Designed metallopeptides as tools in Chemical Biology

Designed metallopeptides as tools in Chemical Biology Ph.D. 2017 Ghofrane Barka COMPOSTELA Y , PR SANTIAGO DE COMPOSTELA, CERTIFICAN: que la memoria adjunta titulada “Designed Metallopeptides as Tools in Chemical Biology” , presenta Dra. Materiales Moleculares (CIQUS) de la Universidad de Santiago de Compostela. Considerando que constituye trabajo de Universidad de Santiago de Compostela. , se expide el presente certificado en Santiago de Compostela a 11 de Septiembre de 2017. Fdo. Prof. Mig Director y tutor Fdo. Pr Codirector Acknowledgements I would particularly like to thank my supervisors Eugenio and Miguel for their excellent cooperation and for all of the opportunities I was given to conduct my research and further my dissertation. I strongly acknowledge all their support and patience with my everyday work as a PhD student. I am also very thankful with Eu-Metalic (II) Erasmus scholarship for giving me afterwards the opportunity to expand my research experience. Special thanks to the international office Team for their help and advice. I also want to express my deep appreciation for my colleagues, especially Iria, Gustavo, Diego, David, Jacobo, Sonia, Lidia, Renata, Cristina, Jessica, Sergio and Arcadio, for the help and support you have offered me on my recent project. I feel so grateful to you for taking time out of your busy schedule to answer so many questions. Without your help, I would not have been able to complete the project in such a proficient and timely manner. Out of the lab, and for making my time in Santiago enjoyable, I would like to thank Irene, Marouan, Ahmad, Anas, Salam and Diana, for their friendship and good time. Finally, I must express my very profound gratitude to my parents for providing me with unfailing support and continuous encouragement throughout my years of study. Thank you. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 7 Index Acknowledgements .......................................................................................... 5 Index ................................................................................................................. 7 Abbreviations ................................................................................................... 9 Abstract .......................................................................................................... 11 General Introduction ..................................................................................... 15 Chemical Biology: origins and relevance ...................................................... 15 Sequence-Specific DNA Recognition with artificial peptides ....................... 16 Introduction ............................................................................................... 16 DNA recognition from a supramolecular perspective ................................ 17 DNA-binding peptides ............................................................................... 20 Stimuli-responsive DNA-binding peptides................................................. 25 Interaction of Rh(II) and Ru(II) complexes with DNA.................................. 40 Objetive .......................................................................................................... 45 Discusion......................................................................................................... 47 Abstract ........................................................................................................ 47 Introduction .................................................................................................. 49 Results and discussion .................................................................................. 52 Metallopeptide synthesis ............................................................................ 52 DNA binding studies with synthetic oligonucleotides ................................ 54 Circular dichroism studies ......................................................................... 60 Cell internalization studies by fluorescence microscopy ............................ 62 Conclusions .................................................................................................... 65 Experimental Section ..................................................................................... 67 General ......................................................................................................... 67 Synthetic procedures ..................................................................................... 69 Synthesis of the unnatural coordinating residue Fmoc-βAla-bpy-OH (1) ... 69 Synthesis of peptide ligands and metallopeptides....................................... 69 GHOFRANE BARKA 8 General procedure for peptide cleavage-deprotection ................................ 70 Mass spectra and HPLC chromatograms of the Ru(II) metallopeptides (3 to 6) ............................................................................................................... 71 Fluorescence studies ..................................................................................... 76 ) H “w ll- ” -DNA binding studies ................................... 76 b) Double- “w - ” “ ” -DNA binding studies ........................................................................................................ 91 UV-vis studies ............................................................................................ 130 ) H “w - ” -DNA binding studies ................................. 130 b) Double- “w - ” “ ” -DNA binding studies ...................................................................................................... 132 CD studies .................................................................................................. 161 ) H “w - ” -DNA binding studies ................................. 161 b) Double- “w - ” “ ” -DNA binding studies ...................................................................................................... 163 Fluorescent microscopy studies .................................................................. 191 DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 9 Abbreviations δ chemical shift ε extinction coefficient λ wavelength μL microliter μM micromolar μm micrometer Ac acetyl AcOH acetic acid a.u. arbitrary units Boc tert-butoxycarbonyl Bzl bencyloxycarbonyl cm centimeter c.p.s. counts per second d doublet dd double doublet ds double stranded DIEA N,N-diisopropylethylamine DMF dimethylformamide dmol decimol DMSO dimethylsulfoxide DNA deoxyribonucleic acid em emission equiv equivalents ESI electrospray ionization Et3N triethylamine Et2O diethyl ether EtOAc ethyl acetate EtOH ethanol exc excitation Fmoc 9-fluorenylmethoxycarbonyl h hour J coupling constant KD dissociation constant l optical path lenght M molar m multiplet mAU absorbance miliunits Me methyl MeCN acetonitrile MeOH methanol mg miligram MHz megahertz min minute mL mililiter mM milimolar mmol milimol mre molar ellipticty per residue MS mass spectrometry m/z mass charge relation nM nanomolar nm nanometer PBS phosphate-buffered saline PhSiH3 phenylsilane RMN nuclear magnetic resonance rt room temperature s secon, singlet SPPS solid phase peptide synthesis t-Bu tert-butyl TCEP tris(2-carboxyethyl)phosphine TFA trifluoroacetic acid THF tetrahydrofuran TIS triisopropylsilane TMS tetramethylsilane TNBS 2,4,6-trinitrobenzene sulfonic acid UV ultraviolet GHOFRANE BARKA 16 century of a new discipline: Biochemistry. While this discipline is more focused to the study of biological processes and the behaviour of biomolecules and biopolymers from a biological perspective, Chemical Biology deals with the development of chemical tools to further understand the molecular bases of biology as well as to modify them in a selective manner.4 hl ’s synth s s of a, wh ch p s nts a landma k ach v m nt n o an c synthesis and biological chemistry. This new field of science has rapidly established in the last years as demonstrated by the launching of several specialized journals like Chemistry & Biology, ChemBioChem, Current Opinion in Chemical Biology, ACS Chemical Biology, Organic & Biomolecular Chemistry or Nature Chemical Biology. Sequence-Specific DNA Recognition with artificial peptides Introduction Gene transcription in eukaryotic cells is largely controlled by the interaction of certain proteins called transcription factors (TFs) with specific DNA sequences,5 which modify the basal levels of transcription, either enhancing or repressing their expression.6 It is known that misregulation of TFs is at the origin of a number of diseases, including cancer,7 and thus there is growing interest in understanding the molecular basis of specific DNA recognition, as well as in developing designed DNA binding agents. Furthermore, in addition to the potential applications of such synthetic binders, the development of artificial DNA binding agents represents an unmet challenge at the crossroads between supramolecular and biological chemistry and, beyond its biological relevance, it might find future applications in combination with DNA nanotechnology.8 Most 4 G. von Kiedrowski, Chembiochem, 2001, 2, 597–598. 5 A. H. Brivanlou, J. E. Darnell Jr, Science 2002, 295, 813–818. 6 a) A.-L. Todeschini, A. Georges, R. A. Veitia, Trends Genet. 2014, 30, 211–219; b) M. Ptashne, Nature 1986, 322, 697–701. 7 P. P. Pandolfi, Oncogene 2001, 20, 3116–3127. 8 A. R. Chandrasekaran, Nanoscale 2016, 8, 4436–4446. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 17 artificial DNA binding agents described to date are small molecules, including coordination compounds, as well as small organic molecules, which typically bind to the DNA through minor groove insertion or intercalation mechanisms, and have been the subject of a number of excellent reviews.9 10 In addition to those classic DNA-binding agents, researchers, inspired by natural TFs, have also explored the potential of designed peptides for the specific recognition of DNA, developing increasingly sophisticated systems that not only display excellent binding properties, but also are endowed with new properties not found in their natural counterparts, such as luminescence, photocontrol, nuclease activity, or stimuli-responsive binding, which we will review in the following pages.11 DNA recognition from a supramolecular perspective The B-DNA, the most relevant DNA conformation under physiological conditions, is characterized by a rather uniform right-handed double helix formed by two antiparallel oligodeoxyribonucleotide chains, held together by the combination of stacking interactions between the base pairs (bps), and hydrogen bonds between complementary Watson-Crick bps.12 The asymmetry of the deoxyribonucleotides generates two grooves in B-DNA double helix: the major groove, which is the wide side (~11.7 Å between phosphates across the groove) facing away the sugar-phosphate backbone, and the minor groove, which is the narrow side (~5.7 Å) of the bps facing towards the backbone (Figure 1). Importantly, the B-DNA conformation exposes the polar sugar/phosphate backbone while shielding the aromatic surface of the bases from the aqueous environment, thus giving rise to a monotonous 9 a) S. Neidle, Nat. Prod. Rep. 2001, 18, 291–309; b) A. Ali, S. Bhattacharya, Bioorg. Med. Chem. 2014, 22, 4506–4521; c) W. Han Ang, P. J. Dyson, Eur. J. Inorg. Chem. 2006, 2006, 4003–4018; d) C. Moucheron, New J. Chem. 2009, 33, 235–245; e) B. M. Zeglis, V. C. Pierre, J. K. Barton, Chem. Commun. 2007, 4565–4579; f) C. Y. Majmudar, A. K. Mapp, Curr. Opin. Chem. Biol. 2005, 9, 467–474; g) P. B. Dervan, R. W. Bürli, Curr. Opin. Chem. Biol. 1999, 3, 688–693; h) P. B. Dervan, Bioorg. Med. Chem. 2001, 9, 2215–2235. 10 a) J. B. Chaires, Curr. Opin. Struct. Biol. 1998, 8, 314–320; b) I. Romero-Canelón, P. J. Sadler, Inorg. Chem. 2013, 52, 12276–12291; c) A. C. Komor, J. K. Barton, Chem. Commun. 2013, 49, 3617–3630; d) G. S. Khan, A. Shah, Zia-ur-Rehman, D. Barker, J. Photochem. Photobiol. B 2012, 115, 105–118. 11 a) E. Pazos, J. Mosquera, M. E. Vázquez, J. L. Mascareñas, ChemBioChem 2011, 12, 1958–1973; b) M. E. Vázquez, A. M. Caamaño, J. L. Mascareñas, Chem. Soc. Rev. 2003, 32, 338–349. 12 V. A. Bloomfield, D. M. Crothers, I. Tinoco, Nucleic Acids: Structures, Properties, and Functions, Sterling Publishing Company, 2000; b) C. R. Calladine, H. Drew, Understanding DNA: The Molecule and How It Works, Academic Press, 1997. GHOFRANE BARKA 18 physicochemical landscape, where particular base pairs can only be distinguished by relatively minor differences in their hydrogen bond donor/acceptor patterns at the bottom of the grooves (A·T/T·A, and G·C/C·G bps are indeed degenerate from the minor groove). Thus, B-DNA poses a great challenge for the development of specific binders, which given the geometric “ ” major groove. Despite the apparent regularity in the B-DNA, it is now recognized that the local properties of the B-DNA double helix are highly dependent on the base pair sequence, which can be considered not only a linear code, but also a structural code that influences both the conformation and molecular dynamics.13 For example, A·T-rich tracts are known to induce bending of the DNA double helix, which is required for the activity of some regulatory sites.14 A·T-rich sequences also display a narrower minor groove,15 with a distinct hydration pattern along the floor of the groove,16 and is also characterized by a much more negative electrostatic potential,17 which can influence both reactivity,18 and specific recognition of such sequences.19 Therefore, specific DNA recognition involves two types of interactions: direct readout through the formation of specific hydrogen bonds, polar interactions, and hydrophobic contacts between amino acids side chains and nucleotide functional groups—typically at the exposed edges of the bps in the DNA major groove,20 and indirect readout, which is a more subtle mechanism related to the unique physicochemical properties of specific DNA sequences, such as readout of the stiffness of DNA double helix, its conformational flexibility, our particular electrostatic potential.21 These effects can be even observed in the 13 R. E. Dickerson, J. Mol. Biol. 1983, 166, 419–441. 14 H. C. Nelson, J. T. Finch, B. F. Luisi, A. Klug, Nature 1987, 330, 221–226. 15 D. G. Alexeev, A. A. Lipanov, Skuratovskii IYa, Nature 1987, 325, 821–823. 16 a) S. K. Pal, L. Zhao, A. H. Zewail, Proc. Natl. Acad. Sci. USA 2003, 100, 8113–8118; b) M. L. McDermott, H. Vanselous, S. A. Corcelli, P. B. Petersen, ACS Cent. Sci. 2017, DOI 10.1021/acscentsci.7b00100. 17 B. Jayaram, K. A. Sharp, B. Honig, Biopolymers 1989, 28, 975–993. 18 E. P. Bishop, R. Rohs, S. C. J. Parker, S. M. West, P. Liu, R. S. Mann, B. Honig, T. D. Tullius, ACS Chem. Biol. 2011, 6, 1314–1320. 19 V. Iyer, K. Struhl, EMBO J. 1995, 14, 2570–2579. 20 S. A. Coulocheri, D. G. Pigis, K. A. Papavassiliou, A. G. Papavassiliou, Biochimie 2007, 89, 1291–1303. 21 a) R. Rohs, S. M. West, P. Liu, B. Honig, Curr. Opin. Struct. Biol. 2009, 19, 171–177; b) R. Rohs, S. M. West, A. Sosinsky, P. Liu, R. S. Mann, B. Honig, Nature 2009, 461, 1248– 1253; c) M. Y. Zhitnikova, A. V. Shestopalova, J. Biomol. Struct. Dyn. 2016, 1–14. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 19 DNA binding preferences of small molecules and, for example, it has been found that simple intercalators display some sequence selectivity due to the local deformability and conformational preferences of particular DNA sequences.22 Evidently, natural DNA-binding proteins exploit both strategies to achieve high affinity and selectivity. Unfortunately, artificial systems are still much more rudimentary, and rational implementation of indirect readout in designed systems is still extremely complex. Figure 1. a) Classic Dickerson-Drew B-DNA dodecamer (PDB: 4C64) highlighting the major structural elements: sugar-phosphate backbone in orange, exposed bp edges in the major groove in dark blue, and exposed edges of the bps in the minor groove in light blue. b) structure of the G·C and A·T bps showing the pattern of hydrogen bond donors (d) and acceptors (a).23 Recognition of specific DNA sequences in eukaryotes not only relies on the readout mechanisms that have been just outlined that mediate the interactions between the DNA and each protein, but is also orchestrated through specific protein-protein interactions that lead to the formation of higher order assemblies of multiple TFs that cooperatively bind to the DNA. This allows the integration 22 a) I. Haq, J. Ladbury, J. Mol. Recognit. 2000, 13, 188–197; b) J. B. Chaires, Biopolymers 1997, 44, 201–215. 23 L. Lercher, M. A. McDonough, A. H. El-Sagheer, A. Thalhammer, S. Kriaucionis, T. Brown, C. J. Schofield, Chem. Commun. 2014, 50, 1794–1796. GHOFRANE BARKA 20 of multiple signaling pathways into complex networks of TFs,24 and also expands the number of unique DNA binding sites that can be addressed with a given number of TFs, which can combine in different ways to bind to composite sequences, thus, allowing the complex spatio-temporal control of the >30,000 human genes with a limited set of 2,000-3,000 TFs.25 Despite the relevance of TF cooperativity, the complexities involved in the implementation of such effects have hampered the design of artificial systems that display some level of cooperativity in their DNA binding. DNA-binding peptides GCN4, the Jack of all trades, master of designed DNA-binding peptides Transcription factors are grouped in families according to the fold of their DNA binding domains.26 The GCN4 is an archetypical member of the Basic Leucine Zipper (bZIP) family of TFs. As all bZIP TFs, GCN4 binds to its target / (5′-ATGAC GTCAT-3′) 1/ (5′-ATGA(c/g)TCAT-3′) sites as a leucine zipper-mediated dimer of uninterrupted α-helices. The specific contacts with the edges of the bases exposed in the major groove of the DNA take place through the N-terminal basic region (br), which folds into an α-helix upon DNA binding (Figure 2).27 Importantly, it has been shown that monomeric bZIP TFs typically display low DNA binding affinity in the µM range,28 and that dimerization is required for DNA binding by bZIP proteins, because of the high entropic cost involved in the folding of the basic region into the α-helical conformation.29 Thus bZIP TFs behave as intrinsically disordered proteins that are largely unstructured under physiological conditions and display folding coupled DNA binding. This allows bZIP proteins to recognize their target DNA sites with exquisite selectivity while still forming relatively weak, and readily reversible complexes—in contrast with rigid molecules, in which stability of 24 a) S. Neph, A. B. Stergachis, A. Reynolds, R. Sandstrom, E. Borenstein, J. A. Stamatoyannopoulos, Cell 2012, 150, 1274–1286; b) T. Ravasi, H. Suzuki, C. V. Cannistraci, S. Katayama, V. B. Bajic, K. Tan, A. Akalin, S. Schmeier, M. Kanamori-Katayama, N. Bertin, et al., Cell 2010, 140, 744–752. 25 a) L. Chen, Curr. Opin. Struct. Biol. 1999, 9, 48–55; b) A. Reményi, H. R. Schöler, M. Wilmanns, Nat. Struct. Mol. Biol. 2004, 11, 812–815. 26 C. W. Garvie, C. Wolberger, Mol. Cell 2001, 8, 937–946. 27 a) T. E. Ellenberger, C. J. Brandl, K. Struhl, S. C. Harrison, Cell 1992, 71, 1223–1237; b) W. Keller, P. König, T. J. Richmond, J. Mol. Biol. 1995, 254, 657–667. 28 X. Wang, W. Cao, A. Cao, L. Lai, Biophys. J. 2003, 84, 1867–1875. 29 a) C. Park, J. L. Campbell, W. A. Goddard, J. Am. Chem. Soc. 1996, 118, 4235–4239; b) M. Zhang, B. Wu, H. Zhao, J. W. Taylor, J. Pept. Sci. 2002, 8, 125–136. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 21 their complexes usually correlates with their specificity—and allows increased rates of macromolecular associations within complex interaction networks.30 Figure 2. a) Structure of the DNA binding domain of the GCN4 TF bound to the AP1/GCRE site, highlighting the main structural elements and the key residues for specific DNA recognition. The leucine zipper in light green, and the br in blue. b) Details of the specific interactions between the GCN4 br with the AP1/GCRE half-site. Hydrophobic contacts (Ala238, Ala239, and Ser242) with white dot lines, polar/electrostatic contacts (Asn235, and Arg243) with black dot lines. Bases that do not participate in specific contacts are outlined as slabs for clarity. Artificial GCN4 peptide dimers The structural simplicity of bZIP TFs, added to the wealth of functional, biophysical and structural information about GCN4, made it into the preferred platform for the development of peptide-based DNA binders. A key development was reported in 1990 by the group of Peter S. Kim, who demonstrated in a seminal paper that the complete leucine zipper region of the GCN4 DNA binding domain could be replaced by a disulfide bond, and that the minimized 34-residue peptide corresponding largely to the GCN4 between residues 222 and 253 (plus a short Gly-Gly-Cys linker) could bind to the natural target site CREB with high (nM) affinity at 4 ºC in the form of a disulfide dimer (Figure 3a).31 Additional studies reported two years later helped to identify the sequence of the minimal GCN4 basic region fragment that displays specific DNA binding in the form of disulfide dimer. Thus, a 23-residue peptide featuring a C-terminal Gly-Gly-Cys linker (AcDPAALKRARNTEAARRSRARKLQ-GGC) binds to both ATF/CREB and 30 a) M. Miller, Curr. Protein Pept. Sci. 2009, 10, 244–269; b) J. Habchi, P. Tompa, S. Longhi, V. N. Uversky, Chem. Rev. 2014, 114, 6561–6588. 31 R. V. Talanian, C. J. McKnight, P. S. Kim, Science 1990, 249, 769–771. GHOFRANE BARKA 22 AP1/GCRE sites in a sequence-specific manner with high affinity in its oxidized form as a dimer.32 Figure 3. Schematic representation of the GCN4 dimer showing the helices as tubes, and the strategy to generate artificial GCN4 dimers by removing the leucine zipper and adding sp c f c l nk s b tw n th b f a m nts a) P K m’s d s lf d d m ,27,28 b) A Sch pa tz’s transition metal coordination compound linker,29 c) T Mo ’s ch al l nk d v d f om trans-9,10-dihydrophenanthrene-9,10-diol,31 d) T Mo ’s s p amol c la l nk bas d on a β-cyclodextrin/adamantane complex,33,34 ) A Madd ’s st o d l nk ,36,37 f) A Madd ’s cyclodextrin dimer,35 ) J L Masca ña’s c s/t ans azob nz n photosw tch l nk ,39 h) A. P acock’s m tal switch linker,30 ) A P acock’s photocont oll d anth ac n d m 53 w K ’ w years the replacement of the leucine zipper with other functionalities. The group of Alanna Schepartz reported in 1993 the use of a set of transition metal complexes as linkers to systematically explore the effect of the geometry of the linker—and thus, of the relative geometry of the attached basic regions—on the DNA binding properties of the resulting dimers (Figure 3b). Unsurprisingly, they found that dimerization was not sufficient for DNA binding, and that the geometry of the metal complexes influenced not only the binding affinity of the dimers, but also the sequence selectivity.33 The effect of the geometry in metal complexes was also exploited by the group of A. Peacock, who in 2014 reported 32 R. V. Talanian, C. J. McKnight, R. Rutkowski, P. S. Kim, Biochemistry 1992, 31, 6871– 6875. 33 B. Cuenoud, A. Schepartz, Science 1993, 259, 510–513. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 23 4 w 2 2′:6′ 2′′-terpyridine linker. This linker would undergo a conformational change upon metal ion coordination, which in turn would affect the relative orientation of the basic regions, and influence the DNA binding of the dimer. CD and UV-vis spectroscopy demonstrated that coordination od this metal-chelating GCN4 dimer to Cu(II) or Zn(II) ions promoted DNA binding (Figure 3h).34 The influence of geometric effects on the DNA binding properties of GCN4 dimers were also studied by T. Morii and Y. Sugiura who, based on earlier studies using a 14-residue DNA-binding peptide derived from the basic region of the helix-loop-helix transcription factor MyoD,35 synthesized various GCN4 dimers connected by C2-symmetric, trans-9,10-dihydro phenanthrene-9,10-diol linkers with different chiralities (Figure 3c). In this case, the authors found that the stability of the different complexes was unaffected by the chirality of the linkers, which was consistent with the small effect of the chirality on the relative orientation of the attached basic regions. Interestingly, the authors also reported dimers of the GCN4 br through the N-terminus, which recognized (5′-TCATC GATGA-3′) w polarity of each half-site of the parent ATF/CREB) site was reversed.36 The group of T. Morii was the first to report the use of a non-covalent linker between GCN4 basic regions; for this, they synthesized two peptides by alkylation of a C-terminal Cys attached to the core GCN4 br peptide with N- (bromoacetyl)-1-adamantanemethylamine and mono-6-deoxy-6-iodoβcyclodextrin. Both peptides form a heterodimer, mediated by formation of an inclusion complex between β-cyclodextrin, and an adamantyl group, which / (5′-ATGAC GTCAT-3′) formation of the complex was inhibited by the addition of free β-cyclodextrin, once again demonstrating that dimerization of the GCN4 basic regions is required for specific DNA binding (Figure 3d).37 Interestingly, their design relies on the high stability of the complex between adamantane derivatives and β-cyclodextrins (KD ≈ 1 3 µ ) w and cooperative DNA binding was still possible if the original adamantane was replaced with norbornyl, KD(Nb/β- ) ≈ 3 1 µ 34 E. Oheix, A. F. A. Peacock, Chem. Eur. J. 2014, 20, 2829–2839. 35 T. Morii, M. Simomura, S. Morimoto, I. Saito, J. Am. Chem. Soc. 1993, 115, 1150–1151. 36 T. Morii, Y. Saimei, M. Okagami, K. Makino, Y. Sugiura, J. Am. Chem. Soc. 1997, 119, 3649–3655. 37 M. Ueno, A. Murakami, K. Makino, T. Morii, J. Am. Chem. Soc. 1993, 115, 12575–12576. GHOFRANE BARKA 24 KD(NrA/β- ) ≈ 2 7 µ ) w w cyclohexyl group, which displays a much lower binding affinity for βcyclodextrin with a KD(Ch/β- ) ≈ 14 µ 38 A recent paper by A. Madder also makes use of (α, β, γ)-cyclodextrins for dimerization (Figure 3f). In their case, the cyclodextrins were not used as receptors, but as scaffolds for covalent attachment of the two GCN4 basic regions. The dimers were readily synthesized by a straightforward coppercatalyzed azide/alkyne cycloaddition (CuAAC) between the GCN4 br peptide bearing a C-terminal propargylglycine, and the corresponding diazido cyclodextrins. Curiously, while the α-cyclodextrin and β-cyclodextrin GCN4 br dimers display rather similar binding affinities for the ATF/CREB site with KD(α-CD/DNA) = 50 ± 20 nM and KD(β-CD/DNA) = 30 ± 20 nM, the γcyclodextrin analog binds with weaker affinity to the same DNA with a KD(γCD/DNA) = 100 ± 60 nM, and appears to give rise to non-specific complexes in Electrophoretic Mobility Shift Assays (EMSA), suggesting that the distance between the two GCN4 basic regions in the γ-cyclodextrin dimer is not optimal for their simultaneous insertion as required in the specific complex.39 ’ group has also reported the application of steroid scaffolds for the homodimerization of GCN4 basic regions,40 as well as for the heterodimerization of the basic regions of related c-Myc/Max basic Helix-LoopHelix transcription factors, a class of TFs related to the bZIP family, containing an additional loop between the leucine zipper and the basic region.41 The use of a steroidal cholic acid moiety as dimerizer element was justified because it provides a rigid, and synthetically-accessible scaffold to attach the peptides with reported benefits for improving the cell uptake of the conjugates (Figure 3e). In addition to bZIP dimers that replace the leucine zipper dimerization with other functionalities, in 2012 J. L. Mascareñas reported a derivative of an heterodimeric cFos/cJun complex in which the basic region of the cFos bZIP TF 38 Y. Aizawa, Y. Sugiura, M. Ueno, Y. Mori, K. Imoto, K. Makino, T. Morii, Biochemistry 1999, 38, 4008–4017. 39 J Z ěš ý K A. Madder, Org. Biomol. Chem. 2015, 13, 5273–5278. 40 L. L. G. Carrette, T. Morii, A. Madder, Eur. J. Org. Chem. 2014, 2014, 2883–2891. 41 a) D. Verzele, A. Madder, Eur. J. Org. Chem. 2013, 2013, 673–687; b) Y. R. García, Y. Vladimir Pabon-Martinez, C. I. Edvard Smith, A. Madder, Chem. Commun. 2017, 53, 6653– 6656. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 25 was replaced by a small DNA-binding organic molecule.42 Similarly to the role of cFos in the natural complex, the bisbenzamidine hybrid of the Fos leucine zipper ( → 157 to Leu179) stabilized the cJun/DNA interaction but, in contrast with the natural cFos TF, which is bound to the DNA major groove, the cFos leucine zipper domain was delivered from the adjacent DNA minor groove. Furthermore, the dimer between cJun and the cFosbisbenzamidine conjugate targeted a the composite DNA sequence different from the ATF/CREB binding site targeted by the natural cJun/cFos heterodimer (5′-ATGAC G AAATTT-3′) w ained the natural ATF/CREB half-site (ATGAC) next to the A·T-rich site preferred by the bisbenzamidine with an extra G as spacer between both recognition elements (Figure 4). Figure 4. Left: Schematic representation of the cFos/cJun/DNA complex. Right: proposed interaction between the cFos-bisbenzamidine hybrid and cJun, binding as a heterodimer to a composite DNA site containing the consensus recognition sites for cJun and the bisbenzamidine. Stimuli-responsive DNA-binding peptides Light-responsive systems A relevant subset of GCN4 dimers are those whose DNA binding can be modulated by light. In the year 2000 the group of J. L. Mascareñas reported the first artificial DNA binding peptide that displayed light-induced DNA binding.43 Light responsiveness was achieved by incorporating an azobenzene unit, a well42 M. I. Sánchez, O. Vázquez, J. Martínez-Costas, M. E. Vázquez, J. L. Mascareñas, Chem. Sci. 2012, 3, 2383. 43 A. M. Caamaño, M. E. Vázquez, J. Martínez-Costas, L. Castedo, J. L. Mascareñas, Angew. Chem. Int. Ed Engl. 2000, 112, 3234–3237. GHOFRANE BARKA 32 of a light-sensitive histidine building block for Fmoc/tBu solid-phase peptide synthesis in which the imidazole side chain is protected with a ruthenium complex. This building block was used for the synthesis of a photoactivatable Arg-Gly-His tripeptide (RGH), which has been shown to be an efficient metalchelating sequence (KD < 10–16 M), endowed with DNA binding and endonuclease properties in the presence of Ni(II) ions and oxidizing agents.60 We demonstrated that the caged His analog was unable to coordinate the Ni(II) required form the catalytic metallopeptide. Moreover, we also showed that DNA binding and nuclease activity could be recovered upon irradiation at 455 nm, and uncaging of the His residue (Figure 9). Figure 9. Caged RGH peptide does not bind to Ni(II). Irradiation with red ligt at 455 releases the metal-chelating peptide, which folds and binds into the DNA minor groove, where it can exert its nuclease activity. Metal coordination and dynamic DNA-binding peptides In contrast with the static designs which represent the majority of DNA-binding peptides reported until now, one of the first steps into the development of stimuli-responsive peptides derived from transcription factors was reported by Y. Sugiura in 2004.61 In this work, the Fos leucine zipper is modified with a pair of metal-chelating iminodiacetic acid derivatives of lysine (Ida) at positions i, 60 a) Q. Liang, P. D. Eason, E. C. Long, J. Am. Chem. Soc. 1995, 117, 9625–9631. 61 S. Futaki, T. Kiwada, Y. Sugiura, J. Am. Chem. Soc. 2004, 126, 15762–15769. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 33 and i+2. Incubation with Fe(III), and coordination of this metal ion, destabilizes the alpha helical conformation of the Fos(Ida)2 peptide, thus preventing the formation of the heterodimer with the Jun leucine zipper. Unfortunately, the authors did not pursue this concept for modulating the DNA binding of the FosJun network of homoand heterodimers. In a related work, a successful DNA binding switch in response to a metal ion was reported by the group of Futaki in 2009, who expressed a recombinant GCN4 DNA binding domain (56-mer peptide) featuring two pairs of Cys residues at relative i and i+2 positions in the leucine zipper domain. These residues were selectively derivatized using N-(2tosylthioethyl)iminodiacetic acid to turn them into metal-chelating residues. The resulting tetra-Ida-modified GCN4 DNA binding domain displayed significant helical structure, and high DNA binding affinity for the AP1/GCRE site (KD = 22 ± 3.0 nM) similar to that of the natural GCN4 dimer (KD = 15 ± 2.6 nM). Addition of excess of Co(II) to this metal-binding peptide induced a 33% decrease in its helical content, and also a drastic reduction in its DNA binding affinity (KD > 1.0 µM). As expected, addition of EDTA to the mixture—and sequestering of the Co(II) ions—resulted in the recovery of the initial binding affinity in absence of the metal ion (KD = 22 ± 1.1 nM). Curiously, the switching effect was not observed in control peptides that contained a single pair of Ida residues in their leucine zipper domains.62 The effect to metal ion coordination can be combined with other orthogonal signals, such as redox processes to yield more complex designs that respond to multiple stimuli. This was exemplified in 2013, when we described the synthesis of a GCN4 basic region fragment that contained a redox-sensitive Cys residue on its N-terminus, and a metal5 5′′-dimethyl-2 2′:6′ 2′′- terpyridine ligand orthogonally attached to a C-terminal Lys231 residue (Cys)br(tpy).63 Incubation of this peptide with Ni(ClO4)2 in the presence of the target ATF/CREB site (5'-ATGA CG TCAT-3') gave rise to slow-migrating bands consistent with the formation of a specific peptide-DNA complex between the nickel-mediated dimer of the basic region and the DNA. Under the same conditions, the peptide did not display significant affinity for the inverted sequence (5'-TCAT CG ATGA-3'). In contrast, the N-terminal disulfide dimer formed by oxidation of the peptide with DTNB, 5,5'-dithiobis-(2-nitrobenzoic 62 Y. Azuma, M. Imanishi, T. Yoshimura, T. Kawabata, S. Futaki, Angew. Chem. Int. Ed. 2009, 48, 6853–6856. 63 J. Mosquera, A. Jiménez-Balsa, V. I. Dodero, M. E. Vázquez, J. L. Mascareñas, Nat. Commun. 2013, 4, 1874. GHOFRANE BARKA 34 acid), only showed binding to the inverted site (Figure 10). As expected, reduction of the disulphide dimer by treatment TCEP (tris(2-carboxyethyl) phosphine) recovered the monomeric (Cys)br(tpy) peptide, and promoted the disassembly of the complex with the inverted sequence. Curiously, the disulfide dimer required the presence of Ni(II) ions to bind to its target inverted site. Figure 10. Dynamic DNA binding selection to multiple sites. The reduced monomeric (Cys)br(tpy) peptide in the presence of Ni(ClO4) binds to the consensus DNA (5'-ATGA CG TCAT-3') as a metal-mediated homodimeric complex (KD ≈ 670 nM at 4ºC) Ox dat on of th N-terminal Cys side chain with DTNB yields the N-terminal disulfide dimer, which binds to the inverted polarity sequence 5'-TCAT CG ATGA-3' in the presemnce of Ni(II) salts (KD ≈ 758 nM). The original C-terminal complex can be recovered by reduction with TCEP (tris(2carboxyethyl)phosphine). In both cases the binding of the peptides for their non-target DNAs were in the µM range). DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 35 Artificial GCN4 basic region monomers Monomeric GCN4 basic regions do not bind to their target DNA half-site with high affinity due to the entropic cost associated to the folding of the peptide chain into the appropriate α-helical conformation, but residue grafting is a powerful strategy to obtain stabilized α-helices and thus obtain high-affinity DNA binding monomeric GCN4 peptides.64 In this approach, selected residues that create the desired binding epitope are inserted (grafted) into a stable threedimensional peptide scaffold. The group of A. Schepartz described in 1999 successful application of this strategy to obtaining high-affinity monomeric DNA-binding peptides.65 Their design involved the introduction of the GCN4 residues mediating DNA binding in the basic region into the α-helix of the avian pancreatic polypeptide (aPP), a stable miniprotein consisting of a single α-helix stabilized by hydrophobic interactions with a type II polyproline helix. The resulting chimera was capable of recognizing the half site of the GCN4 DNA t (5’-ATGA-3’) w KD of 1.5 nM under physiological ionic strength. However, the mutations introduced in the aPP sequence for achieving DNA binding resulted in the disruption of the aPP hydrophobic core, and reduced structural stability of the construct. Further refinements of this initial binder using phage libraries resulted in a new peptide with extraordinary DNA affinity at 4 ºC (KD ~ 23 pM), which even retained high affinity at 25 ºC (KD ~ 1.6 nM).66 Following the successful implementation of the grafting strategy to the synthesis of simplified versions of a bZIP transcription factor (GCN4), the Schepartz group demonstrated the versatility of this approach by obtaining miniature homeodomain proteins, again by dissecting the key DNA contacting residues and introducing them into the structure of the versatile aPP peptide scaffold.67 The group of G. L. Verdine demonstrated in 1995 that it was possible to drive specific DNA recognition by a monomeric GCN4 basic region if the peptide was delivered into the major groove intramolecularly.68 Attaching the GCN4 br to the DNA significantly increased its effective concentration, and reduced the entropic loss associated with intermolecular binding, thereby allowing the formation of the desired (intramolecular) complex. The design required a Gly64 a) J. A. Robinson, ChemBioChem 2009, 10, 971–973; b) J. Fernandez-Carneado, D. Grell, P. Durieux, J. Hauert, T. Kovacsovics, G. Tuchscherer, Biopolymers 2000, 55, 451–458. 65 N. J. Zondlo, A. Schepartz, J. Am. Chem. Soc. 1999, 121, 6938–6939. 66 J. W. Chin, A. Schepartz, J. Am. Chem. Soc. 2001, 123, 2929–2930. 67 J. K. Montclare, A. Schepartz, J. Am. Chem. Soc. 2003, 125, 3416–3417. 68 D. Stanojevic, G. L. Verdine, Nat. Struct. Biol. 1995, 2, 450–457. GHOFRANE BARKA 36 Gly-Cys connector to connect the C-terminus of the GCN4 br to an Adenine 5′ 1/ - (5′-A(c)TCAT-3′ modified base in bold) through a disulfide bond. Taking the concept of intramolecular delivery for specific DNA binding one step further, the group of J. L. Mascareñas reported in 2001 the synthesis of conjugates between the GCN4 br and a small minor groove binding molecule that was capable of sequence-specific DNA recognition.69 The role of the minor groove binding agent (a distamycin derivative) was analogous that of the ’ x: supramolecular anchor that, upon binding to its target sequence in the DNA (5′-AAATT-3′) w 4 — the ATF/CREB half- (5′-gTCAT-3′)—in the adjacent major groove. Based on the X-ray structures of the GCN4 dimer and distamycin bound to their respective DNA sites,70 the authors built a hypothetical model of the simultaneous interaction of the GCN4 basic region and the small DNA binding agent bound to contiguous sites, which allowed the identification of Arg245 as the ideal position for installing short aminoalkyl linkers connecting with the distamycin bound to the adjacent minor groove. Unlike isolated GCN4 basic regions, the resulting conjugate displayed tight (low nM affinity at 4 ºC) and selective binding to a composite site containing the consensus sequences for both the GCN4 and the distamycin (5′-gTCAT-AAATT-3′) expected, control experiments with DNAs lacking the target sequence of the distamycin anchor did not bind to the DNA, thus confirming the key role of the accessory interactions in the minor groove for stabilizing the complex. Following that initial report, the group of J. L. Mascareñas demonstrated that the conjugation strategy could be generally applied for the stabilization of TF fragments, describing a variety of peptide hybrids that recognized extended sites containing the sequence of both the peptides and the distamycin. Thus, for example, the DNA binding helix of the Skn-1 transcription factor could be conjugated through its Lys232 side chain to distamycin, and the resulting hybrid 5′-AAAA-TCAT-3′ 71 Importantly, this strategy could also be extended to other transcription factor families, so that conjugation 69 M. E. Vázquez, A. M. Caamaño, J. Martínez-Costas, L. Castedo, J. L. Mascareñas, Angew. Chem. Int. Ed. 2001, 40, 4723–4725. 70 M. Coll, C. A. Frederick, A. H. Wang, A. Rich, Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 8385–8389. 71 J. B. Blanco, M. E. Vázquez, L. Castedo, J. L. Mascareñas, ChemBioChem 2005, 6, 2173– 2176. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 37 to distamycin restored the DNA binding properties of a fragment of the GAGA zinc finger transcription factor, which selectively bound to the composite 5′-TTTT-GAGAG-3′ · -rich site for distamycin (5′-TTTT-3′) x (5′- GAGAG-3′) 72 Similar conjugates with GCN4 and GAGA fragments, and even with homeodomain fragments,73 were also described using bisbenzamidines as minor groove binding ligands,74 further demonstrating the modular nature and general applicability of this strategy for obtaining sequence-selective DNA binding peptides derived from transcription factors (Figure 13).75 The thermodynamic stabilization of FT fragments to obtain high-affinity DNA binders does not require the use of artificial DNA binding agents such as distamycin or bisbenzamidines as described before, but can also be achieved with fully peptidic anchors. The AT-Hook is a short cationic peptide (RKPRGRPKK) found in eukaryotic HMG-I(Y) nuclear proteins.76 Although AT-Hooks bind to their target DNA sites with poor affinity (in the millimolar range),77 HMG-I(Y) proteins exploit the cooperative effect of three AT-Hook repeats to achieve high DNA binding affinity.78 NMR and crystallography studies have provided a detailed structural picture of the interaction of the ATHook with the DNA, and have shown that its central RGR core is deeply inserted into the minor groove in an extended conformation, while the various lysines in the sequence introduce additional electrostatic contacts with the 72 O. Vázquez, M. E. Vázquez, J. B. Blanco, L. Castedo, J. L. Mascareñas, Angew. Chem. Int. Ed Engl. 2007, 46, 6886–6890. 73 J. Mosquera, J. Rodríguez, M. E. Vázquez, J. L. Mascareñas, ChemBioChem 2014, 15, 1092–1095. 74 a) J. B. Chaires, J. Ren, D. Hamelberg, A. Kumar, V. Pandya, D. W. Boykin, W. D. Wilson, J. Med. Chem. 2004, 47, 5729–5742; b) M. Munde, M. A. Ismail, R. Arafa, P. Peixoto, C. J. Collar, Y. Liu, L. Hu, M.-H. David-Cordonnier, A. Lansiaux, C. Bailly, et al., J. Am. Chem. Soc. 2007, 129, 13732–13743; c) O. Vázquez, M. I. Sánchez, J. MartínezCostas, M. E. Vázquez, J. L. Mascareñas, Org. Lett. 2010, 12, 216–219. 75 a) J. B. Blanco, M. E. Vázquez, J. Martinez-Costas, L. Castedo, J. L. Mascareñas, Chem. Biol. 2003, 10, 713–722; b) J. B. Blanco, O. Vázquez, J. Martínez-Costas, L. Castedo, J. L. Mascareñas, Chem. Eur. J. 2005, 11, 4171–4178. 76 a) C. Crane-Robinson, A. I. Dragan, P. L. Privalov, Trends Biochem. Sci. 2006, 31, 547– 552; b) L. Aravind, D. Landsman, Nucleic Acids Res. 1998, 26, 4413–4421; c) R. Reeves, M. S. Nissen, J. Biol. Chem. 1990, 265, 8573–8582; d) T. Lund, K. H. Dahl, E. Mørk, J. Holtlund, S. G. Laland, Biochem. Biophys. Res. Commun. 1987, 146, 725–730. 77 C.-C. Cheng, Y.-H. Jian, C.-J. Lo, J.-W. Cheng, J. Chinese Chemical Soc 1998, 45, 619– 624. 78 B. H. Geierstanger, B. F. Volkman, W. Kremer, D. E. Wemmer, Biochemistry 1994, 33, 5347–5355. GHOFRANE BARKA 38 phosphates of the DNA backbone.79 We recently reported the use of an ATHook as minor groove binding element in combination with the GCN4 br. The peptidic nature of the AT-Hook allows the straightforward synthesis of the GCN4 br/AT-Hook chimera following exclusively solid-phase peptide synthesis methods, and the lower DNA binding affinity of the AT-Hook compared to bisbenzamidines or distamycin results in better selectivity profile.80 Figure 13. Examples of minimized TF stabilized by small minor groove binding agents. a) Original GCN4 basic region conjugate with distamycin,73 b) GAGA/distamycin conjugate,76 c) GCN4/bisbenzamidine hybrid, d) GAGA/bisbenzamidine hybrid; e) GCN4/AT-Hook chimera,84 f) engrailed homeodomain/bisbenzamidine conjugate. 79 a) J. R. Huth, C. A. Bewley, M. S. Nissen, J. N. Evans, R. Reeves, A. M. Gronenborn, G. M. Clore, Nat. Struct. Biol. 1997, 4, 657–665; b) E. Fonfría-Subirós, F. Acosta-Reyes, N. Saperas, J. Pous, J. A. Subirana, J. L. Campos, PLoS One 2012, 7, e37120. 80 J. Rodríguez, J. Mosquera, J. R. Couceiro, M. E. Vázquez, J. L. Mascareñas, Chem. Sci. 2015, 6, 4767–4771. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 39 A related example, recently published by the group of S. Roy in 2017, further demonstrates that the combination of short minor groove anchors and TF fragments is a robust approach to developing sequence-specific DNA binding w ’ -binding tail from the Serum Response Factor (SRF) in combination with the DNA-binding helix of Elk1 to yield a dominant negative of the two transcription factors, which upon DNA binding to the DNA upregulate the oncogene cFos.81 The authors explored different linkers, such as GlyProGlyProGly, Ahx3 (Ahx = aminohexanoic acid), Gly-Ahx3, Gly2-Ahx3, and Gly3-Ahx3, between both DNA binding units, and studied as well conformationally-stabilized versions of the DNA contacting helix containing Aib residues, which are known to stabilize the α-helical conformation by restricting the available backbone conformations.82 Importantly, in contrast with other conjugates that appear to get stuck inside endosomes upon internalization, the Gly2-Ahx3 chimera was readily internalized into the nuclei of A549 lung adenocarcinoma cells, and induced a specific response, down-regulating its targeted gene. In an alternative approach to dominant negative DNA-binding peptides we described the synthesis of a dominant negative of the oncogenic c-Jun protein as an inactive complex by forcing its heterodimerization with a c-Fos-DNA conjugate.83 This oligonucleotide-peptide receptor was based on the X-ray structure of the the c-Fos/c-Jun heterodimer bound to the AP1 site,84 and included a fragment of the c-Fos leucine zipper (Arg158 to Lys192) to induce cJ 5′ (5′- TGACTCATCCATTGCGCG-3′) 1 -site (in bold) for cJun binding. The oligonucleotide provides increased affinity and selectivity for c-Jun, as demonstrated by its tight binding constant (KD ≈ 200 ) w it to efficiently compete with the natural c-Fos and disassemble the natural cFos/c-Jun heterodimer bound to the DNA. 81 M. Chakraborty, S. Roy, Chem. Commun. 2017, 53, 376–379. 82 De Filippis V., De Antoni F, M. Frigo, Polverino de Laureto P, A. Fontana, Biochemistry 1998, 37, 1686–1696. 83 E. Pazos, C. Portela, C. Penas, M. E. Vázquez, J. L. Mascareñas, Org. Biomol. Chem. 2015, 13, 5385–5390. 84 J. N. Glover, S. C. Harrison, Nature 1995, 373, 257–261. GHOFRANE BARKA 40 Interaction of Rh(II) and Ru(II) complexes with DNA De novo designed DNA-binding metallopeptides are relatively uncommon, and typically contain an octahedral metallo-intercalator complex that binds in the DNA major groove attached to a short peptide that modulates its DNA recognition or internalization properties. Prominent examples of this general design have been reported by J. Barton since the 90s, when they described conjugates between short 13-mer peptides and photoactive [Rh(phi)2( ′)]3+ (phi = 9,10- ; ′ = 5-(amidoglutaryl)-1,10phenanthroline) complexes. The coordination compounds intercalate through the DNA major groove with high affinity, and upon photoactivation, induce DNA strand scission. The DNA site specificity was dependent on the sequence of the appended peptide, and the authors found that a single glutamate at position was essential in directing DNA site5′- CCA-3′ 85 Later examples demonstrated the general applicability of this strategy by inserting different peptides with alternative sequence preferences, such as the DNA recognition helix of the phage 434 repressor (preferentially targeting the 5′-ACAA-3′) 86 Related designs featured α-helical metallopeptides with nuclease activity tethered to the [Rh(phi)2( ′)]3+ complex. Thus, the peptide sequence DPDELEHAAKHEAAAK, contains two His residues (in bold) in positions i, i+4 that create a zinc coordination site on one face of the αhelix. A number of residues were included in the sequence to increase the αhelical content, such as a C-terminal E-K salt bridge, and a number of Ala residues. The resulting metallointercalator-peptide nuclease chimera was found to convert a supercoiled pBR322 DNA plasmid to both the nicked and linear forms. Control experiments did not show any cleavage when the DNA was incubated with the unmodified rhodium intercalator, or the peptide nuclease without the intercalator, thus demonstrating the cooperative nature of the construct.87 Similar effects were observed with rhodium conjugates with metallopeptide hairpin nucleases.88 A relatively recent example of these metallointercalator-peptide hybrids explored the effect of the peptide sequence in the internalization and 85 a) N. Y. Sardesai, S. C. Lin, K. Zimmerman, J. K. Barton, Bioconjug. Chem. 1995, 6, 302– 312; b) C. A. Hastings, J. K. Barton, Biochemistry 1999, 38, 10042–10051. 86 N. Y. Sardesai, J. K. Barton, J. Biol. Inorg. Chem. 1997, 2, 762–771. 87 M. P. Fitzsimons, J. K. Barton, J. Am. Chem. Soc. 1997, 119, 3379–3380. 88 K. D. Copeland, M. P. Fitzsimons, R. P. Houser, J. K. Barton, Biochemistry 2002, 41, 343– 356. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 41 intracellular localization.89 [ ( )( ′)( )2]2+ complex ( ′ = 4-(3-carboxypropyl)-4′-methyl-2 2′-bipyridine) was attached to a wellknown (D)-octaarginine (arg8) peptide transporter, and to that same peptide modified with a fluorescein tag (arg8-Flu) (Figure 11). Interestingly, they found that while the simple Ru-[arg8] conjugate was internalized by endocytosis, and remained in the cytoplasm in a punctuate pattern, the Ru-[arg8]-Flu derivative was readily internalized into the cell nucleus under the same conditions for which the complex without fluorescein was excluded. This difference in intracellular localization is consistent with other studies that demonstrate the role of charge and hydrophobicity compensation in peptide transporters.90 This result demonstrates the potential role of the peptide appendages not only in the DNA recognition process, but also for the modification of other properties, such as cell transport. [R (ph n)(bpy′)(dppz)2]2+ arg8 conjugates display different transport properties depending on their N-terminal modification with the hydrophobic fluorescein fluorophore. [R (ph n)(bpy′)(dppz)2]2+-arg8 is internalized in endosomes, while the analog featuring an N-terminal fluorescein is efficiently transported into the cell nucleus. In addition to the modification of discrete metallointercalators, peptides have also been used as integral structural elements in de novo DNA binding metallopeptides without any resemblance to natural transcription factors. In this context, in 2014 we described the application of solid-phase peptide synthesis 89 C. A. Puckett, J. K. Barton, Bioorg. Med. Chem. 2010, 18, 3564–3569. 90 a) C. N. Carrigan, B. Imperiali, Anal. Biochem. 2005, 341, 290–298; b) W. B. Kauffman, T. Fuselier, J. He, W. C. Wimley, Trends Biochem. Sci. 2015, 40, 749–764. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 49 Introduction Over the past few decades there has been an increased interest in the application of transition metal complexes for the development of DNA-based probes, reactive agents, and drugs.93 However, severe side effects, such as nephrotoxicity, neurotoxicity, or drug-induced resistance, have hampered their clinical application, and fueled intense efforts to develop safer alternatives with reduced side effects and improve DNA binding.94 In this context, polypyridyl ruthenium(II) complexes have been exhaustively studied as metal-based DNA binders,95 These complexes usually contain one large heteroaromatic ligand that penetrates into the DNA through the major groove. Unfortunately, these metallointercalators suffer from the same lack of sequence specificity typical of organic intercalators.96 Despite this limitation, metal complexes offer extraordinary opportunities for the modulation of the DNA binding properties, by taking advantage of ancillary ligands and stereochemical effects, not accessible to organic DNA binders. While these effects have been extensively explored in other areas, such as catalyst optimization,97 their study in the context of DNA recognition by metal complexes must still be explored in depth. However, the functionalization of the intercalating/ancillary ligands in polypyridyl Ru(II) DNA binders implies the use demanding synthetic procedures, that complicate the access to multiple structural variants required for SAR studies, as well as the access to single systems endowed with specific biological properties. In this context, a promising and alternative approach to this end is to decorate this class of complexes with peptide sequences. Peptides are easy to synthesize98 and they are highly versatile systems from a biological perspective, being also capable of endowing with versatile biological capabilities (i.e. transmembrane/cellular organelle delivery, protein/nucleic acid 93 a) C. Moucheron, New. J. Chem. 2009, 33, 235; b) K. L. Haas K. J. Franz, Chem. Rev. 2009, 109, 4921. 94 a) L. Kelland, Nat. Rev. Cancer. 2007, 7, 573; b) A. Cisnetti, A. Gautier, Angew. Chem. Int. Ed. 2013, 52, 11976; c) S. Komeda, A. Casini, Curr. Top. Med. Chem. 2012, 12, 219. 95 M. R. Gill, J. Garcia-Lara, S. J. Foster, C. Smythe, G. Battaglia, J. A. Thomas, Nat. Chem. 2009, 1, 662. 96 H.-K. Liu, P.J. Sadler, Acc. Chem. Res. 2011, 44, 349. 97 D. J. Gorin, B.D. Sherry, F. D. Toste, Chem. Rev. 2008, 108, 3351. 98 a) K. Heinze, K. Hempel, Chem. Eur. J. 2009, 15, 1346; b) B. Jung, A. G. Beck-Sickinger, Angew. Chem. Int. Ed. 1992, 31, 367; c) M. Nitz, K. J. Franz, R. L. Maglathlin, B. Imperiali, ChemBioChem 2003, 4, 272; d) G. Dirscherl, B. Koenig, Eur. J. Org. Chem. 2008, 597. GHOFRANE BARKA 50 …) 99 including Ru(II), Rh(III) or Ir(III) polypyridyl derivatives.100,101 On the other hand, a DNA mismatch is a defect that takes place when two noncomplementary bases are faced one to each other in a duplex DNA.102 Mismatches appear in the genome as a consequence of errors during DNA replication,103 or due to DNA damage caused by ionizing radiation or genotoxic chemicals.104 These mistakes are almost always corrected by the enzymatic mismatch repair machinery of the cell,105 but if they are not fixed, they accumulate in the genome, often driving to genetic mutations which can lead to serious consequences, including cancer.106 The selective recognition of single mismatches in duplex DNA by small molecules, and particularly if these systems are capable of emitting light as a result of such recognition, is a powerful strategy for the development of new theranostic drugs in cancer research. Although there are some examples in bibliography about the use of organic molecules for spectrofluorimetric mismatch detection,107 the 99 E. Pazos, J. Mosquera, M. E. Vázquez, J. L. Mascareñas, ChemBioChem 2011, 12, 1958. 100 a) I. Gamba, I. Salvadó, R. F. Brissos, P. Gamez, J. Brea, M. I. Loza, M. E. Vázquez, M. Vázquez López, Chem. Commun., 2016, 52, 1234; b) I. Salvadó, I. Gamba, J. Montenegro, J. Martínez-Costas, J. M. Brea, M. I. Loza, M. Vázquez López, M. E. Vázquez, Chem. Commun., 2016, 52 11008; ) - J - J , M. E. Vázquez, Chem. Eur. J. 2013, 19, 13369. 101 a) U. Neugebauer, Y. Pellegrin, M. Devocelle, R. J. Forster,W: Signac, N. Moran, T. E. Keyes, Chem. Commun., 2008, 5307; b) J. Kuil, P. Steunenberg, P. T. K. Chin, J. Oldenburg, K. Jalink, A. H. Velders,F. W. B. van Leeuwen, ChemBioChem, 2011, 12, 1897; c) N. Y. Sardesai, K. Zimmermann, J. K. Barton, J. Am. Chem. Soc., 1994, 116, 7502; d) M. P. Fitzsimons, J. K. Barton, J. Am. Chem. Soc., 1997, 119, 3379; e) K. D. Copeland, M. P. Fitzsimons, R. P. Houser, J. K. Barton, Biochemistry, 2002, 41, 343; f) C. Sissi, P. Rossi, F. Felluga, F. Formaggio, M. Palumbo, P. Tecilla, C. Toniolo, P. Scrimin, J. Am. Chem. Soc., 2001, 123, 3169-3170; g) A. B. Tabor, Tetrahedron, 1996, 52, 2229. 102 P. Modrich, Annu. Rev. Biochem., 1987, 56, 435. 103 M. F. Goodman, S. Creighton, L. B. Bloom, J. Petruska, J. Crit. Rev. Biochem. Mol. Biol. 1993, 28, 83-126. 104 C. Kunz, Y. Saito, P. Schär. Cell. Mol. Life Sci., 2009, 66, 1021. 105 P. Modrich, J. Biol. Chem. 2006, 281, 30305. 106 a) I. I. Arizamanoglou, F. Gilbert, H. R. Barber, Cancer 1998, 82, 1808; b) L. A. Loeb, K. R. Loeb, J. P. Anderson, Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 776. 107 a) A. Granzhan, M.-P. Teulade-Fichou, Chem. Eur. J. 2009, 15, 1314; b) Y. Sato, A. Honjo, D. Ishikawa, S. Nishizawa, N. Teramae, Chem. Commun. 2011, 47, 5885. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 51 development of DNA-mismatch fluorescent probes based on metal complexes is still poorly developed.108,109 In 2009, Barton et al. reported the mismatch binding properties of the classic metallointercalator [Ru(bpy)2dppz)]2+,110 but this complex binds with almost the same strength both well-matched and mismatched DNA sites, even if they are in the same oligonucleotide sequence.111,112 Later, the same research group showed that through structural modification of the inserting dppz ligand it is not possible to increase the specificity towards mismatched sequences,109 but with the incorporation of methyl groups into the ancillary ligands of [Ru(bpy)2dppz)]2+ it is possible to get a 26-fold increase in the binding affinity for the mismatch compared to the well-matched duplex DNA, opening a window of opportunity to design improved mismatch-specific luminescent binding agents.113 In this context, we are currently embarking into a research program which aims to study the DNA-binding, cell-internalization properties and biological activity of artificial metallopeptides synthesized by SPPS methods and derived from the metal2 2’-bipyridine.100,114 In particular, we are especially interested in studying in which way the presence of an oligoarginine sequence 108 Barton et al. developed a large family of octahedral Rh(III) polypyridyl complexes equipped with sterically expansive planar ligands which selectively recognise DNA mismatches and, upon photoactivation, cause the cleavage of the DNA skeleton in a highly efficient way, but this class of complexes are not emissive. a) K. M. Boyle, J. K. Barton, Inorg. Chim. Acta, 2016, 452, 3; b) A. C. Komor, J. K. Barton, Chem. Commun., 2013, 49, 3617. 109 The Ru( ) ’ ( ) 108 are not emissive at ambient temperature, despite being highly selective for DNA mismatches. A. J. McConnell, M. H. Lim, E. D. Olmon, H. Song, E. E. Dervan, J. K. Barton, Inorg. Chem. 2012, 51, 12511. 110 Polypyridyl ruthenium(II) complexes have been deeply studied since more than 30 years ago due to their ability to bind the DNA in conjunction with their kinetic stability and convenient optical properties. From these, dipyrido[3,2-a:2',3'-c]phenazine (dppz) Ru(II) x “ - w ” -binding, as they are practically non-emissive in water media but show a significant enhance in the emission when interacting with DNA: a) A. E. Friedman, J. C. Chambron, J. P. Sauvage, N. J. Turro, J. K.Barton, J. Am. Chem. Soc. 1990, 112, 4960; b) E. Amouyal, A. Homsi, J.-C. Chambron, J.-P. Sauvage, J. Chem. Soc., Dalton Trans., 1990 184; ) H , J. Am. Chem. Soc., 1993, 115, 3448. 111 M. H. Lim, H. Song, E. D. Olmon, E. E. Dervan, J. K. Barton, Inorg. Chem. 2009, 48, 5392. 112 H. Song, J. T. Kaiser, J. K. Barton, Nat. Chem. 2012, 4, 615-620. 113 A. N. Boynton, L. Marcélis, J. K. Barton, J. Am. Chem. Soc. 2016, 138, 5020. 114 I. Gamba, G. Rama, E. Ortega-Carrasco, J.-D. Maréchal, J. Martínez-Costas, M. E. Vázquez, M. Vázquez López, Chem. Commun., 2014, 50, 11097. GHOFRANE BARKA 52 in the structure of the complexes modulate their DNA-binding and biological properties. We have yet reported the influence of this functionalization in the binding properties of B-DNA groove binders, but the same has never been studied for B-DNA metallointercalators115 as, for example, dppz-derived Ru(II) complexes.116,117 Results and discussion Metallopeptide synthesis We first designed two peptidic ligands equipped with a single βAla-bpy coordinating unit (1,100 scheme 1): 2-R8 (scheme P1) is equipped with a Cterminal octarginine (R8) sequence, and ligand 2, which lacks the octargine tail. The ligands were synthesized following standard Fmoc/tBu solid-phase protocols.118 Once completed the ligand sequence, the peptides still attached to the solid support were reacted with the Ru(II) source ([Ru(DMSO)4Cl2]),119 following by the coordination of the intercalating dppz moiety and the necessary second ancillary ligand.120 To this end, the resins were reacted sequentially with 1,10-phenanthroline (phen) and later with dppz to achieve the desired Ru-dppz “ - w ” [ (2)(phen)(dppz)]2+ (3, scheme P1) and [Ru(2R8)(phen)(dppz)]2+ (4, scheme P1). Acidic cleavage from the support, followed 115 Barton et al. suggested in a very preliminary way that an oligoarginine functionalization may increase the nonspecific DNA binding affinity in chrysy-derived Rh(III) intercalators. However, this issue has never been studied in detail: J. Brunner, J. K. Barton, Biochemistry 2006, 45, 12295. 116 Barton et. al. reported studies on the influence of an oligoarginine functionalization in the cell-internalization properties of a classic Ru(II)-dppz intercalator, but the DNA-binding properties of such metallopeptide conjugate have never been studied so far: a) C. A. Puckett, J. K. Barton, J. Am. Chem. Soc., 2009, 131, 8738; b) C. A. Puckett, J. K. Barton, Bioorg. Med. Chem., 2010, 18, 3564. 117 Barton et al. “ - w ” ( )-dppz peptide conjugates. In this study they suggested that the peptide functionalization slightly modulate the DNA-binding affinity of the Ru(II)-dppz intercalators and that the presence of positive charged residues in the peptide sequence has a quite discreet but positive effect on such affinity due the establishment of electrostatic interactions with the DNA polyphosphate backbone. However, the studies were made with call-thymus DNA and the peptide functionalizations studied were sequences of 5/6 residues (the maximum number of positively charged residues in the sequences is 3 and none of which was an octoarginine sequence: K. D. Copeland, A. M. K. Lueras, E. D. A. Stemp, J. K. Barton, Biochemistry, 2002, 41, 12785. 118 I. Coin, M. Beyermann, M. Bienert, Nat. Protoc., 2007, 2, 3247. 119 P. Evans, A. Spencer, G. Wilkinson, J. C. S. Dalton, 1973, 204. 120 J. E. Dickenson, L. A. Summers, Aust. J. Chem., 1970, 23, 1023. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 53 by reverse-phase HPLC purification, afforded the Ru(II) metallopeptides as diasteromeric mixtures. It has to be noted that these class of metallopeptides have four different isomers (scheme S1): the arrangement of ligands around the metal center can be ∆ or Λ “ x ” “ ” .117 Scheme P1. Solid-phase synthesis of the peptidic ligand 2 and its derived Ru(II) metallopeptides 3 and 5 as well as their oligoarginine derivatives 2-R8, 4 and 6. The first step of the route (in blue) takes place only for the synthesis of the oligoarginine derivatives. GHOFRANE BARKA 54 DNA binding studies with synthetic oligonucleotides Recognition of canonical dsDNA Once we had the target Ru(II)-dppz derivatives at hand, the second step was to exploit their solvatochromic properties for studying their interaction with synthetic oligonucleotides, which would allow us to obtain quantitative information about their binding affinity towards particular well-matched dsDNA sequences: AAAATTT, GAATTC and GGCCC (see table S1 for the entire sequences). Thus, the addition of successive aliquots of a stock solution of the B-DNA hairpin oligonucleotides to 2 M solutions of 3 in Tris-HCl buffer at pH 7.5, resulted in a progressive increase of the intensity of emission band of the Ru(II) complex, which could be fitted to a 1:1 binding model corresponding to the formation of specific 3/B-DNA adducts (figure P1 and figures S1-S4). The resulting apparent dissociation constants obtained suggest that 3 interacts indistinctly and with almost the same strength with the three oligonucleotides. The DNA affinity constants calculated for 3 are in line with those previously reported for related Ru(II)-dppz complexes121 and peptide conjugates (table S2).117 After that, we repeated these studies with the oligoarginine derivative 4. Interestingly, the titrations studies carried out indicated that this metallopeptide shows a more complex and interesting behavior than 3. In particular, the fluorescence titration profiles at λem= 630 nm clearly suggest the existence of a two-step DNA-binding process. During the first additions of the DNA stock solution, the intensity of the emission band increases rapidly (and at a much higher rate than in the case of 3). In fact, the emission intensity reaches its maximum when the [Ru]/[DNA] ratio is c.a. 8.0. After that, the emission intensity experiences first a slight decrease and then a very little pronounced but steady rise up until the end of the titration experiment (Figure P1 and Figures S5-S7). This multiphasic profile has been previously observed in reverse titrations of cationic proteins with DNA,122 and also in titrations of bisbenzamidine-peptide hybrids with dsDNA,123 and in all these cases it has been explained in terms of the competitive formation of multiple 121 a) I. Haq, P. Lincoln, D. Suh, B. Nordén, B. Z. Chowdry, J. B. Chaires, J. B., J. Am. Chem. Soc., 1995, 117, 4788; b) P. Lincoln, A. Broo, B. Nordén, J. Am. Chem. Soc., 1996, 118, 2644. 122 a) J. J. Hollenbeck, M. G. Oakley, Biochemistry 2000, 39, 6380; b) C. Portela, F. Albericio, R. Eritja, L. Castedo, J. L. Mascareñas, ChemBioChem, 2007, 8, 1110; c) A. V. Fedorova, I.-S. Chan, J. A. Shin, Biochim. Biophys. Acta 2006, 1764, 1252. 123 M I. Sánchez, O. Vázquez, M. E. Vázquez, J. L. Mascareñas, Chem. Eur. J., 2013, 19, 9923. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 55 nonspecific complexes with the DNA oligomers at high ligand/DNA ratios and the subsequent formation of specific 1:1 complexes at low ligand/DNA ratios. However, in the particular case of the metallopeptide 4 it should be noted that the increase in the emission intensity must be ascribed solely to the intercalation of the dppz moiety into the DNA double helix and that this intercalative binding mode cannot be considered as a nonspecific interaction. Thus, this unusual profile could be interpreted as the result of the existence of two different intercalative binding modes at different [Ru]/[DNA] ratios. In particular, we suggest the occurrence of an intercalative process of multiple metallopeptide units within a single DNA molecule in the first steps of the titration experiment, when the ratio of the DNA vs complex concentration in the media is very low. Later, in a second stage, when the concentration of DNA rises up, the metallopeptide units rearrange, and intercalate in other DNA molecules. Moreover, we were able to calculated a Kd of 1.09(0.26) μM for the second binding process of 4 with the GAATTC oligonucleotide (the fit was made only for the points represented by white circles). In the other two cases (AAATTT and GGCCC) the second part of the curve is too shallow to make the fit (Figure P1). This interpretation implies the existence of a greater affinity of 4 for the BDNA with respect to that of 3 although the affinities of both metallopeptides cannot be quantitatively compared. The B-DNA binding behaviour of 4 is also quite different to those shown by related Ru-dppz peptide conjugates:117 everything seems to indicate that the presence of such a high number of positive charged residues in the peptide tail of the Ru(II)-dppz complex is responsible for this unusual behavior. GHOFRANE BARKA 56 Figure P1. Up, luminescence spectra of a 2.0 μM solution of 3 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of AAAATTT oligonucleotide solution until saturation. Back, profile of the titration experiments of 4 with AAAATTT (left), GAATTC (middle) and GGCCC (right) oligonucleotides at λem = 630 nm (emission intensity vs. concentration of DNA in the media). We were able to make the fit for the second part of the titration experiment only for the case of the GAATTC oligonucleotide [Kd= 1.09(0.26) μM]. Absorption spectroscopy is a very useful technique to study the interaction of small molecules with the DNA.124 In the particular case of polypyridyl DNA metallointercalators, the binding process usually involves both bathocromic and hypsochromic phenomenas of the MLCT band of the complex.125 The 124 J.-G. Liu, Q.-L. Zhang, X.-F. Shi, L.-N. Ji, Inorg. Chem., 2001, 40, 5045. 125 - , S. Blasco, S. A. Bright, J. M. Kelly, D. Clive Williams, T. Gunnlaugsson, Dalton. Trans., 2016, 45, 18208. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 57 hypochromicity may be attributed to the interaction between the electronic states of the complex and those of the DNA bases,126 while the bathochromic shift is related with the decrease in the energy gap between the HOMO/LUMO molecular orbitals after the binding process.127 It has been suggested that the extent of hypochromism in the MLCT band of the complexes is related with the intercalative binding strength of the binder128 and/or with different levels of penetration of the dppz ligand into the B-DNA base stack.124 In this context, we studied the interaction of the metallopeptides 3 and 4 with the AAAATTT hairpin oligonucleotide by UV-vis spectroscopy. The intercalative binding mode of these metallopeptides proposed on the basis of the fluorescence studies are also obvious from the UV-data. In particular, the absorption spectra of these complexes are characterized by a MLCT band centered at 443 nm that suffers of both a bathochromic and a hypochromic phenomenon after the addition of the AT-rich B-DNA oligonucleotide (figures S51 and S52; table S4), indicating that 4 binds to B-DNA in an intercalative fashion.124,129 Interestingly, the decrease in the absorbance at 443 nm is more pronounced in 3 (37.5%) than in 4 (19.0%), which in this case could reflect different levels of penetration of the dppz ligand into the B-DNA base stack, probably due to the steric hindrance caused by the oligoarginine tail of 4.124 Recognition of mismatched DNAs The next step in our research program was to investigate the affinity of the Ru(II)-dppz metallopeptides 3 and 4 for a set of B-DNA oligonucleotides with different number of mismatches in their sequences (A: 1’: ; : 2’: w / ; : 3’: / / ; table S1 for the entire sequences). For comparison, parent well-matched (WM) Bw ( : ’) successive aliquots of a stock solution of these mismatched (MM) B-DNA oligonucleotides to 2 µM solutions of 3 or 4 in Tris-HCl buffer at pH 7.5, resulted in a progressive increase of the intensity of emission band of the Ru(II) complex, which could be fitted to a 1:1 binding model corresponding to the formation of specific metallopeptide/MM B-DNA adducts (figures S15-S19 and 126 B. D. Wang, Z. Y. Yang, P. Crewdson, D. Q. Wang, J. Inorg. Biochem., 2007, 101, 1492. 127 F. Arjmand, M. Aziz, Eur. J. Med. Chem., 2009, 44, 834. 128 J. K. Barton, J. J. Dennenberg, J. B. Chaires, Biochemistry, 1993, 32, 2573. 129 a) A. K. F. Martensson, P. Lincoln, Dalton. Trans., 2015, 44, 3604; b) T. Very, S. Despax, P. Hébraud, A. Monari, X, Assfeld, Phys. Chem. Chem. Phys., 2012, 14, 12496. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 65 Conclusions We report herein a systematic study about the effect of the presence of an oligoarginine functionalization in the B-DNA and mismatched B-DNA binding properties of a Ru(II)-dppz metallointercalator. In particular, the fluorescence and UV-vis studies carried out suggested that the functionalization of a Ru(II)- dppz complex with a octaarginine tail causes an increase of its affinity for wellmatched B-DNA. However, this peptide tail does not seem to have any influence on their binding properties for mismatched B-DNA. In the same way, the role of the non-peptidic ancillary ligand of these complexes over the B-DNA binding properties of these complexes to the entire set of studied oligonucleotides seems to be also negligible. Moreover, CD studies suggest that there is no preferential binding of any of the possible 4 isomers of this class of metallopeptides to the well-matched and mismatched B-DNA and also that their intercalation to the double helix cause a slight destabilization of the same. Finally, the R8 functionalization endowed these Ru(II) metallopeptides with cell-internalization properties and, moreover, with appreciable cytotoxic capabilities. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 67 Experimental Section General All reagents were acquired from commercial sources: Dimethyl sulfoxide (DMSO), Dimethylformamide (DMF) and Trifluoroacetic acid (TFA) were purchased from Scharlau, EtOH and CH2Cl2 from Panreac and CH3CN from Merck. All peptide synthesis reagents, namely the coupling agents HBTU (OBenzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate) and HATU (2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium), and Fmoc amino acid derivatives were purchased from GL Biochem (Shanghai) Ltd. Fmoc-Rink amide AM resin was purchased from Iris Biotech. The oligonucleotides were purchased from Thermo Fisher Scientific GmbH. All other chemicals were purchased from Alfa-Aesar, Sigma-Aldrich or Fluka. All solvents were dry and of synthesis grade, unless specifically noted. RuCl3.3H2O was purchased from Matthews Chemicals. Reactions were followed by analytical RP-HPLC with an Agilent 1100 series LC/MS using a Luna C18 (250 x 4.60 mm) analytical column from Phenomenex. Standard conditions for analytical RP-HPLC consisted on a linear gradient from 30% to 95% of solvent B for 30 min at a flow rate of 1 mL/min (A: water with 0.1% TFA, B: acetonitrile with 0.1% TFA). Compounds were detected by UV absorption at 222, 254 and 310 nm. High-Performance Liquid Chromatography (HPLC) was performed using an Agilent 1100 series Liquid Chromatograph Mass Spectrometer system. Analytical HPLC was run using a Luna C18 (250 x 4.60 mm) reverse phase analytical column; compounds were detected by UV absorption at 222, 254 and 310 nm. The purification of the peptides was performed on a Luna C18 (250 x 10 mm) semi-preparative reverse phase column from Phenomenex. The standard gradient used for analytical and semi-preparative HPLC was 70:30 to 5:95 over 30 min (water/acetonitrile, 0.1% TFA). Compounds were detected by UV absorption (222 nm) and by ESI+−MS. The fractions containing the products were freeze-dried, and their identity was confirmed by ESI+−MS and MALDI-TOF. Electrospray Ionization Mass Spectrometry (ESI/MS) was performed with an Agilent 1100 Series LC/MS model in positive scan mode using direct injection of the purified peptide solution into the MS. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) was performed with a Bruker Autoflex GHOFRANE BARKA 68 MALDI/TOF model in positive scan mode by direct irradiation of the matrixabsorbed peptide. Luminescence experiments were made with a Jobin-Yvon Fluoromax-3 fluorescence spectrometers (DataMax 2.20), coupled to a −3751 were made with a Hellma semi-micro cuvette (114F-QS) at 20 ºC. CD experiments were made with a Jasco J-715 coupled to a Neslab RTE-111 thermostarted water bath at 20ºC. UV-vis absorption experiments were performed in a Jasco V-630 spectrophotometer coupled to a Jasco ETC-717 temperature controller at 20ºC. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 69 Synthetic procedures Synthesis of the unnatural coordinating residue Fmoc-βAla-bpyOH (1) The coordinating residue Fmoc-βAla-bpy-OH (1) was synthesized following a procedure previously reported by our research group.100 Synthesis of peptide ligands and metallopeptides Synthesis of the peptide ligands 2 and 2-R8 C-terminal amide peptides were synthesized by following standard SPPS protocols on a 0.1 mmol scale using a 0.45 mmol/g Fmoc-Rink-amide resin. Arginines were coupled, in 10-fold excess (vs. mmol of resin load), by using O- (benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) as an activating agent. Fmoc-βAlaBpy-OH (1) was coupled in 5-fold excess using O-(7Azabenzotriazol-1-yl)-N,N,N’,N’- tetramethyluronium hexafluorophosphate (HATU) as activating agent. Couplings were conducted for 1 h. Deprotection of the temporal Fmoc protecting-group was performed with 20% piperidine in DMF for 15 min. Test cleavages were performed at a 1 mg scale for 2 h with CH2Cl2 (50 µL), H2O (25 µL), TIS (triisopropylsilane, 25 µL), and TFA (900 µL) (~1 mL of cocktail for 20 mg of resin). Synthesis of the Ru(II) metallopeptides 3-6 Common step Once the peptide ligands 2 and 2-R8 were synthesized, 222 mg of the resin with the corresponding peptide anchored was suspended in 3 mL of EtOH:DMF (1:1) in the dark and the resulting mixture were purged with Ar for 15 min. 54.6 mg (1.3 eq) of [Ru(DMSO)4Cl2]102 was added and the resulting mixture was stirred under argon for 24 hours at 80 ºC. Then, the resin was washed with DMF (5 x 10 mL, 10 min) and dried under vacuum. Phen-dppz Ru(II) metallopeptides (3 and 4) The resin was then suspended in 3 mL of EtOH:DMF (1:1) in the dark and the x w w 15 18 0 (1 0 ) 1 10’- phenantroline was added and the resulting mixture was stirred under argon for GHOFRANE BARKA 70 24 hours at 80 ºC. Then, the resin was washed with DMF (5 x 10 mL, 10 min) and dried under vacuum. Finally, the resin was suspended in 3 mL of EtOH:DMF (1:1) in the dark and the resulting mixture were purged with Ar for 15 min. 28.2 mg (1.0 eq) of dipyrido[3,2-a:2',3'-c]phenazine (dppz)17 was added and the resulting mixture was stirred under argon for 24 hours at 80 ºC. Then, the resin was washed with DMF (5 x 10 mL, 10 min) and dried under vacuum. Double-dppz Ru(II) metallopeptides (5 and 6) The resin was then suspended in 3 mL of EtOH:DMF (1:1) in the dark and the resulting mixture were purged with Ar for 15 min. 56.5 mg (2.0 eq) of dipyrido[3,2-a:2',3'-c]phenazine (dppz)103 was added and the resulting mixture was stirred under argon for 24 hours at 80 ºC. Then, the resin was washed with DMF (5 x 10 mL, 10 min) and dried under vacuum. General procedure for peptide cleavage-deprotection The resin was filtered, washed with DMF and CH2Cl2 and dried. The metallopeptide was cleaved with 5 mL of the standard TFA cocktail (TIS, H2O, CH2Cl2 and TFA) over 2.5 hours. After that, the resin was filtered and washed with TFA (1 x 2 mL) and the filtrate was concentrated until 1 mL of volume with a N2 stream. Then, 4 mL of H2O and 5 mg of NH4PF6 were added to this solution. The red orange solid precipitated was separated by centrifugation, washed with H2O (1 x 4 mL) and purified by semi-preparative HPLC to give the desired product. [Ru(DMSO)Cl2] and dipyrido[3,2-a:2',3'-c]phenazine (dppz) were synthesized following reported procedures.102,103 DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 71 Mass spectra and HPLC chromatograms of the Ru(II) metallopeptides (3 to 6) a) [Ru(2)(phen)(dppz)]2+ (3) MALDI-TOF: m/z calculated for C43H35N11O3Ru: 891.2, found: 890.2 HPLC chromatogram: 1-75 %B, tR=24.3´ GHOFRANE BARKA 72 b) [Ru(2-R8)(phen)(dppz)]2+ (4) MALDI-TOF: m/z calculated for C94H131N43O11Ru: 2140.0, found: 2141.1 HPLC chromatogram: 1-75 %B, tR=19 3’ DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 73 c) [Ru(2)(dppz)2]2+ (5) MALDI-TOF: m/z calculated for C52H37N13O3Ru: 993.2, found: 993.2 HPLC chromatogram: 1-75 %B, tR=28.3´ GHOFRANE BARKA 80 Figure S4. Profile of the titration experiments of 3 with AAAATTT (black diamonds), GAATTC (green triangles) and GGCCC (red circles) oligonucleotides at λem = 630 nm (emission intensity vs. concentration of DNA in the media) with the corresponding best fits (black, green and red lines, respectively). DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 81 Figure S5. Top, luminescence spectra of a 2.0 μM solution of 4 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of AAAATTT oligonucleotide solution until saturation; bottom, profile (black triangles) of the fluorescence titration experiment of 4 with AAAATTT oligonucleotide at λem = 630 nm (emission intensity vs. concentration of DNA in the media). GHOFRANE BARKA 82 Figure S6. Top, luminescence spectra of a 2.0 μM solution of 4 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of GAATTC oligonucleotide solution until saturation; bottom, profile (black triangles) of the fluorescence titration experiment of 4 with GAATTC oligonucleotide at λem = 630 nm (emission intensity vs. concentration of DNA in the media). DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 83 Figure S7. Top, luminescence spectra of a 2.0 μM solution of 4 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of GGCCC oligonucleotide solution until saturation; bottom, profile (black triangles) of the fluorescence titration experiment of 4 with GGCCC oligonucleotide at λem = 630 nm (emission intensity vs. concentration of DNA in the media). GHOFRANE BARKA 84 Figure S8. Luminescence spectra of a 2.0 μM solution of 5 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of AAAATTT oligonucleotide solution until saturation. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 85 Figure S9. Luminescence spectra of a 2.0 μM solution of 5 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of GAATTC oligonucleotide solution until saturation. GHOFRANE BARKA 86 Figure S10. Luminescence spectra of a 2.0 μM solution of 5 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of GGCCC oligonucleotide solution until saturation. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 87 Figure S11. Profile of the titration experiments of 5 with AAAATTT (blue circles), GAATTC (red squares) and GGCCC (black triangles) oligonucleotides at λem = 630 nm (emission intensity vs. concentration of DNA in the media) with the corresponding best fits (blue, red and black lines, respectively). GHOFRANE BARKA 88 Figure S12. Top, luminescence spectra of a 2.0 μM solution of 6 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of AAAATTT oligonucleotide solution until saturation; bottom, profile (black triangles) of the fluorescence titration experiment of 6 with AAAATTT oligonucleotide at λem = 630 nm (emission intensity vs. concentration of DNA in the media). DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 89 Figure S13. Top, luminescence spectra of a 2.0 μM solution of 6 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of GAATTC oligonucleotide solution until saturation; bottom, profile (black triangles) of the fluorescence titration experiment of 6 with GAATTC oligonucleotide at λem = 630 nm (emission intensity vs. concentration of DNA in the media). GHOFRANE BARKA 96 Figure S20. Luminescence spectra of a 2.0 μM solution of 3 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of al q ots of AA m smatch d B:B ’ oligonucleotide solution until saturation. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 97 Figure S21. Luminescence spectra of a 2.0 μM solution of 3 in Tris-HCl buffer (20 mM), NaCl ( 00 mM), pH 7 5 and vol t on pon add t on of al q ots of CC m smatch d C:C ’ oligonucleotide solution until saturation. GHOFRANE BARKA 98 Figure S22. Luminescence spectra of a 2.0 μM solution of 3 in Tris-HCl buffer (20 mM), NaCl ( 00 mM), pH 7 5 and vol t on pon add t on of al q ots of TT m smatch d D:D ’ oligonucleotide solution until saturation. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 99 Figure S23. Profile of the titration experiments of 3 w th GG m smatch d (A A ’; black d amonds), AA m smatch d (B:B ’; d t an l s), CC m smatch d (C:C ’; bl c cl s) and TT m smatch d (D:D ’; n sq a s) ol on cl ot d s at λem = 630 nm (emission intensity vs. concentration of DNA in the media) with the corresponding best fits (black, red, blue and green lines, respectively). GHOFRANE BARKA 100 Figure S24. Top, luminescence spectra of a 2.0 μM solution of 4 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and vol t on pon add t on of al q ots of “w ll-match d” A:A’ oligonucleotide solution until saturation; bottom, profile (black triangles) of the fluorescence titration experiment of 4 w th “w ll-match d” A:A’ ol on cl ot d at λem = 630 nm (emission intensity vs. concentration of DNA in the media). DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 101 Figure S25. Luminescence spectra of a 2.0 μM solution of 4 in Tris-HCl buffer (20 mM), NaCl ( 00 mM), pH 7 5 and vol t on pon add t on of al q ots of GG m smatch d A:A ’ oligonucleotide solution until saturation. GHOFRANE BARKA 102 Figure S26. Luminescence spectra of a 2.0 μM solution of 4 in Tris-HCl buffer (20 mM), NaCl ( 00 mM), pH 7 5 and vol t on pon add t on of al q ots of GG/AA m smatch d A:A2’ oligonucleotide solution until saturation. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 103 Figure S27. Luminescence spectra of a 2.0 μM solution of 4 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of GG/GG/AA mismatched A:A3’ ol on cl ot d sol t on nt l sat at on GHOFRANE BARKA 104 Figure S28. Profile of the titration experiments of 4 with GG m smatch d (A A ’; n sq a s), GG/AA m smatch d (A:A2’; black c cl s) and GG/GG/AA m smatch d (A:A3’; d triangles) oligonucleotides at λem = 630 nm (emission intensity vs. concentration of DNA in the media) with the corresponding best fits (green, black and red lines, respectively). DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 105 Figure S29. Luminescence spectra of a 2.0 μM solution of 4 in Tris-HCl buffer (20 mM), NaCl ( 00 mM), pH 7 5 and vol t on pon add t on of al q ots of AA m smatch d B:B ’ oligonucleotide solution until saturation. GHOFRANE BARKA 112 Figure S36. Luminescence spectra of a 2.0 μM solution of 5 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of GG/GG/AA mismatched A:A3’ ol on cl ot d sol t on nt l sat at on DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 113 Figure S37. Profile of the titration experiments of 5 w th “w ll match d” (A:A’; n diamonds), GG m smatch d (A A ’; d t an l s), GG/AA m smatch d (A:A2’; black c cl s) and GG/GG/AA m smatch d (A:A3’; cyan sq a s) ol on cl ot d s at λem = 630 nm (emission intensity vs. concentration of DNA in the media) with the corresponding best fits (green, red, black and cyan lines, respectively). GHOFRANE BARKA 114 Figure S38. Luminescence spectra of a 2.0 μM solution of 5 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7 5 and vol t on pon add t on of al q ots of AA m smatch d B:B ’ oligonucleotide solution until saturation. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 115 Figure S39. Luminescence spectra of a 2.0 μM solution of 5 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of al q ots of CC m smatch d C:C ’ oligonucleotide solution until saturation. GHOFRANE BARKA 116 Figure S40. Luminescence spectra of a 2.0 μM solution of 5 in Tris-HCl buffer (20 mM), NaCl ( 00 mM), pH 7 5 and vol t on pon add t on of al q ots of TT m smatch d D:D ’ oligonucleotide solution until saturation. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 117 Figure S41. Profile of the titration experiments of 5 w th GG m smatch d (A A ’; bl c cl s), AA m smatch d (B:B ’; n d amonds), CC m smatch d (C:C ’; d sq a s) and TT m smatch d (D:D ’; black t an l s) ol gonucleotides at λem = 630 nm (emission intensity vs. concentration of DNA in the media) with the corresponding best fits (blue, green, red and black lines, respectively). GHOFRANE BARKA 118 Figure S42. Top, luminescence spectra of a 2.0 μM solution of 6 in Tris-HCl buffer (20 mM), NaCl ( 00 mM), pH 7 5 and vol t on pon add t on of al q ots of “w ll-match d” A:A’ oligonucleotide solution until saturation; bottom, profile (black triangles) of the fluorescence titration experiment of 6 w th “w ll-match d” A:A’ ol on cl ot d at λem = 630 nm (emission intensity vs. concentration of DNA in the media). DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 119 Figure S43. Luminescence spectra of a 2.0 μM solution of 6 in Tris-HCl buffer (20 mM), NaCl ( 00 mM), pH 7 5 and vol t on pon add t on of al q ots of GG m smatch d A:A ’ oligonucleotide solution until saturation. GHOFRANE BARKA 120 Figure S44. Luminescence spectra of a 2.0 μM solution of 6 in Tris-HCl buffer (20 mM), NaCl ( 00 mM), pH 7 5 and vol t on pon add t on of al q ots of GG/AA m smatch d A:A2’ oligonucleotide solution until saturation. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 121 Figure S45. Luminescence spectra of a 2.0 μM solution of 6 in Tris-HCl buffer (20 mM), NaCl (100 mM), pH 7.5 and evolution upon addition of aliquots of GG/GG/AA mismatched A:A3’ ol on cl ot d sol t on nt l sat at on GHOFRANE BARKA 128 Table S2. Dissociation constants of the Ru(II) metallopeptides (3-6) with selected oligonucleotides. NC = Not calculated. Complex B-DNA oligonucleotides AAAATTT GAATTC GGCCC 3 0.32 (0.01) 0.19 (0.01) 0.18 (0.02) 4 NC NC NC 5 0.07 (0.006) 0.17 (0.006) 0.11 (0.03) 6 NC NC NC DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 129 Table S3. Dissociation constants of the Ru(II) metallopeptides (3-6) with selected oligonucleotides. NC = Not calculated. GHOFRANE BARKA 130 UV-vis studies To a solution of the selected Ru(II) metallopeptide (3-6) in Tris-HCl buffer (20 mM), pH 7.5 and NaCl (100 mM), an aliquot of the selected B-DNA stock solution (in water) was added in order to reach a [Ru]/[DNA] ratio of 0.5. The absorption spectra of the metallopeptides were recorded before and after the addition of the corresponding oligonucleotide. The sequences of the oligonucleotides studied are listed in table S1. a) Hairpin “well-matched” B-DNA binding studies Figure S51. UV-vis absorption spectra of metallopeptide 3 (2.9 μM) before (red line) and after (black line) the addition of a solution of AAAATTT oligonucleotide. DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 131 Figure S52. UV-vis absorption spectra of metallopeptide 4 (2.7 μM) before (red line) and after (black line) the addition of a solution of AAAATTT oligonucleotide. GHOFRANE BARKA 132 b) Double-stranded “well-matched” and “mismatched” B-DNA binding studies Figure S53. UV-vis absorption spectra of metallopeptide 3 (3.4 μM) before (red line) and aft (black l n ) th add t on of a sol t on of “w ll match d” A:A’ ol on cl ot d DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 133 Figure S54. UV-vis absorption spectra of metallopeptide 3 (4.4 μM) before (red line) and aft (black l n ) th add t on of a sol t on of GG m smatch d A:A ’ ol on cl ot d GHOFRANE BARKA 134 Figure S55. UV-vis absorption spectra of metallopeptide 3 (2.5 μM) before (red line) and aft (black l n ) th add t on of a sol t on of GG/AA m smatch d A:A2’ ol on cl ot d DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 135 Figure S56. UV-vis absorption spectra of metallopeptide 3 (3.0 μM) before (red line) and after (black line) the addition of a solution of GG/GG/AA mismatched A:A3’ ol on cl ot d GHOFRANE BARKA 136 Figure S57. UV-vis absorption spectra of metallopeptide 3 (2.9 μM) before (red line) and aft (black l n ) th add t on of a sol t on of AA m smatch d B:B ’ ol on cl ot d DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 137 Figure S58. UV-vis absorption spectra of metallopeptide 3 (3.2 μM) before (red line) and aft (black l n ) th add t on of a sol t on of CC m smatch d C:C ’ ol on cl ot d GHOFRANE BARKA 144 Figure S65. UV-vis absorption spectra of metallopeptide 4 (3.6 μM) before (red line) and aft (black l n ) th add t on of a sol t on of CC m smatch d C:C ’ ol on cl ot d DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 145 Figure S66. UV-vis absorption spectra of metallopeptide 4 (3.7 μM) before (red line) and aft (black l n ) th add t on of a sol t on of TT m smatch d D:D ’ ol gonucleotide. GHOFRANE BARKA 146 Figure S67. UV-vis absorption spectra of metallopeptide 5 (3.7 μM) before (red line) and aft (black l n ) th add t on of a sol t on of “w ll match d” A:A’ ol on cl ot d DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 147 Figure S68. UV-vis absorption spectra of metallopeptide 5 (2.0 μM) before (red line) and aft (black l n ) th add t on of a sol t on of GG m smatch d A:A ’ ol on cl ot d GHOFRANE BARKA 148 Figure S69. UV-vis absorption spectra of metallopeptide 5 (3.6 μM) before (red line) and after (black line) the addition of a solution of GG/AA m smatch d A:A2’ ol on cl ot d DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 149 Figure S70. UV-vis absorption spectra of metallopeptide 5 (3.7 μM) before (red line) and aft (black l n ) th add t on of a sol t on of GG/GG/AA m smatch d A:A3’ ol on cl ot d GHOFRANE BARKA 150 Figure S71. UV-vis absorption spectra of metallopeptide 5 (3.5 μM) before (red line) and aft (black l n ) th add t on of a sol t on of AA m smatch d B:B ’ ol on cl ot d DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 151 Figure S72. UV-vis absorption spectra of metallopeptide 5 (4.0 μM) before (red line) and after (black line) the add t on of a sol t on of CC m smatch d C:C ’ ol on cl ot d GHOFRANE BARKA 152 Figure S73. UV-vis absorption spectra of metallopeptide 5 (3.1 μM) before (red line) and aft (black l n ) th add t on of a sol t on of TT m smatch d D:D ’ ol on cl ot d DESIGNED METALLOPEPTIDES AS TOOLS IN CHEMICAL BIOLOGY 153 Figure S74. UV-vis absorption spectra of metallopeptide 6 (3.6 μM) before (red line) and aft (black l n ) th add t on of a sol t on of “w ll match d” A:A’ ol on cl ot d