Non-enantioselective bio-oxidation of sec-alcohols

Through the development of biochemistry, molecular biology and microbiology, biocatalysis has emerged as an alternative technology for organic synthesis, complementing other areas of catalysis. Key-asset of biocatalysis is the outstanding selectivity of the enzymes, displaying excellent regio-, enantio-, or chemo-selectivity. Together with their simple mode of preparation via fermentation on large scale, biocatalysis has become a viable green alternative for chemistry. The integration of several biocatalytic transformations in a multi-enzyme cascade system is particularly appealing since it offers economic as well as environmental benefits, because the amount of chemicals/solvents required for extraction/purification of intermediates is minimized. prim-Amines, especially allylic amines, are an important class of compounds as intermediates in synthesis. Much progress has been made recently in chemical catalytic enantioselective synthesis of allylic amines. However, a direct stereoselective transformation of easily accessible, racemic allylic alcohols to unprotected, optically active primary allylic amines has not been reported, yet. In this project titled Non-enantioselective bio-oxidation of sec-alcohols for stereoselective amination cascades to optically pure amines, our aim is to construct an artificial multi-enzyme cascade to access enantiopure allylic amines starting from the corresponding racemic alcohols. Since chemical methods with transition metals as catalysts require harsher conditions compared to enzymes, the biocatalytic amination of alcohols would represent an excellent alternative. However, no single enzyme is known to catalyze the direct amination of an alcohol; consequently various multienzyme systems have been designed to convert alcohols to the corresponding amines. All reported methods are based on alcohol oxidation to the corresponding ketone/aldehyde intermediate which is aminated subsequently. To date, only few examples of oxidation-reduction enzymatic cascades have been described for sec-alcohols which are all hampered either by the equilibrium which does not allow full conversion in most cases and/or the necessity to use purified enzymes. Since allylic alcohols represent a special challenge for chemical amination methods, due to the two functional groups in the molecule, this type of alcohol will be the focus of this study. The objective of the project is (i) the development of a oxidase for allylic alcohols, which oxidizes both enantiomers of the substrate, (ii) the investigation of the compatibility of the oxidase with aminating enzymes and (iii) the development of simple and general methods for the preparation of optically pure -chiral allylic amines from readily available allylic alcohols; a general protocol using enzymes is expected to have a major impact, since methods to prepare allylic amines are highly desired. The proposed sequence is not limited by the thermodynamic equilibrium and should be broadly and general applicable.

prim-Amines, especially allylic amines are an important class of compounds in chemistry. Much progress has been made recently in chemical enantioselective synthesis of allylic amines. However, a direct stereleoelective transformation of racemic allylic alcohols to unprotected optically active prim-allylic amines has not been reported, yet. Enzymes are highly selective catalysts, which minimize the formation of side products. In addition, they are environmentally degradable ("Green"), since they are made of amino acids. Specially by using enzymes, reaction conditions are mild, which reduces the need of energy. These features make enzyme catalysis a versatile and easy-to-use platform for production of valuable building blocks for organic synthesis and medicinal chemistry. Since chemical methods with transition metals as catalysts require harsher conditions compared to enzymes, the biocatalytic amination of alcohols represents an excellent alternative. In this project, a one-pot two-enzyme cascade was constructed to access enantiopure allylic amines starting from the corresponding racemic alcohols. The objectives of the project were (i) finding enzymes which can oxidize both enantiomers of racemic substrates, (ii) the investigation of the compatibility of the oxidizing enzymes in the first step of the cascade with the aminating enzymes in the second step and (iii) the development of simple and general methods for the preparation of optically pure -chiral allylic amines from readily available allylic alcohols. A general protocol using enzymes is expected to have a major impact, since methods to prepare allylic amines are highly desired. The proposed sequence is not limited by the thermodynamic equilibrium and should be broadly and general applicable. During the project, a biocatalytic alternative to chemical process for the synthesis of optically active prim-allylic amines was developed. First of all, suitable enzymes (various oxidases and PQQ-dependent dehydrogenase) were identified to oxidize racemic sec-allylic alcohols. In the next step, stereoselective amination of alpha,beta-unsaturated ketone, which represent a special challenge for chemical amination methods, due to the two functional groups in the molecule was established using -transaminases and different amine donors. High conversion level and high enantiomeric excess was obtained for different allylic alcohols.