J. Am. Chem. Soc. 1993, 115. 11341-11352
11341
Synthesis, Characterization, DNA Binding Properties, and Solution Thermochromism of Platinum( 11) Complexes of the Ethidium Cation: Regiospecificity in a DNA-Promoted Reaction Tong Ren, Daniel P. Bancroft, Wesley I. Sundquist, Axel Masschelein, Michael V. Keck, and Stephen J. Lippard' Contribution from the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received June 14. 1993'
Abstract: The reaction of [Pt(NH3)nCl+n](*2) ( n = 1, 2, and 3) complexes with 1 equiv of the ethidium (Etd) cation afforded cis- [Pt (NH3) 2( Etd)Cl] 2+, trans- [Pt( NH3)2( Etd)Cl]2+, cis- [Pt( NH3) (Etd)C12]+,and [Pt( NH3)3 (Etd)]3+, in which Etd is coordinated to platinum through one of its two exocyclic amino groups. These N3 and N8 linkage isomers were separated by reverse-phase HPLC. The platinum coordination sites on ethidium were determined with the use of both 195Pt and 1H N M R spectroscopy as well as by single-crystal X-ray diffraction studies of the N3 and N8 isomers of cis- [Pt(NH3)2(Etd)Cl]C12. Solutions of the acetate salts of all the complexes displayed temperature-dependent optical spectroscopic changes in which a transition at -490 nm (orange) diminished in intensity with concomitant growth of a band at -640 nm (blue) as the temperature increased. This reversible thermochromic phenomenon was shown to arise from proton transfer between the coordinated ex9cyclic amino group and the acetate counterion. Temperature-dependent electronic spectral changes resulting from this reaction were fit to the appropriate equilibrium expressions by global analysis, yielding the corresponding thermodynamic parameters, AHo and M0.Acidity constants for the deprotonation of the coordinated exocyclic amino groups were determined from such equilibrium studies of most of the complexes in methanol and converted to the corresponding values in aqueous solution. These experiments revealed the effects of overall charge, linkage isomerism, and platinum geometric isomerism on the pK, values of the coordinated amino group. Through a Fenske-Hall molecular orbital analysis, the 490-nm band was assigned as a charge-transfer transition from the lone pair orbital of the uncoordinated exocyclic amino nitrogen to the K* orbital (LUMO) of the phenanthridinium ring. The blue band at -640 nm was best described as a charge-transfer transition from a K orbital delocalizedover the Pt-N(coordinated exocyclic amine) linkage to the X' orbital of the phenanthridinium ring. The ability to assign the relative pK, values of the linkage isomers has been used to demonstrate that the previously reported DNA-promoted reaction between cis-DDP and ethidium is regiospecific, with the platinum reacting primarily at the N8 position of the Etd ring.
For more than a decade, cis-diamminedichloroplatinum(I1) (cis-DDP) has been used clinically as a potent anticancer agent against a variety of tumor systems.' Correspondingly, there has been intense interest in the coordination chemistry of platinum ammine complexes with nucleobases, oligonucleotides, and DNA.2 Intercalators, such as ethidium (Etd) bromide, induce changes in both the mode and exonuclease I11 sensitive sites of binding of cis-DDP with DNA, suggesting the occurrence of an intercalator/drug interaction.3~4 Although cis-DDP does not react
cis-DDP
Ethidium Bromide, Etd Br
appreciably with ethidium in dilute (- 50 pM) aqueous solution, in the presence of duplex DNA a significant reaction occurs in which a DNA-Pt-ethidium ternary complex is The *Abstract published in Advance ACS Absrracrs, October 15, 1993. (1) Loehrer, P. J.; Einhorn, L. H.Ann. Intern. Med. 1984, 100, 704. (2) Sundquist, W. I.; Lippard, S. J. Coord. Chem. Reu. 1990, 100, 293. (3) Tullius, T. D.; Lippard, S.J. Proc. Narl. Acad. Sci. U.S.A. 1982, 79,
-ldR9 .--.
(4) Merkel, C. M.; Lippard, S.J. Cold Spring Harbor Symp. Quanr. Biol. 1983, 47, 355. (5) Malinge, J.-M.; Leng, M. Proc. Nail. Acad. Sci. U.S.A. 1986, 83, 6317.
0002-7863/93/1515-11341$04.00/0
structure of this ternary complex has been elucidated from spectroscopic studies employing model complexes formed in the reaction of cis-DDP with Etd in dimethylformamide.7 In particular, a cis-(Pt(NH3)#+ moiety bound monofunctionally to DNA, presumably at the N7 position of either a guanine or adenine nucleobase, is coordinated to one of the amino groups of Etd. In this reaction, DNA serves as a template, facilitating complex formation by holding the reactants in close proximity, resulting in a t least a 60-fold increase in the rate of formation of the Pt-Etd bond.7 Recently, a second example of this type of reaction was reported.6 In addition to their unique ability to form in a DNA-promoted reaction, platinum ethidium complexes exhibit unusual thermochromism in solution not observed with either cis-DDP or ethidium alone. Thermochromism, or the reversible dependence of color on temperature, occurs in a wide variety of both organic and inorganic compounds.9JJ Here we present the synthesis and characterization of an expanded series of platinum-ethidium complexes, including the cis and trans isomers of [Pt(NH3)z(Etd)C1]2+, cis-[Pt(NH3)(Etd)C12]+, and [Pt(NH&(Etd)]S+. Linkage isomers have been identified and the solution thermochromism of the acetate salts (6) Malinge, J.-M.; Schwartz, A.; Leng, M. Nucl. Acids Res. 1987, 15, 1779.
(7) Sundquist, W. I.; Bancroft, D. P.; Chassot, L.; Lippard, S.J. J. Am. Chem. SOC.1988, 110, 8559. (8) Malinge, J.-M.; Sip, M.; Blacker, A. J.; Lehn, J.-M.; Leng, M. Nucl. Acids Res. 1990, 18, 3887. (9) Day, J. H. Chem. Reu. 1963,63, 65. (IO).Bloomquist, D. R.; Willett, R. D. Coord. Chem. Rev. 1982,47, 125.
0 1993 American Chemical Society
11342 J . Am. Chem. SOC.,Vol. 11 5, No. 24, 1993
Ren et ai.
has been investigated. Solutions of the complexes also exhibit pH-dependent color changes when coordinated to duplex DNA, a property which has been exploited to yield structural information about the cis-DDP/Etd/DNA ternary complex. Finally, we describe the results of molecular orbital analyses which allow the assignment of the optical transitions responsible for the thermochromism.
Experimental Section Reagents. Ethidium bromide was purchased from Sigma Chemical Co. and used without further purification. Ethidiumnitrate was obtained by reactionofthe bromidesalt with AgNO3. Dimethylformamide (DMF) was vacuum distilled from BaO and stored over molecular sieves prior to use. All other solvents were of reagent grade or better. cis- and truns-DDP,L1J2(Ph4P) [Pt(NH3)Cl3] ,13and [Pt(NHs),CI] (N03)14were prepared according to published procedures. Instrumentation and Analytical Methods. Unless stated otherwise, a Varian VXR5OO NMR spectrometer (500 MHz) was used to record oneand two-dimensional correlated (COSY) 'H NMR spectra for the compounds. IH NMR spectra were obtained in CDSOD, with chemical shifts (6) referenced to internal tetramethylsilane. The same instrument equipped with a broad band probe was used to measure 107.25-MHz 195PtNMR spectra. 195Ptchemical shifts were referenced to an external standard of 0.1 M K2PtC14 in 0.1 M DCI/D20 (-1624 ppm) and are reported relative to H2PtC16 in D20 (0 ppm). Broad band IH decoupling was not used during acquisition of the 195PtNMR spectra. Platinum analyses were made by flameless atomic absorption spectroscopy on a Varian AA1475 instrument equipped with a GTA95 graphite furnace. Positiveion fast atom bombardment mass spectra (FABMS) wereobtained in glycerol/water or 3-nitrobenzyl alcohol matrices. Separation of Isomeric Platinum-Ethidium Complexes by ReversePhase HPLC. Mixtures of platinum-ethidium linkage isomers, prepared as described below, were separated into isomerically pure products by reverse-phase HPLC on a Waters 600E liquid chromatograph equipped with a Waters 484 tunable absorbance detector operating at 450 nm and a Hewlett Packard 3396A integrator. A 2.2 X 25 cm Whatman Partisil ODs-111 column run with aqueous ammonium acetate (0.1 M, pH 5.2)/ acetonitrile gradients over 30-60 min was used to effect the separations. Solutionsof the separated isomers were immediately frozen and lyophilized todrynessaftercollectionfromthecolumn. h a typicalinjection, 100150 mg of crude material could be purified. Synthesis of cis-[Pt(NH3)2(Etd)Cl](OAc)2. cis-DDP (1.OO g, 3.33 mmol) was dissolved in 30 mL of DMF and allowed to react with AgNO3 (0.566 g, 3.33 mmol) at ambient temperature in the dark with stirring for 24 h. After centrifugation to remove AgCI, the solution was mixed with ethidium nitrate (1.51 g, 4.0 mmol). The mixture was then stirred at ambient temperature in the dark for 72 h. Subsequent removal of DMF in vacuo yielded a red-orange oil containing the crude mixture of cis- [Pt(NH~)2(Etd)Cl](NO3)2linkageisomers. Theoilwas washedwith 50 mL of diethyl ether and triturated with 100 mL of chloroform to solidify the product and remove excess ethidium nitrate. The solid product was isolated by centrifugation, washed withanother 100 mLof chloroform followed by 50 mL of diethyl ether, and dried in air. The yield of crude product was 1.97 g (85% based on cis-DDP). Separation of N3 and N8 linkage isomers from the crude product mixture was achieved by reversephase HPLC as outlined above. Spectroscopic data for cis- [Pt(NH3)2(N3-Etd)CI](OAc)2(la): IH NMR (CD3OD) 6 8.86 (d, H1, 'J= 9.16 Hz), 8.75 (d, H10, 'J= 9.16 Hz), 8.30 (d, H4, 4J, unresolved), 7.91 (dd, H2, 3 J = 8.97 Hz, 4 J = 1.57 Hz), 7.81 (m, phenyl, unresolved), 7.65 (m, phenyl, H9, unresolved), 6.59 (d, H7, 4J = 2.42 Hz), 4.75 (q, CH2, 'J = 7.26 Hz), 1.90 (s, 6 H, acetate), 1.59 (t, CHI, = 7.26 Hz). 195PtNMR (MeOH) 6 -2342. UV-vis spectral data [A, in nm, (e in M-' cm-')] H20, 444 (4710); MeOH, 480 (6700). FABMS (3-nitrobenzyl alcohol, NBA) M / z : 578, [M - H+]+ (matches theoretical isotope distribution); 561, [M - H+ NHa]+; 543, [M - H+ - CI]+; 314, [Etd]+. Spectroscopic data for cis-[Pt(NH3)2(NB-Etd)CI](OAc)2 (lb): IH NMR (CD3OD) 6 8.80 (d, H10, 3J 9.07 Hz), 8.79 (d, H1, 3J = 9.77 Hz), 8.10 (dd, H9, 3J = 9.10 Hz, 4J = 2.18 Hz), 7.77 (m, phenyl, unresolved), 7.66 (m, phenyl, unresolved), 7.45 (d, H4, 4J = 1.99 Hz), 7.45 (dd, H2, 3J = 9.53 Hz, 4J = 2.04 Hz), 7.32 (d, H7, 4J = 2.16 Hz),
-
(11) Dhara, S. C. Indian J . Chem. 1970, 8, 193. (12) Kaufman, G. B.; Cowan, D. 0. Inorg. Synrh. 1963, 7, 239. (13) Abrams, M. J.; Giandomenico, C. M.; Vollano, J. F.;Schwartz, D. A. Inorg. Chim.Acta 1987, 131, 3.
(14) Lepre, C. A. Ph.D. Thesis, Massachusetts Institute of Technology, 1989.
4.69 (9, CH2, 'J = 7.24 Hz), 1.90 (s, 6 H, acetate), 1.59 (t, CH3, 3J = 7.20 Hz). lssPt NMR (MeOH) 6 -2356. UV-vis spectral data [A,. in nm, (e in M-I cm-I)] H20,456 (7970); MeOH, 492 (7610). FABMS (3-nitrobenzyl alcoho1,NBA) M/z: 578, [M -H+]+ (matches theoretical isotopedistribution); 561, [M-H+-NH3]+; 543, [M-H+-CI]+; 314, [Etd] +. Synthesis of cis-[Pt(NH,),(N3Etd)~~I2 (IC). An excess of ammonium chloride (15 mg, 280 pmol) was added to an aqueous solution ofcis-[Pt(NH3)2(N3-Etd)CI](OAc)2(5mg;8.0pmolin 1.5mLof H20). The solution was immediately frozen and lyophilized to dryness. The solid was washed with 5 mL of ethanol to remove ammonium acetate and excess ammonium chloride and again lyophilized to dryness. Crystalline material was obtained by refrigeration at 4 OC of a methanol solution of cis- [R(NH,)2(N3-Etd)CIl2+ containing excess ammonium chloride. CI]C was I ~also The N8 linkage isomer, cis- [ P ~ ( N H ~ ) z ( N ~ - E ~ ~ )(la), crystallized in this manner from an aqueous solution. Synthesis of traas-[Pt(NH~)2(Etd)Cl](OAc)2. Starting from transwas DDP (1.00 g, 3.33 mmol), crude rr~ns-[R(NH3)2(Etd)CIj(NO,)~ obtained by the procedure identical to that used for the cis compound. Yield: 1.57 g (67% based on trans-DDP). The N3 and N8 linkage isomers were separated with acetate as the counterion by reverse phase HPLC. Spectroscopic data for trans- [Pt(NH3)2(N3-Etd)CI] (OAC)~ (2p): IH NMR (measured on a Varian XL300,300 MHz, CD3OD) 6 8.89 (d, HI, 3J = 9.13 Hz), 8.74 (d, H10, 3J = 9.13 Hz), 8.39 (d, H4, 4J = 1.82 Hz), 7.93 (dd, H2, 3J = 8.70 Hz, 4J = 1.81 Hz), 7.80 (phenyl, unresolved), 7.64 (m, phenyl, H9 unresolved), 6.58 (d, H7, 'J = 2.43 Hz), 4.77 (q, CH2),1.92(~,6H,acetate), 1.53 (t,CH3). '95RNMRspectrum(MeOH) 6 -2360. UV-vis spectral data A[, in nm, (P in M-I cm-I)]: H20,456 (4540); MeOH, 524 (4790). FABMS (glycerol/water) M/z: 578, [M - H+]+(matchestheoretical isotopedistribution); 561, [M-H+-NH3]+; 543, [M - H+ - CI]+; 314, [Etd]+. Spectroscopicdata for trans- [R(NH3)2(NB-Etd)Cl](0Ac)z (2b): lH NMR (CD30D) 6 8.88 (d, H10, 3J = 9.28 Hz), 8.81 (d, H1, 3J = 9.77 Hz), 8.16 (dd, H9, 3 J = 9.03 Hz, 4J = 2.20 Hz), 7.81 (m, phenyl, unresolved), 7.65 (dd, phenyl, 3J = 8.06 Hz, 4J = 1.22 Hz), 7.46 (s, H4), 7.46 (dd, H2, unresolved), 7.23 (d, H7, 4J = 1.95 Hz), 4.68 (q, CHI, 3J = 7.20 Hz), 1.90 (s, 6 H, acetate), 1.54 (t, CH3, 'J = 7.08 Hz). 195Pt NMR (MeOH) 6 -2377. UV-vis spectral data A[, in nm, (e in M-1 cm-I)]: H20,454 (5850);MeOH,491(7390). FABMS (glycerol/water) M/z: 578, [M - H+]+ (matches theoretical isotope distribution); 561, [M - H+ - NH3]+; 543, [M - H+ - CI]+; 314, [Etd]+. Synthesisofcis[Pt(NHa)(Etd)C12](OAc). (Ph,P)[Pt(NH3)C13] (1.50 g, 2.28 mmol) was allowed to react with AgN03 (0.39 g, 2.3 mmol) in 30 mL of DMF at room temperature for 24 h. After centrifugation to remove AgCI, the solution was added to ethidium nitrate (0.95 g, 2.3 mmol). The mixture was then gently stirred at room temperature for 72 h. Subsequent removal of the solvent in vacuo yielded a red-orange oil. The oily product mixture was washed with diethyl ether (25 mL) and triturated with chloroform (50 mL) to solidify the product and remove excess ethidium nitrate. The solidified crude product mixture, which contained insoluble (Ph4P)NO3, was collected by centrifugation and washed with chloroform and diethyl ether and then air dried. The yield was 1.94 g (77% based on (Ph#)[Pt(NH3)C13], and assuming that the crude product was a 1:l mixture of the desired Pt complex and (Ph4P)N o d . Separation of the isomers was achieved through reverse-phase HPLC as described above. Spectroscopic data for cis- [P~(NHS)(N~-E~~)CI~](OA~) ( 3 4 : 1H NMR (CD3OD) 6 8.88 (d, H1, 'J = 8.79 Hz), 8.73 (d, H10, 3J 9.28 Hz), 8.35 (d, H4, 4J = 1.46 Hz), 7.94 (dd, H2, 3J = 8.79 Hz, 4J = 1.46 Hz), 7.79 (m, phenyl, unresolved), 7.64 (m, phenyl, H9, unresolved), 6.59 (d, H7, 4J = 2.44 Hz), 4.77 (q, CHI, 3J = 7.08 Hz), 1.92 (s, 3 H, acetate), 1.59 (t, CH,, 3J = 7.08 Hz). lssPt NMR (MeOH) 6 -2093. UV-vis spectral data A[, in nm, (e in M-l cm-I)]: HzO, 449 (8300); MeOH, 486 (8300). FABMS (glycerol/water) M/z: 596, [MI+ (matches theoretical isotope distribution); 3 14, [Etd]+. Spectroscopic data for cis- [Pt(NH3)(N8-Etd)Clz](OAc) (3b): 1H NMR (CD3OD) 6 8.80 (2d, H1, H10, unresolved), 8.1 1 (dd, H9, 3J = 9.03 Hz, 4J = 2.20 Hz). 7.78 (m, phenyl, unresolved), 7.66 (m, phenyl, unresolved), 7.46 (m, H2, H4, unresolved), 7.28 (d, H7, 'J = 1.95 Hz), 4.69 (q, CH2, 'J = 7.45 Hz), 1.93 (s, 3 H, acetate), 1.54 (t, CH3, 3J = 7.45 Hz). Is5Pt NMR (MeOH) 6 -2109. UV-vis spectral data A[, innm, (einM-"rl)]: H20,452(8720); MeOH,486(7310). FABMS (glycerol/water) M / z : 596, [MI+ (matches theoretical isotope distribution); 314, [Etd]+. Synthesis of [Pt(NH3)3(Etd)](OAc),. [Pt(NH3)pCI]C1(0.634g, 2.0 mmol) was allowed to react with AgNO3 (0.700 g, 4.1 mmol) in DMF
Study of the Pt(II) Complexes of the Ethidium Cation
J. Am. Chem. SOC.,Vol. 115, No. 24, 1993 11343
at room temperature for 24 h. The mixture was centrifuged to remove Table I. Experimental Details of the X-ray Diffraction Studies of ICand Id precipitated AgCl and ethidium nitrate (0.83 g, 2.2 mmol) was added. The solution was stirred for 72 h, evaporated to dryness in vacuo, and lc.2.5H20 ld.2H20 washed with diethyl ether. A red-orange solid containing a mixture of formula PtC21H2702.sNsCh N3 and N8 linkage isomers was isolated after trituration with chloroform. fw 690.92 The yield was 1.47 g (98% based on [Pt(NHs)3Cl]CI). The N3 and N8 cryst syst triclinic triclinic isomers wereseparated withacetateascounterion by reverse-phaseHPLC. space group Pi Pi Alternatively, [Pt(NH3)3Cl](NOs) was used as starting material. 7.939(2) 11.547(2) a, A Spectroscopic data for [Pt(NH3)3(N3-Etd)](OAc)3( 4 4 : IH NMR 12.919(4) 12.906(3) b, A (CDsOD) 6 8.56 (d, H1, ' J = 9.28 Hz), 8.53 (d, H10, 3J 9.28 Hz), 14.886(6) 9.384(2) c, A 7.82 (s, H4), 7.76 (m, phenyl, unresolved), 7.68 (d, H2, 3J = 8.79 Hz), 113.69(3) 105.92(2) a,deg 7.59(m,phenyl,unresolved),7.52(dd,H9,3J=9.03H~,~J=2.20Hz), B. deg 94.66(3) 93.69(1) 6.41 (d, H7, 4J = 1.95 Hz), 4.64 (q, CH2, ' J = 7.08 Hz), 1.90 (s, 9 H, 90.21(3) 74.84(2) 7 7 deg Z acetate), 1.51 (t, CH3, 3J = 7.08 Hz). lPJPtNMR (MeOH) 6 -2532. L 2 v,A3 1392.5(8) 1292.9(4) in nm, (e in M-I ~ m - ~ ) ]H20, : 443 (4370); UV-vis spectral data [A, 1.640 1.744 MeOH, 632 (1 1860). FABMS (glycerol/water) M/z: 558, [M - 2H+]+ 173 173 (matches theoretical isotope distribution); 524, [M - 2H+ - 2NH3]+; no. of reflctns collctd 1768 4554 507, [M - 2H+ - 3NH3]+; 314, [Etd]+. data collctn range, deg 4 5 2e 5 35 3 I2e 5 50 Spectroscopic data for [Pt(NH3)3(Nt?-Etd)](OAC)3 (4b): IH NMR data limits +h,ik,il +h,ik,kl (CDjOD) 6 8.82 (d, H10, ' J = 9.28 Hz), 8.78 (d, H1, ' J 9.77 Hz), 1459 no. of unique datae 3380 8.16 (dd, H9, sJ = 9.03 Hz, 4J= 2.20 Hz), 7.81 (m, phenyl, unresolved), no. of parameters 182 30 1 7.63 (m, phenyl, unresolved), 7.46 (dd, H2, unresolved), 7.45 (s, H4), p(Mo Ka), cm-1 54.02 57.96 7.23 (d, H7, 4J = 2.44 Hz), 4.67 (q, CH2, 3J = 7.08 Hz), 1.87 (s, 9 H, transmission coeff 0.76-1.24d 0.7&1.00 acetate), 1.53 (t, CH3, 3J = 7.08 Hz). Ig5PtNMR (MeOH) 6 -2563. Rb 0.063 0.052 UV-vis spectral data A[, in nm, (e in M-I cm-I)]: H20, 454 (5900); 0.076 0.070 RWC largest shift/esd, final 0.01 MeOH, 490 (3970). FABMS (glycerol/water) M/z: 558, [M - 2H+]+ 0.06 largest peak, e/A3 1.35c 2.47e (matches theoretical isotope distribution); 524, [M - 2H+ - 2NH#; 507, [M - 2H+ - 3NH3]+; 314, [Etd]+. Visible Absorption Spectra and DNA Binding of Platinum-Ethidium Complexes. Spectra were obtained on a Perkin-Elmer Lambda 7 e Near C1- counterion. spectrophotometer equipped with a data station unless otherwise specified. Extinction coefficientswereobtained using solutions in which the platinum in our laboratory.IJ Absorption was taken into account by use of the concentration was determined by atomic absorption spectroscopy. Spectra program DIFABS.I6 The lattice contained a region of partially occupied for pH-dependent studies were obtained by titrating aqueous solutions and/or disordered solvent which was included in the refinement as five ofthe complexeswith 0.1 N NaOH and monitoring thevisibleabsorption partially occupied H20 molecules; the presence of additional disordered spectrum at various pH values. An Orion Research Model 23 1 pH meter solvent is not ruled out. Other information is provided in Table I and as supplementary material (Tables S l S 6 ) . equipped with a semimicro Ross combination pH electrode was used to measure the solution pH in the cell immediately before a q u cis-[Pt(NH3)2(N8-Etd)CI]C12.2HzO. An orange crystal of dimensions 0.075 X 0.167 X 0.225 mm was mounted on a glass fiber with epoxy each spectrum. Standardized buffer solutions were used to calibrate the cement. The crystal quality was good as judged by open counter w-scans pH meter prior to each titration. For pH-dependent spectra of ternary on several strong, low-angle reflections which revealed an average width complexes generated from the reactions of l a and l b with DNA, 1 mL at half height of 0.27O. Unit cell parameters were obtained by a leastof a 2.5 mM solution of calf thymus DNA was used. The complexes were squares fit of 25 well-centered reflections, and space group P l was chosen added from freshly prepared stock solutions at a formal Pt:nucleotide and confirmed by successful refinement of the structure. The crystal did ratio (rf) of 0.1. The platinum complexes were allowed to react with the not decay appreciably ( 3.3 eV for 1,2, and 3, and AE = 1.6 eV for 4). Instead, the band appears more likely to involve charge transfer from the HOMO, and possibly also the SHOMO, to the LUMO. Such an assignment is based on the fact that the HOMO of all [BL] complexescontainssignificant contributions (-20%) from both the pI orbital of the coordinated amine and the pI orbitals of C1, C2, and C4. A significant transition dipole moment is therefore expected. Thus, deprotonation shifts the donor orbital involved in charge transfer from the uncoordinated to the coordinated exocyclic amino group of the ethidium ligand, now a major contributor to the HOMO, the acceptor (LUMO) remaining the T* orbital of the ring in both cases. From the discussion above it is evident that the HOMO-LUMO gaps for [BL] complexes are always smaller than the SHOMO-LUMO gaps in the corresponding [OR] forms, which accounts for the observed red shift of the absorption maximum upon deprotonation. It should be pointed out that, despite the consistency in the qualitative assignments just made, none of the calculated (S)HOMO-LUMOgaps matches the experimentally determined transition energies (orange forms at 480 nm, ca. 2.58 eV; blue forms, 640 nm,ca. 2.06 eV). Thedifferencebetweenthe transition energies of the [OR] and [BL] forms also does not correspond quantitatively to the observed red-shift of ca. 0.5 eV. These discrepanciesare not unexpected, however, since it is well-known that the single Slater determinant Hartr-Fock method tends to overestimate the Coulombic repulsion of the unoccupied orbitals, and consequently a larger HOMO-LUMOgap is always found in such calculations.42 In addition, we have not been able to correlate the results of the present MO calculations with the observed trend in the acidity of the Pt-Etd complexes, which is not surprising since acidity has rarely been addressed by SCFMO methods. Effect of DNA on the Linkage Isomer Obtained during Formationof a Ternary Complex amongEthidium, Platinum, and a Nucleobase. As previously reported, DNA can serve as a template to facilitate the reaction of cis-DDP and ethidium to form ternary ethidium-Pt-DNA complexes.5-' The nature of these complexes was revealed when it was demonstrated that covalent binding of cis- [Pt(NH3)2(N3-Etd)Cl](OAC)~ and cis[Pt(NH3)2(N8-Etd)Cl](OAc)2 to DNA affords species which are spectroscopically indistinguishable from ternary complexes formed by addition of cis-DDP and ethidium to DNA.' The ternary complexes therefore consist of a (Pt(NH3)2)2+moiety covalently bonded to the 3-amino (or 8-amino) group of ethidium as well as to the N7 position of a purinenucleobase. Remarkably, the reaction rate of platinum with the ethidium exocyclic amino group is much more rapid in the presence of DNA than in its (41) Ballhausen,C.J. MolecularElectronicStructuresofTransitionMetal Complexes; McGraw-Hill: London, 1979. (42) Connolly,J. W. D. InsemiempiricalMethodsof ElectronicStructure Calculation.Part A: Techniques;G. A. Segal, Ed.; Plenum Press: New York, 1977.
Ren et al.
11352 J. Am. Chem. SOC.,Vol. 115, No. 24, I993 0.5
0.4
-/
A
a
O
b
* '
I
,.""
to the N8 exocyclic amino group. This result provides the first example of regiospecificity in a DNA-promoted reaction, further supporting the concept that the double helix serves to bind and orient the two reactants in much the same manner as occurs in enzyme-catalyzed condensation reactions.
Concluding Remarks In this paper we have thoroughly characterized a series of complexes formed between platinum(I1) ammines and the ethidium cation. Platinum binding occurs at both exocyclic amino groups of ethidium forming linkage isomers that can be separated chromatographically. The structures of these complexes have been determined for representative examples by X-ray crystalA* lography in the solid state, in solution by N M R spectroscopy, and in the gas phase by mass spectrometry. The orange-to-blue ;*oe: , thermochromism of solutions of the acetate salts of the complexes O * has been shown to arise from deprotonation of the exocyclic amino 6 7 8 9 10 11 12 13 group to which the platinum atom is coordinated, the presence of the positively charged metal ion at this position lowering the PH pKa of this group. The change in pK. is affected by the overall Figure 11. Plot of absorbance versus pH for (a) cis-[Pt(NHp)z(Njlcharge on the complex, the linkage isomer, and the stereoisomer Etd)]-df thy"DNA, (b) ~fs-[Pt(NH3)z(N&Etd)]-4fthp~DNA, (cis versus trans) in a manner dictated by the electronic properties and (c) Pt-Etd-calf thymus DNA ternary complex generated in situ. Rf of the resulting acid anion. From Fenske-Hall molecular orbital is 0.1. The absorbances were monitored at 612 nm. calculations it has been possible to assign the visible spectral transition of the orange and blue species. The former arises from absence. Possibly, the DNA double helix serves both to increase charge transfer involving the lone pair on the unplatinated the collision frequency of the freely diffusing Etd+ moiety with ethidium amino group and the T* orbital of the ring (LUMO), the covalently anchored cis-diamminechloroplatinum(I1) fragwhereas in the blue complexes, the optical transition is from an ment and to orient the two reagents, optimizing the stereochemorbital (HOMO) localized primarily near the coordinated, now istry required for nucleophilic displacement of the chloride ion. deprotonated, imino functionality to the same acceptor orbital. If this model is correct, then DNA might also be expected to Finally, the solution thermochromism of these complexes has influence the regioselectivity of the reaction of cis-diammibeen used to identify the regiospecificity of the ternary complex nechloroplatinum(I1) with ethidium. In particular, our previous formed between cis-diamminedichloroplatinum(II),ethidium, and model building studies suggested that platinum might react calf thymus DNA. This DNA-promoted reaction leads prepreferentially at the N8 rather than the N3 position of the dominantly to the single regioisomer in which DNA-bound phenanthridium ring.7 platinum is coordinated to the 8-amino group of the ethidium Until now, however, it has not been possible to assess the ring. regioselectivity of this DNA-promoted reaction because the absorption spectra of ternary complexes formed by the N3 and Acknowledgment. This work was supported by U.S. Public N8 isomers are nearly identi~al.~ The discovery that the different Health Service Grant CA34992 (to S.J.L.) from the National linkage isomers have distinctly different pKa values, however, Cancer Institute and N I H National Research Service Award provides a spectroscopic means of assessing which amino group GM11880-01 from the National Institute of General Medical of the ethidium ion is coordinating to platinum in the DNASciences (to D.P.B.). A.M. thanks the Human Frontier Science promoted reaction. As shown in Figure 11, the color change that Program Organization for a fellowship and research funding. accompanies deprotonation of the platinated exocyclic amino FAB mass spectra were obtained with the assistance of Dr. C. group in the cis-{Pt(NH3)2(N3-Etd))'+and cis- (Pt(NH&(N8Costello at the facility supported by NIH Grant R R 00317 Etd)]3+ DNA adducts occurs a t a much lower pH value for the (Principal Investigator, Prof. K.Biemann) from the Biotechnology former than the latter. This result nicely parallels the relative Resources Branch, Division of Research Resources. We also pKa values of the corresponding chloro complexes of these two thank Drs. L. Chassot and J. G. Bentsen for helpful discussions. cations. Moreover, the figure reveals that the titration curve for the ternary complex closely mimics that for the adduct made SupplementaryMaterial Available: Tables of atomic positional with the N8 isomer. These data indicate that the reaction of parameters for non-hydrogen atoms, anisotropic temperature cis-DDP and ethidium on a DNA template is regiospecific, with factors, bond distance and angles, and torsion angles for ICand platinum reacting at the N8-amino position of ethidium. This Id (Tables SI,2,4-8, 1&12), the Appendix, figures of overlay regiospecificityreflectstheasymmetric binding of Etd with respect and extracted spectra for complexes la, 2a, 2b, and 4a (Figures to the pseudo-2-fold axis that passes between adjacent base pairs S1-8), and tabulation (Table S13) of upper valence molecular of the double helix.7,43*'4 Thus, DNA serves not only to increase orbitals from Fenske-Hall calculations (3 1 pages); listing of thereaction rateofcisplatin withEtd but also todirect thereaction calculated and observed structure factors (Tables S3 and S9) (42 pages). Ordering information is given on any current masthead (43) Tsai, C.C.; Jain, S . C. J. Mol. Biol. 1977, l I 4 , 317. (44) Lybrand, T.; Kollman, P . Biopolymers 1985, 24, 1863. page-
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