S-Adenosyl-L-methionine (AdoMet) is the major methyl donor for biological methylation reactions catalyzed by methyltransferases. We report the first chemical synthesis of AdoMet analogs with extended carbon chains replacing the methyl group and their evaluation as cofactors for all three classes of DNA methyltransferases. Extended groups containing a double or triple bond in the beta position to the sulfonium center were transferred onto DNA in a catalytic and sequence-specific manner, demonstrating a high utility of such synthetic cofactors for targeted functionalization of biopolymers.

The 2.0 A crystal structure of the N6-adenine DNA methyltransferase M.TaqI in complex with specific DNA and a nonreactive cofactor analog reveals a previously unrecognized stabilization of the extrahelical target base. To catalyze the transfer of the methyl group from the cofactor S-adenosyl-l-methionine to the 6-amino group of adenine within the double-stranded DNA sequence 5'-TCGA-3', the target nucleoside is rotated out of the DNA helix. Stabilization of the extrahelical conformation is achieved by DNA compression perpendicular to the DNA helix axis at the target base pair position and relocation of the partner base thymine in an interstrand pi-stacked position, where it would sterically overlap with an innerhelical target adenine. The extrahelical target adenine is specifically recognized in the active site, and the 6-amino group of adenine donates two hydrogen bonds to Asn 105 and Pro 106, which both belong to the conserved catalytic motif IV of N6-adenine DNA methyltransferases. These hydrogen bonds appear to increase the partial negative charge of the N6 atom of adenine and activate it for direct nucleophilic attack on the methyl group of the cofactor.

Pass and click: Protein methylation is an important posttranslational modification. Because the methyl group is a poor reporter group, new methods are needed to analyze methyltransferase substrates. A S‐adenosyl‐L‐methionine‐based cofactor was synthesized and used for the site‐specific functionalization of proteins with alkynes by methyltransferases (first step) and subsequent labeling through CuAAC click chemistry (second step; see scheme).

Here we describe a one-step synthetic procedure for the preparation of S-adenosyl-L-methionine (AdoMet) analogs with extended carbon chains replacing the methyl group. These AdoMet analogs function as efficient cofactors for DNA methyltransferases (MTases), and we provide a protocol for sequence-specific transfer of extended side chains from these AdoMet analogs to DNA by DNA MTases. Direct chemoselective allylation or propargylation of S-adenosyl-L-homocysteine (AdoHcy) at sulfur is achieved under the acidic conditions needed to protect other nucleophilic positions in AdoHcy. The unsaturated bonds in beta position to the sulfonium center of the resulting AdoMet analogs are designed to stabilize the transition state formed upon DNA MTase-catalyzed nucleophilic attack at the carbon next to the sulfonium center and lead to efficient transfer of the extended side chains to DNA. Using these protocols, sequence-specific functionalized DNA can be obtained within one to two weeks.