Biomedical subjects
Publications and source records attributed to Hartmut Laatsch.
A new alpha-methylanthraquinone glucoside, laccaic acid D 8-o-beta-D-glucoside, and six known anthraquinones have been isolated from the aqueous alcoholic extract of Emex spinosus (L.) Campd. (Polygonaceae), together with two known flavonol glycosides. The structures were determined by conventional analytical methods and confirmed by MS and NMR spectral analysis, including 2D experiments for the new compound.
The hallucinogenic mushroom Inocybe aeruginascens contains several typical Psilocybe alkaloids including psilocybin. We have now elucidated the structure of a further indole derivative named aeruginascin as the quaternary ammonium compound N, N, N-trimethyl-4-phosphoryloxytryptamine. Aeruginascin is closely related to the frog skin toxin bufotenidine (5-HTQ), a potent 5-HT3 receptor agonist, and has been found exclusively in Inocybe aeruginascens so far.
Cluster analysis of gas-chromatographic (GC) data of ca. 500 bacterial isolates was used as an aid in detection and identification of new natural compounds. This approach reduces the number of GC/MS analysis (dereplication) and concomitantly improves the selection of samples with high probability to contain unknown natural products. Lipophilic bacterial extracts were derivatized and analyzed by GC under standardized conditions. A program was developed to convert chromatographic data into a two-dimensional matrix. Based on the results of hierarchical cluster analysis samples were selected for further investigation by GC/MS and NMR. This approach avoided unnecessary analysis of similar samples. By this method, the unusual oligoprenylsesquiterpenes 1 and 2 as well as new aromatic amides 7 and 8 were identified.
The ethyl acetate extract of the Streptomyces sp. isolate GW99/1572 exhibited significant biological activity against Gram-positive bacteria and delivered kettapeptin (1), a new hexadepsipeptide antibiotic of the azinothricin type. The structure was elucidated by various 1D and 2D NMR techniques, mass spectrometry and by comparison of the NMR data with those of closely related antibiotics. The absolute configuration of the compound was derived by crystal structure analysis and by comparison with the optical rotation data of related compounds.
In our screening of micro-organisms for novel bioactive natural products, a new staurosporinone, N-carboxamido-staurosporine (1c), and a new sesquiterpene, (5S,8S,9R, 10S)-selina-4(14),7(11)-diene-8,9-diol (2a), were isolated from the culture broth of the marine-derived Streptomyces sp. QD518. Their structures were determined by spectroscopic methods and by comparison of the NMR data with those of structurally related known natural products, which were isolated from the same strain.
Melatonin had previously been shown to reduce up to four 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) cation radicals (ABTS*+) via a scavenger cascade ending with N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK). However, when melatonin is added to the reaction system in much lower quantities than ABTS*+, the number of radicals scavenged per melatonin molecule is considerably higher and can attain a value of ten. Under conditions allowing for such a stoichiometry, novel products have been detected which derive from AFMK (1). These were separated by repeated chromatography and the major compounds were characterized by spectroscopic methods, such as mass spectrometry (HPLC-MS, EI-MS and ESI-HRMS), 1H nuclear magnetic resonance (NMR) and 13C NMR, heteronuclear multiple bond connectivity (HMBC) correlations. The identified substances are formed by re-cyclization and represent 3-indolinones carrying the side chain at C2; the N-formyl group can be maintained, but deformylated analogs seem to be also generated, according to MS. The primary product from AFMK (1) is N-(1-formyl-5-methoxy-3-oxo-2,3-dihydro-1H-indol-2-ylidenemethyl)-acetamide (2), which is obtained after purification as E- and Z-isomers (2a, 2b); a secondary product has been identified as N-(1-formyl-2-hydroxy-5-methoxy-3-oxo-2,3-dihydro-1H-indol-2-ylmethyl)-acetamide (3). When H2O2 is added to the ABTS*+ reaction mixture in quantities not already leading to substantial reduction of this radical, compound 3 is isolated as the major product, whereas 2a and 2b are virtually absent. The substances formed differ from all previously known oxidation products which derive from melatonin and are, among these, the first 3-indolinones. Moreover, the aliphatic side chain at C2 is reminiscent of other substances which have been synthesized in the search for melatonin receptor ligands.
The melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK; 1), which was previously shown to be a potent radical scavenger, was oxidized using the ABTS cation radical [ABTS = 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)]. Several new oxidation products were obtained, which were separated by repeated chromatography and characterized by spectroscopic methods such as mass spectrometry (ESI-MS and ESI-HRMS), 1H-NMR and 13C-NMR, HMBC, HSQC, H,H COSY correlations and IR spectroscopy. The main products were oligomers of 1 (3 dimers, 1 trimer and 2 tetramers). In all cases, the amino group N2 was involved in the reactions. Two of the dimers turned out to be cis (2a) and trans (2b) isomers containing an azo bond. In the other dimer (3a), the nitrogen atom N2 was attached to atom C5 of the second aromatic amine, with loss of the methoxy group. In the trimer (5), one N2 formed a bridge to C5 of unit B, as in the respective dimer, while this one of C had bridged to C6 of B. One of the tetramers (6) was composed of a trimer 5 attached to N2 of a fourth 1 molecule via an azo bond as in 2a/b. In the other tetramer (7), an additional C-C bond between rings B and C in 6 is assumed. Although oligomers of AMK may only attain low in vivo concentrations, the types of reactions observed shed light on the physiologically possible metabolism of AMK once reacted with a free radical. The displacement of a methoxy group, rarely seen in the oxidation of methoxylated biomolecules, underlines the reactivity of AMK (1). Preliminary data show that, in the presence of ABTS cation radicals, AMK (1) can interact with side chains of aromatic amino acids, a finding which may be crucial for understanding to date unidentified protein modification by a melatonin metabolite detected earlier in experiments with radioactively labeled melatonin.
Streptomyces diastaticus var. 108, a newly isolated strain, was recently characterized as a producer of two polyene macrolide antibiotics (rimocidin and CE-108), and the biosynthetic gene cluster was partially characterized. When the producer strain was genetically modified by transformation with some engineered SCP2*-derived vectors carrying the ermE gene, two previously uncharacterized macrolides were detected in the fermentation broth of the recombinant strain and chemically characterized as the amides of the parental polyene carboxylic acids. The biological activity and some in vitro toxicity assays showed that this chemical modification resulted in pharmaceuticals with improved biological properties compared with the parental products.
We recently characterized rimocidin B (3b) and CE-108B (4b) as two polyene amides with improved pharmacological properties, produced by genetically modified Streptomyces diastaticus var. 108. In this work, genetic and biochemical analysis of the producer strain show that the two amides are derived from the parental polyenes rimocidin (3a) and CE-108 (4a) by a post-PKS modification of the free side chain carboxylic acid. This modification is mediated by an amidotransferase activity operating after the biosynthesis of rimocidin (3a) and CE-108 (4a) are completed. Two polyenes, intermediates of the biosynthetic pathway of rimocidin (3a) and CE-108 (4a), were also isolated and shown to have some improved pharmacological properties compared with the final products.
In our screening of marine Streptomycetes for bioactive principles, two novel antitumor antibiotics designated as chinikomycins A (2a) and B (2b) were isolated together with manumycin A (1), and their structures were elucidated by a detailed interpretation of their spectra. Chinikomycins A (2a) and B (2b) are chlorine-containing aromatized manumycin derivatives of the type 64-pABA-2 with an unusual para orientation of the side chains. They exhibited antitumor activity against different human cancer cell lines, but were inactive in antiviral, antimicrobial, and phytotoxicity tests.
In our screening of microorganisms for new natural products, the fungus Penicillium notatum delivered further members of the sorbicillin family, namely, the sohirnones A [3, 1-(2,4-dihydroxy-5-methylphenyl)hex-4-en-1-one], B [4a, 1-(2,4,5-trihydroxy-3,6-dimethylphenyl)hexa-2,4-dien-1-one], and C [5, 1-(2,4,5-trihydroxy-3,6-dimethylphenyl)hex-4-en-1-one]. A stable tautomer of oxosorbicillinol (7) was characterized as 6, and the recently described 7-deacetoxyyanuthone (8) was reisolated. The additionally isolated rezishanones A-D (12-13c) are the first natural Diels-Alder products of sorbicillinol (1) with dienophiles not related with 1. The monomers and dimers showed weak antibacterial activity, but were inactive against fungi and algae. The structures were determined by spectroscopic methods and by comparison of the NMR data with those of the structurally related 2',3'-dihydrosorbicillin (2) and, in the case of 4a, by transformation into the known sorrentanone (4b).
Cultivation of the marine-derived streptomycete isolate B8005 delivered three known antibiotics, resistomycin (1), resistoflavin (3a) and tetracenomycin (4), and a further member of the rare resistomycin class, the weakly antibiotically active 1-hydroxy-1-norresistomycin (2). From a related marine strain B4842, 1 and resistoflavin methyl ether (3b) have been isolated. The formation of 2 is of interest from a biosynthetic point of view.
The melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) was found to be unstable in air when adsorbed on a thin-layer silica gel chromatography plate, a result that is in good agreement with the relatively high reactivity of this compound. Three novel main products were separated from the reaction mixture and identified by mass spectrometry and nuclear magnetic resonance data as: (i) 3-acetamidomethyl-6-methoxycinnolinone (AMMC), (ii) 3-nitro-AMK (AMNK, N1-acetyl-5-methoxy-3-nitrokynuramine), and (iii) N-[2-(6-methoxyquinazolin-4-yl)-ethyl]-acetamide (MQA). AMMC and AMNK are shown to be nonenzymatically formed also in solution, by nitric oxide (NO) in the first case, and by a mixture of peroxynitrite and hydrogen carbonate, in the second one. The use of three different NO donors, PAPA-NONOate, S-nitroso-N-acetylpenicillamine and sodium nitroprussiate led to essentially the same results, with regard to a highly preferential formation of AMMC; AMNK was not detected in these reaction systems. Competition experiments with the NO scavenger N-acetylcysteine indicate a somewhat lower reactivity compared with the competitor. Peroxynitrite led to AMNK formation in the presence of physiological concentrations of hydrogen carbonate at pH 7.4, but not in its absence, indicating that nitration involves a mixture of carbonate radicals and NO2, formed from the peroxynitrite-CO2 adduct. No AMMC was detected after AMK exposure to peroxynitrite. Both AMNK and AMMC exhibited a much lower reactivity toward 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) cation radicals than did AMK. In a competition assay for hydroxyl radicals, AMMC showed prooxidant properties, whereas AMNK was a moderate antioxidant. AMMC and AMNK should represent relatively stable physiological products, although their rates of synthesis are still unknown and may be low. Formation of these compounds may contribute to the disappearance of AMK from tissues and body fluids.
The biosynthesis of the potent marine antibiotic, pentabromopseudilin (1), was investigated. Feeding studies with Alteromonas luteoviolaceus were performed on a defined medium. D,L-[5-(13)C]proline was incorporated symmetrically, demonstrating that the pyrrole ring of pentabromopseudilin is derived from proline.
A new actinomycete strain, designated US80 and producing antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi, was isolated from Tunisian oasis soil. Cultural characteristic studies strongly suggested that this strain belongs to the genus Streptomyces. The nucleotide sequence of the 16S rRNA gene (1517 pb) of Streptomyces sp. strain US80 exhibited close similarity (97-98%) with other Streptomyces 16S rRNA genes. Similarity of 98% was obtained with the 16S rRNA gene of Streptomyces roseoflavus, which produces the aminoglycoside antibiotic flavomycin. Study of the influence of different nutritional compounds on production of bioactive molecules showed that the highest antimicrobial activities were obtained when glucose at 1% (w/v) was used as sole carbon source in the presence of magnesium. Extraction of fermentation broth of Streptomyces sp. strain US80 and various separation and purification steps led to isolation of three pure active molecules. The chemical structure of these three compounds, named irumamycin (1a), X-14952B (1b) and 17-hydroxy-venturicidin A (1c), was established on the basis on their IR, ESI-MS, 1H and 13C/APT NMR data and by comparison with reference data from the literature.
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Six quinones were isolated from the chloroform extract of the roots of Diospyros sylvatica and identified as 2-methyl-anthraquinone, plumbagin, diosindigo, diospyrin, isodiospyrin and microphyllone. The effect of the root extract on the orientation and survival of the subterranean termite, Odontotermes obesus was tested. In addition, four of these quinones were tested on the survival of the subterranean termite. In a direct-choice experiment, exposure to an extract-treated filter disc had a significantly repellent effect over the solvent-treated filter disc. The no-choice experiment revealed the toxic property of the extract as well as the tested quinones and showed high mortality of the O. obesus workers after 48 h on forced exposure. The major termiticidal components identified were plumbagin, isodiospyrin and microphyllone while diospyrin was not toxic to termites at the concentration tested. All the quinones are reported for the first time from D. sylvatica.
Two new caprolactones, (R)-10-methyl-6-undecanolide (1) and (6R,10S)-10-methyl-6-dodecanolide (2), were identified in the lipid extract of a marine streptomycete (isolate B6007). Their structures were proposed on the basis of GC-MS experiments and proved by synthesis. The absolute configuration of the compounds was established by comparison of the natural and synthetic stereoisomers using chiral gas chromatography. These caprolactones show a moderate phytotoxicity and a promising activity against cancer cells with concomitant low general cytotoxicity.