Letter pubs.acs.org/OrgLett
Nam7 Hydroxylase Is Responsible for the Formation of the Naphthalenic Ring in the Biosynthesis of Neoansamycins Juanli Zhang,†,‡,∥ Shanren Li,†,∥ Xingkang Wu,† Zhixing Guo,† Chunhua Lu,*,† and Yuemao Shen†,§ †
Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012, P. R. China ‡ Department of Pharmacy, Xijing Hospital, The Fourth Military Medical University, Changle West Street 15, Xi’an, Shaanxi 710032, P. R. China § State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, P. R. China S Supporting Information *
ABSTRACT: Ten new benzenic ansamycins, 5,10-seco-neoansamycins A−J (1−10), were isolated from the nam7-disrupted mutant strain SR201nam1OEΔnam7. These are the benzenic counterparts of the neoansamycins, which provide direct evidence that the putative hydroxylase Nam7 is involved in the formation of naphthalenic ring in neoansamycin biosynthesis and connect benzenic and naphthalenic ansamycins for the first time.
A
disrupting the nam7 gene in the SR201nam1OE strain, which was obtained by overexpression of the LuxR-type transcriptional regulatory gene nam1 in Streptomyces sp. LZ35 (Figure S1).5,11,14 Surprisingly, disruption of nam7 resulted in the production of diverse metabolites but with the elimination of neoansamycins (Figure S2). Large-scale fermentation of the SR201nam1OEΔnam7 strain led to the isolation of 10 ansamycins (1−10). All of them were fully characterized on the basis of analysis of the HR ESIMS and NMR data (Tables S2−S9) and determined to be new benzenic ansamycins, namely 5,10-seco-neoansamycins A−J (1−10) (Figure 1), respectively. The stereochemistry of 8 and 10 was determined by single-crystal X-ray diffractions (see Figures 3 and 4). The molecular formula of 1 was established as C30H43NO7 by high-resolution ESIMS m/z 530.313 [M + H]+. The IR spectra indicated the absorption bonds for hydroxyl (3433 cm−1), carbonyl (1664 cm−1), and benzenic (1613, 1563 cm−1) groups, respectively. Interpretation of the 1D and 2D NMR spectroscopic data of 1 exhibited the presence of a metatrisubstituted benzene ring that was found to be an AHBA residue by analyzing the HMBC correlations. A two-carbon fragment and two seven-carbon fragments from C-7 to C-8, C12b to C-16, and C-19 to C-20e were established on the basis of COSY correlations (Figure 2 and Table S1). The connectivity between C-7 and C-6 was established on the basis of analysis of the HMBC correlations from H-7 (δH 4.97) to C-1, C-5, and C-6 (Table S1). The HMBC correlations from H-7, H-10, and from H-12 to the corresponding carbons suggested the presence of the fragment from C-9 to C-11, and that linked the fragments C-7 to C-8 and C-12b to C-16
nsamycins are a family of macrocyclic lactams that include the antituberculous drug rifamycin1 and antitumor agents geldanamycin2 and maytansine,3 whose molecular architecture comprises an aromatic chromophore bridged at nonadjacent positions by an aliphatic chain (ansa chain) through an amide linkage.4 This family shows remarkable potential for drug development, but only about 200 members have been reported. We have approached novel ansamycins by PCR screening 3amino-5-hydroxybenzoic acid (AHBA) synthase genes of plantassociated and marine-derived actinomycetes,5 which resulted in the isolation of new hygrocins,6,7 divergolides,8,9 juanlimycins,10 and neoansamycins A−C.11 Neoansamycins A−C are novel naphthalenic octaketide ansamycins isolated by genome mining and activation of the cryptic nam gene cluster.11 Octaketide ansamycins represented by geldanamycins and maytansinoids have benzenic aromatic chromophores. On the basis of the structures of their aromatic chromophores, conventionally, ansamycins are classified into benzenic and naphthalenic (rifamycin) types. However, the formation of the naphthalenic ring was only investigated and proposed in the biosynthesis of rifamycin to be catalyzed by Rif-Orf19, a putative 3-(3-hydroxyphenyl)propionate hydroxylase-like protein.12,13 Inactivation of the rif-orf19 gene in Amycolatopsis mediterranei S699 resulted in the loss of rifamycin production but did not affect the formation of SY4b and desacetyl-SY4b, two linear tetraketide intermediates of rifamycins. Disruption of rifamycin production was found to be due to premature polyketide chain termination from the PKS, but no anticipated benzenic precursors of rifamycins were isolated and detected, which left the formation of the naphthalenic ring in ansamycins still uncharacterized. The nam gene cluster contains a homologue of the rif-orf19 gene, namely nam7 (GenBank accession no. KJ590158). In this study, the SR201nam1OEΔnam7 strain was produced by © 2017 American Chemical Society
Received: April 10, 2017 Published: April 25, 2017 2442
DOI: 10.1021/acs.orglett.7b01083 Org. Lett. 2017, 19, 2442−2445
Letter
Organic Letters
11 by dehydration in 7, which was confirmed by the HMBC correlations from H-7 (δH 5.61) to C-11 (δC 180.7), and the olefinic bond formed between C-7 and C-8 by dehydration in 8, which was confirmed by the 1D and 2D NMR spectroscopic data, respectively. The large coupling constants (J > 15.0 Hz) between H-7 and H-8 led to assignment of the 7(8)-E configuration for 8. Finally, the absolute configuration of 8 was fully determined by X-ray diffraction analysis of a single crystal obtained from acetone (CCDC no. 1038770) (Figure 3).
Figure 1. Structures of 1−10, novel benzenic ansamycins from the mutant strain SR201nam1OEΔnam7.
Figure 3. Single-crystal X-ray structure for 8.
Compound 9 has the same molecular formula of C30H43NO7 as that of 1 determined by HR ESIMS m/z 530.3092 [M + H]+. NMR comparison revealed that the planar structure of 9 was the same as that of 1. The 18(19)-E configuration in 9 was supported by the NOE correlations between Me-18a and H-20, which were different from the 18(19)-Z configuration in compounds 1−8 determined by the strong NOE correlations between Me-18a and H-19. The molecular formula of 10 was determined to be C30H41NO6 on the basis of analysis of the HR ESIMS at m/z 512.303 [M + H]+. The NMR data of 10 were similar to those of 1 but differed in the formation of a 4-methyl-2,4-dienal ketone residue by dehydration and cyclization between C-17 and the amide NH. Finally, the structure of 10 with absolute configuration was fully confirmed by single-crystal X-ray analysis (CCDC no. 1038787) (Figure 4).
Figure 2. Selected HMBC (→) and COSY () correlations of 1.
(Figure 2). The linkage between the two seven-carbon fragments was determined on the basis of the HMBC correlations from the methyl protons at δH 2.00, H-16 and H-19 to the corresponding carbons (Figure 2). The presence of the amide bond was deduced on the basis of the chemical shifts of C-2 (δC 140.3) and C-21 (δC 173.7) together with the HR ESIMS at m/z 530.313. Interpretation of the HR ESIMS and NMR spectroscopical data (Supporting Information) revealed that compounds 2−4 had similar structures as that of 1 except for the different side chains at C-20 (Figure 1). The spectroscopic data of 5 were similar to the data for 1, except that the C-18 was substituted by a hydroxymethyl group (δH 4.33 and 4.51), which was further supported by the HR ESIMS data at m/z 546.3046 [M + H]+. The molecular formula of 6 was determined to be C36H51NO13 on the basis of the HR ESIMS data at m/z 706.3420 [M + H]+. Interpretation of the NMR spectroscopic data indicated that compound 6 had the same ansa skeleton as that of 1 but had an extra six-carbon unit deduced to be a βglucuronic acid (GlcA) moiety located at C-4, which was confirmed by the HMBC correlations from H-1′ (δH 5.11, d, J = 7.5 Hz) to C-4 (δC 158.9) (Table S6). HR ESIMS data indicated that compounds 7 and 8 had the same molecular formula C30H41NO6 ([M + H]+, m/z 512.2996 and 512.2987, respectively). The NMR data revealed that each featured a largely conserved 1 skeleton. The apparent difference was the formation of a six-membered ring between C-7 and C-
Figure 4. Single-crystal X-ray structure for 10.
Compounds 1, 6−8, and 10 were assayed for their antimicrobial activity against Staphylococcus aureus ATCC 25923, Mycobacterium smegmatis mc2 155, and Candida albicans 5314 but were found to have no activity. Compounds 1, 3, 4, 7, 8, and 10 showed moderate antiproliferative activity against human cancer SW480, MDA-MB-231, HeLa, HepG2, and HL7702 cell lines (Table S1). In addition, 1, 3, 4, and 10 inhibited the secretion of SPI-1 effectors of SipA/B/C/D in a dose-dependent manner (Figure S4). 2443
DOI: 10.1021/acs.orglett.7b01083 Org. Lett. 2017, 19, 2442−2445
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Organic Letters Scheme 1. Proposed Biosynthetic Pathway for Benzenic and Naphthalenic Neoansamycins
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On the basis of the structures of aromatic chromophores, ansamycins can be sorted into benzenic and naphthalenic types. Interestingly, for the known ansamycin types classified according to the length of polyketide chains each has only either benzenic or naphthalenic chromophores except divergolides, which have both,8,9,15,16 supporting the on-PKS formation hypothesis of the naphthalenic ring proposed for rifamycin biosynthesis (Scheme 1).12,13,15 However, deletion of the rif-orf19 gene did not produce the anticipated benzenic precursors of rifamycins in A. mediterranei S699, which left this hypothesis pending. In this study, disruption of the rif-orf19 homologous gene, nam7, resulted in the production of 10 benzenic ansamycins (1−10), which implies that NamB, NamC, NamD, and NamE may have a broader substrate specificity than that of module 4 of RifB, which may only chain extend a naphthalenic tetraketide.13 Indeed, RifB, RifC, RifD, and RifE did show substrate promiscuity in the production of a linear benzenic bromo undecaketide by supplementing an AHBA(−) mutant strain of A. mediterranei with 3-amino-4bromobenzoic acid.17 In essence, this study provided direct evidence that the putative hydroxylase Nam7 is involved in the formation of the naphthalenic ring in neoansamycin biosynthesis and connects benzenic and naphthalenic ansamycins for the first time (Scheme 1). Additionally, more unusual extender units were found in compounds 3 and 4, which confirmed that the relaxed substrate specificity of neoansamycins could facilitate the bioengineering of novel ansamycins.18−20 This study obviously has implications for future efforts to produce modified ansamycins by genetic engineering of the biosynthetic process.21−23
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01083. Complete description of methods, additional tables and figures, including structure elucidation and NMR data for compounds 1−10 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Chunhua Lu: 0000-0002-3261-1020 Yuemao Shen: 0000-0002-3881-0135 Author Contributions ∥
J.Z. and S.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported in part by the National Natural Science Foundation of China (81673317, 81530091, 81602979), the Science Foundation of Two sides of Strait (U1405223), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT13028). 2444
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REFERENCES
(1) Sensi, P.; Margalith, P.; Timbal, M. T. Farmaco. Sci. 1959, 14, 146−147. (2) DeBoer, C.; Meulman, P. A.; Wnuk, R. J.; Peterson, D. H. J. Antibiot. 1970, 23, 442−447. (3) Kupchan, S. M.; Komoda, Y.; Court, W. A.; Thomas, G. J.; Smith, R. M.; Karim, A.; Gilmore, C. J.; Haltiwanger, R. C.; Bryan, R. F. J. Am. Chem. Soc. 1972, 94, 1354−1356. (4) Kang, Q.; Shen, Y.; Bai, L. Nat. Prod. Rep. 2012, 29, 243−263. (5) Wang, H. X.; Chen, Y. Y.; Ge, L.; Fang, T. T.; Meng, J.; Liu, Z.; Fang, X. Y.; Ni, S.; Lin, C.; Wu, Y. Y.; Wang, M. L.; Shi, N. N.; He, H. G.; Hong, K.; Shen, Y. M. J. Appl. Microbiol. 2013, 115, 77−85. (6) Lu, C.; Li, Y.; Deng, J.; Li, S.; Shen, Y.; Wang, H.; Shen, Y. J. Nat. Prod. 2013, 76, 2175−2179. (7) Li, S.; Lu, C.; Ou, J.; Deng, J.; Shen, Y. RSC Adv. 2015, 5, 83843− 83846. (8) Li, S. R.; Zhao, G. S.; Sun, M. W.; He, H. G.; Wang, H. X.; Li, Y. Y.; Lu, C. H.; Shen, Y. M. Gene 2014, 544, 93−99. (9) Zhao, G.; Li, S.; Guo, Z.; Sun, M.; Lu, C. RSC Adv. 2015, 5, 98209−98214. (10) Zhang, J.; Qian, Z.; Wu, X.; Ding, Y.; Li, J.; Lu, C.; Shen, Y. Org. Lett. 2014, 16, 2752−2755. (11) Li, S.; Li, Y.; Lu, C.; Zhang, J.; Zhu, J.; Wang, H.; Shen, Y. Org. Lett. 2015, 17, 3706−3709. (12) Xu, J.; Wan, E.; Kim, C. J.; Floss, H. G.; Mahmud, T. Microbiology 2005, 151, 2515−2528. (13) Yu, T. W.; Shen, Y.; Doi-Katayama, Y.; Tang, L.; Park, C.; Moore, B. S.; Richard Hutchinson, C.; Floss, H. G. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 9051−9056. (14) Li, S.; Wang, H.; Li, Y.; Deng, J.; Lu, C.; Shen, Y.; Shen, Y. ChemBioChem 2014, 15, 94−102. (15) Xu, Z.; Baunach, M.; Ding, L.; Peng, H.; Franke, J.; Hertweck, C. ChemBioChem 2014, 15, 1274−1279. (16) Ding, L.; Maier, A.; Fiebig, H. H.; Gorls, H.; Lin, W. H.; Peschel, G.; Hertweck, C. Angew. Chem., Int. Ed. 2011, 50, 1630−1634. (17) Bulyszko, I.; Drager, G.; Klenge, A.; Kirschning, A. Chem. - Eur. J. 2015, 21, 19231−19242. (18) Song, Y. N.; Jiao, R. H.; Zhang, W. J.; Zhao, G. Y.; Dou, H.; Jiang, R.; Zhang, A. H.; Hou, Y. Y.; Bi, S. F.; Ge, H. M.; Tan, R.-X. Org. Lett. 2015, 17, 556−559. (19) Le, T. C.; Yang, I.; Yoon, Y. J.; Nam, S. J.; Fenical, W. Org. Lett. 2016, 18, 2256−2559. (20) Wilson, M. C.; Nam, S. J.; Gulder, T. A.; Kauffman, C. A.; Jensen, P. R.; Fenical, W.; Moore, B. S. J. Am. Chem. Soc. 2011, 133, 1971−1977. (21) Shi, G.; Shi, N.; Li, Y.; Chen, W.; Deng, J.; Liu, C.; Zhu, J.; Wang, H.; Shen, Y. ACS Chem. Biol. 2016, 11, 876−881. (22) Tian, Y.; Jiang, N.; Zhang, A. H.; Chen, C. J.; Deng, X. Z.; Zhang, W. J.; Tan, R.-X. Org. Lett. 2015, 17, 1457−1460. (23) Eichner, S.; Knobloch, T.; Floss, H. G.; Fohrer, J.; Harmrolfs, K.; Hermane, J.; Schulz, A.; Sasse, F.; Spiteller, P.; Taft, F.; Kirschning, A. Angew. Chem., Int. Ed. 2012, 51, 752−757.
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