Beyond Thiol‐Dependent Cyclization of Peptide Libraries: Assessing a Photochemical Alternative

Cyclic peptides are powerful scaffolds for chemical biology and drug discovery. Genetically encoded display (phage, ribosome, mRNA, and yeast) enables high-throughput screening of peptide libraries. Yet expanding their accessible chemical space depends not only on library size but also on the cyclization chemistry, which selects the conformations the library samples and makes linkage choice a primary design variable. Cyclization most often relies on cysteine through disulfides, thiol-reactive linkers, or metal-thiolate bonds. Enzymatic head-to-tail ligation and noncanonical amino acids (ncAAs) provide cysteine-free macrocyclization through amide bonds, but each imposes practical limits such as motif constraints or reliance on genetic code expansion. In this perspective, we examine photoinduced aromatic crosslinking as a complementary, cysteine-independent strategy. Recent fluorotryptophan photochemistry shows that irradiation drives diaryl bond formation between fluorinated tryptophan and proximal phenylalanine or tyrosine, generating intrinsically fluorescent macrocycles and enabling an optical readout of cyclization yield. Fluorotryptophan is a conservative modification that can be introduced at native Trp codons, which may avoid the genetic code expansion. Antimicrobial peptides offer a demanding test case: their function depends on backbone rigidity, amphipathic face presentation, and membrane interaction, all of which an aromatic crosslink could influence.