Protein-protein interactions are defined by interfacial secondary structural motifs that impart a high degree of selectivity. Peptides can be designed to bind at these sites, though they are invariably unstructured, a shortcoming that can be addressed by introducing a covalent linker to define the required geometry. Imaging of these constrained peptides requires a fluorescent tag, however, classic constraints introduced by metathesis, lactamisation and ‘click’ chemistry lack this property and as such it must be introduced separately. Here we repurpose a protein cross-linker, dibromobimane, as a peptide constraint to define secondary structure and introduce fluorescence.
Ac-HcyAHcy-CONH2 (Hcy = homocysteine) and Ac-CARAAARC-CONH2 were prepared by SPPS and reacted with dibromobimane under biologically compatible conditions (PBS, pH 7-8) to give a new class of fluorescent peptides with a defined secondary structure. Unlike existing methods, this does not require incorporation of unusual or expensive amino-acids into the sequence for cyclisation (c.f. metathesis & ‘click’). The reaction is carried out using a peptide concentration of 0.5 mg/ml and 1 equivalent of dibromobimane. These conditions were used to prepare i-i+2 and i-i+7 bimane-cyclised peptides that display beta-strand and helical conformation respectively. An i-i+4 cyclised analogue did not display appreciable structure. All secondary structures were characterised by NMR and CD. Macrocyclisation with dibromobimane was also achieved on solid support using peptides of up to 8 residues in length. The fluorescent properties of the resultant constrained peptides were investigated via plate photometry. Analysis at an excitation wavelength of 385 nm and emission at 477 nm in PBS, demonstrated that the fluorescent peptide can be detected at concentrations as low as 10 nM. Our new fluorescent peptide linker is introduced in an efficient manner using natural amino-acid sequences, and allows the design of new protein-protein interaction inhibitors that do not require further functionalization for in vivo studies.