Selective methylation and alkylation using methyl transferases

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Background: Methyl groups can have a significant beneficial effect on the physicochemical andbiological properties of bioactive compounds. This so called "methyl group effect", has beenwidely exploited in medicinal chemistry to increase lipophilicity and bioavailability, as well asprotecting compounds in vivo from enzymatic degradation, thus offering a method for finetuning adrug candidate's metabolism and efficacy in therapeutic applications.1 Methylation can also alterthe conformations of small molecules, through stereoelectronic and steric effects. However, it isvery difficult to achieve selective methylation reactions using traditional synthetic approaches andtypically, toxic reagents such as methyl iodide are used. Enzymatic methylation provides a veryvaluable alternative method using methyl transferases (MTs) which have received comparativelylittle attention to date in biocatalytic syntheses.2 They are particularly useful enzymes for theregioselective methylation of compounds as well as the diversification of compound libraries.In recent work we have started to investigate the cloning and use of MTs, together with otherenzymes to drive the equilibrium towards the desired product, as well as generating the expensiveco-factor S-adenosyl methionine (SAM) in situ.3,4 In preliminary studies we have used differentMTs with dopamine and analogues for selective methylations, together with 5'-methylthioadenosine/S-adenosyl homocysteine nucleosidase (MTAN). Furthermore, we haveused a methionine adenosyltransferase (MAT) for the formation of SAM and SAM analogues toexpand the approach, enabling other selective alkylations.2,3Aims: In this interdisciplinary project we will develop MTs and the cofactor supply/recyclingsystems in enzymatic reaction cascades with a range of compounds for the selective methylationof O-, N- and C-groups. In addition, we aim to integrate the in situ alkylation of enzymaticallygenerated hydroxyl or amino groups formed via other enzymes to ensure the construction of2highly integrated new reaction cascades towards heterocyclic compounds using SAM and SAManalogues.Project: During the training rotations in Chemistry (with Helen Hailes) and BiochemicalEngineering (with the co-supervisor John Ward) the student will develop skills in enzymeexpression, enzyme screening, substrate synthesis, assays, product isolation andcharacterisation. They will also use available MTs with alkaloids5 or alcohols and amines inbiocatalytic reactions to establish initial regio and stereoselectivities. This will include the use ofMAT for the formation of SAM in situ and MTAN to drive the reaction towards the product Theywill then focus on establishing an initial transaminase (TAm) + MT cascade with a view tooptimising the cascade with a methylation/ethylation step and incorporation of MAT and MTAN. Inaddition they will identify new O-, N-, C-MTs using metagenomic and bioinformatics strategies andscreen screen these to establish the substrate scope. In the later half of the PhD they will use ofMTs with other-SAM analogues such as allyl-SAM, synthesised in situ from MTAN and allylmethionine,will be explored to give a 3- enzyme one pot allylation reaction with alkaloids that canbe prepared in situ as previously described.5 Also, the use of amino alcohols for allylation andsubsequent chemical cyclisations to produce a chemoenzymatic cascade. If required MTmutagenesis will be carried out to enhance enzyme performance. In the final year, building uponsuccessful results, extension to other enzyme cascades and