ABSTRACT
Ruthenium polypyridyl complexes which can sensitise the photo-oxidation of nucleic acids and other biological molecules show potential for photo-therapeutic applications. In this article a combination of transient visible absorption (TrA) and time-resolved infra-red (TRIR) spectroscopy are used to compare the photo-oxidation of guanine by the enantiomers of [Ru(TAP)2(dppz)]2+ in both polymeric {poly(dG-dC), poly(dA-dT) and natural DNA} and small mixed-sequence duplex-forming oligodeoxynucleotides. The products of electron transfer are readily monitored by the appearance of a characteristic TRIR band centred at ca. 1700 cm-1 for the guanine radical cation and a band centered at ca. 515 nm in the TrA for the reduced ruthenium complex. It is found that efficient electron transfer requires that the complex be intercalated at a G-C base-pair containing site. Significantly, changes in the nucleobase vibrations of the TRIR spectra induced by the bound excited state before electron transfer takes place are used to identify preferred intercalation sites in mixed-sequence oligodeoxynucleotides and natural DNA. Interestingly, with natural DNA, while it is found that quenching is inefficient in the picosecond range, a slower electron transfer process occurs, which is not found with the mixed-sequence duplex-forming oligodeoxynucleotides studied.
ABSTRACT
Key to the development of DNA-targeting phototherapeutic drugs is determining the interplay between the photoactivity of the drug and its binding preference for a target sequence. For the photo-oxidising lambda-[Ru(TAP)2 (dppz)]2+ (Λ-1) (dppz=dipyridophenazine) complex bound to either d{T1 C2 G3 G4 C5 G6 C7 C8 G9 A10 }2 (G9) or d{TCGGCGCCIA}2 (I9), the X-ray crystal structures show the dppz intercalated at the terminal T1 C2 ;G9 A10 step or T1 C2 ;I9 A10 step. Thus substitution of the G9 nucleobase by inosine does not affect intercalation in the solid state although with I9 the dppz is more deeply inserted. In solution it is found that the extent of guanine photo-oxidation, and the rate of back electron-transfer, as determined by pico- and nanosecond time-resolved infrared and transient visible absorption spectroscopy, is enhanced in I9, despite it containing the less oxidisable inosine. This is attributed to the nature of the binding in the minor groove due to the absence of an NH2 group. Similar behaviour and the same binding site in the crystal are found for d{TTGGCGCCAA}2 (A9). In solution, we propose that intercalation occurs at the C2 G3 ;C8 I9 or T2 G3 ;C8 A9 steps, respectively, with G3 the likely target for photo-oxidation. This demonstrates how changes in the minor groove (in this case removal of an NH2 group) can facilitate binding of RuII dppz complexes and hence influence any sensitised reactions occurring at these sites. No similar enhancement of photooxidation on binding to I9 is found for the delta enantiomer.
Subject(s)
Coordination Complexes/chemistry , DNA/chemistry , Inosine/chemistry , Oxidants, Photochemical/chemistry , Ruthenium/chemistry , Base Sequence , Binding Sites , Electron Transport , Guanine/chemistry , Intercalating Agents/chemistry , Models, Molecular , Molecular Structure , Oxidation-Reduction , Spectrophotometry, Ultraviolet/methods , Spectroscopy, Fourier Transform Infrared/methods , Stereoisomerism , Structure-Activity Relationship , ThermodynamicsABSTRACT
Four new Ru(ii) polypyridyl complexes that contain an extended aromatic moiety derived from pyrazino[2,3-h]dipyrido[3,2-a:2',3'-c]phenazine and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) have been synthesized, their solid state X-ray crystal structure determined and their photophysical and biological properties evaluated. Their interactions with DNA have been studied, and they have been tested for their potential as photodynamic therapeutic (PDT) agents in the treatment of cancer. A practical modification of a method by Carter, Rodriguez and Bard has been introduced and used to calculate binding parameters for the complexes which show a strong affinity for DNA with binding constants in the order of 107 M-1 (in 10 mM phosphate buffer). The complexes containing phen as an ancillary ligand become emissive upon binding to DNA ("light switch effect"), but do not show selective cytotoxicity upon light irradiation. On the other hand, the TAP complexes, which show an inverse "light switch effect" (emission quenched upon binding to DNA), are strongly photo-toxic suggesting their use in Photodynamic Therapy (PDT). In HeLa cells the best PDT agent shows an IC50 value (light) = 4 µM vs. IC50 value (dark) = 62 µM.
