The Cu(II) complexes of cyclen and two of its heterosubstituted analogues were
shown to be efficient oxidative DNA cleavers. The reactivity strongly depends
on the heteroatom inserted into the macrocycle (O > S > N)

Deibel, Naina

Hormann, Jan

Kulak, Nora

Lentz, Dieter

Perera, Chrischani

Sarkar, Biprajit

Institutional Repository of the Freie Universität Berlin

DaltonTransactionsCOMMUNICATIONCite this: Dalton Trans., 2013, 42, 4357Received 28th November 2012,Accepted 30th January 2013DOI: 10.1039/c3dt32857kwww.rsc.org/daltonStraightforward approach to eﬃcient oxidative DNAcleaving agents based on Cu(II) complexes ofheterosubstituted cyclens†Jan Hormann,a Chrischani Perera,a Naina Deibel,a,b Dieter Lentz,a Biprajit Sarkaraand Nora Kulak*aThe Cu(II) complexes of cyclen and two of its heterosubstitutedanalogues were shown to be eﬃcient oxidative DNA cleavers. Thereactivity strongly depends on the heteroatom inserted into themacrocycle (O > S > N).The key to success of organic life is the stability of deoxyribo-nucleic acid (DNA). The half-life of its phosphodiester bondunder physiological conditions is estimated to be in betweenten to one hundred billion years.1 This stability is due to therepulsion of potential nucleophiles by its negatively chargedbackbone. Nevertheless, cleavage of DNA is of high impor-tance: during replication and transcription of DNA the enzymetopoisomerase cleaves one or both of the DNA strands, rever-sing supercoiling and enabling the reading process of DNA.2Restriction enzymes guard the genome against viral DNA bycutting the alien DNA out.3 Manipulation of DNA by mankindremains a big challenge, though. Up to the present developedartificial nucleases can by far not catch up with naturallyoccurring enzymes, although DNA cleaving agents promisenumerous applications in medicine and biotechnology.4Hydrolytically and oxidatively cleaving nucleases can be distin-guished. Whereas the first group may be used to design restric-tion agents in biotechnology that act via hydrolysis of thephosphodiester bond, the latter ones would be suitable as chemo-therapeutic agents cleaving nuclear DNA by catalysing oxi-dation reactions that break down the sugar moiety of DNA.4,5Since their first synthesis in 1961 macrocyclic polyaminessuch as 1,4,7,10-tetraazacyclododecane, the so called cyclenligand or [12]aneN4, have been widely used in bioinorganicand medicinal chemistry due to their ability to form stablecomplexes with transition metals and rare earth metals.6Diﬀerent concepts were used to develop DNA-cleaving agentsbased on the cyclen ligand. Hydrolytically cleaving complexesbased on Zn(II) and Co(III) as well as oxidatively cleaving com-plexes based on Cu(II) were synthesised.4,7–9 It is well knownthat Cu(II) cyclen complexes are able to cleave DNA via an oxi-dative pathway, thus they may represent potential anticancerdrug candidates.10 Recent eﬀorts have focused on further deri-vatisation of the simple Cu(II) cyclen complex to increase theaﬃnity to DNA. This was achieved by introducing DNA-inter-calating agents, adding positively charged groups or synthesisingmultinuclear complexes.7–9 The straightforward approach ofchanging the tetraaza ligand itself, however, by replacing thedonor atoms and its impact on DNA cutting activity has neverbeen studied to our knowledge. This is in contrast to appli-cations in protein and RNA cleavers where the oxygen ana-logue of cyclen is a well established scaﬀold.11 Our approachwould possibly lead to changes in the redox behaviour of thecopper centre, thus resulting in a change of cutting activitydepending on the introduced heteroatom.The three ligands [12]aneN4, [12]aneN3O and [12]aneN3Swere synthesised using previously described protocols.12 Thecorresponding Cu(II) complexes 1, 2 and 3 (Fig. 1) were syn-thesised and characterised (cf. S-1, S-2†). While the crystalstructure of [Cu([12]aneN4)(NO3)](NO3) 1 has already been pub-lished,13 the crystal structures of the nitrato complexes 2 and 3are presented in this communication (Fig. 2). All three com-plexes show a distorted square-pyramidal environment withCu–N bond lengths of nearly 2 Å. The Cu–O bond (2.231 Å) ofcomplex 2 and the Cu–S bond (2.328 Å) of complex 3 are,however, elongated. Yet another diﬀerence exists in the C–N,Fig. 1 Structures of complexes 1, 2 and 3.†Electronic supplementary information (ESI) available: Experimental details andanalytical data. CCDC 911568 and 911569. For ESI and crystallographic data inCIF or other electronic format see DOI: 10.1039/c3dt32857kaInstitut für Chemie und Biochemie, Freie Universität Berlin, Fabeckstr. 34/36,D-14195 Berlin, Germany. E-mail: nora.kulak@fu-berlin.de; Fax: +49 30 838 52440;Tel: +49 30 838 54697bUniversität Stuttgart, Institut für Anorganische Chemie, Pfaﬀenwaldring 55,70569 Stuttgart, GermayThis journal is © The Royal Society of Chemistry 2013 Dalton Trans., 2013, 42, 4357–4360 | 4357Published on 15 February 2013. Downloaded by Freie Universitaet Berlin on 16/03/2015 11:48:42. View Article OnlineView Journal  | View IssueC–O and the C–S bond lengths. While the C–N bond length ofcomplex 1 (1.453 Å) and the C–O bond length of complex 2(1.435 Å) diﬀer only slightly, the C–S bond length of complex 3(1.818 Å) is considerably longer. The hapticity of the nitratoligand of complexes 1 and 3, where it coordinates inη1-fashion with a typical apical Cu–O bond length15 of 2.183 and2.160 Å, respectively, diﬀers from complex 2, where it showsη2-like coordination with Cu–O bond lengths of 2.031 and2.470 Å. Such an anisodentate chelation of nitrate in Cu(II)cyclen analogues has been observed by Spiccia et al.15 before.They have assumed that the long Cu–O bond indicates an elec-trostatic rather than a coordinative interaction.The cleavage activities of 1, 2 and 3 toward pBR322 super-coiled plasmid DNA were studied under approximate physio-logical conditions at 37 °C and ascorbic acid at 0.32 mMconcentration as a reducing agent. Agarose gel electrophoresiswas used to monitor the conversion of supercoiled (I) pBR322into its nicked (II) and linear (III) form. Fig. 3 shows a com-parison between the DNA cleavage activities of the complexes1, 2 and 3.After two hours incubation and at a complex concentrationof 0.04 mM gel electrophoresis shows that complex 2 was morereactive than complexes 1 and 3. Complex 2 cleaved super-coiled pBR322 plasmid DNA almost completely to form II andadditionally it was able to generate about 4% of form III. Com-pared to complex 1, the DNA cleavage eﬃciency of complex 3was two times and of complex 2 even four times higher.Further investigations of the cleaving ability were carried outperforming a series of optimisation experiments includingvariations of concentration, pH value and reaction time. Theconcentration dependent investigation shows that complex 2exhibited an approximately linear relationship betweencomplex concentration and DNA cleavage activity (Fig. 4). At0.08 mM concentration form I DNA was cleaved into thenicked form almost completely. As opposed to that complexes1 and 3 did not cleave plasmid DNA to that extent and did notshow a linear relationship between complex concentration andcleavage activity. In contrast the Cu([12]aneN4) complex 1shows a maximum in cutting activity at 0.02 mM and theCu([12]aneN3S) complex 3 at 0.16 mM. After passing these con-centrations cleavage activities of 1 and 3 decrease. A similarbehaviour has been described for Cu([9]aneN3) complexesprior to our study by Burstyn et al. and was attributed to theformation of bis-(μ-hydroxo)-bridged dimers.16 As hydroxo-bridged dimer formation should also be favoured by a higherhydroxide concentration we conducted a pH dependent study,proving that the DNA cleavage activity decreases with increas-ing pH values (cf. S-3,2†).Furthermore, the eﬀect of reaction time and temperatureon the cleavage of pBR322 plasmid DNA was studied. Increas-ing DNA cleavage activity was observed when both temperatureand reaction time were raised (cf. S-3,3 and S-3,4†).To elucidate the mechanism of DNA cleavage promoted bythe complexes 1, 2 and 3 the incubation was conducted underan argon atmosphere and under addition of scavengers forreactive oxygen species (ROS) like hydroxyl radicals (t-BuOHand DMSO),17 singlet oxygen (NaN3),18 hydrogen peroxide (cata-lase)19 and superoxide (superoxide dismutase, SOD).18 Underanaerobic conditions cleavage was reduced, indicating thatoxygen plays a role in the cleavage mechanism (Fig. 5A). In thecase of 2 even traces of oxygen are enough to promote DNAcleavage indicating a high catalytic activity of 2. As shownexemplarily for complex 3 in part B of Fig. 5 DMSO and cata-lase show a strong and NaN3 a weak inhibition eﬀect on DNAcleavage. These results prove the mechanism of DNA cleavageto be oxidative involving hydroxyl radicals, hydrogen peroxideand to some extent singlet oxygen as ROS.To gain further insight into the redox reactions involvedelectrochemical studies were carried out. Complexes 1–3display a one-electron reduction wave in their cyclicFig. 2 ORTEP14 diagrams of the molecular structure of 2 (left) and 3 (right).Thermal ellipsoids are drawn at a probability level of 50%. The counter ions areomitted for clarity.‡Fig. 3 Cleavage activities of complexes 1, 2 and 3 (0.04 mM) on pBR322(0.025 μg μL−1) in Tris-HCl buﬀer (100 mM, pH 7.4) and ascorbic acid (0.32 mM)at 37 °C for 2 h. Illustrated is the average of two measurements, the standarddeviation is shown as error bars.Communication Dalton Transactions4358 | Dalton Trans., 2013, 42, 4357–4360 This journal is © The Royal Society of Chemistry 2013Published on 15 February 2013. Downloaded by Freie Universitaet Berlin on 16/03/2015 11:48:42. View Article Onlinevoltammogram (cf. S-4,1†). This process is attributed to thereduction of Cu(II) to Cu(I). Whereas this wave is reversible for3, it is electrochemically irreversible for 1 and 2. The irreversi-bility of this wave is attributed to the release of Cu(I) from thecyclen ligands after reduction. This phenomenon has beenobserved earlier20 and in the present case, proof comes fromscanning the cyclic voltammograms multiple times, whichshows an increase in the intensity of the wave attributed toredox processes at the released copper centre (cf. S-4,2†). Thereversible nature of the reduction step for 3 is readily explainedby the higher aﬃnity of Cu(I) for a sulphur donor as for anoxygen or a nitrogen donor. As would be expected, 2 is reducedat a lower negative potential compared to 1, an observationthat is related to the higher electronegativity of “O” comparedto “N”. The reduction of 3 at even lower negative potentialthan 2 is attributed to the better π-donor ability of oxygen com-pared to sulphur. Such a trend has precedence in theliterature.21The release of Cu(I) from macrocycles 1 and 2 as suggestedby the cyclovoltammetric results implies that the free Cu(I)/Cu(II)could be the reactive species for the generation of ROS.22 Thismight explain the high reactivity of complex 2 despite theunfavourable potential for reduction in comparison to com-plexes 1 and 3. Electrochemistry, however, displays an outer-sphere electron transfer process, whereas the reduction of O2generating ROS must be attributed to inner sphere electronFig. 4 Eﬀect of diﬀerent concentrations of complexes 1, 2 and 3 on pBR322 (0.025 μg μL−1) cleavage activity in Tris-HCl buﬀer (100 mM, pH 7.4) and ascorbic acid(0.32 mM) at 37 °C for 2 h. Illustrated is the average of two measurements, the standard deviation is shown as error bars (top). As an example the agarose gel ofcomplex 2 is shown (bottom). See ESI† for gels of complexes 1 and 3 (cf. S-3,1).Fig. 5 Cleavage of pBR322 plasmid DNA (0.025 μg μL−1) in Tris-HCl buﬀer (100 mM, pH 7.4) and ascorbic acid (0.32 mM) for 2 h at 37 °C. The average of twomeasurements is illustrated, the standard deviation is shown as error bars. (A) Cleavage by complexes 1, 2 and 3 (0.04 mM) under aerobic and anaerobic conditions.As an example the agarose gel of complex 1 is shown (bottom). (B) Cleavage by complex 3 in the presence of ROS scavengers. See ESI† for a complete overview ofgels (cf. S-3,5 and S-3,6).Dalton Transactions CommunicationThis journal is © The Royal Society of Chemistry 2013 Dalton Trans., 2013, 42, 4357–4360 | 4359Published on 15 February 2013. Downloaded by Freie Universitaet Berlin on 16/03/2015 11:48:42. View Article Onlinetransfer.23 Therefore, activity in DNA cleavage does not necess-arily correlate with redox potentials. The attenuated cleavageactivity of 1 might be explained by hydroxo-bridged dimer for-mation16 which blocks the coordination sites of Cu(II) leavingno space for coordination of reducing agents, thus inhibitingthe reduction process and the release of Cu(I).In conclusion, we present a series of simple macrocyclicligands for Cu(II)-based DNA cleavers. In contrast to othercyclen-based systems, where activity is increased by conju-gation with moieties enhancing DNA aﬃnity, activity increaseis achieved here by simply changing one out of four donoratoms in the system. Cu(II) oxacyclen is the most eﬃcientcomplex that exceeds the literature-known system Cu(II) cyclenby a factor of four to ten depending on the concentration. Thehigh reactivity of this derivative might be a good starting pointfor developing more eﬃcient DNA cleavers for potential medi-cinal applications.AcknowledgementsWe thank Dr Kai Licha from mivenion GmbH for generousdonations of cyclen.Notes and references‡Crystal data for 2: C8H19CuN5O7, M = 360.82, monoclinic, a = 7.921(19) Å, b =11.979(3) Å, c = 14.757(3) Å, α = 90°, β = 92.587(5)°, γ = 90°, V = 1398.9(6) Å3, T =133(2) K, space group P21/n, Z = 4, μ(MoKα) = 1.604 mm−1, 17 378 reflectionsmeasured, 4238 independent reflections (Rint = 0.0253). The final R1 value was0.0290 (I > 2σ(I)). The final wR2 value was 0.0683 (I > 2σ(I)). The final R1 valuewas 0.0399 (all data). The final wR2 value was 0.0731 (all data). The goodness offit on F2 was 1.089.Crystal data for 3: C8H19CuN5O6S, M = 376.88, monoclinic, a = 8.532(16) Å, b =11.903(2) Å, c = 14.168(3) Å, α = 90°, β = 94.368 (4)°, γ = 90°, V = 1434.7(5) Å3, T =133(2) K, space group P21/c, Z = 4, μ(MoKα) = 1.703 mm−1, 17 105 reflectionsmeasured, 4381 independent reflections (Rint = 0.0143). The final R1 value was0.0241 (I > 2σ(I)). The final wR2 value was 0.0639 (I > 2σ(I)). The final R1 valuewas 0.0264 (all data). The final wR2 value was 0.0649 (all data). The goodness offit on F2 was 1.129.1 F. H. Westheimer, Science, 1987, 235, 1173.2 J. C. Wang, Nat. Rev. Mol. Cell Biol., 2002, 3, 430.3 H. W. Boyer, Annu. Rev. Microbiol., 1971, 25, 153.4 (a) F. Mancin, P. Scrimin, P. Tecilla and U. Tonellato, Chem.Commun., 2005, 2540; (b) F. Mancin, P. Scrimin andP. Tecilla, Chem. Commun., 2012, 48, 5545.5 W. Knapp Pogozelski and T. D. Tullius, Chem. Rev., 1998,98, 1089.6 (a) H. Stetter and K.-H. Mayer, Chem. Ber., 1961, 94, 1410;(b) J. E. Richman and T. J. Atkins, J. Am. Chem. Soc., 1974,96, 2268; (c) M. Suchý and R. H. E. Hudson, Eur. J. Org.Chem., 2008, 4847.7 (a) J. Li, Y. Yue, J. Zhang, Q.-S. Lu, K. Li, Y. Huang,Z.-W. Zhang, H.-H. Lin, N. Wang and X.-Q. Yu, TransitionMet. Chem., 2008, 33, 759; (b) Q. Liu, J. Zhang, M.-Q. Wang,D.-W. Zhang, Q.-S. Lu, Y. Huang, H.-H. Lin and X.-Q. Yu,Eur. J. Med. Chem., 2010, 45, 5302.8 Q.-L. Li, J. Huang, Q. Wang, N. Jiang, C.-Q. Xia,H.-H. Lin, J. Wua and X.-Q. Yu, Bioorg. Med. Chem.,2006, 14, 4151.9 (a) A. Bencini, E. Berni, A. Bianchi, C. Giorgi, B. Valtancoli,D. K. Chand and H.-J. Schneider, Dalton Trans., 2003, 793;(b) Y. Huang, S.-Y. Chen, J. Zhang, X.-Y. Tan, N. Jiang,J.-J. Zhang, Y. Zhang, H.-H. Lin and X.-Q. Yu, Chem. Bio-divers., 2009, 6, 475.10 J. Stubbe and J. W. Kozarich, Chem. Rev., 1987, 87, 1107.11 (a) S. W. Jang and J. Suh, Org. Lett., 2008, 10, 481;(b) C. S. Rossiter, R. A. Mathews and J. R. Morrow, Inorg.Chem., 2005, 44, 9397.12 See ESI† for references of ligand synthesis.13 R. Clay, P. Murray-Rust and J. Murray-Rust, Acta Cryst. B,1979, 35, 1894.14 L. J. Farrugia, J. Appl. Crystallogr., 1997, 30, 565.15 G. Gasser, M. J. Belousoﬀ, A. M. Bond and L. Spiccia, Inorg.Chem., 2007, 46, 3876.16 K. M. Deck, T. A. Tseng and J. N. Burstyn, Inorg. Chem.,2002, 41, 669.17 E. L. Hegg and J. N. Burstyn, Inorg. Chem., 1996, 35,7474.18 A. Sreedhara, J. D. Freed and J. A. Cowan, J. Am. Chem. Soc.,2000, 122, 8814.19 C. A. Detmer III, F. V. Pamatong and J. R. Bocarsly, Inorg.Chem., 1996, 35, 6292.20 L. M. P. Lima, D. Esteban-Gómez, R. Delgado, C. Platas-Iglesias and R. Tripier, Inorg. Chem., 2012, 51, 6916.21 (a) G. A. Fox and C. G. Pierpont, Inorg. Chem., 1992, 31,3718; (b) K. Ray, T. Weyhermüller, F. Neese andK. Wieghardt, Inorg. Chem., 2005, 44, 5345.22 S.-H. Chiou, J. Biochem., 1983, 94, 1259.23 J. Schnödt, M. Sieger, B. Sarkar, S. Strobel, J. Fiedler,J. S. Manzur, C.-Y. Su and W. Kaim, Z. Anorg. Allg. Chem.,2011, 637, 930.Communication Dalton Transactions4360 | Dalton Trans., 2013, 42, 4357–4360 This journal is © The Royal Society of Chemistry 2013Published on 15 February 2013. Downloaded by Freie Universitaet Berlin on 16/03/2015 11:48:42. View Article Online

Straightforward approach to efficient oxidative DNA cleaving agents based on
Cu(II) complexes of heterosubstituted cyclens

DaltonTransactionsCOMMUNICATIONCite this: Dalton Trans., 2013, 42, 4357Received 28th November 2012,Accepted 30th January 2013DOI: 10.1039/c3dt32857kwww.rsc.org/daltonStraightforward approach to eﬃcient oxidative DNAcleaving agents based on Cu(II) complexes ofheterosubstituted cyclens†Jan Hormann,a Chrischani Perera,a Naina Deibel,a,b Dieter Lentz,a Biprajit Sarkaraand Nora Kulak*aThe Cu(II) complexes of cyclen and two of its heterosubstitutedanalogues were shown to be eﬃcient oxidative DNA cleavers. Thereactivity strongly depends on the heteroatom inserted into themacrocycle (O > S > N).The key to success of organic life is the stability of deoxyribo-nucleic acid (DNA). The half-life of its phosphodiester bondunder physiological conditions is estimated to be in betweenten to one hundred billion years.1 This stability is due to therepulsion of potential nucleophiles by its negatively chargedbackbone. Nevertheless, cleavage of DNA is of high impor-tance: during replication and transcription of DNA the enzymetopoisomerase cleaves one or both of the DNA strands, rever-sing supercoiling and enabling the reading process of DNA.2Restriction enzymes guard the genome against viral DNA bycutting the alien DNA out.3 Manipulation of DNA by mankindremains a big challenge, though. Up to the present developedartificial nucleases can by far not catch up with naturallyoccurring enzymes, although DNA cleaving agents promisenumerous applications in medicine and biotechnology.4Hydrolytically and oxidatively cleaving nucleases can be distin-guished. Whereas the first group may be used to design restric-tion agents in biotechnology that act via hydrolysis of thephosphodiester bond, the latter ones would be suitable as chemo-therapeutic agents cleaving nuclear DNA by catalysing oxi-dation reactions that break down the sugar moiety of DNA.4,5Since their first synthesis in 1961 macrocyclic polyaminessuch as 1,4,7,10-tetraazacyclododecane, the so called cyclenligand or [12]aneN4, have been widely used in bioinorganicand medicinal chemistry due to their ability to form stablecomplexes with transition metals and rare earth metals.6Diﬀerent concepts were used to develop DNA-cleaving agentsbased on the cyclen ligand. Hydrolytically cleaving complexesbased on Zn(II) and Co(III) as well as oxidatively cleaving com-plexes based on Cu(II) were synthesised.4,7–9 It is well knownthat Cu(II) cyclen complexes are able to cleave DNA via an oxi-dative pathway, thus they may represent potential anticancerdrug candidates.10 Recent eﬀorts have focused on further deri-vatisation of the simple Cu(II) cyclen complex to increase theaﬃnity to DNA. This was achieved by introducing DNA-inter-calating agents, adding positively charged groups or synthesisingmultinuclear complexes.7–9 The straightforward approach ofchanging the tetraaza ligand itself, however, by replacing thedonor atoms and its impact on DNA cutting activity has neverbeen studied to our knowledge. This is in contrast to appli-cations in protein and RNA cleavers where the oxygen ana-logue of cyclen is a well established scaﬀold.11 Our approachwould possibly lead to changes in the redox behaviour of thecopper centre, thus resulting in a change of cutting activitydepending on the introduced heteroatom.The three ligands [12]aneN4, [12]aneN3O and [12]aneN3Swere synthesised using previously described protocols.12 Thecorresponding Cu(II) complexes 1, 2 and 3 (Fig. 1) were syn-thesised and characterised (cf. S-1, S-2†). While the crystalstructure of [Cu([12]aneN4)(NO3)](NO3) 1 has already been pub-lished,13 the crystal structures of the nitrato complexes 2 and 3are presented in this communication (Fig. 2). All three com-plexes show a distorted square-pyramidal environment withCu–N bond lengths of nearly 2 Å. The Cu–O bond (2.231 Å) ofcomplex 2 and the Cu–S bond (2.328 Å) of complex 3 are,however, elongated. Yet another diﬀerence exists in the C–N,Fig. 1 Structures of complexes 1, 2 and 3.†Electronic supplementary information (ESI) available: Experimental details andanalytical data. CCDC 911568 and 911569. For ESI and crystallographic data inCIF or other electronic format see DOI: 10.1039/c3dt32857kaInstitut für Chemie und Biochemie, Freie Universität Berlin, Fabeckstr. 34/36,D-14195 Berlin, Germany. E-mail: nora.kulak@fu-berlin.de; Fax: +49 30 838 52440;Tel: +49 30 838 54697bUniversität Stuttgart, Institut für Anorganische Chemie, Pfaﬀenwaldring 55,70569 Stuttgart, GermayThis journal is © The Royal Society of Chemistry 2013 Dalton Trans., 2013, 42, 4357–4360 | 4357Published on 15 February 2013. Downloaded by Freie Universitaet Berlin on 24/03/2015 13:23:13. View Article OnlineView Journal  | View IssueC–O and the C–S bond lengths. While the C–N bond length ofcomplex 1 (1.453 Å) and the C–O bond length of complex 2(1.435 Å) diﬀer only slightly, the C–S bond length of complex 3(1.818 Å) is considerably longer. The hapticity of the nitratoligand of complexes 1 and 3, where it coordinates inη1-fashion with a typical apical Cu–O bond length15 of 2.183 and2.160 Å, respectively, diﬀers from complex 2, where it showsη2-like coordination with Cu–O bond lengths of 2.031 and2.470 Å. Such an anisodentate chelation of nitrate in Cu(II)cyclen analogues has been observed by Spiccia et al.15 before.They have assumed that the long Cu–O bond indicates an elec-trostatic rather than a coordinative interaction.The cleavage activities of 1, 2 and 3 toward pBR322 super-coiled plasmid DNA were studied under approximate physio-logical conditions at 37 °C and ascorbic acid at 0.32 mMconcentration as a reducing agent. Agarose gel electrophoresiswas used to monitor the conversion of supercoiled (I) pBR322into its nicked (II) and linear (III) form. Fig. 3 shows a com-parison between the DNA cleavage activities of the complexes1, 2 and 3.After two hours incubation and at a complex concentrationof 0.04 mM gel electrophoresis shows that complex 2 was morereactive than complexes 1 and 3. Complex 2 cleaved super-coiled pBR322 plasmid DNA almost completely to form II andadditionally it was able to generate about 4% of form III. Com-pared to complex 1, the DNA cleavage eﬃciency of complex 3was two times and of complex 2 even four times higher.Further investigations of the cleaving ability were carried outperforming a series of optimisation experiments includingvariations of concentration, pH value and reaction time. Theconcentration dependent investigation shows that complex 2exhibited an approximately linear relationship betweencomplex concentration and DNA cleavage activity (Fig. 4). At0.08 mM concentration form I DNA was cleaved into thenicked form almost completely. As opposed to that complexes1 and 3 did not cleave plasmid DNA to that extent and did notshow a linear relationship between complex concentration andcleavage activity. In contrast the Cu([12]aneN4) complex 1shows a maximum in cutting activity at 0.02 mM and theCu([12]aneN3S) complex 3 at 0.16 mM. After passing these con-centrations cleavage activities of 1 and 3 decrease. A similarbehaviour has been described for Cu([9]aneN3) complexesprior to our study by Burstyn et al. and was attributed to theformation of bis-(μ-hydroxo)-bridged dimers.16 As hydroxo-bridged dimer formation should also be favoured by a higherhydroxide concentration we conducted a pH dependent study,proving that the DNA cleavage activity decreases with increas-ing pH values (cf. S-3,2†).Furthermore, the eﬀect of reaction time and temperatureon the cleavage of pBR322 plasmid DNA was studied. Increas-ing DNA cleavage activity was observed when both temperatureand reaction time were raised (cf. S-3,3 and S-3,4†).To elucidate the mechanism of DNA cleavage promoted bythe complexes 1, 2 and 3 the incubation was conducted underan argon atmosphere and under addition of scavengers forreactive oxygen species (ROS) like hydroxyl radicals (t-BuOHand DMSO),17 singlet oxygen (NaN3),18 hydrogen peroxide (cata-lase)19 and superoxide (superoxide dismutase, SOD).18 Underanaerobic conditions cleavage was reduced, indicating thatoxygen plays a role in the cleavage mechanism (Fig. 5A). In thecase of 2 even traces of oxygen are enough to promote DNAcleavage indicating a high catalytic activity of 2. As shownexemplarily for complex 3 in part B of Fig. 5 DMSO and cata-lase show a strong and NaN3 a weak inhibition eﬀect on DNAcleavage. These results prove the mechanism of DNA cleavageto be oxidative involving hydroxyl radicals, hydrogen peroxideand to some extent singlet oxygen as ROS.To gain further insight into the redox reactions involvedelectrochemical studies were carried out. Complexes 1–3display a one-electron reduction wave in their cyclicFig. 2 ORTEP14 diagrams of the molecular structure of 2 (left) and 3 (right).Thermal ellipsoids are drawn at a probability level of 50%. The counter ions areomitted for clarity.‡Fig. 3 Cleavage activities of complexes 1, 2 and 3 (0.04 mM) on pBR322(0.025 μg μL−1) in Tris-HCl buﬀer (100 mM, pH 7.4) and ascorbic acid (0.32 mM)at 37 °C for 2 h. Illustrated is the average of two measurements, the standarddeviation is shown as error bars.Communication Dalton Transactions4358 | Dalton Trans., 2013, 42, 4357–4360 This journal is © The Royal Society of Chemistry 2013Published on 15 February 2013. Downloaded by Freie Universitaet Berlin on 24/03/2015 13:23:13. View Article Onlinevoltammogram (cf. S-4,1†). This process is attributed to thereduction of Cu(II) to Cu(I). Whereas this wave is reversible for3, it is electrochemically irreversible for 1 and 2. The irreversi-bility of this wave is attributed to the release of Cu(I) from thecyclen ligands after reduction. This phenomenon has beenobserved earlier20 and in the present case, proof comes fromscanning the cyclic voltammograms multiple times, whichshows an increase in the intensity of the wave attributed toredox processes at the released copper centre (cf. S-4,2†). Thereversible nature of the reduction step for 3 is readily explainedby the higher aﬃnity of Cu(I) for a sulphur donor as for anoxygen or a nitrogen donor. As would be expected, 2 is reducedat a lower negative potential compared to 1, an observationthat is related to the higher electronegativity of “O” comparedto “N”. The reduction of 3 at even lower negative potentialthan 2 is attributed to the better π-donor ability of oxygen com-pared to sulphur. Such a trend has precedence in theliterature.21The release of Cu(I) from macrocycles 1 and 2 as suggestedby the cyclovoltammetric results implies that the free Cu(I)/Cu(II)could be the reactive species for the generation of ROS.22 Thismight explain the high reactivity of complex 2 despite theunfavourable potential for reduction in comparison to com-plexes 1 and 3. Electrochemistry, however, displays an outer-sphere electron transfer process, whereas the reduction of O2generating ROS must be attributed to inner sphere electronFig. 4 Eﬀect of diﬀerent concentrations of complexes 1, 2 and 3 on pBR322 (0.025 μg μL−1) cleavage activity in Tris-HCl buﬀer (100 mM, pH 7.4) and ascorbic acid(0.32 mM) at 37 °C for 2 h. Illustrated is the average of two measurements, the standard deviation is shown as error bars (top). As an example the agarose gel ofcomplex 2 is shown (bottom). See ESI† for gels of complexes 1 and 3 (cf. S-3,1).Fig. 5 Cleavage of pBR322 plasmid DNA (0.025 μg μL−1) in Tris-HCl buﬀer (100 mM, pH 7.4) and ascorbic acid (0.32 mM) for 2 h at 37 °C. The average of twomeasurements is illustrated, the standard deviation is shown as error bars. (A) Cleavage by complexes 1, 2 and 3 (0.04 mM) under aerobic and anaerobic conditions.As an example the agarose gel of complex 1 is shown (bottom). (B) Cleavage by complex 3 in the presence of ROS scavengers. See ESI† for a complete overview ofgels (cf. S-3,5 and S-3,6).Dalton Transactions CommunicationThis journal is © The Royal Society of Chemistry 2013 Dalton Trans., 2013, 42, 4357–4360 | 4359Published on 15 February 2013. Downloaded by Freie Universitaet Berlin on 24/03/2015 13:23:13. View Article Onlinetransfer.23 Therefore, activity in DNA cleavage does not necess-arily correlate with redox potentials. The attenuated cleavageactivity of 1 might be explained by hydroxo-bridged dimer for-mation16 which blocks the coordination sites of Cu(II) leavingno space for coordination of reducing agents, thus inhibitingthe reduction process and the release of Cu(I).In conclusion, we present a series of simple macrocyclicligands for Cu(II)-based DNA cleavers. In contrast to othercyclen-based systems, where activity is increased by conju-gation with moieties enhancing DNA aﬃnity, activity increaseis achieved here by simply changing one out of four donoratoms in the system. Cu(II) oxacyclen is the most eﬃcientcomplex that exceeds the literature-known system Cu(II) cyclenby a factor of four to ten depending on the concentration. Thehigh reactivity of this derivative might be a good starting pointfor developing more eﬃcient DNA cleavers for potential medi-cinal applications.AcknowledgementsWe thank Dr Kai Licha from mivenion GmbH for generousdonations of cyclen.Notes and references‡Crystal data for 2: C8H19CuN5O7, M = 360.82, monoclinic, a = 7.921(19) Å, b =11.979(3) Å, c = 14.757(3) Å, α = 90°, β = 92.587(5)°, γ = 90°, V = 1398.9(6) Å3, T =133(2) K, space group P21/n, Z = 4, μ(MoKα) = 1.604 mm−1, 17 378 reflectionsmeasured, 4238 independent reflections (Rint = 0.0253). The final R1 value was0.0290 (I > 2σ(I)). The final wR2 value was 0.0683 (I > 2σ(I)). The final R1 valuewas 0.0399 (all data). The final wR2 value was 0.0731 (all data). The goodness offit on F2 was 1.089.Crystal data for 3: C8H19CuN5O6S, M = 376.88, monoclinic, a = 8.532(16) Å, b =11.903(2) Å, c = 14.168(3) Å, α = 90°, β = 94.368 (4)°, γ = 90°, V = 1434.7(5) Å3, T =133(2) K, space group P21/c, Z = 4, μ(MoKα) = 1.703 mm−1, 17 105 reflectionsmeasured, 4381 independent reflections (Rint = 0.0143). The final R1 value was0.0241 (I > 2σ(I)). The final wR2 value was 0.0639 (I > 2σ(I)). The final R1 valuewas 0.0264 (all data). The final wR2 value was 0.0649 (all data). The goodness offit on F2 was 1.129.1 F. H. Westheimer, Science, 1987, 235, 1173.2 J. C. Wang, Nat. Rev. Mol. Cell Biol., 2002, 3, 430.3 H. W. Boyer, Annu. Rev. Microbiol., 1971, 25, 153.4 (a) F. Mancin, P. Scrimin, P. Tecilla and U. Tonellato, Chem.Commun., 2005, 2540; (b) F. Mancin, P. Scrimin andP. Tecilla, Chem. Commun., 2012, 48, 5545.5 W. Knapp Pogozelski and T. D. Tullius, Chem. Rev., 1998,98, 1089.6 (a) H. Stetter and K.-H. Mayer, Chem. Ber., 1961, 94, 1410;(b) J. E. Richman and T. J. Atkins, J. Am. Chem. Soc., 1974,96, 2268; (c) M. Suchý and R. H. E. Hudson, Eur. J. Org.Chem., 2008, 4847.7 (a) J. Li, Y. Yue, J. Zhang, Q.-S. Lu, K. Li, Y. Huang,Z.-W. Zhang, H.-H. Lin, N. Wang and X.-Q. Yu, TransitionMet. Chem., 2008, 33, 759; (b) Q. Liu, J. Zhang, M.-Q. Wang,D.-W. Zhang, Q.-S. Lu, Y. Huang, H.-H. Lin and X.-Q. Yu,Eur. J. Med. Chem., 2010, 45, 5302.8 Q.-L. Li, J. Huang, Q. Wang, N. Jiang, C.-Q. Xia,H.-H. Lin, J. Wua and X.-Q. Yu, Bioorg. Med. Chem.,2006, 14, 4151.9 (a) A. Bencini, E. Berni, A. Bianchi, C. Giorgi, B. Valtancoli,D. K. Chand and H.-J. Schneider, Dalton Trans., 2003, 793;(b) Y. Huang, S.-Y. Chen, J. Zhang, X.-Y. Tan, N. Jiang,J.-J. Zhang, Y. Zhang, H.-H. Lin and X.-Q. Yu, Chem. Bio-divers., 2009, 6, 475.10 J. Stubbe and J. W. Kozarich, Chem. Rev., 1987, 87, 1107.11 (a) S. W. Jang and J. Suh, Org. Lett., 2008, 10, 481;(b) C. S. Rossiter, R. A. Mathews and J. R. Morrow, Inorg.Chem., 2005, 44, 9397.12 See ESI† for references of ligand synthesis.13 R. Clay, P. Murray-Rust and J. Murray-Rust, Acta Cryst. B,1979, 35, 1894.14 L. J. Farrugia, J. Appl. Crystallogr., 1997, 30, 565.15 G. Gasser, M. J. Belousoﬀ, A. M. Bond and L. Spiccia, Inorg.Chem., 2007, 46, 3876.16 K. M. Deck, T. A. Tseng and J. N. Burstyn, Inorg. Chem.,2002, 41, 669.17 E. L. Hegg and J. N. Burstyn, Inorg. Chem., 1996, 35,7474.18 A. Sreedhara, J. D. Freed and J. A. Cowan, J. Am. Chem. Soc.,2000, 122, 8814.19 C. A. Detmer III, F. V. Pamatong and J. R. Bocarsly, Inorg.Chem., 1996, 35, 6292.20 L. M. P. Lima, D. Esteban-Gómez, R. Delgado, C. Platas-Iglesias and R. Tripier, Inorg. Chem., 2012, 51, 6916.21 (a) G. A. Fox and C. G. Pierpont, Inorg. Chem., 1992, 31,3718; (b) K. Ray, T. Weyhermüller, F. Neese andK. Wieghardt, Inorg. Chem., 2005, 44, 5345.22 S.-H. Chiou, J. Biochem., 1983, 94, 1259.23 J. Schnödt, M. Sieger, B. Sarkar, S. Strobel, J. Fiedler,J. S. Manzur, C.-Y. Su and W. Kaim, Z. Anorg. Allg. Chem.,2011, 637, 930.Communication Dalton Transactions4360 | Dalton Trans., 2013, 42, 4357–4360 This journal is © The Royal Society of Chemistry 2013Published on 15 February 2013. Downloaded by Freie Universitaet Berlin on 24/03/2015 13:23:13. View Article Online

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Straightforward approach to efficient oxidative DNA cleaving agents based on Cu(II) complexes of heterosubstituted cyclens

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