Project Summary Methyl groups are an important structural modification in natural products and are often essential for the effectiveness of natural products as antibacterial and/or antitumor agents. In nature, these methyl groups are most often appended by S-adenosylmethionine (SAM)-dependent methyltransferases (MTs) in a regioselective and stereoselective manner. This proposal focuses on transferring fluoromethyl and other fluorine-containing alkyl groups to natural products, because fluorine atoms have been shown to enhance the pharmacological effects of many drugs. In fact, about 20-30% of all drugs contain at least one fluorine atom. Strategies for introducing fluorine into biomolecules are therefore at the forefront of organic and medicinal chemistry. In this proposal, the the synthesis of novel fluoromethyl and difluoromethyl donors that have biological relevance is described. Indeed, we show that these fluorine-containing analogs of SAM, Te-adenosyl-(fluoromethyl)- telluromethionine (FMeTeSAM) and Te-adenosyl-(difluoromethyl)-telluromethionine (F2MeTeSAM) are acted upon by SAM-dependent MTs to transfer fluoroalkyl groups to target substrates, including catechol-O- methyltransferase-catalyzed O-fluoromethylation on dihydroxybenzoic acid and O-difluoromethylation on norepinephrine; nicotinamide N-methyltransferase-catalyzed N-fluoromethylation on nicotinamide; NovO- catalyzed C-fluoromethylation on coumarin; and 6-mercaptopurine methyltransferase-catalyzed S- difluoromethylation on 6-mercaptopurine. Ideally, this ability will be leveraged to modify methyl-containing natural products of clinical value (e.g. novobiocin and oxaline) with fluoromethyl groups in a facile manner at a late stage in their biosynthetic pathways, and to assess whether their pharmacological effects are improved. Other aims will focus on the enzymatic synthesis of β-fluoromethyl amino acids, which are found in a number of important antibiotics, as well as the addition of fluoromethyl groups to completely unactivated carbon centers via radical mechanisms.