Photoactivated Proximity Protein Labeling Reveals Enhanced Tumor Retention of a D‐Peptide‐Ruthenium Prodrug Conjugate

Amino acid chirality is known to influence the biological properties of peptide-containing prodrugs. In this work, both Δ and Λ isomers of three cyclic ruthenium-peptide photoactivated chemotherapy (PACT) conjugates [1]Cl₂-[3]Cl₂ are prepared that bear the bidentate peptide Ac-MRGDM-NH₂, Ac-mrGdm-NH₂, or Ac-MrGdM-NH₂, respectively, where M, R, and D are L-amino acids and m, r, and d are their D-isomers. All six PACT compounds show low dark cytotoxicity (EC_50,dark > 30 µM) toward normoxic (21% O₂) and hypoxic (1% O₂) A549 human lung cancer cells. Upon green light irradiation, the peptide is cleaved off via an efficient two-step photosubstitution reaction, which raises the cytotoxicity up to 20-fold in normoxia and 4.5-fold in hypoxia. The Λ-[1]Cl₂, Λ-[2]Cl₂ and Λ-[3]Cl₂ isomers are further studied in A549 human lung xenograft in mice. Strikingly, the D-peptide conjugate Λ-[2]Cl₂ has higher antitumor activity than the two other isomers. For the first time, the fate of the photoactivated PACT prodrug can be tracked in vivo via red phosphorescence resulting from proximity labeling of histidine-containing proteins. Photoactivated Λ-[2]Cl₂ shows higher tumor retention and better clearance from the rest of the body, thereby explaining the excellent antitumor properties of this PACT compound.