Crystal Structure , Supramolecular Self-Assembly and Interaction with DNA of a Mixed Ligand Manganese ( II ) Complex

Um complexo misto de manganês(II), [Mn(sal)(phen)2]ClO4 (1) (sal = salicilaldeído, phen = 1,10-fenantrolina), foi sintetizado e caracterizado por análise elementar e difração de raio-X de monocristais. A análise dos difratogramas revelou que o complexo 1 cristaliza no grupo espacial monoclínico P21/n. A unidade assimétrica do complexo é composta do cátion complexo [Mn(sal)(phen)2] + e do ânion perclorato não coordenado. O manganês(II) está num ambiente de coordenação MnN4O2 de geometria octaédrica bem distorcida. Quatro tipos de empilhamento p-p envolvendo ligantes fenantrolina e salicilaldeído estão envolvidos na auto-montagem supramolecular. Estas interações de empilhamento aromático cooperam com ligações de hidrogênio para a montagem de uma fascinante arquitetura supramolecular 3D com o complexo. Estudos de espectroscopia de absorção eletrônica e de titulação monitorando-se a fluorescência de 1 com DNA de timo de bezerro sugerem a ligação do complexo por intercalação com uma constante de formação igual a 7,97×10 L mol e uma constante de supressão de Stern-Volmer de 1,34×10 L mol.


Introduction
2][3][4][5] Metal complex can bind to DNA in non-covalent way such as by electrostatic interaction, minor or major groove-bound fashion and intercalation. 6,7][10][11][12][13] A metallointercalator binds DNA through the insertion of a planar polycyclic aromatic ligands, p-stacking in between two base pairs. 14,15So the binding site and the intensity of a metallointercalator can be tuned by the metal coordination center and chelate ligand.

Materials and methods
Calf thymus DNA was purchased from the Sigma Company.All other chemicals and solvents were acquired from commercial sources and used as received, unless otherwise noted.Tris-HCl buffer solution (pH 7.66, Tris = tris(hydroxymethyl)aminomethane) was prepared using redistilled water.
The C, H and N microanalyses were performed on a Perkin-Elmer 240 elemental analyzer.The electronic spectrum was measured on a Cary 300 spectrophotometer.The fluorescence spectra were measured with a FP-6200 fluorescence spectrophotometer.

Determination of X-ray crystal structure
The X-ray diffraction experiment for complex 1 was carried out on a Bruker Smart-1000 CCD diffractometer with graphite monochromated Mo K α α(l = 0.71073 Å) radiation at room temperature.Data collection, cell refinement, data reduction and correction for absorption were performed using Bruker programs. 24The structure was solved by direct method and expanded using the Fourier transform package in Shelxl-97 program. 25The nonhydrogen atoms were refined anisotropically by full-matrix least-square calculations on F 2 .The C-bound H atoms were placed in calculated positions, with C-H distance of 0.93 Å, and refined as riding, with U iso (H) = 1.2U eq (C).Detailed information about crystal data and structure determinations is summarized in Supplementary Information (Table S1).

Interaction of complex with DNA
All experiments involving CT-DNA were performed in Tris-HCl buffer solution.Stock solution of the CT-DNA was stored at 4 o C and used in no more than four days.The ratio of UV absorbance at 260 and 280 nm is about 1.9, which indicates that the DNA was sufficiently independent of protein in the buffer. 26The CT-DNA concentration was determined spectrophotometrically using the molar absorptivity of 6600 L mol −1 cm −1 at 260 nm. 27oncentrated stock solution of complex 1 was prepared in DMF and the solution was diluted with Tris-HCl buffer.Spectrophotometric titration was performed by adding increments of the CT-DNA to the complex solution and incubating for 10 min before recording the signal.The fluorescence titration experiment was performed by keeping the concentration of complex 1 constant while increasing the CT-DNA concentration.The fluorescence spectra of complex 1 were recorded exciting at 254 nm and monitoring at 366 nm.Fluorescence quenching experiments were carried out with CT-DNA pretreated with ethidium bromide (EB) in the dark for at least 2 h.Increasing amounts of complex solution were added to the pretreated CT-DNA solution and their effect on emission intensity was measured.The ethidium bromide displacement spectra were recorded exciting at 522 nm and monitoring at 583 nm.

Description of the crystal structure
The asymmetric cell of complex 1 is comprised of [Mn(sal)(phen) 2 ] + complex cation and uncoordinated perchlorate anion.The Oak Ridge thermal ellipsoid plot of complex cation is depicted in Figure 1.Complex 1 crystallizes in the monoclinic space group P2 1 /n.In [Mn(sal)(phen) 2 ] + complex cation, Mn(II) is coordinated by two O atoms from one salicylaldehyde and four N atoms from two phenanthroline ligands.Four atoms, N1, N2, N4 and O2 are located at the equatorial plane with the mean deviation of 0.144 Å.The manganese center is only 0.062 Å displaced from the equatorial plane.][30] Perchlorate anion is not directly coordinated to the metal center but forms a series of hydrogen bonds in the supramolecular structure.

Non-covalent interactions and supramolecular selfassembly
Crystal structure analysis and calculation revealed abundant non-covalent interactions such as p-p stacking interactions and hydrogen bonds in complex 1.2][33][34] Our previous studies demonstrate that 1,10-phenanthroline is one of the most favorable bidentate ligands to display p-p stacking supramolecular interactions in the crystal due to its large aromatic system. 35As dealt with before, 36,37 two stacking phenanthroline molecules are divided into four pyridine (A, A′, C and C′) and two arene (B and B′) segments in order to facilitate the description of this extended p system.The corresponding stacking data such as the interplanar separation (h), dihedral angle (α), centroid-centroid distance (d), centroid displacement (r) and slip angle (q) were calculated (Table S3).The analysis of stacking data indicates four types of intermolecular p-p stacking modes, i.e. (I) phenanthroline-phenanthroline (phen-phen), (II) pyridine-pyridine (py-py), (III) pyridinearene (py-ar) offset face-to-face and (IV) T-shaped stacking were observed in complex 1 (Figure 2).As illustrated in Figure 2a, two N4-bound phenanthroline (N4-phen) molecules (symmetry codes: 2-x, 2-y, -z) give an opposite orientation in stacking mode I.They are parallel to each other with the interplanar separation of 3.520 Å.The centroid-centroid distances of A-C′, C-A′, B-C′, B-B′, C-B′ and C-A′ all fall in the 3.63-4.00Å range, which indicates the p-p interactions involve the extended p system of whole phenanthroline molecule.For two stacking phenanthroline molecules, the centroid displacement is 1.23 Å and the slip angle is 19.32°, similar to those of a perfect face-to-face alignment.Such phenanthrolinephenanthroline stacking mode is rare in the reported complexes. 38Two N1-bound phenanthroline molecules (symmetry codes: 1.5-x, 0.5+y, 0.5-z) stack with each other to give stacking mode II (Figure 2b).The stacking parts are not completely parallel to each other but inclined with the dihedral angle of 11°.Other centroid-centroid distances among the six-membered rings are all longer than 4.0 Å except A′-C for 3.787 Å, suggesting p-p stacking interactions mainly occur in two outer pyridine rings.This mode is comparable to pyridine-pyridine-type approach of stacked quinoline molecules. 38Owing to the large centroid displacement (1.44 Å) and slip angle (22.29 °), the stacking force in complex 1 is apparently weaker than that in the {[Cu(phen)CN][Cu(phen)][Cu(CN) 2 ]} n complex. 35The complex cations are organized into a ladder-like chain through the p-p stacking interactions I and II (Figure 3a).
Another two types of p-p stacking interactions (mode III and IV) involving phenanthroline and salicylaldehyde bear out the severely offset p-stacked geometry with an aromatic attraction rather than a p-p repulsion. 39The stacked geometry of mode IV is very distinct from that of the above three (Figure 2d).Two stacked aromatic systems, C7-bound arene ring of salicylaldehyde and N1A-bound phenanthroline (symmetry codes: 0.5+x, 1.5-y, 0.5+z), do not assume parallel or near parallel face-to-face arrangement but almost perpendicular with the dihedral angle of 95°.The distances of H7 atom-centroid (2.970 Å), H6 … C17A (2.852 Å) and H7 … C13A (2.887 Å) are all smaller than the observed value (3.02 Å), making it clear that mode IV is a typical edge-to-face, namely T-shaped arrangement. 40Such edge-to-face stacking systems are  mainly dominated by p-δ attraction and more stable than offset face-to-face arrangement. 39As displayed in Figure 3b, the complex cations are assembled into a quasi-helical chain along the crystallographic c axis through p-p stacking action III and IV.

Spectrophotometric titration for DNA binding studies
Electronic absorption spectroscopy is an effective method to determine the binding mode and intensity of metal complex with DNA.The hypochromism is associated with the intercalative binding mode involving a strong stacking interaction between the aromatic chromophore of complex and the base pairs of DNA. 43The electronic absorption spectra of complex 1 in absence and presence of CT-DNA are given in Figure 4.As the concentration of CT-DNA increased, the electronic spectra display a progressive decrease at 266 nm, which suggests that complex 1 interacts with CT-DNA through an intercalative association in which the planar aromatic moiety slides in between base pairs by means of p-p stacking interactions. 44The result can be rationally explained as follows: some p* orbitals of the aromatic ligand are coupled with p orbitals of the base pairs when complex 1 intercalates into the base pairs of CT-DNA.The coupled orbitals are partially filled by electrons, which gives rise to the decrease of the p-p* transition energy and the transition probability, and shows hypochromism. 9he intrinsic binding constant K b can be obtained from the spectral titration data by equation 1 to determine quantitatively the DNA binding characteristics of complex 1: 45 [DNA] / (e a − e f ) = [DNA] / (e b − e f ) + 1 / K b (e b − e f ) (1)   where [DNA] is the concentration of CT-DNA; e f , e a and e b correspond to the extinction coefficient for free, added, and fully bound to complex 1, respectively.The plot of [DNA]/(e a −e f ) versus [DNA] gives the K b value as the ratio of slope to intercept.From the plot shown in Figure 4, the intrinsic binding constant K b for complex 1 is calculated as 7.97×10 3 L mol −1 (r = 0.992 for five points) which is smaller than that of [Co(phen) 2 (amtp)](ClO 4 ) 3 (3.23×10 5 L mol −1 ), 46 indicating that it is a moderate binder of CT-DNA.

Fluorescence titration experiments for DNA binding studies
The fluorescence titration experiment has been used to characterize the interaction of complex with DNA following the changes in fluorescence intensity of complex.As shown in Figure 5a, complex 1 is fluorescent exhibiting the most intense peak at 366 nm in Tris-HCl buffer at ambient temperature.The emission intensities gradually decreased with the addition of CT-DNA, indicating an interaction with complex 1.The quenching may be attributed to the electron transfer from the base pairs of CT-DNA to the excited MLCT state of complex. 47n order to further investigate the interaction between complex 1 and CT-DNA, the ethidium bromide (EB) fluorescence displacement experiment has been carried out.It is known that EB can emit intense fluorescence in presence of DNA due to the strong intercalation between the adjacent DNA base pairs.The fluorescence of EB-DNA system can be quenched by addition of a second species that is able to displace the EB molecules.The quenching degree of the EB-DNA system fluorescence is usually used to determine the binding strength. 48,49The fluorescence spectra of EB-DNA system in absence and presence of complex 1 are given in Figure 5b.The fluorescence intensity at 583 nm decreased as a function of the complex concentration, suggesting the displacement of some EB molecules from the base pairs.This behavior was analyzed using the Stern-Volmer equation (2): 50 where I 0 and I represent the fluorescence intensities in absence and presence of quencher, respectively, [Q] is the concentration of quencher, K sv is a linear Stern-Volmer quenching constant.In the quenching plot of I 0 / I versus [complex], K sv value is given by the slope.The K sv value for complex 1 was calculated as 1.34×10 4 L mol −1 (r = 0.995 for eight points).This is smaller than that of complex [Co(phen) 2 (amtp)](ClO 4 ) 3 (7.75×10 5 L mol −1 ), 46 indicating complex 1 is more weakly bound to CT-DNA.The result also matches with the spectrophotometric titration experiments.

Conclusions
A new mixed ligand manganese(II) complex [Mn(sal) (phen) 2 ]ClO 4 was synthesized and its crystal structure resolved by single crystal X-ray diffraction.Some C-H … O hydrogen bonds and four types of p-p stacking interaction modes involving phenanthroline and salicylaldehyde ligands were observed and discussed in detail.A fascinating 3D supramolecular structure is generated by these supramolecular non-covalent interactions.Spectroscopic studies suggest the manganese(II) complex interacts with CT-DNA by means of intercalation mechanism with moderate binding intensity.The results are valuable in understanding the binding modes of the metal complex with DNA as well as laying a foundation for rationally design of novel agents with different binding affinity for probing and targeting nucleic acids.

Figure 3 .
Figure 3.The supramolecular self-assembly architecture of complex 1.(a) The ladder-like chain formed by complex cations through p-p stacking mode I and II.(b) The quasi-helical chain formed by complex cations through p-p stacking mode III and IV.(c) The 3D supramolecular construction by means of p-p stacking interactions and hydrogen bonds.