Synthesis of Isotopically Labeled Fe and S-alkylated Iron-Sulfur Clusters

Abstract

Radical S-adenosylmethionine (SAM) enzymes (RS enzymes) use a 3:1 site-differentiated [Fe₄S₄]⁺ cluster to reductively cleave the SAM cofactor and generate a 5’-deoxyadenosyl radical intermediate (5’-dAdo•) that regio- and stereospecifically abstracts an H-atom from the target substrate. It has been proposed that 5’-dAdo• binds to the unique Fe site before abstracting an Hatom from the substrate. However, due to the transient nature of captured reaction intermediates, their precise structures have yet to be fully elucidated and, therefore, their role in the mechanism of RS enzymes remains unclear. Our group has established reliable methods of synthesizing alkylated [Fe₄S₄] clusters that can serve as models of organometallic intermediates in RS enzyme catalysis. These clusters are competent for radical release and, upon oxidation, undergo an alkyl migration process to yield S-alkylated clusters. A cluster species containing a unique alkylated Fe site with a coordination number greater than four is likely generated in these processes, although a stable cluster of this type has yet to isolated and crystallographically characterized. This work reports the synthesis of α-²H and α-¹³C isotopically labeled Fe and S ethyl ligated [Fe₄S₄] clusters to determine their electron-nuclear hyperfine parameters by ENDOR spectroscopy. These parameters will aid in identification of alkylated [Fe₄S₄] cluster intermediates generated in biological studies. Additionally, in an attempt to synthesize an [Fe₄S₄]⁺³ cluster with a five coordinate, Fe-alkylated site, a series of benzyl and phenyl ligated clusters were prepared and analyzed by NMR and EPR spectroscopies.