Copper-catalyzed diastereo- and enantioselective desymmetrization of cyclopropenes: Synthesis of cyclopropylboronates

This document is the accepted manuscript version of a Published Work that appeared in final form in Journal of American Chemical Society 136.45, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see DOI: 10.1021/ja510419zA novel Cu-catalyzed diastereo- and enantioselective desymmetrization of cyclopropenes to afford nonracemic cyclopropylboronates is described. Trapping the cyclopropylcopper intermediate with electrophilic amines allows for the synthesis of cyclopropylaminoboronic esters and demonstrates the potential of the approach for the synthesis of functionalized cyclopropanesWe thank the European Research Council (ERC-337776) and MINECO (CTQ2012-35957) for financial support. M. T. and A. P. thank MICINN for RyC and JdC contract

Optimizing the biological activity of Fab fragments by controlling their molecular orientation and spatial distribution across porous hydrogels

Immobilization of antibodies (Ab) on hydrogels though long studied is still a challenge on account of the continuous development of new immune technologies. Enabling methodologies for antibody orientation, antibody stability and maximum recognition of their corresponding antigens is an object of intense study. Mini Ab such Fab fragments are less susceptible to conformational changes on surfaces, or unwanted reactivities compared to a whole Ab molecule. Herein, we have developed an immobilization protocol for a Fab anti epsilon toxin from Clostridium perfringens. Fine tuning of variables during immobilization showed a crucial role in the orientation and the spatial distribution of the Fab across the support surface. The final optimized immune-matrices demonstrated quantitative adsorption of antigen (1:1 molar ratio Fab to antigen) meaning that both the Fab biological activity was maintained after immobilization and an optimal orientation was achieved during the immobilization process. Immobilized Fab gained stability after immobilization as demonstrated by real time protein unfolding.We would like to thank IKERBASQUE, Basque foundation for Science for the funding of Fernando López-Gallego, National Agency of Research and Innovation (ANII, Uruguay) and Universidad ORT Uruguay for their financial support.Peer Reviewe

Purification, immobilization and stabilization of a highly enantioselective alcohol dehydrogenase from Thermus thermophilus HB27 cloned in E. coli

Funding This work has been supported by CAM (grants S0505/PPQ/0344) and MEC (grants BIO2004-02671 and CTQ2005-02420/PPQ). An institutional grant from Fundación Ramón Areces to CBMSO is also acknowledged. J.M. Bolívar and J. Rocha-Martín are holders of a PhD fellowship from CAM. Dr. Ángel Berenguer (Universidad de Alicante) is gratefully acknowledged for his kind help during the writing of this paper.This paper shows the purification and immobilization of a very interesting thermophilic alcohol dehydrogenase from Thermus thermophilus HB27 cloned in Escherichia coli. The purification was based on a first thermal treatment of the crude extract, that leaves the target enzyme in the supernatant, followed by the adsorption of most contaminant proteins in a IMAC column (the target protein did not adsorb on these columns due to the poorness of His residues). Final purification factor was around a 9-fold factor (no other protein bands were detected in SDS-PAGE gels) with an overall yield around 80%. Covalent immobilization of the enzyme on very different supports only permitted to improve the enzyme stability by a 5–10-fold factor, very similarly to the results obtained by the adsorption of the enzyme on polyethyleneimine coated supports. This enzyme adsorbed by ionic exchange maintained the activity unaltered during immobilization which was a very rapid process, and was more stable than the covalent preparations in the presence of organic solvents, and the enzyme was quite strongly adsorbed on the support. Therefore, it was proposed as a good option to prepare industrial biocatalysts of the enzyme. This preparation was utilized in the asymmetric reduction of acetophenone to produce (S)-(−)-1-phenylethanol, with an enantiomeric excess of more than 99%.Comunidad de MadridMinisterio de Educación y Ciencia (MEC)Fundación Ramón ArecesDepto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasFac. de Ciencias QuímicasTRUEpu