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Cite this: Dalton Trans., 2015, 44, 6715 Received 9th January 2015, Accepted 11th March 2015
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Mesityl phenanthroline-modified 2’-deoxyuridine for heteroleptic complexes in metal ion-mediated assembly of DNA† Philipp Ensslen and Hans-Achim Wagenknecht*
DOI: 10.1039/c5dt00100e www.rsc.org/dalton
The synthesis of a new DNA building block that bears the metal ion ligand 2,9-bis-mesityl-3-ethynyl-phenanthroline attached to the 5-position of 2’-deoxyuridine is presented. In the presence of Zn2+, Cu2+, Fe2+ and Ni2+ the complex formation of an accordingly modified DNA double strand with a second DNA duplex bearing the 2,2’:6’,2’’-terpyridine ligand was studied by optical spectroscopy. The selective formation of heteroleptic assemblies between the two different DNA pieces was evidenced by denaturing polyacrylamide gel electrophoresis.
DNA with its intrinsic self-assembling properties is not only suitable for the sequence dependent arrangements of two- and three-dimensional DNA objects and origami,1–3 but also a promising scaffold for supramolecular architectures. For the latter purpose it is crucial to develop additional binding motifs, especially with metal ion-binding ligands that allow increasing the complexity and structural variety.4 A broad variety of DNA building blocks has been synthesized for metal ion-mediated base pairing (inside the DNA duplex).5,6 Fewer examples have been published for metal ion-mediated DNA assembly (outside the duplex).4,5 In particular 2,2′:6′,2″-terpyridine (terpy) as a ligand attached to the termini of DNA double strands via flexible alkyl linkers allows the formation of stable complexes with different metal ions and thereby astonishing three-dimensional DNA constructs.4,7,8 Recently, we9 and Hocek with coworkers10 showed that the terpy ligand attached to the 5-position of 2′-deoxyuridine via the rigid acetylene bridge yields metal-mediated DNA assembly of short strands, both “side by side” and “end on end”. However, the application of only a single type of ligand, like terpy, as DNA modification (TP) only allows the formation of homoleptic metal ion
Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany. E-mail: [email protected]; Fax: +49-721-608-44825; Tel: +49-721-608-47486 † Electronic supplementary information (ESI) available: Materials and methods, synthesis and characterization of 3, DNA1 and DNA2, images of NMR and MS spectra and HPLC analyses, and the procedure for the preparation of metal ionmediated DNA assemblies. See DOI: 10.1039/c5dt00100e
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complexes and accordingly mediates DNA assemblies, and thereby significantly limits the control of stepwise DNA assembly to larger objects.4,8 Hence, in this bottom-up approach it is highly desirable to develop DNA modifications for heteroleptic metal ion complexes in order to predictably control the assembly of metal ion-mediated DNA constructs.11 A first attempt for the formation of heteroleptic metal ion complexes in DNA was presented by Sleiman and coworkers.12 Their combination of phenanthroline- and terpy-modified complementary DNA strands gives stable heteroleptic complexes. The DNA assembly to homoleptic and heteroleptic complexes was mainly controlled by the type of metal ion and not by ligand interactions. Schmittel and coworkers published a concept that they called HETTAP (heteroleptic terpyridine and phenanthroline complex formation). Mesityl substituents at the 2- and 9-positions of phenanthroline exclusively lead to the formation of kinetically labile heteroleptic complexes with the terpy ligand in the presence of a variety of metal ions.13 The sterically demanding mesityl substituents not only inhibit the homoleptic complex formation by the phenanthroline ligands but also induce π–π interactions with the terpy ligand (Fig. 1a). In order to extend this concept to aqueous solutions and apply it to supramolecular DNA chemistry, we present herein the synthesis of a new DNA building block MP that bears the bis-2,9-mesityl-substituted phenanthroline attached to the 5-position of 2′-deoxyuridine via an acetylene bridge (Fig. 1b). Furthermore, this DNA building block of MP and the previously described terpy building block TP were used to prepare two single modified oligonucleotides DNA1 and DNA2 (Fig. 1c), respectively. The selective assembly of the corresponding two DNA double strands by heteroleptic complex formation was demonstrated and studied in the presence of four different metal ions. The 2,9-bis-mesityl-3-ethynyl-phenanthroline 1 (Scheme 1), as an important precursor for the new DNA building block MP, was synthesized according to the literature.14 The phenanthroline 1 was attached to 5′-DMT protected 5-iodo-2′-deoxyuridine via a palladium-catalyzed Sonogashira-type reaction (60% yield). The final steps to the DNA building block 3 followed standard phosphoramidite chemistry. Subsequently, we
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Fig. 1 (a) Concept of heteroleptic metal ion–ligand complex formation, (b) DNA building blocks MP and TP, and (c) sequences of DNA1 and DNA2.
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and Fe2+ by MALDI MS. The recorded MS spectra (see ESI†) exclusively show the mass peaks of the heteroleptic complexes MP-M2+-TP and no corresponding peaks of the homoleptic complexes. The metal ion complex formation between the two duplexes (DNA1 and DNA2) was probed in the presence of their fully complementary counterstrands to avoid undesired metal–ion interactions or complexation with the DNA bases. Hence, both double strands were annealed separately. Subsequently, metal ions and afterwards DNA1 were added to DNA2 in aqueous buffer and incubated for 30 min at room temperature. The melting temperature of both duplex parts in the metal-mediated conjugates dropped as compared to the separated double strands. Obviously, metal complexation promotes destacking of the ligands MP and TP from the corresponding DNA duplex parts. The metal ion-mediated DNA assemblies were studied by UV/vis absorption and fluorescence spectroscopy. The metal-ion free double strands of DNA1 and DNA2 do not influence each other in absorption properties, since the corresponding spectrum matches exactly that of a 1 : 1 mixture of both duplexes (Fig. 2a). Outside the nucleic acid absorption range, the TP modification of DNA1 exhibits a typical absorption with a single maximum at 318 nm, whereas the MP modification of DNA1 is characterized by two absorption
Scheme 1 Synthesis of the DNA building block 3 for the preparation of MP-modified oligonucleotides: (i) 5’-DMT-protected 5-iodo-2’deoxyuridine, Pd(PPh3)4, CuI, Et3N, DMF, 60 °C, overnight (60%); (ii) NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2, CH2Cl2, overnight (60%).
synthesized two DNA strands (modified single strands of DNA1 and DNA2) of different lengths to potentially distinguish the desired heteroleptic complex formation from the undesired homoleptic assemblies by different electrical mobilities through gels. The shorter modified single strand of DNA1 with a length of 7 base pairs contains the TP modification at the 5′-end and was synthesized according to our published protocol.9 The longer modified single strand of DNA2 (17 base pairs) contains the new building block MP also at the 5′-terminus. Both modified DNA strands were purified by semi-preparative HPLC and identified by MALDI-TOF mass spectrometry. We firstly probed the complex formation between the nucleosides MP and TP with the metal ions Cu2+, Zn2+, Ni2+
6716 | Dalton Trans., 2015, 44, 6715–6718
Fig. 2 (a) UV/Vis absorption and (b) fluorescence (λexc = 336 nm) of the double strands of DNA1 and DNA2 in the absence and in the presence of metal ions (Cu2+, Zn2+, Ni2+, Fe2+, each 2.75 µM, 2.5 µM duplex in 10 mM Na-Pi buffer pH = 7.2, 20 °C).
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Table 1 UV/Vis absorption data and melting temperatures (Tm) of the DNA1, DNA2 and their complexes in the presence of Cu2+, Zn2+, Ni2+, Fe2+, each 2.75 µM, 2.5 µM duplex in 10 mM Na-Pi buffer pH = 7.2, 20 °C
DNA
λabs [nm]
λemi [nm]
Tm (ΔTm) [°C]
DNA1 DNA2 DNA1-Cu2+-DNA2 DNA1-Zn2+-DNA2 DNA1-Ni2+-DNA2 DNA1-Fe2+-DNA2
318 336, 354 358 347 336, 351 338, 581
412 394 398 400 396 398
21.4 61.1 15 (−6.4)/57.5 (−3.6) 15 (−6.4)/57.8 (−3.3) 16 (−5.4)/57.8 (−3.3) 10 (−11.4)/58.3 (−2.8)
peaks at 335 nm and 354 nm. Upon addition of 1.1 equivalents of metal ions (Cu2+, Zn2+, Ni2+ and Fe2+) the absorption of DNA1 and DNA2 in the range between 300 and 400 nm rises and the maxima are shifted (Table 1). The absorption spectra of the separate double strands, DNA1 or DNA2, in the presence of the different metal ions, are considerably different from those of the DNA1–DNA2 mixture, and thereby indicate the formation of the heteroleptic complex. In particular the MLCTband15 that is observable for DNA1, or DNA2 separately, in the presence of Fe2+ at 581 nm exhibits significantly smaller extinction compared to the DNA1–DNA2 mixture. Moreover, the gel experiments clearly evidence the formation of heteroleptic complexes between DNA1 and DNA2 in the presence of metal ions (vide infra). When excited at 336 nm, the TP-modified DNA1 shows only very weak fluorescence intensity (Fig. 2b). In contrast, the fluorescence intensity of MP in DNA2 is strong and significantly quenched in the presence of each of the four different metal ions (Cu2+: 99%, Zn2+: 88%, Fe2+: 65% and Ni2+: 61%). These results stand in contrast to the recently published terpymediated DNA assemblies that showed significant quenching of the terpy fluorescence in the presence of all four different metal ions.9 In order to evidence not only the existence of the heteroleptic complexes that are formed by DNA1 and DNA2 in the presence of metal ions but also the selectivity, we performed 25% denaturing polyacrylamide gel electrophoresis (PAGE) (Fig. 3a). The references DNA1 and DNA2 without metal ions (lanes 2 and 3) and the mixture of both in the presence of EDTA (lane 4) show two results: (i) DNA1 (lane 2) has a significantly longer retention time compared to its counter-strand, but unexpectedly only a little lower retention time compared to DNA2 (lane 3) although it is significantly shorter. Both results indicate that DNA2 dimerizes by hydrophobic interactions between the terminal TP modifications that persist under denaturing conditions. Similar observations were made with perylenebisimides as 5′-DNA caps.16 (ii) As expected, DNA2 has the same retention time as its counter-strand (lane 3). In the presence of metal ions a slower moving new band appears that can be assigned to the heteroleptic complex and the assembly between DNA1 and DNA2. The yield varies depending on the type of metal ion and is highest in the presence of Ni2+.
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Fig. 3 25% denaturing PAGE analysis. (a) Lanes 1–9: ladder, lane 2: DNA1, lane 3: DNA2, lane 4: DNA1–DNA2 + EDTA, lane 5: DNA1–DNA2 + Cu2+, lane 6: DNA1–DNA2 + Zn2+, lane 7: DNA1–DNA2 + Fe2+, lane 8: DNA1–DNA2 + Ni2+. (b) Control experiments: lane 1: DNA2 + Cu2+, lane 2: DNA2 + Zn2+, lane 3: DNA2 + Fe2+, lane 4: DNA2 + Ni2+, lane 5: DNA1–DNA2 + Ni2+ (identical to Fig. 3a/lane 8), lane 6: DNA1–DNA2 + EDTA (identical to Fig. 3a/lane 4), lane 7: DNA1 + Cu2+, lane 8: DNA1 + Zn2+, lane 9: DNA1 + Fe2+, lane 10: DNA1 + Ni2+.
The gel image of the control experiments (Fig. 3b) excludes that homoleptic complexes between the MP modifications of DNA2 are formed in the presence of any of the four different metal ions (lanes 1–4), likely because of the sterically demanding mesityl substituents. It is clear that homoleptic dimers of DNA1 would exhibit smaller electric mobility on the gel than the corresponding heteroleptic DNA1–2 complexes. In contrast to DNA2, DNA1 forms homoleptic dimers (according to the slower moving band in lanes 8–10) due to metal ion-mediated complexation of the terpy ligand in the presence of Zn2+, Fe2+ and Ni2+, as previously published.9 It is important to note that these homoleptic assemblies of DNA1 are not detected in the mixtures of DNA1 and DNA2 (and metal ions) since the electrical mobility allows one to distinguish between them. This is an important result since it shows that the selective formation of heteroleptic assemblies is controlled by the ligand concept and not by the type of metal ion.
Conclusions In conclusion, we describe the synthesis of a new DNA building block MP that bears 2,9-bis-mesityl-3-ethynyl-phenanthroline attached to the 5-position of 2′-deoxyuridine. The combination of this new modification and our recent TP modification (based on the terpy ligand) now allows the selective formation of metal ion-mediated heteroleptic complexes
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between two different DNA double strands, DNA1 and DNA2, in the presence of Cu2+, Zn2+, Ni2+ and Fe2+. Homoleptic complexes are observed only with DNA1 and not with DNA2 and, most importantly, not in mixtures of DNA1 and DNA2. The selectivity for heteroleptic DNA assemblies is controlled by the ligand concept and not by the type of metal ion. Thus, the HETTAP concept13 has been successfully extended to aqueous solutions and can be applied for the bottom-up approach in supramolecular DNA chemistry in order to control the selective assembly of larger DNA architectures with higher order structures.
Acknowledgements Financial support by the Deutsche Forschungsgemeinschaft (Was 1368/12-2), the Karlsruhe School of Optics and Photonics (KSOP), and KIT is gratefully acknowledged.
Notes and references 1 (a) S. Woo and P. W. K. Rothemund, Nat. Chem., 2011, 3, 620–627; (b) C. E. Castro, F. Kilcherr, D.-N. Kim, E. L. Shao, T. Wauer, P. Wortmann, M. Bathe and H. Dietz, Nat. Methods, 2011, 8, 221–229; (c) F. C. Simmel, Angew. Chem., Int. Ed., 2008, 47, 5884–5887. 2 (a) G. Zhang, S. P. Surwade, F. Zhou and H. Liu, Chem. Soc. Rev., 2013, 42, 2488–2496; (b) E. Stulz, Chem. – Eur. J., 2012, 18, 4456–4469; (c) B. Sacca and C. M. Niemeyer, Angew. Chem., Int. Ed., 2012, 51, 58–66. 3 (a) T. Torring, N. V. Voigt, J. Nangreave, H. Yan and K. V. Gothelf, Chem. Soc. Rev., 2011, 40, 5636–4646; (b) J. Nangreave, D. Han, Y. Liu and H. Yan, Curr. Opin. Chem. Biol., 2010, 14, 608–615; (c) N. C. Seeman, Nano Lett., 2010, 10, 1971–1978. 4 (a) C. K. McLaughlin, G. S. Hamblin and H. F. Sleiman, Chem. Soc. Rev., 2011, 40, 5647–5656; (b) H. Yang, K. L. Metera and H. F. Sleiman, Coord. Chem. Rev., 2010, 254, 2403–2415. 5 S. Ghosh and E. Defrancq, Chem. – Eur. J., 2010, 16, 12780– 12787.
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6 (a) Y. Takezawa and M. Shionoya, Acc. Chem. Res., 2012, 45, 2066–2076; (b) G. H. Clever and M. Shionoya, Coord. Chem. Rev., 2010, 254, 2391–2402; (c) G. Clever, C. Kaul and T. Carell, Angew. Chem., Int. Ed., 2007, 46, 6226–6236. 7 (a) R. B. Martin and J. A. Lissfelt, J. Am. Chem. Soc., 1956, 78, 938–940; (b) R. H. Holyer, C. D. Hubbard, S. F. A. Kettle and R. G. Wilkins, Inorg. Chem., 1966, 5, 622–625; (c) R. Cali, E. Rizzarelli, S. Sammartano and G. Siracusa, Transition Met. Chem., 1979, 4, 328–332; (d) G. U. Priimov, P. Moore, L. Helm and A. E. Merbach, Inorg. React. Mech., 2001, 3, 1–23; (e) S. Leininger, B. Olenyuk and P. J. Stang, Chem. Rev., 2000, 100, 853–907; (f ) L. Zapata, K. Bathany, J. M. Schmitter and S. Moreau, Eur. J. Org. Chem., 2003, 1022–1028. 8 (a) K. M. Stewart and L. W. McLaughlin, Chem. Commun., 2003, 2934–2935; (b) S. Ghosh, I. Pignot-Paintrand, P. Dumy and E. Defrancq, Org. Biomol. Chem., 2009, 7, 2729–2737; (c) J. S. Choi, C. W. Kang, K. Jung, J. W. Yang, Y. G. Kim and H. Y. J. Han, J. Am. Chem. Soc., 2004, 126, 8606–8607. 9 T. Ehrenschwender, A. Barth, H. Puchta and H.-A. Wagenknecht, Org. Biomol. Chem., 2012, 10, 46–48. 10 (a) L. Kalachova, R. Pohl and M. Hocek, Org. Biomol. Chem., 2012, 10, 49–55; (b) L. Kalachova, R. Pohl and M. Hocek, Synthesis, 2009, 105–112. 11 S. De, K. Mahata and M. Schmittel, Chem. Soc. Rev., 2010, 39, 1555–1575. 12 H. Yang, A. Z. Rys, C. K. McLaughlin and H. F. Sleiman, Angew. Chem., Int. Ed., 2009, 48, 9919–9923. 13 V. Kalsani, M. Schmittel, A. Listorti, G. Accorsi and N. Armaroli, Inorg. Chem., 2006, 45, 2061–2067. 14 M. Schmittel, C. Michel, A. Wiegrefe and V. Kalsani, Synthesis, 2001, 1561–1567. 15 P. S. Braterman, J. L. Song and R. D. Peacock, Inorg. Chem., 1992, 31, 555–559. 16 (a) D. Baumstark and H.-A. Wagenknecht, Angew. Chem., Int. Ed., 2008, 47, 2612–2614; (b) F. Menacher, V. Stepanenko, F. Würthner and H.-A. Wagenknecht, Chem. – Eur. J., 2011, 17, 6683–6688.
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Cite this: Dalton Trans., 2015, 44, 6715 Received 9th January 2015, Accepted 11th March 2015
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Mesityl phenanthroline-modified 2’-deoxyuridine for heteroleptic complexes in metal ion-mediated assembly of DNA† Philipp Ensslen and Hans-Achim Wagenknecht*
DOI: 10.1039/c5dt00100e www.rsc.org/dalton
The synthesis of a new DNA building block that bears the metal ion ligand 2,9-bis-mesityl-3-ethynyl-phenanthroline attached to the 5-position of 2’-deoxyuridine is presented. In the presence of Zn2+, Cu2+, Fe2+ and Ni2+ the complex formation of an accordingly modified DNA double strand with a second DNA duplex bearing the 2,2’:6’,2’’-terpyridine ligand was studied by optical spectroscopy. The selective formation of heteroleptic assemblies between the two different DNA pieces was evidenced by denaturing polyacrylamide gel electrophoresis.
DNA with its intrinsic self-assembling properties is not only suitable for the sequence dependent arrangements of two- and three-dimensional DNA objects and origami,1–3 but also a promising scaffold for supramolecular architectures. For the latter purpose it is crucial to develop additional binding motifs, especially with metal ion-binding ligands that allow increasing the complexity and structural variety.4 A broad variety of DNA building blocks has been synthesized for metal ion-mediated base pairing (inside the DNA duplex).5,6 Fewer examples have been published for metal ion-mediated DNA assembly (outside the duplex).4,5 In particular 2,2′:6′,2″-terpyridine (terpy) as a ligand attached to the termini of DNA double strands via flexible alkyl linkers allows the formation of stable complexes with different metal ions and thereby astonishing three-dimensional DNA constructs.4,7,8 Recently, we9 and Hocek with coworkers10 showed that the terpy ligand attached to the 5-position of 2′-deoxyuridine via the rigid acetylene bridge yields metal-mediated DNA assembly of short strands, both “side by side” and “end on end”. However, the application of only a single type of ligand, like terpy, as DNA modification (TP) only allows the formation of homoleptic metal ion
Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany. E-mail: [email protected]; Fax: +49-721-608-44825; Tel: +49-721-608-47486 † Electronic supplementary information (ESI) available: Materials and methods, synthesis and characterization of 3, DNA1 and DNA2, images of NMR and MS spectra and HPLC analyses, and the procedure for the preparation of metal ionmediated DNA assemblies. See DOI: 10.1039/c5dt00100e
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complexes and accordingly mediates DNA assemblies, and thereby significantly limits the control of stepwise DNA assembly to larger objects.4,8 Hence, in this bottom-up approach it is highly desirable to develop DNA modifications for heteroleptic metal ion complexes in order to predictably control the assembly of metal ion-mediated DNA constructs.11 A first attempt for the formation of heteroleptic metal ion complexes in DNA was presented by Sleiman and coworkers.12 Their combination of phenanthroline- and terpy-modified complementary DNA strands gives stable heteroleptic complexes. The DNA assembly to homoleptic and heteroleptic complexes was mainly controlled by the type of metal ion and not by ligand interactions. Schmittel and coworkers published a concept that they called HETTAP (heteroleptic terpyridine and phenanthroline complex formation). Mesityl substituents at the 2- and 9-positions of phenanthroline exclusively lead to the formation of kinetically labile heteroleptic complexes with the terpy ligand in the presence of a variety of metal ions.13 The sterically demanding mesityl substituents not only inhibit the homoleptic complex formation by the phenanthroline ligands but also induce π–π interactions with the terpy ligand (Fig. 1a). In order to extend this concept to aqueous solutions and apply it to supramolecular DNA chemistry, we present herein the synthesis of a new DNA building block MP that bears the bis-2,9-mesityl-substituted phenanthroline attached to the 5-position of 2′-deoxyuridine via an acetylene bridge (Fig. 1b). Furthermore, this DNA building block of MP and the previously described terpy building block TP were used to prepare two single modified oligonucleotides DNA1 and DNA2 (Fig. 1c), respectively. The selective assembly of the corresponding two DNA double strands by heteroleptic complex formation was demonstrated and studied in the presence of four different metal ions. The 2,9-bis-mesityl-3-ethynyl-phenanthroline 1 (Scheme 1), as an important precursor for the new DNA building block MP, was synthesized according to the literature.14 The phenanthroline 1 was attached to 5′-DMT protected 5-iodo-2′-deoxyuridine via a palladium-catalyzed Sonogashira-type reaction (60% yield). The final steps to the DNA building block 3 followed standard phosphoramidite chemistry. Subsequently, we
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Fig. 1 (a) Concept of heteroleptic metal ion–ligand complex formation, (b) DNA building blocks MP and TP, and (c) sequences of DNA1 and DNA2.
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and Fe2+ by MALDI MS. The recorded MS spectra (see ESI†) exclusively show the mass peaks of the heteroleptic complexes MP-M2+-TP and no corresponding peaks of the homoleptic complexes. The metal ion complex formation between the two duplexes (DNA1 and DNA2) was probed in the presence of their fully complementary counterstrands to avoid undesired metal–ion interactions or complexation with the DNA bases. Hence, both double strands were annealed separately. Subsequently, metal ions and afterwards DNA1 were added to DNA2 in aqueous buffer and incubated for 30 min at room temperature. The melting temperature of both duplex parts in the metal-mediated conjugates dropped as compared to the separated double strands. Obviously, metal complexation promotes destacking of the ligands MP and TP from the corresponding DNA duplex parts. The metal ion-mediated DNA assemblies were studied by UV/vis absorption and fluorescence spectroscopy. The metal-ion free double strands of DNA1 and DNA2 do not influence each other in absorption properties, since the corresponding spectrum matches exactly that of a 1 : 1 mixture of both duplexes (Fig. 2a). Outside the nucleic acid absorption range, the TP modification of DNA1 exhibits a typical absorption with a single maximum at 318 nm, whereas the MP modification of DNA1 is characterized by two absorption
Scheme 1 Synthesis of the DNA building block 3 for the preparation of MP-modified oligonucleotides: (i) 5’-DMT-protected 5-iodo-2’deoxyuridine, Pd(PPh3)4, CuI, Et3N, DMF, 60 °C, overnight (60%); (ii) NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2, CH2Cl2, overnight (60%).
synthesized two DNA strands (modified single strands of DNA1 and DNA2) of different lengths to potentially distinguish the desired heteroleptic complex formation from the undesired homoleptic assemblies by different electrical mobilities through gels. The shorter modified single strand of DNA1 with a length of 7 base pairs contains the TP modification at the 5′-end and was synthesized according to our published protocol.9 The longer modified single strand of DNA2 (17 base pairs) contains the new building block MP also at the 5′-terminus. Both modified DNA strands were purified by semi-preparative HPLC and identified by MALDI-TOF mass spectrometry. We firstly probed the complex formation between the nucleosides MP and TP with the metal ions Cu2+, Zn2+, Ni2+
6716 | Dalton Trans., 2015, 44, 6715–6718
Fig. 2 (a) UV/Vis absorption and (b) fluorescence (λexc = 336 nm) of the double strands of DNA1 and DNA2 in the absence and in the presence of metal ions (Cu2+, Zn2+, Ni2+, Fe2+, each 2.75 µM, 2.5 µM duplex in 10 mM Na-Pi buffer pH = 7.2, 20 °C).
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Table 1 UV/Vis absorption data and melting temperatures (Tm) of the DNA1, DNA2 and their complexes in the presence of Cu2+, Zn2+, Ni2+, Fe2+, each 2.75 µM, 2.5 µM duplex in 10 mM Na-Pi buffer pH = 7.2, 20 °C
DNA
λabs [nm]
λemi [nm]
Tm (ΔTm) [°C]
DNA1 DNA2 DNA1-Cu2+-DNA2 DNA1-Zn2+-DNA2 DNA1-Ni2+-DNA2 DNA1-Fe2+-DNA2
318 336, 354 358 347 336, 351 338, 581
412 394 398 400 396 398
21.4 61.1 15 (−6.4)/57.5 (−3.6) 15 (−6.4)/57.8 (−3.3) 16 (−5.4)/57.8 (−3.3) 10 (−11.4)/58.3 (−2.8)
peaks at 335 nm and 354 nm. Upon addition of 1.1 equivalents of metal ions (Cu2+, Zn2+, Ni2+ and Fe2+) the absorption of DNA1 and DNA2 in the range between 300 and 400 nm rises and the maxima are shifted (Table 1). The absorption spectra of the separate double strands, DNA1 or DNA2, in the presence of the different metal ions, are considerably different from those of the DNA1–DNA2 mixture, and thereby indicate the formation of the heteroleptic complex. In particular the MLCTband15 that is observable for DNA1, or DNA2 separately, in the presence of Fe2+ at 581 nm exhibits significantly smaller extinction compared to the DNA1–DNA2 mixture. Moreover, the gel experiments clearly evidence the formation of heteroleptic complexes between DNA1 and DNA2 in the presence of metal ions (vide infra). When excited at 336 nm, the TP-modified DNA1 shows only very weak fluorescence intensity (Fig. 2b). In contrast, the fluorescence intensity of MP in DNA2 is strong and significantly quenched in the presence of each of the four different metal ions (Cu2+: 99%, Zn2+: 88%, Fe2+: 65% and Ni2+: 61%). These results stand in contrast to the recently published terpymediated DNA assemblies that showed significant quenching of the terpy fluorescence in the presence of all four different metal ions.9 In order to evidence not only the existence of the heteroleptic complexes that are formed by DNA1 and DNA2 in the presence of metal ions but also the selectivity, we performed 25% denaturing polyacrylamide gel electrophoresis (PAGE) (Fig. 3a). The references DNA1 and DNA2 without metal ions (lanes 2 and 3) and the mixture of both in the presence of EDTA (lane 4) show two results: (i) DNA1 (lane 2) has a significantly longer retention time compared to its counter-strand, but unexpectedly only a little lower retention time compared to DNA2 (lane 3) although it is significantly shorter. Both results indicate that DNA2 dimerizes by hydrophobic interactions between the terminal TP modifications that persist under denaturing conditions. Similar observations were made with perylenebisimides as 5′-DNA caps.16 (ii) As expected, DNA2 has the same retention time as its counter-strand (lane 3). In the presence of metal ions a slower moving new band appears that can be assigned to the heteroleptic complex and the assembly between DNA1 and DNA2. The yield varies depending on the type of metal ion and is highest in the presence of Ni2+.
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Fig. 3 25% denaturing PAGE analysis. (a) Lanes 1–9: ladder, lane 2: DNA1, lane 3: DNA2, lane 4: DNA1–DNA2 + EDTA, lane 5: DNA1–DNA2 + Cu2+, lane 6: DNA1–DNA2 + Zn2+, lane 7: DNA1–DNA2 + Fe2+, lane 8: DNA1–DNA2 + Ni2+. (b) Control experiments: lane 1: DNA2 + Cu2+, lane 2: DNA2 + Zn2+, lane 3: DNA2 + Fe2+, lane 4: DNA2 + Ni2+, lane 5: DNA1–DNA2 + Ni2+ (identical to Fig. 3a/lane 8), lane 6: DNA1–DNA2 + EDTA (identical to Fig. 3a/lane 4), lane 7: DNA1 + Cu2+, lane 8: DNA1 + Zn2+, lane 9: DNA1 + Fe2+, lane 10: DNA1 + Ni2+.
The gel image of the control experiments (Fig. 3b) excludes that homoleptic complexes between the MP modifications of DNA2 are formed in the presence of any of the four different metal ions (lanes 1–4), likely because of the sterically demanding mesityl substituents. It is clear that homoleptic dimers of DNA1 would exhibit smaller electric mobility on the gel than the corresponding heteroleptic DNA1–2 complexes. In contrast to DNA2, DNA1 forms homoleptic dimers (according to the slower moving band in lanes 8–10) due to metal ion-mediated complexation of the terpy ligand in the presence of Zn2+, Fe2+ and Ni2+, as previously published.9 It is important to note that these homoleptic assemblies of DNA1 are not detected in the mixtures of DNA1 and DNA2 (and metal ions) since the electrical mobility allows one to distinguish between them. This is an important result since it shows that the selective formation of heteroleptic assemblies is controlled by the ligand concept and not by the type of metal ion.
Conclusions In conclusion, we describe the synthesis of a new DNA building block MP that bears 2,9-bis-mesityl-3-ethynyl-phenanthroline attached to the 5-position of 2′-deoxyuridine. The combination of this new modification and our recent TP modification (based on the terpy ligand) now allows the selective formation of metal ion-mediated heteroleptic complexes
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between two different DNA double strands, DNA1 and DNA2, in the presence of Cu2+, Zn2+, Ni2+ and Fe2+. Homoleptic complexes are observed only with DNA1 and not with DNA2 and, most importantly, not in mixtures of DNA1 and DNA2. The selectivity for heteroleptic DNA assemblies is controlled by the ligand concept and not by the type of metal ion. Thus, the HETTAP concept13 has been successfully extended to aqueous solutions and can be applied for the bottom-up approach in supramolecular DNA chemistry in order to control the selective assembly of larger DNA architectures with higher order structures.
Acknowledgements Financial support by the Deutsche Forschungsgemeinschaft (Was 1368/12-2), the Karlsruhe School of Optics and Photonics (KSOP), and KIT is gratefully acknowledged.
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