Hybrid Macrocyclic Peptides - Synthetic Strategies to Diversify and Cyclize Peptide Libraries in Phage Display Selections.

Macrocyclic peptides (MPs) are privileged molecular architectures in drug discovery that combine the target selectivity of biologics with the pharmacological advantages of small molecules. However, peptide binders identified by biological selection platforms such as bacteriophage display often require extensive post-selection medicinal chemistry optimization before entering the clinic. In particular, the incorporation of nonpeptidic scaffolds and synthetic cyclization units is frequently necessary to improve binding affinity, proteolytic stability, membrane permeability, and overall pharmacological performance. In this Review, we provide a comprehensive overview and critical analysis of strategies that enable the direct selection of such hybrid MPs by bacteriophage display. We discuss established cysteine- and lysine-targeting chemistries, recent advances in residue-selective and chemoenzymatic cyclization methods, and emerging approaches that exploit proximity-driven reactivity or the chemical sturdiness of bacteriophages. Collectively, these strategies enable the simultaneous exploration of peptide sequence and synthetic chemical space during biological selections, thereby potentially reducing downstream optimization efforts. Finally, we outline key design principles for phage-compatible macrocyclization reactions and discuss future opportunities at the interface of bioconjugation chemistry, enzyme engineering, and machine-learning-guided peptide discovery to accelerate the development of next-generation peptide therapeutics.