Three diterpene synthases from actinomycetes have been studied. The first enzyme from Streptomyces cattleya produced the novel compound cattleyene. The other two enzymes from Nocardia testacea and Nocardia rhamnosiphila were identified as phomopsene synthases. The cyclisation mechanism of cattleyene synthase and the EIMS fragmentation mechanism of its product were extensively studied by incubation experiments with isotopically labelled precursors. Oxidative transformations expanded the chemical space of these unique diterpenes.
In this work, the synthesis, structural and photophysical characterization of six phosphorescent H 2 O-soluble Pt(II) complexes are reported while addressing their emission maxima, photoluminescence quantum yields (Φ L ), lifetimes (τ), aggregation tendency, and microenvironment sensitivity as a function of the substitution pattern on the main tridentate luminophore. Different ancillary ligands, namely, a trisulfonated phosphane and maltohexaose-conjugated pyridines (with or without amide bridges), were introduced and evaluated for the realization of switch-on-photoluminescent labels reporting on the microenvironment sensed in biofilms of Gram + and Gram − models, namely, Staphylococcus aureus and Escherichia coli. With the aid of confocal luminescence micro(spectro)scopy, we observed that selected complexes specifically interact with the biofilms while leaving planktonic cells unlabeled. By using photoluminescence lifetime imaging microscopy, excited-state lifetimes within S. aureus biofilms were measured. The photoluminescence intensities were drastically boosted, and the excited state lifetimes were significantly prolonged upon binding to the viscous biofilm matrix, mainly due to the suppression of radiationless deactivation pathways upon shielding from physical quenching processes, such as interactions with solvent molecules and 3 O 2 . The best performances were attained for non-aggregating complexes with maltohexaose targeting units and without amide bridges. Notably, in the absence of the maltodextrin, a hydrophobic adamantyl moiety suffices to attain a sizeable labeling capacity. Moreover, photoluminescence studies showed that selected complexes can also effectively interact with E. coli biofilms, where the bacterial cells are able to partially uptake the maltodextrin-based agents. In summary, the herein introduced concepts enable the development of specific biofilm reporters providing spatial resolution as well as lifetime-and spectrum-based readouts. Considering that most theragnostic agents reported so far mainly address metabolically active bacteria at the surface of biofilms but without reaching cells deeply immersed in the matrix, a new platform with a clear structure-property correlation is provided for the early detection of such bacterial arrays.
The absolute configuration of fusaterpenol (GJ1012E) has been revised by an enantioselective deuteration strategy. A bifunctional enzyme with a terpene synthase and a prenyltransferase domain from Aspergillus brasiliensis was characterised as variediene synthase, and the absolute configuration of its product was elucidated. The uniform absolute configurations of these and structurally related di‐ and sesterterpenes together with a common stereochemical course for the geminal methyl groups of GGPP unravel a similar conformational fold of the substrate in the active sites of the terpene synthases. For variediene, a thermal reaction observed during GC/MS analysis was studied in detail for which a surprising mechanism was uncovered.
Drei Diterpensynthasen aus Actinomyceten wurden studiert. Das erste Enzym aus Streptomyces cattleya produzierte die neue Verbindung Cattleyen. Zwei weitere Enzyme aus Nocardia testacea und Nocardia rhamnosiphila wurden als Phomopsensynthasen identifiziert. Der Cyclisierungsmechanismus der Cattleyensynthase und der EI-MS-Fragmentierungsmechanismus ihres Produktes wurden extensiv durch Inkubationsexperimente mit isotopenmarkierten Vorläufern studiert. Oxidative Transformationen erweiterten den chemi-schenStrukturraum dieser einzigartigen Diterpene. Die kationischen Cyclisierungskaskaden zu Te rpenen gehçren zu den komplexesten biosynthetischen Reaktionen in der Natur,d ie durch ein einziges Enzym realisiert werden. [1] Faszinierenderweise sind nur wenige lineare Vorläufer, die Oligoprenyldiphosphate,a usreichend, um eine große Diversitätp olycyclischer Skelette mit multiplen Stereozentren zu erzeugen. Fürd iese Tr ansformationen sind verschiedene Enzymklassen evolviert, einschließlich der Te rpensynthasen (TS) des Ty ps I, die das Substrat durch Abstraktion von Diphosphat (PP) ionisieren. Eine wachsende Anzahl von Enzymen der Klasse II ist bekannt, die die Bildung eines Intermediates fürn achfolgende Umsetzung durch ein Enzym der Klasse Ikatalysieren. Diese Funktionen werden manchmal in bifunktionalen Enzymen wie der ent-Kaurensynthase aus Fusarium fujikuroi oder der Abietadiensynthase aus Abies grandis kombiniert. [2] Eine dritte Klasse von Enzymen mit TS-Aktivitätist zu der Prenyltransferase UbiA verwandt, die an der Ubichinonbiosynthese beteiligt ist und ebenfalls durch PP-Abstraktion agiert. [3] Jüngst wurde in dem Pilz Acremonium strictum ein weiterer TS-Typ (AsR6) fürH umulen identifiziert. [4] Nach der Entdeckung der Fusicoccadiensynthase aus Phomopsis amygdali [5] wurden einige weitere bifunktionale Diterpensynthasen (DTS) mit einer Geranylgeranyl-PP-Synthase-(GGPPS) und einer TS-Domäne aus Pilzen identifiziert, die eine initiale 1,11-und 10,14-Cyclisierung katalysieren. [6] VorK urzem wurde über eine Reihe von bakteriellen monofunktionalen DTS mit demselben initialen Cyclisierungsmodus berichtet, [7] mit der Synthase fürC yclooctat-9-en-7-ol (CotB2) aus Streptomyces melanosporofa-
A full understanding concerning the photophysical properties of a fluorescent label is crucial for a reliable and predictable performance in biolabelling applications. This holds true not only for the choice of a fluorophore in general, but also for the correct interpretation of data, considering the complexity of biological environments. In the frame of a case study involving inflammation imaging, we report the photophysical characterization of four fluorescent S100A9-targeting compounds in terms of UV–vis absorption and photoluminescence spectroscopy, fluorescence quantum yields (ΦF) and excited state lifetimes (τ) as well as the evaluation of the radiative and non-radiative rate constants (kr and knr, respectively). The probes were synthesized based on a 2-amino benzimidazole-based lead structure in combination with commercially available dyes, covering a broad color range from green (6-FAM) over orange (BODIPY-TMR) to red (BODIPY-TR) and near-infrared (Cy5.5) emission. The effect of conjugation with the targeting structure was addressed by comparison of the probes with their corresponding dye-azide precursors. Additionally, the 6-FAM and Cy5.5 probes were measured in the presence of murine S100A9 to determine whether protein binding influences their photophysical properties. An interesting rise in ΦF upon binding of 6-FAM-SST177 to murine S100A9 enabled the determination of its dissociation equilibrium constant, reaching up to KD = 324 nM. This result gives an outlook for potential applications of our compounds in S100A9 inflammation imaging and fluorescence assay developments. With respect to the other dyes, this study demonstrates how diverse microenvironmental factors can severely impair their performance while rendering them poor performers in biological media, showing that a preliminary photophysical screening is key to assess the suitability of a particular luminophore.
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