Metal-mediated diradical tuning for DNA replication PNAS PLUS arrest via template strand scission

Meghan R. Portera, Sarah E. Lindahla, Anne Lietzkeb, Erin M. Metzgera, Quan Wangb, Erik Hencka,b, Chun-Hsing Chenc, Hengyao Niub,1, and Jeffrey M. Zaleskia,1

aDepartment of Chemistry, Indiana University, Bloomington, IN 47405; bDepartment of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405; and cMolecular Structure Center, Indiana University, Bloomington, IN 47405 SEE COMMENTARY Edited by Richard Eisenberg, University of Rochester, Rochester, NY, and approved June 30, 2017 (received for review December 27, 2016)

− 2− A series of M(PyED)·X(X= 2Cl ,SO4 ) pyridine–metalloenediyne damage cellular DNA in a fashion that leaves DNA polymerase complexes [M = Cu(II), Fe(II), or Zn(II)] and their independently unable to either repair the damage or replicate the DNA. synthesized, cyclized analogs have been prepared to investigate their Double-stranded DNA, and more recently the machinery involved potential as radical-generating DNA-damaging agents. All complexes in DNA replication and repair, have been prominent targets for the possess a 1:1 metal-to-ligand stoichiometry as determined by electronic development of novel, metal-based cancer therapeutics. The impor- absorption spectroscopy and X-ray diffraction. Solution structural tance of the /stereochemistry of the metal center cannot be analysis reveals a pπ Cl → Cu(II) LMCT (22,026 cm−1) for Cu(PyED)·2Cl, overstated because this determines the orientation of ancillary ligands indicating three nitrogens and a chloride in the psuedo-equatorial with respect to the DNA duplex and can be used to tune the reactivity plane with the remaining pyridine nitrogen and solvent in axial and binding modes of these metal-containing therapeutics. Metal- positions. EPR spectra of the Cu(II) complexes exhibit an axially elon- loinserters and metallointercalators exploit noncovalent interactions gated . This spectroscopic evidence, together with den- of planar aromatic ligands to inhibit DNA repair because they show sity functional theory computed , suggest six-coordinate unwinding of DNA to π-stack between base pairs, groove binding, or structures for Cu(II) and Fe(II) complexes and a five-coordinate envi- insertion into a destabilized site (12, 13). Covalent interactions can be ronment for Zn(II) analogs. Bergman cyclization via thermal activa- used to directly bind complexes to the DNA duplex whereas the li-

tion of these constructs yields benzannulated product indicative gands may be functionalized for subsequent reactions such as radical- CHEMISTRY of diradical generation in all complexes within 3 h at 37 °C. A sig- induced DNA cleavage (14–19). Complexes using these mechanisms nificant metal dependence on the rate of the reaction is observed often demonstrate inhibition of DNA replication, leading to G2 [Cu(II) > Fe(II) > Zn(II)], which is mirrored in in vitro DNA-damaging arrest to halt progression of the damaged DNA during the cell cycle; outcomes. Whereas in situ chelation of PyED leads to considerable however, many suffer from lengthy reaction times of up to 48 h or degradation in the presence of all metals within 1 h under hyper- inefficient DNA degradation. Thus, development of molecules that thermia conditions, Cu(II) activation produces >50% compromised react on shorter timescales or with higher potency are important to DNA within 5 min. Additionally, Cu(II) chelated PyED outcompetes advance the potential of these DNA-damaging therapeutics. DNA polymerase I to successfully inhibit template strand extension. One of the best-known molecules that produces radical species · = μ BIOCHEMISTRY Exposure of HeLa cells to Cu(PyBD) SO4 (IC50 10 M) results in a G2/M upon interaction with a metal center [i.e., Cu(II), Fe(II)] is arrest compared with untreated samples, indicating significant DNA bleomycin (BLM). Administered as a metal-free ligand, BLM damage. These results demonstrate metal-controlled radical gener- ation for degradation of biopolymers under physiologically relevant Significance temperatures on short timescales. Pharmaceuticals often act within a lock-and-key model whereby metal-mediated radicals | DNA degradation | polymerase inhibition | molecules bind their targets nearly irreversibly, either stalling or enediyne | Bergman cyclization initiating biological processes. Here, the agent itself performs no chemical transformation on its target but rather triggers an pproximately 14.1 million new cancer cases were diagnosed event or cascade. However, unwanted side effects become more Ain 2012 alone (International Agency for Research on Can- likely as the reactivity of these molecules increases. In contrast, cer, WHO) (1), and although extensive research is underway to molecular compounds may irreversibly damage biological tar- combat these statistics additional therapeutic paradigms are still gets using metal-mediated radical chemistry, but controlling the needed. One of the hallmarks of cancerous cells is their high pro- onset and extent of reaction is challenging. Even so, multiple liferation rate due in part to DNA polymerases that are essential for examples of metal-containing or metal-radical paradigms have damage repair and replication (2). Alterations in these repair path- been used clinically for imaging and chemotherapy. Within this ways or inhibition of the polymerases themselves during S phase have framework we report a class of metal-mediated radical genera- been linked with increased treatment response to DNA-damaging tors that attack DNA, outcompete DNA polymerase, and are cytotoxic drugs. Thus, inhibition of DNA polymerases serves as a cytotoxic in short times and modest concentrations. potential chemotherapeutic target for cancer treatment due to their vital role in fundamental cellular processes (3). Author contributions: H.N. and J.M.Z. designed research; M.R.P., S.E.L., A.L., E.M.M., Q.W., At least 15 DNA polymerases have been discovered, each varying and E.H. performed research; C.-H.C. solved the crystallographic structures; and M.R.P., considerably in processivity and repair ability, making the task of S.E.L., E.M.M., H.N., and J.M.Z. wrote the manuscript. designing specific inhibitors to target individual DNA polymerases The authors declare no conflict of interest. an arduous one. Despite being well-developed, currently available This article is a PNAS Direct Submission. drugs, including toxins that reduce dNTP pools (4, 5), nucleotide Data deposition: The crystallography, atomic coordinates, and structure factors have been deposited in the Cambridge Structural Database, Cambridge Crystallographic Data Cen- analogs that directly block chain extension (6, 7), and genotoxins tre, Cambridge CB2 1EZ, United Kingdom (CCDC nos. 1523776 and 1523777). that damage the DNA template and cause replication stalling (8– See Commentary on page 9497. 11), are narrow in their target scope (2). Given the diversity of DNA 1To whom correspondence may be addressed. Email: [email protected] or hniu@ lesions and the pathways repairing them, effort has been spent on indiana.edu. identifying new strategies that can be applied to a broad spectrum This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of DNA targets. This alternative paradigm focuses on the ability to 1073/pnas.1621349114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1621349114 PNAS | Published online July 31, 2017 | E7405–E7414 Downloaded by guest on October 1, 2021 2.5 h at room temperature upon chelation of Mg(II) in methanolic solution (36). In aqueous media, hyperthermal (42.5 °C) treatment of HeLa cells with PyED (20 μM) in the presence of divalent metal ions for 1 h leads to a surviving cell fraction of only 0.5 (37). More- over, this diradical-induced degradation approach is also func- tional against peptidic biopolymer frameworks such as Aβ fibrils, which can be disaggregated upon Cu(II) or Zn(II) chelation within 2 h under physiological conditions (38). Thematically, structure-dependent diradical formation and its influence on substrate activation in real time represents an alter- native therapeutic paradigm and has significant potential for influ- encing kinetically dominant events during cell proliferation. To this end, we report the preparation of PyED–metalloenediyne com- plexes containing Cu(II), Fe(II), and Zn(II) in an effort to evaluate the geometric structure/Bergman cyclization relationship under physiological conditions and probe dynamic arrest of DNA extension processes via diradical degradation. Fig. 1. Raman spectra of Fe(PyED)·Cl2 (red) acquired at 77 K and Fe(PyBD)·Cl2 (blue) acquired at room temperature. The spectrum of Fe(PyED)·Cl2 exhibits a diagnostic alkyne vibration (2,176 cm−1) that is not present in the cyclized Results and Discussion · control complex Fe(PyBD) Cl2. Preparation of Pyridine Metalloenediynes and Cyclized Analogs. The tetradentate enediyne ligand, PyED, and cyclized analog, PyBD, were prepared according to published literature procedures and binds Cu(II) to aid in transportation into the cell, where it is characterized by their 1Hand13C NMR and mass signatures (36). – subsequently replaced by Fe(II) (20). Fe(II) BLM abstracts an A series of air-stable metalloenediyne complexes was synthesized by H-atom from the C4′ deoxyribose sugar to produce a radical, the addition of PyED to a solution of MCl2·xH2OorMSO4·xH2O leading to a gapped DNA site in the presence of O2, subsequent [M = Cu(II), Fe(II), or Zn(II)] in MeOH at 0 °C. After stirring for single- and double-strand DNA breaks (21), and cell cycle arrest at 4 h, the solvent was removed in vacuo, the remaining solid stirred in the G2/M checkpoint (22, 23). Although the BLM analogs phleo- Et2O at the same temperature, and the final product isolated by mycin and zorbamycin have differing DNA sequence selectivity filtration (SI Appendix,SchemeS1). The cyclized PyBD derivatives (24), each acts along a similar metal-chelation, radical-generation were generated under identical conditions to assist in the elucida- pathway, ultimately leading to overreplication following prevention tion of solution structures for the reactive metalloenediynes. The of the G2/M transition by DNA damage and/or incompletely rep- chloride complexes are moderately soluble in water and methanol, licated DNA (25). Although many DNA-damaging reagents display whereas the sulfate derivatives demonstrate excellent water solu- a small degree of G2 arrest as the cell provides itself an opportunity – bility but are insoluble in all organic solvents other than DMSO. to repair the damage (25 27), cellular toxicity may arise from either Magnetic moments of the copper complexes (1.7–1.8 μ )confirmd9 the inducement of apoptosis or permanent cell cycle arrest (26). B Cu(II) centers, whereas those of the iron analogs (chlorides: 4.2–4.6 μ ; In addition to the BLM family of therapeutics, small molecules B sulfates: 2.4–3.2 μ ) suggest that the Fe(II) systems are not purely such as hydralazine, doxorubicin, and ascorbic acid have also been B low- or high-spin at room temperature due to a possible temperature- shown to produce radical oxygen species (ROS) through various dependent spin cross-over for these complexes. mechanisms upon interaction with Cu(II) (28, 29). Controlled ROS The IR spectra for the PyED and PyBD complexes are com- generation can provide a potent means of damage in tumor cells; parable due to the high structural similarity between the ligands; however, when unregulated, it can lead to significant problems in however, the metalloenediyne spectra all contain a diagnostic healthy cells (30). Therefore, an alternative approach harnessing − alkyne stretch at ∼2,100 cm 1 [chlorides: Cu(II) = 2,171, direct H-atom abstraction without the need for ROS formation − Fe(II) = 2,179, Zn(II) = 2,181 cm 1; sulfates: Cu(II) = 2,174, would allow for activation of radical generators in hypoxic envi- = = −1 ronments and circumvent the issues associated with uncontrolled Fe(II) 2,173, Zn(II) 2,180 cm ], confirming the integrity ROS production. In the interest of metal chelation leading to of the enediyne functionality in the isolated products. Although radical generation, Cu(II) is an attractive choice due to the increased Cu(II) levels observed in malignancies (31, 32) arising from Cu(II)- mediated processes for tumorigenesis, such as BRAF signaling (33). This suggests that once inside the cell a ligand would have the opportunity to chelate Cu(II) if needed for activation. Natural products containing a distinctive enediyne framework provide a unique mechanism for damaging DNA. The cyclo- aromatization of the enediyne unit produces a potent 1,4-diradical species capable of H-atom abstraction from the closely positioned ribose ring of DNA. Unfortunately, the ability to control the onset of the radical formation event has proven challenging. Our current approach toward the development of small molecule, chelation- induced diradical generators capable of DNA damage is influenced by metal-mediated therapeutics such as Fe–BLM and hydroxyl radical footprinting reagents that act via Fenton chemistry or H- atom abstraction, leading to oxidative DNA strand scission (34, 35). From this conceptual platform, we have prepared the versatile tetradentate ligand (Z)-N,N-bis[1-pyridin-2-yl-meth(E)-ylidene]oct-

4-ene-2,6-diyne-1,8-diamine (PyED), which demonstrates facile Fig. 2. Electronic spectra of Cu(PyED)·Cl2 (red) and Cu(PyBD)·Cl2 (blue) in Bergman cyclization and 1,4-benzenoid diradical formation within MeOH. (Inset) Visible region is expanded.

E7406 | www.pnas.org/cgi/doi/10.1073/pnas.1621349114 Porter et al. Downloaded by guest on October 1, 2021 PNAS PLUS SEE COMMENTARY

Fig. 3. Proposed solution structures for Cu(PyED)·2Cl and Cu(PyBD)·2Cl. In the chelated ligand, N represents the imine nitrogens and N’ represents the pyridyl nitrogens. A indicates both pyridyl nitrogens and chloride bound to the metal center, however, in solution either the chloride (B) or the axial pyridyl ligand (C) may be displaced by solvent.

Fig. 4. Electronic spectra of Fe(PyED)·Cl2 (red) and Fe(PyBD)·Cl2 (blue) obtained in MeOH with the visible region shown (Inset). the Raman spectrum of Fe(PyED)·2Cl is not as clearly resolved as that of Fe(PyBD)·2Cl even at reduced temperature, the presence − − − of the intact enediyne is confirmed via a weak alkyne vibration at transition is observed at 22,026 cm 1 (e = 1,032 M 1·cm 1)(Fig.2, − 2,176 cm 1 (39) (Fig. 1). Additionally, C = NandC= Cin-plane Inset), confirming the presence of chloride in the equatorial plane − vibrations are observed at 1,554, 1,471, and 1,021 cm 1,whereas (45, 49–53) (Fig. 3A). This LMCT transition is not observed in the − the imine stretches at 1,613 and 1,591 cm 1 are broadened due to Cu(PyBD)·2Cl spectrum, suggesting that the equatorial chloride is overlap with the C = C stretch of the enediyne functional group displacedbyMeOHinsolution(Fig.3B). The smaller size and (39, 40) (Fig. 1). decreased flexibility of the PyBD chelate results in stronger binding Job plots for the stable, cyclized PyBD complexes were generated to the metal center compared with PyED, leading to more tightly using the continuous variation method (41) for determination of coordinated imine donors in the equatorial plane that weaken the CHEMISTRY solution stoichiometry upon binding to Cu(II) and Fe(II) in trans-Cl bond, subsequently increasing substitution by coordinating methanol. A 1:1 metal-to-ligand ratio is observed in solution for solvents. A qualitative analysis correlates the higher-energy absorp- − − − both Cu(PyBD)·2Cl and Fe(PyBD)·2Cl (SI Appendix,Fig.S1)and tion bands centered at 38,461 cm 1 (e = 9,280 M 1·cm 1,PyED)to confirmed using high-resolution electrospray mass spectrome- σ → – the expected p Cl dx2−y2 Cu(II) (49 51, 53) transitions that try (HRMS-ESI) performed immediately after complex dissolution. π → overlap the with lower-energy p (imine) dx2−y2 Cu(II) LMCT (45, − − − Analysis of HRMS-ESI data reveals a 1:1 metal-to-ligand relation- 54) features, whereas the band at 34,722 cm 1 (e = 9,467 M 1·cm 1, ship for the Zn(II) species as well. The chloride derivatives for all π → + PyBD) is attributed to p (imine) dx2−y2 Cu(II) LMCT because no

= BIOCHEMISTRY metals match the composition of [MLCl] (L PyED or PyBD), Cl → Cu(II) LMCT transitions are observed. These charge transfer indicating one chloride ligand bound to the metal center even under transitions are overlaid on the tail end of intense, higher-energy high vacuum. The presence of a bound chloride is supported by σ → features from the p (imine) dx2−y2 Cu(II) LMCT (45, 55, 56) electronic spectroscopy measurements (discussed below) for the and pyridine π–π* (45, 57, 58) (Fig. 2). Overall, the proposed Cu(II) and Fe(II) derivatives and is in good agreement with previ- Cu(N2)(N′2)Cl ligand field environment for both constructs is ously proposed structures of analogous quinoline-containing metal- 1 supported by these spectroscopic signatures, as well as the pre- loenediynes bound to divalent Cu and Fe (42). In contrast, HNMR viously reported, energy-minimized structure for the Mg(II)– spectroscopy of the Zn(II) complexes reveals a highly symmetric pyridine metalloenediyne (36). structure, suggesting the absence of an inner-sphere chloride ligand The electronic absorption spectra of Fe(PyED)·2Cl and Fe in solution. HRMS-ESI of the sulfate products yields a composition · + + – + (PyBD) 2Cl obtained in MeOH display characteristic bands cor- of [ML HSO4] (Cu,Fe)or[MLH] (Zn), indicating a nonco- responding to the π–π* transitions for diimine ligands coordinated + − − − ordinating anion and an overall 2 charge on the metal species. to Fe(II) (59–61) at 36,101 cm 1 (e = 14,777 M 1·cm 1,PyED) − − − and 35,842 cm 1 (e = 14,083 M 1·cm 1, PyBD) (Fig. 4), in agree- Structural Evaluation via Electronic Absorption Spectroscopy. The ment with other Fe(II) complexes containing pyridine and/or diimine wealth of information available in the literature regarding the electronic spectra of Cu(II) with N-donor ligands allows for a ligands (62, 63). Multiple MLCT absorption features are observed in the visible region of the spectrum via excitation of an electron qualitative analysis of the solution structures via their electronic π absorption spectra (43). Ligand field absorption profiles can be from the t2g orbitals on the Fe(II) center to the empty * pyridyl-imine used to assess the coordination number/geometry relationship, because planar complexes display ligand field transitions − in the range of 17,000–20,000 cm 1 with decreasing energies as the geometry varies from square planar to octahedral to tetrahedral (44– 46). The electronic spectra for Cu(PyED)·2Cl and Cu(PyBD)·2Cl acquired in MeOH at room temperature reveal low-energy d–d − transitions centered at 13,300 cm 1, indicative of a six-coordinate, distorted octahedral structure for both complexes (45, 47, 48) (Fig. 2, Inset). Although these ligand field transitions provide information re- garding the solution coordination number, they do not lend insight into the spatial arrangement of the ligands. However, the interac- tion of the equatorial ligand field with the dx2−y2 Cu-centered SOMO is accessible by charge transfer electronic spectroscopy. In Fig. 5. X-band EPR spectra of Cu(PyBD)·2Cl (600 μM, blue trace) and Cu(PyED)·2Cl the electronic spectrum of Cu(PyED)·2Cl, a pπ Cl → Cu(II) LMCT (600 μM, red trace) at 77 K in MeOH. Simulated spectra are shown in black.

Porter et al. PNAS | Published online July 31, 2017 | E7407 Downloaded by guest on October 1, 2021 Table 1. X-band EPR spectral parameters of Cu(PyBD)·2Cl and vein, analysis of the chloride-bound forms of both species suggests Cu(PyED)·2Cl at 77 K in MeOH a tighter and more covalent ligand–Cu(II) interaction for the cyclized complex (g║ = 2.22) compared with the uncyclized ene- AIICu −4 −1 diyne (g║ = 2.25) (66, 67). This higher degree of covalency stems Complex Species gII g┴ (× 10 ), cm GgII/AII Percentage from the constricted binding pocket of the cyclized ligand com- Cu(PyBD)·2Cl 3B 2.25 2.06 157 4.17 143 85 pared with the more flexible chelation of the enediyne analog. 3A 2.22 2.06 150 3.67 148 15 Cu(PyED)·2Cl 3A 2.25 2.07 136 3.57 165 65 Crystallographic and Computational Structure Determination. Obtaining 3B/3C 2.27 2.07 157 3.86 145 35 crystallographic information from stable, cyclized controls is para- mount for insight into the conformations of their highly reactive enediyne-containing counterparts. Within this theme, X-ray crystal- orbital set on the ligand (45, 61, 64, 65). Overlapping with these lographic analysis of Cu(PyBD)·2Cl reveals a five-coordinate ge- intense π–π* ligand transitions are MLCT transitions [PyED: ometry between trigonal bipyramidal and square pyramidal (τ = − − − − 29,498 cm 1, e = 5,246 M 1·cm 1; PyBD: 28,490 cm 1, e = 0.549) containing a chloride, both imines, and one pyridine nitrogen − − 2,740 M 1·cm 1] (Fig. 4). A second MLCT band for each complex bound in a pseudoequatorial plane with the remaining pyridine −1 is observed with a shoulder at higher energy [PyED: 19,880 cm , donor coordinated axially, consistent with other N4-chelated com- − − − − − e = 1,647 M 1·cm 1 and 18,115 cm 1, e = 1,833 M 1·cm 1;PyBD: pounds (74, 75) (Fig. 6). The equatorial Cu–N distances range from − − − − − − 19,305 cm 1, e = 2,672 M 1·cm 1 and 17,857 cm 1, e = 3,478 M 1·cm 1] 1.99 to 2.01 Å, typical for Cu–imine single bonds (76–78), whereas (59) (Fig. 4, Inset). the axial pyridine shows weaker coordination (2.16 Å) (SI Appendix, Table S1). This solid-state geometry provides an open coordination Copper Complex Structure from EPR Spectroscopy. EPR spectra of site trans to the axial pyridine for solvent coordination, forming a Cu(PyED)·2Cl and the cyclized control Cu(PyBD)·2Cl in MeOH tetragonally elongated octahedron that accounts for the low-energy were obtained at 77 K to elucidate the solution geometry and ligand ligand field transitions observed in the electronic absorption spec- binding about the Cu(II) centers (Fig. 5). Both complexes exhibit trum. Additionally, the long Cu–Cl bond (2.29 Å) trans to a short > > axial symmetry (g║ g┴ 2.0023) with a Cu(II) dx2−y2 ground state Cu–imine distance (2.01 Å) correlates well with the absence of Cl → (66–68). The Cu(PyBD)·2Cl spectrum reveals a major species with Cu(II) LMCT in the electronic absorption spectrum due to chloride = = g║ 2.25 and g┴ 2.06, whereas the enediyne analog exhibits a displacement by solvent. The Zn(PyBD)·SO4 crystal structure adopts major species with g║ = 2.25 and g┴ = 2.07 (Table 1) that is con- a weak six-coordinate geometry containing an asymmetric sulfate sistent with aza-aromatic nitrogen-donor ligands such as pyridine chelate with one shorter Zn–O1 bond (2.18 Å), which is in agree- (69). These larger g║ values result from increased ligand anisotropy ment with all other six-coordinate chelated sulfate zinc structures in the xy plane due to the presence of either bound chloride (79–81), and a longer Zn–O2 distance at 2.21 Å. Parallel to the [Cu(PyED)·2Cl] or an electron-withdrawing oxygen from methanolic Cu(PyBD)·2Cl structure described above, the two imine nitrogens solvent [Cu(PyBD)·2Cl] in an equatorial position (Fig. 3 A and B). and one pyridine nitrogen coordinate in the equatorial plane, with The resulting spin Hamiltonian parameters obtained from spec- the remaining pyridine nitrogen in an axial position (Fig. 7). tral simulation for Cu(PyBD)·2Cl show the presence of a minor Due to the absence of crystallographic data on the reactive species (∼15%) with a reduced g║ value (g║ = 2.22). This likely PyED complexes, density functional theory calculations were corresponds to the Cu(PyBD)·2Cl structure with the chloride bound used to probe the structures and predict the Bergman cyclization in the equatorial position (Fig. 3A),becausethetimescalefor reactivity of these complexes. The computed structures of sample preparation (10 min) lies between HRMS analysis (2 min) documenting inner sphere chloride and electronic spectral confir- mation of chloride displacement by MeOH within 30 min (Fig. 2, Inset). Although the chloride-bound Cu(PyED)·2Cl complex EPR spectrum also exhibits a minor species (∼35%), the larger g║ value (g║ = 2.27) indicates a more oxygen-rich coordination environment upon displacement of either chloride (Fig. 3B)ortheweaklyco- ordinated axial pyridine by MeOH (Fig. 3C). From these spin Hamiltonian parameters, G values, G = (g║ − 2.0023)/(g┴ − 2.0023),canbedirectlycalculatedtoshedlightonthe deviation from planarity of the equatorial ligand set. Values greater than 4 signify the equatorial M–L bonds are in the same plane or only slightly misaligned, whereas values less than 4 suggest a sig- nificant dihedral distortion (70). The G value for Cu(PyBD)·2Cl (G = 4.17) containing a pyridyl nitrogen in the axial position reveals only slight dihedral distortion from the xy plane. However, once the chloride has been displaced by solvent the distortion increases (G = 3.67). In contrast, Cu(PyED)·2Cl (G = 3.57) exhibits a larger dis- tortion of the equatorial plane that is relaxed upon replacement of the axial pyridine by solvent (G = 3.86) (Table 1). Distortion of the dihedral angle around the metal center is supported by reduced A║ values, which denote a decrease in direct overlap between the equatorial ligand set and the Cu(II) dx2−y2 SOMO (69, 71, 72). Because deviation of g║ from the free electron value (2.0023) originates from spin-orbit coupling, variations in these values can be used to interpret the ionic vs. covalent character of ligand co- ordination (70). Because g║ values >2.3 are symptomatic of more ionic bonding, the g║ values of both the cyclized and uncyclized Fig. 6. Solid-state X-ray crystallographic structure of Cu(PyBD)·2Cl. Thermal analogs are indicative of a more covalent character (70, 73). In this ellipsoids are illustrated at 20% probability.

E7408 | www.pnas.org/cgi/doi/10.1073/pnas.1621349114 Porter et al. Downloaded by guest on October 1, 2021 PNAS PLUS SEE COMMENTARY

Fig. 8. Computed structures of ligand–metal bonding interactions for M (PyED) complexes [M = Cu(II), Fe(II), and Zn(II)], where d is the interalkynyl − distance. Monodentate ligands are presented as either MeOH or Cl .

− 2− (X = 2Cl ;SO4 ) reveals a highly symmetric splitting pattern that is identical for both analogs, supporting the proposal that in so- lution the anions are noncoordinating. Based on complex solubility in polar solvent and the nature of the coordinated anion in the Zn (PyBD)·SO4 crystal (Fig. 8), a weakly solvated five-coordinate structure is purported for Zn(PyED)·2Cl. Consistent with this proposal, the computed enediyne structure shows a pseudosquare pyramidal geometry (τ = 0.014) containing an axial pyridine. The Zn1–N1 (2.14 Å) and Zn1–O1 (2.31 Å) bonds are elongated, gen- erating a weak axis (SI Appendix,TableS3). As such, the bound

Fig. 7. Crystallographically characterized solid state structure of Zn(PyBD)·SO4. solvent is likely fluxional in solution, allowing the complex to oscillate Thermal ellipsoids are illustrated at 50% probability. between a four- and five-coordinate structure. This fluxionality ac- CHEMISTRY counts for the highly symmetric, time-averaged NMR spectra ob- served for both Zn(PyED)·SO and Zn(PyED)·2Cl and is in line · · 4 Cu(PyBD) 2Cl and Zn(PyBD) SO4 were obtained by minimization with the spectroscopic observables for the cyclized controls. of the crystallographic coordinates to ensure reliable computational From spectroscopic analyses of the enediyne-containing con- methods for modeling the reactive PyED complexes. Geometry op- structs, six-coordinate solution geometries are proposed for timizations, vibrational analyses, and solvation calculations (MeOH, both the Cu(II) and Fe(II) PyED chloride complexes. Uniting dielectric constant e = 32.613) using the PCM model (82–86) were the structural datasets reveals that Cu(PyED)·2Cl exhibits tighter – performed in Gaussian 09 (87) with the (U)BPW91 (88 93)/6-31G** M–L bonding in the equatorial plane and weaker axial coordi- BIOCHEMISTRY density functional/basis set combination and an ultrafine grid, nation relative to Fe(PyED)·2Cl due to the tetragonal elongation applying the SDD (94–98) pseudopotential to all metal atoms. experienced by the d9 Cu(II) center. This distortion leads to Although all calculated bond lengths for both Cu(PyBD)·2Cl compression of the imine bonds in the xy plane [Cu(II): 2.06, 2.16 Å; · and Zn(PyBD) SO4 are slightly overestimated, these minor devi- Fe(II): 2.23, 2.29 Å] and a decrease in the interalkynyl distance ations are anticipated as the computed structures account for of Cu(PyED)·2Cl. Comparatively, the interalkynyl distance of the solvation (SI Appendix,TablesS1andS2). Zn(PyED)·2Cl complex (4.17 Å) is significantly larger than both Although the bond lengths for the computed Cu(PyBD)·2Cl the Cu(II) (3.68 Å) or Fe(II) (3.82 Å) analogs, resulting not from dif- complex are consistent with the X-ray structure, deviations of 11–13° ferences in bond lengths but from changes in bond angles (Fig. 8). in the N3–Cu1–N1 and N3–Cu1–Cl1 bond angles are observed (SI Appendix,TableS1), implying that upon solvation Cu(PyBD)·2Cl adopts a more square pyramidal geometry (τ = 0.338) relative to the solid-state structure (τ = 0.549). This pyramidalization allows for coordination of a sixth ligand from solvent, in line with the electronic absorption spectrum of Cu(PyBD)·2Cl indicating a distorted octa- hedron in solution. In light of this proposed solution structure, computational analysis of a six-coordinate solvated Cu(PyED)·2Cl reveals an axially elongated octahedral complex (Fig. 8). The dif- ferences in coordination number notwithstanding, an axial pyridine is consistent with the X-ray structure of the five-coordinate cyclized analog. However, the Cu1–N3 bond is lengthened by 0.11 Å, indi- cating that PyED acts as a weaker σ donor relative to PyBD. Ad- ditionally, the Cu1–N1 bond is found to lengthen dramatically (∼0.16 Å) in the enediyne-containing compound, consistent with the minor product in the Cu(PyED)·2Cl EPR spectrum where the axial pyridine has been displaced by solvent (Fig. 3C). Unlike Cu(II), d10 Zn(II) centers demonstrate only modest elec- tronic barriers between different coordination numbers and geom- etries. In this vein, the crystallographic and computed structures for Zn(PyBD)·SO4 show a weakly chelated sulfate (SI Appendix,Table S2), implying that in the presence of coordinating solvent the anion can be displaced to generate a four-coordinate or weak five-coordinate Scheme 1. Thermal activation of PyED at physiological temperature (37 °C) 1 complex. Examination of the H NMR spectra for Zn(PyBD)·X to yield cyclized PyBDH via Bergman cyclization. M = Cu(II), Fe(II), or Zn(II).

Porter et al. PNAS | Published online July 31, 2017 | E7409 Downloaded by guest on October 1, 2021 Although all complexes display formation of the cyclized product within 3 h at 37 °C, differences in the extent of cyclization within this time period are observed. Ionizability of the reduced ligands was measured by recording mass spectra from an equimolar solution of PyBDH and PyEDH. Based on those measurements, a simple analysis of the ratio of cyclized product vs. starting material of multiple trials by MS was used to probe these variances. The ad- dition of PyED to a solution of CuCl2 yields no detectable starting material after 3 h at 37 °C. In contrast, reaction of PyED with ZnCl2 leads to ∼50% formation of the cyclized product, whereas the FeCl2 reaction lies somewhere between these two limits, with roughly 74% formation of cyclized product compared with unreacted enediyne. The reduced product formation for cyclization in the presence of Zn(II) relative to Cu(II) aligns well with the suite of structural data, as well as the disaggregation of preformed amyloid-β aggregates at 37 °C within 2 h in the presence of Cu(II) vs. 8 h with Zn(II) (38). The extent of product formation for all metals increases at 42 °C, although under these hyperthermal conditions the enhanced ki- netics of the radical reaction escalates the degree of polymerization and limits detection of the cyclized product. For the MeOH in- soluble, but highly water-soluble sulfate derivatives, cyclized product formation was also confirmed by HRMS for Cu(II), Fe(II), and Zn(II) (SI Appendix,TableS5) with parallel reactivity trends to the chloride salts, although rapid formation of H2 and degradation of NaBH4 to form sodium metaborate in aqueous solution precluded quantitative product analysis.

Metal-Mediated PyED Radical Damage of DNA. Although PyED has been shown to promote HeLa cell death after 1 h at 42.5 °C in the presence of divalent metals (37), the mechanism of toxicity has not been established. To examine the ability of PyED to damage DNA as a potential target of this reaction, supercoiled DNA was treated Fig. 9. (A) ϕX174 RFI supercoiled DNA (100 ng, ∼33 μM nucleotides, 2.9 nM to various concentrations of ligand in the absence or presence of DNA molecules) (NEB) was incubated with PyED or PyBD in the absence or μ presence of M(II) in a buffer system containing 10 mM Tris·HCl (pH 7.4) for 20 M Cu(II), Fe(II), or Zn(II) at 42 °C (Fig. 9A). Efficient nicking 1 h at 42 °C and analyzed by 0.8% agarose gel electrophoresis in TBE buffer [M(II) = Cu(II), Fe(II), or Zn(II)]; nc, nicked circular DNA; sc, supercoiled DNA. (B) Reactions were executed and analyzed as in A except PyED was replaced by phleomycin for chelation to Fe(II). (C) Reactions were performed as in A at 37 °C with Cu(II) as the divalent cofactor.

The five-coordinate geometry of the Zn(II) analog permits a wider enediyne conformation (N2–M–N3 = 146.8°) compared with Cu(II) and Fe(II) (101.9° and 103.0°, respectively), resulting in a larger interalkynyl distance (SI Appendix,TableS3). Because Bergman cyclization requires the formation of a C–C bond, shorter inter- alkynyl distances allow for facile formation of the benzannulated cyclization product. Therefore, comparison of the interalkynyl dis- tances for these reactive PyED complexes isolated at 0 °C suggests that upon metal complexation at elevated temperatures thermal Bergman cyclization rates in solution should be observed in the order of Cu(II) > Fe(II) > Zn(II).

In Situ Chelation-Induced Diradical Reactivity. The addition of free PyED to a solution of MCl2 [M = Cu(II), Fe(II), or Zn(II)] in MeOH results in the formation of the Bergman cyclized product detectable by HRMS within 3 h at 37 °C (SI Appendix, Table S4) for all complexes. This timescale correlates well with the pre- viously published Mg(II) analog that displays in situ cyclization within 2.5 h in MeOH at ambient temperature (36). The soluble organic reaction products were isolated via reduction of the imine ϕ bonds with NaBH4 and extraction of the metal center using an Fig. 10. (A) Reactions containing X174 RFI supercoiled DNA and various EDTA solution (Scheme 1). Formation of the benzannulated ligand amounts of PyED in the presence of Cu(II) (40 μM) were incubated at 42 °C was confirmed by reaction of the independently cyclized PyBD and stopped at the desired time by adding EDTA (50 mM final concentra- tion). A control reaction containing only PyED (1 h) was also included. The complexes under identical conditions (SI Appendix,SchemeS2)and reactions were analyzed as described in Fig. 9A.(B) Reactions were con- the HRMS signatures for the cyclized product were established by ducted as in Fig. 9A; however, the solutions contain 40 μM divalent ions and comparison with the independently prepared amine PyBDH. up to 40 μM PyED or PyBD.

E7410 | www.pnas.org/cgi/doi/10.1073/pnas.1621349114 Porter et al. Downloaded by guest on October 1, 2021 PyED (40 μM) and DNA polymerase I efficiently inhibits primer PNAS PLUS extension in a Cu(II)-dependent manner, leading to the complete inhibition of 50% of the polymerase reaction (Fig. 11A). The polymerase assay was repeated allowing for preincubation of PyED and Cu(II) with either DNA polymerase I or substrate DNA. Interestingly, preincubation of substrate DNA with 50 μM PyED and Cu(II) completely stalls primer extension, whereas preincubation with DNA polymerase I has minimal effect on the extension reaction (Fig. 11B). Consequently, the effective pre- vention of the DNA polymerase reaction by PyED is largely due to its reaction with the DNA substrate itself. Although E. coli DNA SEE COMMENTARY polymerase I is not the most robust of the polymerase family, once engaged it replicates DNA at a speed of 10–20 nucleotides per s. The efficient inhibition of the DNA polymerase reaction upon addition of PyED concurrently with DNA polymerase I suggests the enediyne radical generation event is fast enough to out- compete DNA replication machinery and potentially other DNA metabolic processes. This speed may be an additional attribute

Fig. 11. (A) DNA was mixed with the indicated amount of PyED in the ab- sence or presence of Cu(II) in a system containing 10 mM Tris·HCl, 50 mM NaCl,

10 mM MgCl2, 1 mM DTT, 0.25 mM dNTP each, 0.25 U DNA polymerase I Klenow fragment, and 10 nM Template-Primer DNA substrate at pH 7.9. The reactions were incubated at 37 °C for 30 min and analyzed by 12% denaturing PAGE in TBE buffer. (B) Reactions were run as in A with or without pre- CHEMISTRY incubation of either DNA substrate or DNA polymerase with 50 μMPyEDand 50 μM Cu(II) for 1 h. Reactions were analyzed as described in Fig. 9. Asterisk indicates primer/template duplex DNA that is not fully denatured.

of the supercoiled DNA occurs in the presence of all three divalent ions; however, the extent of the reaction is strongly metal- dependent. Consistent with the structure–activity relationship BIOCHEMISTRY above, the reaction with Cu(II) is the most effective, followed by Fe(II) and Zn(II). The observed DNA damage by Cu(II)-activated PyED compares favorably with that of phleomycin, a chelation- dependent, radical-generating control, with an equivalent response occurring upon exposure to lower concentrations of PyED (Fig. 9B). The activity displayed by the Cu(II) reaction at 37 °C is similar to that demonstrated at 42 °C (Fig. 9C), with 80% DNA degra- dation in the presence of 10 μM PyED and DNA nicking occurring at concentrations as low as 2.5 μM after 1 h. Minimal degradation is detected in the presence of either free PyED (20 μM) or PyBD, indicating that the chelation-induced radical-generation event is responsible for the activity observed. To determine the rate of this radical-induced DNA damage, time-course analyses were conducted by titration of PyED in the presence of Cu(II) (Fig. 10A). Using approximately a 1:1 ratio of PyED (30 μM) to nucleotides (33 μM), over 50% of the DNA is reacted within 5 min. Notably, when PyED is present in excess (40 μM), slower migrating DNA species are also observed corre- sponding to higher molecular weights (Fig. 10B), which may stem from PyED-induced DNA cross-linking. This type of DNA lesion is extremely toxic to human cells due to the multiple strand cleavage events that are required for repair. Further investigation of these species with a higher molecular weight could allow for potentiation of PyED as a cross-linking strategy toward cancer therapy. Many chemotherapeutic agents or antibiotics induce DNA damage by inhibiting cell growth through DNA lesions that stall replication. Therefore, the effect of PyED treatment on DNA replication was examined using an in vitro primer-extension assay Fig. 12. Toxicity of M(PyED)·SO4 [M = Cu(II), Fe(II), or Zn(II)] to HeLa cells. to monitor extension of 49 nucleotides by Escherichia coli DNA HeLa cells were treated with M(PyED)·SO or the nonradical-generating polymerase I large fragment (Klenow). This polymerase I large 4 analog, M(PyBD)·SO4, at varying concentrations for 1 h at 37 °C. Surviving fragment lacks 5′-exonuclease activity but retains full polymerase fraction was measured using a clonogenic assay. Error bars represent stan- and 3′-exonuclease proofreading ability. Simultaneous addition of dard SEM from three independent treatments.

Porter et al. PNAS | Published online July 31, 2017 | E7411 Downloaded by guest on October 1, 2021 Fig. 13. Perturbation of the cell cycle progression in cells treated with Cu(PyED)·SO4.(A) Asynchronous HeLa cells were either untreated (i)ortreatedwith Cu(PyED)·SO4 (100 μM) (ii) and incubated at 37 °C before harvest at indicated time intervals for flow cytometry analysis with PI staining. (B)SynchronizedHeLacells with double thymidine block were either untreated (i) or treated with Cu(PyED)·SO4 (100 μM) at G1/S boundary for 1 h (ii), then released to the drug-free medium for indicated time before harvest for cell cycle analysis.

of PyED as an effective DNA-damaging agent in vivo, because was observed with Cu(PyED)·SO4, whereas survival fractions of most of the genomic DNA is protected by the formation of chro- 23.6 ± 1.5% and 76 ± 3.5% were observed with Fe(PyED)·SO4 matin and is only exposed transiently during DNA replication, and Zn(PyED)·SO4, respectively. Importantly, the nonreactive transcription, and repair. M(PyBD)·SO4 complexes demonstrate minimal toxicity to HeLa cells, indicating that the radical-generating enediyne moiety is es- Radical-Induced in Vivo Cellular Effects. To query whether the differ- sential to cell death. ence in DNA-damaging capabilities between PyED complexed with Cells that have undergone DNA damage, DNA double-strand Cu(II), Fe(II), Zn(II) correlates with their cellular toxicity, clono- breaks in particular, often display G2/M delay upon activation of the genic assays were conducted to measure HeLa cell survival upon DNA damage checkpoint response to aid in repair. Treatment of · = treatment with M(PyED) SO4 [M Cu(II), Fe(II), and Zn(II)] at Cu(PyED)·SO4 generates mostly single-strand breaks on supercoiled 37 °C. Cu(PyED)·SO4, the most effective DNA-damaging agent in DNA in vitro. In cells, however, these single-strand breaks may be vitro, is also the most toxic to HeLa cells among all three compounds converted into double-strand breaks during DNA replication. Thus, tested with a calculated IC50 of 10.5 μM (Fig. 12). Under the maxi- cell cycle assays were conducted to answer whether treatment mum concentration used (100 μM) a survival fraction of <0.5% of HeLa cells with Cu(PyED)·SO4 alters progression through

E7412 | www.pnas.org/cgi/doi/10.1073/pnas.1621349114 Porter et al. Downloaded by guest on October 1, 2021 G2/M phase. Exposure of asynchronous HeLa cell culture to Conclusion PNAS PLUS · Cu(PyED) SO4 at 37 °C causes an increase of the G2/M fraction To gain insight into the development of reactive, yet controllable, from ∼16–31% at 24 h posttreatment, persisting at 48 h posttreat- small molecules for biological activity a series of pyridine–metal- ment (Fig. 13A). To ensure the nearly twofold increase in the G2/M loenediyne complexes and their cyclized analogs have been syn- population is due to a G2/M delay and not because only a fraction thesized and the differences in their solution Bergman cyclization of the cell culture has responded to Cu(PyED)·SO4, the effect of reactivities examined. Electronic and EPR spectroscopic analysis Cu(PyED)·SO4 treatment on cell cycle progression was explored indicates a six-coordinate solution structure for all Cu(II) and using HeLa cells synchronized at the G1/S border using double treat- Fe(II) complexes, each containing three nitrogens and a chloride ment of thymidine, a deoxynucleoside that in excess inhibits DNA in the equatorial plane with a weakly bound pyridine nitrogen and synthesis in a reversible manner. The HeLa cells were first arrested solvent in the axial positions, and a five-coordinate solution structure is determined for the Zn(II) complexes. PyED demon- SEE COMMENTARY at the G1/S border followed by treatment with Cu(PyED)·SO4 for 1 h and then released to cell cycle progression by thymidine removal. strates facile Bergman cyclization within 3 h at 37 °C in the At 8 h after the release from G1/S arrest the majority of both un- presence of divalent Cu, Fe, or Zn with the extent of product formation following the order Cu(II) > Fe(II) > Zn(II). Com- treated (53.8%) and Cu(PyED)·SO4-treated (65.4%) HeLa cells had progressed to G2/M phase (Fig. 13B). However, 10 h after the re- putational investigation shows the source of the reactivity profile lease from G1/S arrest the majority of untreated HeLa cells had to be structural differences that lead to lengthening of the inter- alkynyl distance and subsequent retardation of cyclization in so- progressed through G2/M phase and reached G1 phase (58.2%) and lution. This trend is mirrored in the ability of PyED to damage the fraction of cells at G2/M phase reduced to 27.8% (Fig. 13B). In · DNA by metal-dependent diradical formation within 1 h at 42 °C. contrast, the Cu(PyED) SO4-treated samples demonstrate a majority Remarkably, efficient inhibition of the DNA polymerase reaction of cells (63.7%) remaining at the G2/M phase (Fig. 13B), clearly · in the presence of Cu(II) suggests the radical-generation event is indicating a G2/M delay due to Cu(PyED) SO4 exposure. The fast enough to outcompete the replication process. Treatment of · findings that the cytotoxicity of M(PyED) SO4 correlates with the HeLa cells with Cu(PyED)·SO leads to arrest of the cell cycle at · 4 DNA-damaging efficiency in vitro and Cu(PyED) SO4 treatment the G2/M checkpoint, indicating that this DNA damage is effective causes a G2/M delay in HeLa cells suggest that the cytotoxicity of in vivo, and cellular toxicity is observed with an IC of 10.5 μM. · 50 M(PyED) SO4 is due in part to their DNA-damaging activities. Thus, metal-mediated PyED activation offers unique control of Because these metalloenediynes are highly reactive toward radical the potent diradical generation event and thereby a means for CHEMISTRY generation, the majority of cells receiving treatment in these ex- potentiation of critical DNA metabolic processes under physio- periments are at the G1 phase or the G1/S border. Given that logically relevant temperatures on short timescales. Cu(PyED)·SO4 inhibits DNA replication in vitro, it will be inter- esting to examine whether the S-phase cells are hypersensitive to ACKNOWLEDGMENTS. We thank Dr. Stephen Bell for sharing the reagents for PyED-derived compounds. In addition to DNA single- and double- the primer-extension assays. We thank Christiane Hassel (Indiana University Bloomington Flow Cytometry Core Facility) for assistance. This work was sup- strand breaks, M(PyED)·X radical-induced DNA damage may ported by NSF Grant CHE-1265703 and NIH Grants NIH-5R01, CA196293-02, also generate DNA cross-links and adducts, both of which are and R00 ES021441. Additionally, this research was supported in part by Lilly more toxic DNA lesions. Thus, radical-generating compounds Endowment, Inc., through its support for the Indiana University Pervasive Technology Institute, and in part by the Indiana METACyt Initiative. The BIOCHEMISTRY of this type may eventually serve as an alternative strategy to Indiana METACyt Initiative at Indiana University is also supported in part by DNA interruption of malignant cell proliferation. Lilly Endowment, Inc.

1. International Agency for Research on Cancer, World Health Organization (2016) 17. Meyer A, et al. (2014) Gold(I) N-heterocyclic carbene complexes with naphthalimide GLOBOCAN 2012: Estimated cancer incidence, mortality, and prevalence worldwide in ligands as combined thioredoxin reductase inhibitors and DNA intercalators. 2012 (IARC, Lyon, France). ChemMedChem 9:1794–1800. 2. Puigvert JC, Sanjiv K, Helleday T (2016) Targeting DNA repair, DNA metabolism and 18. Boer DR, Wu L, Lincoln P, Coll M (2014) Thread insertion of a bis(dipyridophenazine) replication stress as anti-cancer strategies. FEBS J 283:232–245. diruthenium complex into the DNA double helix by the extrusion of AT base pairs and 3. Parsons JL, Nicolay NH, Sharma RA (2013) Biological and therapeutic relevance of cross-linking of DNA duplexes. Angew Chem Int Ed Engl 53:1949–1952.

nonreplicative DNA polymerases to cancer. Antioxid Redox Signal 18:851–873. 19. Akhimie RN, White JK, Turro C (2017) Dual photoreactivity of a new Rh2(II,II) complex 4. Bianchi V, Pontis E, Reichard P (1986) Changes of deoxyribonucleoside triphosphate pools for biological applications. Inorganica Chim Acta 454:149–154. induced by hydroxyurea and their relation to DNA synthesis. J Biol Chem 261:16037–16042. 20. Dorr RT (1992) Bleomycin pharmacology: Mechanism of action and resistance, and 5. Cerqueira NMFSA, Fernandes PA, Ramos MJ (2007) Understanding ribonucleotide clinical pharmacokinetics. Semin Oncol 19(2, Suppl 5):3–8. reductase inactivation by gemcitabine. Chemistry 13:8507–8515. 21. Hecht SM (2000) Bleomycin: New perspectives on the mechanism of action. J Nat Prod 6. Cheung-Ong K, Giaever G, Nislow C (2013) DNA-damaging agents in cancer chemo- 63:158–168. therapy: Serendipity and chemical biology. Chem Biol 20:648–659. 22. Arai M, et al. (2004) Selective inhibition of bleomycin-induced G2 cell cycle checkpoint 7. Burke MP, Borland KM, Litosh VA (2016) Base-modified nucleosides as chemothera- by simaomicin α. Biochem Biophys Res Commun 317:817–822. peutic agents: Past and future. Curr Top Med Chem 16:1231–1241. 23. Byrnes RW, Templin J, Sem D, Lyman S, Petering DH (1990) Intracellular DNA strand scission 8. Merrick CJ, Jackson D, Diffley JFX (2004) Visualization of altered replication dynamics and growth inhibition of Ehrlich ascites tumor cells by bleomycins. Cancer Res 50:5275–5286. after DNA damage in human cells. J Biol Chem 279:20067–20075. 24. Chen JK, Yang D, Shen B, Neilan BA, Murray V (2016) Zorbamycin has a different DNA se- 9. Sale JE, Lehmann AR, Woodgate R (2012) Y-family DNA polymerases and their role in quence selectivity compared with bleomycin and analogues. Bioorg Med Chem 24:6094–6101. tolerance of cellular DNA damage. Nat Rev Mol Cell Biol 13:141–152. 25. Hartwell LH, Kastan MB (1994) Cell cycle control and cancer. Science 266:1821–1828. 10. Siddik ZH (2003) Cisplatin: Mode of cytotoxic action and molecular basis of resistance. 26. Jeggo PA, Löbrich M (2006) Contribution of DNA repair and cell cycle checkpoint Oncogene 22:7265–7279. arrest to the maintenance of genomic stability. DNA Repair (Amst) 5:1192–1198. 11. Zhang J, Stevens MFG, Bradshaw TD (2012) Temozolomide: Mechanisms of action, 27. Lukas J, Lukas C, Bartek J (2004) Mammalian cell cycle checkpoints: Signalling path- repair and resistance. Curr Mol Pharmacol 5:102–114. ways and their organization in space and time. DNA Repair (Amst) 3:997–1007. 12. Boyle KM, Barton JK (2016) Targeting DNA mismatches with rhodium metal- 28. Kankala RK, et al. (2017) Overcoming multidrug resistance through co-delivery of loinsertors. Inorganica Chim Acta 452:3–11. ROS-generating nano-machinery in cancer therapeutics. J Mater Chem B 5:1507–1517. 13. Komor AC, Barton JK (2013) The path for metal complexes to a DNA target. Chem 29. Wagner BA, Evig CB, Reszka KJ, Buettner GR, Burns CP (2005) Doxorubicin increases in- Commun (Camb) 49:3617–3630. tracellular hydrogen peroxide in PC3 prostate cancer cells. Arch Biochem Biophys 440:181–190. 14. Pravin N, Raman N (2014) Investigation of in vitro anticancer and DNA strap interactions in live 30. Trachootham D, Alexandre J, Huang P (2009) Targeting cancer cells by ROS-mediated cells using carboplatin type Cu(II) and Zn(II) metalloinsertors. Eur J Med Chem 85:675–687. mechanisms: A radical therapeutic approach? Nat Rev Drug Discov 8:579–591. 15. Raman N, Selvaganapathy M, Sudharsan S (2015) DNA, the biopolymer as a target 31. Zowczak M, Iskra M, Torlinski L, Cofta S (2001) Analysis of serum copper and zinc material for metalloinsertors: From chemistry to preclinical implications. Mater Sci concentrations in cancer patients. Biol Trace Elem Res 82:1–8. Eng C Mater Biol Appl 53:239–251. 32. Gaál A, et al. Comparative in vitro investigation of anticancer copper chelating 16. Mohler DL, et al. (2005) DNA cleavage by the photolysis of cyclopentadienyl metal agents. Microchem J, in press. complexes: Mechanistic studies and sequence selectivity of strand scission by 33. Brady DC, et al. (2014) Copper is required for oncogenic BRAF signalling and tu-

CpW(CO)3CH3. J Org Chem 70:9093–9102. morigenesis. Nature 509:492–496.

Porter et al. PNAS | Published online July 31, 2017 | E7413 Downloaded by guest on October 1, 2021 34. Breen AP, Murphy JA (1995) Reactions of oxyl radicals with DNA. Free Radic Biol Med 65. Byers W, Lever ABP, Parish RV (1968) Stereochemistry of amine oxide metal com- 18:1033–1077. plexes. Inorg Chem 7:1835–1840. 35. Goshe MB, Chen YH, Anderson VE (2000) Identification of the sites of hydroxyl radical 66. Balasubramanian S, Krishnan CN (1986) Synthesis and characterization of five- reaction with peptides by hydrogen/deuterium exchange: Prevalence of reactions coordinate macrocyclic complexes of nickel(II) and copper(II). 5:669–675. with the side chains. Biochemistry 39:1761–1770. 67. Kivelson D, Neiman R (1961) ESR [electron spin resonance] studies on the bonding in + 36. Rawat DS, Zaleski JM (2001) Mg2 -induced thermal enediyne cyclization at ambient copper complexes. J Chem Phys 35:149–155. temperature. J Am Chem Soc 123:9675–9676. 68. Manzur J, et al. (2007) Copper(II) complexes with new polypodal ligands presenting axial- 37. Routt SM, Zhu J, Zaleski JM, Dynlacht JR (2011) Potentiation of metalloenediyne cy- equatorial phenoxo bridges 2-[(bis(2-pyridylmethyl)amino)methyl]-4-methylphenol, 2-[(bis(2- totoxicity by hyperthermia. Int J Hyperthermia 27:435–444. pyridylmethyl)amino)methyl]-4-methyl-6-(methylthio)phenol: Examples of ferromagnetically 38. Porter MR, Kochi A, Karty JA, Lim MH, Zaleski JM (2015) Chelation-induced diradical coupled bi- and trinuclear copper(II) complexes. Inorg Chem 46:6924–6932. formation as an approach to modulation of the amyloid-β aggregation pathway. 69. Linss M, Weser U (1986) The di-Schiff-base of pyridine-2-aldehyde and 1,4-dia- Chem Sci (Camb) 6:1018–1026. minobutane, a flexible Cu(I)/Cu(II) chelator of significant superoxide dismutase mi- 39. Socrates G (2004) Infrared and Raman Characteristic Group Frequencies (Wiley, New York), metic activity. Inorganica Chim Acta 125:117–121. 3rdEd,p366. 70. Hathaway BJ (1987) Copper. Comprehensive Coordination Chemistry: Theory and 40. Alex S, Turcotte P, Fournier R, Vocelle D (1991) Study of the protonation of simple Schiff bases Background, ed Wilkinson G (Pergamon, Oxford), Vol 5, pp 533–774. in solvents of various polarity by means of Raman spectroscopy. Can J Chem 69:239–245. 71. Bencini A, Bertini I, Gatteschi D, Scozzafava A (1978) Single-crystal ESR spectra of 41. Huang CY (1982) Determination of binding stoichiometry by the continuous variation copper(II) complexes with geometries intermediate between a square pyramid and a – method: The Job plot. Methods Enzymol 87:509–525. trigonal bipyramid. Inorg Chem 17:3194 3197. 42. Porter MR, Zaleski JM (2016) The role of ligand covalency in the selective activation of 72. Tabbì G, Giuffrida A, Bonomo RP (2013) Determination of formal redox potentials in aqueous metalloenediynes for Bergman cyclization. Polyhedron 103:187–195. solution of copper(II) complexes with ligands having nitrogen and oxygen donor atoms and – 43. Addison AW, Carpenter M, Lau LK-M, Wicholas M (1978) Coordination sphere flexi- comparison with their EPR and UV-Vis spectral features. J Inorg Biochem 128:137 145. + bility at copper: Chemistry of a unipositive copper(II) macrocycle, [Cu(cyclops)] . Inorg 73. Hathaway BJ, Billing DE (1970) Electronic properties and stereochemistry of mono- – Chem 17:1545–1552. nuclear complexes of the copper(II) ion. Coord Chem Rev 5:143 207. 44. Hathaway BJ (1972) Correlation of the electronic properties and stereochemistry of 74. Bhattacharyya A, et al. (2014) A combined experimental and computational study of mononuclear [CuN ] chromophores. J Chem Soc Dalton Trans 1196–1199. supramolecular assemblies in ternary copper(II) complexes with a tetradentate N4 4-6 – 45. Lever ABP (1984) Inorganic Electronic Spectroscopy, Studies in Physical and Theoret- donor Schiff base and halides. RSC Advances 4:58643 58651. ical Chemistry (Elsevier, Amsterdam), 2nd Ed, Vol 33, p 862. 75. Rahaman SH, Fun H-K, Ghosh BK (2005) A study on copper(II)-Schiff base-azide co- 46. Solomon EI (1984) Inorganic spectroscopy–An overview. Comments Inorg Chem 3:238–249. ordination complexes: Synthesis, X-ray structure and luminescence properties of [Cu = = – 47. Godlewska S, Jezierska J, Baranowska K, Augustin E, Dolega A (2013) Copper(II) (L)(N3)]X (L Schiff bases; X ClO4,PF6). Polyhedron 24:3091 3097. complexes with substituted imidazole and chloro ligands: X-ray, UV-Vis, magnetic and 76. Chattopadhyay S, Drew MGB, Ghosh A (2006) Synthesis, characterization, and anion EPR studies and chemotherapeutic potential. Polyhedron 65:288–297. selectivity of copper(II) complexes with a tetradentate Schiff base ligand. Inorganica – 48. McLachlan GA, Fallon GD, Martin RL, Spiccia L (1995) Synthesis, structure and prop- Chim Acta 359:4519 4525. 77. Lange J, Elias H, Paulus H, Müller J, Weser U (2000) Copper(II) and copper(I) complexes with erties of five-coordinate copper(II) complexes of pentadentate ligands with pyridyl an open-chain N Schiff base ligand modeling CuZn superoxide dismutase: Structural and pendant arms. Inorg Chem 34:254–261. 4 3− spectroscopic characterization and kinetics of electron transfer. Inorg Chem 39:3342–3349. 49. Allen GC, Hush NS (1967) Reflectance spectrum and electronic states of the CuCl5 78. Ray MS, et al. (2003) Synthesis, characterisation and X-ray crystal structure of copper(II) com- ion in a number of crystal lattices. Inorg Chem 6:4–8. plexes with unsymmetrical tetradentate Schiff base ligands: First evidence of Cu(II) catalysed 50. DeKock CW, Gruen DM (1966) Electronic absorption spectra of the gaseous 3d rearrangement of unsymmetrical to symmetrical complex. Polyhedron 22:617–624. transition-metal dichlorides. J Chem Phys 44:4387–4398. 79. Cui J-D, Zhong K-L, Liu Y-Y (2010) Bis(1,10-phenanthroline-κN,N′)(sulfato-κO,O′)zinc(II) 51. Desjardins SR, Penfield KW, Cohen SL, Musselman RL, Solomon EI (1983) Detailed propane-1,3-diol solvate. Acta Crystallogr Sect E Struct Rep Online 66:m564. absorption, reflectance, and UV photoelectron spectroscopic and theoretical studies 80. Zhong K-L (2010) Bis(2,2′-bipyridyl-κN,N′)(sulfato-κO,O′)zinc(II) ethane-1,2-diol sol- of the charge-transfer transitions of tetrachlorocuprate(2-) ion: Correlation of the vate. Acta Crystallogr Sect E Struct Rep Online 66:m131. square-planar and the tetrahedral limits. J Am Chem Soc 105:4590–4603. 81. Zhu Y-M, Zhong K-L, Lu W-J (2006) Bis(1,10-phenanthroline-[κ]2N,N′)(sulfato-[κ]2O,O′)zinc(II) 52. Harris CM, Patil HRH, Sinn E (1967) Nitrogenous chelate complexes of transition metals. IV. 1,2-ethenediol solvate. Acta Crystallogr Sect E Struct Rep Online 62:m2725–m2726. Pseudo-tetrahedral copper(II) complexes containing 2,2′-biquinolyl. Inorg Chem 6:1102–1105. 82. Cossi M, Barone V, Cammi R, Tomasi J (1996) Ab initio study of solvated molecules: A new 53. Willett RD, Liles OL, Jr, Michelson C (1967) Electronic absorption spectra of mono- implementation of the polarizable continuum model. Chem Phys Lett 255:327–335. meric copper(II) chloride species and the electron spin resonance spectrum of the 83. Miertus S, Scrocco E, Tomasi J (1981) Electrostatic interaction of a sulte with a continuum. A direct square-planar CuCl 2− ion. Inorg Chem 6:1885–1889. 4 utilization of ab initio molecular potentials for the prevision of solvent. Chem Phys 55:117–129. 54. Bernarducci E, Bharadwaj PK, Krogh-Jespersen K, Potenza JA, Schugar HJ (1983) 84. Miertus S, Tomasi J (1982) Approximate evaluations of the electrostatic free energy Electronic structure of alkylated imidazoles and electronic spectra of tetrakis(imid- and internal energy changes in solution processes. Chem Phys 65:239–245. azole)copper(II) complexes. Molecular structure of tetrakis(1,4,5-trimethylimidazole) 85. Pascual-Ahuir JL, Silla E, Tuñón I (1994) GEPOL: An improved description of molecular – copper(II) diperchlorate. J Am Chem Soc 105:3860 3866. surfaces. III. A new algorithm for the computation of a solvent-excluding surface. 55. Fawcett TG, Bernarducci EE, Krogh-Jespersen K, Schugar HJ (1980) Charge-transfer J Comput Chem 15:1127–1138. absorptions of copper(II)-imidazole and copper(II)-imidazolate chromophores. JAm 86. Tomasi J, Mennucci B, Cammi R (2005) Quantum mechanical continuum solvation – Chem Soc 102:2598 2604. models. Chem Rev 105:2999–3093. 56. Miskowski VM, Thich JA, Solomon R, Schugar HJ (1976) Electronic spectra of 87. Frisch MJ, et al. (2009) Gaussian 09 (Gaussian, Inc., Wallingford, CT). – substituted copper(II) thioether complexes. J Am Chem Soc 98:8344 8350. 88. Becke AD (1988) Density-functional exchange-energy approximation with correct 57. Kaljurand I, Rodima T, Leito I, Koppel IA, Schwesinger R (2000) Self-consistent spec- asymptotic behavior. Phys Rev A Gen Phys 38:3098–3100. trophotometric basicity scale in acetonitrile covering the range between pyridine and 89. Burke K, Perdew JP, Wang Y (1998) Electronic Density Functional Theory: Recent – DBU. J Org Chem 65:6202 6208. Progress and New Directions, Dobson JF, Vignale G, Das MP (Plenum, New York). 58. Zalis S, et al. (2011) Origin of electronic absorption spectra of MLCT-excited and one-electron 90. Perdew JP (1991) Electronic Structure of Solids ’91 (Akademie, Berlin). ′ – reduced 2,2 -bipyridine and 1,10-phenanthroline complexes. Inorganica Chim Acta 374:578 585. 91. Perdew JP, Burke K, Wang Y (1996) Generalized gradient approximation for the exchange- 59. Krumholz P, Serra OA, De Paoli MA (1975) Coordinate bond. VII. Complexes of dii- correlation hole of a many-electron system. Phys Rev B Condens Matter 54:16533–16539. mines with iron. Synthesis and spectral properties. Inorganica Chim Acta 15:25–32. 92. Perdew JP, et al. (1992) Atoms, molecules, solids, and surfaces: Applications of the generalized 60. Mori K, Kagohara K, Yamashita H (2008) Synthesis of tris(2,2′-bipyridine)iron(II) gradient approximation for exchange and correlation. Phys Rev B Condens Matter 46:6671–6687. complexes in zeolite Y cages: Influence of exchanged alkali metal cations on physi- 93. Perdew JP, et al. (1993) Erratum: Atoms, molecules, solids, and surfaces: Applications cochemical properties and catalytic activity. J Phys Chem C 112:2593–2600. of the generalized gradient approximation for exchange and correlation. Phys Rev B 61. Williams RJP (1955) The absorption spectra of some complex ions of analytical im- Condens Matter 48:4978–4978. portance. J Chem Soc 137–145. 94. Andrae D, Häußermann U, Dolg M, Stoll H, Preuß H (1990) Energy-adjusted ab initio pseu- 62. Linert W, Konecny M, Renz F (1994) Spin-state equilibria in non-aqueous solution and dopotentials for the second and third row transition elements. Theor Chim Acta 77:123–141. quantum-mechanical investigations of iron(II) and nickel(II) complexes with 4- 95. Dolg M, Stoll H, Preuss H (1989) Energy-adjusted pseudopotentials for the rare earth substituted 2,6-bis(benzimidazol-2-yl)pyridines. J Chem Soc, Dalton Trans 1523–1531. elements. J Chem Phys 90:1730–1734. 63. Loeb KE, et al. (1995) Spectroscopic definition of the geometric and electronic 96. Dolg M, Stoll H, Savin A, Preuss H (1989) Energy-adjusted pseudopotentials for the structure of the non-heme iron active site in iron(II) Bleomycin: Correlation with ox- rare earth elements. Theor Chim Acta 75:173–194. ygen reactivity. J Am Chem Soc 117:4545–4561. 97. Dolg M, Wedig U, Stoll H, Preuss H (1987) Energy-adjusted ab initio pseudopotentials 64. Bryant GM, Fergusson JE, Powell HKJ (1971) Charge-transfer and intraligand elec- for the first row transition elements. J Chem Phys 86:866–872. tronic spectra of bipyridyl complexes of iron, ruthenium, and osmium. I. Bivalent 98. Dunning TH, Jr, Hay PJ (1977) Modern Theoretical Chemistry, ed Schafer HF (Plenum, complexes. Aust J Chem 24:257–273. New York), pp 1–28.

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