High-Affinity Sequence-Selective DNA binding by Iridium(III) polypyridyl organometallopeptides

DNA-binding drugs are the workhorse of current anticancer therapies. However, despite their extensive use, many of them suffer from severe side-effects, which has fuelled the search of safer alternatives with improved pharmacological profiles. In the past few years, there has been an increased interest in the application of coordination compounds as DNA-targeted probes, reactive agents and therapeutics, Among them, Ru(II), Os(II) and Rh(III) polypyridyl mononuclear complexes have been exhaustively studied for their kinetic stability and convenient redox and optical properties. In contrast, the potential of classical Werner and organometallic Ir(III) analogues as DNA-binding agents is still largely unexplored, despite being also kinetically inert and displaying excellent photochemical properties with tuneable excited states and long emission wavelengths for sensing and imaging. Indeed, current examples in the literature are practically limited to mononuclear intercalators, with very few reports of di/polynuclear derivatives,1011 or groove-binding agents. We have recently reported the extension of standard solidphase peptide synthesis procedures (SPPS) for the construction of polypyridyl dinuclear Ru(II) complexes. Intrigued by the potential of Ir(III) complexes as DNA binders, we decided to expand the scope of this methodology to the synthesis of Ir(III) organometallopeptides. Considering that the DNA binding properties of these complexes would be highly influenced by their nuclearity, we also synthesized dinuclear and trinuclear derivatives in addition to the mononuclear complexes. Finally, it is also worth noting that there are very few precedents of Ir(III)-peptide conjugates, and limited to examples in which the Ir(III) complexes are attached to the N-terminus, or the side chains of the peptide chain, but never integrated in the peptide backbone structure. Thus, based on our earlier studies with Ru(II) metallopeptides, we designed three peptidic ligands containing one, two or three βAla-bpy coordinating units (1, Scheme 1) to generate mono, di and trinuclear Ir(III) polypyridyl organometallopeptides. The peptidic ligands were synthesized following standard Fmoc/tBu solid-phase protocols. Once fully assembled, the peptides still attached to the solid support were reacted with [Ir(ppy)2Cl]2 (ppy: 2-phenylpyridine) to give the desired cyclometalated complexes Ir, Ir2 and Ir3 (Scheme 1). Acidic cleavage from the support, followed by reversephase HPLC purification, afforded the desired Ir(III) organometallopeptides as diasteromeric mixtures.

We demonstrate the application of solid-phase peptide synthesis methods for the straightforward assembly of polynuclear Ir(III) organometallopeptides, and show that their oligoarginine derivatives exhibit high DNA binding affinity, sequence selectivity, and high cytotoxicicty towards a set of cancer cell lines.
DNA-binding drugs are the workhorse of current anticancer therapies. 1However, despite their extensive use, many of them suffer from severe side-effects, which has fuelled the search of safer alternatives with improved pharmacological profiles. 2In the past few years, there has been an increased interest in the application of coordination compounds as DNA-targeted probes, reactive agents and therapeutics, 3,4,5 Among them, Ru(II), Os(II) and Rh(III) polypyridyl mononuclear complexes have been exhaustively studied for their kinetic stability and convenient redox and optical properties. 6,7In contrast, the potential of classical Werner and organometallic Ir(III) analogues as DNA-binding agents is still largely unexplored, despite being also kinetically inert and displaying excellent photochemical properties with tuneable excited states and long emission wavelengths for sensing and imaging.6e,8 Indeed, current examples in the literature are practically limited to mononuclear intercalators, 9 with very few reports of di/polynuclear derivatives, 10,11 or groove-binding agents. 9e have recently reported the extension of standard solidphase peptide synthesis procedures (SPPS) for the construction of polypyridyl dinuclear Ru(II) complexes.5a Intrigued by the potential of Ir(III) complexes as DNA binders, we decided to expand the scope of this methodology to the synthesis of Ir(III) organometallopeptides.Considering that the DNA binding properties of these complexes would be highly influenced by their nuclearity, 12 we also synthesized dinuclear and trinuclear derivatives in addition to the mononuclear complexes. 13Finally, it is also worth noting that there are very few precedents of Ir(III)-peptide conjugates, and limited to examples in which the Ir(III) complexes are attached to the N-terminus, 14 or the side chains of the peptide chain, but never integrated in the peptide backbone structure.
Thus, based on our earlier studies with Ru(II) metallopeptides, we designed three peptidic ligands containing one, two or three βAla-bpy coordinating units (1, Scheme 1) to generate mono, di and trinuclear Ir(III) polypyridyl organometallopeptides.The peptidic ligands were synthesized following standard Fmoc/tBu solid-phase protocols. 15Once fully assembled, the peptides still attached to the solid support were reacted with [Ir(ppy) 2 Cl] 2 (ppy: 2-phenylpyridine) to give the desired cyclometalated complexes Ir, Ir 2 and Ir 3 (Scheme 1).Acidic cleavage from the support, followed by reversephase HPLC purification, afforded the desired Ir(III) organometallopeptides as diasteromeric mixtures.Having at hand the desired Ir(III) organometallopeptides, we studied their DNA binding by exploiting the intrinsic environment-sensitive luminescence of the Ir(III) complexes.In contrast with the good DNA binding affinity of their Ru(II) analogs, 13 the fluorescence titrations experiments carried out with the Ir, Ir 2 and Ir 3 organometallopeptides did not show any significant affinity for short DNA oligonucleotides, so that addition of increasing amounts of hairpin oligonucleotides containing A/T or G/C-rich sequences did not induce any change in the emissive properties of the organometallopeptides (see ESI †).A possible explanation for the low DNA affinity displayed by these Ir(III) metallopeptides is the reduced charge of [Ir(ppy) 2 bpy] + units (+1 charge in each complex) in comparison to that of the [Ru(bpy) 3 ] +2 DNA complexes (+2).We envisaged that the reduced electrostatic attraction for the negatively charged DNA might be compensated by introducing additional positively charged groups, and thus we decided to synthesize the octaarginine derivatives of Ir, Ir 2 and 3 (named Ir-R 8 , Ir 2 -R 8 and Ir 3 -R 8 , respectively).Indeed, it has been shown that tethering octaarginine domains to DNA binding agents results in conjugates that display increased DNA affinity, 16,17 as well as improved cell internalization and solubility. 18,19We took advantage of the flexibility provided by the SPPS methodology to synthesize these oligocationic metallopeptides as described before, assembling the βAla-bpy coordinating units at the Nterminus of a previously synthesized R 8 peptide.
Once we synthesized the desired oligoarginine conjugates, we studied their DNA binding by fluorescence spectroscopy.Thus, incubation of 0.2 µM solutions of Ir-R 8 , Ir 2 -R 8 and Ir 3 -R 8 with increasing concentrations of DNA hairpins resulted in a progressive increase of the 620 nm emission upon excitation at 320 nm (Fig 1, left).The corresponding titration profiles for each metallopeptide with different oligonucleotides could be fitted to the Bard model (Fig 1, right), 20 which allowed us to determine the affinity constants (Ka) for the different DNA sequences (Table 1).These results show that the binding affinity is dramatically increased with respect to the parent metallopeptides (Ir, Ir 2 and Ir 3 ), and heavily dependent on the nuclearity of the metallopeptides.In fact, the Ka values are approximately in the order of 10 6 , 10 7 and 10 8 M -1 for Ir-R 8 , Ir 2 -R 8 and Ir 3 -R 8 , respectively.Besides, Ir-R 8 shows a slight preference for for A/T-rich sequences, whereas Ir 2 -R 8 and Ir 3 -R 8 display a clear preference for hairpins with high G/C content, perhaps suggesting alternate binding preferences for the two metallopeptides.
Interestingly, the calculated association constants for the interaction of Ir 3 -R 8 for the DNA hairpin are 100 times higher than those typically reported for common mononuclear intercalating complexes (such as Ru(II)/dppz derivatives), 6c and 1000 times stronger than those observed for other nonintercalating DNA-binding metal complexes, 5a and in the order of the binding constants measured for widely used organic DNA minor-groove binders, like Hoechst 33258.17b,21 Therefore, to the best of our knowledge, Ir 3 -R 8 displays the highest DNA affinity observed for a metal complex.In order to explore the role of the Arg 8 appendage we synthesized a scrambled trinuclear Ir(III) organometallopeptide in which the iridium centers are separated by groups of three arginine residues Ir-R 3 -Ir-R 3 -Ir-R 3 .Interestingly, this analog displayed negligible affinity for duplex DNA (see figures S4 and S5 in the ESI †), thus suggesting an important role for the C-terminal R 8 domain beyond simple electrostatic stabilization of the complexes with the DNA.The interaction of Ir 2 and Ir 2 -R 8 with DNA was investigated further using atomic-force microscopy (AFM).These studies were carried out with the relaxed pBR322 plasmid, which allows a direct observation of all possible interactions with the cyclometalated complexes. 22The relaxed DNA molecules dispersed over a mica surface show some circular structures with a number of crossing points that are indicative of supercoiling initiation (  Following the in vitro characterization, we evaluated the cytotoxicity of Ir 2 and Ir 2 -R 8 with a set of tumor cell lines including NCI-H460 (lung carcinoma), MCF-7 (breast cancer) and A2780 cis (ovarian carcinoma) cells using MTT method (detailed methods are reported in the ESI).In agreement with the DNA binding data, Ir 2 -R 8 displayed higher cytotoxic activity than Ir 2 .Thus, Ir 2 has negligible cytotoxic effects, but Ir 2 -R 8 exhibits significant effects on cell viability, so that the observed IC 50 values are comparable to those obtained for cisplatin under the same experimental conditions (Table 2). 23urthermore, the cytotoxicity of the entire set of Ir(III) R 8organometallopeptides was examined with the model doxorubicin-resistance NCI/ADR-RES ovarian cell line, which is particularly useful for the identification of compounds subjected to drug resistance.Interestingly, any of the three Ir(III) R 8 -organometallopeptides caused up to 90 % inhibition of the cell viability of NCI/ADR-RES cells.The IC 50 values estimated for Ir-R 8 , Ir 2 -R 8 and Ir 3 -R 8 (IC 50 = 32 µM, 50 µM, and 13 µM, respectively) are in the same range to that of cisplatin (IC 50 = 14 µM). 24The cytotoxicity of these compounds could be explained by the induction of highly supercoiled DNA-as observed in the AFM studies-and the resulting obstruction of processes requiring the access of proteins to the DNA (e.g., avoiding the formation of the replication fork, or the assembly of the transcriptional machinery). 25

Conclusions
In summary, we have applied a versatile solid phase peptide synthesis approach for the assembly of mono, di and trinuclear polypyridyl Ir(III) organometallopeptides and their octaarginine analogs, which display high DNA binding affinity and sequence selectivity.The DNA binding affinity of the trinuclear Ir 3 -R 8 metallopeptide is in the order of the best known organic DNA minor-groove binders, and, to the best of our knowledge, is one of the highest DNA affinity ever reported for a metal complex.17b,26 Moreover, these Ir(III) R 8 -derivatives are highly cytotoxic against diverse cell lines, including doxorubicinresistance NCI/ADR-RES and display as much activity as cisplatin.

Notes and references
Fig 2a, and Fig S1 in the ESI).Actually, some small supercoiled DNA fragments are observed (Fig S1 in the ESI).Incubation of Ir 2 with the relaxed plasmid for 24 h affected the morphology of the DNA structure, so that significantly more crossing points are noticed, and longer supercoiled fragments are clearly formed (Figs 2b, and S2 in the ESI).In addition, several DNA molecules start to aggregate, which suggest strong interactions of Ir 2 with the biomolecule.In agreement with the fluorescence titrations, Published on 23 November 2015.Downloaded by Universidade de Santiago de Compostela on 23/11/2015 09:56:34.View Article Online DOI: 10.1039/C5CC07619F This%journal%is%©%The%Royal%Society%of%Chemistry%2015! J.!Name.,%2015,%00,%1=3%|%3%incubation of pBR322 with Ir 2 -R 8 revealed even higher affinity for the DNA, with the induction of a major proportion of supercoiled DNA and of increased aggregation; the supercoiled fragments are significantly longer than those observed with Ir 2 , and large open forms, such as those observed in Fig 2a, are not present anymore Furthermore, large complex-induced DNA aggregates are detected (see Fig 2c and Fig S3).Thus, the AFM results confirm the spectroscopic observations, and support the strong DNA-binding properties of Ir 2 -R 8 .Interestingly, this important effect of Ir 2 -R 8 on the degree of DNA coiling aggregation may be compared to proteins that pack the DNA into chromosomes.