Cano, Raul J.

Galazzo, Jorge L.

Lee, May D.

Mauger, Jacob B.

Qureshi, Asfia

English

DigitalCommons@CalPoly

MF-EA-705a & MF-EA-705Jj, New Metabolites from Microbial Fermentation of a Streptomyces sp. ASFIA Q URESHI, JACOB B. MAUGER, RAUL J. CAt-:0, JORGE L. GALAZZO and MAY 0. LEE As part of an antifungal discovery program, the crude extract of a Streptomyces sp. from Ecuador, MF-EA-705, was found to display broad-spectrum antifungal activity against Candida spp. and Aspergillus spp. T he extract was found to contain the known weakly-antibacterial compound actinopyrone A (1 ), 1l previously isolated from Streptomyces pactum. We report herein the production, isolation, structure elucidation and biological activities of two new compounds MF-EA-705a (2) and MF-EA-705/3 (3). A structurally similar compound has been reported in the microbial naturdl product literature, NFAT-133 (4),2> known to be an immunosuppressive agent in vitro. An 8-cm sporulation agar plate was inoculated with a 100 pi portion of frozen MF-EA-705 stock, and incubated at 28°C until growth (medium, beige-white) was observed ( 16 days). Sterile phosphate-buffered saline (6 mJ) was then added. The MF-EA-705 mycelia and spores were gently scraped with a sterile loop, and a 3-ml portion of the resultant suspension was pipetted into a 500-ml flask containing sterile ATCC- 172 media (150 mi. 2% soluble starch, 1% dextrose, 0.5% NZ amine type A, 0.3% Difco beef extract, 0.5% Difco bacto peptone, 0.5% yeast extract, 0.1% CaC03, presterile pH 7.0). The seed culture was incubated for 24 hours at 28°C, 200 rpm, and 85% humidity. Each of five production flasks (2800-ml Fcmbach) containing 500 ml of production media (5% glycerol, 2.5% corn meal, 0.5% llyeast 444, presterile pH 7.0) was inoculated with 20 ml of seed culture prepared Fig. I. Structures ofcompounds. 19 0 OH 0 3 5 HO I 2 ~. ~ 6 7 1 2 OH 0 HO 0 OH 3 4 above and incubated at 28°C, 160 rpm, and 85% humidity for 192 hours (8 days). The fermentation was harvested by centrifugation ( 4,500 rpm, 15 minutes, 4°C) and the supernatant (2200 ml) was decanted and stored at 4°C until used. The pellet was extracted with I 000 ml of 90% aqueous acetone for 2 hours and the extr~ct was separated from the cell debris by centrifugation ( 4,500 rpm, 15 minutes, 4°C) and concentrated in vacuo (30 mbar, 30°C). The remaining aqueous suspension (200 ml) was also stored at 4°C until used. The production of the bioactive components and their purification was monitored by inhibitory activity against Candida albicans in a cut-well agar diffusion assay. The fcnnentation supernatant and the concentrated pellet extract were recombined and adsorbed onto a column (2.5 em i.d.X 22 em L, 110 ml) packed with D iaion® HP20 (Mitsubishi Kasei) that had been equilibrated in water (1 000 ml). The column was sequentially eluted at 5 ml/minute with water (600 ml), a gradient of 0 to 20% acetone (350 ml), 20% acetone (350 ml), a gradient of 20 to 100% acetone (550 ml) and I 00% acetone (550 ml), collecting fractions at 5-minute intervals. The active eluates (60- 80% acetone) were combined and concentrated in vacuo (<30°C), and the residual aqueous solution was lyophi lized to yield 1.8 g of crude material. The sample was dissolved in a mixture of 1.25 ml of buffer A (0. 1 M NH40Ac, pH 4.9) and 3.75 ml of methanol, and was top-loaded on a column (2.2 cmX35 em, ] 35 ml) packed with cl8 silica gel (Amicon) that had been pre-equilibrated with 75% methanol in 0.1 M NH40Ac (pH 4.9). The column was eluted at 5 ml/minute with the same solvent mixture. The active fractions were each analyzed by HPLC (Amicon C 18, 4.6mmX100mm, l.7ml; 75% methanol in 0.1 M NH40Ac, pH 4.9) and the fractions corresponding to separate peaks (as detected by UV) combined accordingly. These pooled active fractions were further purified by reverse-phase HPLC on a Matrex C 18 column (MODcoJ®, 2.12cmX25cm, 90ml) eluted with 75% methanol in 0.1 M NH40Ac pH 4.9, to obtain MF-EA­705a (2, 65 mg), MF-EA-705j3 (3, 20 mg) and actina­pyrone A (1, 12 mg). The physico-chemical properties of compounds 2 and 3 are summarized in Table 1. MF-EA-705a (2) was isolated as an optically-inactive colorless oil. The molecular formula <;0H2p 2, which requires ten degrees of w1saturation, was established by HRFABMS [mlz 295.1688 (M+HtJ and by interpretation of the 1H and 13C NMR data. The IR spectrum contained .typical carboxylic acid bands at 3420 and 1710 em - I, and the UV absorption at 280 nm was consistent with a chromophore arising due to conjugation. The aliphatic region of the 1H NMR spectrum (Table 2) contained two methyl singlets at 8 2.34 (Me-19) and 2.02 (Me-20) and a single methylene signal at 8 3.05 (H-2). Two one-proton coupled signals at 5.10 and 5.27 (H-18a and H-18b) were correlated (HMQC) to a single carbon at 8 117 .2, confirming the presence of a terminal methylene group. From 1H NMR, DEPT and HMQC experiments, the presence of eleven additional methine carbons and five sp2 quaternary carbons, one of which was an acid carbonyl (8 17'7.0), was strongly inferred. ln addition to the data presented above, the COSY experiment allowed the Table 1. Physico-chemical properties ofMF-EA-705 a and j3. MF-EA-705a MF-EA-705p Appearance Molecular Formula HRFABMS Calc. Found UV Arnax (MeOH) nm, £ IR Vrnu (neat) cm·1 Solubility clear oil (M+Ht 295. 1698 295. I 688 3420, 1710 MeOH, CHCI3, ethyl acetate clear oil [M+Ht 297.1855 297.1856 240 (35630) 280 (51330) 3390, 1710 MeOH, CHCI3, ethyl acetate Table 2. 1H NMR (400 MHz, 25°C, CDCI3) and 13C NMR (100 MHz) data ofMF-EA-705a (2). Cno. Oc OH mull, J (Hz) HMBC I 177.0 2 37.7 3.05 d,2H, 7.1 177.0, 123.4, 134.6 3 123.4 5.64 dt, IH, 15, 7.3 177.0, 37.7, 130.3 4 134.6 5.97 dd, IH, 15, II 37.7, 130.6 5 130.3 6.09 dd. I H, 15.6, 11.3 123.4, 134.6, 128.3 6 130.6 5.77 dd, IH, 14.8, II 134.6 7 128.3 6.15 d, IH, I 1.2 130.6, 138.1, 26.2 8 139.5 9 138.1 LO 128.9 6.91 d, 1H, 7.7 139.5, 134.2, 136.7 II 128.4 7.02 dd, IH, 7 .7, 1.1 138.1, 125.5, 21.2 12 136.7 13 125.5 7.40 br s, 1H 138.1, 128.4, 130.8, 21.2 14 134.2 IS 130.8 6.50 d, IH, 15.7 125.5, 130.1 , 137.6 16 130.1 6.71 dd. IH, 15.7, 10.4 134.2, 130.8, 137.6 17 137.6 6.42 dt, IH, 16.8, 10 18a I 17.2 5.10 dd, 1H, 10.1, 0.8 130. 1 18b 5.27 dd, 1H. 16.8, 0.8 130.1, 137.6 19 21.2 2.34 s, 3H 125.5, 136.7, 128.4 20 26.2 2 .02 s, 3H 128.3, 139.5, 138.1 1-0H 5.60 br s, lH construction of several partial structures that could then be interconnected using data obtained from the HMBC experiment. The 1 ,2,4-trisubstituted benzene moiety was assigned on the basis of the coupling constants of the H-1 0, H-11 and H-13 signals and COSY correlations between H­10 and H-11. Key long-range HMBC correlations (Figure 2a) are as follows: H-13 to C-9, C-1 J, C-15 and C-19; H-11 to C-9 and H-10 to C-8, C-14 and C-12. These results are consistent with a trisubstituted aromatic dng. The remainder of the molecule was established as follows. The 1H NMR signals at 8 5.64 (H-3), 5.97 (H-4), 6.09 (H-5), 5.77 (H-6) and 6.15 (H-7), each integrating to one proton, were assigned on the basis of COSY to the hydrogens on a set of three conjugated olefins. The geometries of the C3- C4 and C5-C6 olefins were assigned as trans on the basis of the 1H coupling constants (Table 2) and the NOESY data (Figure 2b). The configuration of the C7-C8 olefin was assigned as shown based on a NOESY correlation between H-7 and H-20. The placement of the C­20 methyl group at C-8 was assigned on the basis ofCOSY and HMBC data. Further HMBC correlations from H-3 to C-1 and C-5, and from H-2 to C-4 established the terminal carboxylic acid portion of the chain. The remaining portion of the molecule, C-15 to C-18, was determined similarly. Fig. 2. (a) Selected HMBC and (b) NOESY correlations for EA-705a (2). (a) HO (b) 0 HO Table 3. 11-l NMR (400 MHz, 25°C, CDCI3) and 13C NMR (100MHz) data ofMF-EA-705a (3). ·---- ------- -- -Cno. oc OH I 177.2 2 37.7 3.06 3 123.0 5.65 4 134.8 5.99 5 129.8 6.09 6 131.0 5.81 7 127.6 6.09 8 140.4 9 138.3 10 128.5 6.94 11 127.4 7.02 12 136.0 13 130.0 7.06 14 135.4 15 127.3 6.20 16 134.4 5.56 17 21.8 2.19 18 14.4 0.97 19 21.2 2.33 20 25.6 1.9& 1-0H. * not observed The C-19 methyl group was placed at C- 12 on the basis of HMBC correlations between CH3- 19 to C-11 and C-13. MF-EA-705{3 (3) was isolated as an optically-inactive colorless oil. Its molecular formula, C20H240 2, was 2 amu heavier than that of MF-EA-705a (2). Analysis of the NMR data (Table 3) revealed that the only structural changes were saturation of the C-1 7 to C- 18 double bond of 2, and a cis geometry of the C 15- C 16 double bond. The signals at De 137.6 and I 17.2 and Ou 6.42, 5. 1 0 and 5.27 in the NMR spectra of 2 were replaced by a methylene carbon signal at 21.8 and an additional methyl signal at Oc 14.4 and oH 0.97 (t, 3H, J 7.5, 7.5 Hz). The new CH3 signal showed HMBC correlations to C-16, and H-17 correlated to C-15. Further HMBC correlations indicated the chain was still attached to C-14 of the benzene ring, although the effect of the change was noted in the chemical shift of H-13 (o 7.40 to 7.06), H-15 (6.50 to 6.20) and H-16 (6.71 to 5.56). The remaining spectral data suggested the rest of the molecule was identical to 2. The purified compounds MF-EA-705a (2) and MF-EA­705{3 (3) were inactive at a concentration of 128)lg/ml against Candida albicans, C. glabrata, C. krusei, Cryptococcus neoformans, and Aspe1gillus fumigatus. The minimum inhibitory concentration (MIC) of (2) against C. albicans was determined to be I mg/ml, suggesting the mulf,J (Hz) HMBC d. 2H, 7 dt, IH, 15,7 dd, IH, 15.2. 11 m, IH dd, IH, 15, II m, IH 177.2, 123.0, 134.8 177.2, 37.7, 129.8 37.7 134.8 138.3, 25.6 d, IH, 7.4 br d, IH. 8.2 140.4, 135.4 I 38.3, I 30.0, 2 I .2 br s, IH 138.3, 127.3, 21.2 d, JH, 11.2 dl , IH, II , 7 q, 2H, 7.5 l, 3H, 7.5 s, 3H s, 3H 130.0, 21.8 135.4, 21.8, 14.4 127.3, 134.4, 14.4 134.4, 21.8 130.0, 136.0 127.6, 140.4, 138.3 unsuitability of this compound as a drug candidate. The activity observed and followed during bioassay-guided fractionation was thus due to high concentrations of the compound being assayed in the cut-well agar plates. Nevertheless, these compounds are interesting novel metabolites, with a similar compound having been isolated only once previously in the microbial natural product literature.21 Acknowledgements We thank UMA VJJAYAM, KAREN LOLANS and KSH!TIJ Moo1 for biological assays and ANDREW STALEY for helpful comments on the manuscript. References I) 	 YANO, K.; K. YOKOI, J. SATO, J. OONO, T. KOUDA, Y. OGAWA & T. NAKASHIMA: Actinopyrones A, B and C, new physiologically active substances. J. Antibiotics 39: 32- 37, 1986 2) 	 BURRES, N. S.; U. PREMACHANDRAN, S. HOSELTON, 0 . CWIK, J. E. HOCHLOWSKI, Q. YE, G. N. SUNGA, J. P. KAROWSKI, M. JACKSON, D. N. WHrTIERN & J. B. McALPINE: Simple aromatics identified with a NFAT­IacZ transcnptton assay for the detection of immunosuppressants. J. Antibiotics 48: 380- 386, 1995 

MF-EA-705\u3cem\u3eα &\u3c/em\u3e MF-EA-705\u3cem\u3eβ\u3c/em\u3e, New Metabolites from Microbial Fermentation of a \u3cem\u3eStreptomyces\u3c/em\u3e sp.

https://digitalcommons.calpoly.edu/cgi/viewcontent.cgi?article=1382&amp;context=bio_fac

MF-EA-705\u3cem\u3eα &\u3c/em\u3e MF-EA-705\u3cem\u3eβ\u3c/em\u3e, New Metabolites from Microbial Fermentation of a \u3cem\u3eStreptomyces\u3c/em\u3e sp.

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