Radical Mechanism and Stereochemical Control in Consecutive C-C Bond Formation by the Nonheme Iron Enzyme Hvm1.

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.