RESUMEN
4-Hydroxycoumarins represents an important structural motif in life sciences molecules with remarkable biological properties. 4-Hydroxycoumarins are important precursors in the synthesis of organic compounds and critical pharmacophores in medicinal chemistry. One of the derivatives of this compound is biscoumarin, which has anticoagulant medicinal properties. Porcine pancreas lipase (PPL) has been used as an efficient green biocatalyst for the synthesis of bis-4-hydroxycoumarin compounds and expands the biocatalytic promiscuity of lipase in organic synthesis. Some aromatic aldehydes have been used in this investigation for screening and examining the ability of enzyme in knoevenagel condensation. Various parameters including temperature, solvent, and the amount of biocatalyst were investigated, ethanol, 45 °C and 10 mg of PPL gave rise to best yields. Bis-4-hydroxy coumarin compounds were synthesized under mild reaction conditions with high conversion yields (81-88%).
Asunto(s)
4-Hidroxicumarinas/biosíntesis , Lipasa/metabolismo , Páncreas/enzimología , 4-Hidroxicumarinas/química , Animales , Biocatálisis , Estructura Molecular , PorcinosRESUMEN
Acyl-CoAs are essential intermediates in the biosynthetic pathways of a number of industrially and pharmaceutically important molecules. When these pathways are reconstituted in a heterologous microbial host for metabolic engineering purposes, the acyl-CoAs may be subject to undesirable hydrolysis by the host's native thioesterases, resulting in a waste of cellular energy and decreased intermediate availability, thus impairing bioconversion efficiency. 4-hydroxycoumarin (4HC) is a direct synthetic precursor to the commonly used oral anticoagulants (e.g. warfarin) and rodenticides. In our previous study, we have established an artificial pathway for 4HC biosynthesis in Escherichia coli, which involves the thioester intermediate salicoyl-CoA. Here, we utilized the 4HC pathway as a demonstration to examine the negative effect of salicoyl-CoA degradaton, identify and inactivate the responsible thioesterase, and eventually improve the 4HC production. We screened a total of 16 E. coli thioesterases and tested their hydrolytic activity towards salicoyl-CoA in vitro. Among all the tested candidate enzymes, YdiI was found to be the dominant contributor to the salicoyl-CoA degradation in E. coli. Remarkably, the ydiI knockout strain carrying the 4HC pathway exhibited an up to 300% increase in 4HC production. An optimized 4HC pathway construct introduced in the ydiI knockout strain led to the accumulation of 935mg/L of 4HC in shake flasks, which is about 1.5 folds higher than the wild-type strain. This study demonstrates a systematic strategy to alleviate the undesirable hydrolysis of thioester intermediates, allowing production enhancement for other biosynthetic pathways with similar issues.
Asunto(s)
4-Hidroxicumarinas/biosíntesis , Escherichia coli/metabolismo , Palmitoil-CoA Hidrolasa/biosíntesis , Escherichia coli/genética , Palmitoil-CoA Hidrolasa/genéticaRESUMEN
OBJECTIVES: Due to the increasing prevalence of drug-resistant Staphylococcus aureus infection, we develop novel 4-hydroxycoumarin derivatives as antimicrobials. METHODS: The antibacterial activity of 4-hydroxycoumarin derivatives against drug-susceptive S. aureus (ATCC 29213) and methicillin-resistant S. aureus (MRSA) were evaluated using minimal inhibitory concentration (MIC) assay; the activity of favourable compound was further observed using bacterial growth curves assay and in the MRSA infection mice. KEY FINDINGS: Compared with dihydropyran derivatives, compound 1 as one of biscoumarins showed most potent activity with MIC values of 4-8 µg/ml and apparently inhibited the growth rate of S. aureusâ ATCC 29213 and USA300 strain in concentrations of both 16 and 32 mg/ml. In the mice infected with MRSA USA300, administration of 5 mg/kg compound 1 improved the animal survival rate to 66.7%, and improved the pathological change in lung tissue compared with the infection model animals. No significant cytotoxicity of compound 1 was observed on the umbilical vein endothelial cells (HUVECs) under the concentration of 800 µg/ml. CONCLUSION: Compared with the dihydropyran derivatives, biscoumarins exhibited more promising activity against both drug-sensitive and drug-resistant S. aureus, and it is efficacious in treating MRSA infections in mouse models with a favourable safety in human cells.
Asunto(s)
4-Hidroxicumarinas/uso terapéutico , Antibacterianos/uso terapéutico , Enfermedades Pulmonares/tratamiento farmacológico , Pulmón/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Infecciones Estafilocócicas/tratamiento farmacológico , 4-Hidroxicumarinas/biosíntesis , 4-Hidroxicumarinas/farmacología , Animales , Antibacterianos/farmacología , Células Endoteliales de la Vena Umbilical Humana , Pulmón/microbiología , Pulmón/patología , Enfermedades Pulmonares/microbiología , Enfermedades Pulmonares/patología , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Infecciones Estafilocócicas/microbiología , Staphylococcus aureusRESUMEN
4-Hydroxycoumarin (4HC) type anticoagulants (for example, warfarin) are known to have a significant role in the treatment of thromboembolic diseases--a leading cause of patient morbidity and mortality worldwide. 4HC serves as an immediate precursor of these synthetic anticoagulants. Although 4HC was initially identified as a naturally occurring product, its biosynthesis has not been fully elucidated. Here we present the design, validation, in vitro diagnosis and optimization of an artificial biosynthetic mechanism leading to the microbial biosynthesis of 4HC. Remarkably, function-based enzyme bioprospecting leads to the identification of a characteristic FabH-like quinolone synthase from Pseudomonas aeruginosa with high efficiency on the 4HC-forming reaction, which promotes the high-level de novo biosynthesis of 4HC in Escherichia coli (~500 mg l⻹ in shake flasks) and further in situ semisynthesis of warfarin. This work has the potential to be scaled-up for microbial production of 4HC and opens up the possibility of biosynthesizing diverse coumarin molecules with pharmaceutical importance.
Asunto(s)
4-Hidroxicumarinas/biosíntesis , Anticoagulantes/metabolismo , Proteínas Bacterianas/biosíntesis , Ligasas/biosíntesis , Profármacos/metabolismo , Pseudomonas aeruginosa/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Cinética , Ligasas/genética , Ligasas/aislamiento & purificación , Ingeniería Metabólica , Redes y Vías Metabólicas , Pseudomonas aeruginosa/genética , Warfarina/metabolismoRESUMEN
The extensive use of anticoagulant rodenticides (ARs) for rodent control has led to widespread secondary exposure in nontarget predatory wildlife species. We investigated exposure rates and concentrations of five ARs in liver samples from five raptors and six owls from Denmark. A total of 430 birds were analysed. ARs were detected in 84-100 % of individual birds within each species. Multiple AR exposure was detected in 73 % of all birds. Average number of substances detected in individual birds was 2.2 with no differences between owls and raptors. Difenacoum, bromadiolone, and brodifacoum were the most prevalent substances and occurred in the highest concentrations. Second-generation ARs made up 96 % of the summed AR burden. Among the six core species (sample size >30), summed AR concentrations were lower in rough-legged buzzard (Buteo lagopus) and long-eared owl (Asio otus) than in barn owl (Tyto alba), buzzard (B. buteo), kestrel (Falco tinnunculus), and tawny owl (Strix aluco). There was a strong tendency for seasonal variations in the summed AR concentration with levels being lowest during autumn, which is probably related to an influx of less-exposed migrating birds from northern Scandinavia during autumn. High hepatic AR residue concentrations (>100 ng/g wet weight), which have been associated with symptoms of rodenticide poisoning and increased mortality, were recorded high frequencies (12.9-37.4 %) in five of the six core species. The results suggest that the present use of ARs in Denmark, at least locally, may have adverse effects on reproduction and, ultimately, population status in some raptors and owls.
Asunto(s)
4-Hidroxicumarinas/metabolismo , Anticoagulantes/metabolismo , Aves/metabolismo , Exposición a Riesgos Ambientales/estadística & datos numéricos , Contaminantes Ambientales/metabolismo , Rodenticidas/metabolismo , 4-Hidroxicumarinas/análisis , 4-Hidroxicumarinas/biosíntesis , Animales , Anticoagulantes/análisis , Dinamarca , Exposición a Riesgos Ambientales/análisis , Femenino , Masculino , Rapaces/metabolismo , Rodenticidas/análisis , Estrigiformes/metabolismoRESUMEN
Coumarin forms in melilotoside (trans-ortho-coumaric acid glucoside)-containing plant species upon cell damage. In moldy melilotoside-containing plant material, trans-ortho-coumaric acid is converted by fungi to 4-hydroxycoumarin, two molecules of which spontaneously combine with formaldehyde to give dicoumarol. Dicoumarol causes internal bleeding in livestock and is the forerunner of the warfarin group of medicinal anticoagulants. Here, we report 4-hydroxycoumarin formation by biphenyl synthase (BIS). Two new BIS cDNAs were isolated from elicitor-treated Sorbus aucuparia cell cultures. The encoded isoenzymes preferred ortho-hydroxybenzoyl (salicoyl)-CoA as a starter substrate and catalyzed a single decarboxylative condensation with malonyl-CoA to give 4-hydroxycoumarin. When elicitor-treated S. aucuparia cell cultures were fed with the N-acetylcysteamine thioester of salicylic acid, 4-hydroxycoumarin accumulated in the culture medium. Incubation of the BIS isoenzymes with benzoyl-CoA and malonyl-CoA resulted in the formation of 3,5-dihydroxybiphenyl which is the precursor of the phytoalexins of the Maloideae.
Asunto(s)
4-Hidroxicumarinas/biosíntesis , Proteínas de Plantas/metabolismo , Sorbus/metabolismo , 4-Hidroxicumarinas/química , 4-Hidroxicumarinas/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cromatografía de Gases y Espectrometría de Masas , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sorbus/genéticaRESUMEN
Due to exceptional reactivity of 4-hydroxycoumarin, the synthesis of new coumarin derivatives of dimer and tetramer type has been carried out. The synthesis was carried out from 4-hydroxycoumarin and various aromatic aldehydes. In this way, compounds of the dimer 3,3'-(benzilidene)bis (4-hydroxycoumarin) type, as well as of the tetramer 3,3',3'',3'''-(1,4-dimethylenphenyl)tetra (4-hydroxycoumarin) type were prepared. The newly synthesized derivatives contain different functional groups, and as such they could exhibit microbiological activity. Therefore, we tested the microbiological activity of these derivatives on various species of bacteria and fungi. The tested compounds have shown different activity in terms of growth inhibition of microorganisms. Newly synthesized derivatives exhibit antibacterial activities, manifested as growth inhibition on Gram-positive bacteria types (Bacillus, Staphylococcus), while the activity against Candida was much weaker. The same compound did not show any antimicrobial activity against two Gram-negative bacteria types (Escherichia coli, Pseudomonas aeruginosa). The compound 1 showed the best microbiological activity. The obtained results confirmed its good antibacterial and antimycotic activities against different microorganisms.