Isabel Sattler

Maremycins C and D, New Diketopiperazines, and Maremycins E and F, Novel Polycyclicspiro-Indole Metabolites Isolated fromStreptomycessp.

New diketopiperazines, named maremycins C1/C2 (1a/1b) and D1/D2 (2a/2b), as well as the novel spiro-indoles, maremycins E (3) and F (4), have been detected alongside the known maremycin B (6) in the culture broth of Streptomyces sp. (strain GT 051237) by chemical screening. The structures have been determined by detailed NMR spectroscopic investigations of the isolated metabolites. Maremycins C1/C2 (1a/1b) as well as D1/D2 (2a/2b) are diastereomers and were identified as mixtures. Structurally, maremycins C1/C2 (1a/1b) are the diastereomers of the sulfur oxidation products of maremycin B (6), while D1/D2 (2a/2b) are the demethylmercapto analogues of maremycins A (5) and B (6), respectively. Maremycins E and F possess a novel structural skeleton, where a spiro moiety is formed between the 6-position of the cyclopenta[f]quinoxaline moiety and the 3′-position of the indol-2-one moiety of the initial diketopiperazine product.

Botryorhodines A–D, antifungal and cytotoxic depsidones from Botryosphaeria rhodina, an endophyte of the medicinal plant Bidens pilosa

An endophytic fungus (Botryosphaeria rhodina) was isolated from the stems of the medicinal plant Bidens pilosa (Asteraceae) that is known for its anti-inflammatory, antiseptic and antifungal effects. The ethyl acetate extract of the fungal isolate exhibits significant antifungal activity as well as potent cytotoxic and antiproliferative effects against several cancer cell lines. Activity-guided fractionation resulted in the isolation of a complex of four depsidones, botryorhodines A-D and the auxin indole carboxylic acid. Botryorhodine A and B show moderate to weak cytotoxic activities against HeLa cell lines with a CC(50) of 96.97 microM and 36.41 microM, respectively. In addition, they also show antifungal activity against a range of pathogenic fungi such as Aspergillus terreus (MIC 26.03 microM for botryorhodine A and 49.70 microM for B) and the plant pathogen Fusarium oxysporum (MIC 191.60 microM for botryorhodine A and 238.80 microM for B). A potential role of the endophyte in modulating fungal populations living within or attacking the host plant is discussed.

Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace® SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.

Gabosines L, N and O: New Carba-Sugars fromStreptomyces with DNA-Binding Properties

In addition to the known gabosines A (4), B (5) and C (6), three new gabosines L (1), N (2) and O (3) were detected by chemical screening as secondary metabolites of Streptomyces (strains GT 041230, GT 051024 and S 1096). The constitutions of 1, 2 and 3 were established by spectroscopic techniques and derivatization reactions. The absolute stereochemistry of 1 and 2 was determined by Helmchens method and has been verified in the case of gabosine N (2) by X-ray analysis. The DNA-binding properties of the gabosines were investigated and analyzed by binding studies using a recently developed thin-layer chromatography technique (bimolecular-chemical screening).

Arousing sleeping genes: shifts in secondary metabolism of metal tolerant actinobacteria under conditions of heavy metal stress

Numerous microbial habitats are strongly influenced by elevated levels of heavy metals. This type of habitat has developed either due to ore mining and metal processing or by pedogenesis above metal-rich base rocks. Most actinobacteria are soil-borne microbes with a remarkable capability for the synthesis of a broad variety of biologically active secondary metabolites. One major obstacle in identifying secondary metabolites, however, is the known phenomenon of sleeping gene clusters which are present, but silent under standard screening conditions. Here, we proceed to show that sleeping gene clusters can be awakened by the induction in heavy metal stress. Both, a chemical and a biological screening with extracts of supernatant and biomass of 10 strains derived from metal contaminated and non-contaminated environments was carried out to assay the influence of heavy metals on secondary metabolite patterns of metal tolerant actinobacteria. Metabolite patterns of cultures grown in complex and minimal media were compared to nickel (or cadmium) spiked parallels. Extracts of some strains grown in the presence of a metal salt displayed intense antibiosis against Escherichia coli, Mycobacterium smegmatis, Staphylococcus aureus and Candida albicans. Contrarily to the widely held opinion of metals as hindrance in secondary metabolism, metals thus can induce or enhance synthesis of possibly potent and medically relevant metabolites in metal tolerant strains. Hence, re-screening of existing strain libraries as well as identification of new strains from contaminated areas are valid strategies for the detection of new antibiotics in the future.

Benzopyrenomycin, a Cytotoxic Bacterial Polyketide Metabolite with a Benzo[a]pyrene‐Type Carbocyclic Ring System

Polyketides form a major class of secondary metabolites of bacteria, fungi, and plants with broad structural diversity as a result of dense functionalization and spatial properties. Many polyphenolic derivatives have found considerable interest as pharmaceuticals, biological tools, and dyes. A hallmark of aromatic polyketides is their common biogenesis from simple acyl and malonyl units through the action of different types of iterative polyketide synthases. The orchestrated assembly and processing of the biosynthetic intermediates gives rise to polyphenolic compounds that differ widely in the number of carbocycles they contain, their topology, and the substitution of the rings. From a structural point of view, it is remarkable that only a limited number of carbocyclic (aromatic) polyketide frameworks occur naturally. Linear and monoangular polyphenolic ring systems are found almost exclusively (Scheme 1); perifused carbocycles, in which rings are fused through more than one face, are rarities. A possible rationale for this observation is the preferred U-shaped folding of a nascent poly-b-keto chain. S-shaped cyclization patterns, as in the biosynthesis of the pentacyclic “discoid” Streptomyces naphthanthrene metabolites resistomycin and resistoflavin, are clear exceptions. Some phenalenes and benz[d,e]anthracenes from plants and fungi are probably also formed by an alternating polyketide folding pattern, whereas the biosynthesis of phenylphenalenones involves intramolecular cycloaddition with a cinnamoyl-derived moiety. Homologous tetracyclic pyrenes thought to be derived from phenanthrenes have been isolated from Uvaria and Juncus spp. An important example of a perylene is altertoxin, which is formed by naphthol dimerization in analogy with the hypericin biosynthetic pathway. To date, however, a significant gap has remained between the pentacyclic pentangular and discoid polyketide structures: the benzo[a]pyrene scaffold. Benzo[a]pyrenes are only known as notorious products of the pyrolysis of organic matter which are transformed into carcinogens upon epoxidation. No example of the biogenesis of a related carbocyclic system has been described. Herein, we report the first discovery of a natural product with a benzo[a]pyrene framework. During the course of metabolic profiling of the ketalin producer Streptomyces lavendulae (strain T< 1668), we noted the formation of minute amounts of a novel aromatic compound with UVabsorptions at lmax= 417, 251, and 217 nm when the strain was cultured on a large scale (2 > 50 L). Highresolution EIMS provided sufficient evidence that the compound had not been described previously. The crude extract was subjected to purification first with amberlite XAD16, then by reversed-phase flash chromatography (RP18) and subsequent open-column chromatography on Sephadex LH20 and silica gel. The new compound 1 (7 mg in total) was isolated as a yellow solid. A series of biological assays with 1 revealed inhibitory activity against various tumor-cell lines. Compound 1 showed strong antiproliferative activity against the cell lines L-929 and K562 with GI50 values of 3.2 mgmL 1 (8.2 mm) and 4.2 mgmL 1 (10.8 mm), respectively, and moderate cytotoxicity against HeLa cells with a CC50 value of 26.4 mgmL 1 (68.0 mm). The structure of the cytotoxic metabolite was resolved fully by MS and NMR spectroscopy. High-resolution EIMS Scheme 1. Structure-based phylogeny of fundamental dito pentacyclic ring systems found in natural aromatic polyketides; no natural product with a benzo[a]pyrene-type skeleton was known previously. Shared faces are highlighted in bold.

Design, synthesis and early structure–activity relationship of farnesyltransferase inhibitors which mimic both the peptidic and the prenylic substrate

Inhibition of the farnesylation of ras proteins has been identified as a promising target in tumor therapy. Only a few farnesyltransferase inhibitors are bisubstrate analogues displaying features of both substrates, the farnesylpyrophosphate and the C-terminal CAAX-tetrapeptide sequence of the ras protein. These known bisubstrate analogues consist of an AAX-tripeptide and a farnesyl residue connected through various linkers. We have developed a class of novel compounds that mimic a bisubstrate inhibitor structure and that differ from the known ones by lacking peptidic or farnesylic substructures. Long chain fatty acids and aryl-substituted carboxylic acids were used as farnesyl surrogates. These structures were linked to isoleucine amide, benzoic acid amide, N-substituted aminobenzenesulfonamides and N(alpha)-aryl-substituted methionine derivatives, respectively, which function as AA- or AAX-mimetics.

Two new constituents from mangrove Bruguiera gymnorrhiza

A lignan and two aromatic compounds were isolated from the branches of the mangrove plant, Bruguiera gymnorrhiza. They were brugunin A (1), bruguierol D (2) and 2,3-dimethoxy-5-propylphenol (3). Among them, 1 and 2 were new compounds; 3 was isolated from a natural source for the first time. The structures of these compounds were determined by NMR spectroscopic studies as well as chemical evidence.

Natural products for lead identification: Nature is a valuable resource for providing tools

About 30% of drugs on the worldwide market are natural products or are derived from natural products. A similar ratio accounts for clinical candidates currently under development. Though recombinant proteins and peptides account for an increasing market volume, the superiority of low-molecular mass compounds in human diseases therapy remains undisputed mainly due to more favorable compliance and bioavailability properties.

Acyl α-L-Rhamnopyranosides, a Novel Family of Secondary Metabolites fromStreptomyces sp.: Isolation and Biosynthesis

In the course of our chemical screening program, the novel acyl α-L-rhamnopyranosides (1-6) were detected as metabolites from five different strains of Streptomycetes. The structures of all these compounds were elucidated by chemical and spectroscopic methods. The biosynthesis of 1 and 3 was established by feeding 13C-labelled acetate, glycerol, and D-glucose to Streptomyces griseoviridis (strain Tu 3634), and resulted in a complete labelling pattern of the 2,4-dimethyl-3-furanylcarbonyl and benzoyl residues, as well as the rhamnose moiety. These results reveal biosynthetic pathways of general importance and give an insight into the generation of the hexose phosphates, from which deoxysugars are formed. The acyl rhamnosides are members of a novel family of microbial metabolites and are considered as rhamnoconjugates from Streptomycetes.