[7.7]Paracyclophane produzierende Cyanobakterien als Quelle biologisch aktiver Naturstoffe

Cyanobakterien sind eine vielversprechende Quelle an strukturell diversen und biologisch hochaktiven Naturstoffen für die Entwicklung neuer Wirkstoffe. Bislang konnte die Strukturklasse der [7.7]Paracyclophane nur in fädigen Cyanobakterien der Gattungen Nostoc und Cylindrospermum nachgewiesen werden. Vorangegangene Arbeiten zeigten, dass gerade die Carbamidocyclophane chemisch und biologisch interessante Verbindungen darstellen. Im Rahmen dieser Arbeit wurden vor allem die Carbamidocyclophane produzierenden Cyanobakterien Nostoc sp. CAVN2 und Nostoc sp. CAVN10 unter besonderer Berücksichtigung der strukturellen Vielfalt an biosynthetisierten Metaboliten sowie deren antimikrobieller Aktivität umfassend charakterisiert. Um das biosynthetische Potenzial auf der metabolischen Ebene zu untersuchen, wurde im Vorfeld eine spezifische [7.7]Paracyclophan-Analytik etabliert, die skalierbare Methoden für alle Aufarbeitungsschritte beinhaltet. Die Optimierung endete in einem validierten sowie arbeits- und zeitsparenden einstufigen Extraktions- und Aufreinigungsverfahren mittels eines Zweiphasensystems und anschließender LC-UV-Analyse, um biologische Proben reproduzierbar zu analysieren und enthaltene Carbamidocyclophane zu quantifizieren. Kultivierungsstudien zum Einfluss der Temperatur an metabolisch aktiven und defizienten Nostoc-sp.-CAVN10-Kulturen ergaben einen direkten Zusammenhang zwischen der Biomassezunahme und der Temperaturerhöhung. Im Gegensatz dazu zeigten die einzelnen Carbamidocyclophan-Gehalte ein eher differenzierteres Bild über die verschiedenen Wachstumsphasen und Temperaturen hinweg. Da nur eine geringe Korrelation zwischen der spezifischen Wachstumsrate und der spezifischen Carbamidocyclophan-Produktionsrate ermittelt werden konnte, ist eine Relevanz dieser Verbindungen für den primären Zellstoffwechsel nicht ersichtlich. Bei Kultivierungsexperimenten an Nostoc sp. CAVN2 hatte der Zusatz von Chlorid- oder Bromid-Ionen eine drastische Erhöhung der Basalrate und Strukturdiversität der [7.7]Paracyclophane zur Folge. Das gleichzeitige Vorhandensein beider Halogenide im Medium zeigte kompetitive Effekte, wobei Chlorid als Substrat für den Halogenierungsprozess favorisiert wurde. Mit Hilfe eigens entwickelter Kultivierungsprozedere und Separierungsstrategien konnten insgesamt 25 Verbindungen aus Stamm CAVN2 isoliert und strukturell aufgeklärt werden. Dabei bilden die Carbamidocyclophane H–U neue chlorierte, bromierte und nicht halogenierte Naturstoffe. Zusätzlich konnten aus Stamm Cylindrospermum stagnale PCC 7417 neben den bekannten Cylindrocyclophanen A, B und D die drei neuen Cylindrofridine A–C erhalten werden. Diese stellen den Cylindrocyclophanen strukturell eng verwandte lineare Mono- und Dialkylresorcinole dar. Die vergleichende Evaluierung der Bioaktivität von 30 Reinsubstanzen ergab, dass viele Verbindungen sehr starke antimikrobielle Aktivität gegen grampositive Bakterien zeigen – besonders gegen Antibiotika-resistente Kokken mit minimalen Hemmkonzentrationen von oftmals deutlich unter 1 µM. Dabei bedingten die verschiedenen Substituenten (Carbamoyl- und Acetoxy-Reste sowie Hydroxygruppen oder Halogene) z.T. signifikante Aktivitätsunterschiede. Die Zytotoxizität der [7.7]Paracyclophane ist vor allem an das Vorhandensein des Makrozyklus gebunden, da lineare Derivate (Cylindrofridine B/C) kaum biologisch aktiv waren. Eine Ausnahme stellt dabei das nicht zytotoxische, aber antimikrobiell aktive Cylindrocyclophan-D-Monomer Cylindrofridin A dar. Die phylogenetische Analyse der 16S-rDNA-Daten bestätigte die morphologisch-taxonomische Identifizierung der Stämme CAVN2 und CAVN10 als Cyanobakterien der Gattung Nostoc und ergab weiterhin, dass alle Carbamido- und Cylindrocyclophane produzierenden Nostoc-Stämme Bestandteil einer monophyletischen Gruppe sind, die phylogenetisch distinkt zu anderen [7.7]Paracyclophan-Produzenten ist. Des Weiteren konnten keine Nukleotidunterschiede zwischen Stamm CAVN2 und CAVN10 auf den untersuchten Markergen-Sequenzen festgestellt werden, was beide auf der phylogenetischen Ebene als identisch erscheinen lässt und sie somit nur metabolisch aufgrund der strukturellen Diversität und Quantität an [7.7]Paracyclophanen differenzierbar sind. Mit Hilfe von molekulargenetischen Analyseverfahren und bioinformatorischer Auswertung konnte in Stamm CAVN2 das Carbamidocyclophan-Biosynthesegencluster mit einer Gesamtgröße von ca. 26,9 kbp identifiziert werden. Dieses beinhaltet 13 offene Leserahmen (cabA-cabM), wobei das Gen cabL für eine putative Carbamoyltransferase codiert. Ein neuer Halogenase-Typ in Verbindung mit einer Tandem-ACP-Domänen-Struktur in der Typ I Polyketidsynthase CabD könnte für die Ausbildung halogenierter Derivate verantwortlich sein. Der Nachweis eines codierenden Bereichs mit Rieske-Domäne (cabM) lässt eine direkte oxidative intermolekulare Makrozyklisierung bei der Assemblierung vermuten.Cyanobacteria are a promising source of structurally diverse and highly biologically active natural products for the development of new drug substances. The structural class of [7.7]paracyclophanes has so far only been found in filamentous cyanobacteria of the genera Nostoc and Cylindrospermum. Previous work showed that the carbamidocyclophanes are chemically and biologically interesting compounds. Within the scope of this thesis, especially the carbamidocyclophane-producing cyanobacteria Nostoc sp. CAVN2 and Nostoc sp. CAVN10 have been investigated extensively with particular reference to the structural diversity of biosynthesized metabolites as well as their antimicrobial activity. In order to study the biosynthetic potential at the metabolic level, a specific [7.7]paracyclophane analysis has been established in advance, which includes scalable methods for all processing steps. The optimization procedure culminated in a validated as well as work- and time-saving single-stage extraction and purification process using a two-phase system and subsequent LC-UV analysis for both the reproducible analysis of biological samples and the quantification of carbamidocyclophanes. Cultivation studies on the influence of temperature on metabolically active and deficient Nostoc-sp.-CAVN10 cultures showed a direct correlation between biomass growth and temperature increase. In contrast, the individual carbamidocyclophane contents showed a more differentiated outcome over the various growth phases and temperatures. Since only a small correlation between specific growth rate and specific carbamidocyclophane production rate could be determined, a biological relevance of these compounds for the primary metabolism is not apparent. Cultivation experiments on Nostoc sp. CAVN2 revealed that the addition of chloride or bromide ions caused a drastic increase in the basal rate and structural diversity of [7.7]paracyclophanes. The simultaneous presence of both halides in the medium showed competitive effects, with chloride being favored as a substrate for the halogenation process. By means of specially developed cultivation procedures and separation strategies, a total of 25 compounds could be isolated from strain CAVN2 and structurally elucidated. Among these derivatives, the carbamidocyclophanes H-U represent new chlorinated, brominated and non-halogenated natural products. In addition to the known cylindrocyclophanes A, B and D, the three novel cylindrofridins A–C could be obtained from strain Cylindrospermum stagnale PCC 7417. These compounds are cylindrocyclophane-related linear mono- and dialkylresorcinols. A comparative evaluation of the bioactivity of 30 pure substances revealed that many compounds exhibit very strong antimicrobial activity against Gram-positive bacteria – especially against antibiotic-resistant cocci with minimal inhibitory concentrations of often well below 1 µM. In this context, the various substituents (carbamate and acetoxy moieties, hydroxy groups or halogen atoms) attached to the [7.7]paracyclophane carbon skeleton partly caused significant differences in activity. The cytotoxicity of [7.7]paracyclophanes is mainly due to the presence of the macrocycle since linear derivatives (cylindrofridine B/C) were hardly biologically active. An outstanding exception is the non-cytotoxic but antimicrobially active cylindrocyclophane D monomer cylindrofridin A. The phylogenetic analysis of the 16S rDNA data confirmed the morphological and taxonomic identification of strains CAVN2 and CAVN10 as cyanobacteria of the genus Nostoc, and further revealed that all carbamidocyclophane- and cylindrocyclophane-producing Nostoc strains are part of a monophyletic group, which is phylogenetically distinct from other [7.7]paracyclophane producers. Furthermore, no nucleotide differences between strain CAVN2 and strain CAVN10 on the investigated marker gene sequences could be determined. Thus, both strains appear to be identical on the phylogenetic level and can be differentiated only metabolically due to the structural diversity and quantity of containing [7.7]paracyclophanes. Using molecular genetic methods and bioinformatics, the carbamidocyclophane biosynthetic gene cluster could be identified in strain CAVN2. It has a total size of approx. 26.9 kpb and comprises 13 open reading frames (cabA-cabM), whereby the gene cabL is encoding for a putative carbamoyltransferase. A novel type of halogenase coupled with a tandem ACP domain structure in the type I polyketide synthase CabD might be responsible for the formation of halogenated derivatives. Furthermore, the detection of a coding region with a Rieske domain (cabM) suggests a direct oxidative intermolecular macrocyclization during carbamidocyclophane assembly

Balticidins A–D, Antifungal Hassallidin-Like Lipopeptides from the Baltic Sea Cyanobacterium Anabaena cylindrica Bio33

Balticidins A–D (<b>1</b>–<b>4</b>), four new antifungal lipopeptides, were isolated from the laboratory-cultivated cyanobacterium Anabaena cylindrica strain Bio33 isolated from a water sample collected from the Baltic Sea, Rügen Island, Germany. Fractionation of the 50% aqueous MeOH extract was performed by bioassay-guided silica gel column chromatography followed by SPE and repeated reversed-phase HPLC. The main fraction containing the compounds exhibited a strong and specific antifungal activity with inhibition zones in an agar-diffusion assay from 21 to 32 mm against Candida albicans, Candida krusei, Candida maltosa, Aspergillus fumigatus, Microsporum gypseum, Mucor sp., and Microsporum canis. The structures were elucidated by multidimensional ¹H and ¹³C NMR spectroscopy, HRESIMS, amino acid analysis, and sugar analysis. Spectroscopic data analysis afforded an unambiguous sequence of R.CHO­(S1).CHOH.CONH-Thr(1)-Thr(2)-Thr(3)-HOTyr(4)-Dhb(5)-d-Gln­(6)-Gly­(7)-NMeThr­(8)­(S2)-l-Gln <i>C</i>OOH­(9), in which Dhb is dehydro­aminobutyric acid, S1 is d­(−)-arabinose-(3-1)-d-(+)-galacturonic acid, S2 is d-(+)-mannose, and R is the aliphatic residue -C₁₃H₂₆Cl or -C₁₃H₂₇. Besides NMeThr, d-<i>allo</i>-Thr, d-Thr, and l-Thr were identified, but the position of the enantiomers in the sequence is not clear. The four balticidins differ in their cyclic (<b>2</b>, <b>4</b>)/linear (<b>1</b>, <b>3</b>) core and the presence (<b>1</b>, <b>2</b>)/absence (<b>3</b>, <b>4</b>) of chlorine in the aliphatic unit

A new strategy for aromatic ring alkylation in cylindrocyclophane biosynthesis

Alkylation of aromatic rings with alkyl halides is an important transformation in organic synthesis, yet an enzymatic equivalent is unknown. Here, we report that cylindrocyclophane biosynthesis in Cylindrospermum licheniforme ATCC 29412 involves chlorination of an unactivated carbon center by a novel halogenase, followed by a previously uncharacterized enzymatic dimerization reaction featuring sequential, stereospecific alkylations of resorcinol aromatic rings. Discovery of the enzymatic machinery underlying this unique biosynthetic carbon–carbon bond formation has implications for biocatalysis and metabolic engineering

Effects of Halide Ions on the Carbamidocyclophane Biosynthesis in Nostoc sp. CAVN2

In this study, the influence of halide ions on [7.7]paracyclophane biosynthesis in the cyanobacterium Nostoc sp. CAVN2 was investigated. In contrast to KI and KF, supplementation of the culture medium with KCl or KBr resulted not only in an increase of growth but also in an up-regulation of carbamidocyclophane production. LC-MS analysis indicated the presence of chlorinated, brominated, but also non-halogenated derivatives. In addition to 22 known cylindrocyclophanes and carbamidocyclophanes, 27 putative congeners have been detected. Nine compounds, carbamidocyclophanes M−U, were isolated, and their structural elucidation by 1D and 2D NMR experiments in combination with HRMS and ECD analysis revealed that they are brominated analogues of chlorinated carbamidocyclophanes. Quantification of the carbamidocyclophanes showed that chloride is the preferably utilized halide, but incorporation is reduced in the presence of bromide. Evaluation of the antibacterial activity of 30 [7.7]paracyclophanes and related derivatives against selected pathogenic Gram-positive and Gram-negative bacteria exhibited remarkable effects especially against methicillin- and vancomycin-resistant staphylococci and Mycobacterium tuberculosis. For deeper insights into the mechanisms of biosynthesis, the carbamidocyclophane biosynthetic gene cluster in Nostoc sp. CAVN2 was studied. The gene putatively coding for the carbamoyltransferase has been identified. Based on bioinformatic analyses, a possible biosynthetic assembly is discussed

Cylindrofridins A–C, Linear Cylindrocyclophane-Related Alkylresorcinols from the Cyanobacterium <i>Cylindrospermum stagnale</i>

A rapid and exhaustive one-step biomass extraction as well as an enrichment and cleanup procedure has been developed for HPLC-UV detection and quantification of closely related [7.7]­paracyclophanes and structural derivatives based on a two-phase solvent system. The procedure has been validated using the biomass of the carbamidocyclophane- and cylindrocyclophane-producing cyanobacterium <i>Nostoc</i> sp. CAVN2 and was utilized to perform a screening comprising 102 cyanobacterial strains. As a result, three new cylindrocyclophane-related alkylresorcinols, cylindrofridins A–C (<b>1</b>–<b>3</b>), and known cylindrocyclophanes (<b>4</b>–<b>6</b>) were detected and isolated from <i>Cylindrospermum stagnale</i> PCC 7417. Structures of <b>1</b>–<b>3</b> were elucidated by a combination of 1D and 2D NMR experiments, HRMS, and ECD spectroscopy. Cylindrofridin A (<b>1</b>) is the first naturally occurring [7.7]­paracyclophane-related monomeric derivative. In contrast, cylindrofridins B (<b>2</b>) and C (<b>3</b>) represent dimers related to <b>1</b>. Due to chlorination at the alkyl carbon atom in <b>1</b>–<b>3</b>, the site of [7.7]­paracyclophane macrocycle formation, the cylindrofridins represent linearized congeners of the cylindrocyclophanes. Compounds <b>1</b>–<b>3</b> were not toxic against nontumorigenic HaCaT cells (IC₅₀ values >25 μM) compared to the respective cylindrocyclophanes, but <b>1</b> was the only cylindrofridin showing moderate activity against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) and <i>Streptococcus pneumoniae</i> with MIC values of 9 and 17 μM, respectively