Investigation Of The Intereactions Of Photoactivatable Anticancer Pt(Ⅳ) Prodrugs With Single-and Double-stranded DNA By Electrospray Ionization Mass Spectrometry

Photoactivated Pt^Ⅳ prodrug trans,trans,trans-[Pt^Ⅳ（N₃）₂（OH）₂（py）₂]（py=pyridine,complex 1）attracts extensive attention due to its high stability in the dark and high cytotoxicity under light.It has high activity against cisplatin-resistant cancer cells,one of its potential targets is considered as DNA and the photochemical mechanisms of complex1 are very involute.In the previous studies on complex 1 intereacting with models such as mono-nucleotide and di-nucleotide,in addition to the coordination of the reduced PtⅡspecies with the bases,complex 1 also underwent photolysis producing the reactive oxygen species and reactive nitrogen species,by which process caused oxidized damage to the bases.At the same time,we also found that compared with mono-nucleotides,di-nucleotides had significantly more types of platinated and oxidation products.The photolysis mechanism and photochemical intereaction of complex 1 were obviously substrate-dependent.Those result indicated that the changes of sequence had significant effects on the photochemical intereaction of complex 1 with DNA.Based on this,we studied the interaction of complex 1 with long single/double-strands DNA in this paper.We selected one DNA single-strand containing only one G base(ODN I:5‘-CT₂CTCTTG₈T₉CT₁₁TCTC-3‘)and two DNA double-strands((duplex Ⅳ:CCTTCTT₇G₈CTTCT₁₃CC-3‘（ODN Ⅱ）,5‘-GGAG₄A₅AGC₈AAGAAGG-3‘（ODN Ⅲ）;duplex Ⅶ:5‘-ATTA₄TTAG₈C₉TAA₁₂TTAA-3‘（ODN V）,5‘-TTAATT₆AG₈CTAA₁₂TAAT-3‘（ODN Ⅵ）)whose one strand only contained one G base to interact with complex 1,respectively,and electrospray mass spectrometry was used to analyze the photochemical intereacting products and identify the platinated sites.Compared with the previous studies,the effects of sequence and length in the interaction of complex 1 with DNA photoreaction were explored.The mass spectrometry results were as follows:（1）In the photoreaction of single-strand ODN I with complex 1,primary mass spectrometry detected a series of mono-,di-,tri-,tetra-,and penta-platinated products containing[Pt^Ⅱ（N₃）（py）₂]⁺,these products indicated that[Pt^Ⅱ（N₃）（py）₂]⁺was the main platinated molecule of complex 1 bounding to bases after photolysis,which was similar to the intereaction of guanosine and di-nucleotides.Since ODN I contained only one G base,the above poly-platinated products indicated that at least four T or C bases were available for platinated binding.In addition,a series of platinated products containing[Pt^Ⅱ（py）₂]²⁺were detected in mass spectrometry,whereas in the intereaction of complex1 with guanosine and di-nucleotides,no obvious products with[Pt^Ⅱ（py）₂]²⁺was observed.Next,tandem mass spectrometry（MS/MS）was applied to break the mono-platinated products{ODN I+[Pt^Ⅱ（N₃）（py）₂]⁺},bi-and tri-platinated products,yielding.We found the platinated sites of complex 1 on ODN I were T₂,G₈,T₉ and T₁₁.These data indicated that in addition to G bases in ODN I,T bases were the main platinated sites for complex1.We were not able to identify any C-base binding sites,possibly due to preferential dissociation of C-bases.（2）Similar to the above intereaction conditions,we used mass spectrometry to study the photochemical intereaction of duplex Ⅳ with complex 1.First,we analyzed the photochemical products of complex 1 with two complementary stands（ODN Ⅱ and ODN Ⅲ）,respectively.Up to seven platinated products containing[Pt^Ⅱ（N₃）（py）₂]⁺were detected in the photoreaction of ODN Ⅱ with complex 1,while only up to five platinated products were detected in the photoreaction of ODN Ⅲ with complex 1.G base in ODN Ⅱ could be oxidized to 8-OH-G,Fapy G and Sp.The MS/MS results indicated that T₇,G₈and A₁₃ were the platinated sites on ODN Ⅱ during the photoreacting process.G on ODN Ⅲ could be oxidized to 8-OH-G,Fapy G and dehydroguanidinolactone（DGh）,which was different from the results of ODN Ⅱ.The results of MS/MS indicated that the platinated sites on ODN Ⅲ were G₄,A₅,C₉ and A₁₀.Next,in the photoreaction of duplex Ⅳ with complex 1,up to tri-platinated products containing[Pt^Ⅱ（N₃）（py）₂]⁺were detected by mass spectrometry,and no related oxidized products were generated,indicating the lower reactivity of duplex Ⅳ compared to single strand.（3）Similarly,the photochemical intereaction between duplex Ⅶ and complex 1was also studied by mass spectrometry.First,we analyzed the photochemical intereaction products of complex 1 and two complementary strands（ODN V and ODN Ⅵ）respectively.In the photochemical intereaction of single-strand ODN V and ODN Ⅵ with complex 1,up to penta-platinated products containing[Pt^Ⅱ（N₃）（py）₂]⁺could be detected,accompanied by obvious products containing[Pt^Ⅱ（py）₂]²⁺.G base in both sequences could be oxidized to 8-OH-G.The results of secondary mass spectrometry indicated that A₄,G₈,C₉ and A₁₂ were platinated sites on ODN V,and T₆,G₈ and A₁₂ were platinated sites on ODN Ⅵ.Next,in the photochemical reaction of duplex Ⅶ with complex 1,only the mono-platinated products containing[Pt^Ⅱ（N₃）（py）₂]⁺detected by mass spectrometry,showing the lower reactivity of the double-strand than the single-strand again,AT-rich duplex Ⅶ was even less reactive than CT-rich duplex Ⅳ,also suggesting a substrate-dependent reactivity of complex 1.In summary,our results showed that when complex 1 interacted with DNA of different sequences,there were obvious differences in the reactivity and oxidized producing forms of different sequences,Double-strands showed significantly lower reactivity than single-strands,which clearly demonstrated the sequence dependence in the photochemical intereaction of the photoactivate PtⅣprodrug with DNA,providing important information for understanding the mechanism of its photochemical intereaction.