DNA Mismatch Recognition by a Hexacoordinate Silicon Sandwich

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DNA Mismatch Recognition by a Hexacoordinate Silicon Sandwich− Ruthenium Hybrid Complex Chen Fu,† Klaus Harms,† Lilu Zhang,† and Eric Meggers*,†,‡ †

Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, 35043 Marburg, Germany College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: The diastereoselective synthesis of two dinuclear Ru−Si complexes is reported, in which silicon(IV) is coordinated in an octahedral fashion by two 1,10-phenanthrolines and one 4,5pyrenediolato ligand and additionally η6-coordinated to a (η5pentamethylcyclopentadienyl)ruthenium(II) moiety through one fused benzene ring of the pyrene ligand. One of these Ru−Si hybrid complexes was found to selectively stabilize DNA duplexes that contain cytosine− cytosine or cytosine−thymine mismatches, and it is proposed that this occurs by a novel dual insertion/intercalation binding mode in which the entire ruthenium sandwich unit is introduced into the DNA π-stacking at the site of the DNA mismatch.

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Scheme 1. Synthesis of the Dinuclear Ru−Si Complexes RuSi1 and RuSi2

ubstitutionally inert octahedral transition-metal complexes are prevalent templates for the design of noncovalent DNA binding agents.1 Beyond groove binding and intercalation, Barton and co-workers recently established a prior unobserved binding mode, termed “insertion”,2 in which a “metalloinsertor” introduces one planar ligand into the DNA π-stacking by ejecting nucleobases so that the planar ligand serves as a replacement of a base pair.3,4 Barton exploited this unique mode of binding for the recognition of mismatched base pairs in duplex DNA. However, the design of completely selective DNA insertors constitutes a significant challenge, since the planar aromatic ligands of DNA insertors might also possess an intrinsic tendency to intercalate into DNA in a more unspecific fashion by locally unwinding the DNA duplex and placing the planar aromatic ligand between two intact Watson−Crick base pairs.4 Recently, our group started to investigate hexacoordinate silicon as a candidate to serve as a structural substitute for octahedral transition metals.5,6 In a proof-of-principle study we were able to demonstrate that octahedral silicon complexes (e.g., Si15 in Scheme 1) can serve as robust, hydrolytically stable scaffolds7,8 for the design of hexacoordinate silicon-based DNA intercalators, displaying DNA affinities comparable to those of typical metallo DNA intercalators. Here we now wish to report how we converted a canonical silicon DNA intercalator into a selective DNA mismatch recognizing agent by introducing a ruthenium sandwich moiety. Accordingly, the reaction of the (4,5-pyrenediolato)silicon(IV) complexes Si1 and Si2 with [Cp*Ru(MeCN) 3 ]PF 6, with Cp* = η 5pentamethylcyclopentadienyl, in dry 1,2-dichloroethane at 60 °C for 40 h afforded in a diastereoselective fashion the sandwich complexes RuSi1 and RuSi2 in yields of 40% and 42%, respectively.9 The dinuclear complexes RuSi1 and RuSi2 © XXXX American Chemical Society

are quite stable and can be purified by standard silica gel column chromatography. Single crystals of RuSi1 suitable for X-ray diffraction were grown by slow evaporation of an acetone/water solution. An ORTEP drawing is presented in Figure 1 and demonstrates that silicon is coordinated in an octahedral fashion by two 1,10phenanthrolines and one 4,5-pyrenediolato ligand and additionally η6-coordinated to a Cp*Ru moiety through a fused benzene ring of the pyrene ligand. The Cp* moiety and η6coordinated pyrene are oriented almost parallel and separated by a distance of 3.520 Å.10 The structure also reveals the reason for the observed diastereoselectivity, since a coordination of the Cp*Ru moiety from the other face of the same fused benzene ring of the pyrene would lead to a steric clash between the methyl groups of the Cp* ring and one of the propeller-like 1,10-phenanthroline ligands. The apparently only limited steric constraint between the Cp* moiety and one 1,10-phenanthroReceived: April 8, 2014

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and we attribute this to steric effects of the bulky methylated 3,4,7,8-tetramethyl-1,10-phenanthroline ligands which must interfere with a proper binding of the complexes to DNA.11 In contrast, the established DNA intercalator Si15 resulted in a strong stabilization of the duplex DNA by an increase of the melting temperature (Tm) by ΔTm = 3.0 °C, whereas the ruthenium−silicon hybrid RuSi1 did not change the Tm value significantly (ΔTm = 0.1 °C). This is not unexpected, since the η6 coordination of the Cp*Ru moiety onto the pyrene-4,5diolato ligand must prevent its intercalation into duplex DNA. However, we were surprised to find that RuSi1 instead stabilizes DNA duplexes containing cytosine−cytosine (CC) and cytosine−thymine (CT) mismatches by ΔTm = 7.2 °C (Table 1 and Figure 2). RuSi1 did not affect the melting point

Figure 1. Crystal structure of the complex RuSi1: ORTEP representation with 50% thermal ellipsoids. Three hexafluorophosphate anions and a solvent molecule (acetone) are omitted for clarity. Selected bond distances (Å) and angles (°): N1−Si1 1.939(4), N4− Si1 1.927(4), N15−Si1 1.933(4), N18−Si1 1.916(4), O1−Si1 1.727(4), O2−Si1 1.717(4), C38−Ru1 2.241(6), C39−Ru1 2.201(6), C40−Ru1 2.210(6), C41−Ru1 2.223(6), C42−Ru1 2.264(5), C43−Ru1 2.266(5), C45−Ru1 2.152(6), C46−Ru1 2.167(6), C47−Ru1 2.180(5), C48−Ru1 2.171(6), C49−Ru1 2.145(6); O1−Si1−O2 92.32(17), O1−Si1−N15 89.59(18), O2− Si1−N1 87.61(17), N15−Si1−N1 90.81(18), N4−Si1−N18 172.12(19).

Figure 2. UV-melting curves (λ 260 nm) of mismatched duplex DNA (2 μM) in Tris-HCl (5 mM, pH 7.4) with NaCl (50 mM) in the absence and presence of the complex RuSi1. Tm values were determined from three independent experiments, and standard deviations of ±0.1−0.5 °C were determined.

line ligand in RuSi1 affects neither any silicon bond length nor the silicon coordination geometry5 but leads to a slight twisting of the coordinated 4,5-pyrenediolato ligand. We next investigated the influence of the complexes Si1,2 and RuSi1,2 on the thermal stability of duplex DNA and therefore monitored the melting of a mixed-sequence 19mer duplex DNA (2 μM) in the presence and absence of 1 equiv of silicon complexes or ruthenium−silicon hybrids by temperature-dependent UV spectroscopy at 260 nm. As shown in Table 1, the methylated derivatives Si2 and RuSi2 displayed overall little influence on the melting behavior of duplex DNA,

of a duplex harboring the less destabilizing cytosine−adenine (CA) mismatch (ΔTm = 0.1 °C). For comparison, the intercalator Si1 increases the thermal stability of both matched and mismatched duplexes by a more narrow range of ΔTm = 0.7−3.0 °C. On the basis of the geometry of the silicon− ruthenium hybrid RuSi1, in which the distance between the planes of the Cp* moiety and the pyrene ring is equal to the distance between two Watson−Crick base pairs in B-form duplex DNA, we propose that RuSi1 binds in a combined intercalation/insertion mode. According to this model, the Cp*Ru(pyrene) sandwich unit inserts itself into the π-stacking of the duplex DNA at the site of the CC or CT mismatch by ejecting the mismatched pyrimidine nucleobases and at the same time locally unwinding the DNA duplex to create additional space for the entire sandwich unit. This combined intercalation/insertion binding mode would also nicely explain the low affinity of RuSi1 for regular duplex DNA. It is noteworthy that a coordinative interaction between RuSi1 and DNA can be excluded, since the hybrid complex is completely hydrolytically stable in the presence of water, as has been verified by 1H NMR (Figure 3). In conclusion, we here report the selective DNA mismatch recognition of an unusual silicon−ruthenium hybrid complex and propose a novel binding mode that consists of a combination of insertion by base ejection and intercalation through local DNA unwinding. These results are potentially of

Table 1. UV-Melting Points (°C) of Matched and Mismatched DNA Duplexes in the Absence and Presence of 1 Equiv of Silicon Complexes or Ruthenium−Silicon Hybridsa

complex none Si1 Si2 RuSi1 RuSi2

X=G 61.8 64.8 62.9 61.9 61.8

(+3.0) (+1.1) (+0.1) (±0)

X=A 53.3 56.2 53.4 53.4 53.7

(+2.9) (+0.1) (+0.1) (+0.4)

X=C 51.0 53.7 51.0 58.2 51.1

(+2.7) (±0) (+7.2) (+0.1)

X=T 50.5 51.2 50.6 57.7 50.9

(+0.7) (+0.1) (+7.2) (+0.4)

a

Changes in UV absorbance upon heating were monitored at 260 nm. Conditions: 5 mM Tris-HCl, 50 mM NaCl, pH 7.4, and 2 μM of each strand. Tm values were determined from three independent experiments, and the mean values were taken. Abbreviations: G = guanine, A = adenine, C = cytosine, T = thymine. B

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= 9.6 Hz, 1H), 8.84 (d, J = 9.6 Hz, 1H), 8.78 (m, 2H), 8.33 (d, J = 9.3 Hz, 1H), 8.18 (d, J = 7.7 Hz, 1H), 8.10 (m, 3H), 8.03 (t, J = 7.7 Hz, 1H), 7.80 (d, J = 9.3 Hz, 1H), 6.78 (d, J = 5.9 Hz, 1H), 6.66 (d, J = 5.9 Hz, 1H), 6.33 (t, J = 5.9 Hz, 1H), 3.21 (s, 3H), 3.01 (s, 3H), 2.99 (s, 3H), 2.98 (s, 3H), 2.82 (s, 3H), 2.46 (s, 3H), 2.35 (s, 3H), 2.34 (s, 3H), 1.09 (s, 15H). 13C NMR (100 MHz, acetone-d6): δ (ppm) 159.5, 158.6, 157.5, 156.7, 149.2, 149.1, 147.3, 147.2, 141.8, 139.1, 138.8, 138.7, 138.3, 134.8, 134.7, 134.6, 134.1, 133.7, 133.1, 132.9, 130.2, 129.7, 129.6, 129.4, 129.1, 128.0, 126.8, 126.5, 126.2, 125.9, 125.8, 125.6, 124.4, 122.1, 121.3, 94.4, 93.2, 91.7, 88.4, 88.3, 85.9, 79.8, 18.8, 18.5, 18.2, 16.6, 16.3, 16.1, 8.9. 29Si NMR (79.5 MHz, 243 K, acetoned6): δ (ppm) −148.6 ppm. IR (neat): ν (cm−1) 1626, 1545, 1443, 1368, 1322, 1269, 1219, 1178, 1100, 1031, 833, 722, 641, 592, 554, 520, 444, 410. HRMS (ESI): calcd for C58H55F12N4O2P2RuSi (M − P F 6 ) + 125 9. 240 4, found 1 25 9.2 43 2. A n al . Calcd f or C58H55F18N4O2P3RuSi: C, 49.61; H, 3.95; N, 3.99. Found: C, 49.37; H, 4.08; N, 4.24. X-ray Diffraction. Single crystals of the complex RuSi1·C3H6O (C53H45F18N4O3P3RuSi, Mr = 1359.00) suitable for X-ray diffraction measurement were obtained by slow evaporation of acetone from a water/acetone solution. The X-ray diffraction study was conducted on a Bruker D8 Quest diffractometer with Mo Kα radiation (λ = 0.71073 Å) at 100(2) K using a crystal with dimensions 0.26 × 0.16 × 0.11 mm3. Cell parameters were obtained by global refinement of the positions of 9832 reflections: monoclinic cell with a = 13.2610(5) Å, b = 13.0303(6) Å, c = 30.4909(13) Å, β = 92.1125(16)°, space group P21/c. With Z = 4, the calculated density was 1.703 Mg/m3 and the absorption coefficient 5.26 cm−1. A total of 25495 reflections were collected, 9787 of which were independent (Rint = 0.038) and 7298 observed (I > 2σ(I)). Intensities were corrected for Lorentz and polarization effects and absorption. The structure was solved by direct methods with SHELXS-9712 and refined by full-matrix least squares on F2 using SHELXL-2013.13 All hydrogen atoms were placed in calculated positions. During refinement, restraints were included for geometry and displacement factors of disordered hexafluorophosphate ions. Refinement of 947 parameters converged to wR2 = 0.181 (all data) and R1 = 0.067 (observed data). The “goodness of fit” was 1.079. CCDC 995005 contains 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. UV-Melting Experiments. The melting studies were carried out in 1 cm path length quartz cells (total volumn 325 μL; 250 μL sample solutions were covered by mineral oil) on a UV/vis spectrophotometer equipped with a thermo-programmer. Melting curves were monitored at 260 nm with a heating rate of 1 °C/min. Melting temperatures were calculated from the first derivatives of the heating curves. Experiments were performed in triplicate, and mean values with standard deviations were determined.

Figure 3. Hydrolytic stability of complex RuSi1 at room temperature in DMSO-d6/D2O (3/1) as verfied by 1H NMR.

significant relevance, as they might lead to a new general concept for the design of selective DNA mismatch recognizing agents which is based on the parity of the distances between two aromatic moieties of organometallic sandwich complexes and two Watson−Crick base pairs of B-form DNA duplexes.

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EXPERIMENTAL SECTION

RuSi1. A suspension of Si15 (100 mg, 0.11 mmol) and [η5Cp*Ru(MeCN)3]PF6 (50 mg, 0.10 mmol) in dry 1,2-dichloroethane (20 mL) was purged with N2 for 15 min and then heated at 60 °C for 40 h. The resulting dark brown mixture was cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography over silica gel, first with acetone and thereafter switched to acetone/H2O/KNO3(saturated) (50/3/1) and finally to acetone/H2O/KNO3(saturated) (10/3/1). The combined product eluents were dried and dissolved in minimal amounts of H2O. The product was precipitated by the addition of excess solid NH4PF6. The collected solid was washed twice with H2O and dried under high vacuum to afford the pure complex RuSi1 (52 mg, 40%). 1H NMR (500 MHz, acetone-d6): δ (ppm) 10.2 (d, J = 4.9 Hz, 1H), 9.81 (s, 1H), 9.80 (s, 1H), 9.53 (d, J = 8.2 Hz, 1H), 9.45 (m, 2H), 8.96 (dd, J = 7.6, 6.0 Hz, 1H), 8.89 (d, J = 9.2 Hz, 1H), 8.83 (d, J = 9.2 Hz, 1H), 8.77 (m, 2H), 8.47 (m, 3H), 8.33 (d, J = 9.2 Hz, 1H), 8.20 (m, 3H), 8.09 (d, J = 7.7 Hz, 1H), 8.03 (t, J = 7.7 Hz, 1H), 7.82 (d, J = 9.3 Hz, 1H), 6.75 (d, J = 6.1 Hz, 1H), 6.69 (d, J = 6.1 Hz, 1H), 6.35 (t, J = 6.0 Hz, 1H), 1.13 (s, 15H). 13C NMR (125 MHz, acetone-d6): δ (ppm) 150.6, 150.4, 148.5, 148.4, 148.1, 147.2, 146.88, 146.86, 141.9, 136.1, 135.9, 135.1, 134.9, 134.7, 133.2, 133.0, 131.6, 131.5, 131.3, 131.1, 130.5, 130.3, 130.1, 130.0, 129.8, 129.5, 129.4, 129.2, 128.2, 126.6, 126.2, 122.4, 121.5, 94.3, 93.5, 91.8, 88.4, 88.2, 85.8, 79.9, 9.2. 29Si NMR (79.5 MHz, 243 K, acetone-d6): δ (ppm) −148.1 ppm. IR (neat): ν (cm−1) 1628, 1529, 1440, 1366, 1326, 1219, 1159, 1098, 1033, 832, 762, 724, 588, 555, 519, 482, 453. HRMS (ESI): calcd for C50H39F12N4O2P2RuSi (M − PF6)+ 1147.1142, found 1147.1177. Anal. Calcd for C50H39F18N4O2P3RuSi: C, 46.48; H, 3.04; N, 4.34. Found: C, 46.65; H, 3.27; N, 4.56. RuSi2. A suspension of Si2 (100 mg, 0.098 mmol) and [η5Cp*Ru(MeCN)3]PF6 (44 mg, 0.088 mmol) in dry 1,2-dichloroethane (20 mL) was purged with N2 for 15 min and then heated at 60 °C for 40 h. The resulting dark brown mixture was cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography over silica gel, first with acetone and thereafter switched to acetone/H2O/KNO3(sat) (50/3/1) and finally to acetone/H2O/KNO3(sat) (10/3/1). The combined product eluents were dried and dissolved in minimal amounts of H2O. The product was precipitated by the addition of excess solid NH4PF6. The collected solid was washed twice with H2O and dried under high vacuum to afford the pure complex RuSi2 (52 mg, 42%). 1H NMR (400 MHz, acetone-d6): δ (ppm) 9.78 (s, 1H), 9.41 (s, 1H), 8.89 (d, J

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ASSOCIATED CONTENT

S Supporting Information *

Text, figures, tables, and a CIF file giving NMR spectra and crystallographic data for RuSi1 and details of the synthesis of silicon complex Si2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail for E.M.: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support was provided by a LOEWE research cluster (SynChemBio) of the Federal State of Hessen, Germany. C

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REFERENCES

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