The ethyl acetate extract of a terrestrial Streptomyces sp. GW 12/3995 exhibited moderate antibacterial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Streptomyces viridochromogenes. It had also antialgal activity against the micro-algae Chlorella vulgaris and Chlorella sorokiniana.
During TLC screening, two polar UV absorbing zones were observed, one band of which was stained to brown by treatment with anisaldehyde/sulphuric acid. The ter-restrial Streptomyces sp. GW 12/2995 was cultivated on M2 medium as 20-liter fer-menter. Four fractions were isolated by using silica gel column chromatography and elution with dichloromethane-methanol. Further purification of fractions II-III led to 3,4-dihydroxybenzoic acid (226) and other known compounds (Figure 101)
GW12/3995 (20 l Fermenter)
Biomass Filtrate
mixing with celite and filtered by filterpress
4 x with EtOAc i.vac 4 x with EtOAc
crude extract (3.1 g)
chrom. on silica gel (DCM-MeOH )
Fraction I Fraction II Fraction III Fraction IV
A1 = PTLC(7% MeOH/CHCl3); A2 = PTLC(10% MeOH/CHCl3);
B1 = Sephadex LH-20 (CHCl3/MeOH; 6:4); B2 = Sephadex LH-20 (MeOH) 2,5-furan-dimethylalcohol 3,4-dihydroxybenzoic acid
A2, B2 A1, B1
Figure 101: Work-up procedure of terrestrial Streptomyces isolate GW12/3995 4.16.1 3,4-Dihydroxy benzoic acid
Compound 226, was isolated as colourless solid from fraction III by PTLC fol-lowed by Sephadex LH-20. It exhibited no colour reaction with the spraying re-agents, pointing to a nitrogen-free compound. The 1H NMR spectrum of compound 226 showed three aromatic 1H signals, displayed as two ortho-protons (dd) at δ 7.37 and δ 6.77 and a third m-coupled proton at δ 7.42 delivering an 1,2,4-trisubstiuted aromatic system.
The 13C/APT NMR spectra of compound 226 showed seven sp2 carbon signals, out of them one at δ 174.2 due to a carboxylic acid derivative. In addition, three
qua-trum of 226 fixed its molecular weight as 154 Dalton. The loss of ∆m/z 17 indicated the expulsion of a hydroxyl group.
According to the revealed spectral data and the molecular weight, two possible structures were proposed: 3,4-dihydroxy benzoic acid (226) and 2,4-dihydroxy ben-zoic acid (227). The observed deep field of the m-coupled proton (δ 7.42) suggested its locality between the carboxylic and hydroxyl groups directing towards 226. How-ever, an existence of the m-coupled proton between two hydroxyl groups as in 227 should be shifted up field (δ < 7), which is not matched with the measured data. This pointed to 3,4-dihydroxy benzoic acid (226), which was further confirmed by 2 D long-range correlations (HMBC) as shown below.
OH
Figure 102: HMBC-correlations of 3,4-dihydroxy benzoic acid (226) 4.17 Roseobacter strains (DFL12, DFL38, DFL16, DFL27, DFL30)
The ethyl acetate extracts of various marine Roseobacter sp. (DFL12, DFL38, DFL16, DFL27 and DFL30) possessed rather weak antimicrobial activity, however,
drew our attention because of the distinguished dark red colour of their colonies.
During TLC analysis, a dark red zone turned blue by treatment with sulphuric acid was detected, pointing to a carotinoid. Chemical screening of the various extracts by TLC and HPLC MS established identical metabolic patterns.
DFL 12& 38 (each 10 l shaker
Biomass Filtrate
mixing with celite and filtered by filterpress
4x with EtOAc i.vac 4 x with EtOAc
Crude extracts (3.1, 2.9 g )
chrom. on silica gel (DCM-MeOH)
Fraction I Fraction II
cis-cyclo(leucyl-prolyl) ciscyclo(phenylalanyl-prolyl)
A = Sap.with KOH/MeOH, B = SC. on Al2O3,
C = Sephadex LH-20 (CHCl3/MeOH3:2), D = PTLC (DCM-MeOH 7 %)
A, B, C C, D
spheroidenone
Figure 103: Work-up procedure of the Roseobacter sp. (isolates DFL12 and DFL38) The strains (DFL 12 and DFL38) were each fermented as 10-liter shaking cul-ture at 28 °C for 3 days. The resulting dark red culcul-ture broths were filtered and ex-tracted and chromatographed using flash silica gel column and elution with di-chloromethane-methanol gradient to give each two fractions. From fraction II, the known compounds cis-cyclo(leucyl-prolyl) (119), cis-cyclo(phenylalanyl-prolyl) (40), in addition to linoleic acid (239) were obtained.
4.17.1 Spheroidenone
Fraction I contained a red compound which turned to blue by treatment with sulphuric acid, pointing to a carotenoid. The compound was isolated by mild saponi-fication using methanolic potassium hydroxide at room temperature[259], and the
re-oxide and Sephadex to give pure 230 as a dark red powder.
The UV spectrum showed a strong broad peak at λmax 493 nm, in addition to three shoulders at λmax 261, 304 and 382, establishing a polyene system with a con-jugated carbonyl group. The molecular weight of compound 230 was established by (+)-ESI mass spectrum as 582 Dalton. HREI MS of 230 gave the corresponding mo-lecular formula C41H58O2 affording 13 double bond equivalents.
The 1H NMR spectrum showed a transoid double bond (J∼15.3 Hz) in conjuga-tion with a carbonyl group due to the presence of two doublets at δ 7.52 and 6.72. In addition, overlapping olefinic proton signals with an intensity of 12H were exhibited between δ 6.68-6.18, in addition to a 1H doublet at δ 5.95. Furthermore, a 2H multi-plet was displayed in the range of δ 5.17-5.09. In accordance, compound 230 possi-bly contained 17 olefinic protons. In the aliphatic region, it displayed at δ 3.22 a singlet of a methyl ether linked to sp3 carbon. Furthermore, four singlets between δ 1.97~1.95 with an intensity of 12 protons and a 12H due to two singlets at δ 1.68 (3H) and 1.58 (9H) were indicating eight methyl groups linked to double bonds. The last singlet (1.32) is attributed to two methyl groups linked possibly to saturated car-bon. The spectrum exhibited further two 4H multiplets between δ 2.10-2.03, which are assigned as of two methylene groups, probably adjacent to sp2 carbons.
Figure 104: 1H NMR spectrum (CDCl3, 300 MHz) of spheroidenone (230)
The 13C/APT NMR spectra of compound 230 showed a deep field quaternary carbon signal at δ 203.0 of a carbonyl group conjugated with an olefinic or aromatic moiety. In addition, 17 olefinic methine signals and seven quaternary sp2 carbons were displayed. In the aliphatic region, the spectrum showed a quaternary
oxygen-ated carbon at δ 81.6, and a methoxy signal at δ 52.2. Moreover, four methylene car-bons were observed at δ ~ 26. Between δ 25.7~12.8, 10 methyl signals were found.
By a search in AntiBase and the DNP, four possible carotinoids were obtained:
1,2-dihydro-1-methoxy-ψ,ψ-caroten-4-one (228), rhodopinol (229), spheroideno-ne[24] (230) and thiothece 474 (231).
Figure 105: 13C NMR spectrum (CDCl3, 125 MHz) of spheroidenone (230)
Compound 229 is an aldehyde and was excluded therefore. The carotenoids 228 and 231 are conjugated ketones, but don't have protons in α,β-position. In addition, the methylene groups in α,β-position to the carbonyl group in 228 and 231 were not displayed (normally found as two triplets). Therefore, compounds 228 and 231 were excluded, pointing to spheroidenone (230) as the sole consistent structure to the data.
Furthermore, the fragment (A) of compound 230 was also confirmed by 2 D correla-tions. In addition, EI mass spectrum showed two characteristic fragments at m/z 73 and 69. The fragment (m/z 73) is due to a terminal tertiary methoxy group ([-C(OCH3)(CH3)2), while the second one (m/z 69) is characteristic for a terminal iso-prene group (-C5H9]), further establishing the spheroidenone (230).
MeO
O
228
O H
OMe
229
O
Figure 106: 2D correlation of partial structure (fragment A) of spheroidenone (230)
C
Figure 107: HH-COSY (H↔H) and HMBC (H → C) correlations of spheroidenone (230)
Figure 108: HMBC NMR spectra and its expanded sections (CDCl3, 125 MHz) of spheroidenone (230)
Spheroidenone (230) was isolated previously from Rhodobacter sphaeroides, a member of the non-sulphur purple facultative[260] photosynthetic proteobacteria, ca-pable to grow under a variety of cultivation conditions. Compound 230 was also ob-tained from the photosynthetic bacteria Rhodopseudomonas spheroides and Erythro-bacter sp.[24,261]. It is structurally related to recently reported methoxylated keto-carotinoids from the purple non-sulphur bacteria Rhodopseudomonas globiformis.
These bacteria belong to the Athiorhodaceae recently renamed as Rhodopirilla-ceae[262]. Additionally, Rhodobacter denitrificans (previously named Erythrobacter sp) synthesized spheroidenone as a major carotenoid under aerobic dark conditions.
However, in illuminating under anaerobic conditions, many yellow pigments ap-peared e.g. 3,4-dihydrospheroidenone, while a considerable amount of spheroide-none disappeared. This pointed to a photo-reduction of the C=C double bond at the 3,4-position of spheroidenone[263].
Previous examination of drilling cores showed that the majority of the carotenes and carotenoids originally present in the various planktonic organisms and photophic bacteria are preserved in sediments and were found to act as indicators of tro-phic changes in Lake Lobsigen during the last 14000 years. Spheroidenone is one of such pigments present in the sedimental bacteria which were collected from deep sea[264].
and other bacteria species, 18 bacterial isolates were selected. These strains showed a characteristic red/orange colouration during their growth on agar plates. The strains were compared with our five Roseobacter sp. (DFL16, DFL30, DFL27, DFL38 and DFL12) worked up previously. However, chemical screening of the selected strain extracts exhibited a different chromatographic behaviour (Rf, colour of bands (yel-low∼red) and no blue colours with sulphuric acid (mostly to brown). This indicated that carotinoides were not present in these strains and confirmed spheroidenone (230) as an taxonomic marker of Roseobacter sp. This conclusion was confirmed addition-ally by subjecting their extracts to HPLC MS analysis, which established their simi-larity.
Table 10: Comparison of the Roseobacter sp. isolates (DFL) with other bacteria sp.
producing coloured metabolites on basis of TLC analysis.
Taxonomy Strain Nr. Agar plat
colour Colour change by H2SO4
Colour by TLC Colour of the Extract Roseob. Hel 28 Red - Yellow/orange Yellow/orange Roseob. Hel 38/983 Red - Yellow Yellow/orange Roseob. Hel 42 orange - Yellow Brown Roseob. Hel 28 Red - Orange Red
Roseob. DFL16 Red Blue Red Red
Roseob. DFL30 Red Blue Red Red
Roseob. DFL27 Red Blue Red Red
Roseob. DFL38 Red Blue Red Red
Roseob. DFL12 Red Blue Red Red
Unknown 1241/Pic. Red - Red Red Unknown 1261/Bio Red - Yellow Yellow Unknown 1266/Bio Red - Orange Brown/red Unknown Bio215 Red - Red Red/brown G-posit. Hel 31 Red - Orange Brown Unknown Bio221 Orange/red - Red Red/brown Unknown STO025 Red - Red Red/brown Unknown 1267/Bio Red - Red Red/brown