Novel anti-inflammatory diketopiperazine alkaloids from the marine-derived fungus Penicillium brasilianum.

Diketopiperazine alkaloids have proven the most abundant heterocyclic alkaloids up to now, which usually process diverse scaffolds and rich biological activities. In our search for bioactive diketopiperazine alkaloids from marine-derived fungi, two novel diketopiperazine alkaloids, penipiperazine A (1) and its biogenetically related new metabolite (2), together with a known analogue neofipiperzine C (3), were obtained from the strain Penicillium brasilianum. Their planar structures and absolute configurations were elucidated by extensive spectroscopic analyses, 13C NMR calculation, Marfey's, ECD, and ORD methods. Compound 1 featured a unique 6/5/6/6/5 indole-pyrazino-pyrazino-pyrrolo system, and its plausible biogenetic pathway was also proposed. Additionally, compounds 1-3 have been tested for their inflammatory activities. 1 and 2 significantly inhibited the release of NO and the expression of related pro-inflammatory cytokines on LPS-stimulated RAW264.7 cells, suggesting they could be attracting candidate for further development as anti-inflammatory agent. KEY POINTS: • A novel diketopiperazine alkaloid featuring a unique 6/5/6/6/5 indole-pyrazino-pyrazino-pyrrolo system was isolated from the marine fungus Penicillium brasilianum. • The structure of 1 was elucidated by detailed analysis of 2D NMR data, 13C NMR calculation, Marfey's, ECD, and ORD methods. • Compounds 1 and 2 significantly inhibited the release of NO and the expression of related pro-inflammatory cytokines on LPS-stimulated RAW264.7 cells.

Secondary metabolites produced by Microbacterium sp. LGMB471 with antifungal activity against the phytopathogen Phyllosticta citricarpa.

The citrus black spot (CBS), caused by Phyllosticta citricarpa, is one of the most important citrus diseases in subtropical regions of Africa, Asia, Oceania, and the Americas, and fruits with CBS lesions are still subject to quarantine regulations in the European Union. Despite the high application of fungicides, the disease remains present in the citrus crops of Central and South America. In order to find alternatives to help control CBS and reduce the use of fungicides, we explored the antifungal potential of endophytic actinomycetes isolated from the Brazilian medicinal plant Vochysia divergens found in the Pantanal biome. Two different culture media and temperatures were selected to identify the most efficient conditions for the production of active secondary metabolites. The metabolites produced by strain Microbacterium sp. LGMB471 cultured in SG medium at 36 °C considerably inhibited the development of P. citricarpa. Three isoflavones and five diketopiperazines were identified, and the compounds 7-O-ß-D-glucosyl-genistein and 7-O-ß-D-glucosyl-daidzein showed high activity against P. citricarpa, with the MIC of 33 µg/mL and inhibited the production of asexual spores of P. citricarpa on leaves and citrus fruits. Compounds that inhibit conidia formation may be a promising alternative to reduce the use of fungicides in the control of CBS lesions, especially in regions where sexual reproduction does not occur, as in the USA. Our data suggest the use of Microbacterium sp. LGMB471 or its metabolites as an ecological alternative to be used in association with the fungicides for the control of CBS disease.

Escherichia coli GutM4 produces 2, 5-diketopiperazines and inhibits human pathogens in vitro

Background: It has been a very common practice to use probiotics or their metabolites as alternative antimicrobial strategies for the treatment and prevention of infections as rampant and indiscriminate use of antibiotics causes the development of antibiotic-resistant pathogens. The objective of this study was to select a potential antimicrobial probiotic strain of Escherichia coli from the human gastrointestinal tract and investigate the production of diketopiperazines that contribute to the antimicrobial activity. Results: E. coli GutM4 was isolated from the feces of a healthy adult. E. coli GutM4 showed significant antagonistic activity against 10 indicator pathogens, and this activity was no less than that of the reference strain E. coli Nissle 1917 against eight of the indicator pathogens. Moreover, E. coli GutM4 produced antagonistic substances containing trypsin-targeted peptide bonds because the inhibitory effects of E. coli GutM4 supernatant significantly decreased upon treatment with trypsin. Consistent with the antagonistic activity and peptide compounds of E. coli GutM4, 14 2,5-diketopiperazines were isolated from the fermented broth of E. coli GutM4, including 12 cyclo(Pro-Phe), 3 cyclo(Pro-Tyr), and 5 cyclo(4-hydroxyl-Pro-Leu), which are reported to have antipathogenic activity. Conclusion: E. coli GutM4 produces 2,5-diketopiperazines that are partly involved in antagonistic action against human pathogens in vitro.

A Simple Defined Medium for the Production of True Diketopiperazines in Xylella fastidiosa and Their Identification by Ultra-Fast Liquid Chromatography-Electrospray Ionization Ion Trap Mass Spectrometry.

Diketopiperazines can be generated by non-enzymatic cyclization of linear dipeptides at extreme temperature or pH, and the complex medium used to culture bacteria and fungi including phytone peptone and trypticase peptone, can also produce cyclic peptides by heat sterilization. As a result, it is not always clear if many diketopiperazines reported in the literature are artifacts formed by the different complex media used in microorganism growth. An ideal method for analysis of these compounds should identify whether they are either synthesized de novo from the products of primary metabolism and deliver true diketopiperazines. A simple defined medium (X. fastidiosa medium or XFM) containing a single carbon source and no preformed amino acids has emerged as a method with a particularly high potential for the grown of X. fastidiosa and to produce genuine natural products. In this work, we identified a range of diketopiperazines from X. fastidiosa 9a5c growth in XFM, using Ultra-Fast Liquid Chromatography coupled with mass spectrometry. Diketopiperazines are reported for the first time from X. fastidiosa, which is responsible for citrus variegated chlorosis. We also report here fatty acids from X. fastidiosa, which were not biologically active as diffusible signals, and the role of diketopiperazines in signal transduction still remains unknown.

Expanding the Chemical Repertoire of the Endophyte Streptomyces albospinus RLe7 Reveals Amphotericin B as an Inducer of a Fungal Phenotype.

During an investigation of the chemistry of the endophytic actinobacterium Streptomyces albospinus RLe7, which was isolated from the roots of the Brazilian medicinal plant Lychnophora ericoides, three new natural products, (2R*,4S*)-2-((1'S*)-hydroxy-4'-methylpentyl)-4-(hydroxymethyl)butanolide (1), (3R*,4S*,5R*,6S*)-tetrahydro-4-hydroxy-3,5,6-trimethyl-2-pyranone (2), and 1-O-(phenylacetyl)glycerol (3), together with known secondary metabolites (S)-4-benzyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (4), (S)-4-isobutyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (5), and the diketopiperazines cyclo(l-Tyr-l-Pro) (6) and cyclo(l-Val-l-Pro) (7), were isolated. The role of isolated natural products in the interaction between S. albospinus RLe7 and the fungus Coniochaeta sp. FLe4, an endophyte from the same plant, was investigated. None of these isolated actinobacterial compounds were able to inhibit the fungus or induce the fungal red pigmentation observed when both endophytes interact. Further investigation using mass spectrometry approaches enabled identifying the well-known antifungal compound amphotericin B (9) as a microbial metabolite of S. albospinus RLe7. Finally, compound 9 was demonstrated as at least one of the agents responsible for both the antifungal activity and induction of red-pigmented fungal phenotype.

Diketopiperazines from Costa Rican endolichenic fungus Colpoma sp. CR1465A.

Three new diketopiperazines (1-3), cyclo(l-Pro-d-trans-Hyp) (1), cyclo(l-Pro-d-Glu) (2), and cyclo(d-Pro-d-Glu) (3) and five known diketopiperazines (4-8) were isolated from the endolichenic fungus Colpoma sp. CR1465A identified from the Costa Rican plant Henriettea tuberculosa (Melatomataceae). The structures of the new compounds 1-3 were elucidated using a combination of extensive spectroscopic analyses, including 2D NMR and HR-MS, and their absolute configurations were determined by a combination of NOESY analysis and Marfey's method. Cyclo(l-Pro-d-allo-Thr) (4) was recently isolated from a South China Sea marine sponge Callyspongia sp., but its NMR spectroscopic data were not reported, and cyclo(l-Pro-l-Asp) (5) was previously reported but only as a synthetic product. The NMR data assignments of compounds 4 and 5 are reported for the first time. All of the isolated compounds were tested for antifungal and antimicrobial properties.

α-Selective glycosylation affords mucin-related GalNAc amino acids and diketopiperazines active on Trypanosoma cruzi.

This work addresses the synthesis and biological evaluation of glycosyl diketopiperazines (DKPs) cyclo[Asp-(αGalNAc)Ser] 3 and cyclo[Asp-(αGalNAc)Thr] 4 for the development of novel anti-trypanosomal agents and Trypanosoma cruzi trans-sialidase (TcTS) inhibitors. The target compounds were synthetized by coupling reactions between glycosyl amino acids αGalNAc-Ser 7 or αGalNAc-Thr 8 and the amino acid (O-tBu)-Asp 17, followed by one-pot deprotection-cyclisation reaction in the presence of 20% piperidine in DMF. The protected glycosyl amino acid intermediates 7 and 8 were, in turn, obtained by α-selective, HgBr2-catalysed glycosylation reactions of Fmoc-Ser/Thr benzyl esters 12/14 with αGalN3Cl 11, being, subsequently, fully deprotected for comparative biological assays. The DKPs 3 and 4 showed relevant anti-trypanosomal effects (IC50 282-124 µM), whereas glycosyl amino acids 1 and 2 showed better TcTS inhibition (57-79%) than the corresponding DKPs (13-25%).