Finally, we were wondering if these macrocyclic peptides with the rigid triple bonds can be potential inhibitors for protein-protein interactions (PPIs). Keap1-Nrf2 was chosen as our target due to its importance in biological pathways related to inflammation and neurodegenerative diseases. Peptides containing a DxETGE motif showed good binding affinities to this target. Therefore, 3 peptide sequences, containing a DAETGE motif and varying size, from 6-mer to 8-mer, were synthesized. Based on our method, we could successfully cyclize the three linear peptides (13w-13y) in moderate yield (Fig ). The binding of cyclic peptides and their corresponding linear precursors to Keap1 were tested using a TR-FRET assay, in which the replacement of a linear Sulfo-Cy5-N3-labeled peptide inhibitor (sequence: Ac-Propargylglycine-Peg2-LDEETGEFL-NH2) was measured. Compared with linear peptides, enhanced binding affinity was observed for all three cyclic peptides (Fig ). Notably, the 8mer cyclic peptide 13y binds to KEAP1 with a 20-fold higher affinity (IC50 = 90 nM) when compared to the linear precursor 2ad (IC50 = 1.8 μM) (Fig ). These results indicated that our cyclization strategy displayed potentials for enhancing the binding affinity for Keap1-Nrf2 PPIs site. Intriguingly, a significant 5.5-fold drop of binding affinity was observed when we reduced the triple bond of 13y to a single bond 16b, highlighting the importance of the rigid alkyne for high binding affinity.