Design of pH-controlled light-activated reagents for efficient cleavage of double-stranded DNA and cancer phototherapy

In Chapter I, the photochemistry of enediynes is reviewed. Photo-Bergman cyclization, C1-C5 photocyclization and other photochemical reactions of alkynes in the enediyne moiety are discussed. DNA cleavage by photochemically activated enediynes as well as photophysical properties of enediynes is summarized.;In Chapter II, we show that double-stranded DNA cleavage of light-activated lysine conjugates is strongly enhanced at slightly acidic pH (<7) suitable for selective targeting of cancer cells. This enhancement stems from the presence of two amino groups of different basicities. The first amino group plays an auxiliary role by enhancing solubility and affinity to DNA whereas the second amino group which is positioned next to the light-activated DNA-cleaver undergoes protonation at the desired pH threshold. This protonation results in two synergistic effects which account for the increased DNA-cleaving ability at the lower pH. First, lysine conjugates show tighter binding to DNA at the lower pH, which is consistent with the anticipated higher degree of interaction between two positively charged ammonium groups with the negatively charged phosphate backbone of DNA. Second, the unproductive pathway which quenches the excited state of the photocleaver through intramolecular electron transfer is eliminated once the donor amino group next to the chromophore is protonated. Experiments in the presence of traps for diffusing radicals show that reactive oxygen species do not contribute significantly to the mechanism of DNA cleavage at the lower pH, which is indicative of tighter binding to DNA under these conditions. This feature is valuable not only because many solid tumors are hypoxic but also because cleavage which does not depend on diffusing species is more localized and efficient. Sequence-selectivity experiments suggest combination of PET and base alkylation as the chemical basis for the observed DNA-damage. The utility of these molecules for phototherapy of cancer is confirmed by the drastic increase in toxicity of five conjugates against cancer cell lines upon photoactivation.;In Chapter III, to determine whether photocytotoxicity of lysine conjugates stems from DNA cleavage, we investigated their uptake in cells and intracellular DNA damage induced by these molecules in LNCap cancer cells using single cell gel electrophoresis (SCGE) assays. The observation of efficient DNA damage confirmed that the lysine acetylene conjugate is capable of cleaving the densely compacted intracelluar DNA. This result provides a key mechanistic link between efficient DNA cleavage and cytotoxicity towards cancer cells for this family of light-activated anticancer agents.;In order to test connection between the alkylating ability and the DNA-damaging properties of these compounds, we investigated the photoreactivity of three isomeric aryl-TFP alkynes with different positions (o-, m- and p-) of amide substituents towards a model pi-system in Chapter IV. Reactions with 1,4-cyclohexadiene (1,4-CHD) were used to probe the alkylating properties of the triplet excited states in these three isomers whereas Stern-Volmer quenching experiments were used to investigate the kinetics of PET. The three analogous isomeric lysine conjugates cleaved DNA with different efficiency (34 %, 15 % and 0 % of ds DNA cleavage for p-, m- and o-substituted lysine conjugates, respectively) consistent with the alkylating ability of the respective acetamides. Significant protecting effect of hydroxyl radical and singlet oxygen scavengers to DNA cleavage was shown only with m-lysine conjugate. All three isomeric lysine conjugates inhibit human melanoma cell growth under photoactivation but the p-conjugate has the lowest CC50 value of 1.49 x 10-7 M.;Chapter V describes the most efficient family of compounds for light-activated double strand DNA cleavage known to date. This family represents the 2 nd generation of a "switchable" molecular system for pH-gated double strand DNA-cleavage. These hybrid compounds consist of two functional parts: an efficient DNA-photocleaving agent and a pH-regulated part derived from a dipeptide (bis-lysine). We used two alternative ways to create the dipeptide moiety, which allowed us to change the relative number of the alpha-amino vs epsilon-amino groups and achieve better control over the reactivity and selectivity of the ds DNA cleavage. Depending on the structure, the conjugates exist as either monocations or dications at the neutral pH but are transformed into trications at the acidic pH. The efficiency of ds-cleavage increases dramatically at the slightly acidic pH (<7) where it exceeds the ds:ss ratio for the most efficient of non-enzymatic ds DNA cleavers, the natural enediyne calicheamicin.