Letter pubs.acs.org/OrgLett
Mycolic Acid Containing Bacterium Stimulates Tandem Cyclization of Polyene Macrolactam in a Lake Sediment Derived Rare Actinomycete Shotaro Hoshino,† Masahiro Okada,† Takayoshi Awakawa,† Shumpei Asamizu,‡ Hiroyasu Onaka,‡ and Ikuro Abe*,† †
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
‡
S Supporting Information *
ABSTRACT: Two novel macrolactams, dracolactams A and B, were identified from a combined-culture of Micromonospora species and a mycolic-acid containing bacterium (MACB). Their structures and stereochemistries were completely assigned, based on spectroscopic analyses and chemical derivatization. Both dracolactams were probably generated from a common macrolactam precursor produced by the Micromonospora species. In this combined-culture system, MACB is likely to activate cryptic oxidase genes in the Micromonospora species and induce the downstream polyene macrolactam cyclization.
A
duced polyene macrolactams with novel skeletons only when it was cocultured with the MACB, Tsukamurella pulmonis TPB05966 (1 and 2, Figure 1A,B), while both the pure and combined-cultures of M. wenchangensis HEK-797 yielded the 26-membered polyene macrolactam 7, which is likely to be the precursor of both 1 and 2 (Figure 1A,B). Herein, we report the isolation, structural elucidation, and biosynthetic insights of two novel macrolactams, named dracolactams A (1) and B (2). To determine the structures of 1 and 2, we prepared a 1.5 L culture of M. wenchangensis HEK-797 with T. pulmonis TPB0596. The mycelium and the HP-20 resin included in the culture broth were collected by centrifugation. The resulting pellet was lyophilized and extracted with methanol. The methanol extract was roughly separated by iterative steps of flash silica gel column chromatography and subjected to HPLC purification using an ODS column. Finally, we obtained pure 1 (15.2 mg of white powder) and 2 (3.2 mg of white powder). The HRTOFMS data of 1 provided a molecular formula of C28H39NO6 (observed [M + H]+ at m/z 486.2859), indicating 10 degrees of unsaturation. The 13C NMR showed 28 resonances, including one amide group (δC 174.5 ppm) and 10 olefinic carbons from 138.2 to 121.7 ppm, explaining the six degrees of unsaturation and suggesting the presence of four ring systems. Analyses of the 1H, 13C, COSY, and HMQC data clearly indicated the presence of two isolated spin systems
ctinomycetes are a group of Gram-positive bacteria with a large potential for the production of secondary metabolites, including pharmaceutically important compounds. Among them, the genus Streptomyces has been recognized as a dominant producer of secondary metabolites, and much effort in natural product discovery has been focused on Streptomyces species.1,2 In contrast, rare actinomycetes (= non-Streptomyces) are a less-exploited resource for natural product isolation, although there are some reports about bioactive compounds isolated from them as drug candidates.3,4 Recent bacterial genome sequencing projects revealed that some rare actinomycetes possess comparable levels of natural product biosynthetic gene clusters in their genomes. However, many biosynthetic genes remain unexpressed under general culture conditions.5 Therefore, the activation of silent genes in rare actinomycetes is a promising pathway toward the isolation of novel secondary metabolites. We previously demonstrated that a “combined-culture”, a coculture with a mycolic acid containing bacterium (MACB), efficiently induces secondary metabolite production in numerous Streptomyces species.6,7 Although several new secondary metabolites were discovered from Streptomyces species with the combined-culture method,7−10 the application to rare actinomycetes was quite limited.11 Thus, we applied the combined-culture method to our collection of rare actinomycetes isolated from various environments. In the course of screening, we found that the lake sediment-derived, rare actinomycete Micromonospora wenchangensis HEK-797 pro© 2017 American Chemical Society
Received: August 13, 2017 Published: September 7, 2017 4992
DOI: 10.1021/acs.orglett.7b02508 Org. Lett. 2017, 19, 4992−4995
Letter
Organic Letters
Considering the chemical shifts and the formation of diacetonide 4 from 1 (Scheme 1), the hydroxyl groups are Scheme 1. Preparation of Acetonide Derivatives of 1 and 13C NMR Signals of Acetonide Groups
also likely to be attached to C11 and C13, although the corresponding signals were not observed. The unassigned nitrogen atom should be included in the pyrrolizidine ring (A/ B ring system in Figure 2A) to account for the remaining site of unsaturation and the chemical shifts of C1, C22, and C25. The geometries of the five C−C double bonds were elucidated to be 3Z, 6E, 14Z, 16E, and 18E on the basis of the NOESY correlations (Figure 2B) and several vicinal coupling constants (3J(H3, H4) = 10.5, 3J(H6, H7) = 15.5, 3J(H14, H15) = 11.5, and 3J(H16, H17) = 15.0 Hz). The HRTOFMS data of 2 provided the same molecular formula as that of 1 (observed [M + H]+ at m/z 486.2860). The 13C NMR also showed 28 resonances, including one unsaturated amide group (δC 166.8) and 14 olefinic carbons from 141.9 to 123.7 ppm, satisfying 8 out of 10 degrees of unsaturation. The 1H, 13C, COSY, and HMQC data showed the presence of five isolated spin systems (fragments I−V in Figure 2A), including four hydroxyl groups (δH 4.53, 4.56, 4.65, and 4.96 ppm in DMSO-d6) and an amide −NH group (δH 7.50 in DMSO-d6). Some signal overlaps and the absence of a COSY correlation of H13/H14 hampered the construction of several assumed connections. However, the unassigned C−C bonds (C1/C2, C6/C7, C13/C14, C17/C18/C19, C20/C21, and C18/C27) were completely established by the HMBC spectrum (Figure 2A). The connection of C1/NH was strongly suggested from the chemical shift of C1 (δC 166.8 ppm). Finally, to explain the remaining degree of unsaturation, the sixth unassigned oxygen atom should be attached to both C11 and C14 to construct the tetrahydrofuran (THF) ring system. All of the C−C double bonds in 2 were assigned to the E configuration, based on the observed NOESY correlations (Figure 2B) and the vicinal coupling constants (3J(H2, H3) = 15.0, 3J(H4, H5) = 15.0, 3J(H15, H16) = 15.5, 3J(H19, H20) = 15.0, and 3J(H21, H22) = 15.0 Hz). Next, we performed a stereochemical analysis of 1, which revealed an unprecedented fused-5/5/6/16 ring system (A−D in Figure 2A). The relative stereochemistries of the conformationally rigid A−C ring system (C2, C5, C20−C23, and C25) were clearly elucidated on the basis of the series of NOESY correlations (H2/H20, H5/H20, H19/H21, H19/H23, H20/
Figure 1. (A) HPLC profiles of M. wenchangensis cocultured with T. pulmonis (top), Micromonospora species pure culture (middle), and T. pulmonis pure culture (bottom), detected by UV absorption at 290 nm. (B) Proposed structures of 1, 2, and 7. Structure 7 is the plausible common precursor of 1 and 2. The stereochemistry of 7 was deduced from those of 1 and 2.
(fragments I and II in Figure 2A). The absence of COSY correlations between H2/H3 and H8/H9 indicated that the
Figure 2. (A) COSY (black bold bonds) and HMBC (red arrows) correlations in 1 and 2. The COSY correlation revealed two isolated spin systems in 1 (I and II) and five isolated spin systems in 2 (I−V). The COSY correlations from the OH/NH groups were only observed in DMSO-d6. (B) Key NOESY correlations (red dashed arrows) of 1 and 2.
corresponding dihedral torsion angles are close to 90°. The remaining C−C connections (C1/C2/C3, C8/C9, C17/C18/ C19, and C18/C27) were fully established, based on the HMBC correlations (Figure 2A). The NMR data in DMSO-d6 showed three exchangeable protons (δH 4.33, 4.38, 4.65) at the three hydroxyl groups bound to C9, C10, and C23. 4993
DOI: 10.1021/acs.orglett.7b02508 Org. Lett. 2017, 19, 4992−4995
Letter
Organic Letters H22, H21/H23, and H22/H28, Figure 2B and Figure S37). The proposed stereochemistries in the A−C rings were also supported by a set of coupling constants (3J(H2, H21) = 10.0, 3 J(H5, H20) = 6.0, 3J(H20, H21) = 10.0, and 3J(H21, H22) = 10.0 Hz). On the other hand, the stereochemical assignment around 16-memebered D ring was hampered by its conformational flexibility. To determine the relative configurations of D ring, we prepared two types of acetonides, 3 and 4, under two different reaction conditions (Scheme 1), and their structures were analyzed by 1D and 2D NMR spectroscopy. To establish the relative stereochemistries of the 1,2- and 1,3-diol groups in 1, we applied Rychnovsky’s and/or Dana’s methods to acetonides 3 and 4 (Scheme 1 and Figure S38).12−14 As a result, both of the diols at the C9/C11 and C11/C13 positions were determined to adopt the 1,3-syn (= 9R*/11S*/13R*) configuration, based on the 13C NMR signals of 3 and 4 at the 1,3-diol acetonides.12,14 In contrast, the diol at the C9/C10 position was determined to adopt the 1,2-anti (= 9R*/10S*) configuration, based on the 13C NMR signals of 4 at the 1,2diol acetonide.13,14 The strong NOESY correlations among H9, H11, and one of the acetonide methyl groups (δH 1.46), and the vicinal coupling constants of H9/H10 (9.0 Hz) and H10/ H11 (9.0 Hz), clearly indicated that the 1,3-dioxane ring in 3 retains the chair conformation (Figure S39). In addition, H5/ H6, H7/H8, H8/H9 and H19/H20 in 3 were deduced to bein an antiperiplanar relationship on the basis of the NOESY correlations (H5/H7, H6/H8, H8/H10, H6/H19 and H20/ H27), vicinal coupling constants (3J(H5, H6) = 9.5 Hz, 3J(H6, H7) = 15.5 Hz 3J(H7, H8) = 9.5 Hz, 3J(H8, H9) = 9.0 Hz and 3 J(H19, H20) = 11.0 Hz), and avoidance of 1,3-allylic strains (between H6 and H8/C9/C26). Under the conformational requirements discussed above, the transannular NOESY correlation of H10/H19 in 3 could be explained only when 1 has the 5R*/9R* configuration, suggesting that both C9 and C20 reside on the same face of the C5−C8 plane system in 3 (Figure S39). Finally, we investigated the orientation of C26 methyl group and the relative configuration of C8. The H26 protons showed NOESY correlations only to H7, H8, and H9 protons, while any transannular NOEs were not observed, strongly indicating that C26 methyl group reside on the “outside” of the D-ring and 1 has the 8S*/9R* configuration (Figure S39). Thus, the relative configurations of the D-ring in 1 were assigned to be 5R*/8S*/9R*/10S*/11S*/13R*/20S*. According to their structural resemblance, 1 and 2 should be generated from a common precursor in their biosynthetic pathway. Thus, most of the stereochemistries of 2 should be identical to those of 1, except for the newly generated chiral center at C14 of 2. The vicinal coupling constant of H13/H14 (approximately 0 Hz) in 2 suggested that the corresponding torsion angle is nearly 90°. A previous molecular dynamics simulation study revealed that the dihedral angle between the cis vicinal protons in a THF ring ranges from −60° to +60°, while the dihedral angle between the trans vicinal protons ranges from |60°| to |180°|.15 Thus, we concluded that H13 and H14 have a 1,2-trans relationship (= 13R*/14S*). Finally, the absolute configurations of 1 and 2 were determined by applying a modified Mosher’s method to the C13 position of 2 (Scheme 2).16 Compound 2 was initially converted to 5 with acetonide protection to reduce the number of free hydroxyl groups. Then, the (S)- or (R)-MTPA ester was introduced to the free hydroxyl group at the C-13 position of 5 to provide the corresponding MTPA esters 6a and 6b. From
Scheme 2. Acetonide and Mosher Derivatization of 2 To Determine the Absolute Configuration of the C13 Position
the calculated Δδ values, we assigned the absolute configuration of C13 as R (Scheme 2). In conclusion, all of the absolute configurations of 2 were assigned as (8S,9R,10R,11S,13R,14S,23R,25S), and those of 1 were deduced as (2S,5R,8S,9R,10S,11S,13R,20S,21S,22S,23R,25S). The unprecedented skeletons of 1 and 2 were predicted to be generated from the 26-membered polyketide macrolactam 7 via epoxidation and subsequent tandem cyclization (Figure 1B and Figure S40). Notably, the plausible precursor 7 has an identical planar structure to that of micromonolactam.17 Micromonolactam was previously isolated from various Micromonospora species. The biosynthetic gene cluster of micromonolactam has also been identified (mml cluster), and it includes a type I modular PKS and accessory genes.17 Since the original study did not mention the stereochemistries of the micromonolactam, we conducted a genetic analysis of the ketoreductase (KR) domains in the mml cluster and predicted the absolute configurations of micromonolactam. Our prediction did not conflict with the stereochemistries of 7 translated from 1 and 2 (Figure 1B and Figure S41).18 These observations strongly suggested that M. wenchangensis HEK-797 also produces micromonolactam, which is converted to 1 and 2 via epoxidation and subsequent cyclization. In fact, UV−HPLC and LC−MS analyses suggested that M. wenchangensis HEK797 produces a compound with the same molecular formula and UV spectrum as those of micromonolactam, and its production level was not affected by MACB (Figure 1A and Figure S42). The production of dracolactams induced by combinedculture and the constant production of the plausible precursor (7) strongly suggested that the addition of MACB induces the expression of latent dracolactam biosynthetic pathways, including those involved in the epoxidation and subsequent cyclization of 7. In this case, MACB should upregulate the expression of genes in the latent pathway present in M. wenchangensis HEK-797. It was unlikely that 7 was converted into 1 and 2 through a simple bioconversion by MACB, because the formation of 1 and 2 was not observed when the crude extract containing 7 was fed to a pure culture of MACB (Supporting Information). Finally, we assessed the antimicrobial activities and cytotoxicities of 1 and 2. However, no bioactivities were observed in our assay system. The biological functions of these molecules remain unknown. 4994
DOI: 10.1021/acs.orglett.7b02508 Org. Lett. 2017, 19, 4992−4995
Letter
Organic Letters
(11) Derewacz, D. K.; Covington, B. C.; McLean, J. A.; Bachmann, B. O. ACS Chem. Biol. 2015, 10, 1998−2006. (12) Rychnovsky, S. D.; Skalitzky, D. J. Tetrahedron Lett. 1990, 31, 945−948. (13) Dana, G.; Danechpajouh, H. Bull. Soc. Chim. Fr. 1980, 395−399. (14) Bock, M.; Buntin, K.; Müller, R.; Kirschning, A. Angew. Chem., Int. Ed. 2008, 47, 2308−2311. (15) Napolitano, J. G.; Gavín, J. A.; García, C.; Norte, M.; Fernández, J. J.; Daranas, A. H. Chem. - Eur. J. 2011, 17, 6338−6347. (16) Seco, J. M.; Quiñoá, E.; Riguera, R. Tetrahedron: Asymmetry 2001, 12, 2915−2925. (17) Skellam, E. J.; Stewart, A. K.; Strangman, W. K.; Wright, J. L. J. Antibiot. 2013, 66, 431−441. (18) Keatinge-Clay, A. T. Chem. Biol. 2007, 14, 898−908. (19) Raju, R.; Piggott, A. M.; Conte, M. M.; Capon, R. J. Org. Biomol. Chem. 2010, 8, 4682−4689.
In conclusion, we applied the combined-culture method to the rare actinomycete M. wenchangensis HEK-797 and successfully obtained two polyketide macrolactams with unprecedented skeletons. Our study confirmed the potential of rare actinomycetes as sources of novel secondary metabolites, and also demonstrated that the combined-culture method enables easy access to cryptic secondary metabolites, not only in Streptomyces species but also in rare actinomycetes. Furthermore, it is noteworthy that the combined-culture induced the occurrence of a new oxidation reaction that significantly amplifies the complexity of the macrolactams. Similar induced oxidations have been reported, implying the existence of common cryptic oxidation pathways in actinomycetes.10,11,19
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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.orglett.7b02508. Experimental procedure, NMR chemical shift tables, NMR spectra of 1, 2, and their chemical derivatives, more detailed stereochemical analysis of 1 and 2, bioinformatic analysis of micromonolactam biosynthetic gene cluster, and HRMS and UV data of 7 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Ikuro Abe: 0000-0002-3640-888X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (JSPS KAKENHI Grant Nos. JP16H06443, JP16K13084, and JP15H01836), and JSPS Research Fellowships for Young Scientists (to S.H.).
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REFERENCES
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DOI: 10.1021/acs.orglett.7b02508 Org. Lett. 2017, 19, 4992−4995
