Development of a Multifunctional Benzophenone Linker for Peptide Stapling and Photoaffinity Labelling

Abstract

Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo‐crosslinking motif and a peptide stapling reagent. Using double‐click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo‐crosslinking amino acid. When applied to a p53‐derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull‐down assays, and can be used to investigate the target selectivity of stapled peptides.

Target identification of natural medicine with chemical proteomics approach: probe synthesis, target fishing and protein identification

“…Frequently used photoaffinity groups include benzophenone, aryl azide, and diazirine, and of these, benzophenone is the most widely applied due to its stability and ease of synthesis. 80,81 In recent years, diazirine-based photoaffinity probe synthesis has attracted much interest due to its small size and Unfortunately, photoaffinity groups can intrinsically bind some nonspecific proteins (e.g., diazirine to voltagedependent anion channels), 82,83 which may affect the accuracy of the results.…”

Strategies to expand peptide functionality through hybridisation with a small molecule component

“…This allowed the development of peptides which preferentially cross-linked to MDM2 in the presence of BSA. 157 Switchable and triggerable small molecule probes are an important class of compounds for chemical biology studies. The methods utilised to make these compounds are now being applied to peptide-based compounds through conjugation of small molecule fragments into peptide structures.…”

Using Peptidomimetics and Constrained Peptides as Valuable Tools for Inhibiting Protein–Protein Interactions

“…A number of two-component stapling techniques have been reported including photocontrollable macrocycles [ 115 , 116 , 117 , 118 ] and the use of bridging motifs like alkyl chains [ 119 , 120 ], aromatics [ 120 , 121 , 122 , 123 , 124 ], perfluoroaryl [ 125 ] and tetrazine [ 126 ]. These examples have been carried out using the natural amino acid cysteine as an attachment point ( Figure 5 ), but other examples have also been reported for two-component stapling using lysine and tryptophan residues [ 127 , 128 ] and non-native amino acids containing alkyne functionalities [ 129 , 130 , 131 , 132 , 133 ]. Cysteine has been exploited for two-component stapling partly due to the high nucleophilicity of the sulfhydryl group which can readily undergo alkylation with suitable electrophiles such as α-halocarbonyls and Michael acceptors [ 134 ].…”