Synthesis of cis-[Cr(diimine)2(1-methylimidazole)2]3+ Complexes and

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Synthesis of cis-[Cr(diimine)2(1-methylimidazole)2]3+ Complexes and an Investigation of Their Interaction with Mononucleotides and Polynucleotides Sarah K. Goforth, Thomas W. Gill, April E. Weisbruch, Kimberlee A. Kane-Maguire, Marian E. Helsel, Katherine W. Sun, Hillary D. Rodgers, Floyd E. Stanley, Samuel R. Goudy, Sandra K. Wheeler,* John F. Wheeler,* and Noel A. P. Kane-Maguire* Department of Chemistry, Furman University, Greenville, South Carolina 29613, United States S Supporting Information *

ABSTRACT: A protocol is presented for the synthesis of chromium(III) complexes of the type cis-[Cr(diimine)2(1methylimidazole)2]3+. These compounds exhibit large excitedstate oxidizing powers and strong luminescence in solution. Emission is quenched by added guanine, yielding rate constants that track the driving force for guanine oxidation. The cis[Cr(TMP)(DPPZ)(1-MeImid)2]3+ species binds strongly to duplex DNA with a preference for AT base sites in the minor groove and may serve as a precursor for photoactivated DNA covalent adduct formation.

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INTRODUCTION We have previously undertaken extensive studies on the interaction of [Cr(diimine)3]3+ complexes with duplex DNA.1−7 These and other recent investigations8−11 have provided evidence that the 2Eg (Oh) excited state of tris(diimine)chromium(III) compounds is capable of photoactivating DNA strand cleavage via direct one-electron guanine base oxidation. Because of the marked inertness to substitution of the bidentate diimine ligands present, the DNA binding of these complexes is noncovalent in nature (occurring primarily in the minor groove and with a predilection for AT base binding sites).1,5,6 In a very recent report by Turro and co-workers, two rhodium systems were described that concurrently exhibit both covalent and noncovalent modes of binding to DNA.12 Covalent adduct formation with DNA is initiated in each instance via the facile photorelease of monodentate ligands from the parent transition-metal complex. Such species and others investigated by the Turro and Dunbar groups13−16 offer considerable promise as dual-action phototherapeutic agents, and we have been encouraged to explore similar behavior for chromium(III) systems related to the [Cr(diimine) 3 ]3+ complexes mentioned above. We report here our initial studies in this area, where we have developed synthetic procedures for the isolation of octahedral chromium(III) complexes with the general formula cis-[Cr(diimine)2(1-MeImid)2]3+, where 1-MeImid is the monodentate ligand 1-methylimidazole. The 1-MeImid molecule features a five-membered aromatic ring system containing an imine N © XXXX American Chemical Society

binding site, which is suggestive of the N7 position in the guanine and adenine bases of DNA (Figure 1).

Figure 1. Structures of 1-MeImid, dGMP, and dAMP.

Chromium(III) complexes of this type may deliver several attractive possibilities: 1. Development of drugs utilizing readily available first-row transition metals that could confer some economic advantages. 2. Light-activated guanine base oxidation because it is probable that the 2 E g (O h ) excited state of cis-[Cr(diimine)2(1-MeImid)2]3+ systems will retain much of the Received: October 12, 2015

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DOI: 10.1021/acs.inorgchem.5b02323 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry high oxidizing power known for [Cr(diimine)3]3+ systems.17 This expectation is based on the electronic similarity of the imine N lone-pair donor site of 1-MeImid to that of the aromatic N atoms of diimine ligands. 3. The monodentate 1-MeImid ligands are likely to be photolabile. Thus, photolysis of these compounds when noncovalently bound to DNA might lead to covalent cis-GG or cis-AG adduct formation reminiscent of that present with square-planar cisplatin.18 An octahedral complex literature precedent is the intrastrand covalent binding reported by Singh and Turro when cis-[Ru(bpy)2(NH3)2]2+ (where bpy = 2,2′-bipyridine) is irradiated in the presence of a ds-15-mer.19 A novel aspect of such cis-GG adduct formation with a bis(diimine)chromium(III) core would be the likelihood that the 2Eg (Oh) excited state of the CrIII center will have an oxidizing power comparable to that for the cis-[Cr(diimine)2(1MeImid)2]3+ parent because of the molecular structural similarities (see Figure 1). Therefore, subsequent light activation after adduct formation may also result in further DNA damage, thus providing a potential additional avenue for enhanced and more selective tumor cell destruction in any future phototherapeutic applications. It is important to note, however, that the likelihood of photoactivated covalent adduct formation with DNA by these new 1-MeImid complexes is predicated by the necessity that 1MeImid ligand photoaquation is a competitive excited-state deactivation process versus the redox pathway involving DNA oxidation. Should photooxidation of DNA by the 2Eg (Oh) excited state of the chromium(III) complex be very efficient and very rapid, then photoadduct formation would be very unlikely. This important caveat is addressed later in the Results and Discussion section. Herein we describe the syntheses of cis-[Cr(diimine)2(1MeImid)2]3+ complexes for the cases where the diimine ligand is either bpy, 1,10-phenanthroline (phen), 3,4,7,8-tetramethyl1,10-phenanthroline (TMP), 5-chloro-1,10-phenanthroline (5Clphen), or dipyridophenazine (DPPZ) (Figure S1). The complex cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ is of particular interest because of the presence of the intercalating DPPZ ligand, which results in an especially strong binding interaction with duplex DNA (vide infra). All of these complexes exhibit strong 2Eg → 4A2g (Oh) phosphorescence in a room temperature aqueous solution, and kinetic information is reported for the quenching of their emission by the mononucleotides 2′-deoxyguanosine 5′-monophosphate (dGMP) and 2′-deoxyadenosine 5′-monophosphate (dAMP). Emission quenching data are also presented for cis[Cr(TMP)(DPPZ)(1-MeImid)2]3+ with calf thymus DNA (CT-DNA). In addition, UV−visible titration data for cis[Cr(TMP)(DPPZ)(1-MeImid)2]3+ with CT-DNA and the polynucleotide poly(dA-dT)·poly(dA-dT) are provided. Likewise, equilibrium dialysis/circular dichroism (CD) studies for this complex with several polynucleotides are reported. Also presented is a representative case study for 1-MeImid ligand photoaquation employing cis-[Cr(phen)2(1-MeImid)2]3+.

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cia Biotech, Inc. Sodium salts of dGMP, dAMP, and CT-DNA were received from Sigma-Aldrich, as were the dihydrochloride salts of distamycin A and netropsin. All other chemicals employed were of reagent-grade quality from commercial sources and were used without further purification. Elemental analyses (C, H, and N) were performed by Midwest Microlab. The following molar absorptivities, ε, were used for concentration determinations: CT-DNA, ε260 = 6600 M−1 cm−1/ base pair; poly(dG-dC)·poly(dG-dC), ε254 = 8400 M−1 cm−1/base pair; poly(dA-dT)·poly(dA-dT), ε262 = 6600 M−1 cm−1/base pair; distamycin A, ε302 = 37000 M−1 cm−1;20 netropsin, ε296 = 21500 M−1 cm−1.21 The DPPZ ligand was synthesized from 1,10-phenanthroline-5,6dione22 using a literature procedure,23a and its purity was verified via 1 H NMR. Precursor homoleptic complexes of the type cis-[Cr(diimine)2Cl2]Cl (diimine = bpy, phen, TMP) were synthesized employing a literature method.24 With the exception of the cis-[Cr(5Clphen)2Cl2]Cl system, cis-[Cr(diimine)2Cl2]Cl compounds were subsequently converted to the corresponding cis-[Cr(diimine)2(CF3SO3)2]CF3SO3 complexes by dissolution in 98% trifluoromethanesulfonic acid for 24 h (while purging the solution with N2 to expel HCl gas), followed by precipitation with ether.6,25,26 Instrumental Methods. UV−visible absorption spectra were recorded on either a HP 8250a diode array or Cary 50 spectrophotometer, while a SPEX Fluorolog 2 or a SLM 8000C fluorimeter employing a red-sensitive Hamamatsu R928 photomultiplier tube was used for steady-state emission studies. Analogous emission lifetime measurements were performed utilizing a Photon Technology International N2 laser (model GL-3300)/dye laser (GL302) system. Pulse excitation was performed at 380 nm using an Excitation Inc. BBQ dye. Data were collected using an OLIS SM-45 EM emission lifetime measurement system and analyzed utilizing OLIS SpectraWorks. 1H NMR spectra were obtained by employing a Varian 300 MHz spectrophotometer. Cyclic voltammetry measurements in an aqueous solution were carried out using a BAS 100B electrochemical analyzer, employing platinum working and auxiliary electrodes and a Ag/AgCl reference electrode, with 0.1 M KCl as the supporting electrolyte. All electrospray ionization mass spectrometry (ESI-MS) studies were performed on acetonitrile solutions of the chromium(III) products using a Waters ZQ LC/MS system. Chiral capillary electrophoresis (CCE) studies of product purity were carried out on a Beckman Instruments MDQ HPCE system with UV−visible detection, via methods presented previously.6 Potassium antimonylL-tartrate, sodium dibenzoyl-L-tartrate (prepared by NaOH titration of commercially available dibenzoyl-L-tartaric acid), or the sodium salt of sulfonated β-cyclodextrin were used as the chiral buffer additive in these CCE studies. Equilibrium dialysis experiments on cis-[Cr(TMP)(DPPZ)(1MeImid)2]3+ with duplex DNA were carried out using procedures identical with those outlined in detail previously.6 Each dialysis cell was separated into two chambers by a 6000 MW cutoff membrane that did not permit passage of the polynucleotides employed. One side was loaded with a buffer solution of racemic cis-[Cr(TMP)(DPPZ)(1MeImid)2]3+/DNA (with or without added distamycin A or netropsin), while the other side contained an equal volume of buffer only. The entire cell was then placed in the dark at room temperature for 96 h to achieve equilibrium. A JASCO 710 spectropolarimeter was used for the subsequent CD measurements. Synthesis of Homoleptic Bis(diimine) Complexes. Synthesis of cis-[Cr(bpy)2(1-MeImid)2](CF3SO3)3·2H2O. A sample of cis-[Cr(bpy)2(CF3SO3)2]CF3SO3 (0.120 g, 1.48 × 10−4 mol) was added to 1-MeImid (93.8 μL, 1.18 × 10−3 mol) dissolved in 15 mL of anhydrous dichloromethane in a 50 mL round-bottomed flask. The mixture was refluxed for 1 h and then refrigerated for 1.5 h. The yellow solid obtained was collected by suction filtration and washed with two 15 mL portions of dichloromethane and two 15 mL portions of ethyl ether. The precipitate was suction-dried for 10 min, leaving a powdery yellow solid. Yield: 0.1203 g (83%). Anal. Calcd for C31H32N8F9S3O11Cr: C, 36.80; H, 3.19; N, 11.07. Found: C, 36.55;

EXPERIMENTAL SECTION

Chemicals. Trifluoromethanesulfonic acid (98%), 1-MeImid, and the diimine ligands bpy, phen, TMP, and 5-Clphen were acquired from Sigma-Aldrich. Likewise, the chiral additive β-sulfonated cyclodextrin (sodium salt) and [Cr(THF)3Cl3]0 were purchased from SigmaAldrich. The synthetic polynucleotides poly(dG-dC)·poly(dG-dC) and poly(dA-dT)·poly(dA-dT) were provided by Amersham PharmaB


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Inorganic Chemistry H, 2.95; N, 10.90. ESI-MS: intense M3+ parent ion peak (for the Cr-52 isotope) at m/z 176.3. Synthesis of cis-[Cr(phen)2(1-MeImid)2](CF3SO3)3·1.5H2O. A sample (88.9 μL, 1.11 × 10−3 mol) of 1-MeImid was added to 50 mL of anhydrous dichloromethane in a 100 mL round-bottomed flask. To this solution was added cis-[Cr(phen)2(CF3SO3)2]CF3SO3 (0.1207 g, 1.40 × 10−4 mol), and the solution was refluxed for 1.5 h. Within 15 min, the color of the solution changed from red to yellow. The yellow precipitate obtained was collected by vacuum filtration, washed with two 15 mL aliquots of dichloromethane and two 15 mL aliquots of anhydrous ethyl ether, and suction-dried. Yield: 0.124 g (86%). Anal. Calcd for C35H31N8F9S3O11.5Cr: C, 40.00; H, 2.97; N, 10.66. Found: C, 39.66; H, 2.81; N, 10.90. ESI-MS: intense M3+ parent ion peak (for the Cr-52 isotope) at m/z 192.2. Synthesis of cis-[Cr(TMP)2(1-MeImid)2](CF3SO3)3·H2O. A sample of cis-[Cr(TMP)2(CF3SO3)2]CF3SO3 (0.1204 g, 1.23 × 10−4 mol) was added to a solution of 1-MeImid (39.2 μL, 4.94 × 10−4 mol) in 15 mL of anhydrous dichloromethane in a 50 mL round-bottomed flask. The red solution obtained was refluxed for 2.5 h, which resulted in the color changing to a deep yellow. The yellow solution was evaporated to dryness under a gentle N2 stream. A 2 mL aliquot of H2O was then added to the oily yellow solid, and the suspension was sonicated for several minutes. The resulting yellow solid was collected by vacuum filtration, washed with 15 mL of ether, and suction-dried for 10 min. Yield: 0.1053 g (73%). Anal. Calcd for C43H46N8F9S3O10Cr: C, 44.75; H, 4.02; N, 9.71. Found: C, 44.87; H, 3.94; N, 9.49. ESI-MS: intense M3+ parent ion peak (for the Cr-52 isotope) at m/z 229.3. Synthesis of cis-[Cr(5-Clphen)2Cl2]Cl. In a 25 mL round-bottomed flask, [Cr(THF)3Cl3]0 (0.675 g, 1.80 × 10−3 mol) was added to 5 mL of anhydrous methanol. The free 5-Clphen ligand (1.000 g, 4.66 × 10−3 mol) was then added to the purple mixture, whereupon the color of the solution mixture changed from purple to green. After 4 h of reflux, followed by a brief cooling, the resulting olive-green precipitate obtained was collected by vacuum filtration and washed with three 10 mL aliquots of ethyl ether. Yield: 0.105 g. The dark-green filtrate was stored in the refrigerator overnight, and the additional olive-green precipitate obtained was suction-filtered and washed with three 10 mL aliquots of ethyl ether. Yield: 0.604 g. Total yield: 0.709 g (67%). ESIMS: intense M+ parent ion peak (for the Cr-52 isotope) at m/z 551.9. Synthesis of cis-[Cr(5-Clphen)2(1-MeImid)2](CF3SO3)3·2H2O. In a 25 mL round-bottomed flask, [Cr(5-Clphen)2Cl2]Cl (0.200 g, 3.40 × 10−4 mol) was added to 25 mL of anhydrous dichloromethane. AgCF3SO3 (0.267 g, 1.04 × 10−3 mol) was then added. The mixture was covered in foil and refluxed for 23 h. After Celite was stirred into the mixture, the solid byproduct (consisting of AgCl and excess AgCF3SO3) was removed by suction filtration and washed with 15 mL of anhydrous dichloromethane. To the 20 mL of deep-red filtrate (∼3.4 × 10−4 mol of [Cr(5-Clphen)2(CF3SO3)2]CF3SO3) was added 1-MeImid (0.324 mL, 4.05 × 10−4 mol). The solution was stirred at room temperature for 2 h with a drying tube attached at the top of the condenser. After reflux, a yellow precipitate was present, and the mixture was sonicated. The yellow solid was collected by vacuum filtration and washed with anhydrous ethyl ether. The final solid was a canary-yellow powder. Yield: 0.057 g (30%). Anal. Calcd for C35H30N8F9S3O10Cr: C, 37.34; H, 2.68; N, 9.93. Found: C, 37.24; H, 2.33; N, 9.74. ESI-MS: intense M3+ parent ion peak (for the Cr-52 isotope) at m/z 214.9. Synthesis of Heteroleptic Bis(diimine) Complexes. Synthesis of cis-[Cr(phen)(TMP)(1-Melmid)2](CF3SO3)3·3H2O. In a 10 mL round-bottomed flask, cis-[Cr(phen)(TMP)(CF 3 SO 3 ) 2 ]CF3 SO 3 (0.0507 g, 5.54 × 10−5 mol) was combined with 1-MeImid (15.6 μL, 1.96x 10−4 mol) in 5 mL of anhydrous chloroform. The mixture was refluxed for 2.5 h. The bright-yellow precipitate that formed was collected by vacuum filtration, washed with ethyl ether (≈30 mL), and suction-dried. Yield: 0.0479 g (80.1%). Anal. Calcd for C39H42N8F9S3O12Cr: C, 41.31; H, 3.73; N, 9.88. Found: C, 41.27; H, 3.65; N, 9.51. ESI-MS: intense M3+ parent ion peak (for the Cr-52 isotope) at m/z 211.2. Synthesis of [Cr(DPPZ)Cl3DMF]0. A vacuum-dried, finely ground sample of CrCl3·6H2O (1.51 g, 5.68 mmol) was dissolved in 16 mL of

hot N,N-dimethylformamide (DMF) with stirring to give a dark-green solution. To this was added DPPZ (1.01 g, 3.59 × 10−3 mol), and the mixture was stirred for 30 min on a hot plate. A green precipitate formed, and after the reaction mixture was cooled to room temperature, the solid was vacuum-filtered, washed with 30 mL of ethyl ether, and suction-dried. Yield: 1.49 g (81.0%). Synthesis of cis-[Cr(TMP)(DPPZ)Cl2]CF3SO3. A sample of [Cr(DPPZ)Cl3(DMF)]0 (1.00 g, 1.95 × 10−3 mol) was placed in a 100 mL round-bottomed flask with Ag(CF3SO3) (0.50 g, 1.95 × 10−3 mol) and 45 mL of anhydrous acetonitrile. The reaction mixture was refluxed for 5 h to give a dark-green solution and a white AgCl precipitate. AgCl was removed by filtering the mixture through Celite. After the Celite was washed with 10 mL of acetonitrile, the filtrate volume (including washings) was reduced to 13 mL by heating gently on a hot plate for ≈20 min. The mixture was added to a 50 mL Erlenmeyer flask containing TMP (0.382 g, 1.62 × 10−3 mol) and then heated on a hot plate for 35 min, yielding a green precipitate in a brown solution. Approximately 20 mL of acetonitrile was added during this heating in order to prevent the mixture from going to dryness. The mixture was placed in the refrigerator for 15 min, and the green precipitate was collected by vacuum filtration, washed with ethyl ether (≈30 mL), and suction-dried. The filtrate was also placed in the refrigerator and then filtered to give a small amount of additional product. Yield: 0.5126 g (51.3%). ESI-MS: intense M+ parent ion peak (for the Cr-52 isotope) at m/z 640.6. Synthesis of cis-[Cr(TMP)(DPPZ)(CF3SO3)2]CF3SO3. A sample of cis[Cr(TMP)(DPPZ)Cl2]CF3SO3 (0.6347 g, 8.03 × 10−4 mol) was reacted at room temperature with 10 mL of 98% CF3SO3H while bubbling the reaction mixture with N2 gas for 25 h in the hood. The reaction solution was then poured into a 600 mL beaker, and 250 mL of ethyl ether was added progressively with scratching to give a yelloworange precipitate. The product was collected through vacuum filtration, washed with ethyl ether (≈100 mL), suction-dried, and stored in a desiccator. Yield: 0.681 g (83.3%). Synthesis of cis-[Cr(TMP)(DPPZ)(1-MeImid)2](CF3SO3)3·3H2O. In a 25 mL round-bottomed flask, cis-[Cr(TMP)(DPPZ)(CF3SO3)2]CF3SO3 (0.0991 g, 9.74 × 10−5 mol) was combined with 1-MeImid (31.2 μL, 3.91 × 10−4 mol) in 10 mL of anhydrous chloroform. The mixture was refluxed for 2 h. The yellow precipitate that formed was collected by vacuum filtration, washed with ethyl ether (≈20 mL), and suction-dried. Yield: 0.0840 g (73.0%). Anal. Calcd for C45H44N10F9S3O12Cr: C, 43.73; H, 3.59; N, 11.33. Found: C, 43.50; H, 3.20; N, 11.27. ESI-MS: intense M3+ parent ion peak (for the Cr-52 isotope) at m/z 245.3.

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RESULTS AND DISCUSSION Syntheses of cis-[Cr(diimine)2(1-MeImid)2]3+ complexes. These syntheses usually involved refluxing a precursor cis-[Cr(diimine)2(CF3SO3)2]CF3SO3 complex with 1-MeImid in a noncoordinating (or a very weakly coordinating) solvent. The choice of cis-[Cr(diimine)2(CF3SO3)2]+ complexes as starting reagents was based on the excellent leaving-group characteristics of the trifluoromethanesulfonate anion ligand, CF3SO3−.6,25−30 Good elemental analyses (% C, H, and N) were obtained for the six cis-[Cr(diimine)2(1-MeImid)2]3+ complexes isolated in the present study. Further evidence for sample purity was obtained from ESIMS and CCE studies on these compounds. ESI-MS data for the 1-MeImid complexes were provided in the Experimental Section. In all cases, the mass spectrum was dominated by an intense ion peak with a m/z value matching that predicted for the target M3+ molecular ion containing the Cr-52 isotope. The triply positive charge of this ion was confirmed from the m/z differences detected between the various isotope satellite peaks. A representative example is shown for cis-[Cr(TMP)(DPPZ)(1-MeImid)2](CF3SO3)3 in Figure S2. C


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Inorganic Chemistry

intense DPPZ ligand π → π* transition, and the marked hypochromism observed upon double-helical DNA addition has been widely attributed to DPPZ/DNA intercalation.6,9,23,36 In the present study, this interaction has been examined for the polynucleotides CT-DNA and poly(dA-dT)·poly(dA-dT). A representative example of the spectral data for the case of CTDNA as the titrant is presented in Figure 4.

We have noted elsewhere the value of CCE for assessing the sample purity for chiral paramagnetic chromium(III) complexes.6,25,26,31−34 Two representative cis-[Cr(diimine)2(1MeImid)2]3+ electropherograms are shown in Figures 2 and

Figure 2. Electropherogram of cis-[Cr(TMP)(DPPZ)(1-MeImid)2](CF3SO3)3 using 130 mM potassium antimonyl-D-tartrate in 25 mM phosphate buffer (pH 5.0). Conditions: 60 cm capillary column (52 cm to detection); field strength = 167 V cm−1; injection time = 8 s at 0.5 psi; detection wavelength = 280 nm; T = 25 °C.

S3 for the species cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ and cis-[Cr(phen)2(1-MeImid)2]3+, respectively. Both complexes exhibit two peaks of equal area, which confirms the expected cis disposition of the monodentate 1-MeImid ligands. These two peaks are assigned to the Λ and Δ optical isomers of the chiral complexes. UV−Visible Absorption Studies. The UV−visible absorption spectra in an aqueous solution for the 1-MeImid compounds are very reminiscent of those observed for related [Cr(diimine)3]3+ complexes. Examples are provided in Figures 3 and S4 for the complexes cis-[Cr(TMP)(DPPZ)(1-

Figure 4. UV−visible spectral titration of a 50 mM Tris-HCl/100 mM NaCl buffer solution (pH 7.4) of 6.0 × 10−5 M cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ in the presence of increasing concentrations of CT-DNA (0−4.5 × 10−4 M). The inset is the Scatchard plot of r/Cf versus r using absorbance data at 360 nm, where r is the average number of chromium(III) complexes per base pair and Cf is the concentration of the unbound chromium(III) over the 20−80% range for bound chromium(III).

The DNA binding constants with CT-DNA and poly(dAdT)·poly(dA-dT) were obtained from their respective Scatchard plots based on the nonlinear least-squares fits using the noncooperative model of the McGhee−von Hippel equation.36 This procedure yielded a KDNA value of 6.0 ± 0.5 × 104 M−1 (n = 2.9) for CT-DNA as the titrant, where n is the number of base pairs per chromium(III) complex. This KDNA value lies within a factor of 3−5 of those we have previously reported for a range of [Cr(diimine)2(DPPZ)]3+ complexes measured under similar conditions.6 For both cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ and the related [Cr(diimine)2(DPPZ)]3+ complexes, the strongest contribution to the overall DNA binding strength is expected to be associated with intercalation by the DPPZ ligand. We have observed for [Cr(diimine) 2 (DPPZ)] 3+ systems6 that changes in the ancillary diimine ligands have relatively modest effects on the overall KDNA values. For the case of cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+, one of these diimine ancillary ligands has been replaced by two 1-MeImid groups. Decreased hydrogen-bonding/van der Waals interactions by the two 1-MeMid ancillary ligands with DNA residues may be responsible for the lower KDNA value observed for this complex with CT-DNA. It is also worth noting that our KDNA values for cis[Cr(TMP)(DPPZ)(1-MeImid)2]3+ and the related [Cr(diimine)2(DPPZ)]3+ systems are substantially smaller than those commonly reported for other DNA intercalating metal complexes. A contributing factor is that the present studies were carried out in a 50 mM Tris-HCl/100 mM NaCl buffer solution. In contrast, low ionic strength conditions have normally been utilized for related [Ru(diimine)2(DPPZ)]2+ systems (for example, see Hiort et al.).23a Finally, it is noted that in the case of poly(dA-dT)·poly(dAdT) as the titrant (see Figure S5), the corresponding KDNA value determined for cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+

Figure 3. UV−visible spectrum of cis-[Cr(TMP)(DPPZ)(1Melmid)2](CF3SO3)3 in a 3.1 × 10−4 M aqueous solution (0.100 cm cell).

MeImid)2]3+ and cis-[Cr(phen)2(1-MeImid)2]3+, respectively. For both complexes, a very intense diimine π → π* transition is observed in the 250−300 nm region, whereas the low-intensity shoulder observed in both cases in the 410−450 nm area involves contributions of d−d origin [corresponding to the symmetry-forbidden 4A2g → 4T2g (Oh) transition].5,17b,c,35 In addition, the cis-[Cr(TMP)(DPPZ)(1-Melmid)2]3+ spectrum exhibits an intense absorption in the 350−380 nm region, which is associated with a DPPZ ligand π → π* transition.6,23,25,26 UV−Visible Titration of cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ with Duplex DNA. The DPPZ-containing complex cis-[Cr(TMP)(DPPZ)(1-MeImid) 2 ] 3+ is an attractive chromium(III) system for study because of the expectation of a strong duplex DNA binding interaction.6,9 Furthermore, DNA binding constants, KDNA, can be readily assessed by monitoring the large absorption spectral changes that occur in the near-visible region upon the addition of duplex polynucleotides. This spectral region is dominated by an D


Article

Inorganic Chemistry was 6.5 ± 0.6 × 104 M−1 (n = 2.6), which is slightly larger than the corresponding value reported earlier for CT-DNA. However, in view of the significant error component in both KDNA values, the data provide no clear guidance as to the compound’s base-pair preferences. We believe the related equilibrium dialysis studies presented below for this chromium(III) species yield a more reliable indicator of any such selectivity. Equilibrium Dialysis Studies of cis-[Cr(TMP)(DPPZ)(1MeImid)2]3+ with Duplex DNA. In prior investigations of DPPZ-containing complexes of the type [Cr(diimine)2(DPPZ)]3+, we have shown that equilibrium dialysis/CD analyses are valuable probes for assessing binding details of these chromium(III) species with double-helical DNA. Such studies provided compelling evidence that DPPZ intercalation occurs from the minor groove. Furthermore, these chromium(III) compounds exhibit a predisposition for AT base-site binding, and in most cases, binding involves Λ optical isomer enantioselectivity.2,6 The results of an analogous investigation of the cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ complex are presented below and reveal binding preferences very similar to those observed for the [Cr(diimine)2(DPPZ)]3+ systems. Following 96 h of equilibration,6 the concentrations of the retentate (initial chromium(III)/DNA side of cell membrane) and dialysate (initial buffer only side) were determined via UV absorption spectral analysis (see footnote b in Table 1). The

initial dialysis solution, furnish information as to whether chromium(III) binding occurs primarily in the minor or major groove of DNA. The CD values at 280 nm, Δε280, for the dialysate solutions are provided in column 5 of Table 1, which afford information on the degree of enantiomeric stereoselectivity in chromium(III) binding. The relative sizes of the [R]/[[D] ratios observed in experiments 1, 4, and 5 for the three different polynucleotides examined provide strong evidence for a significant binding preference for AT base sequences. The intermediate [R]/[[D] value of 5.5 recorded for CT-DNA is in keeping with the ∼40% GC character of this duplex DNA. The preference by cis[Cr(TMP)(DPPZ)(1-MeImid)2]3+ for AT binding sites in duplex DNA is comparable to what we have previously observed for [Cr(diimine)2(DPPZ)]3+ systems.6 It is also noted that the Barton group has reported a predisposition for AT binding by the ruthenium(II) complex [Ru(phen)2(DPPZ)]2+.20 In the additional CT experiments 2 and 3, distamycin A and netropsin were also present in the initial chromium(III)/DNA side of the dialysis cell. Both of these additives are strongly AT base-selective minor groove binders21,37,38 and have been used as competitive binding agents to help determine the drug affinity for minor groove binding.20,39 The marked decrease in the [R]/[[D] ratios observed in their presence offers strong support for the view that DNA binding occurs primarily via the minor groove. The larger [R]/[[D] ratio found for netropsin relative to distamycin is consistent with the smaller DNA binding constant of the former.37 In similar equilibrium studies of [Cr(diimine)2(DPPZ)]2+ systems with CT-DNA, we have also obtained support for DPPZ intercalation from the minor groove.6 It is worth noting that literature solution studies of duplex DNA binding by [Ru(diimine)2(DPPZ)]2+ species have reached conflicting conclusions as to whether DPPZ intercalation occurs via the DNA minor or major groove.20,40−42 However, very recent atomic resolution X-ray crystallographic studies with a variety of duplex oligonucleotides have provided conclusive evidence that DPPZ intercalation occurs preferentially from the minor groove.43−46 It has been commented that the energetic differences between DPPZ intercalation via the minor versus major groove may, in fact, be small.44,47 Finally, CD studies on dialysate solutions in experiments 1, 4, and 5 revealed substantial optical activity induction in the cis[Cr(TMP)(DPPZ)(1-MeImid)2]3+ present. This is illustrated, for example, in experiment 1 studies with CT-DNA, where a representative dialysate CD spectrum is shown in Figure 5. Two CD bands of opposite sign are seen in the 250−300 nm region of the long-axis-polarized diimine π → π* absorption transition (shown earlier in Figure 3).

Table 1. Dialysis Results for Racemic cis[Cr(TMP)(DPPZ)(1-MeImid)2]3+ Equilibrated 96 h with Duplex DNA at Room Temperature in 100 mM Tris-HCl/ 100 m M NaCl (pH 7.4)a expt no. 1 2 3 4 5

DNA type CT CT CT poly(dA-dT)·poly(dAdT) poly(dG-dC)·poly(dGdC)

minor groove binder added

[R]/ [D]b

Δε280c

none netropsin distamycin A none

5.5 1.9 1.3 9.4

−28 ND ND −42

none

3.4

−16

a

All experiments were conducted with a molar ratio of DNA base pairs: Cr3+ of 5:1 in 100 mM Tris-HCl/100 mM NaCl buffer (pH 7.4). In studies where the competitive binding agents distamycin A and netropsin were also present, their concentrations were 50% that used for the DNA. ND = nondetectable. bThe chromium(III) retentate [R] and dialysate [D] molar concentrations were determined using the wavelengths and corresponding molar absorptivities noted below. The molar absorptivities employed for retentate concentration calculations are those associated with the 387 nm isosbestic wavelength observed in chromium(III)/DNA titrations (see Figure 4). Dialysate: ε360 = 13900 M−1 cm−1. Retentate: ε387 = 5300 M−1 cm−1. cThe Δε CD numbers reported at 280 nm (in M−1 cm−1 units) are those calculated from the experimentally observed ellipticity values (in mdeg, mθ) employing the formula Δε280 = mθ/(32980)bc, where b = cell path length (in cm) and c = chromium(III) molarity.

ratios of the retentate and dialysate concentrations, [R]/[D], are given in column 4 of Table 1 and provide a measure of the relative DNA binding strengths under different experimental conditions. A large [R]/[D] value denotes a strong DNA association, whereas no binding would yield a [R]/[D] ratio of unity. Experiments 2 and 3, which include the additional presence of distamycin A or netropsin, respectively, in the

Figure 5. CD spectrum of a dialysate solution of cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ (4.68 × 10−5 M; 0.20 cm cell) after 96 h of equilibration with CT-DNA. E


Article

Inorganic Chemistry Exciton theory6,48,49 applied to diimine metal complex systems predicts two such CD bands of opposite sign for cis[M(diimine)1(diimine)2X2]n+ systems such as cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ (see case C in ref 41). Exciton coupling theory also predicts for these two CD bands that the one at longer wavelength (in this case at 280 nm) will have a positive sign for the Λ optical isomer. Therefore, the negative sign for this component in Figure 5 is attributed to the presence of an excess of the Δ enantiomer in the dialysate (and thus a Λisomer binding selectivity for CT-DNA). The same Λenantiomer binding preference was likewise observed when poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) were employed as nucleotides (experiments 4 and 5). On the basis of the relative magnitudes of the Δε280 CD values shown in Table 1, the Λ-chirality preference is seen to be most strikingly exhibited by nucleotides with AT base-rich sequences. Because the Δε280 value for the optically pure cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ is not presently known, we cannot quantitatively calculate the optical purity of the dialysate solutions examined. It is noted that the dialysate CD spectra are very similar to that for the related Δ-[Cr(TMP)2(DPPZ)]3+ complex (see Figure 5 in ref 6), for which we have reported a Δε280 value of −155 M−1 cm−1 for the optically pure isomer. Emission Studies. The previously noted resemblance of the CrIIIN6 d−d chromophores of these new complexes with those of related [Cr(diimine)3]3+ species is complemented by similar correspondences in their emission spectra. All of the 1MeImid complexes investigated exhibit strong room temperature emission in aqueous solution. A typical spectrum is provided in Figure 6 for cis-[Cr(phen)2(1-Melmid)2]3+ upon

ligands occur at substantially shorter wavelength (the emission peak for [Cr(NH3)6]3+, for example, is detected at 675 nm).50 The associated emission lifetimes of the 2Eg (Oh) excited state of these cis-[Cr(diimine)2(1-MeImid)2]3+ complexes in an airsaturated room temperature solution are approximately a factor of 10 shorter than those for related [Cr(diimine)3]3+species but are still substantially longer-lived than the emissive triplet metal-to-ligand charge-transfer excited states of the corresponding [Ru(diimine)3]2+ systems. It is also observed that the emission lifetimes (and the corresponding steady-state emission intensities) of the 1-MeImid complexes are markedly less sensitive to dissolved oxygen than their [Cr(diimine)3]3+ counterparts. As a final comment, it is noted that all six emission spectra (Figures 6 and 6S) reveal the presence of a distinctive shoulder (or peak) at a wavelength approximately 20 nm longer than that of the intense signal associated with the 2Eg → 4A2g (Oh) pure electronic transition (for example, this secondary band appears at 739 nm for cis-[Cr(phen)2(1-Melmid)2]3+). The presence of this emission component is in interesting contrast to its absence under identical conditions for the related [Cr(phen)3]3+ species (Figure S7). Its size relative to that of the 0−0 transition remains invariant during steady-state emission quenching experiments (Figures 8 and 10), with the excitation wavelength employed (360−460 nm), upon sample conversion to the PF6− salt, and with the use of different samples of the same complex. Furthermore, emission lifetime studies on cis[Cr(phen)2(1-MeImid)2]3+ using narrow detection slits (5 nm bandpass) yielded identical lifetimes for these two emission signals (Figure S8). We, therefore, believe it is unlikely that this longer wavelength component is associated with an impurity and tentatively assign the signal to a 2Eg → 4A2g (Oh) vibronic transition involving a low-frequency vibration common to these imidazole systems. Electrochemistry. With the exception of cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+, the cyclic voltammograms of the new complexes display a reversible Cr3+/Cr2+ couple, from which are obtained the values listed in Table 2 for their ground-state standard reduction potentials, E°(Cr3+/Cr2+). The dependence of E°(Cr3+/Cr2+) on the diimine ligand type is in accordance with trends previously observed with [Cr(diimine) 3 ] 3+ species.6,17,25,26,29,30 It is also noted that the E°(Cr3+/Cr2+) values are approximately 0.16 V more negative than those observed for comparable [Cr(diimine)3]3+ compounds. This distinction is illustrated in the superimposed cyclic voltammograms of [Cr(phen)3]3+ and cis-[Cr(phen)2(1-MeImid)2]3+ depicted in Figure 7. It is concluded that the ability of 1MeImid ligands to help stabilize the [Cr(diimine)2(1MeImid)2]2+ reduced species is somewhat less than that of diimine ligands (but markedly superior to saturated amine ligands such as ammonia or ethylenediamine). The calculated oxidizing powers of the 2Eg (Oh) excited state of the 1-MeImid systems, E°(*Cr3+/Cr2+), are also provided in column 6 of Table 2 (listed in decreasing order of magnitude). These E°(*Cr3+/Cr2+) values were estimated from the differences between the emission energies in electronvolt units (column 3) and the corresponding E°(Cr3+/Cr2+) values (column 5). As anticipated earlier, these compounds exhibit 2Eg (Oh) excited-state oxidizing powers that meet or exceed the threshold energy estimated for guanine nucleobase oxidation.53 The trends in E°(*Cr3+/Cr2+) observed in Table 2 are in accordance with earlier observations on [Cr(diimine)3]3+

Figure 6. Steady-state emission spectrum of cis-[Cr(phen)2(1Melmid)2](CF3SO3)3 in a 7.3 × 10−4 M aqueous solution (excitation wavelength = 360 nm).

360 nm excitation, while those for the remaining imidazole systems recorded under identical conditions are depicted in Figure S6. By analogy with their [Cr(diimine)3]3+ relatives, the dominant emission signal at 717 nm is assigned to the 2Eg → 4 A2g (Oh) pure electronic (0−0) transition.5,6,17,35 The emission wavelength maxima for the 2Eg → 4A2g (Oh) pure electronic transition for the new complexes (as well as their emission lifetimes in air- and argon-saturated aqueous solutions) are collated in Table 2. The wavelengths listed are slightly shorter (∼10 nm) than those observed for related [Cr(diimine)3]3+ species, an observation that is consistent with a somewhat smaller nephelauxetic contribution by imidazoletype ligands. It is worth noting that the corresponding emission maxima for CrIIIN6 complexes containing saturated N donor F


Article

Inorganic Chemistry Table 2. Photophysical Data for [Cr(diimine)2(1-MeImid)2]3+ Species in an Aqueous Solution at 23 °C emission lifetime (μs) chromium(III) complex 3+

cis-[Cr(TMP)(DPPZ)(1-MeImid)2] cis-[Cr(5-Clphen)2(1-MeImid)2]3+ cis-[Cr(phen)2(1-MeImid)2]3+ cis-[Cr(bpy)2(1-MeImid)2]3+ cis-[Cr(phen)(TMP)(1-MeImid)2]3+ cis-[Cr(TMP)2(1-MeImid)2]3+

emission max (nm)

emission energy (V)

air

argon

721 719 717 717 719 722

1.72 1.72 1.73 1.73 1.72 1.71

6.0 4.6 7.3 7.3 6.9 17.5

7.6 4.9 7.5 7.4 8.3 28.5

E°(Cr3+/Cr2+) vs SHEa (V) E°(*Cr3+/Cr2+) vs SHE (V) −0.34b −0.39 −0.44 −0.45 −0.46 −0.48

1.38 1.33 1.29 1.28 1.26 1.23

a

The abbreviations Cr3+ and Cr2+ indicate the overall charges, n+, of the oxidized and reduced forms of the cis-[Cr(diimine)2(1-Imid)2]n+ complexes. Recent studies reveal that, for the related [Cr(diimine)3]n+ compounds, the reductive processes are ligand-based and that [Cr(diimine)3]2+ molecules are best represented as chromium(III) species antiferromagnetically coupled to a diimine− radical.51,52 Potentials versus SHE were calculated using a potential of 200 mV for the Ag/AgCl reference electrode. bEstimate only (because an irreversible reduction wave is observed).

Figure 8. Steady-state emission spectrum of an air-saturated 3.4 × 10−4 M solution of cis-[Cr(phen)2(1-Melmid)2]3+ in a 0.05 M TrisHCl buffer solution (pH 7.4) in the presence of increasing concentrations of dGMP (0−1.0 × 10−2 M). Excitation wavelength = 360 nm.

Figure 7. Cyclic voltammograms of 0.42 mM [Cr(phen)3]3+ (dashed line) and 0.80 mM cis-[Cr(phen)2(1-Melmid)2]3+ (solid line) in a 100 mM KCl aqueous solution (vs Ag/AgCl reference electrode). Scan rate = 100 mV s−1.

systems, where it was found that replacing a phen ligand by DPPZ or Clphen results in an enhanced excited-state oxidation potential, while the reverse trend occurs upon substitution of a phen ligand by TMP.6,17,25,26,29 Quenching of the Emission of cis-[Cr(diimine)2(1MeImid)2]3+ Complexes by Mononucleotides. Of the four nucleotide bases in DNA, guanine is known to be the most readily oxidized, with an approximate 1.2 V threshold value required for direct one-electron oxidation.53 For this reason, dGMP was the primary quencher employed in our emission investigations. A. dGMP Studies. Stern−Volmer (SV) quenching studies were performed in 50 mM Tris-HCl buffer (pH 7.4) in the presence of increasing concentrations of dGMP. Quenching of both the steady-state emission and emission lifetime of the complexes by added dGMP was observed. A representative example of steady-state emission quenching for the case of cis[Cr(phen)2(1-MeImid)2]3+ is provided in Figure 8. A subsequent SV kinetic analysis of these steady-state and lifetime quenching data yielded straight-line plots. These steady-state and lifetime plots are depicted in Figures 9 and S9, respectively, for the five phen-derived cis-[Cr(diimine)2(1MeImid)2]3+ complexes studied. The bimolecular quenching rate constants, kq, obtained from such SV studies are provided in Table 3. For related [Cr(diimine)3]3+ species, where direct one-electron transfer is implicated, the rate constants observed (with the exception of [Cr(TMP)3]3+) were found to have values at or near the diffusion-controlled limit (see entries 1

Figure 9. Steady-state SV plots for emission quenching by dGMP of several cis-[Cr(diimine)2(1-Melmid)2](CF3SO3)3 systems in an airsaturated 0.05 M Tris-HCl buffer solution at pH 7.4: (1) cis-[Cr(5Clphen)2(1-MeImid)2]3+; (2) cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+; (3) cis-[Cr(phen) 2 (1-MeImid)2 ] 3+ ; (4) cis-[Cr(TMP)2 (1-MeImid)2]3+; (5) cis-[Cr(phen)(TMP)(1-MeImid)2]3+.

and 2 in Table 3).1,2,6,8 In contrast, the kq values for all of the 1MeImid systems investigated are significantly below the diffusion-controlled limit, an observation in accordance with their lower 2Eg (Oh) excited-state oxidation potentials, E°(*Cr3+/Cr2+), in Table 3. The good agreement between the steady-state- and lifetimederived kq values for each 1-MeImid complex is indicative of emission quenching being dominated by a collisional G


Article

Inorganic Chemistry Table 3. Data for Chromium(III) 2Eg (Oh) Excited-State Emission Quenching by dGMP chromium(III) complex

a

E°Q,Q+ (V vs SHE)

E°*Cr3+,Cr2+ (V vs SHE)

E°net rn (V)

[Cr(phen)2(DPPZ)] [Cr(phen)3]3+ cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+

−1.2 −1.2 −1.2

+1.49 +1.42 +1.38

+0.29 +0.22 +0.18

cis-[Cr(5-Clphen)2(1-MeImid)2]3+

−1.2

+1.33

+0.13

cis-[Cr(phen)2(1-MeImid)2]3+

−1.2

+1.29

+0.09

cis-[Cr((bpy)2(1-MeImid)2]3+

−1.2

+1.28

+0.08

cis-[Cr((phen)(TMP)(1-MeImid)2]3+

−1.2

+1.26

+0.06

cis-[Cr(TMP)2(1-MeImid)2]3+

−1.2

+1.23

+0.03

3+

kqa (M−1 s−1) 2.4 2.2 2.5 2.8 5.0 4.8 7.6 7.7 4.7 4.3 1.9 1.9 1.2 1.1

× × × × × × × × × × × × × ×

R2d

9b

10 109 c 108 (τ) 108 (ss) 108 (τ) 108 (ss) 107 (τ) 107 (ss) 107 (τ) 107 (ss) 107 (τ) 107 (ss) 107 (τ) 107 (ss)

0.9921 0.9988 0.9973 0.9996 0.9981 0.9976 0.9988 0.9969 0.9844 0.9900 0.9840 0.9934

τ = S−V lifetime data; ss = steady-state emission intensity data. bReference 2. cReference 1. dThe R2 value is the square of the correlation coefficient

deactivation process.1,2,6,54 Assuming this pathway is associated with chromium(III) 2Eg (Oh) excited-state oxidation of dGMP, the thermodynamic driving force for the net redox reaction (expressed in volts, E°net rn) can be estimated from a summation of the electrochemical data provided in columns 2 and 3 of Table 3, i.e., E°net rn = EQ,Q+ + E°*Cr3+/Cr2+, where EQ,Q+ is the potential for dGMP oxidation and E°*Cr3+/Cr2+ is the calculated oxidation potential of the chromium(III) 2Eg (Oh) excited state. Strong support for the redox quenching pathway postulated comes from the finding that, in general, the quenching rate constants in Table 3 for the 1-MeImid series of complexes track the thermodynamic driving force for this net reaction. However, one exception is apparent. Despite the cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ complex appearing to have a higher net driving force for redox quenching, the observed rate constant is lower than that for the cis-[Cr(5-Clphen)2(1MeImid)2]3+ system. This discrepancy may be associated with differing contributions from electronic factors such as orbital overlap. B. dAMP Studies. Adenine is known to be the second most easily oxidized nucleobase, with an approximate 1.4 V threshold value required for direct one-electron oxidation.53 The complex cis-[Cr(5-Clphen)2(1-MeImid)2]3+ is estimated to have only a slightly disfavored ΔG° for the photooxidation of dAMP (E°net rn ≈ −0.07 V). In fact, emission quenching of this 1MeImid complex by dAMP is experimentally observed, albeit with a markedly smaller quenching rate constant of 4.5 × 105 M−1 cm−1. Quenching of the Emission of cis-[Cr(TMP)(DPPZ)(1MeImid)2]3+ by CT-DNA. Changes in the steady-state emission spectrum of cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ in Tris-HCl buffer in response to the sequential addition of CT-DNA are shown in Figure 10. The data were obtained using 387 nm excitation, which is the wavelength at which an isosbestic point is observed in the corresponding UV−visible titration curve (Figure 4). Employment of this wavelength eliminates contributions to emission quenching due to a decrease in absorption associated with the presence of added DNA. An unusual feature is observed in the steady-state emission quenching depicted above relative to that previously reported for related [Cr(diimine)2(DPPZ)]3+ systems.2,6,9 A significant component (approximately 19%) of the original emission signal

Figure 10. Steady-state emission spectrum of an air-saturated 3.4 × 10−5 M solution of cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ in 0.05 M Tris-HCl buffer (pH 7.4) with increasing concentrations of CT-DNA (0−2.48 × 10−3 M). Excitation wavelength = 387 nm.

appears to be essentially unquenchable, despite the presence of a final DNA/chromium(III) molar ratio of 73:1. In contrast, for example, greater than 98% emission quenching has been reported for the corresponding [Cr(phen)2(DPPZ)]3+ complex.56 The unusual situation noted above for cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ is also observed during emission lifetime experiments on the same solutions. As a result, the associated emission intensity and lifetime SV plots exhibit quite atypical behavior, where the development of a plateau region at high DNA concentration becomes apparent (Figure 11). Normal expectations are that the lifetime SV plot will display straight-line behavior in accordance with dynamic (collisional) quenching. Likewise, the corresponding steady-state SV plot is expected to exhibit marked upward curvature with increasing DNA concentration because of static quenching associated with the formation of DNA-bound chromium(III), which is normally considered to be nonluminescent.2,6,8 Support for the presence of an unquenchable emission component for the complex cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ is also provided using data available during emission lifetime studies involving pulse excitation. The intensity of the initial pulse signal prior to subsequent emission signal decay, I0, may normally be taken as a reliable measure of the amount of unbound chromium(III) present if the bound complex is nonemissive. This initial I0 value will then proceed to zero at sufficiently high DNA H


Article

Inorganic Chemistry

state species. In addition, it is important to note that the presence of a significant percentage of nonquenchable DNAbound cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ may provide an opportunity for photoaquation to be competitive with electron transfer as a deactivation process at those particular binding sites. Wojdyla et al.8 have previously discussed several possible explanations for the nearly 100% emission quenching observed with [Cr(diimine)2(DPPZ)]3+ systems despite the fact that 34% of CT-DNA binding sites consist of only AT base pairs (both of which are poor candidates for photooxidation).53 Of the explanations offered, the two that appear to be the most relevant for the present discussion are that (a) the [Cr(diimine)2(DPPZ)]3+ 2Eg (Oh) excited states are sufficiently long-lived for the complex to leave a binding site and relocate within its lifetime and (b) the complex remains at a binding site but is capable of oxidizing a guanine at a nearby site. Neither of these options would be as viable for the cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ species because of its significantly shorter lifetime and the smaller excited-state oxidizing power of its 2Eg (Oh) excited state. A possible candidate for a bound but nonquenchable cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ complex might be one that is bound to DNA at strongly AT-rich sequences, where access to oxidizable guanine base sites may be further restricted. These AT-rich regions may be among the more likely sites for any covalent adduct formation following chromium(III) photoexcitation. Finally, it is noted that at low DNA concentrations the SV lifetime plot shows reasonable linearity (Figure S11), from which a bimolecular quenching rate constant can be extracted. The value obtained, kq = 5.8 × 107 M−1 s−1, is comparable to those that have been previously reported for [Cr(diimine)2(DPPZ)]3+ species, where quenching is believed to be diffusion-controlled.6,8 This similarity in the rate constants is to be contrasted with those obtained when dGMP was the candidate for photooxidation (Table 3). In this latter case, the emission quenching rate constant obtained for cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ was a factor of 10 slower than that measured for [Cr(phen)2(DPPZ)]3+ (which is a value at the diffusion-controlled limit). However, literature studies indicate that guanine in DNA has a less negative potential for oxidation than the mononucleotide,53 which might account for the diffusion-controlled rate constant value estimated above for cis[Cr(TMP)(DPPZ)(1-MeImid)2]3+ with CT-DNA. Photoaquation of cis-[Cr(phen)2(1-MeImid)2]3+ in 50 mM Tris-HCl buffer (pH 7.4). In any future DNA covalent adduct studies of these new cis-[Cr(diimine)2(1-MeImid)2]3+ complexes, and in particular cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+, an essential prerequisite for such formation would be the facile release of the monodentate 1-MeImid ligands. UV−visible spectral investigations in aqueous solution indicate that imidazole ligand aquation is a slow thermal process at room temperature (t1/2 > 7 days). An attractive alternative avenue for imidazole hydrolysis is via photoactivated ligand loss, such as has been employed successfully by Singh and Turro for DNA covalent adduct formation via ammonia ligand photorelease from cis-[Ru(bpy)2(NH3)2]2+.19 In the present study, this particular approach has been case tested for the complex cis-[Cr(phen)2(1-MeImid)2]3+, for which extensive spectral literature information is available for the anticipated aquated products.55 The visible absorption changes accompanying 420 nm photolysis of this compound in 50 mM TrisHCl buffer (pH 7.4) are displayed in Figure 13 A.

Figure 11. SV plots for emission quenching of an air-saturated 3.4 × 10−5 M solution of cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ in 0.05 M Tris-HCl buffer (pH 7.4) by CT-DNA: (●) steady-state emission intensity data, I0/I; (◆) lifetime emission data, τ0/τ. Note: For visual clarity, a curved-line “fit” has been drawn via eye inspection through the steady-state and lifetime data points.

concentration. A representative example of the application of this procedure for the calculation of I0 for the cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ emission quenching experiment is provided in Figure S10 for the case where the CT quencher concentration was 4.96 × 10−4 M. The corresponding SV plot utilizing the pulse I0 values for all of the DNA concentrations involved in Figure 10 is depicted in Figure 12.

Figure 12. SV plot using the initial laser pulse signal, I0, for a 3.4 × 10−5 M solution of cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ in 0.05 M Tris-HCl buffer (pH 7.4). Note: For visual clarity, a curved-line “fit” has been drawn via eye inspection through the experimental data points.

A very clear plateau region is apparent at high [DNA] in Figure 12, from which it is calculated that approximately 22% of the chromium(III) initially present still remains emissive with a lifetime of 4.8 μs (although almost certainly bound at the DNA/chromium(III) molar ratio of 73:1 present in the final solution). It is noted that this percentage agrees well with the 19% value reported above for the steady-state emission quenching data presented in Figure 10. The pulsed emission decay curve for cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ in the presence of the highest CT-DNA concentration employed (2.48 × 10−3 M) is displayed in Figure S12. Also shown in Figure S12 is the fit of this curve to monoexponential decay kinetics. The good fit observed provides support for a single binding mode for the nonquenchable chromium(III) excitedI


Article

Inorganic Chemistry

emission signal remains unquenchable, even in the presence of a very high DNA/chromium(III) molar ratio. This unquenchable bound chromium(III) component (with an excited-state lifetime in the microsecond range) may then prove to be susceptible to photoactivated 1-MeImid ligand release, which might thus provide an avenue for covalent adduct formation with DNA. Another future experiment of interest would be the use of the complex cis-[Cr(phen)2(1-MeImid)2]3+ in studies to address the literature failure to date to obtain transient absorption evidence for the formation of reduced [Cr(diimine)2(DPPZ)]2+ and oxidized guanine products following nanosecond or picosecond pulse excitation of [Cr(diimine)2(DPPZ)]3+ and guanine-containing nucleotides. This failure has been attributed to a very rapid back-electrontransfer reaction.7,8,56,57 Our kinetic data in Table 3 for the emission quenching of cis-[Cr(phen)2(1-MeImid)2]3+ by dGMP reveal a rate constant factor of 100 slower than that observed for [Cr(phen)2(DPPZ)]3+ (consistent with the 110 mV higher excited-state oxidation potential for the latter). Should the reverse back electron transfer also be slower, this may allow for the expected transient radicals to be detected.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b02323. Structure of DPPZ, an electropherogram, ESI-MS, UV− visible, and steady-state emission spectra, emission lifetime details, lifetime SV plots, and emission decay profiles (PDF)

Figure 13. (A) Visible spectral changes during 420 nm photolysis for 20 min (8 lamps, RPR-100 Rayonet photochemical reactor) of a 4.5 × 10−4 M solution of cis-[Cr(phen)2(1-MeImid)2]3+ in 50 mM Tris-HCl buffer (pH 7.4). A 1 cm glass emission cell. (B) Final reaction spectrum after acidification.

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The spectrum of the final solution following acidification (Figure 13B) shows a d−d absorption maximum at 497 nm and a minimum at approximately 458 nm, which are excellent matches for those reported for the anticipated diaquo product, cis-[Cr(phen)2(H2O)2]3+.55

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected].

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Notes

The authors declare no competing financial interest.

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CONCLUSION We have described above versatile synthetic protocols for the isolation of a range of chromium(III) complexes containing 1MeImid ligands. All of these compounds exhibit strong room temperature solution emission, with emission lifetimes in the microsecond range. We have taken advantage of these emission characteristics to demonstrate their potential as photooxidants of the mononucleotides dGMP and dAMP via emission quenching experiments. In general, their quenching rate constants with dGMP tracked well with their 2Eg (Oh) excited-state oxidation potentials. Intercalation by the complex cis-[Cr(TMP)(DPPZ)(1-MeImid)2]3+ leads to a large DNA binding constant, as determined from UV−visible titration analysis, while complementary equilibrium dialysis studies established a strong binding preference for AT base sites within the minor groove. A photolysis study on cis-[Cr(phen)2(1-MeImid)2]3+ in a 0.05 M Tris-HCl solution at pH 7.4 demonstrated that these complexes are labile toward photoaquation involving release of the 1-MeImid ligands, which is a prerequisite for their potential use in future photoactivated DNA covalent adduct investigations. For the case of cis[Cr(TMP)(DPPZ)(1-MeImid)2]3+, emission quenching studies with CT-DNA revealed that approximately 20% of the

ACKNOWLEDGMENTS The authors gratefully acknowledge financial support of this work provided by Furman University, the National Institutes of Health (NIH-INBRE Grant P20 RR-016461), and the Howard Hughes Medical Foundation through an Undergraduate Science Education Award. The authors also graciously thank Kyle F. Martin and Dr. Jared A. Pienkos for their invaluable assistance with the figures.

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Synthesis of cis-[Cr(diimine)2(1-methylimidazole)2]3+ Complexes and

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