Tomasz Janeczko

Microbial transformation of (+)-nootkatone and the antiproliferative activity of its metabolites

Six metabolites were obtained as a result of microbial transformation of (+)-nootkatone (1) by the fungal strains: Botrytis, Didymosphaeria, Aspergillus, Chaetomium and Fusarium. Their structure were established as (+)-(4R,5S,7R,9R)-9α-hydroxynootkatone (2), (+)-(4R,5S,7R)-13-hydroxynootkatone (3) and (+)-(4R,5S,7R,9R,11S)-11,12-epoxy-9α-hydroxynootkatone (4), (+)-(4R,5S,7R,11S)-11,12-epoksynootkatone (5), (+)-(4R,5S,7R)-11,12-dihydroxynootkatone (6) and (+)-(4R,5S,7R)-7,11,12-trihydroxynootkatone (7) on the basis of their spectral data. Two products: (4) and (7) were not previously reported in the literature. The antiproliferative activity of (+)-nootkatone (1) and isolated metabolites (2-7) of its biotransformation has been evaluated.

Biotransformations of steroid compounds by Chaetomium sp. KCH 6651

Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14alpha-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6beta-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6beta position. Progesterone was transformed to a single product-6beta,14alpha-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7alpha-hydroxy derivative, which was subsequently converted to 7alpha-hydroxyandrost-4-en-3,17-dione.

Microbial Transformations of 7-Hydroxyflavanone

Microbial transformations of racemic 7-hydroxyflavanone using strains of genus Aspergillus (A. niger KB, A. niger 13/5, A. ochraceus 456) and the species Penicillium chermesinum 113 were studied. The products of O-methylation, O-methylation along with hydroxylation at C-3′ and C-4′, reduction of the carbonyl group, reduction of the carbonyl group along with hydroxylation at C-5, and dehydrogenation of C-2 and C-3 were obtained. Most of the products (with the exception of the O-methylation one) have stronger antioxidant properties than the initial substrate.

Synthetic Flavanones Augment the Anticancer Effect of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as the most promising anticancer agent in the TNF superfamily because of its selective cytotoxicity against tumor cells versus normal primary cells. However, as more tumor cells are reported to be resistant to TRAIL-mediated death, it is important to develop new therapeutic strategies to overcome this resistance. Flavonoids have been shown to sensitize cancer cells to TRAIL-induced apoptosis. The aim of this study was to examine the cytotoxic and apoptotic activities of TRAIL on HeLa cancer cells in combination with two synthetic compounds: 6-hydroxyflavanone (6-HF) and its derivative 6-propionoxy-flavanone (6-PF) and to determine the mechanism by which the flavanones overcome the TRAIL-resistance. The cytotoxicity was measured by MTT and LDH assays. The apoptosis was detected by annexin V-FITC fluorescence staining in flow cytometry and microscopy. Death receptor (TRAIL-R1/DR4 and TRAIL-R2/DR5) expression were analysed using flow cytometry. Mitochondrial membrane potential was evaluated using DePsipher staining by fluorescence microscopy. The synthetic flavanones enhanced TRAIL-induced apoptosis in HeLa cells through increased expression of TRAIL-R2 death receptor and reduction of mitochondrial membrane potential. Our study indicates that the 6-HF and 6-PF augmented the anticancer effects of TRAIL and confirm a potential use of flavanones in TRAIL-based anticancer therapy and prevention.

Microbial transformation of selected flavanones as a method of increasing the antioxidant properties.

Antioxidant properties of substrates [fl avanone (1), 6-hydroxy- (2), 7-hydroxy- (3), 5,7,4’- trihydroxy- (5), and 7-methoxyfl avanone (4)] and products of their microbial transformations, comprising hydroxylation, O-methylation, stereospecifi c reduction, dehydrogenation, and C-ring cleavage of the benzo-γ-pyrone system, were determined. Measurements of the antiradical activity (expressed as IC50 value) of both the substrates and the products led to the determination of the impact of type and location of substituents in the tested fl avonoids on changes in their antioxidant activities.

Microbial hydroxylation of chiral bicyclic enones by Chaetomium sp.1 and Didymosphaeria igniaria cultures

The biotransformations of (R)-(−)-methyloctalone and (S)-(+)-methyloctalone were investigated using Chaetomium sp.1 KCH 6651 and Didymosphaeria igniaria KCH 6670 as biocatalysts, yielding mostly 6β- and 7β-hydroxy derivatives. During the incubation of (R)-methyloctalone with the Chaetomium sp.1 culture, three products were obtained: the trans-7β-hydroxy derivative in 50% yield, trans-6β-hydroxy derivative in 30% yield and cis-8α-hydroxy derivative in 6% yield. The (S)-enone was hydroxylated mainly in the 6α-position (with 60% yield). 6β- and 7β-hydroxy derivatives are new compounds, not previously described in the literature. The biotransformation of the two enantiomers of methyloctalone with D. igniaria also afforded monohydroxy derivatives. In both cases the main product of transformation was the allylic hydroxy derivative (27–35% yield). D. igniaria transformed the (R)-methyloctalone more rapidly whereas Chaetomium sp. 1 preferred the (S)-methyloctalone.

Biotransformation of dehydroepiandrosterone (DHEA) by environmental strains of filamentous fungi

Microbial transformations of steroids are an important method of obtaining new steroid derivatives of potential pharmaceutical activity, which follows the principles of green chemistry. We studied the ability of selected filamentous fungus species to transform dehydroepiandrosterone (DHEA) and interesting DHEA derivatives were obtained. Twenty-five strains of filamentous fungi were isolated from soil and air in Wroclaw and they were used as biocatalysts. Strains of the genus Penicillium transformed the substrate into the products of Baeyer–Villiger lactonization in the D ring. Biotransformation of DHEA by Fusarium acuminatum KCh S1 strain led to a stereoselective hydroxylation at 7α (the product was obtained with 97% conversion). Androst-5-ene-3β,7α,17α-triol, compound with high anticancer activity towards glioblastoma and lymphoma cell lines was obtained with 65% yield in the culture of Mucor hiemalis KCh W2 strain. In the culture of Aspergillus versicolor KCh TJ1 strain, DHEA was transformed into androst-1,4-diene-3,17-dione (ADD) with high isolated yield.

Glycosylation of 6-methylflavone by the strain Isaria fumosorosea KCH J2

Entomopathogenic fungi are known for their ability to carry out glycosylation of flavonoids, which usually results in the improvement of their stability and bioavailability. In this study we used a newly isolated strain of the entomopathogenic filamentous fungus Isaria fumosorosea KCH J2 as a biocatalyst. Our aim was to evaluate its ability to carry out the biotransformation of flavonoids and to obtain new flavonoid derivatives. The fungus was isolated from a spider’s carcass and molecularly identified using analysis of the ITS1-ITS2 rDNA sequence. As a result of biotransformation of 6-methylflavone two new products were obtained: 6-methylflavone 8-O-β-D-(4”-O-methyl)-glucopyranoside and 6-methylflavone 4’-O-β-D-(4”-O-methyl)-glucopyranoside. Chemical structures of the products were determined based on spectroscopic methods (1H NMR, 13C NMR, COSY, HMBC, HSQC). Our research allowed us to discover a new species of filamentous fungus capable of carrying out glycosylation reactions and proved that I. fumosorosea KCH J2 is an effective biocatalyst for glycosylation of flavonoid compounds. For the first time we describe biotransformations of 6-methylflavone and the attachment of the sugar unit to the flavonoid substrate having no hydroxyl group. The possibility of using flavonoid aglycones is often limited by their low bioavailability due to poor solubility in water. The incorporation of a sugar unit improves the physical properties of tested compounds and thus increases the chance of using them as pharmaceuticals.

Microbial transformations of 6- and 7-methoxyflavones in Aspergillus niger and Penicillium chermesinum cultures.

A detailed study of the biotransformation of 6- and 7-methoxyfl avones by four fungal strains (Aspergillus niger strains MB, KB, and SBP; Penicillium chermesinum 113) was carried out. Products of demethylation and also demethylation combined with hydroxylation at C-4’ were identifi ed. The biotransformation products were stronger antioxidants than the substrates.

Biotransformation of steroids by entomopathogenic strains of Isaria farinosa

BackgroundSteroid compounds are very interesting substrates for biotransformation due to their high biological activity and a high number of inactivated carbons which make chemical modification difficult. Microbial transformation can involve reactions which are complicated and uneconomical in chemical synthesis, and searching for a new effective biocatalyst is necessary. The best known entomopathogenic species used in steroid modification is Beauveria bassiana. In this study we tested the ability of Isaria farinosa, another entomopathogenic species, to transform several steroids.ResultsTwelve strains of the entomopathogenic filamentous fungus Isaria farinosa, collected in abandoned mines located in the area of the Lower Silesian Voivodeship, Poland, from insects’ bodies covered by fungus, were used as a biocatalyst. All the tested strains effectively transformed dehydroepiandrosterone (DHEA). We observed 7α- and 7β-hydroxy derivatives as well as changes in the percentage composition of the emerging products. Due to the similar metabolism of DHEA in all tested strains, one of them was selected for further investigation. In the culture of the selected strain, Isaria farinosa KCh KW1.1, transformations of androstenediol, androstenedione, adrenosterone, 17α-methyltestosterone, 17β-hydroxyandrost-1,4,6-triene-3-one and progesterone were performed. All the substrates were hydroxylated with high yield and stereoselectivity. We obtained 6β-hydroxyandrost-4-ene-3,11,17-trione, 15α,17β-dihydroxy-6β,7β-epoxyandrost-1,4-diene-3-one and 6β,11α-dihydroxyprogesterone. There is no evidence of either earlier microbial transformation of 17β-hydroxyandrost-1,4,6-triene-3-one or new epoxy derivatives.ConclusionsIsaria farinosa has a broad spectrum of highly effective steroid hydroxylases. The obtained 7-hydroxydehydroepiandrosterone has proven high biological activity and can be used in Alzheimer’s disease and as a key intermediate in the synthesis of aldosterone antagonists. Transformation of progesterone leads to high yield of 6β,11α-dihydroxyprogesterone and it is worth further study.