The marine Streptomyces sp. B8300 had previously been investigated by Bia-bani in our group and afforded several metabolites with high bioactivities[322]. How-ever, further compounds were isolated with insufficient amounts for analysis. Hence, the strain was re-cultivate to isolate a further new bioactive components.
TLC of the crude extract obtained from a 20 L shaker culture using M2+ medium exhibited a number of yellow, UV absorbing bands. Three further UV absorbing bands turned blue or reddish-brown on spraying with anisaldehyde/sulphuric acid. In the antimicrobial assay, the extract showed a strong activity against Bacillus subtilis, Escherichia coli, Streptomyces viridochromogenes (Tü 57) and Candida albicans, moderate activity against Chlorella vulgaris and Chlorella sorokiniana, while it ex-hibited no activity against either Mucor miehei (Tü 284) or Staphylococcus aureus (Table 73).
XAD-16 (MeOH/H2O), EtOAC B8300
(20 l Shaker)
Biomass Filtrate
mixing with cilite and filtered by filterpress
4 x with EtOAC and 1x with acetone i.vac Crude extract
(4.66 g)
chrom. on silica gel (CH2Cl2-MeOH)
Fraction I (0.65 g)
5 days
Fraction II (1.4 g)
A1 = Sephadex LH-20 (CH2Cl2/40% MeOH) Fraction III (1.15 g)
A2, B1, A1
Fat (1.8 g) Phencomycin methyl ester
KST8 (10.6 mg)
methy ester; KST3 (3.7 mg)
2,5-Furandimethanol KST7 (35.7 mg)
Adenine 2A3
A2, B2, B3, B4
ββββ-Indomycinone Saptomycin-A
Figure 117: work-up procedure of marine Streptomyces sp. B8300
Purification of the extract was carried out by silica gel column chromatography and PTLC followed by size exclusion chromatography (Figure 117). As a result, eight compounds were isolated: 2,5-furandimethanol, dihydrophencomycin methyl ester (212), phencomycin methyl ester (215), β-indomycinone (219), saptomycin A (220), adenine[152], adenosine[152] and 2'-deoxy-adenosine[152].
5.8.1 Dihydrophencomycin methyl ester
Compound 212 was obtained as a low polar reddish-brown solid which exhib-ited no colour change on treatment with diluted solution of sodium hydroxide, ex-cluding peri-hydroxyquinones. The EI mass spectrum exhibited a molecular weight of 298 Dalton. The molecular ion showed a loss of methanol and CO to give the fragment ions at m/z 266 and m/z 238.
The 1H NMR spectrum showed one broad singlet of an acidic proton (δ 8.74), three downfield 1H signals (δ 6.98~6.13) belonging to a 1,2,3-trisubstitued aromatic ring, and the singlet methoxy group at δ 3.82. 13C NMR & HMQC spectra of com-pound 212 displayed 8 carbon signals, of which three sp2 methines, three quaternary carbon signals and a CO signal of an acid derivative (δ 168.4) in addition to the methoxy signal at δ 51.7 was found. These NMR patterns were indicative for a dihy-drophenazine skeleton, consisting of two symmetrical 1,2,3-trisubstituted aromatic residues, each containing a carboxymethyl group. So, two possible structures were suggested: 5,10-Dihydrophenazine-1,6-dicarboxylic acid dimethyl ester (212) and 5,10-dihydro-phenazine-1,9-dicarboxylic acid dimethyl ester (213).
NH NH O O
C H3
O O CH3
1
3 5 7
9 10
NH NH O C O
H3 O O
CH3
1
3 5 7
9 10
212 213
The structure of 5,10-dihydrophencomycin methyl ester (212) was finally con-firmed by direct comparison with authentic spectra from our collection[139]. 5,10-Dihydrophencomycin methyl ester (212) was isolated previously in our research group from an unidentified marine Streptomyces sp. The compound had weak anti-bacterial activity against E. coli and B. subtilis.
O O
O O
NH NH
CH3 C
H3
1
3 5
7 9 10
Figure 118: H,H COSY (↔) and HMBC ( ) couplings of Dihydrophencomycin methyl ester (212).
Table 30: 13C and 1H NMR spectroscopic data for compounds 212 and 215 in CDCl3
(J in [Hz]).
Position Dihydrophencomycin methyl ester (212) Phencomycin methyl ester (215) δC (125 MHz) δH (300 MHz) δC (75 MHz) δH (300 MHz)
1, 6 108.7 - 131.1 -
1, 6-CO 168.4 - 166.8 -
OCH3 51.7 3.82 (s) 52.7 4.11 (s)
2, 7 122.5 6.98 (dd, 8.3, 1.4) 132.9 8.29 (dd, 6.9, 1.4)
3, 8 119.3 6.29 (t, 8.3) 129.5 7.89 (dd, 8.8, 6.9)
4, 9 134.3 6.13 (dd, 8.3, 1.3) 134.3 8.48 (dd, 8.8, 1.4)
4a, 9a 132.6 - 142.9 -
5, 10 - 8.74 (brs) - -
5a, 10a 138.4 - 140.9 -
5.8.2 Phencomycin methyl ester
Compound 215 was isolated as low polar yellow crystals from fraction I using PTLC and purified on Sephadex LH-20. Compound 215 showed also no colour col-our change on treatment with sodium hydroxide, excluding peri-hydroxyquinones as in 212. The molecular weight of 215 was determined by EI MS as 296 Dalton, 2 amu less than compound 212. The molecular ion showed an expulsion of a methoxy group, affording a fragment ion at m/z 265. The latter fragment displayed a further splitting of carbonyl group to give a peak at m/z 238 as in 212, as indicative of the presence of phenazine skeleton in 215. HRESI MS confirmed the molecular formula as C16H12N2O4.
The 1H NMR spectrum of 215 showed the same spectral pattern as 212 except that the acidic NH protons were missing. So, a 1,2,3-trisubstitued aromatic ring, an aromatic bound methoxy group (δ 4.11) were fixed.
The 13C NMR spectrum of 215 showed three sp2 methines (δ 134.3, 132.9, 129.5), two signals of quaternary sp2 carbons (δ 142.9, 140.9), and one CO signal (δ 166.8) of an acid derivative, besides those of the methoxy signal (δ 52.7). This pointed to phencomycin methyl ester, and the identity was indeed confirmed by comparison with data of an authentic sample[139]. This is the first time that the methyl ester 215 was isolated as a natural product.
O O
O O N
N
R'' R'
1
3 5 7
9 10
214: R' = CH3, R'' = H 215: R' = R'' = CH3
216: R' = R'' = H
O O
O O
N N
CH3 C
H3
1
3 5 7
9 10
Figure 119: H,H COSY (↔) and HMBC ( ) couplings of Phencomycin methyl ester (215).
To exclude the possibility that 212 and 215 were formed as artefacts during iso-lation, the extracts were worked up carefully avoiding any methanol as solvent. Even under these conditions, both esters were isolated again, indicating that 212 and 215 are natural products indeed.
Phenazine-1,6-dicarboxylic acid (216) inhibits xanthine oxidase, shows antibi-otic properties, and is an intermediate in the biosynthesis of lomofungin (217)
pro-duced by Pseudomonas and Streptomyces sp.[105,279]. Phencomycin (214) was isolated from Streptomyces sp. HIL Y-9031725[280].
N N
OH O
OH OH
O H
O H
NH NH
O O
O O
R
R
217 218: R = H, 212: R = CH3
Due to the interesting biological activities of naturally occurring phenazines[139,
323-326], the synthesis of poly-substituted phenazines was developed by Holliman and co-workers[327] from the reductive cyclization of o-nitrodiphenylamines. However, the yield was poor due to the competitive cyclization. N-arylation has become more accessible owing to the advent of a new methodology developed by Hartwig and Buchwald[328]. The synthesis of phenazines were developed using sequential aniline arylation (palladium(II)-catalyzed intramolecular amination)[329].
5.8.3 ββββ-Indomycinone
From sub-fraction II, compound 219 was isolated as a yellow solid. It was UV absorbing at 254 nm and showed a red fluorescence at 366 nm, and turned to red-violet by treating with 2N NaOH as indication of a peri-hydroxyquinone.
The 1H NMR spectrum of 219 revealed singlets at δ 12.85 (OH) and δ 8.08, three ortho-coupled aromatic protons at δ 7.83, 7.70 and 7.38, a 1H singlet at δ 6.56.
In addition, two multiplets of an olefinic double bond were detected (δ 5.74 and 5.38), and a methyl singlet at δ 3.02. Two methylene protons of an ABX system were observed ( 2.91 and 2.78), together with two methyl (1 s, 1 d) linked to sp2 or oxy-genated carbons ( 1.72 and 1.64).
The ESI mass spectrum resulted in a molecular weight of 219 as 404 Dalton. A search in AntiBase resulted in -Indomycinone (219). -Indomycinone (219) belongs to the pluramycins, a group of structurally highly diverse reactive agents, possessing antimicrobial and anticancer activity[330].
O
CH3
CH3 O C
H3 OH
O O
OH
5
1 3
4
9 7
12a
13 14 15
17 19
11
219 5.8.4 Saptomycin-A
A second peri-hydroxyquinone was obtained as yellow solid from sub-fraction II by PTLC followed by Sephadex LH-20. It showed similar physiochemical proper-ties as 219. The ESI mass spectrum fixed the molecular weight of compound 220 as 404 Dalton as an isomer of ß-indomycinone (219).
The 1H NMR spectrum showed nearly the same signal pattern as -Indomycinone (219). A singlet of a peri-hydroxy group at δ 12.64 was exhibited. In the aromatic region, a 1H singlet at 8.09, three aromatic protons ( 7.83, 7.69 and 7.36) of an 1,2,3-trisubstituted aromatic ring, and a singlet of H-3 ( 6.28) were shown. In addition, a singlet of a peri-methyl group ( 3.02) among with a multiplet of an olefinic double bond (2 H, 5.40) was observed. Moreover, two methine pro-tons were displayed at 5.40 and 2.98. The first methine group was oxygenated, while the second methine was flanked probably two sp2 carbons. Finally, two methyl doublets were detected ( 1.71 and 1.45), whereof the first was attached to an sp2 carbon.
The corresponding molecular formula of compound 220 was determined as C24H20O6, according to the Rule of 13[331]. Accordingly, compound 220 exhibited close structural similarity to the hydroxyquinone mentioned above. Based on the spectroscopic data, a search in AntiBase, as well as by comparison with authentic spectra and the literature, compound 220 was established as saptomycin A[332].
Pluramycins are metabolites containing the 4H-anthra[1,2-b]pyran-4,7,12-trione nucleus to which amino sugars typically are attached at C-8 and C-10 positions. They are frequently isolated from terrestrial Streptomycetes sp.[322,333,334]. Pluramycins are