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

ABSTRACT 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 (GI 50 = 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.