Aims:We aimed to expand the microbial biocatalyst platform to generate essential oxyfunctionalized standards for pharmaceutical, toxicological and environmental research. In particular, we examined the production of oxyfunctionalized nonsteroidal anti-inflammatory drugs (NSAIDs) by filamentous-fungi. Methods and Results: Four NSAIDs; diclofenac, ibuprofen, naproxen and mefenamic acid were used as substrates for oxyfunctionalization in a biocatalytic process involving three filamentous-fungi strains; Beauveria bassiana, Clitocybe nebularis and Mucor hiemalis. Oxyfunctionalized metabolites that are major degradation intermediates formed by Cytochrome P450 monooxygenases in human metabolism were produced in isolated yields of up to 99% using 1 g l À1 of substrate. In addition, a novel compound, 3 0 ,4 0dihydroxydiclofenac, was produced by B. bassiana. Proteomic analysis identified CYP548A5 that might be responsible for diclofenac oxyfunctionalization in B. bassiana. Conclusions: Efficient fungi catalysed oxyfunctionalization was achieved when using NSAIDs as substrates. High purities and isolated yields of the produced metabolites were achieved. Significance and Impact of the Study: The lack of current efficient synthetic strategies for oxyfunctionalization of NSAIDs is a bottleneck to perform pharmacokinetic, pharmacodynamic and toxicological analysis for the pharmaceutical industry. Additionally, oxyfunctionalized derivatives are needed for tracking the fate and impact of such metabolites in the environment. Herein, we described a fungi catalysed process that surpasses previously reported strategies in terms of efficiency, to synthesize oxyfunctionalized NSAIDs.
11 12 Abbreviations: GC, gas chromatography; HPLC, high-performance liquid 13 chromatography; MS, mass spectrometry; NMR, nuclear magnetic resonance; NSAID, 14 non-steroidal anti-inflammatory drug; PEG, potato-extract-glucose; P450, cytochrome 15 P450 monooxygenase; P450 BM3, cytochrome P450 from Bacillus megaterium; P450 16RhF, cytochrome P450 from Rhodococcus sp. strain NCIMB 9784. 17 Abstract 18The synthesis of valuable metabolites and degradation intermediates of drugs, like non-19 steroidal anti-inflammatory drugs (NSAIDs), are substantially for toxicological and 20 environmental studies, but efficient synthesis strategies and the metabolite availability are 21 still challenging aspects. To overcome these bottlenecks filamentous fungi as microbial 22 biocatalysts were applied. Different NSAIDs like diclofenac, ibuprofen, naproxen and 23 mefenamic acid could be oxyfunctionalized to produce human metabolites in isolated 24 yields of up to 99% using 1 g L -1 of substrate. Thereby the biotransformations using 25Beauveria bassiana, Clitocybe nebularis or Mucor hiemalis surpass previous reported 26 chemical, microbial and P450-based routes in terms of efficiency. In addition to different 27 hydroxylated compounds of diclofenac, a novel metabolite, 3',4'-dihydroxydiclofenac, has 28 been catalyzed by B. bassiana and the responsible P450s were identified by proteome 29 analysis. The applied filamentous fungi present an interesting alternative, microbial 30 biocatalysts platform for the production of valuable oxyfunctionalized drug metabolites. 31 32 Importance 33The occurrence of pharmaceutically active compounds, such as diclofenac and its 34 metabolites, in the environment, in particular in aquatic systems, is of increasing concern 35 because of the increased application of drugs. Standards of putative metabolites are 36 therefore necessary for environmental studies. Moreover, pharmaceutical research and 37 development requires assessment of the bioavailability, toxicity and metabolic fate of 38 potential new drugs to ensure its safety for users and the environment. Since most of the 39 reactions in the early pharmacokinetics of drugs are oxyfunctionalizations catalysed by 40 P450s, oxyfunctionalized metabolites are of major interest. However, to assess these 41 metabolites chemical synthesis often suffer from multistep reactions, toxic substances, 42 polluting conditions and achieve only low regioselectivity. Biocatalysis can contribute to 43 this by using microbial cell factories. The significance of our research is to complement or 44 even exceed synthetic methods for the production of oxyfunctionalized drug metabolites. 45 46Approximately 80% of all reactions in the early pharmacokinetics of drugs (phase I 59 reactions) are P450-catalyzed (1, 2). Therefore, oxyfunctionalized metabolites are often 60 major degradation intermediates of potential toxic endogenous and exogenous compounds 61 in mammalians and nature. For further approval of new drugs, knowledge about potential 62 metabolism intermediates and thei...
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