Engineered cytochrome P450 monooxygenase variants are reported as highly active and selective catalysts for the bioorthogonal uncaging of propargylic and benzylic ether protected substrates, including uncaging in living E. coli. observed selectivity is supported by induced-fit docking and molecular dynamics simulations. This proof-of-principle study points towards the utility of bioorthogonal enzyme/protecting group pairs for applications in the life sciences.
The investigation of stereoselective biocatalytic transformations at a single-cell level is to date an unsolved challenge. Here, we report the development of an integrated microfluidic device which enables the analytical characterization of enantioselective reactions at nanoliter scale by combining whole-cell catalyzed on-chip syntheses, chiral microchip electrophoresis, and label-free detection of enantiomers by deep UV time-resolved fluorescence. Using Escherichia coli expressing recombinant Aspergillus niger epoxide hydrolase as the model enzyme for various enantioselective reactions, we evaluated the approach for downscaling the reaction to a few hundred cells. Our work is thus an important step toward the analysis of single-cell stereoselective biocatalysis.
Acetamide and thioacetamide react with the superacid solutions HF/MF 5 (M = As, Sb) under formation of the corresponding salts [H 3 CC(OH)NH 2 ] + MF 6 and [H 3 CC(SH)NH 2 ] + MF 6 -(M = As, Sb), respectively. The reaction of DF/AsF 5 with acetamide and thioacetamide lead to the corresponding deuterated salts [H 3 CC(OD)ND 2 ] + AsF 6 and [H 3 CC(SD)ND 2 ] + AsF 6 -, respectively . The salts are characterized by vibrational and NMR spectroscopy, and in the case of [H 3 CC(OH)NH 2 ] + AsF 6 and [H 3 CC(SH)NH 2 ] + AsF 6 also by singlecrystal X-ray analyses. The [H 3 CC(OH)NH 2 ] + AsF 6 -(1) salt crys-* Prof. Dr. A. Kornath
Wir berichtenü ber die Verwendung evolvierter Varianten von Cytochrom-P450-Monooxygenasen als hochaktive und hochselektive Katalysatoren für die bioorthogonale Aktivierung Propargylether-u nd Benzylether-maskierter Substrate,e inschließlich der erfolgreichen Entschützung in lebenden E. coli. Docking-Studien und Moleküldynamik-Simulationen stützen die beobachtete Selektivität. Diese Studie veranschaulichtd en mçglichen Nutzen von bioorthogonalen Paaren aus Enzym und Schutzgruppe für Anwendungen in den Lebenswissenschaften.
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