Indi an Journal of Chemistry Yol. 42A, September 2003, pp. 2 185-2190

Synthesis, crystal structure and nuclease activity of bis( dipyridoquinoxaline )copper(I) perchlorate

Pattubala A N Reddy, Bidyut K Santra, Munirathinam Nethaji & Akhil R Chakravarty*

Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, Indi a

E-mail: arc@ipc.iisc.ernet.in

Received 26 November 2002

A copper(l ) complex [Cu(dpqh l(CI04) (1), where dpq is dipyrido-[3,2-d:2',3'-j]-quinoxaline. is prepared and structurally characterized. The complex crystallizes in the orthorhombic space group Pnna with the unit cell parameters ({ = 7.7797(9) A, b = 20.185(2) A, c = 16.593(2) A, V = 2605.6(5) A3, Z = 4. The structure was refined to R = 0.07 13 for 2525 reflec ti ons with 11 27 having I > 2cr(1) and 192 parameters. The cationic complex has the copper(I) center bonded to two N.N-donor dpq ligands giving a distorted tetrahedral coordination geometry with a Cu-N distance of 2.05 A.. The cry tal structure di spl ays extensive 1t-1t stack ing interactions involving the planar aromatic rings of dpq . Such an in teract ion g ives ri se to the formation of chains and the perchlorate anions are located in-between the chains. The complex is diamagneti c and exhibits a metal to ligand charge transfer band at 442 nm (E, 6000 M""I cm- I

) in MeCN and a perchlorate stretch at 1088 cm- I in the JR spectrum in KBr phase. It shows a quasi-reversible cyclic voltammetric response at 0. 17 V (L'.Ep = 120 mY) at 50 mY S- I in DMF for the Cu(Il)/Cu(J) couple. Complex 1 is a chemical nuclease. It shows efficient nuclease acti vity on treatment with supercoiled DN A in presence of hydrogen peroxide. The nuclease activity of 1 is found to be better than [Cu(phen)~r under similar reaction conditions. Mechani stic studies have shown minor groove binding of the compl ex and the in volvement of hydroxyl radicals in the DNA cleavage reaction s.

Introduction Redox active transition metal complexes that cleave DNA in an oxidative manner are of importance for their diverse applications like footprinting, sequence­specific binding to nucleic acids, new structural probes and therapeutic agents l

-15

. Metallointercalators having pl anar N-donor heterocycl ic bases are useful as photochemical and chemical reagents in nucleic ac ids chemistry. Among redox-based pseudonucleases, bis( I , IO-phenanthroline)copper(l ) complex [Cu(phenht has been found to cleave DNA efficiently in the presence of hydrogen peroxide l

.

Extensive studies have shown that the complex binds to DNA in the minor groove and the active species formed from the reaction with H20 2 leads to strand sciss ion l 6

. It has also been observed that the DNA strand cleavage effected by the copper-based chemi cal nucleases proceeds via proton abstraction from the deoxyribose C-4' or C-I' positions by the

. . 1718 act t ve species . . We have recently reported the chemical nuclease

activity of monoaquo-bis(dipyridoquinoxaline)­copper(II ) complex in presence of ascorbic ac id (H2A) as a reducing agent l9

. This complex is found to be a better cleaver of DNA than its analogolls copper(II) phen species. The present work stems from our

interest to prepare and explore the DNA cleavage properties of bi s( di pyridoqui noxal ine )copper( I) complex. It has been propOSed that the nuclease ac ti vity of [Cu(phenht is related to the partial intercalation or binding of one phen ligand in the minor groove while the other phen makes favou rable contact within the groove ll

. For an efficient nuclease act ivity, the phen to copper ratio should be greater than 2: 1 (refs 11 ,21). The dpq li gand with its extended planar quinoxaline moi ety is expected to have enhanced binding ability to DNA than phen. Here in we report the synthesis, crystal structure and nuclease activity of [Cu(dpqh ](CI04 ) (1).

Materials and Methods All reagents and chemicals were purchased from

commercial sources. Solvents used for chemical and spectroscopic studies were purified by standard procedures. Superco iled(SC) pUC19 DNA was purchased from Bangalore Genei (India) . Agarose (molecul ar biology grade), distamycin , ethidiul11 bromide (EB) and other reagents used for electrophoretic studies were from Sigma (USA). Tri s- HCI buffer solution was prepared uSing deioni sed, sonicated triple di stilled water. The ligand dipyrido-[3,2-d:2',3-j]-quinoxaline (dpq) anc! the

2186 INDIAN J CHEM, SEC A, SEPTEMBER 2003

bis(phen)copper(I) perchlorate were prepared by literature methods '4,21. The preparative procedure for the precursor complex [Cu(dpqh(H20)](Cl04h .H20 has earlier been reported '9 .

Synthesis of [Cur dpqh}(CL04) (1 ) The complex was prepared by reacting

[Cu(dpqh(H20 )](Cl04h- H20 (0.2 g, 0.26 mmol) in C H2C I2 (20 mL) with a methanolic solution (5 mL) of ascorbic acid (0.03 g, 0 .17 mmol) under din itrogen atmosphere using a Schlenk line. On completion of the reactio n after 15 min, the solvents were removed under vacuum. The solid thus obtained was thoroughly washed with cold deoxygenated water to remove any unreacted ascorbic acid and finally dried ill vacuo over P40 l0 (yield, 65 %). Anal. Found: C, 53.45; H, 2.64; N, 17 .74; Calcd. for C2gH I6Ng0 4CICu: C 53.58; H, 2 .55; N, 17 .86%. IR (cm- I, KBr pellet) : 3065w, 1576m, 1469m, 1406m, 1386s, 1088vs, 8 14m, 727m, 620m, 432m (vs, very strong; s, strong; m, medium; w, weak). UV-vis, Amax, nm (E, If' cm- I) in MeCN: 442 (6,000), 338 (9,950), 287 (47,000), 253 ( 1.16,000), 22 1 (69,500). I H NMR (DMSO-d6, ppm):

8 = 9.7 1, 9 .34 , 9.17, 8.22. Complex 1 is soluble in MeCN, CH2Cl2, DMF, DMSO and MeOH. It is sparingly soluble in water. Cautioll! Perchlorate salts of metal co mplexes having organic ligands are potentially explos ive and should be handled in small quant ity with care!

Physica/measurements Elemental analysis was done using Heraeus CHN-

o Rapid instrument. fR , NMR and electronic spectra l daia were obtai ned from Bruker Equinox 55, Bruker AMX400 and Hitachi U3400 spectrometers, respecti ve ly. Electrochemical measurements were

made at 25°C on a EG&G PAR Model 253 Versa Stat potentiostat/galvanostat with elec trochem ical analys is so ftware 270 for vo ltammetric work usi ng a three­elect rode cell setup comprising of a g lassy carbon working, platinum wire auxiliary and a saturated ca lome l reference (SCE) e lectrode. The electrochemical data were uncorrected for junction potentia ls. Ferrocene was used as a standard showing the Fe(IIl)/Fe(JI ) couple at 0.38 V (vs SCE) under ~ imilar experimental conditions in MeCN containing 0. 1 M tetrabutylammonium perchlorate (TBA P).

X-ray crystallographic procedures Si ngle c rystal s of 1 with 0.5H 20 as solvent of

crys tallization were grown by a diffusion techniq ue in

which petroleum ether was layered above a C H2Cl e solution of the complex under dinitrogen atmosphere. The source of water could be the solvents used for crystallization. A single crystal of size 0 .22 x 0.16 x 0.08 mm3 was mounted on a glass fibre with epoxy cement. The cell parameters and the intensi ty data were obtained using a Bruker SMART APEX CCD di ffrac tometer, equipped with a fine focus 1.75 kW sealed tube Mo-Ka X-ray source (A, 0.71073 A) with

increasing w (width of 0.3 deg. per fra me) at a scan speed of 10 sec/frame. The SMART software was used for data acqui sition and the SA INT software for data extraction22. Absorption corrections were done usi ng SADABS23 . The metal atom posi ti on was obtained by direct method . The other atoms were generated from subsequent difference Fourier maps. The structure solution was done using SHELX programs24. All the hydrogen atoms belo nging to the cationic complex in 1 were in fixed pos iti ons and were refined using a riding model. All non-hydrogen atoms were refined anisotropically except the disordered oxygen ato ms. Positional disorder was observed in the oxygen atoms of the perchlorate anion. The water oxygen showed reduced elec tron densi ty and was modeled for a hemihydrate.

Crystal structure formula C2 H I7C ICuN 0~ .5 ' M = 636.49, orthorhombic, space group PIlIW (no . 52), a = 7.7797(9) A, b = 20. 185(2) A, c = 16.593(2) A. V = 2605.6(5) A3

, Z = 4 , T = 293(2) K, J-l = 0.997 mm- I. F(OOO) = 1292, P = 1.62 g cm-3

; I 127 observed

reflections with I> 2cr(1) in the limit 1.59 < e < 26.0°. number of parameters 192, RI = 0.07 13 [R(all ) = 0.1653], wR = 0.1758 [wR (a ll ) = 0.2 168]; highest shi ft/e .s.d. = 0.0, goodness-of-fit = 0.935 , highest peak in the final di fference Fourier map = 0.456 e

A-3; weighting scheme applied: w = 1/[cT(Fn2) +

(0. 1112P)2 + O.OP] , where P = (F} + 2Fc2 )/3. Perspective view of the molecule was obtained by ORTEp25. Detai led crysta ll ographic data in the C IF format have been depos ited with the Cambridge Crystallographic Data Centre, CCDC No. 196742. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road. Cambridge CB2 lEZ, UK (Fax: +44- 1223-336033; e­mail : deposit @ccdc.cam. ac. uk o r www: http://w\Vw.ccdc/cam.ac.uk) .

DNA cleavage experiments The extent of cleavage of ON A by the copper( I )

complex was monitored by agarose gel electrophoresis. Supercoiled pUCI9 DNA (6 ~lL.

REDDY et al. : STUDY OF BIS(DIPYRIDOQUINOXALINE)COPPER(I) PERCHLORATE 2187

-500 ng) in SO mM Tris-HCI buffer (PH 7.2) containing SO mM NaCl was treated with the metal complex (20 ~ followed by dilution with the Tris­

HCI buffer to a total volume of 20 ~L. The samples after incubation for 1 h at 37°C were added to the loading buffer containing 25% bromophenol blue,

0.25 % xylene cyanol, 30% glycerol (3 ~L) and the solution was finally loaded on 0 .8% agarose gel

containing l.0 ~g/mL ethidium bromide. Electrophoresis was carried out for 3 h at 40 V in TBE buffer. Bands were visualized by UV light and photographed. The extent of DNA cleavage was measured from the intensities of the bands using UVITEC Gel Documentation System. Due correction was made for the low level of nicked circular (NC) form present in the original SC DNA sample and for the low affi nity of ethidium bromide binding to supercoiled DNA compared to nicked circular and linear forms of DNA26. In the inhibition reactions ,

di stamycin (75 ~M) or DMSO (4 ~L) was added initially to pUC19 DNA (-500 ng) in SO mM Tris~ HClI aCI buffer and incubation was done for 15 min at 37 °C prior to the addition of the complex. The mixture was diluted with the buffer to a total volume of 20 ~L. After a further incubation of 1 h at 37 °C, the samples were subjected for ge l electrophoresi s using procedures as described above.

Results and Discussion Bis-(dipyridoquinoxaline)copper(I) perchlorate (1)

has been prepared in high yield by reacting [Cu(dpqh(H20 )](CI04)2 with ascorbic acid . While the complex is moderately stable in the solid state, it is air sensitive and unstable in solution. The brown coloured solution of the complex in MeCN shows a metal to ligand charge transfer transition at 442 nm. The complex di splays the perchlorate stretch at 1088 cm- I in its IR spectrum. The IH NMR spectrum in DMSO-d6 exhibits signals that are characteristic of the dpq ligand . Complex 1 in DMF-O.l M TEAP shows a quasi-reversible cyclic voltammetric response due to the Cu(Il)/Cu(l) couple at 0 .17 V with a ipali pc ratio of unity and a L1Ep value of 120 m V at a scan rate of SO mV S- I (Fig. I) .

Complex 1 has been characterized by single crystal X-ray diffraction method. An ORTEP view of the comp lex is shown in Fig . 2. Selected bond distances and angle are given in Table 1. The structure consists of a mononuclear copper(I) complex, a perchlorate anion and a hemihydrate lattice molecule. The complex has a CuN4 chromophore showing a

2 5

<t: :l. ~ -c Q)

~ · 2 5

~

0 ( iii )

·5 0

600 400 200 - 200 - 400 - 600

E (m V )vs SCE

Fig. l-Cyclic voltammograms of [Cu(dpq)21(Cl04 ) in DMF -0. 1 M TBAP at scan rates (i) 50, (ii) 100 and (iii ) 200 mY S- I.

C2

Fig . 2-An ORTEP view of the cationiC complex in [Cu(dpq)21(CI04).0.5H20 showing 50% probabil ity thermal e llipsoids and the atom numbering scheme.

distorted tetralledral geometry . The Cu(l)- ( I) and

Cu(l )-N(2) di stances are -2.05 A. The dpq ligand shows N(l )-Cu(l )-N(2) bite angle of 81.9(2)°. The acute nature of this angle has made the other angles significantly higher than the idealized tetrahedral angle. These angles vary in the range 109° to 150°. The structural features in 1 are si milar to those observed for [Cu(phen)2](CI04) having a bite angle of _81° and Cu-N distance of - 2.05 A21.

The planar dpq bases in 1 are involved in TI -TI

stacking interactions forming a chain-type of structure. Both the dpq li gands in 1 are involved in thi s non-covalent interaction showing a di stance of -3.5 A. The dpq li gands are in an essentially planar

confi guration givi ng a dihedral angle of 58° between two planes. The packing diagram shows the formati on of planes consisting of such chains with the perchlorate anions located in-between the planes (Fig. 3).

The nuclease activi ty of complex 1 has been studied by gel electrophoresis uo' ing su percoi led

2 188 INDIAN J CHEM, SEC A, SEPTEMBER 2003

Table I-Selected bond di stances (A) and angles (deg.) for [Cu(dpqh l(Cl04).0.5H20 (1) with e.s.d.s in the parenthesis

Cu( I)-N( I) Cu( I )-N(2) N(l )-C( I) N( I)-C( II ) N(2)-C( IO) N(2)-C( 12) N(3)-C(l 3) N(3)-C(5) N(4)-C(l 4) N(4)-C(6) C( 1)-C(2) C(2)-C(3) N( I )-Cu(l )-N( 1)# I N( I )-Cu( I )-N(2)# I N( I )-Cu(l )-N(2) N(2)# I-Cu( I )-N(2) C( I)-N(l )-C( II ) C( 1)-N( I)-Cu(l) C( II )-N( I)-Cu(l) C( 1O)-N(2)-C( 12) C( 1O)-N(2)-Cu( I) C( 12)-N(2)-Cu(l) C(l3)-N(3)-C(5) C( 14)-N(4)-C(6) N( I )-C( I )-C(2) C(3)-C(2)-C(l ) C(2)-C(3 )-C( 4) C( I I )-C( 4 )-C(3) C( 11)-C(4)-C(5) C(3)-C(4)-C(5) N(3)-C(5)-C(6)

2.045(5) 2.046(5) 1.31 6(8) 1.366(7) 1.31 3(7) 1.364(7) 1.336(8) 1.354(7) 1.332(9) 1.334(7) 1.383(9) 1.350(9) 136.2(3) 109.63(19) 8 1.85( 19) 149.8(3) 11 7.8(5) 129.9(5) 112.2(4) 116.8(5) 130.4(4) 111 .9(4) 11 5. 1(6) 115. 1(6) 123.4(7) 11 8.8(7) 11 9.7(6) 11 8. 1(6) 11 9.6(6) 122.3(6) 12 1.4(6)

C(3)-C(4) C(4)-C( 11 ) C(4)-C(5) C(5)-C(6) C(6)-C(7) C(7)-C(l2) C(7)-C(8) C(8)-C(9) C(9)-C( 10) C( 11 )-C(l2) C( 13)-C( 14)

N(3)-C(5)-C(4) C(6)-C(5)-C(4) N(4)-C(6)-C(5) N(4)-C(6)-C(7) C(5)-C(6)-C(7) C( 12)-C(7)-C(8) C( 12)-C(7)-C(6) C(8)-C(7)-C(6) C(9)-C(8)-C(7) C(8)-C(9)-C( I 0) N(2)-C(l 0)-C(9) N( I)-C( II )-C(4) N( I )-C( II )-C( 12) C( 4 )-C(ll )-C(l2) N(2)-C( 12)-C(7) N(2)-C( 12)-C( I I) C(7)-C( 12)-C( II ) N(3)-C( 13)-C( 14) N( 4 )-C(l 4)-C( 13)

1.400(8) 1.372(7) 1.456(8) 1.41 6(8) 1.468(8) 1.383(8) 1.386(7) 1.350(8) 1.393(8) 1.447(8)

1.363( 10)

11 8.9(6) 11 9.6(5) 12 1.8(6) 11 8.4(6) 11 9.7(5) 11 8.2(5) 11 9. 1(5) 122.7(5) 120.0(6) 11 8.1 (5) 124.3(5) 122. 1(5) 11 6.6(5) 12 1.3(6) 122.4(5) 11 7.0(5) 120.6(5) 122.6(7) j 23.9(7)

Sy mmetry transformations used to generate equi valent atoms: #1 x,-y+1 /2,-z+1 /2

Fi g. 3--Pack ing diagram viewed along a ax is show ing the chain formation involving the cati onic complexes and the location of the perchlorate anions (perch lorate anion has the positi onally disordered oxygen atoms).

pUC 19 DNA in 50 mM Tri s- HC 1/50 mM NaC I buffer (PH 7 .2) in the presence of hydrogen peroxide. Contro l experiments usi ng only DNA or DNA with H20 2 do not show any apparent c leavage. At the

concentration of 20 /-tM, compl ex 1 is able to convert 64% of the ini tial SC to NC form whereas [Cu(phenh t can convert 42% of the SC (Fig. 4) . The enhanced nuclease ac ti vity of 1 in comparison to its phen analogue is a signi ficant observation of this study . The dpq ligand with its extended aromatic ring could have better propensity for intercalative b ind ing to the DNA molecule than phen.

The mechani stic aspects of the c leavage reactions have been probed by inhibi tion tud ies using di stamycin and DMSO. The redox-active copper co mplexes are known to c leave DNA by ox idative attack at the sugar moiety. Although the true identity of the ox idizing species is yet to be conclusivel y establi shed, the acti ve species is proposed to be either a hydroxy l rad ical or a copper(HT) bound oxo or hydroxo species27

.28

. We have st d ied the DNA cleavage reactions using minor groove binder di stamycin and hydroxyl radica l scavenger DMSO.

REDDY et al.: STUDY OF BIS(DIPYRIDOQUINOXALINE)COPPER(I) PERCHLORATE 2 189

II

Fig. 4--Cleavage of SC pUC 19 DNA (0.5 ~g) by

rC u(dpq )2](CI04) and the ICu(phenh l (CI04) complexes (20 ~M, 2 pL) in the presence o f hydrogen peroxide: lane I , DNA control ; lane 2. DNA + H20 ]: lane 3, DNA + [Cu(phen)2t+ H20 2; lane 4, DNA + [Cu(dpqh t + H20 2. Forms I and II are supercoiled and nicked c ircular DNA respec tively .

2 3 4 5 6 7

- ~~-~ . - II ---... .--_ .. Fig. )-Cleavage o f SC pUCI9 DNA (0.5 pg) by [Cu(dpqh t (20

pM. 2 ~L) using different inhibition reagents: lane I, DNA cOl1lro l; lane 2, DNA + H20 2: lane 3, DNA + distamycin ; lane 4, DNA + DMSO; lane 5. DNA + [Cu(dpq)2t + H20 2; lane 6, DNA + di stamycin + [Cu(dpq)2t + H20 2; lane 7, DNA + DMSO + ICu(dpq)2r + H20 2. Forms I and II are supercoiled and nicked c ircular DNA respectively. The form I and II percentages for lanes 1-7 are: 92. 8: 88. 12; 90, 10; 89, II ; 29, 71 ; 92, 8; 89, I 1.

Gel electrophoresis done in presence of distamycin shows a complete inhibition of DNA cleavage. This suggests that complex 1 binds to DNA in the minor groove (Fig. 5). Again, when the reaction is carried out in presence of DMSO, complete inhibition is observed. This predicts the involvement of hydroxyl radical in the scission reaction .

Conclusion A new copper(l) complex [Cu(dpqh](CI04) having

NN-donor heterocyclic base dipyridoquinoxaline has been prepared and structurally characterized . It has a te trahedral structure with a CuN4 chromophore. The complex shows novel 1t-1t stacking interactions involving the dpq ligands. Complex 1 exhibits better nuclease activity in presence of H20 2 than its phen analogue. Mechanistic investigations suggest minor groove binding for 1 and the involvement of hydroxyl radical in the DNA cleavage reactions.

Acknowledgement We thank the Department of Science &

Technology , Government of India, for financial

support and for the CCD facility . Thanks are due to the Alexander von Humboldt Foundation , Germany. for donation of an electroanalytical system and the Bioinformatics Centre of IISc, Bangalore. fo r database search . PANR thanks the CSIR, New Delhi. for a fellowship.

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