Fast HPLC analysis of omeprazole, 5-hydroxyomeprazole and omeprazole sulfone in liquid culture medium using a monolithic column for application in biotransformation studies with fungi

A fast liquid chromatography method was developed and validated for the simultaneous determination of omeprazole (OMZ), 5-hydroxyomeprazole (5-HOMZ) and omeprazole sulphone (OMZ SUL) in liquid culture medium for application in biotransformation studies employing phytopathogenic and endophytic fungi. The separation was achieved using a monolithic Chromolith Fast gradient RP 18 endcapped column, using a mobile phase consisting of 0.15% (v/v) trifluoroacetid acid (TFA) in water (solvent A) and 0.15% (v/v) TFA in acetonitrile (solvent B), under linear gradient of 5 to 90% of B in 1 min, flow rate of 1.0 mL min-1, temperature at 30 ºC and detection at 220 nm. Sample preparation was performed by liquid-liquid extraction, with recoveries in the range of 62.3 to 76.6% for all analytes. The method was linear in the range of 0.2 to 10.0 µg mL-1 (r &#8805; 0.995). The values for intra- and inter-day precision (% coefficient of variation) and accuracy (% relative error) were < 15% for all analytes. The validated method was used to evaluate OMZ biotransformation to their mammalian metabolites by selected fungi. In general, the phytopathogenic fungi studied were more efficient to biotransform OMZ. The sulfonation reaction was more prevalent for all studied fungi.Um método rápido por cromatografia líquida foi desenvolvido para a determinação simultânea de omeprazol (OMZ), 5-hidroxiomeprazol (5-HOMZ) e omeprazol sulfona (OMZ SUL) em meio de cultura líquido, para aplicação em estudos de biotransformação empregando fungos fitopatogênicos e endofíticos. A separação foi realizada empregando uma coluna monolítica Chromolith Fast gradient RP 18 com a fase móvel constituída por ácido trifluoroacético (TFA) 0,15% (v/v) em água (solvente A) e TFA 0,15% (v/v) em acetonitrila (solvente B). Foi empregado um gradiente linear de 5 a 90% de B em 1 minuto, vazão de 1,0 mL min-1, temperatura de 30 ºC e detecção em 220 nm. A extração líquido-líquido foi empregada na preparação das amostras, com recuperações na faixa de 62,3-76,6% para todos os analitos. O método foi linear na faixa de 0,2-10,0 µg mL-1 (r &#8805; 0,995). Os valores de precisão e exatidão intra- e inter-dias (coeficiente de variação e erro relativo) foram inferiores a 15% para todos os analitos. O método validado foi utilizado para avaliar a biotransformação do OMZ em seus principais metabólitos humanos pelos fungos selecionados. Em geral, os fungos fitopatogênicos foram mais eficientes para biotransformar o OMZ. A reação de sulfonação foi mais prevalente em todos os fungos estudadosFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Biocatalytic preparation and absolute configuration of enantiomerically pure fungistatic anti-2-benzylindane derivatives. Study of the detoxification mechanism by Botrytis cinerea

Enantiomerically pure 2-benzylindane derivatives were prepared using biocatalytic methods and their absolute configuration determined. (1R,2S)-2-Benzylindan-1-ol ((1R,2S)-2) and (S)-2-benzylindan-1-one ((S)-3) were produced by fermenting baker’s yeast. Lipase-mediated esterifications and hydrolysis of the corresponding racemic substrates gave rise to the enantiopure compounds (1S,2R)-2-benzylindan-1-ol ((1S,2R)-2) and (1R,2S)-2-benzylindan-1-ol ((1R,2S)-2), respectively. The antifungal activity of these products against two strains of the plant pathogen Botrytis cinerea was tested. The metabolism of anti-(±)-2-benzylindan-1-ol (anti-(±)-2) by B. cinerea as part of the fungal detoxification mechanism is also described and revealed interesting differences in the genome of both strains

Genetic and molecular basis of botrydial biosynthesis: connecting cytochrome P450-encoding genes to biosynthetic intermediates

Over two hundred species of plants can be infected by the phytopathogenic fungus Botrytis cinerea under a range of different environmental conditions. In response to these, the fungus produces unique terpenoid and polyketide metabolites. Parts of the plants may be killed by the phytotoxin botrydial, enabling the fungus to feed on the dead cells. In this paper, we describe the genetic and molecular basis of botrydial biosynthesis and the function of the five genes of the genome of B. cinerea that together constitute the botrydial biosynthetic gene cluster. Genes BcBOT3 and BcBOT4, encoding two cytochrome P450 monooxygenases, were inactivated by homologous recombination and were shown to catalyze regio- and stereospecific hydroxylations at the carbons C-10 and C-4, respectively, of the presilphiperfolan-8β-ol skeleton. The null mutants, bcbot3Δ and bcbot4Δ, accumulated key intermediates in the botrydial biosynthesis enabling the complete genetic and molecular basis of the botrydial biosynthetic pathway to be established. Furthermore, the bcbot4Δ mutant overproduced a significant number of polyketides, which included, in addition to known botcinins, botrylactones and cinbotolide A, two new botrylactones and two new cinbotolides, cinbotolides B and C

Relevance of trichothecenes in fungal physiology: Disruption of tri5 in Trichoderma arundinaceum

Trichothecenes are sesquiterpenoid mycotoxins produced mainly by Fusarium species. Harzianum A (HA), a non-phytotoxic trichothecene produced by Trichoderma arundinaceum, has recently been found to have antagonistic activity against fungal plant pathogens and to induce plant genes involved in defense responses. In the present work, we have shown that disruption of the T. arundinaceum tri5 gene, which encodes a terpene synthase, stops the production of HA, alters the expression of other tri genes involved in HA biosynthesis, and alters the expression of hmgR, dpp1, erg9, erg1, and erg7, all genes involved in terpene biosynthetic pathways. An increase in the level of ergosterol biosynthesis was also observed in the tri5 disrupted transformant in comparison with the wild type strain. The loss of HA also resulted in a drastic reduction of the biocontrol activity of the transformants against the phytopathogenic fungi Botrytis cinerea and Rhizoctonia solani. Finally, the effect of tri5 gene disruption on the regulation and balance of intermediates in terpene biosynthetic pathways, as well as the hypothetical physiological role of trichothecenes, both inter- and intracellularly, on regulation and biocontrol, are discussed

Synthesis of Trichodermin Derivatives and Their Antimicrobial and Cytotoxic Activities

Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used in the design of new useful bioactive molecules. In Trichoderma brevicompactum, the first specific step in trichothecene biosynthesis is carried out by a terpene cyclase, trichodiene synthase, that catalyzes the conversion of farnesyl diphosphate to trichodiene and is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin, a trichothecene-type toxin, which is a valuable tool in preparing new molecules with a trichothecene skeleton. In this work, we developed the hemisynthesis of trichodermin and trichodermol derivatives in order to evaluate their antimicrobial and cytotoxic activities and to study the chemo-modulation of their bioactivity. Some derivatives with a short chain at the C-4 position displayed selective antimicrobial activity against Candida albicans and they showed MIC values similar to those displayed by trichodermin. It is important to highlight the cytotoxic selectivity observed for compounds 9, 13, and 15, which presented average IC50 values of 2 g/mL and were cytotoxic against tumorigenic cell line MCF-7 (breast carcinoma) and not against Fa2N4 (non-tumoral immortalized human hepatocytes)

Overexpression of the Trichoderma brevicompactum tri5 Gene: Effect on the Expression of the Trichodermin Biosynthetic Genes and on Tomato Seedlings

Trichoderma brevicompactum IBT 40841 produces trichodermin, a trichothecene-type toxin that shares most of the steps of its biosynthesis with harzianum A, another trichothecene produced by several Trichoderma species. The first specific step in the trichothecene biosynthesis is carried out by a terpene cylcase, trichodiene synthase, that catalyzes the conversion of farnesyl pyrophosphate to trichodiene and that is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin production, but also in an increase in tyrosol and hydroxytyrosol production, two antioxidant compounds that may play a regulatory role in trichothecene biosynthesis, and also in a higher expression of three trichothecene genes, tri4, tri6 and tri10, and of the erg1 gene, which participates in the synthesis of triterpenes. The effect of tri5 overexpression on tomato seedling disease response was also studied

Chemically Induced Cryptic Sesquiterpenoids and Expression of Sesquiterpene Cyclases in Botrytis cinerea Revealed New Sporogenic (+)-4-Epieremophil-9-en-11-ols

The sequencing of the genomes of the B05.10 and T4 strains of the fungus Botrytis cinerea revealed an abundance of novel biosynthetic gene clusters, the majority of which were unexpected on the basis of the previous analyses of the fermentation of these and closely related species. By systematic alteration of easy accessible cultivation parameters, using chemical induction with copper sulfate, we have found a cryptic sesquiterpenoid family with new structures related to eremophil-9-ene, which had the basic structure of the sesquiterpene (+)-5-epiaristolochene ((+)-4-epieremophil-9- ene). An expression study of the sesquiterpene cyclase genes present in the Botrytis cinerea genome, under culture conditions, is reported. In general, a 3 day delay and a higher BcSTC genes expression were observed when copper (5 ppm) was fed to the fermentation broth. In addition, to the observed effect on the BcBOT2 (BcSTC1) gene, involved in the biosynthesis of the botrydial toxin, a higher expression level for BcSTC3 and BcSTC4 was observed with respect to the control in the strain B05.10. Interestingly, under copper conditions, the BcSTC4 gene was the most expressed gene in the Botrytis cinerea UCA992 strain. In vitro evaluation of the biological role of these metabolites indicates that they contributed to the conidial development in B. cinerea and appear to be involved in self-regulation of the production of asexual spores. Furthermore, they promoted the formation of complex appressoria or infection cushions

nor-Mevaldic acid surrogates as selective antifungal agent leads against Botrytis cinerea. Enantioselective preparation of 4-hydroxy-6-(1-phenylethoxy)tetrahydro-2H-pyran-2-one

Solvent-free desymmetrisation of meso-dialdehyde 1 with chiral 1-phenylethan-1-ol, led to preparation of 4-silyloxy-6-alkyloxytetrahydro-2H-pyran-2-one (+)-3a with a 96:4 dr Deprotected lactone (+)-19a and the related racemic lactones 16a–18a present a lactone moiety resembling the natural substrate of HMG-CoA reductase and their antifungal properties have been evaluated against the phytopathogenic fungi Botrytis cinerea and Colletotrichum gloeosporioides. These compounds were selectively active against B. cinerea, while inactive against C. gloeosporioides

Terpenoid biotransformations by Mucor species

Terpenoids are natural products of great interest due to their widespread use in agrochemicals, drugs, fragrances, flavouring and pigments. Biocatalysts are increasingly being used in the search for new derivatives with improved properties especially to obtain structurally novel leads for new drugs which are difficult to obtain using conventional organic chemical methods. This review, covering up to the end of 2012, reports on the application of Mucor species as catalysts in terpenoid biotransformation to obtain new drug targets, enhance pharmacological activity or decrease the unwanted effects of starting material