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J. Phys. Chem. B 2010, 114, 11942–11948
Potential Cross-Linking Transition Metal Complexes (M ) Ni, Cu, Zn) in the Ligand-Modified LNA Duplexes Pipsa Hirva,*,† Anne Nielsen,‡ Andrew D. Bond,‡ and Christine J. McKenzie‡ Department of Chemistry, UniVersity of Eastern Finland, Joensuu Campus P.O. Box 111, FI-80101, Joensuu, Finland, and Department of Physics and Chemistry, UniVersity of Southern Denmark, CampusVej 55, 5230 Odense M, Denmark ReceiVed: June 16, 2010; ReVised Manuscript ReceiVed: August 4, 2010
Options for interstrand DNA duplex linkages have been studied by incorporating transition metal ions in the ligand-functionalized LNA (locked nucleic acid) duplexes. The effect of first-row transition metal ions (M ) Ni2+, Cu2+, and Zn2+) on the geometries and formation energies of mono- and dimetallic model complexes was calculated by DFT methods, and the results were compared with available experimental data. The results showed a clear preference for the formation of copper complexes over the corresponding nickel and zinc complexes, in agreement with the trends observed in the denaturation temperatures of the ligand-functionalized LNA duplexes. In addition, dichloride bridged dimeric complex, [LLNACu(µ-Cl)2CuLLNA]2+, in which LLNA is N,N-bis(2-pyridylmethyl)-β-alanyl functionalized LNA, was found energetically very stable, providing a potential structural option for an interstrand duplex linkage. The model complex and its simpler structural analogues were synthesized and structurally characterized. Comparison of the dimeric linker introduced into duplex tetramer strands, which provided a computational model for a double helix with two closely located LNA units, with a similar model for mononuclear Cu(LLNA)22+ linker also showed a clear preference of the dichloride-bridged option, suggesting that the [LLNACu(µ-Cl)2CuLLNA]2+ complex produced a chemically realistic model to explain duplex stabilization in the presence of Cu2+ and excess Cl-. Introduction Metal ions, usually in the form of their aquo and ammine complexes, interact with the phosphate backbone and nucleobases of DNA to influence stability, hybridization, and topology of the DNA duplex.1-3 Attempts to exploit these types of interactions have been made through design and synthesis of bifunctional natural or artificial nucleic acids incorporating binding sites for metal ions. Complexes of such molecules with transition metal ions are especially interesting because of their potential applications in medicine and molecular electronics. There are several approaches to modification of duplex oligomers. For example, Watson-Crick base pairing can be replaced with coordination bonds to metal cations within the core of the double helix.4-8 Alternatively, DNA can be chemically modified with a chelating ligand or complex that may be located in the major or minor groove or at the termini. In this case, Watson-Crick base pairing and mismatch discrimination is generally maintained, and metal ions might be introduced and removed without full denaturation. This methodology has been used to create ribozyme mimics,9-18 radiolabeled systems,19 and electrochemical probes20 and to control duplex topology and folding.21-25 Using the approach of chemical modification, it has been found that the duplex stability of oligonucleotides containing N,N-bis(2pyridylmethyl)-β-alanyl functionalized “locked nucleic acid (LNA)”, (LLNA, Scheme 1) is increased in the presence of nickel, copper, and zinc ions, introduced as their M(II) chloride salts in the presence of ∼10 000 equiv of sodium chloride in aqueous solution.24 The denaturation temperatures are raised by +6 to +28 °C relative to * Corresponding author. E-mail:
[email protected]. † University of Eastern Finland. ‡ University of Southern Denmark.
SCHEME 1: (a) N,N-Bis(2-pyridylmethyl)-βalanyl-2′-amino-LNA (LLNA), (b) N,N-Bis(2-pyridylmethyl)β-alanine Methyl Ester (Lester), and (c) N,N-Bis(2-pyridylmethyl)-β-alanyl Amide (Lamide)
unmodified duplexes with the same sequences, with the most dramatic duplex stabilization observed in the presence of Cu2+. The structures of the metal-LLNA duplex conjugates that may be responsible for the observed duplex stabilization are unknown. Metal complex linker structures of the bis-ligand type have been proposed.24
10.1021/jp105528y 2010 American Chemical Society Published on Web 08/20/2010
Cross-Linking Transition Metal Complexes Recently, computational tools at different levels of theory have also been applied to shed light on the various possibilities to enhance the stability of the DNA duplexes. Detailed DFT studies with small molecular models have been employed to investigate, for example, the direct interaction of Cu+ and Cu2+ ions26 and Zn ions27 with isolated DNA base pairs, magnetic interactions between Cu2+ ions when the natural base pairs are replaced by Cu2+ mediated hydroxypyridone nucleabases,28 and reaction mechanisms for cleavage of phosphodiester linkages between nucleotides in DNA mediated by zinc complexes with tris(pyridylmethyl)amine ligands.29 More extensive models have been utilized in docking calculations and MD simulations of the noncovalent binding of Cu(Phen)2 complexes into the minor and major grooves of DNA, modeled with a 15-mer.30 MD studies have been carried out in which an octahedral metal coordination environment furnished by the six N donors of two dipyridylamine units of two N,N-bis(2-pyridylmethyl)-β-alanyl-LNA groups was allowed to minimize while the structure of the DNA duplex was constrained.24 This resulted in unrealistically long M-N distances of around 3 Å, indicating the importance of full relaxation of the duplex structure in the formation of the interstrand linkages. Another approach was taken more recently for closely related terpy-modified LNA systems.25 In this case, both the bis-terpyLNA metal complex linker and the duplex were constrained, in the former case using M-N distances from the X-ray crystal structure of [Ni(terpy)2]2+. The resulting model indicates that a [Ni(terpy-LNA)2]2+ complex can, in principle, strap between the strands of a duplex, but it does not address adequately its likelihood and stability in an aqueous environment containing an excess of chloride relative to the metal ions and oligomer duplexes. In an effort to clarify the possible mechanisms of duplex stabilization in oligomers containing LLNA, we have sought to isolate and characterize relevant metal-LLNA complexes that would provide a realistic structural basis for more extensive computational studies. In the present study, we have chosen to employ quantum chemical DFT methods using finite molecular models of the various structural options for the interstrand duplexes linkages. We were principally interested to characterize complexes with Cu2+, since this gives the most dramatic stabilizing effect, but any model should also account for the experimentally observed relative stability order of Cu2+ > Ni2+ > Zn2+. Furthermore, to explain the remarkable duplex stabilization observed with copper and an excess of Cl- ions in the solution, we have synthesized and computationally investigated chloride-bridged dimeric M-LLNA complexes and simple analogues and compared the interaction between the linker and a tetrameric model of the duplex structure with the corresponding bis-LLNA complex. Tests for the Method The selection of the computational method was based on a previous study on transition metal carbonyl complexes, in which hybrid density functional PBE1PBE (also called PBE0) in conjunction with the Stuttgard and Dresden quasirelativistic basis set for the metal atoms was found to produce the best compromise between reliability of the results and the computational requirements.31 Since our major interest was in the effect of an excess of chloride ions in the formation of the transition metal containing interstrand complex, we chose to test the method with Ni, Cu, and Zn complexes containing the LLNA and chloride ligands. A potential option for the duplex linkage in the presence of chloride ions would be a dimeric dichlorobridged structure with the [LLNAM(µ-Cl2)MLLNA] rhombus core.
J. Phys. Chem. B, Vol. 114, No. 36, 2010 11943
Figure 1. The cation [LLNACu(µ-Cl2)CuLLNA]2+ in the crystal structure of the model complex with displacement ellipsoids shown at 50% probability. H atoms are omitted.
In the present study, this dimeric structure was selected as a model for testing the reliability of the computational method. For comparison, the dichloride bridged copper complex was synthesized, and the structure was solved by synchrotron X-ray diffraction (Figure 1). Since the LLNA ligand has a long and flexible structure, full conformational analysis by high-level DFT methods is impractical. On the other hand, applying slightly less demanding QM/MM methods for the conformational analysis would have required rigorous testing to ensure reliable performance of the different MM force fields. Since our focus was on the coordination sphere and geometry of the metal ions, we chose to employ a smaller model for the LLNA ligand; namely, N,N-bis(2-pyridylmethyl)-β-alanylamide (Lamide, Scheme 1c). The possible influence of using the complete LLNA ligand on the minimized structure of the [M(Cl)2M] core was tested by taking the conformation obtained from the structural analyses as the initial geometry and performing full optimization. It was confirmed that increasing the ligand size had a negligible effect on the [M(Cl)2M] core, and we conclude, therefore, that the Lamide model was sufficiently large to obtain reliable results. This model was used in successive calculations. Similar Ni2+ and Zn2+ were also optimized, and the results were compared with related structures found in the literature. The key geometrical parameters are listed in Table 1. The overall geometry of the dinuclear complexes is very well reproduced by the selected computational method. Especially, the experimentally observed asymmetry of the M-Cl distances is well dictated by the optimized structures. It is notable that distortion of the rhombus core is also observed for the nickel and zinc systems in the computational and crystallographic models, despite the fact that a Jahn-Teller distortion is not expected in these cases. It should be noted that the slightly larger difference in the optimized Cu(1)-Cl(2) distance as compared with the experimental crystal structure, leading to a more symmetric CuCl2Cu core in the optimized model, is most likely due to packing effects in the solid state, since the copper complex with LLNA ligands crystallizes in a 1D polymer-like motif that arises through H-bonding between the dangling pyrimidine bases in adjacent molecules (Supporting Information, Figure S1). Consequently, an even closer consistency within the Cu-Cl distances can be found when comparing the optimized model structure with a crystal structure of [LesterCu(µCl2)CuLester] complex, in which Lester is a smaller N,N-bis(2pyridylmethyl)-β-alanine methyl ester (Scheme 1b and Supporting Information Table S1).
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Hirva et al.
TABLE 1: Selected Computationally Optimized Parameters for the [LamideM(µ-Cl)2MLamide]2+ Complexesa in Gas Phase and in Aqueous Solution along with Distances Found in the Single Crystal Structures of Closely Related Systems Ni2+
M
Cu2+
Zn2+
solvent
gas
H 2O
expb
gas
H 2O
expc
gas
H 2O
expd
M(1)-Cl(1) M(1)-Cl(2) M(1)-O(1) M(1)-N(1) M(1)-N(2) M(1)-N(3) M(2)-Cl(2) M(2)-Cl(1) M(2)-O(3) M(2)-N(4) M(2)-N(5) M(2)-N(6) M(1).. .M(2) Cl(1)-M(1)-Cl(2) Cl(1)-M(2)-Cl(2) M(1)-Cl(1)-M(2) M(1)-Cl(2)-M(2)
2.402 2.465 2.090 2.079 2.133 2.076 2.402 2.465 2.091 2.075 2.133 2.079 3.562 85.93 85.93 94.07 94.06
2.411 2.514 2.089 2.071 2.113 2.072 2.414 2.517 2.090 2.070 2.111 2.069 3.624 85.39 85.27 94.66 94.66
2.411 2.478 2.017 2.096 2.091 2.143 2.411 2.478 2.017 2.143 2.091 2.096 3.644 83.67 83.67 96.33 96.33
2.284 3.052 2.296 2.015 2.103 2.008 2.284 3.052 2.296 2.008 2.103 2.015 3.920 86.55 86.56 93.46 93.44
2.303 3.117 2.334 2.016 2.072 2.013 2.302 3.109 2.325 2.011 2.071 2.016 3.936 87.90 88.10 92.09 91.90
2.260 3.562 2.369 1.992 2.067 2.033 2.245 3.628 2.286 2.005 2.057 2.025 4.129 94.07 92.58 85.74 87.54
2.353 2.598 2.160 2.122 2.275 2.114 2.353 2.598 2.160 2.114 2.275 2.122 3.572 87.79 87.79 92.21 92.21
2.366 2.709 2.147 2.109 2.234 2.107 2.365 2.712 2.147 2.108 2.230 2.108 3.703 86.61 86.55 93.38 93.47
2.346 2.623 2.185 2.111 2.194 2.100 2.346 2.613 2.185 2.100 2.194 2.111 3.536 89.46 89.46 90.54 90.54
a M ) Ni, Cu, Zn; Lamide ) N,N-bis(2-pyridylmethyl)-β-alanylamide (Scheme 1c). b Values from the X-ray structure of [Ni2Cl2L2]2+; L ) 2,4-bis-tert-butyl-6-((6-methyl-2-pyridylmethyl)(2-pyridylmethyl)amino)methylphenolate.32 c Values from the X-ray structure of [Cu2Cl2L2]2+; L ) N,N-bis(2-pyridylmethyl)-β-alanyl-2′-amino-LNA (this work). d Values from the X-ray structure of [Zn2Cl2L2]2+; L ) 1,8-bis(bis(2-pyridylmethyl)amino)-3,6-dihydroxyxanthone.33
Figure 2. Optimized structures for the model complexes (M ) Cu): (a) [(Lamide)M(µ-Cl)2M(Lamide)]2+, (b) [(Lamide)ClM(µ-Cl)MCl(Lamide)]+, (c) [(Lamide)2M]2+, (d) [(Lamide)M(H2O)2]2+, (e) [(Lamide)MCl(H2O)]+, and (f) [(Lamide)MCl2]. The structures represent optimizations in aqueous solution.
Models To establish information on the relative stability of the different metal-LLNA complexes, we modeled several structures for potential interstrand linkages comprising coordination complexes of N,N-bis(2-pyridylmethyl)-β-alanyl-LNA (LLNA) conjugates. Considering the chemical environment in which these linkages may form (an aqueous solution containing a large excess of chloride relative to metal ions and oligomer duplexes), two feasible dinuclear complexes ([LM(µ-Cl)2ML]2+ and [LClM(µ-Cl)MClL]+) and four mononuclear complexes ([LMCl2], [LMCl(H2O)]+, [LM(H2O)2]2+, and [L2M]2+) can be suggested. The optimized structures of the different options are shown in Figure 2. In all calculations, the LLNA ligand was replaced by a smaller Lamide ligand, as discussed above in the Tests section. In the figure, the structure of the copper complexes is presented, but also the corresponding Ni and Zn complexes optimized in the same overall structures. Both the dinuclear [(Lamide)M(µ-Cl)2M(Lamide)]2+ complex and the mononuclear complexes exhibited 6-coordinated metal ions
Figure 3. The double helix structure of the nonamer showing the location of the LNA sites, which were involved in formation of the interstrand complexes (red line).
in their optimized structures. However, the dimeric [(Lamide)ClM(µ-Cl)MCl(Lamide)]+complex optimized with two different coordination modes for the copper ions when one of the Cu-carbonyl interactions was lost, resulting in one 5-coordinated copper (Figure 2b). It should be noted that the other two metal ions, Ni and Zn, did not show this behavior, but in the corresponding monochloride-bridged complexes, both carbonyl contacts with the β-alanyl “tails” were maintained. The model for the duplex structure included a tetramer in a base pair sequence of ATLAT-TATLA, in which the central thymines included the ligand-functionalized LNA modifications (Figure 3). The initial structure of the model was cut from the experimental crystal structure of a nonamer.
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TABLE 2: Relative Reaction Energies (kJ mol-1) According to Reactions 1-6 for Model M-Lamide Complexes in Aqueous Solution reaction 1 2 3 4 5 6
complex 2+
[LM(µ-Cl)2ML] [LClM(µ-Cl)MClL]+ [L2M]2+ [LM(H2O)2]2+ [LMCl(H2O)]+ [LMCl2]
Ni2+
Cu2+
Zn2+
-679 -644 -385 -358 -355 -328
-1533 -1499 -782 -743 -752 -731
-471 -451 -276 -232 -252 -230
gas phase reaction 1 2 3 4 5 6
2+
complex
Ni 2+
[LM(µ-Cl)2ML] [LClM(µ-Cl)MClL]+ [L2M]2+ [LM(H2O)2]2+ [LMCl(H2O)]+ [LMCl2]
61 76 25 26 28 61
Zn
2+
177 173 88 54 77 73
toluene 2+
water 2+
2+
Ni
Zn
Ni
Zn2+
598 528 247 231 248 272
690 679 335 335 326 341
1007 855 398 385 397 403
1062 1048 506 511 500 501
a The differences in reaction energies are referenced corresponding Cu2+ complexes, which have been set to zero.
Results and Discussion Stability of the Isolated Complexes. The relative stabilities of two feasible dinuclear complexes ([(Lamide)M(µ-Cl)2M(Lamide)]2+ and [(Lamide)ClM(µ-Cl)MCl(Lamide)]+) and four mononuclear complexes ([(Lamide)MCl2], [(Lamide)MCl(H2O)]+, [(Lamide)M(H2O)2]2+, and [(Lamide)2M]2+) were compared by calculating reaction energies according to the formation reactions 1-6:
2M2+ + 2Cl- + 2Lamide f [(Lamide)M(µ-Cl)2M(Lamide)]2+ (1) 2M2+ + 3Cl- + 2Lamide f [(Lamide)ClM(µ-Cl)MCl(Lamide)]+
(2)
M2+ + 2Lamide f [(Lamide)2M]2+
(3)
M2+ + 2H2O + Lamide f [(Lamide)M(H2O)2]2+
(4)
M2+ + H2O + Cl- + Lamide f [(Lamide)MCl(H2O)]+
(5) M2+ + 2Cl- + Lamide f [(Lamide)MCl2]
TABLE 3: The Effect of Solvent on the Relative Energies (kJ mol-1) According to Reactions 1-6 for Model M-Lamide Complexesa
(6)
Table 2 lists the reaction energies obtained from optimizing each compound in aqueous solution. All reactions are calculated to be exothermic. According to the relative energies, the dinuclear Cu2+ complexes, [(Lamide)M(µ-Cl)2M(Lamide)]2+ and [(Lamide)ClM(µ-Cl)MCl(Lamide)]+, are significantly more stable than all of the mononuclear species. We therefore conclude that chloride-bridged dimetallic complexes are the most probable option for linkages between strands in duplexes containing LLNA in the presence of Cu2+ and excess Cl- in aqueous solution. The corresponding Ni2+ and Zn2+ complexes also show stabilization relative to their monomeric derivatives, but to a much lesser degree. On the other hand, the formation energy of the monochloride bridged complex [(Lamide)ClM(µ-Cl)MCl(Lamide)]+ is only ∼30 kJ mol-1 less exothermic than that of the dichloride bridged complex [(Lamide)M(µ-Cl)2M(Lamide)]2+. This relatively small energetic difference is corroborated experimentally by the observation that the two cations [(bpa)ClCu(µ-Cl)CuCl(bpa)]+ and [(bpa)Cu(µ-Cl)2Cu(bpa)]2+ (where bpa ) N,N-bis(2-pyridylmethyl)methylamine) have been observed to coexist in crystalline form.34 Thus, the monochloride bridged structural type can potentially also be involved in interstrand linkages, in addition to the dichloride bridged complex. Indeed, under certain circumstances, likely dependent on chloride concentration, conversion between the dichloride and monochloride bridged interstrand linkages might be expected.
to
The difference between the formation energies of the various monomeric complexes is much smaller. In aqueous solution in the absence of chloride, the most stable monomeric structure for all three metal ions is calculated to be [(Lamide)2M]2+, in which the two L ligands are coordinated to the metal cation via their three N atoms and the carbonyl group remains uncoordinated (Figure 2c). Optimized structures in which the pyridyl N atoms lie in a cis arrangement were 20-30 kJ mol-1 more stable than the optimized trans isomers, depending slightly on the identity of the metal ion. With all of the other optimized mononuclear complexes, the preference for a cis or trans arrangement of pyridyl donors varied, and especially, copper complexes favored the trans conformers. This contrasts with the dimeric dichloride-bridged complexes, for which the trans arrangement of pyridyl N atoms was calculated to be clearly more stable (see Figure 2 and Table S2). Potential solvent effects were assessed by calculation of the relative reaction energies in the gas phase and in the nonpolar solvent toluene. The difference in formation energies of the nickel and zinc complexes relative to the corresponding copper complexes (which were set to ∆E ) 0) is shown in Table 3. In all cases, the stability follows the order Cu > Ni > Zn, but the difference is very much enhanced in polar solvents, especially for the dimetallic complexes. Duplex Linkages. Stability of the complexes does not solely govern the ability to bind to the different coordination sites on the LNA modified duplex, but the complex must adopt feasible geometry to fit the minor or major groove to enhance the interaction between the binding sites. We compared the possible formation of dinuclear dichloride bridged [LLNACu(µ-Cl)2CuLLNA]2+ and mononuclear [(LLNA)2Cu]2+moieties on a tetrameric duplex model with suitably located LNA modifications. These options were selected because they exhibited the most favorable stabilities among the separate dimeric and monomeric complexes, respectively (Table 2). The optimized structures of the resulting copper complexes are shown in Figure 4. Figure 3 shows the location of the complexes at the ligand modified LNA sites. Full geometry optimization showed that both of the complexes can be accommodated without disrupting the double helix structure or significantly affecting the Watson-Crick base pairing. A reasonable adjustment of the distance between the LNA sites was, however, observed with the limited tetrameric model, enhancing the N-N distance of the two coordinating nitrogen sites from the initial 10.2 to 12.8 and 12.3 Å for the dinuclear and mononuclear complex, respectively. Therefore, both of the complexes were found to fit quite nicely into the (minor) groove between the two strands. Energetically, the dichloride-bridged complex was found to be much more stable (∼2700 kJ mol-1) than the bis-ligand complex, when the
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Hirva et al. TABLE 4: Selected Bond Lengths (Å) and angles (°) in the Optimized Computational Models of Doubly Bridged [(Lamide)Cu(µ-Cl)2Cu(Lamide)]2+, Singly Bridged [(Lamide)ClCu(µ-Cl)CuCl(Lamide)]+, Mononuclear [(Lamide)2Cu]2+, and the Corresponding Interstrand Complexes (Figures 4 and 5) in the Gas Phase complex- complex- complex- interstrand- interstrand(µ-Cl)2 (µ-Cl) L2 (µ-Cl)2 L2
Figure 4. Computationally optimized structure of a duplex-stabilizing interstrand linkages on a tetrameric ATLAT-TATLA model: (a) [LLNACu(µ-Cl)2CuLLNA]2+ and (b) [(LLNA)2Cu]2+. The systems were fully optimized by a QM/QM approach with the PBE1PBE functional. For details, see the Computational Details section.
Figure 5. Enlarged view of the interstrand complexes in the duplex models shown in Figure 4: (a) [LLNACu(µ-Cl)2CuLLNA]2+ and (b) [(LLNA)2Cu]2+.
formation of the complexes was referenced to the duplex model with two noninteracting ligands at the LNA sites. This is in line with the larger stability of the chloride-containing individual complexes. Naturally, the interaction energies were highly overestimated because solvent effects could not be taken into account. Furthermore, owing to the huge size of the calculated systems, the level of the theory had to be kept quite limited. Since both of the complexes optimized in very similar manner and with favorable but largely differing interaction energies, it can be concluded that both the monometallic [(LLNA)2Cu]2+ complex and especially the [LLNACu(µ-Cl)2CuLLNA]2+ complex are able to form an interstrand linkage between two closely located LNA sites in the double helix structure. Figure 5 presents more detailed optimized geometries of the interstrand complexes enlarged separately from the duplex model. The structures can be compared to the freely optimized model complexes shown in Figure 2. Selected geometrical parameters are collected in Table 4. Although the double helix structure remains more or less intact, the geometry of the [Cu(µ-Cl)2Cu] moiety is perturbed during the optimization: one of the chloride bridges shortens, and one of the Cu-L(carbonyl) bonds is broken to give one five-coordinated copper ion, while the other one remains effectively six-coordinated. Opening up one coordination site
Cu(1)-Cl(1) Cu(1)-Cl(2) Cu(1)-O(1) Cu(1)-N(1) Cu(1)-N(2) Cu(1)-N(3) Cu(2)-Cl(2) Cu(2)-Cl(1) Cu(2)-O(3) Cu(1)-N(4) Cu(1)-N(5) Cu(1)-N(6) Cu(1) · · · Cu(2) Cl(1)-Cu(1)-Cl(2) Cl(1)-Cu(2)-Cl(2) Cu(1)-Cl(1)-Cu(2) Cu(1)-Cl(2)-Cu(2)
2.284 3.052 2.296 2.015 2.103 2.008 2.284 3.052 2.296 2.008 2.103 2.015 3.920 86.5 86.6 93.5 93.4
2.263 2.690 2.545 2.028 2.124 2.011 2.579
2.008 2.127 2.026 5.100 97.3
150.9
2.066 2.491 2.094
2.408 2.157 2.067
2.281 3.005 2.457 1.934 2.224 1.978 2.248 2.406 2.032 2.012 1.988 3.240 88.9 106.9 87.4 74.6
2.231 2.165 2.004
2.324 2.144 2.022
would enable an additional chloride ion to bind, eventually leading to a singly bridged complex resembling [LClCu(µCl)CuClL]+ (Figure 2b). This is in accordance with the small energy difference of the singly and doubly chloride bridged dinuclear complexes and shows that they are both potential models for the interstrand linkages. The presence of oligonucleotide strands leads to a smaller Cu-Cl-Cu angle and to slightly different torsional behavior of the ligands, but effectively, the resulting interstrand complex has a double chloride bridge comparable to [LCu(µ-Cl)2CuL]2+. The geometry in the interstrand complex is somewhat more asymmetric, probably because of the missing carbonyl interaction in one of the copper ions. This can also be seen in the Cu-Cl distances: whereas in the doubly bridged dimeric complex [LCu(µ-Cl)2CuL]2+, the distances Cu(1)-(µ-Cl) and Cu(2)-(µ-Cl) are clearly different (2.28 and 3.05 Å, respectively), in [LClCu(µ-Cl)CuClL]+, the two Cu-(µ-Cl) distances are similar (2.69 and 2.57 Å). In the interstrand complex, two of the bridging Cu-Cl distances are shorter and similar (2.28 and 2.41 Å), but the other two are different (3.01 and 2.25 Å), indicating formation of an asymmetric, fairly strong double chloride bridge. Inclusion of solvent effects in the calculation might have some impact on the optimized geometries, but as was shown in the case of the separate dinuclear chloro-bridged complex (Table 1), the effect of the solvent on the overall geometry is minimal. In the case of the bis-ligand complex, the structure of the interstrand linkage resembles closely the separately optimized [(Lamide)2Cu]2+, and no major differences could be found in the coordination sphere of the metal ion, as in the case of the chloride-bridged systems. Both gas phase optimizations led to rather unsymmetric Cu-N bond distances, probably because of steric hindrance around the copper ion formed by the bulky N,N-bis(2-pyridylmethyl) ligands in cis conformation. On the other hand, the cis conformer was found to be about 9 kJmol-1 (25 kJ/mol in the water solvent) more favorable than the trans conformer in the optimization of [(Lamide)2Cu]2+ (Table S2). Furthermore, the trans conformer would not fit as well into the groove of the oligonucleotides because of the larger ligand core. Obviously, the optimized interstrand complexes do not correspond to the global minima, since the optimization was performed only from the most likely initial geometries (although
Cross-Linking Transition Metal Complexes finding the most likely ones required several attempts). Nevertheless, the successfully optimized structures and the favorable energies verify that the dichloride bridged [LLNAM(µ-Cl)2MLLNA]2+ complex is a potential option for the interstrand linkage responsible for the duplex stabilization, bearing in mind the environment of the reaction including copper ions and an very large excess of chloride ions. Conclusions A potential option for a duplex stabilizing interstrand complex is suggested on the basis of the computational DFT calculations. The computational geometry agrees very well with the synthesized [LLNACu(µ-Cl)2CuLLNA]2+ model complex, which is the first reported example of a metal complex of an LNA-modified ligand. The complex contains a doubly bridged [M(µ-Cl)2M] core, and we suggest that this and the singly bridged relative [ClM(µ-Cl)MCl] represent chemically feasible structures to participate in interstrand linkages between LNA-modified DNA duplexes. Optimized models verify that such a link does not disrupt Watson-Crick base pairing in the double-helix structure, and the extra covalent and electrostatic bonding between the strands that this can provide may explain the observed increased thermal stability of ligand modified duplexes geometrically capable of forming such links in the presence of divalent metal ions and large excesses of chloride.24 This type of interstrand link could show some geometric flexibility by interconversion between the mono and dibridged chloride complexes controlled by chloride concentration, leading to speculation as to whether these types of linker units could be employed for molecular control of DNA structural switching. Comparison through DFT calculations of the relative stabilities of chloride bridged dimetallic complexes, [(Lamide)M(µCl)2M(Lamide)]2+ and [(Lamide)ClM(µ-Cl)MCl(Lamide)]+, and mononuclear complexes containing combinations of Lamide, chloride and water ligands shows that the chloro-bridged dinuclear complexes are significantly more stable, especially for copper ions. The computational reaction energies show a relative stability order of Cu(II) > Ni(II) > Zn(II), which becomes even more pronounced in polar solvents. These results are entirely consistent with previously reported measurements of the denaturation temperatures of 9-mer N,N-bis(2-pyridylmethyl)-βalanyl-LNA functionalized DNA made in the presence of copper, nickel, and zinc with a large excess of chloride. Our model is also chemically and structurally reasonable in light of the fact that it takes into account the chemical environment, apart from a potential chelating ligand, that is, quantity and type of potentially coordinating solvent and anions, which is often overlooked, when predicting the formation and structure of supramolecular systems based coordination bonding. This is clearly extremely important to keep in mind when the study of motifs of the type described here move from in vitro to in vivo situations. Experimental Section General: Elemental analyses were performed at the Chemistry Department at Copenhagen University, Denmark. IR spectra of the complexes in KBr discs were measured using a Hitachi 270-30 IR spectrometer. Electrospray ionization mass spectra (ESI MS) were obtained using a Q-Star Pulsar quadrupole timeof-flight mass spectrometer (Applied Biosystem/MDS Sciex) equipped with a nanospray ion source. Syntheses: N,N-Bis(2-pyridylmethyl)-β-alanyl-2′-amino-LNA (LLNA)35 and N,N-bis(2-pyridylmethyl)-β-alanine36 were prepared as described previously. Perchlorate salts of metal complexes
J. Phys. Chem. B, Vol. 114, No. 36, 2010 11947 are potentially explosive and should be handled with caution in small quantities. [Cu2Cl2(LLNA)2](ClO4)2 · 2CH3OH H2O (1). A few milligrams of crystals of 1 suitable for X-ray crystallographic analysis were prepared by slow evaporation of a mixture of LLNA (4.6 mg, 9 µmol) in methanol (0.5 mL), CuCl2 · 2H2O (1.9 mg, 11 µmol) in methanol (0.25 mL), and NaClO4 · H2O (1.9 mg, 14 µmol) in methanol (0.25 mL). A larger sample of 1 was prepared by adding solid CuCl2 · 2H2O (106.5 mg, 204 µmol) and NaClO4 · H2O (34.2 mg, 201 µmol) to LLNA (28.4 mg, 202 µmol) in methanol (3 mL). After 4 days, the product was deposited as blue microcrystals (54 mg, 37%). The X-ray powder diffraction pattern of the bulk sample was consistent with the pattern calculated from the structure of 1. ESI MS, IR and elemental analysis were performed on the larger sample. ESI-MS (CH3CN) m/z: 620.1 ([CuCl(LLNA)]+, 100%), 584.1 ({[Cu(LLNA)] - H}+, 24%), 492.1 ({[Cu(LLNA)] - C6H6N (pyridyl) - H}+, 3%), 459.1 ({[Cu(LLNA)] - C5H5N2O2 (thyminyl) - H}+, 72%), 399.1 ({[Cu(LLNA)] - 2 × C6H6N 2 H}+, 4%). IR (KBr) ν (cm-1): 1695 (CdO, vs), 1121, 625 (ClO4-). Anal. Calcd. (%) for C54H66N12O23Cl4Cu2 [Cu2Cl2(LLNA)2](ClO4)2 · 3H2O C, 41.75; H, 4.45; N: 11.23. Found C, 41.78; H, 4.35; N, 11.01. [Cu2Cl2(Lester)2](ClO4)2 (2) and [Cu2Cl2(Lester)2](ClO4)2 · CH3OH (3). Simple analogues of 1, complexes 2, and 3, were synthesized for comparisons of mass and IR spectra, since only a milligram quantity of 1 was available. Detailed synthesis of 2 and 3 has been included in Supporting Information. Single-Crystal X-ray Diffraction. Crystals of 1 were examined at the microcrystal diffraction beamline I711 at the MAX-II storage ring, MAXLab, University of Lund, Sweden. Diffraction data for 2 was collected using a Bruker-Nonius X8 APEX-II instrument. The structures were solved by direct methods, and refinements were carried out with full-matrix leastsquares methods on all F2 data using SHELXTL. Crystal data for 1: [C52H60Cl2Cu2N12O12](ClO4)2 · 2CH3OH · H2O; M ) 1524.10; monoclinic; C2 (no. 5); a ) 35.166(2), b ) 8.308(5), c ) 22.479(1) Å; β ) 101.477(4)°; V ) 6436(4) Å3; Z ) 4; T ) 180(2) K; µ(λ ) 0.8970 Å) ) 0.915 mm-1; Fcalc ) 1.573 g cm-3; 2θmax ) 64.0°; 19 176 reflections measured; 9719 unique (Rint ) 0.114); R1 ) 0.108 (7973 reflections with I > 2σ(I)); wR2 ) 0.3342 (all data); goodness of fit (GOF) ) 1.21; 859 parameters, 25 restraints (Cl-O and O · · · O distances in perchlorate anions). Crystal data for 2: [C32H38Cl2Cu2N6O4](ClO4)2; M ) 967.56; monoclinic; P21/c (no. 14); a ) 8.0644(3), b ) 19.3905(6), c ) 12.4390(4) Å; β ) 99.560(1)°; V ) 1918.11(11) Å3; Z ) 2; T ) 180(2) K; µ (Mo KR) ) 1.457 mm-1; Fcalc ) 1.675 g cm-3; 2θmax ) 53.0°; 16 853 reflections measured; 3897 unique (Rint ) 0.041); R1 ) 0.056 (2956 reflections with I > 2σ(I)); wR2 ) 0.1189 (all data); goodness of fit (GOF) ) 1.13; 281 parameters, 19 restraints (Cl-O and O · · · O distances in perchlorate anions. CCDC-733243-733245 contain(s) the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Computational Details. All calculations were carried out using the Gaussian03 program package.37 DFT level of theory with the hybrid GGA density functional PBE1PBE was selected for the quantum chemical studies38 The basis set comprised the Stuttgart-Dresden effective small core potential39 for the metal atoms (SDD) and a standard all-electron basis set 6-31G* for C and H atoms. For interacting Cl, O, and N atoms, a standard 6-311+G(d) basis set was used. Frequency analysis with no
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scaling was performed to ensure optimization to minima. Solvent effects were included in the optimizations with a polarized continuum model (PCM).40 In the PCM method, the solute is placed in a cavity within the solvent reaction field, described mainly by the dielectric constant of the solution. The solute cavity is created via a set of overlapping spheres located at the nuclei. To obtain reliable reaction energies, the spin state of each complex was optimized. In the dinuclear complexes, the lowest-energy spin states are quintet for Ni2+, triplet for Cu2+, and singlet for Zn2+. For the mononuclear complexes, the spin state was optimized for the [ML2]2+ complexes, then other mononuclear complexes were calculated within the spin state obtained: triplet for Ni2+, doublet for Cu2+, and singlet for Zn2+. Because of the huge size of the tetrameric ATLAT-TATLA model for the double helix structure, the systems shown in Figure 4 were described with a QM/QM method: the high level contained the [LCu(µ-Cl)2CuL] and [L2Cu] complexes, described using the same basis functions as for the isolated molecular complexes; the low level contained the tetrameric model for the duplex structure, where Huzinaga’s MINI basis set41 was used for C, O, and N atoms, 6-31G(d) for P and 6-31G for H. Full optimization was performed with the PBE1PBE functional. Acknowledgment. We are grateful to Dr. B. Ravindra Babu and Professor Jesper Wengel, Nucleic Acid Center, University of Southern Denmark, for providing a sample of LLNA. We acknowledge The Academy of Finland (Grant 129772), The Danish Natural Sciences Research Council and Nordforsk for financial support, and MAXLab, University of Lund, Sweden for synchrotron access. Supporting Information Available: Crystallographic data for [LCu(µ-Cl2)CuL]2+ model complexes (L ) N,N-bis(2pyridylmethyl)-β-alanyl-2′-amino-LNA (1) or L ) N,N-bis(2pyridylmethyl)-β-alanine methylester (2 and 3)) in CIF format. ESI-MS of 1. Synthesis methods of all synthesized complexes. Comparison of relative energies of the computationally optimized cis and trans conformers of dinuclear and mononuclear M-LLNA complexes (M ) Ni, Cu, Zn), in the gas phase and in aqueous solution. This information is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Duguid, J.; Bloomfield, V. A.; Benevides, J.; Thomas, G. J., Jr. Biophys. J. 1993, 65, 1916–1928. (2) Martin, R. B. Acc. Chem. Res. 1985, 18, 32–38. (3) Eichorn, G. L.; Shin, Y. A. J. Am. Chem. Soc. 1968, 90, 7323– 7328. (4) Tanaka, K.; Shionoya, M. Coord. Chem. ReV. 2007, 251, 2732– 2742. (5) He, W.; Franzini, R. M.; Achim, C. In Progress in Inorganic Chemistry; Karlin, K. D., Ed.; John Wiley & Sons, Inc.: New York, 2007, Vol. 55; pp 545-611. (6) Zimmermann, N.; Meggers, E.; Schultz, P. G. J. Am. Chem. Soc. 2002, 124, 13684–13685. (7) Miyake, Y.; Togashi, H.; Tashiro, M.; Yamaguchi, H.; Oda, S.; Kudo, M.; Tanaka, Y.; Kondo, Y.; Sawa, R.; Fujimoto, T.; Machinami, T.; Ono, A. J. Am. Chem. Soc. 2006, 128, 2172–2173. (8) Clever, G. H.; Polborn, K.; Carell, T. Angew. Chem., Int. Ed. 2005, 44, 7204–7208. (9) Bashkin, J. K.; Frolova, E. I.; Sampath, U. J. Am. Chem. Soc. 1994, 116, 5981–5982. (10) Trawick, B. N.; Daniher, A. T.; Bashkin, J. K. Chem. ReV. 1998, 98, 939–960. (11) Sakamoto, S.; Tamura, T.; Furukawa, T.; Komatu, Y.; Ohtsuka, E.; Kitamura, M.; Inoue, H. Nucleic Acids Res. 2003, 31, 1416–1425.
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