Selective methylation and alkylation using methyl transferases

Background: Methyl groups can have a significant beneficial effect on the physicochemical and
biological properties of bioactive compounds. This so called "methyl group effect", has been
widely exploited in medicinal chemistry to increase lipophilicity and bioavailability, as well as
protecting compounds in vivo from enzymatic degradation, thus offering a method for finetuning a
drug candidate's metabolism and efficacy in therapeutic applications.1 Methylation can also alter
the conformations of small molecules, through stereoelectronic and steric effects. However, it is
very difficult to achieve selective methylation reactions using traditional synthetic approaches and
typically, toxic reagents such as methyl iodide are used. Enzymatic methylation provides a very
valuable alternative method using methyl transferases (MTs) which have received comparatively
little attention to date in biocatalytic syntheses.2 They are particularly useful enzymes for the
regioselective 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 other
enzymes to drive the equilibrium towards the desired product, as well as generating the expensive
co-factor S-adenosyl methionine (SAM) in situ.3,4 In preliminary studies we have used different
MTs with dopamine and analogues for selective methylations, together with 5'-
methylthioadenosine/S-adenosyl homocysteine nucleosidase (MTAN). Furthermore, we have
used a methionine adenosyltransferase (MAT) for the formation of SAM and SAM analogues to
expand the approach, enabling other selective alkylations.2,3
Aims: In this interdisciplinary project we will develop MTs and the cofactor supply/recycling
systems in enzymatic reaction cascades with a range of compounds for the selective methylation
of O-, N- and C-groups. In addition, we aim to integrate the in situ alkylation of enzymatically
generated hydroxyl or amino groups formed via other enzymes to ensure the construction of
2
highly integrated new reaction cascades towards heterocyclic compounds using SAM and SAM
analogues.
Project: During the training rotations in Chemistry (with Helen Hailes) and Biochemical
Engineering (with the co-supervisor John Ward) the student will develop skills in enzyme
expression, enzyme screening, substrate synthesis, assays, product isolation and
characterisation. They will also use available MTs with alkaloids5 or alcohols and amines in
biocatalytic reactions to establish initial regio and stereoselectivities. This will include the use of
MAT for the formation of SAM in situ and MTAN to drive the reaction towards the product They
will then focus on establishing an initial transaminase (TAm) + MT cascade with a view to
optimising the cascade with a methylation/ethylation step and incorporation of MAT and MTAN. In
addition they will identify new O-, N-, C-MTs using metagenomic and bioinformatics strategies and
screen screen these to establish the substrate scope. In the later half of the PhD they will use of
MTs 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 can
be prepared in situ as previously described.5 Also, the use of amino alcohols for allylation and
subsequent chemical cyclisations to produce a chemoenzymatic cascade. If required MT
mutagenesis will be carried out to enhance enzyme performance. In the final year, building upon
successful results, extension to other enzyme cascades and