in 38% aqueous CH3CN). These processes were repeated four times, and 1 (60 mg), 2 (5.2 mg), and 3 (1.3 mg) were obtained from 10 L of culture broth, respectively. We performed all processes from cultivation to purification under dark conditions and ice cooling. Niizalactam A (1): yellowish powder; [α]25D −2767.2 (c 0.015, MeOH− CHCl3, 3:1); UV (CH3CN) λmax 227, 263, 327 nm; 1H and 13 C NMR data, see Table 1; HR-MALDIMS m/z 520.2289 [M + Na]+ (calcd for C28H35NO7, 520.2311). Niizalactam B (2): yellowish powder; [α]25D −890.6 (c 0.071, MeOH); UV (CH3CN) λmax 227, 263, 327 nm; 1H and 13C NMR data, see Table 1; HR-MALDIMS m/z 504.2368 [M + Na]+ (calcd for C28H35NO6, 504.2362). Niizalactam C (3): white powder; [α]25D −127.8 (c 0.020, MeOH); UV (CH3CN) λmax 223, 279 nm; 1H and 13C NMR data, see Table 1; HR-MALDIMS m/z 504.2356 [M + Na]+ (calcd for C28H35NO6, 504.2362). Acetonide Protection of Niizalactam A. A 2.8 mg (5.6 μmol) amount of 1 and 2.0 mg of PPTS (8.0 μmol) were dissolved in 1.0 mL of 50:50 DMP−methanol and stirred for 1 h at room temperature without light. The reaction mixture was poured into 10 mL of water and extracted with 10 mL of ethyl acetate three times. After evaporating, 2.9 mg (5.4 μmol, 96%) of 4 was obtained as a yellowish powder. By repeating these processes, 10 mg of 4 was obtained for preparing their MPA or MTPA derivatives. F

DOI: 10.1021/acs.jnatprod.5b00804 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Antimicrobial Assay. For disk diffusion assays, Candida albicans, Bacillus subtilis, and Saccharomyces cereviciae were precultured in LB broth. The resulting broths (1 mL) were directly added to culture mediums (20 mL) of agar plates (Candida albicans: 4.0% glucose, 1.0% Bacto peptone (Difco), pH = 5.7; Bacillus subtilis: LB plate; Saccharomyces cereviciae: 5.0% glucose, 0.5% Bacto peptone, 0.2% yeast extract, 0.4% K2HPO4, 0.2% KH2PO4, and 0.02% MgSO4·7H2O, pH = 5.6). Paper disks (6 mm) containing 1−3 (10, 5, 2.5, and 1.25 μg/disk) were put on the agar plates. All plates were incubated at 37 °C 1 to 2 days and checked for the presence of inhibition rings around the paper disks. Cytotoxicity Assay. To test the cytotoxicities of 1−3 against P388 murine leukemia cells, we conducted the methyl thiazoletetrazolium (MTT) assay.23 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. DMSO solutions of 1 and 2 and methanol solution of 3 were added to a 96-well microplate containing 100 μL of a 1 × 105 cells/mL P388 cell suspension (final concentration: 100, 50, 25, 12.5, 6.3, 3.1, 1.6, and 0.78 μM), and the cells were incubated for 4 days at 37 °C under a 5% CO2 atmosphere. After the cultivation, 50 μL of MTT solution (1 mg/ mL, dissolved in DMSO) was added to each well and incubated for 4 h at the same condition. The mixtures were separated by centrifugation, and supernatant was removed. The resulting precipitate was dissolved in DMSO (50 μL), and the absorbance at 570 nm was measured with a microplate reader.

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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.5b00804. NMR spectroscopic data for 1−7b and the partial base sequence of 16S rRNA gene of Streptomyces sp. NZ-6 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81-3-3818-2532. Fax: +81-3-5841-4744. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by Grants-in-Aid from the MEXT, Japan. We thank Dr. T. Ozaki and Dr. S. Asamizu (The University of Tokyo) for their helpful advice and for conservation of T. pulmonis TP-B0596.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00804 J. Nat. Prod. XXXX, XXX, XXX−XXX