The second part of this article describes the photo-induced cleavage of pGEM-7zf-NIS super-coiled DNA by Cu(II)-meso-tetrakis-(n-N-methylpyridiniumyl)porphyrins (n = 2, 3, 4 referred to as o-, m- and p-CuTMPyP, respectively) and their binding mode. M-CuTMPyP was the most efficient in cleavage compared to o- and p-CuTMPyP isomers. Cleavage was suppressed by N2 bubbling, suggesting that the cleavage occurred by an oxidative cleavage mechanism. Sodium azide, an 1O2 quencher, and DMSO, a hydroxyl radical scavenger, inhibited cleavage, indicating that hydroxyl radicals and singlet oxygen were likely reactive species responsible for the cleavage. Reduced linear dichroism spectroscopy showed angles of o-CuTMPyP’s electric transition moments, in which the periphery pyridinium ring was prevented from free rotation, of 59° and 61° with respect to the local DNA helix axis. The spectra of m- and p-CuTMPyP complexed with pGEM-7zf-NIS DNA were characterized by large negative LD signals in the Soret band, coincident with those of known intercalated porphyrins.
The conditions for the measurement of linear dichroism (LD) can be adjusted so as to solely reflect the length and the flexibility of DNA. The real-time detection of the EDTA•Fe2+-induced oxidative cleavage of double-stranded native and synthetic DNAs was performed using LD. The decrease in the magnitude of the LD at 260 nm, which reflects an increase in the flexibility and a decrease in the length of the DNA, can be described by the sum of two or three exponential curves in relation to the EDTA•Fe2+ concentration. The fast component was assigned to the cleavage of one of the double strands, inducing an increase in the flexibility, while the other slower component was assigned to the cleavage of the double strand, resulting in the shortening of DNA. The decrease in the magnitude of the LD of poly[d(A-T)2] was similar to that of poly[d(I-C)2], while that of poly[d(G-C)2] was found to be the slowest, indicating that the resistance of poly[d(G-C)2] against the Fenton-type reagent was the strongest. This observation suggests that the amine group in the minor groove of the double helix may play an important role in slowing the EDTA•Fe2+-induced oxidative cleavage.
In the last part, a series of structurally related X-benzyl-[Cu(dipicolyl-amine)(NO3)2] complex. (where X = methoxy-, methyl-, H-, fluoro- and nitro-group) were synthesized and their activity on the DNA cleavage were investigated. Replacement of H atom at the dipicolylamine ligand caused a large enhancement in the efficiency of oxidative cleavage of both super-coiled DNA and double stranded DNA as it was evident by the electrophoresis pattern and faster decrease in the LD intensity at 260 nm for the benzyl-[Cu(dipicolylamine)(NO3)2] complex. Further modification of the benzyl group by introducing various substituents at the para-position, the efficiency in DNA cleavage was also altered. When the methyl group is attached, the cleavage efficiency appeared to be the largest. The order of efficiency in DNA cleavage was methyl > methoxy H > fluoro nitro group: when electron withdrawing group was introduced, the cleavage efficiency remarkably decreased. The reactive oxygen species involved in the cleavage process were superoxide radical and singlet oxygen. Possible mechanism for this variation in the DNA cleavage efficiency is proposed.
The third part of this study deals with synthesis of two binuclear Cu(II) complexes of N-functionalized macrocycle ligands namely, 1,3-bis(1,4,7-triaza-1-cyclonomyl)-propane and 1-(3-(1,4,7-triazonan-1-yl) propyl-1,4,7,10-tetraazacyclo-dodecane. Their ability to hydrolytic cleavage of supercoiled plasmid DNA (pBR322) was compared with structurally related non-functionalized mononuclear Cu(II) complexes. The former, the binuclear Cu(II) complex of symmetrical ligand showed significantly enhanced double-strand cleavage activity compared to other three complexes at the same concentration. In contrast, the latter binuclear complex with unsymmetrical macrocylic ligand did not give rise to double-strand DNA cleavage. The linear DNA formation induced by mononuclear Cu(II) 1,4,7,10-tetraazacyclo-dodecane complex is realized via a non-random double-stranded scission process. The different cleavage activity is discussed in relation to the dimer formation, effective cooperation and coordination environment of metal center. The hydrolytic cleavage by the copper complexes without H2O2 is supported by the evidence from anaerobic reaction, free radical quenching, and nitro blue tetrazolium assay. Conversely, both the binuclear complexes cleave supercoiled DNA very efficiently to form III (linearized DNA) in the presence of the hydrogen peroxide, indicating nuclearity is a crucial parameter in oxidative cleavage. The radical scavenger inhibition study and nitro blue tetrazolium assay suggest the involvement of hydrogen peroxide and superoxide ion in the oxidative cleavage of DNA by binuclear complexes.
Effect of the central metal ion on the DNA cleavage was also investigated in this study. The metal complexes of dipyridylamine based ligand with benzo quinoline moiety containing divalent metal ions Co(II), Cu(II), Ni(II), Zn(II) and Cd(II) have been structurally characterized and their electrochemical properties has also been studied. The interaction of the five metal complexes to calf thymus DNA has been investigated with aid of UV absorption and linear dichroism studies, and the mode of CT-DNA binding for the complexes have been proposed. The experimental results show that they can efficiently induce considerable oxidative DNA cleavage in the presence hydrogen peroxide and dioxygen. The Zn(II) complex does not show any appreciable cleavage activity, Ni(II) and Cd(II) compleses are moderately active, while Cu(II) and Co(II) shows formation of a significant quantity of linear DNA, resulting from double-strand breaks. Mechanistic studies reveal the involvement of •OH and superoxide anion as the reactive species in the scission process under an aerobic medium. A mechanism involving either the Fenton or the Haber–Weiss reaction is proposed for the DNA cleavage mediated by these complexes. The Cu(II) complex can also cleave the double stranded CT-DNA in the presence of activator most probably via an oxidative mechanism, whereas the activity of the other complexes is negligible under similar reaction conditions. The kinetic aspects of double-stranded CT-DNA cleavage with the Cu(II) complex are explained in detail.