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 properites 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 ( 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 × 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 2nd 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 ε-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.