Abstract

Conformational precision is critical for peptide therapeutic design, yet the quantitative link between cross-linker geometry and in vivo performance remains unclear. We present a programmable platform using eight bi-triazine cross-linkers with varied bond length, angle, aromaticity, and symmetry to establish multilevel structure–conformation–biology relationships. Using cyclic RGD peptides targeting integrin αvβ3 and dimeric KTLLPTP peptides targeting Plectin-1 as complementary models, we integrated binding assays, cell studies, and in vivo 68Ga-PET/CT imaging to systematically evaluate linker-induced conformational effects. Two design paradigms emerged: in cyclic peptides, aromaticity and symmetry govern conformational locking, with nonmirror-symmetric naphthalene linkers enhancing protein affinity and tumor uptake. In dimeric systems, bond length and angle enable geometric matching via the “molecular ruler” effect, with a 120° benzene linker enabling optimal bivalent binding. This work not only identifies the lead candidate [68Ga]Ga-8a with high tumor contrast (∼5 %ID/mL) but also provides a generalizable framework for precision peptide engineering.