242
B) Shaker Culture
peri-Hydroxy-quinones are very common orange to red coloured microbial me-tabolites, and characterised by the bathochromic shift of their UV absorption in alka-line solution. According to our experience, coloured metabolites are preferably formed in shaker cultures rather than fermenters. Therefore, the strain B8904 was re-cultivated on a 25-liter scale for 7 days at 28 °C as shaker culture (110 rpm). After filtration, the dark red broth was extracted with ethyl acetate. The resulting extract was chromatographed on flash silica gel column using chloroform-methanol gradi-ent. Further separation of the fractions containing the reddish-orange peri-hydroxyquinones (II-IV) delivered seven compounds: the known compounds, ζ-pyrromycinone (243), η-pyrromycinone (244), musettamycin (246), cinerubin B (248), in addition to the three new peri-hydroxyquinones, cinerubin M (254), islamomycin A (257) and islamomycin B (258) (Figure 114).
Biomass Filtrate Mixing with celite and filtered by filterpress
3x with EtOAc and 1x with acetone 4 x with EtOAc
SC. on Silica gel (CHCl3-MeOH ) i.vac
Crude extract ( 3.2 g)
A1= PTLC( 4 % MeOH/CHCl3), A2 = PTLC(10 % MeOH/CHCl3); A3= PTLC(13 % MeOH/CHCl3);
B1 = Sephadex LH-20 (DCM/MeOH, 3:2) B2 = Sephadex LH-20 (MeOH)
Fraction I Fraction II Fraction III Fraction IV
musettamyin; cinerubine B A1, B1
A2, B2
A3, B2 ξ-pyrromycinone
η-pyrromycinone islamomycin A
islamomycin B
cinerubin M
Figure 114: Work-up scheme of the marine Streptomyces isolate B 8904 (shaker) 4.20.3 ζ-Pyrromycinone; Galirubinone C
During the work-up of the fast mobile fraction II, compound 243 was isolated as low polar and major product. It was isolated as red-orange solid by applying the frac-tion to PTLC and Sephadex. The compound showed an orange fluorescence zone under UV light at 366 nm which turned to violet by treating with sodium hydroxide as indication of a peri-hydroxyquinone.
The 1H NMR spectrum of 243 showed three singlets each of 1H at δ 13.02, 12.61, and 12.28, which are characteristic for peri-hydroxy groups in a quinone moi-ety. In the aromatic region, one singlet of 1H at δ 7.69, and two AB systems each of 1H at δ 7.29 and 7.28 were detected, as two ortho-coupled protons in 1,2,3,4-tetra-substituted phenolic ring. In addition, a 1H singlet was observed at δ 3.96, which could be assigned as oxymethine or as methine group flanked by two sp2 carbons.
One methoxy group was observed at δ 3.74 (s). Four multiplets were displayed (each of 1 H), of two vicinal methylene groups, at δ 3.07 (7-HA), 2.86 (7-HB), 2.30 (8-HA) and 1.95 (8-HB). The first methylene group (δ 3.07 and 2.86) should be next to a sp2 carbon, while the other one (δ 2.30 and 1.95) should be next to an oxygenated
qua-ternary sp3 carbon. Furthermore, a third methylene group was giving two multiplets between δ 1.70-1.58, which could be adjacent to a methyl group, observed as triplet at δ 1.09, to constitute an ethyl group.
The (-)-ESI mode exhibited two quasi-molecular ions at m/z 845 ([2 M +Na – 2 H]-) and 411 ([M - H]-), establishing the molecular weight of compound as 412 Dal-ton. It was further established by a CI mass spectrum which exhibited an ion peak at m/z 430 [M + NH4]+. Based on the above chromatographic and spectroscopic data of compound 243 and searching in AntiBase, one possible structure was suggested, ζ-pyrromycinone (243). The compound 243 was further confirmed by direct compari-son of the spectral data with authentic one.
OH 4.20.4 η-Pyrromycinone; Ciclacidine
Along with 243, compound 244 was isolated from fraction II as a red-orange solid. It showed an orange UV fluorescence and turned to violet by treatment with sodium hydroxide, as indication of a peri-hydroxyquinone. The 1H NMR spectrum of 244 showed three chelated peri-hydroxyl groups at δ 13.85, 13.12 and 12.32. In the aromatic region, it showed a deep field AB system at δ 8.53 and 7.61 indicating an 1,2,3,4-tetrasubstituted aromatic system. Additionally, a second AB system was ob-served at δ 7.29 of another 1,2,3,4-tetrasubstituted aromatic system. Moreover, one deep field of 1Hsinglet was observed at δ 8.27 due to a third aromatic ring. In the aliphatic region, a methoxy group was observed at δ 4.13. Finally, an ethyl group was established, due to the quartet of a methylene group at δ 2.85, and its vicinal triplet methyl group at δ 1.35. The observed down field shift of the last ethyl group referred to its link to an sp2 carbon atom.
The molecular weight of compound 244 was established as 392 Dalton using EI MS. Applying the spectral data of compound 244 to AntiBase led to its identification
4.20.5 Musettamycin
Additionally, from fraction III, compound 246 was obtained as a red-orange solid. The compound showed orange UV fluorescence and turned to violet by sodium hydroxide, as indication of another peri-hydroxyquinone.
The 1H NMR spectrum of compound 246 exhibited three peri-hydroxy groups at δ 12.93, 12.81 and 12.22. In the aromatic region, a singlet was observed at δ 7.68 indicating a penta-substituted aromatic ring. Furthermore, a 2H AB system was ob-served at δ 7.28 (2/3-H). In the sugar region, it showed three oxymethines at δ 5.51 (s br.), 5.28 (m), and 5.07 (d), establishing the presence of at least two anomeric pro-tons. Eight additional oxymethines were exhibited between δ 4.47-3.61, containing a singlet at δ 4.13 (H-10). A methoxy group was observed at δ 3.70, an ethyl group was present in the compound because of the methyl triplet at δ 1.08 and the adjacent methylene signals at δ 1.72 and 1.51. Furthermore, two splitted multiplets of a me-thylene group were observed at δ 2.52 (8-Heq) and 2.32 (8- Hax). This led to establish the pyrromycinone chromophore. The spectrum exhibited a singlet of 6 protons at δ 2.16, assigning two equivalent methyl groups linked most likely to a heteroatom (N(CH3)2). Moreover, a 2H multiplet of a methylene group was observed at δ 2.09-1.94 as present e.g. in desoxy sugars. Two methyl doublets were observed at δ 1.27 and 1.18.
Figure 115: 1H NMR spectrum (CDCl3, 600 MHz) of musettamycin (246)
The 13C NMR spectrum of compound 246 exhibited 35 carbon signals. Two quinone carbonyl carbons were observed at δ 190.7 (C-5), and 185.8 (C-12), besides the carbonyl of an acid derivative at δ 171.3 (C-15), three phenolic C atoms at δ 162.3, 158.4 and 157.9, and further 6 quaternary and 3 sp2 methine carbon atoms, establishing the aromatic part of pyrromycinone chromophor. In the sugar region, two methine carbons were observed at δ 101.6 and 99.1 and assigned as two ano-meric carbons. This pointed to the presence of two sugar moieties. Also, eight me-thine carbons were observed between δ 74.1-61.5 which were assigned as oxy- and /or nitrogenous methine carbons. Additionally, a quaternary sp3 oxy-carbon was ob-served at δ 71.7. A further methine carbon was obob-served at δ 57.2, which could be assigned as nitrogenous methine or flanked by two sp2 carbons. The methyl ester carbon of pyrromycinone chromophore was established at δ 52.5, and a carbon signal of two equivalent methyl carbons was observed at δ 42.9 corresponding to NMe2. Furthermore, it exhibited four methylene carbons at δ 33.8, 32.2, 29.3 and 27.0, from which the first two carbons were established of C-8 and C-13 of the pyrromycinone moiety. Finally, three methyl carbons were observed at δ 17.9, 16.0 and 6.7, from which the last carbon (δ 6.7) is characteristic for C-14 in the pyrromycinone skele-ton. The first two carbon signals (δ 17.9 and 16.0) were assigned as two methyl car-bons present in the sugar moieties. Compound 246 has the skeleton of ζ-pyrromycinone (243), except that the methylene group at position-7 (normally at δ 20.7) is replaced by CH-O observed at δ 65.3.
The mass spectrum of compound 246 was fixed as 715 Dalton by ESI mass spectra.
(+)-ESI MS2 of the molecular ion led to a base peak at m/z 586 as a result from the loss of 2-deoxyfucose (A) to afford pyrromycin (245). The latter one (245) showed a further fragmentation due to the expulsion of rhodosamine (B), followed by aromati-sation by loss of two water molecules to give a peak at m/z 393.1 ([M- (2-deoxyfucose + rhodosamin + 2 H2O) + H]+) (Figure 116).