Kurzbeschreibung
(Englisch)
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Artificial metalloenzymes result from incorporation of a catalyst within a protein environment. In this context, supramolecular anchoring by means of the biotin-streptavidin technology has proven most versatile. Within this project, it is proposed to rely on a dual biotin anchoring strategy to activate a latent catalyst, inactive in the absence of the host protein. To achieve this goal, we will rely on in silico docking simulations to design suitable biotinylated bidentate ligands towards the activation of biotinylated piano stool complexes. Thermodynamic studies and X-ray analysis will be carried out to characterize the resulting artificial metalloenzymes. These will be tested towards the reduction of prochiral imines
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Abstract
(Englisch)
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Building on the findings described in the previous report, we completed the engineering and the optimization of an artificial zymogen. This work was recently pusblished in Angewandte Chemie Intenational Edition as a hot paper fZ. Liu, V. Lebrun, T. Kitanosono, H. Mallin, V. Köhler, D. Häussinger, D. Hilvert, S. Kobayashi, T. R. Ward, Angew. Chem. Int. Ed. 2016, DOl: 10.1 002/anie.201 605010). This artificial metalloenzyme is based on a biotinylated [Cp*lrL3] complex incorporated in a streptavidin (Sav) scaffoid. This construct displays very limited transfer hydrogenation activity. Addition of an iridium-binding tripeptide leads to an active artificial transfer hydrogenase. After optimizing independently the host protein and the tripeptide ligand, combinations yielded constructs dispiaying high activity (up to > 2000 TON) and/or high enantioselectivity (up to 81 % ee) for the salsolidine precursor. The activating tripeptide, as weil as the Factor Xa recognition sequence, were encoded at the C-terminus of the Sav. After expression, addition of the inactive biotinyiated cofactor yields the artificiai zymogen. Its latent imine transfer hydrogenase (ATHa5) activity is unleashed upon cleavage by Factor Xa, a natural protease. Upon up-regulation, the zymogen unleashes its activity (both in terms of enantioselectivity and turnover number) obtained with the chemically synthesized peptide.
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