Elansolid A3, a Unique p‐Quinone Methide Antibiotic from Chitinophaga sancti

Abstract

The genus Chitinophaga (Flexibacter) [1] is well known as a producer of peptides with antibacterial activity. Only recently, elansolid A was isolated as the first macrolide antibiotic from Chitinophaga sancti (comb. nov. = new combination), strain GBF13. Remarkably, elansolid A occurred as two, stable, separable atropisomers, A1 (1) and A2 (1*). Detailed NMR studies combined with molecular modeling revealed that the atropisomerism is caused by the rigidity of two differently folded macrolide rings with either C7 or C6 "folded-in" the lactone ring. Importantly, the isomers differ in their biological activity. Whereas 1* showed antibiotic activity against Gram-positive bacteria in the range of 0.2-64 mg mL À1 and cytotoxicity against L929 mouse fibroblast cells with a value for IC 50 of 12 mg mL À1, the other atropisomer 1 was clearly less active.[2]Our observation of a metabolite production depedent on the cultivation conditions of Chitinophaga sancti and the unaccounted occurrence of elansolids B1 (3) and B2 (4), which are assumed to be artifacts from treatment with water or methanol, required an in-depth analysis. We now report on the isolation and characterization of two further unique metabolites of which the exceptional, polyketide-based quinone methide antibiotic elansolid A3 (2) constitutes the major fraction (Scheme 1).For the production of 2, C. sancti was cultivated under slightly acidic conditions (pH 5.85) in the presence of Amberlite XAD 16 resin. After 150 h the neutral adsorber resin along with the adhering cell mass was harvested by sieving and then immediately frozen at À30 8C.Rapid HPLC-UV-HRMS analysis of a fresh acetone extract from the XAD resin additionally revealed a more polar peak of 2 at R t 5.4 min. The elemental composition was determined to be C 37 H 48 O 6 , which is identical to 1 (R t 8.7 min) and 1* (R t 7.6 min). However, isomer 2 differed in the UV spectrum with a new band at 324 nm. During attempted workup under mild conditions, the isolation of this new metabolite (2) turned out to be impossible as it readily reacted with water or methanol, thus yielding elansolid variants 3 and 4.[2] Because pH control did not stabilize the new metabolite 2 sufficiently, an unorthodox isolation process had to be developed. Unexpectedly, the final workup procedure had to be performed by carefully avoiding any contact of the fermentation products with water; this was achieved by using nitrogen as inert gas and all chromatographic steps were limited to the use of dry aprotic organic solvents. The freeze-dried Amberlite XAD 16 was extracted with dry acetone and 2 was isolated by silica-gel column chromatography, which finally yielded about 10 mg of 2 per plate by thick-layer chromatography.The NMR spectra were recorded in dry [D 6 ]acetone (see the Supporting Information, Table S1) [3] and clearly showed[a] Dr.

Pinensins: The First Antifungal Lantibiotics

“…[1] Furthermore,a ntibacterial compounds,a swell as inhibitors of mammalian topoisomerase and elastase have been reported for different Flexibacter species. [2][3][4][5] In our screening for bioactive secondary metabolites, strain C. pinensis DSM 28390 (= DSM 2588) exhibited strong antifungal activity.T hrough activity-guided fractionation, the antifungal activity could be assigned to two unknown peptides,p inensins A( 1)a nd B( 2).…”

Molecular Basis of Elansolid Biosynthesis: Evidence for an Unprecedented Quinone Methide Initiated Intramolecular Diels–Alder Cycloaddition/Macrolactonization

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Elansolids A1/A2 (1) [1] and B1-B3 (2-4) and the structurally unusual and highly reactive elansolid A3 (5) [2] are new metabolites from the gliding bacterium Chitinophaga sancti (formerly Flexibacter spec. ; Scheme 1).

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“…While elansolid A2 (1*) shows antibiotic activity against Gram-positive bacteria in the range of 0.2 to 64 mg mL À1 and cytotoxicity against L929 mouse fibroblast cells with an IC 50 value of 12 mg mL À1 , the atropisomer elansolid A1 (1) is significantly less active. [2,3] The elansolids feature a bicyclo-[4.3.0]nonane core which in the case of elansolids A1/A2 is part of a 19-membered macrolactone. Elansolid B1 is the corresponding seco acid of elansolids A1/A2, while the elansolids B2 and B3 are workup artifacts that result from nucleophilic addition of methanol and NH 3 , respectively, to…”

Molekulare Grundlage für die Biosynthese von Elansolid: Beweise für eine einzigartige, durch ein Chinonmethid initiierte intramolekulare Diels‐Alder‐Cycloaddition/Makrolactonisierung

“…Die Elansolide A1/A2 (1/1*), [1] B1-B3 (2-4) und das strukturell ungewöhnliche und hochreaktive Elansolid A3 (5; Schema 1) [2] sind Metaboliten des gleitenden Bakteriums Chitinophaga sancti (ehemals Flexibacter spec.). Während Elansolid A2 (1*) antibiotische Aktivität gegenüber Gram-positiven Bakterien im Bereich von 0.2 bis 64 mg mL À1 und auch cytotoxische Eigenschaften gegenüber L929-Mausfibroblasten mit einem IC 50 -Wert von 12 mg mL À1 zeigt, ist das Atropisomer Elansolid A1 (1) deutlich weniger aktiv.…”

“…Während Elansolid A2 (1*) antibiotische Aktivität gegenüber Gram-positiven Bakterien im Bereich von 0.2 bis 64 mg mL À1 und auch cytotoxische Eigenschaften gegenüber L929-Mausfibroblasten mit einem IC 50 -Wert von 12 mg mL À1 zeigt, ist das Atropisomer Elansolid A1 (1) deutlich weniger aktiv. [2,3] Die Elansolide enthalten einen Bicyclo [4.3.0]nonan-Kern, der im Fall der Elansolide A1/A2 Teil eines 19-gliedrigen Makrolactonrings ist. Elansolid B1 ist die seco-Säure der Elansolide A1/A2.…”