The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia sp. cf. C. flammea. Culture on a biomalt medium supplemented with sea salt led to the isolation of two new, most unusual N-methylated peptides, i.e., the tetrapeptides endolide A and B (1 and 2). Both of these contain the very rare amino acid 3-(3-furyl)-alanine. In radioligand binding assays endolide A (1) showed affinity to the vasopressin receptor 1A with a Ki of 7.04 μM, whereas endolide B (2) exhibited no affinity to the latter receptor, but was selective toward the serotonin receptor 5HT2b with a Ki of 0.77 μM.

A marine-derived fungus of the genus Stachylidium was isolated from the sponge Callyspongia cf. C. flammea. Chemical investigation of the bioactive fungal extract led to the isolation of the novel phthalimidine derivatives marilines A(1) (1a), A(2) (1b), B (2), and C (3). The absolute configurations of the enantiomeric compounds 1a and 1b were assigned by a combination of experimental circular dichroism (CD) investigations and quantum chemical CD calculations. The skeleton of marilines is most unusual, and its biosynthesis is suggested to require uncommon biochemical reactions in fungal secondary metabolism. Both enantiomers, marilines A(1) (1a) and A(2) (1b), inhibited human leukocyte elastase (HLE) with an IC(50) value of 0.86 μM.

The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C ␤-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC ␤-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the K m values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid-or a chromosome-encoded class C enzyme toward ␤-lactam antibiotics are mainly due to the overproduction of the ␤-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded ␤-lactamases.␤-Lactams illustrate the growing phenomenon of bacterial resistance to antibiotics. Since the beginning of their utilization, over 50 years ago, the introduction of new compounds of this family has resulted in increasingly rapid evolution in the microbial world (4,12,19). The principal bacterial defense mechanism is the production of ␤-lactamases, enzymes which produce a biologically inactive product by hydrolyzing the ␤-lactam ring (20). On the basis of their primary structures, ␤-lactamases are grouped into four classes (A, B, C, and D) (8), and the various enzymes exhibit specific activity spectra. Enzymes of classes A, C, and D are active-site serine enzymes, whereas the activities of class B enzymes are zinc dependent (14). The genes coding for the ␤-lactamases of classes A, B, and D are located either on transferable plasmids or on the bacterial chromosome (14). Initially, class C ␤-lactamases (AmpC) were described as chromosomal enzymes (6). However, over the last 20 years, more than 20 plasmid-encoded class C enzymes have been identified (18). These proteins may be classified into six subgroups based on amino acid sequence similarities. Each subgroup contains plasmid-borne class C enzymes and their closest chromosomal relatives (18). Clinical isolates harboring plasmidic AmpC exhibit high resistance toward ␤-lactam antibiotics, such as cephamycins and monobactams.The present work was focused on an extensive characterization of four plasmid-borne class C ␤-lactamases; ACT-1 and MIR-1 (subgroup 2), CMY-2 (subgroup 1), and CMY-1 (subgroup 6), in order to determine if their kinetic properties were significantly different from those of the chromosome-encoded enzymes, as suggested before. As will be discussed below, this paper provides the first biochemical data showing that these enzymes have not yet significantly improved their catalytic mechanisms and thus do not deserve the appellation of extended-spectrum ␤-lactamases, as previously suggested by some authors (1, 7). MATERIALS AND METHODSAntibiotics and other chemicals. Nitrocefin was from Oxoid (Basingstoke, Hants, United Kingdom). Ampicillin, benzylpenicillin, ...

The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia cf. C. flammea. Four new, putatively tyrosine-derived and O-prenylated natural products, stachylines A -D (1 -4), were obtained from the fungal extract. The structures of 1 -4 were elucidated based on extensive spectroscopic analyses. The absolute configuration of compound 2 was established by Mosher's method. Stachyline A (1) possesses a rare terminal oxime group and occurs as an interchangeable mixture of E/Z-isomers.The marine environment harbours approximately half of the global biodiversity and is estimated to contain between 3 and 500 million different species, offering an almost infinite resource for novel compounds. 1 Among these organisms marine-derived fungi became known as prolific producers of structurally most intriguing compounds. 2 In general, tyrosine derivatives have only rarely been reported from fungi, and in most cases such compounds were obtained from strains originating from environmentally extreme habitats, e.g. tyrosol carbamate which was isolated from the deep-water fungus Arthrinium sp. 3 Phytomyces sp., producing O-prenylated tyrosine derivatives, is an extremophile collected from an acid mine waste rich in toxic metals. 4 Another unusual case is aspergillusol A, an α-glucosidase inhibitor obtained from the sponge-derived fungus Aspergillus aculeatus which is reported to be the only known fungal tyrosine derivative to possess an oxime group. 5 Secondary metabolites with an oxime substituent are rare, and most of the reported examples have potent bioactivity, e.g. the actinomycete-derived nocardicins displayed strong antibiotic activity, 6 and brevioxime from Penicillium brevicompactum inhibited the biosynthesis of insect juvenile hormones. 7 P. olsonii produced 2-(4-hydroxyphenyl)-2-oxoacetaldehyde oxime (PHBA) which regenerated phosphorylated cholinesterase. 8 The oxime geometrical isomers collismycins A and B were isolated from Streptomyces sp. MQ22 which inhibited dexamethasone glucocorticoid receptor binding. 9During our search for new cytotoxic natural products an extract of the marine-derived fungus Stachylidium sp. was found to be active. During chromatographic separations it became clear that this fungus produces a vast array of secondary metabolites with intriguing structural features, among them the four novel, putatively tyrosine-derived and O-prenylated natural products, stachylines A -D (1 -4). Stachyline A (1) is distinguished by an oxime terminal group, probably derived through biosynthetic reactions similar to those known for cyanogenic glycosides and nocardicin A formation. [10][11][12][13][14] The molecules were evaluated in a number of biological assays, to date however no considerable activity was detected.* To whom correspondence should be addressed. Results and DiscussionThe RP-18 HPLC chromatogram of 1 contained two peaks (ratio 1:1), which when reinjected after their individual isolation, again resulted in the same chromatogram. This result suggested that compound 1 exi...