Radical Mechanism andStereochemical Control in ConsecutiveC–C Bond Formation by the Nonheme Iron Enzyme Hvm1

Abstract Iron(II)/2-oxoglutarate-dependent (Fe/2OG) enzymes catalyze consecutive C–C bond formations to assemble complex heterobicyclic ring systems and generate three new stereocenters in piperazine alkaloids helvamide B and the arizonamides through C(sp3)–H activation─a transformation that remains challenging in synthetic chemistry. Here, we report a comprehensive mechanistic study of this unique transformation catalyzed by the Fe/2OG enzyme Hvm1, using a combination of deuterated substrates, substrate analogs bearing electron-withdrawing substituents, and multiple spectroscopic methods (LC-MS, X-ray crystallography, CD spectroscopy, and NMR). The reaction proceeds via consecutive radicaloid C–C (C3′–C2 and C3–C3″) couplings, involving sequential radical attack on the olefin and benzoyl group (Minisci variant), initiated by a C3′ radical generated through C3′ pro-S hydrogen atom transfer (HAT). The first C–C bond (C3′–C2) is formed on the Si-face of C2 with retention of C3′ configuration. The second C–C bond (C3–C3″) formation can proceed with either of two stereochemical senses─antarafacial or suprafacial─relative to the first newly formed C3′–C2 bond: the antarafacial pathway leads to helvamide B, while the suprafacial pathway affords the previously unreported epimer, helvamide A. Crystal structure analysis identifies Y67 as a key residue governing the partitioning of stereochemical outcomes in the second C–C bond formation. Furthermore, the conclusive stereochemical assignment of helvamide B corrects the prior misassignment of the C3′ configuration in the arizonamides.