Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials

Abstract

The advent of antibody-drug conjugates as pharmaceuticals has fuelled a need for reliable methods of site-selective protein modification that furnish homogeneous adducts. Although bioorthogonal methods that use engineered amino acids often provide an elegant solution to the question of selective functionalization, achieving homogeneity using native amino acids remains a challenge. Here, we explore visible-light-mediated single-electron transfer as a mechanism towards enabling site- and chemoselective bioconjugation. Specifically, we demonstrate the use of photoredox catalysis as a platform to selectivity wherein the discrepancy in oxidation potentials between internal versus C-terminal carboxylates can be exploited towards obtaining C-terminal functionalization exclusively. This oxidation potential-gated technology is amenable to endogenous peptides and has been successfully demonstrated on the protein insulin. As a fundamentally new approach to bioconjugation this methodology provides a blueprint toward the development of photoredox catalysis as a generic platform to target other redox-active side chains for native conjugation.

Figure 1:. Photoredox-catalyzed decarboxylative functionalization as a novel electron transfer mechanism towards site- and chemoselective bioconjugation.

Current methods targeting natural amino acid motifs rely on the intrinsic availability of targeted residues on the protein surface to achieve homogeneously modified products. We postulated that photoredox-catalyzed decarboxylation could capitalize on the inherent differences in oxidation potentials between the more abundant Asp and Glu residues vs. the lone terminal carboxylate to obtain exclusive C-terminal-functionalized products.

Figure 2:. Proposed mechanism for the C-terminal-selective photoredox decarboxylative conjugate addition.

After optimization studies, the most effective photocatalysts are riboflavin tetrabutyrate, (1a), and lumiflavin, (1b). The mechanism is proposed to proceed via one-electron oxidation of 3 by the excited photocatalyst, 2, to furnish α-amino radical 4 after decarboxylation. Addition into Michael acceptor 5 provides adduct 6, which is reduced by reduced photocatalyst 8 and protonated to give C-terminal modified product 7.

Figure 3:. Survey of functional group tolerance for the C-terminal-selective photoredox decarboxylative conjugate addition.

The scope of decarboxylative functionalization proves quite general with respect to the various functional groups present in the canonical amino acids at physiological pH. Importantly, no reaction was detected at the Asp or Glu site in tetramers containing these competing acid functionalities. Enhanced reactivity is observed using a pH 3.5 buffer. ^a30 mol% photocatalyst 1b. Yields reported as a % conversion as determined from reverse phase HPLC and are an average of three independent trials.

Figure 4.. Scope of the photoredox decarboxylative conjugate addition applied to endogenous peptides.

A variety of fully unprotected peptides from 8-mers to 58-mers can be site-selectively modified in this transformation, a, Generalized reaction scheme; b—d, substrate scope. All peptides are commercially available. In the case of the fibronectin binding inhibitor peptide (entry 17), the N-terminal Phe residue was added only for ease of analysis when initially developing the quantitative HPLC assay. Notably, longer peptides with nascent secondary structure (c) and peptides bearing high ratios of internal Asp and Glu residues (d) furnish C-terminal modified products exclusively. For entries 15–19 and 21–22, yields are reported as a % conversion as determined from reverse phase HPLC and are an average of three independent trials. ^aUsing photocatalyst lb. ^bRepresentative structure derived from the crystal structure of the full-length protein preproadrenomedullin. ^c3 equiv. photocatalyst lb and 3-methylene-2-norbomanone as the Michael acceptor. ^dYield reported as a combined % conversion after re-subjection of recovered starting material (see Supplementary Information).

Figure 5:. Photoredox-mediated decarboxylative functionalization of human insulin.

a, Human insulin was functionalized using our decarboxylative photoredox methodology to furnish highly selective mono-alkylation at the C-terminus of the A chain exclusively. b, Functionalization of human insulin with a Michael acceptor incorporating desthiobiotin as an affinity tag. Reaction conditions: 1 equiv. insulin (500 nmol), 10 equiv. of the respective Michael acceptor, 3 equiv. photocatalyst 1b, 95:5 pH 3.5 cesium formate buffer:glycerol (1 mM), 34 W blue LED, 8 h. Yield is reported as a % conversion as determined from reverse-phase HPLC. Current work exploring alkyne and azide bearing Michael acceptors is on-going.