Stereoretentive Post-Translational Protein Editing

Abstract

Chemical post-translational methods allow convergent side-chain editing of proteins without needing to resort to genetic intervention. Current approaches that allow the creation of constitutionally native side chains via C–C bond formation, using off-protein carbon-centered C· radicals added to unnatural amino acid radical acceptor (SOMOphile, singly occupied molecular orbital (SOMO)) “tags” such as dehydroalanine, are benign and wide-ranging. However, they also typically create epimeric mixtures of d /l-residues. Here, we describe a light-mediated desulfurative method that, through the creation and reaction of stereoretained on-protein l-alanyl Cβ· radicals, allows Cβ–Hγ, Cβ–Oγ, Cβ–Seγ, Cβ–Bγ, and Cβ–Cγ bond formation to flexibly generate site-selectively edited proteins with full retention of native stereochemistry under mild conditions from a natural amino acid precursor. This methodology shows great potential to explore protein side-chain diversity and function and in the construction of useful bioconjugates.

Carbon-Centered Radicals in Protein Manipulation

“…This scaffold can then be photocatalytically activated to undergo desulfurization creating a stereoretained "on-protein" alanyl radical. 123 Prosthetic Attachment for Initiation at Other Sites (Met). As discussed above, lower copy number residues provide better access to site-selectivity.…”

“…Indeed in “traceless” ligation methods oxidative buffering prior to refolding can be necessary, or, instead, the use of Cys-free targets is adopted. It has also been shown that eliminative mechanisms can compete via other phosphine-mediated desulfurization pathways. , Nonetheless, these examples highlight possible strategies to enable C–S bond scission, for example from intermediate noncanonical amino acid precursors (see below, Figure ), to allow access to on-protein alanyl radicals as productive intermediates.…”

“…Recently, our group has demonstrated site-selective installation of a photolabile tetrafluoropyridyl moiety via Cys arylation (200 equiv perfluoropyridine, 30 min r.t., pH 7.4). This scaffold can then be photocatalytically activated to undergo desulfurization creating a stereoretained “on-protein” alanyl radical . These generated radicals were successfully trapped with various radical acceptors to create C–H, C–O, C–Se, C–B, and C–C bonds to give side chains with native l -amino acid stereochemistry (200 equiv radical acceptors or 1000 equiv B 2 Cat 2 , 100 equiv 4-methylbenzenethiol, UV, 60 min, 4 °C, pH 8.0).…”

“…This demonstrated potential of Cβ−Sγ scission in proteins via the creation of intermediates that allow effective trapping of on-protein sidechain radicals, coupled with known pathways for direct Cα−Sβ scission in Cys (see above), has prompted a recent study (Figure 21). 123 Site-selective installation of a photolabile tetrafluoropyridyl moiety via direct S N 2Ar Cys arylation to create pefluoropyridylcysteine (Pfc) (200 equiv perfluoropyridine, 30 min r.t., pH 7.4) appears to be highly selective and can be monitored advantageously via characteristic 19 F protein NMR signals. This scaffold can then be photocatalytically activated to undergo desulfurization creating a stereoretained "on-protein" alanyl radical via apparent intriguing chargetransfer complexes (proposed to be 'electron donor•acceptor' (EDA) complexes).…”

“…Figure 21. Exploitation of C−S bond scission in an arylated Cys noncanonical amino acid Fpc and alanyl radical trapping in a proposed method for so-called stereoretentive protein editing 123. …”