Halogenated Tryptophans Improve Integrin αVβ6 Affinity of Cyclic RGD Peptides and Provide Handles for Late-Stage Derivatization.
Selective targeting of integrin αVβ6 is of high relevance for molecular imaging and therapeutic applications, yet ligands combining high affinity with structural and functional versatility remain limited. A structure-activity relationship (SAR) study of the nonapeptide cyclo-(-Asp-Leu-Ala-Phe-d-Pro-NMeLys-Phe-Arg-Gly-) was performed through site-specific substitution of phenylalanine residues by tryptophan and halogenated tryptophan derivatives, revealing a pronounced position-dependent effect on integrin recognition. Substitution at position i+3 relative to the RGD sequence with halotryptophans is identified as an affinity hotspot, enabling picomolar binding to αVβ6. In contrast, modification at position i+6 preserves high αVβ6 affinity while providing exceptional tolerance toward structural diversification and enabling late-stage functionalization of unprotected peptides via palladium-catalyzed cross-coupling reactions. This establishes i+6 modification as a versatile platform for chemoselective derivatization without compromising affinity. Halotryptophans serve both as pharmacophores and synthetic handles. Notably, an anthracenyl-functionalized derivative combines sub-nanomolar αVβ6 affinity with water solubility and intrinsic fluorescence, demonstrating that even bulky π-systems can be accommodated at discrete positions without loss of potency. These integrin ligands are compatible with cellular applications and enable receptor-specific detection by flow cytometry (FACS) as well as visualization by confocal fluorescence microscopy. This work defines a robust design for αVβ6-targeting functional probes for molecular imaging and fluorescence-guided tumor detection.