Synthetic reagents for in vitro enzymatic and protein alkylation

Developing novel methods for selective bond formation is a persistent pursuit in organic chemistry. Alkylation is the chemical process that introduces sp3 C-X bonds into organic compounds to synthesize complex molecules from simple fragments. With excellent efficiency and selectivity, enzymatic alkylation has emerged as an alternative strategy in organic synthesis. In particular, S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs) catalyze alkylation reactions using SAM and its derivatives as alkyl donors. Besides being a methyl donor, SAM is also a multifunctional compound that donates methylene, aminocarboxypropyl, adenosyl, and amino moieties during natural product biosynthetic reactions. Thus, the production and utilization of SAM and its derivatives have gained increased importance and attention, which are the focus of this thesis.
As SAM is relatively unstable and expensive for stoichiometric use as a methyl donor, regenerating SAM in situ is a feasible solution to overcome the impediment for large-scale biocatalysis applications of SAM-dependent MTs. The initial design of halide methyltransferase (HMT)-catalyzed SAH methylation for SAM regeneration depended on a volatile, highly toxic, and comparatively high-cost reagent methyl iodide (MeI). In this thesis, we have exploited several non-volatile sulfate- and sulfonate-based methylation reagents for SAH methylation by different promiscuous thiopurine methyltransferase (TPMT) homologs. A simple method combining a TPMT homolog and a synthetic methylation reagent for in vitro SAM regeneration from SAH shows similar efficiency to the MeI-dependent process.
SAH as the precursor of SAM comes with a hefty price tag, especially for preparative-scale production. We have developed a three-enzyme cascade for in vitro stereoselective synthesis of SAH from two low-priced reagents. The production of SAH at a hundred-milligram scale has been achieved with a high isolated yield, showing great potential for industrial applications. Moreover, we describe an all-enzymatic strategy for synthesizing a methyl-edited SAM derivative, β-methyl SAM. An unnatural methylated amino acid has been generated enzymatically from β-methyl SAM, indicating the feasibility of using biocatalysts for methyl-editing diverse bioactive compounds.
In addition to enzymatic alkylation of small molecules, we have ventured towards protein alkylation using novel supramolecular reagents. Selenoimidazolium salts enable selective N-alkylation of native proteins via supramolecular interactions. In contrast to leaving groups of conventional alkylation agents, the selenoimidazole moiety plays a non-innocent role in promoting specific alkylation.