A unique π-expanded reaction cavity tethering a polycyclic moiety which provides a platform for substrate binding was constructed within the robust β-barrel structure of nitrobindin (NB). NB variants with cavities of different sizes and shapes are coupled with N-(1-pyrenyl)maleimide (Pyr) to prepare a series of NB-Pyr conjugates. The orientation of the pyrene moiety is fixed within the cavity by the coupling reaction. The fluorescent quenching analysis of NB-Pyr indicates that azachalcone (aza), which is a dienophile for a Diels-Alder (DA) reaction, is efficiently incorporated within the pyrene-linked reaction cavity by the aromatic interaction. The DA reaction between aza and cyclopentadiene proceeds within the reaction cavity of NB-Pyr in the presence of Cu ion in high yield and high enantio- and regioselectivity.
Directed evolution of Cp*RhIII‐linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high‐throughput screening (HTS) platform that involves an affinity purification step employing a starch‐agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP‐tagged biohybrid catalysts in a 96‐well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII‐linked NB(T98H/L100K/K127E) variant with a 4.9‐fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.
A unique π‐expanded reaction cavity tethering a polycyclic moiety which provides a platform for substrate binding was constructed within the robust β‐barrel structure of nitrobindin (NB). NB variants with cavities of different sizes and shapes are coupled with N‐(1‐pyrenyl)maleimide (Pyr) to prepare a series of NB‐Pyr conjugates. The orientation of the pyrene moiety is fixed within the cavity by the coupling reaction. The fluorescent quenching analysis of NB‐Pyr indicates that azachalcone (aza), which is a dienophile for a Diels–Alder (DA) reaction, is efficiently incorporated within the pyrene‐linked reaction cavity by the aromatic interaction. The DA reaction between aza and cyclopentadiene proceeds within the reaction cavity of NB‐Pyr in the presence of CuII ion in high yield and high enantio‐ and regioselectivity.
Ein Pyren‐verknüpfter Proteinhohlraum zieht zwei Substratmoleküle an – ungefähr so wie der Duft einer Blume Schmetterlinge anlockt. A. Onoda, T. Hayashi und Mitarbeiter demonstrieren in ihrer Zuschrift auf S. 13806, dass eine polycyclische Pyrengruppe, die an das starre Proteingerüst der β‐Fass‐Struktur von Nitrobindin geknüpft ist, aromatische Wechselwirkungen mit einem Substrat bereitstellt. Eine asymmetrische Diels‐Alder‐Reaktion zwischen Azachalkon und Cyclopentadien verläuft glatt mit hoher Stereoselektivität innerhalb des Reaktionsgerüsts.
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