Note pubs.acs.org/jnp
Ralfuranone Is Produced by an Alternative Aryl-Substituted γ‑Lactone Biosynthetic Route in Ralstonia solanacearum Julia Pauly,† Markus Nett,‡ and Dirk Hoffmeister*,† †
Department of Pharmaceutical Microbiology at the Hans-Knöll-Institute, Friedrich-Schiller-Universität, Beutenbergstrasse 11a, 07745 Jena, Germany ‡ Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Beutenbergstrasse 11a, 07745 Jena, Germany S Supporting Information *
ABSTRACT: The aryl-substituted γ-lactones ralfuranones A and B were isolated after feeding L-[1-13C]-phenylalanine to a liquid culture of the plant pathogenic bacterium Ralstonia solanacearum. 13C NMR analysis demonstrated specific enrichment of the label at position 2 of the γ-lactone. This labeling pattern is consistent with a biosynthetic mechanism that includes direct cyclization of two monomeric phenylpyruvate precursors into an α,β-substituted lactone, but incompatible with a terphenylquinone intermediate. As the latter was shown as an intermediate in allantofuranone biosynthesis, we conclude that aryl-substituted γ-lactones can be assembled via divergent biosynthetic routes.
I
carboxylic acid function would result in a hemiacetal, which upon elimination of benzaldehyde, decarboxylation, and reductive steps could be converted into ralfuranone A. The resulting mixture of isotopomers would be distinguished by the occurrence of three isotopically enriched carbon atoms. In route B (Scheme 1), the linkage of the two phenylpyruvate building blocks begins with an aldol condensation, followed by an intramolecular lactonization. Hydrolysis then gives the intermediate ralfuranone I, which is subject to a decarboxylation reaction. Ralfuranone B, which carries only a single carbon label, is obtained after addition of water onto the exocyclic double bond. Eventually, benzaldehyde would be eliminated via a retro-aldol cleavage to yield the final product, ralfuranone A. To investigate the allantofuranone biosynthetic principle for the ralfuranones (i.e., via a terphenylquinone intermediate), a stable-isotope labeling experiment was carried out by feeding L[1-13C]-phenylalanine to R. solanacearum cultures. After 1 day of incubation, the L-[1-13C]-phenylalanine-fed cultures were extracted. The rate of 13C incorporation was analyzed by LCHRESIMS using the crude extract and following the method by Bode et al.12 The detected masses for ralfuranones A and B were in accordance with the enrichment of a single carbon atom (Figure 1). These compounds were then purified to homogeneity via HPLC, and the 13C NMR spectrum recorded were identical to published data.4,5 However, the spectrum showed one enriched signal of a quaternary carbon at 175.6 and 176.7 ppm, respectively, which corresponds to C-2 of both molecules (Supporting Information). The result of only one single 13C label is consistent with route B, which does not involve a quinone intermediate.
n recent years, the secondary metabolism of the destructive plant pathogen Ralstonia solanacearum has been the subject of genetic and chemical investigations, which has led to the discovery of various natural products,1−3 among them the ralfuranones A, B, and I (Chart 1).4−6 Aryl-substituted γlactones represent a diverse family of naturally occurring bioactive and ecologically relevant compounds. Representatives include butyrolactone I7 and allantofuranone,8 the pulvinic acids (e.g., variegatic acid),9 and the gymnoascolides (Chart 1).10 Biosynthetically, these compounds originate from phenylpyruvate or p-hydroxyphenylpyruvate.5,7−9 Incorporation studies with isotopically labeled precursors demonstrated that allantofuranone assembly proceeds through the terphenylquinone polyporic acid.8 A rapid tautomerization of this symmetric intermediate was postulated to account for the randomized labeling pattern after feeding L-[1-13C]-phenylalanine, while an oxidative ring contraction gives rise to the γ-lactone scaffold of allantofuranone.8 In the case of the structurally closely related xenofuranones A and B, reverse labeling experiments suggested a mechanistically distinct biosynthesis that does not involve a terphenylquinone intermediate.11 Although all ralfuranone carbons originate from L-phenylalanine (via phenylpyruvate),5 no final evidence exists as to the biosynthetic route. Two different biosynthetic routes discernible through 13C-labeling experiments are conceivable, both of which are initiated by oxidative deamination of phenylalanine to phenylpyruvate by the transaminase RalD (Scheme 1).5 Route A parallels allantofuranone biosynthesis up to polyporic acid formation and its oxidative ring cleavage. Consistent with the precedent, tautomerization of the intermediate randomizes the labeling pattern at this point. The following steps, however, deviate from the known allantofuranone pathway,8 thereby creating the unique substitution pattern of ralfuranones. An intramolecular lactonization reaction mediated by a vinylogous © 2014 American Chemical Society and American Society of Pharmacognosy
Received: March 23, 2014 Published: July 17, 2014 1967
dx.doi.org/10.1021/np500263r | J. Nat. Prod. 2014, 77, 1967−1971
Journal of Natural Products
Note
Chart 1
quinone or γ-lactone synthetase, i.e., NRPS-like biosynthetic enzymes that share an identical domain layout. This Note presents evidence for the RalA-dependent biosynthetic route from L-phenylalanine via ralfuranones I and B to the ultimate product ralfuranone A. We previously showed that ralfuranone I forms adducts with nucleophilic thiols, including glutathione,6 and interference with host plant signaling may be one of its biological functions. Due to its reactivity, we hypothesize that ralfuranone I represents the biologically relevant product, which may also exert other bioactivities. Therefore, we tested it for antimicrobial properties; Bacillus subtilis, Staphylococcus aureus, and S. aureus (MRSA) minimum inhibitory concentrations of 1.56 μg/mL (5.3 μM) were found, while Mycobacterium vaccae and Enterococcus faecalis (VRE) showed MICs of 3.12 and 6.25 μg/mL (10.6 and 21.2 μM), respectively. Germination of Penicillium notatum conidia was inhibited at 12.5 μg/mL (42.4 μM). In contrast, ralfuranone B did not show antimicrobial activity against the above species at concentrations of
