A convenient synthetic route to (2,4)-methylproline and its exploration for protein engineering of thioredoxin.

4-Substituted prolines, especially 4-fluoroprolines, have been widely used in protein engineering and design. Here, we report a robust and stereoselective approach for the synthesis of (2,4)-methylproline starting from (2)-pyroglutamic acid. Incorporation studies with both (2,4)- and (2,4)-methylproline into the Trx1P variant of the model protein thioredoxin of show that the stereochemistry of the 4-methyl group might be a key determinator for successful incorporation during ribosomal synthesis of this protein.