Sequence Inversion Dictates the Antiproliferative Activity of Bidirectional Tryptophan-Containing Dipeptide Libraries

Two complementary series of novel tryptophan-derived dipeptides (Trp-X and X-Trp) were designed and synthesized to systematically evaluate how backbone sequence inversion alters chemical accessibility and in vitro antineoplastic profiles against a human solid tumor cell line panel (A549, HeLa, MIA PaCa-2, SW1573, T-47D, and WiDr). Reversing the peptide connectivity revealed prominent sequence-dependent chemical reactivities and biological variations. Steric repulsion at the β-carbon limited basic hydrolysis during X-Trp precursor assembly, whereas the Trp-X series allowed straightforward chemical couplings. Phenotypic screening demonstrated that the Trp-X configuration is biologically superior to the X-Trp layout. The conformationally restricted L-proline conjugate (Trp-Pro) emerged as the lead architecture, exhibiting consistent, single-digit sub-micromolar growth inhibition across all histotypes (GI₅₀ = 1.36-2.06 μM) and effectively outperforming clinical standards cisplatin and 5-fluorouracil in resistant models. Interestingly, an inversion of structure-activity relationships was observed in the reverse series, where specific residues like L-phenylglycine and L-tyrosine experienced a prominent rescue of potency upon sequence relocation. These results confirm that the antiproliferative profile of these peptidomimetics is strictly dictated by a highly directional, sequence-specific molecular topology rather than aggregate lipophilicity alone.