Yasemin Kopkalli

A Hand-held Fiber-optic Implement for the Site-specific Delivery of Photosensitizer and Singlet Oxygen

We have constructed a fiber optic device that internally flows triplet oxygen and externally produces singlet oxygen, causing a reaction at the (Z)‐1,2‐dialkoxyethene spacer group, freeing a pheophorbide sensitizer upon the fragmentation of a reactive dioxetane intermediate. The device can be operated and sensitizer photorelease observed using absorption and fluorescence spectroscopy. We demonstrate the preference of sensitizer photorelease when the probe tip is in contact with octanol or lipophilic media. A first‐order photocleavage rate constant of 1.13 h−1 was measured in octanol where dye desorption was not accompanied by readsorption. When the probe tip contacts aqueous solution, the photorelease was inefficient because most of the dye adsorbed on the probe tip, even after the covalent ethene spacer bonds have been broken. The observed stability of the free sensitizer in lipophilic media is reasonable even though it is a pyropheophorbide‐a derivative that carries a p‐formylbenzylic alcohol substituent at the carboxylic acid group. In octanol or lipid systems, we found that the dye was not susceptible to hydrolysis to pyropheophorbide‐a, otherwise a pH effect was observed in a binary methanol‐water system (9:1) at pH below 2 or above 8.

Studies of Quadruplex Folding using a New Fluorescent Porphyrin Derivative

Small molecules that bind selectively to guanine-rich telomeric DNA sequences (quadruplex interactive agents; QIAs) can promote folding to a four-stranded quadruplex conformation. Such quadruplexes have been shown to deactivate telomerase, a key activating enzyme in many cancers, making QIAs promising targets for development of potential cancer drugs. Porphyrin-based QIAs offer several desirable properties as they are non-toxic and found in many oxidation-reduction proteins of the cell. In this study we have examined the effects of a new fluorescent pyropheophorbide porphyrin derivative (PPP-D) on the folding of model human telomeric DNA sequences (HT4). PPP-D demonstrates enhanced lipid solubility (log P = 6.80 ± 1.62) over other quadruplexed-DNA binding porphyrins, including meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and N-methyl mesoporphyrin IX (NMM), and can facilitate cellular delivery (log P = 6.80 ± 1.62) through the cell membrane. Unlike TMPyP4 and NMM, PPP-D is non fluorescent in buffer solutions and highly fluorescent in organic solvents and non-polar environments. CD studies suggest that PPP-D successfully aids in the folding of HT4 sequences in addition to stabilizing the quadruplexed DNA conformation. Furthermore, PPP-D demonstrates selectivity for the quadruplexed (q-DNA) over duplexed DNA (d-DNA) conformation. The significance of these results for anti-tumorigenesis will be discussed. Supported by NIH-SCORE grants ISCI-GM093830-01 (AG) and S06-GM076168-01 (LD).

Fluorescence Studies of Local Environmental Heterogeneity Around Guanine Residues of the Telomeric DNA Quadruplex Conformation

Formation and stabilization of G-quadruplexed DNA formed by the guanine-rich human telomeric sequence d(TTAGGG)4 (HT4), has been shown to inhibit the abnormal activity of telomerase in tumor cells, making stabilization of this DNA secondary structure a target for potential cancer therapeutics. To investigate the effect of local environment on the guanine residues of the G-quadruplex, we previously designed several fluorescence analogs of the HT4 sequence with incorporation of 6-methyl-8-(2-deoxy-D-ribofuranosyl) isoxanthopterin (6MI) at varying guanine-tetrad positions: G1, G4, and G11, which show fluorescence sensitivity to quadruplex folding. In the current studies, we have collected decay-associated spectra (DAS) for the fluorescence lifetime components (τlong, τmedium, and τshort) resolved for each of the mono-labeled sequences. Multi-exponential decay profiles suggest local environmental heterogeneity surrounding the guanine residues in both the folded and unfolded states. Interestingly, the DAS for τlong observed for guanine positions G1 and G11, which are located at the 5’- and 3’-ends, respectively, of the quadruplex-forming sequence, showed significant (10nm) red-wavelength shifts on quadruplex folding. In contrast no DAS shifts are observed for the G4 position, located in the loop region of the folded quadruplex. Thus, in addition to base stacking of the guanine residues with neighboring bases, guanine positions located at G1 and G11, on average, may also be exposed to a more polar environment with quadruplex folding, possibly due to conformational heterogeneity as they are located near the termini of the telomeric sequence. The solvent accessibility of these positions may make them key docking sites for designing ligands that can stabilize the folded conformation of the G-quadruplex.This work was supported by NIH SCORE Grant S06-GM076168-01 (LD).