Near-UV and Visible Light Promote Iron-Catalyzed Site-Specific Radical Transformation of Threonine to Alanine in a Monoclonal Antibody Fc Domain (IgG1-Fc)

Near-UV and visible-light photodegradation is a concern for the development of stable and efficacious protein therapeutics, but mechanistic information on the underlying processes is scarce. We previously reported on the photoconversion and photofragmentation of IgG1-Fc, IgG1, and an IgG1-based fusion protein in the CH₂ domain. Here, we report a novel site-specific photodegradation mechanism, converting Thr into Ala catalyzed by Fe(III). Site-directed mutagenesis and spin-trapping in conjunction with high pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis reveal that this novel photoconversion proceeds via the generation of intermediary protein glycyl and methyl (•CH₃) radicals analogous to metal-catalyzed cross-coupling reactions observed in synthetic organic processes.