RESUMEN
25-Hydroxy-Grundmann's ketone is a key building block in the chemical synthesis of vitamin D3 and its derivatives through convergent routes. Generally, the chemical synthesis of this compound involves tedious procedures and results in a mixture of several products. Recently, the selective hydroxylation of Grundmann's ketone at position C25 by cytochrome P450 (CYP) 154E1 from Thermobifida fusca YX was described. In this study a recombinant whole-cell biocatalyst was developed and applied for hydroxylation of Grundmann's ketone. Biotransformation was performed by Escherichia coli cells expressing CYP154E1 along with two redox partner systems, Pdx/PdR and YkuN/FdR. The system comprising CYP154E1/Pdx/PdR showed the highest production of 25-hydroxy-Grundmann's ketone and resulted in 1.1mM (300mgL(-1)) product concentration.
Asunto(s)
Bacillus subtilis/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Escherichia coli/enzimología , Cetonas/metabolismo , Pseudomonas putida/enzimología , Bacillus subtilis/metabolismo , Biocatálisis , Escherichia coli/citología , Escherichia coli/metabolismo , Cetonas/química , Conformación Molecular , Oxidación-Reducción , Pseudomonas putida/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
C-3-substituted 25-hydroxyvitamin D3 analogues were synthesized as tools to directly measure levels of vitamin D in biological samples. The strategy involves vinyloxycarbonylation of the 3ß-hydroxy group and formation of a carbamate bond with a hydroxyl or amino group at the end of the alkyl chain. Biotinylated conjugates of synthesized derivatives were generated to be linked with vitamin D binding protein (DBP). The spacer group present in the alkyl chain is important in the binding of antibodies to the analogue-DBP complex. When compared to 25-hydroxyvitamin D3-DBP, the binding of some antibodies to the analogue-DBP complex of the 25-hydroxyvitamin D3 derivative 10 that posses an 8-aminoctyl alkyl chain is significantly reduced, but this analogue displaced [26,27-(3)H]-25-hydroxyvitamin D3 from DBP. In contrast, the 8-hydroxyoctyl alkyl chain analogue 9 showed less displacement.
Asunto(s)
Calcifediol/síntesis química , Vitamina D/sangre , Calcifediol/análogos & derivados , Calcifediol/sangre , Calcifediol/química , Humanos , Conformación MolecularRESUMEN
A new enzymatic electrochemical biosensor based on disposable transducers, namely screen-printed carbon electrodes, has been developed for the determination of the antiepileptic drug levetiracetam. Horseradish peroxidase was immobilized onto the carbon working electrode previously modified by an aryl diazonium salt. The formation of amide bonds between the amino and carboxylic groups of the enzyme surface, catalyzed by hydroxysuccinimide and carbodiimide, leads to the electrode functionalization. This orientated enzymatic modification results in high reproducibility, with an associated relative standard deviation of 6.21% for the slopes of several calibration curves in the calibration range from 0.10 to 0.83mM. Experimental variables that can affect levetiracetam chronoamperometric response, such as hydrogen peroxide concentration, pH, and applied potential, were optimized to perform a selective determination. An average limit of detection of 1.75x10(-5)M (alpha=beta=0.05) was obtained. The biosensors were finally applied to the determination of levetiracetam in complex matrices such as pharmaceutical drugs, yielding successful results.
Asunto(s)
Anticonvulsivantes/análisis , Técnicas Biosensibles/instrumentación , Peroxidasa de Rábano Silvestre/metabolismo , Microelectrodos , Piracetam/análogos & derivados , Calibración , Carbono , Compuestos de Diazonio , Técnicas Electroquímicas , Enzimas Inmovilizadas/metabolismo , Peróxido de Hidrógeno , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Levetiracetam , Modelos Lineales , Piracetam/análisis , Reproducibilidad de los Resultados , Plata , Propiedades de Superficie , TransductoresRESUMEN
Vitamin D, through its hormonally active form 1α,25-dihydroxyvitamin D3 [1α,25- (OH)2-D3], exhibits a much broader spectrum of bio logical activities than expected in the endocrine system. However, 1α,25-(OH)2-D3 causes hypercalcemia a t pharmacologically r elevant doses wh ich forms a major obstacle in the clinical development of this compound. As a result, considerable effort has been made toward the synthesis of potential chemotherapeutic structurally related congeners with dissociation of beneficial effects from their toxic effects. Most of the analogues prepared have modifications on the upper side chain, more accessible from a synthetic point of view. Modifications of the A-ring are less extensive, although A-ring analogues exhibit a unique biological profile. This seco steroid can undergo a rotation around the 6,7 carbon-carbon single bond generating a wide array of molecular shapes, extending from the 6-s-cis to the more stable extended 6-s-trans conformation, which plays an important role in modulating the different biological activities of vitamin D. We review here, the synthetic strategies for the preparation of Vitamin D analogues with modific ations on the A-ring, including 6-s-cis locked derivatives that became of interest to further probe the less well investigate membrane actions of 1α,25-(OH)2-D3 for structure-activity relationship studies.