Site‐Selective Modification of Peptides and Proteins via Interception of Free‐Radical‐Mediated Dechalcogenation

Abstract

The development of site‐selective chemistry targeting the canonical amino acids enables the controlled installation of desired functionalities into native peptides and proteins. Such techniques facilitate the development of polypeptide conjugates to advance therapeutics, diagnostics, and fundamental science. We report a versatile and selective method to functionalize peptides and proteins through free‐radical‐mediated dechalcogenation. By exploiting phosphine‐induced homolysis of the C−Se and C−S bonds of selenocysteine and cysteine, respectively, we demonstrate the site‐selective installation of groups appended to a persistent radical trap. The reaction is rapid, operationally simple, and chemoselective. The resulting aminooxy linker is stable under a variety of conditions and selectively cleavable in the presence of a low‐oxidation‐state transition metal. We have explored the full scope of this reaction using complex peptide systems and a recombinantly expressed protein.

Carbon-Centered Radicals in Protein Manipulation

“…In peptidic systems alanyl-radicals generated in this way have shown exciting promise by taking similar advantage of phosphine to activate the C β −S γ bond. 120 The presence of an alanyl radical could be promisingly inferred by trapping with TEMPO-derivatives in C−O bond formation (100 equiv TCEP, 20 equiv Mn(OAc) 3 , 5 equiv functionalized TEMPO-based traps, 2 h, 50 °C, pH 6.5) (Figure 14). 120 Such reactions including prior strategies for desulfurization at cysteine, cystine, or selenenylcysteines proceed via a seemingly complex or possibly multiple-manifold process 121 involving the likely intermediate formation of thiophosphoranyl radical adducts as precursors to C• radicals formed upon C−S bond homolysis via β-scission.…”

“…A possible mechanism of TCEP-mediated desulfurization of Cys, via thiyl radicals, 115 forming C• alanyl radicals that can be trapped by TEMPO derivatives. 120 borylation, 137 and alkynylation. 138 Nevertheless, this methodology has to date been limited to short oligopeptides, in part due to the lack of selectivity with respect to unprotected sidechains (i.e., Asp and Glu), as well as the additional operational complexity, due to the required RAE conversion/generation step.…”

“…Interestingly it has been proposed for over 60 years that C• alanyl radicals may be intermediates in observed Cys desulfurization reactions. , Such desulfurizations are exploited in so-called “traceless native chemical ligation” − to convert Cys to desulfurized Ala residues. In peptidic systems alanyl-radicals generated in this way have shown exciting promise by taking similar advantage of phosphine to activate the C β –S γ bond . The presence of an alanyl radical could be promisingly inferred by trapping with TEMPO-derivatives in C–O bond formation (100 equiv TCEP, 20 equiv Mn­(OAc) 3 , 5 equiv functionalized TEMPO-based traps, 2 h, 50 °C, pH 6.5) (Figure ).…”

“…A possible mechanism of TCEP-mediated desulfurization of Cys, via thiyl radicals, forming C• alanyl radicals that can be trapped by TEMPO derivatives …”

Cysteine‐Selective Modification of Peptides and Proteins via Desulfurative C−C Bond Formation

“…Recently, we described an effective C(sp 3 )−C(sp 3 ) bond forming method that involves the interception of visible‐light‐mediated desulfurization using N ‐modified allylamines to install native lysine (Lys) sidechains carrying a broad range of PTMs (Figure 1B). [38] The reaction proceeds via the generation and interception of an alanyl radical [39–40] . This technique is operationally simple, it can be run on the bench under ambient atmosphere using inexpensive LEDs, and the product of the reaction retains the native stereochemistry at the α‐centre of the target amino acid.…”