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1.
Chembiochem ; 22(13): 2262-2265, 2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-33851500

RESUMEN

Aromatic nitration reactions are a cornerstone of organic chemistry, but are challenging to scale due to corrosive reagents and elevated temperatures. The cytochrome P450 TxtE nitrates the indole 4-position of l-tryptophan at room temperature using NO, O2 and NADPH, and has potential to be developed into a useful aromatic nitration biocatalyst. However, its narrow substrate scope (requiring both the α-amino acid and indole functionalities) have hindered this. Screening of an R59 mutant library of a TxtE-reductase fusion protein identified a variant (R59C) that nitrates tryptamine, which is not accepted by native TxtE. This variant exhibits a broader substrate scope than the wild type enzyme and is able to nitrate a range of tryptamine analogues, with significant alterations to the aromatic and aminoethyl moieties.


Asunto(s)
Nitratos/metabolismo , Triptófano/metabolismo , Sitios de Unión , Sistema Enzimático del Citocromo P-450/metabolismo , Estructura Molecular , Oxidación-Reducción , Especificidad por Sustrato
2.
Angew Chem Int Ed Engl ; 57(42): 13821-13824, 2018 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-30138551

RESUMEN

N-Functionalized amino acids are important building blocks for the preparation of diverse bioactive molecules, including peptides. The development of sustainable manufacturing routes to chiral N-alkylated amino acids remains a significant challenge in the pharmaceutical and fine-chemical industries. Herein we report the discovery of a structurally diverse panel of biocatalysts which catalyze the asymmetric synthesis of N-alkyl amino acids through the reductive coupling of ketones and amines. Reactions have been performed on a gram scale to yield optically pure N-alkyl-functionalized products in high yields.


Asunto(s)
Aminoácidos/química , Biocatálisis , Alquilación , Aminoácidos/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Humanos , Cetonas/química , Pseudomonas/enzimología , Estereoisomerismo
3.
J Org Chem ; 80(2): 950-6, 2015 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-25495724

RESUMEN

P450-BM3 and mutants of this monooxygenase generated by directed evolution are excellent catalysts for the oxidative α-hydroxylation of ketones with formation of chiral acyloins with high regioselectivity (up to 99%) and enantioselectivity (up to 99% ee). This constitutes a new route to a class of chiral compounds that are useful intermediates in the synthesis of many kinds of biologically active compounds.


Asunto(s)
Proteínas Bacterianas/química , Productos Biológicos/química , Sistema Enzimático del Citocromo P-450/química , Cetonas/química , NADPH-Ferrihemoproteína Reductasa/química , Proteínas Bacterianas/metabolismo , Biocatálisis , Catálisis , Sistema Enzimático del Citocromo P-450/metabolismo , Alcoholes Grasos , Hidroxilación , Estructura Molecular , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxidación-Reducción , Estereoisomerismo
4.
Angew Chem Int Ed Engl ; 53(33): 8659-63, 2014 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-24590553

RESUMEN

Regio- and stereoselective oxidative hydroxylation of achiral or chiral organic compounds mediated by synthetic reagents, catalysts, or enzymes generally leads to the formation of one new chiral center that appears in the respective enantiomeric or diastereomeric alcohols. By contrast, when subjecting appropriate achiral compounds to this type of C-H activation, the simultaneous creation of two chiral centers with a defined relative and absolute configuration may result, provided that control of the regio-, diastereo-, and enantioselectivity is ensured. The present study demonstrates that such control is possible by using wild type or mutant forms of the monooxygenase cytochrome P450 BM3 as catalysts in the oxidative hydroxylation of methylcyclohexane and seven other monosubstituted cyclohexane derivatives.


Asunto(s)
Proteínas Bacterianas/metabolismo , Ciclohexanos/química , Sistema Enzimático del Citocromo P-450/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Bacillus megaterium/enzimología , Proteínas Bacterianas/química , Sitios de Unión , Biocatálisis , Carbono/química , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/química , Hidrógeno/química , Hidroxilación , NADPH-Ferrihemoproteína Reductasa/química , Oxidación-Reducción , Estereoisomerismo
5.
J Am Chem Soc ; 135(5): 1665-8, 2013 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-23075382

RESUMEN

Catalytic asymmetric reduction of prochiral ketones of type 4-alkylidene cyclohexanone with formation of the corresponding axially chiral R-configurated alcohols (up to 99% ee) was achieved using alcohol dehydrogenases, whereas chiral transition-metal catalysts fail. Reversal of enantioselectivity proved to be possible by directed evolution based on saturation mutagenesis (up to 98% ee (S)). Utilization of ketone with a vinyl bromide moiety allows respective R- and S-alcohols to be exploited as key compounds in Pd-catalyzed cascade reactions.


Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Alcoholes/metabolismo , Cetonas/metabolismo , Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/genética , Alcoholes/química , Biocatálisis , Cetonas/química , Modelos Moleculares , Estructura Molecular , Oxidación-Reducción , Estereoisomerismo , Thermoanaerobacter/enzimología
6.
Chembiochem ; 13(10): 1465-73, 2012 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-22711296

RESUMEN

Directed evolution of the monooxygenase P450-BM3 utilizing iterative saturation mutagenesis at and near the binding site enables a high degree of both regio- and enantioselectivity in the oxidative hydroxylation of cyclohexene-1-carboxylic acid methyl ester. Wild-type P450-BM3 is 84% regioselective for the allylic 3-position with 34% enantioselectivity in favor of the R alcohol. Mutants enabling R selectivity (>95% ee) or S selectivity (>95% ee) were evolved, while reducing other oxidation products and thus maximizing regioselectivity to >93%. Control of the substrate-to-enzyme ratio is necessary for obtaining optimal and reproducible enantioselectivities, an observation which is important in future protein engineering of these mono-oxygenases. An E. coli strain capable of NADPH regeneration was also engineered, simplifying directed evolution of P450 enzymes in general. These synthetic results set the stage for subsequent stereoselective and stereospecific chemical transformations to form more complex compounds, thereby illustrating the viability of combining genetically altered enzymes as catalysts in organic chemistry with traditional chemical methods.


Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Evolución Molecular Dirigida , Biocatálisis , Carbono/química , Hidrógeno/química , Hidroxilación , Mutagénesis , NADP/metabolismo , Oxidación-Reducción , Ingeniería de Proteínas , Estereoisomerismo , Especificidad por Sustrato
7.
Chembiochem ; 13(1): 80-4, 2012 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-22134988

RESUMEN

We report here the synthesis and biochemical properties of a new peptidyl activity-based probe 1 for SUMO proteases, SENPs. The activity-based probe has at its C terminus a glycine-derived fluoromethylketone moiety as a reactive group designed to target the active-site cysteine of SENPs. Based on a study of the interactions between SENPs and SUMOs, we introduced further design elements that allow the activity-based probe to selectively target SENPs at low micromolar to high nanomolar concentrations. Moreover, 1 out-competes SUMO1 from the reversible SUMO1-SENP1 complex, thus suggesting that 1 and SUMO1 share a common binding site on SENP1.


Asunto(s)
Endopeptidasas/química , Colorantes Fluorescentes/química , Glicina/química , Cetonas/química , Sitios de Unión , Endopeptidasas/biosíntesis , Endopeptidasas/metabolismo , Activación Enzimática , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Glicina/análogos & derivados , Glicina/síntesis química , Glicina/metabolismo , Células HEK293 , Humanos , Cetonas/síntesis química , Cetonas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
8.
Org Biomol Chem ; 10(23): 4516-23, 2012 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-22543859

RESUMEN

We present here a new, general, solid phase strategy for the synthesis of sequence independent peptidyl-fluoromethyl ketones using standard Fmoc peptide chemistry. Our method is based on the synthesis of bifunctional linkers which allows the incorporation of amino acid fluoromethyl ketone unit at the C-terminal end of peptide sequences. Application of this approach for the synthesis of activity based probes for SENPs is also described.


Asunto(s)
Compuestos de Flúor/síntesis química , Cetonas/síntesis química , Péptidos/química , Metilación , Estructura Molecular
9.
Biomacromolecules ; 12(10): 3400-5, 2011 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-21875057

RESUMEN

Self-assembled inorganic-protein arrays with well-defined and controllable size and structure were obtained through the Fe(II) complexation of protein-conjugated terpyridine units (ligand). The atom-level control of the ligand is obtained through residue-specific conjugation between the complexing unit (terpy) containing an activity-based probe and a corresponding active enzyme (papain). The Fe(II)-based self-assembly performed on this unique building block (ligand) leads to chemical species of unprecedented constitution. The first example presented herein opens the way to a shape and size regime usually reserved to polymers.


Asunto(s)
Biotecnología/métodos , Iones/química , Metales/química , Papaína/metabolismo , Análisis por Matrices de Proteínas/métodos , Piridinas/química , Electroforesis en Gel de Poliacrilamida , Compuestos Ferrosos/química , Iones/metabolismo , Ligandos , Metales/metabolismo , Modelos Moleculares , Estructura Molecular , Papaína/química , Piridinas/metabolismo
10.
Inorg Chem ; 50(17): 8132-43, 2011 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-21806046

RESUMEN

Orthopalladated complexes derived from (Z)-2-aryl-4-arylidene-5(4H)-oxazolones have been prepared by reaction of the oxazolone with palladium acetate in acidic medium. The reaction is regioselective, only the ortho C-H bond of the arylidene ring being activated, producing a six-membered ring. The scope and reaction conditions of the orthopalladation are dependent on the acidity of the solvent. In CF(3)CO(2)H a large number of oxazolones can be metalated under mild conditions. As acidity decreases a lesser number of oxazolones can be efficiently palladated and harsher conditions must be used to achieve similar yields. The C-H bond activation in acidic medium agrees with an ambiphilic mechanism, as determined from kinetic measurements at variable temperature and pressure for different oxazolones substituted at the arylidene ring. The mechanism has been confirmed by density functional theory (DFT) calculations, where the formation of the six-membered ring is shown to be favored from both a kinetic and a thermodynamic perspective. In addition, the dependence of the reaction rate on the acidity of the medium has also been accounted for via a fine-tuning between the C-H agostic precoordination and the proton abstraction reaction in the overall process occurring on coordinatively saturated [Pd(κ(N)-oxazolone)(RCO(2)H)(3)](2+).


Asunto(s)
Acetatos/química , Compuestos Organometálicos/química , Oxazolona/química , Teoría Cuántica , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Estructura Molecular , Compuestos Organometálicos/síntesis química , Oxazolona/síntesis química , Estereoisomerismo , Termodinámica
11.
J Biotechnol ; 293: 56-65, 2019 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-30690098

RESUMEN

N-Alkylated-α-amino acids are useful building blocks for the pharmaceutical and fine chemical industries. Enantioselective methods of N-alkylated-α-amino acid synthesis are therefore highly valuable and widely investigated. While there are a variety of chemical methods for their synthesis, they often employ stoichiometric quantities of hazardous reagents such as pyrophoric metal hydrides or genotoxic alkylating agents, whereas biocatalytic routes can provide a greener and cleaner alternative to existing methods. This review highlights the occurrence of the N-alkyl-α-amino acid motif and its role in nature, important applications towards human health and biocatalytic methods of preparation. Several enzyme classes that can be used to access chiral N-alkylated-α-amino acids and their substrate selectivities are detailed.


Asunto(s)
Aminoácidos/biosíntesis , Aminoácidos/química , Animales , Biocatálisis , Humanos , Naturaleza
13.
Chem Commun (Camb) ; 51(12): 2208-24, 2015 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-25483552

RESUMEN

Cytochrome P450 enzymes (CYPs) have been used for more than six decades as catalysts for the CH-activating oxidative hydroxylation of organic compounds with formation of added-value products. However, it has not been possible to control regio- and stereoselectivity in a general manner, which is necessary for wide (industrial) applications. Especially directed evolution as a Darwinian approach to protein engineering has changed the situation for the better during the last 3-4 years leading to extensive progress, which is summarized and analysed in this Feature Article.


Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Compuestos Orgánicos/metabolismo , Biocatálisis , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Evolución Molecular , Hidroxilación , Simulación de Dinámica Molecular , Compuestos Orgánicos/química , Oxidación-Reducción , Polimorfismo Genético , Estructura Terciaria de Proteína , Estereoisomerismo , Testosterona/química , Testosterona/metabolismo
14.
Chem Commun (Camb) ; 50(92): 14310-3, 2014 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-25168091

RESUMEN

Mutants of P450-BM3 evolved by directed evolution are excellent catalysts in the CH-activating oxidative hydroxylation of 1-tetralone derivatives and of indanone, with unusually high regio- and enantioselectivity being observed. Similar results were achieved in the oxidative hydroxylation of tetralin and indane. The products are useful building blocks in the synthesis of a number of biologically active compounds.


Asunto(s)
Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , NADPH-Ferrihemoproteína Reductasa/química , Tetralonas/química , Hidroxilación , Indanos/química , Oxidación-Reducción , Estereoisomerismo
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