In order to put the previously proposed concept of directed evolution of hybrid catalysts (proteins that harbor synthetic transition-metal catalysts or organocatalysts) into practice, several prerequisites must be met. The availability of a robust host protein that can be expressed in sufficiently large amounts, and that can be purified in a simple manner is crucial. The thermostable enzyme tHisF from Thermotoga maritima, which constitutes the synthase subunit of a bi-enzyme complex that is instrumental in the biosynthesis of histidine, fulfills these requirements. In the present study, fermentation has been miniaturized and parallelized, as has purification of the protein by simple heat treatment. Several mutants with strategically placed cysteines for subsequent bioconjugation have been produced. One of the tHisF mutants, Cys9Ala/Asp11Cys, was subjected to bioconjugation by the introduction of a variety of ligands for potential metal ligation, of a ligand/metal moiety, and of several organocatalytic entities that comprise a flavin or thiazolium salts. Characterization by mass spectrometry and tryptic digestion was achieved. As a result of this study, a platform for performing future directed evolution of these hybrid catalysts is now available.
Abstract. Herein, we report a detailed study on the electrophilic alkynylation of cyclic keto-esters and amides with ethynylbenziodoxolone (EBX) reagents. The structure and stability of this class of reagents is first described more in details. DSC experiments showed a strong exothermic decomposition with EBX reagents, leading to guidelines for the safe use of these compounds. The extension of the method to aromatic alkynes and a broad range of benziodoxol(on)e reagents is then reported. Based on our preliminary results using cinchona-based phase-transfer catalysts, the enantioselective alkynylation of cyclic keto-esters could be achieved. Binaphthyl-derived ammonium catalysts developed by Maruoka and co-workers gave the highest asymmetric induction with up to 79% ee for an indanone-derived keto-ester.Throughout this work, asymmetric induction was observed only in the case of benziodoxolone reagents, demonstrating their superiority over conventional alkynyliodonium salts. The deeper understanding gained about the factors leading to higher asymmetric induction will be very useful in the future to develop a truly general and highly enantioselective alkynylation method.
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