An enzymatic equivalent to the Friedel-Crafts Alkylation

Carbon-carbon bond formations built a class of most important reactions in the synthetic organic chemistry. The classical Friedel-Crafts reaction has two major disadvantages, i.e. it results in product mixtures of multiply substituted aromatics and it suffers from rearrangements of prim- to sec- and tert-alkyl groups. In addition, the catalysts are hazardous and very corrosive. Overall, this reaction should be substituted by an environmentally benign (bio)process. According on reaction mechanism methyltransferases are enzymes suitable to perform and/or catalyze electrophilic substitution to aromatic systems. S-Adenosyl-L-methionine (SAM) is the major methyl donor for biological methylation reactions catalyzed by methyltransferases (Mtases). During previous investigations on two Mtases found in Streptomyces strains which are involved in the biosynthetic pathway of antibiotics, it could be shown that the substrate scope of the enzymes are rather relaxed to some extent. The Mtases under investigation are able to catalyze the transfer of alkyl groups from artificial cofactors to aromatic compounds. This is astonishing because enzymes are normally very selective regarding their cofactors. The Mtases under investigation transfered the alkyl groups with high regioselectivity and only monosubstitutions were detected, even though the cofactors were supplied in huge access. Some of the alkylated products cannot be synthesized in such a straight forward manner using chemical procedures. This concept could serve as a starting point for a green and selective Friedel-Crafts alkylation. The investigations have come to a stage - the results show this clearly - that fundamental and basic research will help solving the open questions and bring the research to a mature state. More detailed investigations towards the substrate and cofactor scope of the Mtases are necessary. Biochemical characterization is needed to determine the active site of the enzymes and enable proposals for reaction mechanism. A proposed concept for cofactor regeneration shall bring a solution for the use of these cofactor-dependent enzymes in biocatalysis. The regeneration is based on solid-phase synthesis. It is actually the immobilization of a part of the cofactor which is not consumed. After alkyl transfer which is a biotransformation catalyzed by methyltransferase the immobilized residue of the cofactor can be separated easily from the reaction mixture by simple filtration and regenerated in a chemical alkylation step to give active cofactor for the next cycle.

Nature uses S-adenosyl-L-methionine (SAM or AdoMet) dependent methyltransferases for the transfer of methyl groups onto organic substrates. In this project the natural enzymes CouO and NovO have been investigated as suitable catalysts for such biocatalytic alkylation reactions. In collaboration with the structural biologist Prof. Karl Gruber (University of Graz) the structure of CouO has been determined and the reaction mechanism elucidated. The immobilization of the cofactor SAM to a solid support has been investigated, which could lay the path for addressing the problem of cofactor regeneration.