Establishing an integrated biocatalytic Friedel-Crafts platform for the alkylation of aromatic small molecules

The Friedel-Crafts alkylation is one of the fundamental pillars of synthetic organic chemistry and is used extensively in both academia and industry to alkylate arenes and heteroarenes. Despite the central importance of this reaction in organic synthesis, both alkylated and acylated Friedel-Crafts versions suffer from a number of significant shortfalls. Firstly, these reactions typically require the use of stoichiometric amounts of strong Lewis acid catalysts (e.g. BF3, AlCl3, FeCl3, SnCl4, and TiCl4) and protic acids (such as HF and H2SO4) to accelerate this reaction. Secondly, toxic alkyl halide reagents are also required, producing large amounts of harmful metal by-products. Finally, monoalkylation of arenes and heteroarenes can be difficult to control, producing alkylated mixtures which can be challenging to purify. Conversely, there is a pressing need to develop regioselective, catalytic and more environmentally benign variants of F-C reactions that could be potentially applied on either small or large scale.

A departure from accessing C-alkylated products produced via a chemical F-C process is to explore a biocatalytic route. In Nature, methylation of aromatic substrates can be achieved catalytically using methyl transferases (Mtases) and the cofactor S-adenosylmethionine (SAM) as the methylating agent. This C-methylation could be considered a Friedel-Crafts-like process, thereby opening up opportunities for further exploitation in the regiospecific alkylation of aromatic small molecules. Our collaboration is interested in exploring the potential of expanding the substrate scope of the methyl transferases NovO (Streptomyces rishiriensis) and CuoO (Streptomyces spheroides). These enzymes are known to transfer methyl groups using the SAM cofactor to cuomarin antibiotics coumermycin A (CouO) and novobiocin (NovO). Initial investigations have shown that both NovO and CouO can be overexpressed in E.coli and purified. Furthermore, both NovO and CouO have been shown to be active in our hands, C-methylating a number of coumarin scaffolds present in our compound library. One of the current limitations that we have identified during the course of this work is the need to prepare SAM and alkylated SAM analogues by current chemical synthetic routes. SAM and alkylated analogues are expensive to prepare and are unstable under aqueous neutral and alkaline conditions. What is now required to progress this programme of research forward is a robust biocatalytic platform to couple the formation of SAM cofactors (Step 1) with alkyl transfer to a library of coumarin scaffolds by NovO/CouO (Step 2).

Aims

(i) To explore the substrate scope of NovO and CuoO to accept alkylated and fluoroalkylated SAM analogues.

(ii) To identify engineered variants - by a combination of directed evolution and site-directed mutagenesis - of the wildtype SalL that possess enhanced thermostability, solvent tolerance and can catalyse the formation of alkylated SAM analogues.

(iii) To investigate the compatibility of engineered SalL variants identified in Aim 2 to be coupled with a biocatalytic F-C reaction of cuomarin scaffolds using NovO/CuoO