Preferential Binding of a Long-Arm Porphyrin Ligand to Higher Order G-Quadruplex under Crowding Conditions

Under conditions that are close to the real cellular environment, the human telomeric single-stranded overhang (∼200 nt) consisting of tens of TTAGGG repeats tends to form higher order structures of multiple G-quadruplex (G4) blocks. On account of the higher biological relevance of higher order G4 structures, ligand compounds binding to higher order G4 are significant for the drug design toward inhibiting telomerase activity. Here, we study the interaction between a cationic porphyrin derivative, 5,10,15,20-tetra{4-[2-(1-methyl-1-piperidinyl)propoxy]phenyl}porphyrin (T4), and a human telomeric G4-dimer (AG₃(T₂AG₃)₇) in the mimic intracellular molecularly crowded environment (PEG as a crowding agent) and K⁺ or Na⁺ solution (i.e., K⁺-PEG and Na⁺-PEG), by means of multiple steady-state and time-resolved spectroscopic techniques. It is revealed that the long-armed T4 selectively binds to the K⁺-PEG G4-dimer by intercalating into the cleft pocket between the two G4 blocks, since the two G4 monomers with parallel-stranded topology are stacked by head-to-tail arrangement and can offer π-stacking interface binding with T4. In contrast, the Na⁺-PEG G4-dimer with antiparallel-stranded topology adopts side-by-side arrangement of G-quartets, resulting in a lack of π-π binding sites to stabilize T4 within the cleft, and no obvious binding characteristics are observed. Interestingly, it is observed that protonation of T4 is facilitated upon binding with the K⁺-PEG G4-dimer, which can occur under physiological pH, due to the π-π stacking with two G-quartet planes that enhances the electron-rich character of the central porphyrin core of T4. Afterward, the protonated T4 displays dramatically different spectral characteristics (Soret band, Q-band, fluorescence band, and lifetime), which in turn serves as a spectral reporter for characterizing the DNA-binding event. These findings provide a mechanistic basis for developing targeted ligands that can specifically interact with higher order physiological G4 structures.