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Platensimycin (1) and platencin (2) are novel antibiotic leads against multi-drug resistant pathogens. The production of 2 in Streptomyces platensis MA7339 is under the control of ptnR1, a GntR-like transcriptional regulator. Inactivating ptnR1 afforded S. platensis MA7339 mutant strain SB12600 that overproduces 2 at titer ~100-fold greater than that from the wild-type strain and accumulates platencin A1 (3) and eight new congeners platencin A2–A9 (4–11). The isolation, structural elucidation, and antibacterial activity of 4–11, in comparison to 1–3, are described.
The recently discovered platensimycin (1) and platencin (2) represent one of only a few new classes of antibiotics that have been discovered since the early 1960’s.1–4 They potently inhibit the growth of a range of Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus and Enterococcus sp., making them important antibiotic leads against drug-resistant pathogens. Although both compounds work by inhibiting fatty acid biosynthesis, they have slightly different mechanisms of action – 1 specifically inhibits FabF whereas 2 synergistically inhibits FabF and FabH subunits of the bacterial type II fatty acid synthase.1,2 To determine structure-activity relationships (SAR) of 1 and 2, libraries of analogs featuring the 1 and 2 scaffold have been generated by organic synthesis.5–12 Congeners of 1 and 2 have also been isolated from the wild-type producers upon large-scale fermentations.13–17 Of the analogs reported to date, only platencin A1 (3) and its methyl ester retain the carbon scaffold of 2 (Figure 1).14
We recently reported engineered strains from Streptomyces platensis MA7327, the wild-type dual producer of 1 and 2, that are capable of overproducing 1 and 2 with ~100 fold improved titers.18 We now report an engineered strain from S. platensis MA7339, the wild-type exclusive producer of 2, that overproduces 2, 3, and eight new congeners platencin A2–A9 (4–11) (Figure 1). The isolation, structural elucidation, and antibacterial activity of 4–11 in comparison to 1–3 are described.
The gene cluster encoding 2 production in S. platensis MA7339 contains ptnR1, a closely related homolog to the pathway specific repressor ptmR1 characterized from the 1 and 2 dual producer S. platensis MA7327; inactivation of ptmR1 previously afforded recombinant strains that overproduce 1 and 2.18 We similarly inactivated ptnR1, using REDIRECT Technology,19 in S. platensis MA7339 to afford the desired mutant strain SB12600, the genotype of which was confirmed by PCR and Southern analyses (see Supporting Information).
To determine the effect of ptnR1 inactivation on 2 production, SB12600 was fermented in two different production media with the wild-type strain as a control. In the standard production medium (PTNM),18 the titer of 2 increased from ~0.05 mg/L in the wild-type to ~22 mg/L in SB12600. In addition, HPLC analysis of the crude extract from SB12600 revealed many other metabolites with similar UV-Vis spectra to 2 (Figure 2, panel i and Table 1). In the second medium (SLY) that was previously used for 1 and 2 overproduction by the S. platensis MA7327 dual producers,18 no improvement in 2 titer was seen; however, the additional metabolites were produced in increased titers (Figure 2, panel ii and Table 1) (see Supporting Information).
To isolate the overproduced metabolites, SB12600 was cultured in both PTNM (7.2-L) and SLY (4.4-L) media, and the crude extracts made from the combined fermentation culture were subjected to mutiple steps of chromatography, affording 2 and nine additional metabolites (3–11) (Figure 1) (see Supporting Information).
Compound 2 was confirmed as platencin by comparision with an authentic standard.2,4,18 Compound 3 was identified as platencin A1 by spectroscopic comparisons with literature, including a NOESY correlation between H-13 and H3-17 that verified the stereochemistry of the hydroxyl group at C-13.14
Compounds 4 and 5 share the same molecular formula of C24H27NO7 as established by HiResMALDI-FTMS that yielded [M+Na]+ ions at m/z 464.16586 and 464.16605, respectively (calcd for C24H27NO7Na, 464.16797). Each was predicted to be a hydroxylated analog of 2 based on the 16 mass increase and the initial 1H NMR spectra confirming the presence of an exomethylene characteristic of 2. Similar to compound 3, the loss of a methylene carbon signal and the appearance of unique oxygenated methine signals in compounds 4 and 5 suggested hydroxylation of the terpene moiety. The hydroxyl group in 4 was assigned to C-12 on the basis of a clear gCOSY correlation between the downfield-shifted H-12 and H-11. C-10 could be excluded as there is a gCOSY correlation between its two protons with both H-9 and H-11. A NOESY correlation between H-12 and H3-17 determined the relative stereochemistry of the hydroxy group at C-12; 4 can be assigned as 12(S)-hydroxyplatencin (i.e. platencin A2) if the absolute stereochemistry is assumed to be the same as in 2.4 The hydroxy group on 5 could be placed on C-14 by the presence of an oxygenated methine proton (H-14) with a singlet peak at δH 3.97. This was corroborated by HMBC correlations of C-14 with H2-16 and with H-7. Thus, like 4, 5 was similarly assigned as 14(S)-hydroxyplatencin (i.e., platencin A3) by a clear NOESY correlation between H-14 and H-9.
HiResMALDI-FTMS analysis of 6 gave an [M+Na]+ ion at m/z 467.24902, consistent with a molecular formula of C25H36N2O5 (calcd for C25H36N2O5Na, 467.25164). The UV-Vis spectrum of 6 contained a single peak at 229 nm indicating an enone moiety, but lacked the smaller 300 nm peak characteristic of the benzoic acid moiety common to 2 and other congeners. 13C NMR analysis confirmed the presence of 25 carbons, and, combining gHSQC and 13C NMR, 6 was shown to contain all of the carbon signals expected from the terpenoid moiety of 2 with the addition of one methyl, three methylenes, two methines, and two carbonyl carbons. 1H NMR along with gCOSY, TOCSY, gHSQC and gHMBC data suggested the presence of glutamine, similar to homoplatensimide.16 This accounted for all extra carbon signals except for one methylene, one methine, and one methyl carbon. gCOSY spectra clearly established the three carbons as a CH3-CHR-CH2R′ unit. The chemical shifts of the methine carbon and its proton were indicative of a position α to a carbonyl group. gHMBC showed connectivity between the amide carbon (C-1″) and all of the protons in the new three-carbon fragment. gCOSY correlations between the exocyclic methylene carbons C-2/C-3 and C-2/C-1 confirmed that the extra carbons were located in the flexible linker between the amide and the cyclic region, affording the final assignment of 6 as platencin A4 with the relative stereochemistry of the methyl group at C-2″ undetermined.
Solving the chemical structure of 6 greatly facilitated the structural elucidation of compounds 7 and 8. HiResMALDI-FTMS analysis of 7 and 8 yielded [M+Na]+ ions at m/z 483.24382 and 483.24395, respectively, and the 16 mass increase suggested that they are hydroxylated-6 isomers with the same molecular formula of C25H36N2O6 (calcd for C25H36N2O6Na, 483.24656). Using the same correlations that were used to determine the regiochemistry and stereochemistry of the hydroxy groups in 3–5, 7 and 8 were assigned as platencin A5 and A6 with a 13(R)- and 14(S)-hydroxyl group, respectively.
The molecular formula of compound 9 was deduced as C20H30O6 based on the HiResMALDI-FTMS spectrum that gave an [M+Na]+ ion at m/z 389.19346 (calcd for C20H30O6Na, 389.19339). The 1H and 13C NMR spectra showed similar signals to those produced by the terpenoid moiety of 7, with the notable differences being the loss of signals representing the C-15/C-16 vinyl group. Combination of gHSQC and 13C NMR analyses revealed new signals for an oxygenated methine and an oxygenated methylene, suggesting the presence of two hydroxy groups on C-15/C-16. This was confirmed by HMBC correlations between H2-16 and C-15, C-11, and C-14. A NOESY correlation between H-16 and H-12 determined the stereochemistry of C-15, allowing the final assignment of 9 as platencin A7 with a 15(S)-hydroxy group.
The molecular formula of C25H38N2O6 for compound 10 was established on the basis of HiResMALDI-FTMS analysis that afforded an [M+Na]+ ion at m/z 485.26221 (calcd for C25H38N2O6Na, 485.25856). 1H and 13C NMR spectra of 10 readily revealed a glutamine moiety similar to 6–8, subtraction of which results in a predicted terpene moiety with a molecular formula of C20H29O3, bearing one less degree of unsaturation compared to 6–8. A significant difference between the spectra from 6–8 and that of 10 is the presence of the carbon signal at δC 181.5 indicating a carboxyl functional group. On the basis of extensive gHMBC and gCOSY correlations (Figure 3A), 10 was established as N-[15,18-dicarboxy-ent-copalyl]-glutamine (i.e., platencin A8). NOESY correlations (Figure 3B) allowed the assignment of the relative stereochemistry around most of the ring system, although the lack of any correlations to the C-19 methyl protons prevented us from determining the C-4 stereochemistry.
HiResMALDI-FTMS analysis of compound 11 yielded an [M+Na]+ ion at m/z 656.21335, predicting a molecular formula of C31H39NO11S (calcd for C31H39NO11SNa, 656.21361). The presence of a sulfur atom was supported by the isotopic distribution of the parent ion. The UV-Vis spectrum of 11 displayed an altered absorbance pattern in the 300 nm range compared with other 2 congeners, with a peak at 280 nm and a pronounced shoulder at 320 nm, signifying changes in the benzoic acid moiety. The 1H and 13C NMR spectra were identical to corresponding spectra of 3, except for the presence of seven additional carbons, one methyl signal and six oxygenated resonances, and associated protons. The presence of intact 3 was confirmed by gCOSY, gHSQC, and gHMBC. Additionally, the 1H and 13C NMR data suggested a hexose moiety, which was confirmed by a 1D-TOCSY experiment and verified to be a glucoside by gHSQC, gHMBC, and TOCSY correlations. The anomeric proton H-1″′ (δH 5.07) showed a gHMBC correlation to C-5″′, consistent with a pyran sugar moiety. The large coupling constants of all protons in the sugar moiety revealed equatorial placement of all hydroxy groups also suggestive of a glucoside. The gHMBC correlations of H-1″′ with C-5′ (δC 158.1) established the glycosidic linkage at C-5′. This was further supported by the NOESY correlations between H-6′ and H-1″′, which also showed correlations to H-3″′ and H-5″′, confirming the location of the β-glucoside. The sugar is suspected to be the β-D-glucoside as this is the most prevalent glucoside enantiomer in nature. Lastly, gHMBC correlations between the CH3-S methyl protons (δH 2.50) and the carbonyl carbon C-1′ (δC 196.6) and H-7′ (δH 7.88) with C-1′ (δC 196.6) indicated the presence of a methyl thioester, in place of the free acid found in 2 and related analogs, hence the final assignment of 11 as platencin A9.
When stored in methanol, 11 readily degraded to a new compound (12), which migrated faster than 11 during C18 HPLC and displayed a distinct UV-Vis spectrum (see Supplementary Information). LC-ESI-MS analysis of 12 gave an M− ion at m/z 612.2, corresponding to 16 mass decrease compared to 11. Additionally, 12 no longer produced the sulfur-specific isotope distribution, suggesting that the thioester bond was cleaved by solvolysis in methanol to afford the corresponding methylester. 1H NMR data finally confirmed the assignment of 12 as platencin A10, showing the expected downfield shift of the CH3-S methyl at δH 2.50 in 11 to δH 3.91 for the CH3-O methyl in 12.
Rational metabolic engineering for titer improvement of Streptomyces secondary metabolites is an attractive means of enabling the production and characterization of new natural products.20 Here, we applied knowledge gained from studying the regulation of 1 and 2 dual production in S. platensis MA732718 to quickly generate improved strains from the 2-producing S. platensis MA7339. The improved titers of 2–11 in the engineered S. platensis SB12600 strain relative to the wild-type MA7339 (Table 1) illustrates the utility and effectiveness of such an approach for lead production and optimization.
The new congeners reported here add to the list of 1 and 2 analogs generated thus far for SAR studies and constitute the majority of analogs possessing the 2-scaffold. The newly isolated 2 congeners (4–11) were therefore assayed for antibacterial activities against selected S. aureus strains, in comparison to 1–3 (Table 2). Similar to the naturally isolated amino acylated 1-like diterpenes,13,16 6–10 did not show any bioactivity confirming the importance of benzoate-to-FabH/F active site binding interactions.5 Congeners 4 and 5 were more intriguing, as the chemical structures and binding interactions of 1 and 2 to FabF suggest that a hydrogen-bond acceptor in the otherwise non-polar terpene moiety can strengthen binding,1 and the previously described regioisomer 3 inhibits bacterial fatty acid synthase in a cell free assay with an IC50 of 7.12 µg/mL.14 Indeed, 5 displayed the best activity of all congeners tested, with a MIC of 8 µg/mL against the methicillin resistant S. aureus strain. Although significantly less active than 2, the MIC for 5 is similar to that of the clinical drug Linezolid.2 Compound 11 is a highly modified congener of 2, with a C-13 hydroxylation, an O-5′ glucosidation, and a methyl thioester on C-1′. Although the first two modifications have been seen previously in 314 and platencimycin B415, respectively, this is the first reported example of a sulfur-containing member of the platensimycin/platencin family of compounds. Compound 11 lacked activity due most likely to its glycosylation, as platensimycin B4 similarly showed no antibacterial activity.15
We thank Dr. Sheo Singh, Merck Research Laboratories, Rahway, NJ, for providing the S. platensis MA7339 strain, the Analytical Instrumentation Center of the School of Pharmacy, UW-Madison for support in obtaining MS and NMR data, and the John Innes Center, Norwich, United Kingdom, for providing the REDIRECT Technology kit. This work is supported in part by the MERC program, UW-Madison. M.J.S. is supported in part by NIH grant T32 GM08505.
Supporting Information Available Experimental procedures, MS and 1H and 13C NMR data of 4–12. This material is available free of charge via the Internet at http://pubs.acs.org.