Asymmetric ruthenium (Ⅱ ) Complexes of DNA Synthesis and Mode of Study

1、 Asymmetric ruthenium ( ) Complexes of DNA Synthesis and Mode of StudyAuthor: tube Xiao-Yan, Liu Jun, He Li-Xin, Lu, Home Economics, Wen-Jie Mei Abstract Objective and methods of synthesis of a new ruthenium ( ) polypyridine complexes Ru (4,7 dmp) 2 (PYNI) (ClO4) 2 4,7 dmp = 4,7 2-methyl 1,10 phenan

2、throline, PYNI = 2 (2 pyridine) naphthyl imidazole, using elemental analysis, electrospray mass spectrometry, nuclear magnetic resonance spectra of the complexes were characterized by; electronic absorption spectra, fluorescence spectra , viscosity, melting point determination of DNA testing and res

3、earch of the role of the complex with DNA. Results and Conclusion complexes Ru (4,7 dmp) 2 (PYNI) (ClO4) 2 and the DNA by inserting between the mode of binding. Keywords: ruthenium ( ) complexes; DNA; spectral properties; viscosity test Abstract: Objectiveand Methods A new Ruthenium ( ) polypyridyl

4、complex Ru (4,7 dmp) 2 (PYNI) (ClO4) 2 4,7 dmp = 4,7 dimethyl 1,10 phenanthroline, PYNI = 2 (2 pyridyl) naphthoimidazo imidazole) has been synthesized and characterized by elemental analysis, ES MS and 1H NMR. The DNA binding behaviors were investigated by electronic absorption spectroscopy, fluores

5、cent spectroscopy, viscosity measurement and DNA melting experiment. Results and Conclusion The results showed the complex Ru (4,7 dmp) 2 (PYNI) 2 binding to DNA through intercalation mode. Keywords: ruthenium (Ru) complex; DNA; spectral properties; viscosity measurement In recent years, people are

6、small molecules of transition metal polypyridyl complexes with DNA macromolecules gradually in-depth study of the interaction, this research has become very active in the field of bio-inorganic chemistry study. Octahedral polypyridyl ruthenium metal complexes and the role of DNA has aroused great in

7、terest 1,2, as compared with other metal complexes, such complexes for both easy to construct a rigid brings chiral octahedral configuration, and the thermodynamic stability complexes, photochemical and photophysical information-rich, either as DNA structural probes 3, but also can serve as a DNA mo

8、lecular light switch, DNA-mediated electron transfer, DNA breakage reagent 4 and so on. Studies have shown that these complexes can insert, electrostatic and groove to cover the three kinds of ways and the role of DNA, which insert mode is most important, because many of the complex nature of the co

9、mbination with the insert. Synthesized a new ruthenium ( ) polypyridine complexes Ru (4,7 dmp) 2 (PYNI) (ClO4) 2 4,7 dmp = 4,7 2-methyl 1,10 phenanthroline, PYNI = 2 (2 pyridine) naphthyl imidazole, synthetic route shown in Figure 1. By elemental analysis (EA), electrospray ionization mass spectrome

10、try (ES MS), nuclear magnetic resonance (1H NMR) of the complexes characterized in detail. Electronic absorption spectra, fluorescence spectroscopy, viscosity, melting point determination of DNA testing and research complexes Ru (4,7 dmp) 2 (PYNI) (ClO4) 2 and the DNA effect, the results showed that

11、 Ru (4,7 dmp ) 2 (PYNI) 2 by the insertion of DNA binding way. An experimental part of the 1.1 Reagents and instruments Reagents used in the experiment were analytically pure, prior to use without further purification, the buffer solution prepared with double distilled water; DNA were purchased from

12、 China and the United States company, with Tris (hydroxymethyl 3-amino methane) buffer, dubbed in a certain concentration of stock solution in refrigerator preservation; complexes with 5 mmol / L Tris HCl, 50 mmol / L NaCl (pH = 7.0) buffer solution preparation. LCQ electrospray ionization mass spec

13、trometer (ESMS, Finnigan), fast atom bombardment mass spectrometry (FAB MS) using the VG ZAB HS mass spectrometer, Elemental Vario EL elemental analyzer, Bruker ARX 500 NMR instrument, Shimadu UVPC 3000 UV Vis spectroscopy, fluorescence spectroscopy with Rf 4500 fluorescence spectrometer testing, vi

14、scosity testing using the Ubbelohde viscometer. 1.2 complexes Ru (4,7 dmp) 2 (PYNI) (ClO4) 2 Preparation Weigh 0.312 g cis Ru (4,7 dmp) 2Cl2 * 2H2O 5 (0.5 mmoL), 0.123 g PYNI 6 (0.5 mmoL) by adding a mixture of ethylene glycol 15 mL, heated to 150 argon gas under the protection of reaction 8 h, was

15、a red clear liquid. Cooled to room temperature, filter, add 40 mL water, diluted by adding saturated solution of NaClO4 that generate a large number of red precipitate. Leaching, water, dry ether, after washing several times. After drying the crude product dissolved with a small amount of acetonitri

16、le, using neutral alumina (200 mesh) column separation. In the V (toluene): V (acetonitrile) = 1:3 mixed solvent of elution to collect the red component, and then vacuum, steaming to the solvent, was a red solid, yield 62%. Elemental analysis, measured values (%): C 54.96, H 3.64, N 10.23; calculate

17、d values (%): C 54.95, H 3.67, N 10.19. 1HNMR (DMSO D6, 500 MHz) : 8.34 (d, 1H, J = 8.8 Hz), 8.23 (d, 1H, J = 8.5 Hz), 8.19 (d, 2H, J = 8.6 Hz), 8.03 (d, 1H, J = 8.5 Hz), 7.88 (t, 2H, J = 7.8 Hz), 7.75-7.79 (m, 2H), 7.67 (d, 1H, J = 8.8 Hz), 7.47 (d, 2H, J = 8.0 Hz), 7.45 (d, 2H, J = 8.2 Hz), 7.23 (

18、t, 1H, J = 7.6 Hz), 7.12-7.17 (m, 2H), 7.05-7.09 (m, 2H), 7.03 (d, 1H, J = 8.4 Hz), 6.81 (d, 1H, J = 8.5 Hz), 5.00 (s, 1H), 2.05 (s, 6H), 2.01 (s, 6H). ES MS (CH3OH): m / z 762 (M 2ClO4 H), 382 (M 2ClO4 2). 1.3 Experimental Methods In the UV-Vis spectrometer, the reference pool by adding 3 mL of buf

19、fer solution, in the sample pool by adding the same volume of 20 mol / L of the complex solution, with micro-plus injector for each sample to the reference pool and the pool, respectively Add the same volume of DNA storage solution, so that the concentration of DNA and the complex ratio (CDNA / CRu)

20、 according to a certain percentage increase, up to saturation, the absorption peak is no longer subtractive. Each uniform mixture of about 5 min after the range of 200 800 nm to monitor the complex changes in the electronic absorption spectra. Complexes Ru (4,7 dmp) 2 (PYNI) (ClO4) 2 and the DNA bin

21、ding constants obtained according to the following equation 7 DNA a- b = DNA a- f 1Kb ( b- f) DNA on behalf of DNA concentration a, f, b are complex with DNA binding, the free complexes and complexes with DNA binding reached saturation when the molar absorption coefficient, Kb for the complex with D

22、NA binding constants, Kb from the fitting a straight line intersects with the Y-axis intercept obtained. Fluorescence studies, the preparation of ruthenium complex solution (2 mol / L), with micro-plus for each kind of device to the sample DNA pool by adding the same volume of reservoir fluid, so th

23、at the concentration of DNA and the complex ratio (CDNA / CRu) according to a certain the proportion of incremental, until saturated. Each is about 5 min after mixing evenly, with Rf 4500 fluorescence spectrometer in the range of 500 800 nm to monitor changes in the fluorescence spectra of complexes

24、. With 450 nm excitation light sources, recording light emission peaks and luminous intensity. Viscosity measurements using Ubbelohde viscometer. Measuring viscosity, the temperature constant at (28 ± 0.1) . A fixed DNA concentration, and gradually increase the concentration of Ru complexes. Re

25、lative viscosity determined by the formula: = (t-t0) / t0 The formula, t0 the buffer through the capillary flow time, t for the DNA solution (containing varying concentrations of Ru complexes) the time required to flow through the capillary. To ( / 0) 1 / 3 ratio of r (r = Ru / DNA) mapping. 0 plus

26、ruthenium complexes as not, when the relative viscosity of DNA solution. Determination of DNA melting experiments by UV Vis spectroscopy to monitor DNA complex solution, with or without the presence of the absorption at 260 nm at peak intensity changes. Reposted elsewhere in the paper for free downl

27、oad 2 Results and discussion 2.1 The role of the complex with DNA electronic absorption spectra of Complexes MLCT (metal to ligand charge transfer transition) absorption peak intensity of DNA in the presence of a certain decrease, which is generally considered one of the characteristics into the rol

28、e. Hypochromic effect generated due to DNA base pairs for the insertion occurred between ligand electron stacking, so that the latters * orbital on the air there are certain e-filling, thus reducing the chances of MLCT transitions. The hypochromic effect of the strength, but also the ability to refl

29、ect the size of the insert, insert the ability to get stronger and stronger hypochromic effect. Figure 2 (a small figure that complex with DNA binding constants) shows that with the calf thymus DNA (CT DNA) joined the electronic absorption spectra of the complex took place and some degree of redshif

30、t Hypochromic, metal to ligand charge transfer ( MLCT) peak (462 nm), at room temperature, Hypochromic rate was 21.7%, redshift 4 nm. Complexes with DNA-binding constant Kb = 2.83 * 104 M-1. Complex with DNA binding constants similar to the previously reported complex with DNA binding constants 6. =

31、 20 mol / L. Arrow shows the absorption change upon the increase of DNA concentration 2.2 Fluorescence Fluorescence spectroscopy is a more sensitive test method is widely used to study the interaction of complex with DNA. If ruthenium ( ) polypyridine complexes in fluorescent under certain condition

32、s, when the joined DNA, the fluorescence enhancement, indicating complex with DNA taken place one way or another combination. Fluorescence Enhancement range in size, generally reflect the role of the complex with the strength of DNA. Complexes Ru (4,7 dmp) 2 (PYNI) 2 and the DNA changes in the role

33、of fluorescence in Figure 3, with the accession of DNA complexes increased 4.0-fold fluorescence. The role of complex with DNA fluorescence enhancement is due to the effective protection of the DNA complexes, so that the water molecules from attack. 2.3 Viscosity Test Fluid viscosity method is to st

34、udy the Law of the complex with DNA mode of action of another one of the most powerful experimental method, because it more sensitive to changes in the length of DNA. In general, if the complex with DNA to insert the way the role of the insertion of ligand into the base pairs of DNA, causing DNA dou

35、ble-helix chain elongation, thereby making the relative viscosity of DNA increases; when the complex with DNA with inserted DNA would result in DNA double-helix chain, bending or folding, the length decreases, so its viscosity decreases. When the complexes to the electrostatic interaction with the D

36、NA binding, DNA solution, the viscosity is almost constant. Figure 4 is the complex Ru (bpy) 2 (dppz) 2 ( ), Ru (4,7 dmp) 2 (PYNI) 2 ( ) and Ru (bpy) 3 2 ( ) and the role of the viscosity of DNA variation. Known Ru (bpy) 2 (dppz) 2 is a typical DNA insertion reagents, and the role of DNA solution, t

37、he solution viscosity was significantly higher; Ru (bpy) 3 2 is based on electrostatic interaction with the DNA binding, its the viscosity of DNA solution, no significant change; when the complexes Ru (4,7 dmp) 2 (PYNI) 2 when combined with the DNA solution, DNA solution viscosity increased steadily

38、, similar to the complexes Ru (bpy) 2 (dppz ) 2 and the role of DNA, but the role of intensity than the latter, the Ru (4,7 dmp) 2 (PYNI) 2 and the role of DNA is a combination of insert mode. 2.4 DNA melting point Determination of DNA melting point is to study the transition metal complexes interac

39、t with DNA is an important way, through the determination of DNA melting experiments can measure the melting point of DNA. DNA in the heating process, the hydrogen bonds between base pairs of the destruction, DNA dissociation of the two chains gradually degenerated to a temperature of abscissa, to 2

40、60 nm at absorption rates for the vertical axis, said curve mapping to be for the thermal denaturation curve, when the temperature reached the melting point Tm, there are 50% DNA double-strand change from single-stranded. DNA insertion can serve as agents of some natural or synthetic organic compoun

41、ds and metal-organic complexes with DNA, the role of the insertion of reagents and the DNA base pairs can occur between the stacking, so that DNA double helix has become stable, and thus to make DNA, the melting point (Tm) was significantly higher 8-10. Figure 5 shows the availability of DNA complex

42、es Ru (4,7 dmp) 2 (PYNI) 2 the case of absorption intensity at 260 nm with temperature changes. DNA test measured the melting point (74.3 ± 0.5) , in the complexes Ru (4,7 dmp) 2 (PYNI) 2 exists, DNA increases the melting point of 5.3 , that the complex with DNA between the way through the comb

43、ination of insertion. 3 Conclusion This paper a new asymmetric synthesis of ruthenium ( ) polypyridine metal complexes of Ru (4,7 dmp) 2 (PYNI) (ClO4) 2, and characterized by elemental analysis, electrospray mass spectrometry and 1HNMR spectra. Spectroscopy experiments, testing and DNA melting visco

44、sity changes show that the complex Ru (4,7 dmp) (PYNI) 2 and the DNA is through intercalation combination. References 1 HALL DB, HOLMLIN RE, BARTON J K. Oxidative DNA damage through long-range electron transfer J. Nature, 382:731. 2 DANDLIKER PJ, HOLMLIN RE, BARTON JK, et al. Oxidative thymine dimer repair in the DNA helix J. Science, 1997,275:1464. 3