We recently reported the cultivation and phylogenetic characterization of a new group of obligate marine actinomycete bacteria that is widely distributed in ocean sediments.[1] Analogous soil-derived actinomycetes have been the single most significant source of naturally occurring microbial antibiotics, [2] thus the discovery of a major new group of these bacteria in marine sediments suggests that the ocean represents an overlooked habitat from which to isolate these important microorganisms. Given that the rate of discovery of new biologically active compounds from common soil actinomycetes has been falling, [3] obligate marine actinomycetes represent a new resource for structurally diverse secondary metabolites.To date, we have isolated in excess of 2500 strains belonging to this new taxon for which we have proposed the genus name ™Salinospora∫ (a formal taxonomic description is in progress). ™Salinospora∫ strains, with previously undescribed 16S rRNA gene sequences, have been recovered from five distinct tropical/subtropical ocean systems [1] and from depths as great as À1100 m, which indicates that they represent a widely distributed and taxonomically diverse group of sediment bacteria. All isolates display an obligate requirement of ionic sodium for growth, thus indicating a high level of marine adaptation.In preliminary screening, a high percentage of the organic extracts of cultured ™Salinospora∫ strains possessed antibiotic and anticancer activities, which suggests that these bacteria are an excellent resource for drug discovery. Herein we report the results of our first chemical investigation of a member of the ™Salinospora∫ group and show that strain CNB-392 produces the chemically unique and highly bioactive metabolite salinosporamide A (1, Scheme 1). Salinosporamide A exhibits potent cancer cell cytotoxicity and appears to exert its cytotoxic effects through inhibition of the 20S proteasome.™Salinospora∫ strain CNB-392 was isolated from a heattreated marine sediment sample that was plated on a seawater-based agar nutrient medium. Liquid shake flask cultivation of this strain, followed by solid-phase extraction with Amberlite resin (XAD-16) and elution with acetone, resulted in a crude extract that was highly cytotoxic in vitro toward HCT-116 human colon carcinoma (IC 50 ca. 80 ng mL À1 ). Cytotoxicity-guided fractionation of the crude extract led to the isolation of salinosporamide A (1) as a colorless crystalline solid (yield: 7 mg L À1 ). The complete structural assignment of 1 was accomplished by spectral analysis and by a single-crystal X-ray diffraction study.Analysis of the low-resolution mass spectrum of salinosporamide A showed a characteristic [Mþ2] þ peak indicative of the presence of a chlorine atom. High-resolution massspectral analysis provided the molecular formula Comprehensive analysis of 2D NMR data, including the results of COSY, HMQC, and HMBC experiments, enabled the complete planar structure of salinosporamide A to be assigned, as in 1, to a 2-aza-6-oxabicyclo[3.2.0]heptane-3,7...

Starke und selektive Cytotoxizität gegenüber einer Reihe von Krebszelllinien kennzeichnen Salinosporamid A (1), das aus dem Rohextrakt eines einzigartigen marinen Aktinomyceten der Gattung Salinospora isoliert wurde.

We recently reported the cultivation and phylogenetic characterization of a new group of obligate marine actinomycete bacteria that is widely distributed in ocean sediments.[1] Analogous soil-derived actinomycetes have been the single most significant source of naturally occurring microbial antibiotics, [2] thus the discovery of a major new group of these bacteria in marine sediments suggests that the ocean represents an overlooked habitat from which to isolate these important microorganisms. Given that the rate of discovery of new biologically active compounds from common soil actinomycetes has been falling, [3] obligate marine actinomycetes represent a new resource for structurally diverse secondary metabolites.To date, we have isolated in excess of 2500 strains belonging to this new taxon for which we have proposed the genus name ™Salinospora∫ (a formal taxonomic description is in progress). ™Salinospora∫ strains, with previously undescribed 16S rRNA gene sequences, have been recovered from five distinct tropical/subtropical ocean systems [1] and from depths as great as À1100 m, which indicates that they represent a widely distributed and taxonomically diverse group of sediment bacteria. All isolates display an obligate requirement of ionic sodium for growth, thus indicating a high level of marine adaptation.In preliminary screening, a high percentage of the organic extracts of cultured ™Salinospora∫ strains possessed antibiotic and anticancer activities, which suggests that these bacteria are an excellent resource for drug discovery. Herein we report the results of our first chemical investigation of a member of the ™Salinospora∫ group and show that strain CNB-392 produces the chemically unique and highly bioactive metabolite salinosporamide A (1, Scheme 1). Salinosporamide A exhibits potent cancer cell cytotoxicity and appears to exert its cytotoxic effects through inhibition of the 20S proteasome.™Salinospora∫ strain CNB-392 was isolated from a heattreated marine sediment sample that was plated on a seawater-based agar nutrient medium. Liquid shake flask cultivation of this strain, followed by solid-phase extraction with Amberlite resin (XAD-16) and elution with acetone, resulted in a crude extract that was highly cytotoxic in vitro toward HCT-116 human colon carcinoma (IC 50 ca. 80 ng mL À1 ). Cytotoxicity-guided fractionation of the crude extract led to the isolation of salinosporamide A (1) as a colorless crystalline solid (yield: 7 mg L À1 ). The complete structural assignment of 1 was accomplished by spectral analysis and by a single-crystal X-ray diffraction study.Analysis of the low-resolution mass spectrum of salinosporamide A showed a characteristic [Mþ2] þ peak indicative of the presence of a chlorine atom. High-resolution massspectral analysis provided the molecular formula Comprehensive analysis of 2D NMR data, including the results of COSY, HMQC, and HMBC experiments, enabled the complete planar structure of salinosporamide A to be assigned, as in 1, to a 2-aza-6-oxabicyclo[3.2.0]heptane-3...

[structure: see text] Analysis of the fermentation broth of a strain of the marine actinomycete Salinispora tropica has led to the isolation of two unprecedented macrolides, sporolides A (1) and B (2). The structures and absolute stereochemistries of both metabolites were elucidated using a combination of NMR spectroscopy and X-ray crystallography.

An extensive study of the secondary metabolites produced by the obligate marine actinomycete Salinispora tropica (strain CNB-392), the producing microbe of the potent proteasome inhibitor salinosporamide A (1), has led to the isolation of seven related gamma-lactams. The most important of these compounds were salinosporamide B (3), which is the deschloro-analogue of 1, and salinosporamide C (4), which is a decarboxylated pyrrole analogue. New SAR data for all eight compounds, derived from extensive testing against the human colon carcinoma HCT-116 and the 60-cell-line panel at the NCI, indicate that the chloroethyl moiety plays a major role in the enhanced activity of 1.

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.

Geldanamycin, a polyketide natural product, is of significant interest for development of new anticancer drugs that target the protein chaperone Hsp90. While the chemically reactive groups of geldanamycin have been exploited to make a number of synthetic analogs, including 17-allylamino-17-demethoxy geldanamycin (17-AAG), currently in clinical evaluation, the "inert" groups of the molecule remain unexplored for structure-activity relationships. We have used genetic engineering of the geldanamycin polyketide synthase (GdmPKS) gene cluster in Streptomyces hygroscopicus to modify geldanamycin at such positions. Substitutions of acyltransferase domains were made in six of the seven GdmPKS modules. Four of these led to production of 2-desmethyl, 6-desmethoxy, 8-desmethyl, and 14-desmethyl derivatives, including one analog with a four-fold enhanced affinity for Hsp90. The genetic tools developed for geldanamycin gene manipulation will be useful for engineering additional analogs that aid the development of this chemotherapeutic agent.

Complete 1 H and 13 C spectral assignments are reported for lupeol (1a) and two derivatives where the C-30 methyl group is replaced by CH 2 OH (1b) and HC O (1c). Compound 1c shows conformationally dependent substituent effects on 1 H chemical shifts. It also shows line broadening of some 13 C signals at 25°C, suggesting hindered rotation of the side-chain group. This is confirmed by low-temperature spectra which show splitting of broadened peaks into pairs in a ca 2 : 1 area ratio. The free energy of activation of hindered rotation is estimated as 13.5 kcal mol 1 . By contrast, 1a shows no evidence of hindered rotation down to 40°C although NOE data suggest the presence of two conformers. Spartan molecular mechanics calculations confirm the presence of two stable conformers for 1a and 1c but overestimate the rotational barrier in 1a. The additional barrier in 1c probably reflects loss of conjugative stabilization during rotation. since these had been determined for much higher sample concentrations and it was anticipated that there would be some dilution shifts. This was considered important since we wished to have chemical shifts measured under comparable conditions to probe the effect of changes in the side-chain group on remote chemical shifts.While carrying out spectral assignments, we noted that the 13 C spectrum of 1c, but not for 1a and 1b, showed clear evidence for some type of slow conformational process involving the side-chain group. Therefore, we decided also to investigate the origins of the phenomenon.