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1.
Bioorg Med Chem Lett ; 19(11): 3059-62, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19394221

RESUMO

Combinatorial biocatalysis was applied to generate a diverse set of dihydroxymethylzearalenone analogs with modified ring structure. In one representative chemoenzymatic reaction sequence, dihydroxymethylzearalenone was first subjected to a unique enzyme-catalyzed oxidative ring opening reaction that creates two new carboxylic groups on the molecule. These groups served as reaction sites for further derivatization involving biocatalytic ring closure reactions with structurally diverse bifunctional reagents, including different diols and diamines. As a result, a library of cyclic bislactones and bislactams was created, with modified ring structures covering chemical space and structure activity relationships unattainable by conventional synthetic means.


Assuntos
Zearalenona/química , Biocatálise , Desenho de Fármacos , Enzimas/metabolismo , Lipase/metabolismo , Relação Estrutura-Atividade , Zearalenona/biossíntese
2.
Drug Metab Dispos ; 36(10): 1998-2004, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18606743

RESUMO

A novel reaction system was developed for the production of metabolites of poorly water-soluble parent compounds using mammalian liver microsomes. The system includes the selection and use of an appropriate hydrophobic polymeric resin as a reservoir for the hydrophobic parent compounds and its metabolites. The utility of the extractive biotransformation approach was shown for the production of a low-yielding, synthetically challenging 32-hydroxylated metabolite of the antibiotic rifalazil using mouse liver microsomes. To address the low solubility and reactivity of rifalazil in the predominantly aqueous microsomal catalytic system, a variety of strategies were tested for the enhanced delivery of hydrophobic substrates, including the addition of mild detergents, polyvinylpyrrolidone, glycerol, bovine serum albumin, and hydrophobic polymeric resins. The latter strategy was identified as the most suitable for the production of 32-hydroxy-rifalazil, resulting in up to 13-fold enhancement of the volumetric productivity compared with the standard aqueous system operating at the solubility limit of rifalazil. The production process was optimized for a wide range of reaction parameters; the most important for improving volumetric productivity included the type and amount of the polymeric resin, cofactor recycling system, concentrations of the biocatalyst and rifalazil, reaction temperature, and agitation rate. The optimized extractive biotransformation system was used to synthesize 32-hydroxy-rifalazil on a multimilligram scale.


Assuntos
Antibacterianos/síntese química , Rifamicinas/síntese química , Animais , Antibacterianos/farmacocinética , Biotransformação , Cromatografia Líquida de Alta Pressão , Feminino , Masculino , Espectrometria de Massas , Camundongos , Microssomos Hepáticos/metabolismo , Rifamicinas/farmacocinética , Solubilidade
3.
Eur J Med Chem ; 64: 121-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23644195

RESUMO

The structures of the two predominant metabolites (M4 and M5) of RVX-208, observed both in in vitro human and animal liver microsomal incubations, as well as in plasma from animal in vivo studies, were determined. A panel of biocatalytic systems was tested to identify biocatalysts suitable for milligram scale production of metabolite M4 from RVX-208. Rabbit liver S9 fraction was selected as the most suitable system, primarily based on pragmatic metrics such as catalyst cost and estimated yield of M4 (∼55%). Glucuronidation of RVX-208 catalyzed by rabbit liver S9 fraction was optimized to produce M4 in amounts sufficient for structural characterization. Structural studies using LC/MS/MS analysis and (1)H NMR spectroscopy showed the formation of a glycosidic bond between the primary hydroxyl group of RVX-208 and glucuronic acid. NMR results suggested that the glycosidic bond has the ß-anomeric configuration. A synthetic sample of M4 confirmed the proposed structure. Metabolite M5, hypothesized to be the carboxylate of RVX-208, was prepared using human liver microsomes, purified by HPLC, and characterized by LC/MS/MS and (1)H NMR. The structure was confirmed by comparison to a synthetic sample. Both samples confirmed M5 as a product of oxidation of primary hydroxyl group of RVX-208 to carboxylic acid.


Assuntos
Quinazolinas/isolamento & purificação , Quinazolinas/metabolismo , Animais , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Quinazolinas/química , Quinazolinonas , Coelhos
4.
J Am Chem Soc ; 124(19): 5254-5, 2002 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-11996551

RESUMO

Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques.


Assuntos
Lipase/química , Subtilisina/química , Termolisina/química , Catálise , Ativação Enzimática , Estradiol/química , Estradiol/metabolismo , Ligantes , Lipase/metabolismo , Paclitaxel/química , Paclitaxel/metabolismo , Pseudomonas/enzimologia , Solubilidade , Subtilisina/metabolismo , Termolisina/metabolismo , Água/química
5.
Biotechnol Bioeng ; 78(5): 567-75, 2002 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-12115126

RESUMO

Arylsulfotransferase (AST, EC 2.8.2.22), an enzyme capable of sulfating a wide range of phenol-containing compounds was purified from a Clostridium innocuum isolate (strain 554). The enzyme has a molecular weight of 320 kDa and is composed of four subunits. Unlike many mammalian and plant arylsulfotransferases, AST from Clostridium utilizes arylsulfates, including p-nitrophenyl sulfate, as sulfate donors, and is not reactive with 3-phosphoadenosine-5'-phosphosulfate (PAPS). The enzyme possesses broad substrate specificity and is active with a variety of phenols, quinones and flavonoids, but does not utilize primary and secondary alcohols and sugars as substrates. Arylsulfotransferase tolerates the presence of 10 vol% of polar cosolvents (dimethyl formamide, acetonitrile, methanol), but loses significant activity at higher solvent concentrations of 30-40 vol%. The enzyme retains high arylsulfotransferase activity in biphasic systems composed of water and nonpolar solvents, such as cyclohexane, toluene and chloroform, while in biphasic systems with more polar solvents (ethyl acetate, 2-pentanone, methyl tert-butyl ether, and butyl acetate) the enzyme activity is completely lost. High yields of AST-catalyzed sulfation were achieved in reactions with several phenols and tyrosine-containing peptides. Overall, AST studied in this work is a promising biocatalyst in organic synthesis to afford efficient sulfation of phenolic compounds under mild reaction conditions.


Assuntos
Arilsulfotransferase/biossíntese , Arilsulfotransferase/química , Clostridium/enzimologia , Fenóis/química , Sulfatos/química , Animais , Arilsulfotransferase/classificação , Arilsulfotransferase/isolamento & purificação , Catálise , Gatos , Bovinos , Linhagem Celular , Cães , Ativação Enzimática , Fezes/microbiologia , Humanos , Coelhos , Sensibilidade e Especificidade , Esgotos/microbiologia , Especificidade por Substrato
6.
Protein Eng ; 16(4): 303-9, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12736374

RESUMO

Formation of enzyme-oligoamine complexes was suggested as an approach to obtain biocatalysts with enhanced resistance towards inactivation in water-organic media. Complex formation results in broadening (by 20-40% v/v ethanol) of the range of cosolvent concentrations where the enzyme retains its catalytic activity (stabilization effect). At moderate cosolvent concentrations (20-40% v/v) complex formation activates the enzyme (by 3-6 times). The magnitude of activation and stabilization effects increases with the number of possible electrostatic contacts between the protein surface and the molecules of oligoamines (OA). Circular dichroism spectra in the far-UV region show that complex formation stabilizes protein conformation and prevents aggregation in water-organic solvent mixtures. Two populations of the complexes with different thermodynamic stabilities were found in alpha-chymotrypsin (CT)-OA systems depending on the CT/OA ratio. The average dissociation constants and stoichiometries of both low- and high-affinity populations of the complexes were estimated. It appears that it is the low-affinity sites on the CT surface that are responsible for the activation effect.


Assuntos
Quimotripsina/química , Quimotripsina/metabolismo , Poliaminas/química , Solventes/química , Catálise , Dicroísmo Circular , Ativação Enzimática , Estabilidade Enzimática , Etanol , Cinética , Conformação Proteica , Eletricidade Estática , Termodinâmica , Água
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