Proximity Histidine Labeling by Umpolung Strategy Using Singlet Oxygen

Abstract

While electrophilic reagents for histidine labeling have been developed, we report an umpolung strategy for histidine functionalization. A nucleophilic small molecule, 1-methyl-4-arylurazole, selectively labeled histidine under singlet oxygen (1O2) generation conditions. Rapid histidine labeling can be applied for instant protein labeling. Utilizing the short diffusion distance of 1O2 and a technique to localize the 1O2 generator, a photocatalyst in close proximity to the ligand-binding site, we demonstrated antibody Fc-selective labeling on magnetic beads functionalized with a ruthenium photocatalyst and Fc ligand, ApA. Three histidine residues located around the ApA binding site were identified as labeling sites by liquid chromatography–mass spectrometry analysis. This result suggests that 1O2-mediated histidine labeling can be applied to a proximity labeling reaction on the nanometer scale.

An overview of chemo- and site-selectivity aspects in the chemical conjugation of proteins

“…Chemoselective modification of this residue is challenging due to the presence of more nucleophilic groups on proteins that can outcompete the imidazole ring for substitutions and to the absence of a particular reactive feature of this aromatic ring that could be used to label it selectively—such as phenols’ facile generation of C -centred radicals, or indoles' high molar absorptivity and propensity to undergo photoionization, for example. Consequently, general, broadly applicable strategies for histidine modifications have regularly showed chemoselectivity issues [ 263 ], circumscribing site-selective reactions to only a few examples.…”

Umpolung strategies for the functionalization of peptides and proteins

“…8A ). 51 In the presence of a 1-methyl-4-arylurazole nucleophile 52, the oxidized His intermediate is trapped, leading to a C5 conjugated product 53. This strategy was exemplified by labeling peptides and antibodies with azide handles for fluorescent tagging; modifications occurred rapidly (∼15 min) at the embedded His residues under biocompatible conditions.…”

“…It is envisaged that the efficiency of this approach may also allow for the extension of the strategy to intracellular applications. 51 Notably, this His labeling strategy does not maintain the imidazole structure in the conjugated product 53. This method therefore represents a non-classical umpolung approach as the majority of umpolung strategies preserve the primary structural features of the starting amino acids in their conjugated products.…”

“…The latter approach has recently been explored by Sato and co-workers for labeling at the C5 position of the imidazole ring in peptides and proteins. 51 This strategy was inspired by prior reports which identified His–His, His–Cys and His–Lys cross-linked side chains in the analytical analyses of monoclonal antibodies that were attributed to a photooxidative cross-linking mechanism. 52 Capitalizing on this pathway, the His labeling approach was developed through the photocatalytic generation of singlet oxygen, which subsequently undergoes a Diels–Alder reaction with the His imidazole side chain to afford an electrophilic endoperoxide intermediate 51 ( Fig.…”

Direct Near Infrared Light–Activatable Phthalocyanine Catalysts

“…The oxidative dimerization of benzylamine and the phosphonylation reaction from phenylhydrazine [28] were also achieved. Singlet-oxygen-mediated reactions have been studied in synthetic chemistry and chemical biology; [29] hence, this photocatalytic system expands their applications based on the high penetration depth of NIR light. This NIR-reaction system also permits the scalable synthesis with batch reactors, [7b] which requires in industry.…”