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Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich. This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively. Covering: 1988 to 2012 This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

β-N-methylamino-L-alanine (BMAA) is an excitotoxic neurotoxin that has been implicated in the etiology of Amyotrophic Lateral Sclerosis-Parkinsonism-Dementia Complex in the South Pacific island of Guam. BMAA has been reported to be present in a wide range of cyanobacteria taxa and aquatic invertebrates. However, there are large uncertainties in the human health risks associated with BMAA exposure because of the analytical challenges associated with the detection and quantification of BMAA. Here we describe an improved liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for the analysis of BMAA in aquatic invertebrate sample matrices, and present initial findings for oysters and blue crabs collected in the southeastern United States. The LC-MS/MS method is linear over a wide range of BMAA concentrations (0.4−153.2 pg µL−1, r2 = 0.997), and analytically precise, with a relative standard deviation (RSD) of 2.4%. The on-column limit of detection (LoD) and limit of quantification (LoQ) values for this method were determined to be 5 pg (0.2 pg µL−1) and 17 pg (0.8 pg µL−1), respectively. We observed a weak ion enhancement effect of ~10% in a mollusk matrix that tested negative for BMAA. Analyte recovery for the method averaged 92% (8% RSD). BMAA concentrations for oysters and blue crabs ranged from 5 to 47 µg g−1 dry weight sample and are substantially lower than the concentrations reported previously with a nonspecific BMAA method that was based on liquid chromatography-fluorescence detection (LC-FL). We conclude that BMAA analysis via LC-MS/MS methodologies is absolutely necessary for obtaining reliable data.

This paper overviews several examples of important public health impacts by marine microbes and directs readers to the extensive literature germane to these maladies. These examples include three types of dinoflagellates (Gambierdiscusspp.,Karenia brevis, andAlexandrium fundyense), BMAA-producing cyanobacteria, and infectious microbes. The dinoflagellates are responsible for ciguatera fish poisoning, neurotoxic shellfish poisoning, and paralytic shellfish poisoning, respectively, that have plagued coastal populations over time. Research interest on the potential for marine cyanobacteria to contribute BMAA into human food supplies has been derived by BMAA's discovery in cycad seeds and subsequent implication as the putative cause of amyotrophic lateral sclerosis/parkinsonism dementia complex among the Chamorro people of Guam. Recent UPLC/MS analyses indicate that recent reports that BMAA is prolifically distributed among marine cyanobacteria at high concentrations may be due to analyte misidentification in the analytical protocols being applied for BMAA. Common infectious microbes (including enterovirus, norovirus,Salmonella,Campylobacter,Shigella,Staphylococcus aureus,Cryptosporidium, andGiardia) cause gastrointestinal and skin-related illness. These microbes can be introduced from external human and animal sources, or they can be indigenous to the marine environment.

Two homotyrosine-bearing cyanopeptolins are described from Planktothrix agardhii CYA 126/8. The compounds feature a common homotyrosine-containing cyclohexadepsipeptide and differ by sulfation of an exocyclically located 2-O-methyl-d-glyceric acid residue. In addition we describe two anabaenopeptins, which contain two homotyrosine residues, one of which is N-methylated. The anabaenopeptins have a common cyclopentapeptide portion and differ in the amino acid linked to it via an ureido bond, arginine and tyrosine, respectively.

Microcystins (MCs) are toxic heptapeptides found in cyanobacteria and share the common structure cyclo(-d-Ala(1)-l-X(2)-d-isoMeAsp(3)-l-Z(4)-Adda(5)-d-isoGlu(6)-Mdha(7)). The letters X and Z in the general formula above represent a wide range of l-amino acids that occupy positions 2 and 4, respectively. In general the variation in structural variants is due to the exchange of amino acids in position 7, 2, and 4. In the present work we report two homotyrosine (Hty)-containing microcystin variants, [d-Asp(3),(E)-Dhb(7)]-MC-HtyY (1) and [d-Asp(3),(E)-Dhb(7)]-MC-HtyHty (2), which were isolated from strain No80 of Planktothrix rubescens. Their structures were elucidated using amino acid analysis as well as 1D and 2D NMR techniques. The adenylation domains of McyABC involved in amino acid activation in positions 7, 2, and 4 of the microcystin molecule, respectively, were compared with corresponding genes of Planktothrix strain CYA126/8 producing [d-Asp(3),Mdha(7)]-MC-RR and [d-Asp(3),Mdha(7)]-MC-LR. While the adenylation domain comparison of McyAB between the two Planktothrix strains revealed considerable DNA recombination, the adenylation domain of McyC showed only a single amino acid substitution, which was correlated with the replacement of Arg by Hty in position 4 of the microcystin molecule.

The mode of incorporation into nicotinamide of label from13C-labeled samples of D-glucose, in Escherichia coli and Saccharomyces cerevisiae, was determined by means of13C NMR spectroscopy. The results, which confirm and extend early studies with radioactive tracers, permit a definitive choice to be made between alternative biogenetic proposals.Key words: nicotinic acid, nicotinamide, biosynthesis, glucose incorporation,13C NMR.

1-Deoxy-D-xylulose (= 1-deoxy-D-theropentulose) is a precursor of thiamin (Vitamin B1) and of pyridoxine (Vitamin B6) in bacteria. The synthesis of a [2,3-13C2] bond-labeled sample of the compound, to be used for investigations of the biosynthesis of the two vitamins, is described. In aqueous solution 1-deoxy-D-xylulose exists mainly as the open chain ketone. In methanol solution the compound exists as a mixture of the open chain ketone and the two corresponding epimeric furanoses. In acid solution the compound yields a dimeric anhydride, di-β-1-deoxy-D-xylulofuranose 2,3′:3,2′-dianhydride, whose structure was established by X-ray crystallography. Keywords: [2,3-13C2]-1-deoxy-D-xylulose, di-β-1-deoxy-D-xylulofuranose 2,3′:3,2′-dianhydride, 5-O-benzyl-3,4-O-isopropylidene-1-deoxy-D-xylulose, 4-O-benzyl-2,3-O-isoproylidene-D-threose.

Borophycin (1) is the potent cytotoxin in the lipophilic extract of a marine strain of the blue-green alga (cyanobacterium) Nostoc linckia (Roth) Bornet ex Bornet & Flahault (UH isolate GA-5-23). The gross structure of this boron-containing compound was determined by spectral methods and ita relative stereochemistry established by X-ray crystallography. Borophycin is made up of two identical halves with an overall structure reminiscent of the ionophoric antibiotics boromycin (2) and aplasmomycin (3). The biosynthesis of 1 differs from the biosynthesis of 2 and 3. All three compounds are acetate-derived polyketides that utilize a C3 precursor for the starter unit and methionine for the methyl branches on the polyketide chain. Whereas phosphoglycerate or phosphoenolpyruvate has been suggested to be the C3 starter unit in the biosynthesis of 2 and 3, the C3 starter unit for the biosynthesis of 1 is derived from acetate and methionine, but not propionate. In evaluating lipophilic and hydrophilic extracts of laboratory-cultured blue-green algae (cyanobacteria) for in uitro antitumor activity,' the lipophilic extract of a marine strain of Nostoc Zinckia (Roth) Bornet ex Bomet & Flahault (UH isolate GA-5-23)2 was found to display appreciable cytotoxicity against LoVo (MIC 0.066 pg/mL) and KB (MIC 3.3 pg/mL), two human tumor cell lines used routinely in our laboratory for screening.3 In the more meaningful Corbett and Valeriote assays,4 however, the extract was neither solid tumor selective nor tumor selective, suggesting that further investigation of this extract might not lead to a drug that would offer any distinct advantage over drugs already in clinical use for the treatment of human cancer. Nevertheless, this algal extract was among the most LoVo-cytotoxic found in screening extracts of 665 blue-green algae,l and this

[reaction: see text] There are conflicting reports in the literature concerning the absolute sterochemistry at C-3 of the common plant polyacetylene oxylipin (+)-falcarindiol. We have employed olefin cross-metathesis using Grubbs' second generation catalyst and ethylene gas to degrade falcarindiol to the symmetrical 1,9-decadiene-4,6-diyne-3,8-diol. The reaction is completely selective for net removal of the aliphatic side chain. Degradation of (+)-falcarindiol from Tetraplasandra hawaiiensis yields a meso product as shown by chiral HPLC. Hence, (+)-falcarindiol from this source has a (3R,8S)-configuration.

Anabaenopeptins (AP) are bioactive cyclic hexapeptides synthesized nonribosomally in cyanobacteria. APs are characterized by several conserved motifs, including the ureido bond, N-methylation in position 5, and d -Lys in position 2. All other positions of the AP molecule are variable, resulting in numerous structural variants. We have identified a nonribosomal peptide synthetase (NRPS) operon from Planktothrix agardhii strain CYA126/8 consisting of five genes ( apnA to apnE ) encoding six NRPS modules and have confirmed its role in AP synthesis by the generation of a mutant via insertional inactivation of apnC . In order to correlate the genetic diversity among adenylation domains (A domains) with AP structure variation, we sequenced the A domains of all six NRPS modules from seven Planktothrix strains differing in the production of AP congeners. It is remarkable that single strains coproduce APs bearing either of the chemically divergent amino acids Arg and Tyr in exocyclic position 1. Since the A domain of the initiation module (the ApnA A 1 domain) has been proposed to activate the amino acid incorporated into exocyclic position 1, we decided to analyze this domain both biochemically and phylogenetically. Only ApnA A 1 enzymes from strains producing AP molecules containing Arg or Tyr in position 1 were found to activate these two chemically divergent amino acids in vitro . Phylogenetic analysis of apn A domain sequences revealed that strains with a promiscuous ApnA A 1 domain are derived from an ancestor that activates only Arg. Surprisingly, positive selection appears to affect only three codons within the apnA A 1 gene, suggesting that this remarkable promiscuity has evolved from point mutations only.

Mutants of Escherichia coli (pdx B and pdx C) which are blocked in the biosynthesis of pyridoxol (vitamin B6) showed a growth response to 4-hydroxy-l-threonine. This observation constitutes the first direct evidence in support of the view that 4-hydroxy-l-threonine is implicated in the biosynthesis of vitamin B6. 1-Aminopropan-2,3-diol, the decarboxylation product of 4-hydroxy-l-threonine, does not support the growth of these mutants. Deuterium from deuterium-labelled 1-aminopropan-2,3-diol was not incorporated into pyridoxol.

Extracts of a marine Pseudoalteromonas sp. (CMMED 290) isolated from the surface of a nudibranch collected in Kaneohe Bay, Oahu, displayed significant antimicrobial activity against methicillin-resistant Staphylococcus aureus. Bioassay-guided fractionation of the lipophilic extract led to the isolation and structure elucidation of two new highly brominated compounds, 2,3,5,7-tetrabromobenzofuro[3,2-b]pyrrole (1) and 4,4′,6-tribromo-2,2′-biphenol (2). In addition, we have identified the known compounds pentabromopseudilin and bromophene. We describe the isolation and structure elucidation of the compounds 1 and 2 together with their antimicrobial activities against methicillin-resistant Staphylococcus aureus.

The cancer chemopreventive and cytotoxic properties of 50 extracts derived from Twilight Zone (50-150 m) sponges, gorgonians and associated bacteria, together with 15 extracts from shallow water hard corals, as well as 16 fractions derived from the methanol solubles of the Twilight Zone sponge Suberea sp, were assessed in a series of bioassays. These assays included: Induction of quinone reductase (QR), inhibition of TNF-alpha activated nuclear factor kappa B (NFkappaB), inhibition of aromatase, interaction with retinoid X receptor (RXR), inhibition of nitric oxide (NO) synthase, inhibition 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH), and inhibition of HL-60 and MCF-7 cell proliferation. The results of these assays showed that at least 10 extracts and five fractions inhibited NFkappaB by greater than 60%, two extracts and two fractions inhibited DPPH by more than 50%, nine extracts and two fractions affected the survival of HL-60 cells, no extracts or fractions affected RXR, three extracts and six fractions affected quinone reductase (QR), three extracts and 12 fractions significantly inhibited aromatase, four extracts and five fractions inhibited nitric oxide synthase, and one extract and no fractions inhibited the growth of MCF-7 cells by more than 95%. These data revealed the tested samples to have many and varied activities, making them, as shown with the extract of the Suberea species, useful starting points for further fractionation and purification. Moreover, the large number of samples demonstrating activity in only one or sometimes two assays accentuates the potential of the Twilight Zone, as a largely unexplored habitat, for the discovery of selectively bioactive compounds. The overall high hit rate in many of the employed assays is considered to be a significant finding in terms of "normal" hit rates associated with similar samples from shallower depths.

The rim of the tunic of the flatworm Pseudoceros indicus is characterized by blue dots on a white background. The isolation and structure elucidation of the blue pigment is reported. It is shown by extensive analysis of spectroscopic data to be an indolic azafulvene, which has been named pseudocerosine.

In the course of work aimed at the discovery of new pharmaceutical lead compounds from marine bacteria, a lipophilic extract of the bacterium Pseudoalteromonas rubra displayed significant cytotoxicity against SKOV-3, a human ovarian adenocarcinoma cell line. Bioassay-directed fractionation of this extract resulted in the isolation of a series of known and new prodiginine-type azafulvenes. The structure of the major metabolite was elucidated by interpretation of spectroscopic data as a 2-substituted prodigiosin, which we named 2-(p-hydroxybenzyl)prodigiosin (HBPG).

Studies on the biosynthesis of cylindrospermopsin (1), a potent hepatotoxin associated with the cyanobacterium Cylindrospermopsis raciborskii, indicate that 1 is an acetogenin with guanidinoacetic acid serving as the starter unit of the polyketide chain. Feeding experiments show that C14 and C15 of 1 are derived from C1 and C2 of glycine, respectively, and C4 through C13 arise from five contiguous acetate units attached head to tail. The methyl carbon on C13 originates from the C(1) pool. The starter unit, established by the incorporation of [guanidino-(13)C,alpha-(15)N]-guanidinoacetic acid into N16 and C17 of 1, does not appear to be formed from glycine by known amidination pathways. The origin of the NH-CO-NH segment in the uracil ring is also unknown.

Thirty-seven Rhizobium isolates obtained from the nodules of leguminous trees ( Leucaena spp.) were selected on the basis of their ability to catabolize mimosine, a toxin found in large quantities in the seeds, foliage, and roots of plants of the genera Leucaena and Mimosa . A new medium containing mimosine as the sole source of carbon and nitrogen was used for selection. The enzymes of the mimosine catabolic pathway were inducible and were present in the soluble fraction of the cell extract of induced cells. On the basis of a comparison of the growth rates of Rhizobium strains on general carbon and nitrogen sources versus mimosine, the toxin appears to be converted mostly to biomass and carbon dioxide. Most isolates able to grow on mimosine as a source of carbon and nitrogen are also able to utilize 3-hydroxy-4-pyridone, a toxic intermediate of mimosine degradation in other organisms.

The cancer chemopreventive and cytotoxic properties of 50 extracts derived from Twilight Zone (50–150 m) sponges, gorgonians and associated bacteria, together with 15 extracts from shallow water hard corals, as well as 16 fractions derived from the methanol solubles of the Twilight Zone sponge Suberea sp, were assessed in a series of bioassays. These assays included: Induction of quinone reductase (QR), inhibition of TNF-α activated nuclear factor kappa B (NFκB), inhibition of aromatase, interaction with retinoid X receptor (RXR), inhibition of nitric oxide (NO) synthase, inhibition 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH), and inhibition of HL-60 and MCF-7 cell proliferation. The results of these assays showed that at least 10 extracts and five fractions inhibited NFκB by greater than 60%, two extracts and two fractions inhibited DPPH by more than 50%, nine extracts and two fractions affected the survival of HL-60 cells, no extracts or fractions affected RXR, three extracts and six fractions affected quinone reductase (QR), three extracts and 12 fractions significantly inhibited aromatase, four extracts and five fractions inhibited nitric oxide synthase, and one extract and no fractions inhibited the growth of MCF-7 cells by more than 95%. These data revealed the tested samples to have many and varied activities, making them, as shown with the extract of the Suberea species, useful starting points for further fractionation and purification. Moreover, the large number of samples demonstrating activity in only one or sometimes two assays accentuates the potential of the Twilight Zone, as a largely unexplored habitat, for the discovery of selectively bioactive compounds. The overall high hit rate in many of the employed assays is considered to be a significant finding in terms of “normal” hit rates associated with similar samples from shallower depths.

A new alkaloid, nomofungin, has been isolated from the fermentation broth of an unidentified endophytic fungus obtained from the bark of Ficus microcarpa L. The structure of nomofungin was determined by application of spectroscopic methods. The absolute stereochemistry of nomofungin was assigned by using the exciton chirality method. Nomofungin disrupts microfilaments in cultured mammalian cells and is moderately cytotoxic with minimum inhibitory concentrations (MICs) of 2 and 4.5 microg/mL against LoVo and KB cells, respectively. The ring system of nomofungin is unprecedented.

The mode of incorporation of 2H from [3,3-2H2]cadaverine into lupanine (2) and 13α-hydroxylupanine (3) in Lupinusangustifolius was determined by 2H nmr spectroscopy. Lupanine carried deuterium at positions 4α, 4β, 8α, 8β, 13α, and 13β. These six sites were approximately equally enriched. Similarly, 13α-hydroxylupanine carried label approximately equally at five sites, 4α, 4β, 8α, 8β, and 13β. [3,3-2H2]Cadaverine was prepared by borodeuteride reduction in D2O of the p-toluene-sulfonylhydrazone of N-benzoylpiperid-4-one, followed by degradation of the resulting N-benzoyl[4,4-2H2]piperidine into 1,5-dibromo[3,3-2H2]pentane, conversion into 1,5-diphthalimido[3,3-2H2]pentane, and acid hydrolysis.

Summary Aeruginosins represent a group of peptide metabolites isolated from various cyanobacterial genera and from marine sponges that potently inhibit different types of serine proteases. Members of this family are characterized by the presence of a 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety. We have identified and fully sequenced a NRPS gene cluster in the genome of the cyanobacterium Planktothrix agardhii CYA126/8. Insertional mutagenesis of a NRPS component led to the discovery and structural elucidation of two glycopeptides that were designated aeruginoside 126A and aeruginoside 126B. One variant of the aglycone contains a 1-amino-2-( N -amidino-Δ 3 -pyrrolinyl)ethyl moiety at the C terminus, the other bears an agmatine residue. In silico analyses of the aeruginoside biosynthetic genes aer A- aer I as well as additional mutagenesis and feeding studies allowed the prediction of enzymatic steps leading to the formation of aeruginosides and the unusual Choi moiety.

The H–D exchange of 17-oxosparteine (1b) has been studied under homoenolization conditions. We find that 1b undergoes H–D exchange at the bridgehead carbon C7 at 130 °C, but forcing conditions (240 °C) using the medium (CD3)3COK/(CD3)3COD are required to induce exchange at C11, C15, and other sites. C15-Ha exchanges only marginally faster than C15-Hc, indicating that the β-lactam carbanion is stabilized primarily by the dipolar effect of the amide group. Keywords: protium–deuterium exchange, 17-oxosparteine, homoenolization.