Ein Hochdurchsatz‐Fluoreszenz‐Assay zur Bestimmung der Aktivität von Tryptophan‐Halogenasen

Abstract

Die Anwendung von Tryptophan-Halogenasen für die enzymatische Halogenierung hat noch gravierende Limitierungen. Diese kçnnten mittels gerichteter Evolution überwunden werden, jedoch bedarf die Durchmusterung großer Mutantenbibliotheken eines robusten Hochdurchsatz-fähigen Testsystems.I mH inblickd arauf wurdee ine Suzuki-Miyaura-Kreuzkupplung als quantitativer Halogenase-Assaya uf Basis der Bildung eines fluoreszierenden Aryltryptophans entwickelt. Die Methode wurde fürdie Anwendung im E.-coli-Lysat ohne intermediäre Aufarbeitung optimiert und ermçglicht es, halogenierte Tryptophanderivate mit hoher Spezifitäti nd er Mikrotiterplatte quantitativ nachzuweisen. Unter Verwendung dieses Verfahrens wurde eine thermostabile Tryptophan-6-Halogenase gefunden, indem mittels fehlerbehafteter PCR eine Mutantenbibliothek erzeugt wurde,d ie mithilfe der fluorogenen Kreuzkupplung auf erhçhte Thermostabilitätd urchmustert wurde.D iese Methode lieferte eine verbesserte Enzymvariante mit deutlichg esteigerter Stabilitäts owie 2.5-fachv erbesserter Aktivität.

Peptide stapling by late-stage Suzuki–Miyaura cross-coupling

“…Besides addressing the indole C 2 , the regioselective enzymatic halogenation at C5, C6, or C7 using FAD-dependent tryptophan halogenases opens a broad area of Pd-catalysed late-stage diversifications [53][54][55]. It has been proven that Pd-catalysed cross-couplings are very versatile tools for selective and bio-orthogonal modifications of haloindoles, halotryptophans and halotryptophan-containing peptides as well as natural products [56][57][58][59][60][61][62][63][64][65][66][67][68][69][70]. Additionally, halotryptophans were incorporated in pentapeptides as building blocks for macrocyclisation by Suzuki-Miyaura cross-coupling (SMC) aiming at the preparation of bicyclic peptides [71].…”

Synthesis of 7‐Phenylindole Derivatives through Rhodium‐Catalyzed Dehydrogenative Coupling of 2‐(Acetylamino)‐1,1’‐biphenyls with Alkynes

“…[1] A variety of synthetic methods for preparing functionalized indoles have been developed. [2] Among such indoles, 7-phenylindole derivatives have attracted much attention because of their utility for anti-HIV, [3] anticancer, [4] and other bioactivities [5] as well as for fluorescent [6] and anion receptor abilities. [7] Meanwhile, transition-metal-catalyzed CÀ H functionalization has been recognized as a powerful synthetic tool from the atom-and step-economical points of view.…”

“…A variety of synthetic methods for preparing functionalized indoles have been developed [2] . Among such indoles, 7‐phenylindole derivatives have attracted much attention because of their utility for anti‐HIV, [3] anticancer, [4] and other bioactivities [5] as well as for fluorescent [6] and anion receptor abilities [7]…”

Substrate‐Independent High‐Throughput Assay for the Quantification of Aldehydes

“…Recently, Schnepel et al published a fluorescence-based quantitative method for the determination of tryptophan halogenase activity in crude E. coli lysate after derivatization of their halogenated products via a Suzuki-Miyaura coupling. [14] Yan et al developed an elegant method where product formation was directly analyzed on agar plates by using a qualitative multicomponent analytical imaging method. [15] Lee et al developed a growth dependent selection system for novel threonine aldolases based on the consumption of toxic acetaldehyde.…”