Structural and functional characterization of a complex of Zn(ii)-substituted pheophorbide a and all-parallel G-quadruplex DNA

Abstract

Zn(II)-substituted pheophorbide a (ZnPhed a), a chlorophyll derivative, exhibited strong binding affinity with all-parallel G-quadruplex DNA (d[(TTAGGG)]₄, 6mer). Fluorescence spectroscopy, ¹H nuclear magnetic resonance (NMR) spectroscopy, and computational analysis were applied to characterize the resulting ZnPhed a–6mer complex. Fluorescence titration experiments revealed that the apparent binding constant (K_app) of the ZnPhed a–6mer complex was (1.8 ± 0.39) × 10⁷ M⁻¹, approximately 60 times greater than that of a complex of metal-free pheophorbide a (Phed a) with 6mer (∼0.3 × 10⁶ M⁻¹). ¹H NMR spectroscopy demonstrated selective binding of ZnPhed a to the 3′-terminal G-quartet of G-quadruplex DNA, forming a stable complex. On the basis of a computational analysis, the high binding affinity was attributed to the localized positive charge of the Zn(II) center, which enhanced the electrostatic interactions with the electron-rich G-quartet. Upon red light (680 nm) irradiation of the ZnPhed a–6mer complex under both aerobic and anaerobic conditions, the G-quadruplex DNA was degraded, indicating the ability of ZnPhed a to induce DNA damage through both oxygen-dependent and independent mechanisms. This dual photoreactivity suggested that ZnPhed a can serve as an effective photosensitizer for photodynamic therapy (PDT), particularly under hypoxic conditions that are unsuitable for traditional reactive oxygen species (ROS)-mediated techniques. These findings provide valuable insight for the design of next-generation photosensitizers targeting G-quadruplex structures.