Inorganica Chimica Acta 469 (2018) 484–494

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Inorganica Chimica Acta

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Research paper

Interaction with calf-thymus DNA and photoinduced cleavage of pBR322by rhodium(III) and iridium(III) complexes containing crown thioetherligands

https://doi.org/10.1016/j.ica.2017.10.0050020-1693/� 2017 Elsevier B.V. All rights reserved.

⇑ Corresponding author.E-mail address: jisook-kim@utc.edu (J. Kim).

Jisook Kim ⇑, Ashley D. Cardenal, Hendrik J. Greve, Weinan Chen, Hitesh Vashi, Gregory Grant,Titus V. AlbuDepartment of Chemistry and Physics, University of Tennessee at Chattanooga, Chattanooga, TN 37403, USA

a r t i c l e i n f o

Article history:Received 15 August 2017Received in revised form 3 October 2017Accepted 5 October 2017Available online 10 October 2017

Keywords:PhotoactivationPhotonucleaseCisplatin9S3RhodiumIridium

a b s t r a c t

In this report, we present our investigation on the photoinduced cleavage of plasmid pBR322 and thebinding interactions with calf-thymus (CT) DNA by a series of thioether metal complexes. The complexesof interest are rhodium and iridium complexes containing thiacrown ligands 1,4,7-trithiacyclononane(9S3) and 1-oxa-4,7-dithiacyclononane (9S2O), and the complexes are abbreviated as [Rh(9S3)Cl3], [Rh(9S2O)Cl3], and [Ir(9S3)Cl3]. In the nicking assay, pBR322 was treated with each complex and irradiatedat 254 and 350 nm, respectively, in concentration- and time-dependent studies. The nicking assayrevealed that, under exposure to 254-nm radiation, [Rh(9S3)Cl3] and [Rh(9S2O)Cl3] cleaved pBR322 effi-ciently forming a nicked form, while [Ir(9S3)Cl3] was least efficient. For the 350-nm irradiation, a similartrend was observed, with [Rh(9S3)Cl3] being the most efficient one, however with a lower efficiency thanat 254 nm. An ethidium bromide displacement assay was also carried out to evaluate the binding inter-action of each compound with CT-DNA by titrating the pre-equilibrated complex of CT-DNA and EB withthe investigated complexes. The efficient concentration to achieve a 50% loss in fluorescent emission wasfound to be 24 lM for [Rh(9S3)Cl3] and 35 lM for [Rh(9S2O)Cl3], while [Ir(9S3)Cl3] was an ineffectiveDNA binder.

� 2017 Elsevier B.V. All rights reserved.

1. Introduction

Cisplatin has been a popular anticancer drug and is commonlyused for treating various types of cancers such as ovarian/cervicalcancer, bladder cancer, and/or melanoma [1–3]. The mechanism ofits action involves several steps such as cellular delivery of cis-platin, dissociation of Cl� inside the cells, binding of cisplatin tonitrogen (N-7 position) in guanines, formation of intra-strandcross-linkage to the target DNA, and apoptosis triggered by DNAdamage [1,3,4]. In spite of its efficiency as an anticancer agent,the usage of cisplatin comes with issues such as potential deactiva-tion in cells, cytotoxicity, and drug resistance [1–5]. In an effort toovercome drawbacks of cisplatin, there have been great efforts infinding new anticancer inorganic compounds. Several studiesspearheaded by photo-activation approaches showed promisingresults, focusing mainly on Ru complexes and also including Fe,Cu, Rh, and Ir complexes [2,6–28]. The idea of utilizing light ason/off switch to activate a drug for targeting DNA in cancer cells

is attractive since it is possible to design a drug which can be acti-vated only in the presence of light, while remaining inert or lessactive without irradiation. Furthermore, it is possible to tune theeffectiveness of a photoactivated inorganic compound by control-ling a time lapse for light exposure to the localized target tissueor by choosing an effective irradiating wavelength selectively.

Whether photoactivated or not, the mechanism of DNA modifi-cations caused by inorganic compounds can be complex, and itinvolves binding interactions with DNA that can be covalent,non-covalent, tight binding (via intercalation or groove binding),loose binding in a nonspecific manner, oxygen-mediated, or oxy-gen-independent [2,23,28,29]. The most likely results of interac-tions between inorganic complexes and DNA are either adductformation or DNA strand cleavage. The adduct formation withDNA was observed for platinum containing complexes such as cis-platin, carboplatin, and satraplatin [1,3,23,30], while the DNAcleavage was observed mostly in ruthenium complexes. The corestructure of the Ru complexes is [Ru(bpy)nL3-n]2+, where bpy =2,20-bipyridine and L can be ferrocene/non-ferrocene conjugatedimidazole phenol ligand [6], a series of o-, m-, or p-(nitrophenyl)imidazo[4,5-f] [1,10]phenanthroline) [7], 1,12-diazaperylene (i.e.

J. Kim et al. / Inorganica Chimica Acta 469 (2018) 484–494 485

DAP) [2,8], dipyrido[3,2-a:20,30-c]phenazine (i.e. dppz) [9,10], or4,5,9,16-tetraaza-dibenzo[a,c]naphthacene (i.e. dppn) [11,12].

In addition to extensively studied Pt- and Ru-containing com-plexes, a number of studies were focused on the biological activityof complexes with other metal centers such as Fe [19,21], Cu[15,19,31], Rh [16,20], or Ir [17,18]. Rh(III) and Ir(III) complexesdid not receive as much attention until recently since theses com-plexes were perceived to be relatively less reactive due to theirchemical stability and slow solvent exchange rate [18,32,33]. Thestability of Rh(III) and Ir(III) are in part due to the low spin state,with a d6 electron configuration, and an octahedral geometry[18,32,33]. Interestingly, some studies focus on activating Rh(III)and Ir(III) complexes by adopting suitable ligands upon irradiation.Examples are [Rh(bpy)2Cl2]+ [34], [Rh(phen)2Cl2]+ (phen, 1,10-phenanthroline) [33,34], [RhCl(bpy)9S3]2+ (9S3 = 1,4,7-trithiacy-clononane) [35,36], [IrLn]3+ where L is a terpyridyl-like ligand[17], and [(g5-Cp⁄)Ir(phen)Cl] (Cp⁄ = tetramethyl(phenyl)cy-clopentadiene) [18]. The presence of one or more chloride ionscoordinated to Rh(III) or Ir(III) metal center appears to be essentialin photoactivation of the complexes in these cases. Importantly,many of the complexes above were also shown to be active asDNA cleaving agents upon irradiation, and this finding is promisingin utilizing phototherapy for selective and efficient cancertreatment.

Herein, we present the results of our investigation on the bio-logical activities of three complexes [Rh(9S3)Cl3] 1, [Rh(9S2O)Cl3]2, and [Ir(9S3)Cl3] 3 toward DNA, for both plasmid and calf-thymus(CT) DNA. As shown in Fig. 1, each investigated complex has threechloride ions bound to a metal center, and a facially coordinatingthiacrown ligand to complete the octahedral structure (Fig. 1).These compounds were chosen for this study since the compoundsof interest have chloride ions as ligand, which was found to be crit-ical for photoinduced reactivity for the known Rh(III) and Ir(III)complexes. Moreover, a similar complex, [Rh(9S2N)Cl3] (9S2N =1-aza-4,7-dithiacyclononane) was shown to be active against ovar-ian cancer [37]. We carried out a photonuclease activity assay bytreating plasmid pBR322 with compounds 1, 2, and 3 in a concen-tration- and a time-dependent manner, upon irradiation at 254 and350 nm, respectively. For evaluating the DNA binding properties ofeach complex, we conducted an ethidium bromide displacementassay (EBDA) by exciting the complex of [DNA + EB ± 1, 2, or 3] at520 nm, and monitored the fluorescence emission in the 540–800 nm range. Additionally, an electronic structure theory studyof these compounds was carried out and is reported here. The find-ings in this study will advance the knowledge of the interactionbetween Rh(III)/Ir(III) complexes and DNA.

2. Experimental

2.1. General methodologies and instrumentation

All chemicals were purchased from Fisher Scientific and are ofreagent grade unless specified otherwise. The water used in thestudy was deionized water (dI-H2O) purified by a Millipore system(Milli-Q water). pBR322 was purchased from Fermentas (SD0041),

Fig. 1. Structures of compounds 1–3.

and CT-DNA was purchased from Rockland (MB-102-0100). Gelswere imaged using a Spectroline UV Transilluminator equippedwith a Fotodyne Foto Analyst Apprentice system and a PanasonicDMC-FX580 digital camera. Then, the stained gel images were sub-mitted to quantitation using ImageJ Software to compare the per-cent of nicked plasmid vs. supercoiled plasmid. Fluorescenceanalysis was carried out using a Horiba Jobin Yvon Fluorolog-3spectrophotometer equipped with polarization accessories and afull-spectrum xenon lamp. UV–Vis spectra were obtained using aShimadzu Biospec-1601 spectrophotometer, and data collectionwas obtained using UV Probe 2.3 Software by Shimadzu.

2.2. Synthesis of [Rh(9S3)Cl3], [Rh(9S2O)Cl3], and [Ir(9S3)Cl3]

The syntheses of the three complexes were carried out by fol-lowing the published procedures [38,39] with minor modifications.A representative description is as follows.

a. Preparation of [Rh(9S3)Cl3]: A mixture of RhCl3�3H2O(250.0 mg, 0.949 mmol) and 9S3 (171 mg, 0.949 mmol)were placed in a 100 mL round bottom flask. To this mixturewas added 34.2 mL of EtOH. The solution was refluxed for1.5 h while stirring. As the solution was cooled to room tem-perature, a clear supernatant formed with a solid yellow pre-cipitate. The product is filtered, and the gooey precipitatewas washed with ethanol (3 � 15 mL) followed by ether(3 � 15 mL) to make the product dry and clean. The productof [Rh(9S3)Cl3] weighed 351 mg with a 95.0% yield.

b. Preparation of [Rh(9S2O)Cl3]: A mixture of RhCl3�3H2O(35.0 mg, 0.210 mmol) and 9S2O (55.9 mg, 0.210 mmol)were placed in a 50 mL round bottom flask. To this mixturewas added 10 mL of MeOH and 5 mL of DI H2O. The solutionwas refluxed for 3 h. The reagent 9S2O is a liquid at roomtemperature, so it was pipetted into the flask. The maroonsolution turned bright orange when heated and becamecloudy. The orange suspension was then filtered to recoveran insoluble orange solid weighed at 55 mg, with a 69.4%yield. A fraction of the product was submitted to solubilitytest in various solvents or solvent mixtures (DMSO, iso-propanol, ethanol, methanol, chloroform, acetonitrile, water,trifluoroacetic acid); the mixture of DMSO and H2O appearedto be the best system for dissolving [Rh(9S2O)Cl3].

c. Preparation of [Ir(9S3)Cl3]: The preparation of [Ir(9S3)Cl3]was carried out similarly to the procedure for [Rd(9S3)Cl3]with IrCl3�3H2O, instead of RhCl3�3H2O, following the proce-dure described by Timonen and coworkers [38].

2.3. Preparation of CT-DNA

The CT-DNA (30 mg) was hydrolyzed in filtered phosphate buf-fer (3.0 mL, 50 mM phosphate, 50 mM NaCl, pH 7.0) overnight.Then, the hydrolyzed DNA solution was submitted to dialysis (�3) over 24 h in saline phosphate buffer (i.e. SPB, 50 mM phosphate,50 mM NaCl, pH 7.0). The solution above was diluted with SPB forassays and the DNA concentration per nucleotide was determinedby A260 obtained at UV–Vis spectroscopic scans using the molecu-lar extinction coefficient value of 6600 M�1 cm–1 [40]. Then, ali-quots were stored at �80 �C and diluted freshly prior to eachethidium bromide displacement assay (EBDA).

2.4. Preparation of stock solutions of complexes

Stock solutions of 5.0 mM 1 and 2 were prepared by adding theweighed amount of the complex to DMSO. A stock solution of 2.5mM 2was prepared by adding the weighed amount of the complexto the mixture of 1:1 DMSO:dI H2O by volume. The 5.0 mM solu-

486 J. Kim et al. / Inorganica Chimica Acta 469 (2018) 484–494

tion of 2 was not possible due to poor solubility of 2 in DMSO. Thestock solutions were stored at �80 �C and diluted freshly followedby 10-min sonication, prior to each assay.

2.5. Time-lapse UV–Vis monitoring of complexes

The samples for UV–Vis scanning were prepared similar to theabove-mentioned stock solution in phosphate buffer (50 mM, pH7.0). A diluted solution of 1, 2, and 3 at 100 lM, respectively, uponequilibration at 20 �C, was submitted to UV–Vis scanning in a 1-mL, 1-cm quartz cuvette every 24 h up to 4 days. The compoundswere kept in the dark except every time the sample was submittedto UV–Vis scanning.

2.6. pBR322 photocleavage experiments

pBR322 (0.55 lg) was incubated with 1, 2, and 3, respectively,in 10 lL of 10 mM Tris-1 mM Na2EDTA buffer (pH 7.2, i.e., TE buf-fer), in a time- and concentration-dependent manner at 30�C. Thetime-dependent incubation reactions were carried out for 1-, 5-,and 30-min irradiation at a complex concentration of 10 lM. Theconcentration-dependent incubation reactions were carried outfor 10 min at complex concentrations of 0, 10.0, 50.0, and 100lM, respectively. For both studies, irradiation occurred at 254and 350 nm, respectively. Photoinduced DNA cleavage reactionswere quenched by turning off the irradiating light from the UV–Vis reactor followed by cooling the samples immediately in anice bath at 0 �C. The incubation samples were then analyzed on0.8% agarose gel electrophoresis for 150 min at 100 V in a buffermade of Tris (18.0 mM), boric acid (18.0 mM), and EDTA (0.50mM). Gels were stained with 0.4 lg/mL ethidium bromide, submit-ted to scanning under UV light of the transilluminator FotodyneAnalyst Apprentice system. Each gel image was then submittedto quantitation analysis using ImageJ.

2.7. Ethidium bromide displacement assay (EBDA)

The DNA binding trend of the investigated complexes weredetermined by EBDA using the Horiba fluorimeter at 37 �C. A solu-tion of CT-DNA (2.0 lM) was incubated with EB (2.0 lM) in SPB ina quartz cuvette (1 mL volume, 1 cm path length). The incubationwas carried out at room temperature, in the dark, for 15 min priorto titration with the compounds 1, 2, and 3, respectively. To thepremixed solution of CT-DNA and EB in the quartz cuvette, com-plex 1, 2, or 3 was added at final concentrations of 5.0, 10, 25,50, 75, 100, 125, 175, 200 lM, respectively. The titrated mixturewas equilibrated at 37 �C for 10 min prior to fluorescence scanning.The samples were examined using an excitation wavelength of520 nm. All emission spectra were recorded over the 540–800nm range in increments of 1 nm, with a band pass of 2 nm for bothexcitation and emission, were corrected for the lamp and thedetector response, and were normalized to a constant fluorescenceintensity in the 785–800 nm range. After the fluorescence detec-tion of the titrated samples, each sample was submitted to UV–Vis scanning using the Shimadzu spectrophotometer.

2.8. Electronic structure theory calculation details

Electronic structure theory calculations were carried out forcomplexes 1, 2, and 3 presented in this study. The results reportedhere were obtained using the Hartree–Fock (HF, ab initio) method[41] and the hybrid density functional theory (DFT) mPW1PW91method [42]. We employed the LANL2DZ effective-core potentialbasis set [43,44] for Rh and Ir, and one of the following three differ-ent basis sets for all other atoms, 6-31G(d,p), 6-311G(d,p), or 6-311+G(d,p), respectively. The SCF procedure was carried out using

quadratic convergence methodology. All systems were closed-shellsystems, and we employed restricted wave function calculations.The geometries of these complexes were fully optimized, and theminima-energy structures were verified to be characterized byonly positive frequencies. The Cartesian coordinates for all com-plexes are given in the Supplementary Material section. All elec-tronic structure calculations were carried out using the Gaussian09 program [45], and the molecular orbitals were visualized usingAvogadro software [46].

3. Results and discussion

3.1. Electronic structure theory calculations

Electronic structure theory computations were carried out inorder to determine important geometric and energetic parametersfor these complexes. These parameters are listed in Table 1. Withthe six theoretical methods used in this study, it was found thatthe optimized geometries for complexes 1 and 3 show either a C3

symmetry or a slightly distorted C3 symmetry. The metal–sulfur(M-S) and metal-Cl (M-Cl) distances in the optimized structuresare slightly different depending on the theoretical method used,either ab initio (i.e., HF) or hybrid density functional theory (i.e.,mPW1PW91), but they were very little dependent on the basisset used. The calculated Rh–S distances using mPW1PW91method, around 1.33 Å, are more consistent with Rh–S distancesin known Rh-(9S3) compounds for which X-ray structures areavailable [47–50]. Comparing the geometric parameters of 1 and3, it was found that the Rh–S distance is slightly larger than theIr–S distance while the Rh–Cl distance is shorter than the Ir–Cl dis-tance. For 2, it was found that one Rh–Cl distance (i.e., the oneopposite to the O of 9S2O) is shorter than the other two Rh–Cl dis-tances, which are either equal or almost equal depending on thetheoretical method used. Also, not surprising, the Rh–O distanceis shorter than the Rh–S distances.

Table 1 lists also the calculated energies of HOMO and LUMO forthe investigated complexes. For both HF and mPW1PW91 meth-ods, calculated HOMO energies are little dependent on the basisset used. For HF method, calculated LUMO energies gets lower asmore basis are added to the computation but, for mPW1PW91method, the addition of diffuse function in the basis set has anopposite effect by slightly increasing the energy of LUMO. For 1and 3, LUMO has essentially the same energy as LUMO+1, beingalmost degenerate. Fig. 2 shows the HOMO and LUMO for 1, deter-mined at the mPW1PW91/6-31G(d,p) level of theory. HOMO andLUMO for 2 and 3 have same character and look similar as the orbi-tals for 1, at the same level of theory.

For HF method, the HOMO-LUMO energy gap slightly decreasesas the basis set gets bigger, while for the mPW1PW91 method, thisenergy gap is very little dependent on the basis set. Also, the den-sity functional theory method, mPW1PW91, gives consistently alower energy gap than the HF method. For all theoretical methodsexcept one method, the energy gap was found to be slightly smal-ler for 2 than 1, and to be the highest for 3. This trend is consistentwith experimentally determined UV–Vis absorption data. TheHF/6-311+G(d,p) method gives an unphysically low LUMO energyfor 3, resulting in a lower gap than calculated with all the othermethods.

3.2. Time-lapse UV–Vis monitoring of complexes

Rh(III) and Ir(III) complexes are typically known for slow sol-vent exchange rate and chemical stability in neutral conditions.However, it has been shown that the presence of halide or pseudo-halide in Rh(III)/Ir(III) complexes could lead to halide displacement

Table 1Calculated key geometric and energetic parameters for the investigated complexes.

Property Theoretical Methoda [Rh(9S3)Cl3] [Rh(9S2O)Cl3] [Ir(9S3)Cl3]

M-S (and M-O) distancesb (Å) HF/6-31G(d,p) 2.449 2.461, 2.461, (2.229) 2.402HF/6-311G(d,p) 2.441 2.455, 2.455, (2.218) 2.397HF/6-311+G(d,p) 2.443 2.455, 2.454, (2.226) 2.399mPW1PW91/6-31G(d,p) 2.335 2.348, 2.349, (2.215) 2.314mPW1PW91/6-311G(d,p) 2.334 2.348, 2.349, (2.206) 2.312mPW1PW91/6-311+G(d,p) 2.332 2.343, 2.344, (2.207) 2.312

M-Cl distances (Å) HF/6-31G(d,p) 2.370 2.358, 2.357, 2.333 2.404HF/6-311G(d,p) 2.374 2.362, 2.361, 2.334 2.409HF/6-311+G(d,p) 2.371 2.359, 2.359, 2.329 2.404mPW1PW91/6-31G(d,p) 2.365 2.346, 2.346, 2.307 2.388mPW1PW91/6-311G(d,p) 2.368 2.350, 2.349, 2.307 2.392mPW1PW91/6-311+G(d,p) 2.367 2.349, 2.349, 2.305 2.389

LUMO (hartree) HF/6-31G(d,p) 0.03892 0.03532 0.07872HF/6-311G(d,p) 0.03549 0.03161 0.07216HF/6-311+G(d,p) 0.01977 0.02266 0.01864mPW1PW91/6-31G(d,p) �0.08285 �0.09073 �0.05337mPW1PW91/6-311G(d,p) �0.08846 �0.09567 �0.05966mPW1PW91/6-311+G(d,p) �0.08644 �0.09387 �0.05785

HOMO (hartree) HF/6-31G(d,p) �0.36436 �0.36574 �0.35052HF/6-311G(d,p) �0.36680 �0.36727 �0.35410HF/6-311+G(d,p) �0.36638 �0.36703 �0.35259mPW1PW91/6-31G(d,p) �0.24324 �0.24247 �0.23329mPW1PW91/6-311G(d,p) �0.24882 �0.24796 �0.23908mPW1PW91/6-311+G(d,p) �0.24752 �0.24670 �0.23750

Gap (cm�1) HF/6-31G(d,p) 8.85(+4)c 8.80(+4) 9.42(+4)HF/6-311G(d,p) 8.83(+4) 8.75(+4) 9.36(+4)HF/6-311+G(d,p) 8.48(+4) 8.55(+4) 8.15(+4)mPW1PW91/6-31G(d,p) 3.52(+4) 3.33(+4) 3.95(+4)mPW1PW91/6-311G(d,p) 3.52(+4) 3.34(+4) 3.94(+4)mPW1PW91/6-311+G(d,p) 3.54(+4) 3.35(+4) 3.94(+4)

a The basis set is for all atoms except the central metal, Rh or Ir, for which LANL2DZ basis set was used.b M stands for central metal, Rh or Ir.c 8.85(+4) � 8.85 � 104.

(A) (B)Fig. 2. Representations of HOMO (A) and LUMO (B) for 1 determined at mPW1PW91/6-31G(d,p) level of theory.

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with or without photoactivation [34,39,51]. Since the compoundsof interest have chloride ions coordinated to each metal centerand the reactions with DNA were performed in buffered H2O, weevaluated the chemical stability of complexes 1, 2, and 3 in a buf-fered aqueous system by UV–Vis spectroscopy. As seen in Fig. 3,there is a weak absorption in the range of 300–400 nm for both 1and 2, and a very week absorption around 300 nm for 3, with eachband appearing with a shoulder which tailed into the longer wave-lengths in the visible region. The rank order in absorbance was inline with the visually observed color of each complex with both1 and 2 being strong yellow color, while 3 complex being pale yel-

low, and with the calculated HOMO-LUMO energy gaps. For allthree compounds, a high background on UV–Vis spectra wasdetected at the first scan (0 day) with a semi-opaque yellowish fea-ture in the cuvette upon visual inspection. This high backgroundwas more apparent for 1 and 2, and deceased in intensity as timeprogressed from 0 to 4 day. The decrease of the high backgroundwas accompanied by the formation of an isosbestic point for eachcomplex, even though the change was smaller for 3. These isos-bestic points were monitored at 284 nm later for 1, 264 nm for 2,and 272 nm for 3, respectively. With the formation of isosbesticpoints, UV–Vis spectral feature changed in time noticeably for both

Fig. 3. Time-lapse UV–Vis spectral feature of 1–3 (100 lM) in phosphate buffer (50. mM, pH 7.0) at 20 �C at 0, 1, 2, 3, and 4 days.

488 J. Kim et al. / Inorganica Chimica Acta 469 (2018) 484–494

1 and 2, while there were only minor changes for 3 within thesame time period. The formation of the isosbestic points suggestseach complex is changed into new compound due, most likely, tochloride displacement and photoaquation. Photoaquation of Rh(III) was demonstrated with [RhL2XY] (L = bpy, en, phen; X =halide; Y = pseudohalide) in an earlier study and reported as anefficient process [34]. Similarly, photoaquation of [IrCl4(phen)] to[IrCl3(H2O)(phen)] was observed to occur within minutes withirradiation while the same conversion in the dark took 24 h evenwith boiling [51]. These reported findings together with our UV–Vis observation are important in terms of developing photothera-peutic agents which could be active only upon irradiation, yet inertin the dark.

To better understand these spectral observations, electronicstructure theory computations were carried out for complexes 1–3 having one chloride ion replaced by either a water molecule ora hydroxide ion. The results show that chloride replacement bythe hydroxide ion is more likely as it leads to a neutral complex.The replacement process is endothermic with zero-point-exclusiveenergy of reaction values of 18.5 kcal/mol, 15.9 kcal/mol, and 18.3kcal/mol for 1, 2, and 3, respectively, calculated at themPW1PW91/6-31G(d,p) level of theory. These values show thatthe halide replacement is slightly more facile for complex 2, andthat these energies of reaction are well below the energies of pho-tons in UV or visible regions. For comparison, the chloride replace-ment by water has a zero-point-exclusive energy of reaction ofabout 100 kcal/mol for all three complexes. More details of thesecalculations including fully optimized structures of complexeswith chloride replaced and results obtained at the HF/6-31G(d,p)level of theory are given in the Supplementary Material section.

(A)

(C)

(E)

254 nm .CN 1 5 30 (min)

(B

(D

(F

Fig. 4. Time-dependent photocleavage of pBR322 ± 1–3 (10 lM) upon irradiation for 1, 5pBR322 only without light exposure. (A) pBR322 ± [Rh(9S3)Cl3] 1 at 254 nm, (B) pBR322[Rh(9S2O)Cl3] 2 at 350 nm, (E) pBR322 ± [Ir(9S3)Cl3] 3 at 254 nm, (F) pBR322 ± [Ir(9S3)C

3.3. Photoinduced nuclease activity

Fig. 4 shows the time-dependent photocleavage results ofpBR322 in the absence and the presence of 1–3, respectively,where CN represents the control pBR322 without exposure to lightin the absence of the compounds, while other lanes represent incu-bations treated with 1–3 at 10 lM for 1, 5, and 30 min, under 254and 350 nm, respectively. In addition to the incubation reactions oflight + pBR322 + 1–3, we carried out control experiments for theseries of time-dependent photocleavage reactions, and the controlexperiments were carried out by exposing pBR322 at 254 and 350nm for 1, 5, and 30 min, respectively, without the presence of thecompounds of interest (Fig. 5). In the gel images, two bands wereobserved with different mobility corresponding to two differentforms of plasmid DNA. The one appearing at the top representsthe nicked or cleaved plasmid with low mobility, and the oneappearing at the bottom represents the supercoiled plasmid withhigh mobility.

As seen in Fig. 4A/C/E, the thickness for the nicked plasmidbecame bigger for the incubations of pBR322 + 1–3 at 254 nm, asexposure time increased from 1 to 30 min. On the contrary, irradi-ation at 350 nm (Fig. 4B/D/F) did not appear to generate a recogniz-able band corresponding to the nicked form for 1 and 5 minincubations except the nicked band observed for pBR322 incubatedwith 1 for 30 min (Fig. 4B). Fig. 6A/B shows the graphs of% cleavageof light + pBR322 ± 1–3 in comparison with controls where CNavg

represents averaged% cleavage of the control pBR322 withoutexposure to light in the absence of the compounds and CTY repre-sents the control pBR322 upon irradiation with the wavelength atY nm for 1, 5, and 30 min, respectively. CNavg contained 9% nicked

350 nm .CN 1 5 30 (min)

)

)

)

, and 30 min, respectively, at 30 �C in TE buffer. CN lanes represent the incubation of± [Rh(9S3)Cl3] 1 at 350 nm, (C) pBR322 ± [Rh(9S2O)Cl3] 2 at 254 nm, (D) pBR322 ±l3] 3 at 350 nm.

(A) (B)

254 nm .1 5 30 (min)

350 nm .1 5 30 (min)

Fig. 5. Time-dependent photocleavage of pBR322 upon irradiation in the absence of1–3, for 1, 5, and 30 min, respectively. (A) pBR322 at 254 nm, (B) pBR322 at 350 nm.

CNavg

CT254

Rh(9S3)Cl3Rh(9S2O)Cl3Ir(9S3)Cl3

CNavg

CT350

Rh(9S3)Cl3Rh(9S2O)Cl3Ir(9S3)Cl3

(A)

(B)

Fig. 6. % Cleavage of time-dependent incubations of pBR322 ± 1–3 upon irradiation.(A) pBR322 ± 1–3 at 254 nm, (B) pBR322 ± 1–3 at 350 nm.

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plasmid, while% cleavage of CT254 increased from 10% to 95% uponincreasing irradiation time from 1 to 30 min (Fig. 6A), and% cleav-age of CT350 remained almost similar compared to% cleavage ofCNavg upon increasing irradiation time (Fig. 6B). Fig. 4A shows thatthe rank order of photocleaving efficiency is 1 > 2 > 3 for 1 and 5min irradiation under 254 nm, however 30 min irradiation wastoo strong, resulting in a complete cleavage for control irradiation(95%) as well as for Rh(III)/Ir(III) treated plasmid. Fig. 5B shows thatthere was no recognizable difference in% cleavage for 1 and 5 minirradiation at 350 nm, while 30 min irradiation lead to 21% cleav-age induced by 1, and almost no cleavage detected for 2 and 3.

Fig. 7 shows the gel image for the concentration-dependentphotocleavage results of pBR322 in the absence and the presenceof 1–3, where CN represents the control pBR322 without exposureto light in the absence of the compounds, while CAY (CA254, expo-sure at 254 nm; CA350 exposure at 350 nm) represents the controlpBR322 with light exposure at Y nm for 10 min in the absence ofthe compounds of interest. The 10 min exposure time was chosenfor the concentration-dependent incubations since the time-dependent data shows 5 min did not give enough cleavage espe-cially at 350 nm, while 30 min exposure resulted in a complete

photocleavage even without the presence of the compounds ofinterest. Control experiments across the panel A to F in Fig. 7revealed that CN lanes with no light exposure had on average 9%nicked DNA and mostly uncleaved DNA. However, CA254/350 laneswith light exposure for 10 min showed on average 42% photoin-duced cleavage of pBR322 at 254 nm and 9% cleavage at 350 nmin the absence of Rh(III) and Ir(III) complexes (Fig. 8A/B). Therefore,the data shows UV light at 254 nm itself can nick DNA under theincubation conditions, however 350 nm by itself did not result incleavage of pBR322 in the absence of the compounds.

Fig. 7 shows photocleavage results of pBR322 ± 1 (0, 10, 50, and100 lM) irradiated at 254 nm (Panel A) and at 350 nm (Panel B),respectively, for 10 min. Fig. 7A together with Fig. 8A revealedthe disappearance of the supercoiled form with accompanied for-mation of the nicked form as a major form even at 10 lM of 1, with92% cleavage. 100% cleavage of pBR322 was achieved both at 50and 100 lM of 1. For 350 nm + pBR322 + 1 (Fig. 7B/8B), the pres-ence of the nicked form as well as the supercoiled form wasobserved over all concentration range, with the cleavage percentof 15, 24, and 23% at 10, 50, and 100 lM of 1, respectively. Thisobservation suggests that 1 is very efficient in cleaving pBR322upon exposure at 254 nm, and mildly active upon exposure at350 nm.

Fig. 7 shows photocleavage results of pBR322 ± 2 (0, 10, 50, and100 lM) irradiated at 254 nm (Panel C) and at 350 nm (Panel D),respectively, for 10 min. On Fig. 7C, showing exposure of pBR322to 254 nm, one can see the disappearance of the supercoiled formwith the accompanied formation of the nicked form as the concen-tration of 2 increased similar to the results for 1. Incubationwith10 lM of 2 at 254 nm lead to 63% cleavage, and 100 lM leadto 96% cleavage of pBR322 (Fig. 8A). For 350 nm irradiation inthe presence of 2 (Fig. 7D), the formation of the nicked formincreased slightly as the concentration of 2 increased. The% cleav-age with 350 nm irradiation were 10, 12, and 13% at 10, 50, and100 lM of 2, respectively (Fig. 8B). This observation suggests that2 is efficient in cleaving pBR322 upon exposure to 254 nm in a sim-ilar manner to 1, however almost 2-fold less active than 1 whenirradiated with 350 nm with very low level cleavage even at 100lM.

Fig. 7 shows photocleavage results of pBR322 ± 3 (0, 10, 50, and100 lM) irradiated at 254 nm (Panel E) and at 350 nm (Panel F),respectively, for 10 min. Panel 7E (pBR322 ± 3 + 254 nm) showsvery similar feature between the CA254 lane and 3 treatedpBR322, as confirmed by the cleavage percent values of 56, 47,and 51% at 10, 50, and 100 lM of 3, respectively (Fig. 8A). However,when irradiated with 350 nm, there was no difference in the bandfeature, and on average 9% cleavage was observed over all range ofthe concentrations of 3 (Fig. 8B). This finding suggests that 3 mightbe slightly active when irradiated at 254 nm, since there wasapproximately10% increase in cleavage percent value compare tothe value of CA254. However, 3 was not activated upon irradiationat 350 nm, looking at the same cleavage percent value and the sim-ilar band feature to CA350.

In summary, both time-dependent and concentration-depen-dent experiments implies 1 is the most efficient photocleaver bothat 254 and 350 nm, while 3 was the least efficient one with a mildefficiency of 29% observed at 254 nm and 10 lM for 5 min irradia-tion. Considering the photocleaving power of 254 nm itself, 350nm irradiation combined in the presence of 1–3 could offer amilder and selective approach for phototherapeutic applications.

3.4. Fluorescence behavior of the complexes in the absence of CT-DNA

To assess the possibility of using complex itself as a fluores-cence probe for the DNA binding study, we monitored the fluores-cence behavior of 1–3 in the absence of CT-DNA. Samples

Fig. 7. Concentration dependent photocleavage of pBR322 ± 1–3 (0, 10, 50, 100 lM) at 254 and 350 nm for 10 min, respectively, at 30 �C in TE buffer. CN lanes represent theincubation of pBR322 without light exposure in the absence of the compounds. CAY lanes represent the incubation of pBR322 with light exposure at Y nm for 10 min in theabsence of the compounds. (A) pBR322 ± [Rh(9S3)Cl3] 1 at 254 nm, (B) pBR322 ± [Rh(9S3)Cl3] 1 at 350 nm, (C) pBR322 ± [Rh(9S2O)Cl3] 2 at 254 nm, (D) pBR322 ± [Rh(9S2O)Cl3] 2 at 350 nm, (E) pBR322 ± [Ir(9S3)Cl3] 3 at 254 nm, (F) pBR322 ± [Ir(9S3)Cl3] 3 at 350 nm.

Fig. 8. % Cleavage of concentration-dependent incubations of pBR322 ± 1–3 uponirradiation. (A) pBR322 ± 1–3 at 254 nm, (B) pBR322 ± 1–3 at 350 nm.

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containing each complex with concentrations of 100 or 500 lM inphosphate buffer (50 mM, pH 7.0) was excited with an excitationwavelength (kEXC) of 250, 270, 300, 330, 350, 370, and 400 nm,respectively. This exploration revealed that all emission spectrashowed no recognizable fluorescence signal that could come fromthe complex so the compounds of interest were not fluorescentunder the studied conditions.

3.5. Ethidium bromide displacement assay for DNA binding study

To evaluate the binding of 1–3 toward DNA, we carried outethidium bromide displacement assay (EBDA) by monitoring thedecrease in fluorescence emission of samples containing CT-DNAand EB in the absence and the presence of 1, 2, or 3, respectively,with complex concentration increasing from 0 to 200 lM.

It is known that EB binds to different types of DNA or RNAwhether in their duplex or triplex form, through intercalation,and EB’s binding to DNA leads to strong fluorescence emissionupon excitation at 520 nm [52–55]. In terms of binding sequenceselectivity, it was reported that a A/T-rich region is a preferredbinding site for EB, while the region with positively charged cyto-sine is a poor binding site because of charge repulsion [55–57]. Dueto its DNA binding capacity and the resulted strong fluorescencesignal upon binding to DNA, EB is a great fluorescent probe forEB displacement assay (EBDA) or fluorescence quenching assay.EBDA has been utilized for studying the interaction of DNA and anovel drug candidate, in an effort to evaluate the binding naturewhether via intercalation or groove binding [29,54–57]. In a typicalEBDA, a competitive binder against EB is added to the pre-incu-bated solution containing the complex of [DNA + EB]. As the con-centration of the competitive binder increases, it will displace EB,resulting in decrease in fluorescence emission since unbound EBis not fluorescent mostly due to surrounding quenchers like sol-vent molecules. While EBDA is a commonly performed assay formeasuring DNA binding efficiency of many intercalating drug can-didates [29,54–57], it has been also utilized for quantifying DNAbinding efficiency of groove-binding agents [29,54,56,58]. Anyphysicochemical events happening in a region of DNA triggeredby an interacting ligand can generate a conformational change inother region of the DNA, whether in the major/minor grooves orin the space between stacked bases [57,59–61]. This conforma-tional change induced by the pre-bound ligand through noncova-lent interactions could impact the binding interactions of anincoming molecule at the subsequent step or vice versa, whetherit is an intercalator or a groove-binder [57,59–61].

DNA binding of the complexes of interest would likely to occurthrough groove-binding than through intercalation, since all threecompounds lack the presence of a ligand that could intercalate toDNA. As in the proposed mechanism (Fig. 9), one can postulate fourpossible pathways for displacing EB as the compound of interestbinds to DNA. In the pathway A, the compound displaces EBthrough intercalation of the thioether crown moiety. However, thisis impossible since it would require the collapse of the nonplanarthioether crown structure to fit into the space between the stack-ing base pairs, which is narrow with the rise distance of 0.34 nmfor B-DNA [62,63]. In the pathway B and C, the compound bindsto either the major groove or the minor groove of DNA, and thebinding of the compound triggers a conformational change unfa-vorable to [DNA+ EB] association, resulting in EB dissociation fol-lowed by loss in fluorescence. In the pathway D, the compoundneeds to be photoactivated to form an electronically unstable state

Fig. 9. Proposed EB displacement mechanism of 1–3. M = Rh(III) or Ir(III); X = S or O in [M(9S2X)Cl3].

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or to generate 1O2 through a redox reaction [2]. Subsequently, itwill lead to DNA strand break and dissociation of [DNA + EB] binarycomplex since EB is known to bind to double stranded (ds) DNAnot ssDNA and is strongly fluorescent only upon binding to DNA[54–56].

Consequently, we performed EBDA with 1, 2, and 3, respec-tively, where the binary complex of [DNA + EB] was treated witheach compound of interest. For the assay, we followed fluorescenceemission behavior of the compounds of interest in the presence of[DNA + EB] in SPB (50 mM phosphate, 50 mM NaCl, pH 7.0). Sepa-rately, we also monitored fluorescence behavior of [DNA + 1, 2, or3] in the absence of EB (data not shown) to ensure that the binarycomplex of [DNA + 1, 2, or 3] does not contribute to the fluores-cence background or interfere with the fluorescence emission ofinterest corresponding to [DNA + EB] with the presence of 1, 2, or3, when excited at k = 520 nm.

Fig. 10 shows the emission spectra of the solution of [DNA +EB] in the absence and the presence of the compounds of inter-est upon increasing concentrations of each compound graduallyfrom 0 lM to 200 lM. Incubations of [DNA + EB] with 1 and 2resulted in decrease of emission as the concentration of thecompound increased. The plots of percent integrated intensityin the 575–675 nm range vs. the concentration of the Rh(III)complex (Fig. 10 insets) revealed that both Rh(III) compounds1 and 2 can displace EB through efficient binding to DNA.The efficient concentration to achieve a 50% loss in fluorescentemission (ECFL50) was found to be 24 lM for 1 and 35 lM for 2,respectively. Passing 125 lM for both compounds, the fluores-cence emission intensity plateaued with about 80% loss in theintensity. In conclusion, the fluorescence behavior of 1 and 2were very similar, with 1 being slightly more efficient binder.In contrast to Rh(III) compounds, 3 exhibited very weak bind-ing, resulting in only 27% loss in fluorescence emission at

200 lM which was the highest concentration used for EBDAtitration.

Combined with the proposed mechanism, our EBDA resultsshow the compounds 1 and 2 were able to displace EB based onfluorescence emission decrease, while 3 being a poor binder. Thescan time for fluorescence assay took less than 1 min, which isshorter than the time used for the photocleavage assay conditionsin the UV–Vis reactor. In the photocleavage assay, 1 min incubationin the UV–Vis reactor had almost similar level of cleavage as a con-trol experiment (see Section 3.3). In addition, the kext used forEBDA was 520 nm which is weaker in energy than 254 and 350nm used for photocleavage assay. Therefore, it is likely that theobserved fluorescence decrease was indeed due to displacementof EB caused by binding interaction between DNA and the Rh(III)compounds rather than due to photoinduced breakage of ds DNAwithin the given condition. EBDA results revealed that 2 has aslightly less binding interaction over the same concentration rangecompared to 1. The binding interaction could be affected by fewfactors including polarity or electrostatic interaction. Our compu-tational study utilizing mPW1PW91 method revealed that thedipole moment values of 1–3 do not differ significantly, with thevalues of 16.6 D for 1, 16.0 D for 2, and 16.9 D for 3, respectively.Consequently, our study suggests that dipole moment might notbe a contributing factor in binding. It is possible that the bindinginteraction is influenced more by a repulsive electrostatic interac-tion between the O atom of 2 and the negative charge of phosphategroups on the interacting groove, resulting in a slightly weakerbinding of 2 to DNA compared to 1. Dipole moments calculatedat HF/6-31G(d,p) and mPW1PW91/6-31G(d,p) levels of theory forcomplexes 1–3 and the complexes obtained by replacing chlorideion by hydroxide ion are given in Supplementary Material.

Overall, our study shows that the recognizable binding interac-tion of the Rh(III) compounds to DNA is promising since the role of

Fig. 10. Fluorescence emission of the binary complex of CT-DNA and EB ([CT-DNA]= 2 lM, [EB] = 2 lM) in the presence of 1–3 (0, 5, 10, 25, 50, 75, 100, 125, 150, 175,200 lM) in SPB. (A) CT-DNA + EB ± [Rh(9S3)Cl3] 1, (B) CT-DNA + EB ± [Rh(9S2O)Cl3]2, (C) CT-DNA + EB ± [Ir(9S3)Cl3] 3. Insets show the percent fluorescence integratedin the 575–675 nm range vs the concentration of the complex.

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neutral inorganic complexes on DNA received little attention. Dueto the negative charges on DNA phosphate backbone, a positivecharge on any DNA targeting molecule was thought to be essentialin order to induce cooperative electrostatic interaction with DNA

[64,65]. Such examples are found in many DNA binding proteins;DNA polymerase, nuclease, histone, and transcription factors thatare positively charged at physiological pH [64,65]. However, DNAtargeting molecules with neutral charges may have advantage interms of intracellular delivery since they could be deliveredthrough simple diffusion without requiring the action of a proteincarrier used for transporting charged molecules [64,66]. In thisregard, our investigation offers better insight on designing a seriesof inorganic compounds which can target DNA and be efficient interms of intracellular delivery to the target tissue.

3.6. UV–Vis scanning of samples after EBDA detection

After monitoring fluorescence emission decrease in each samplefor EBDA, the same sample containing [CT-DNA + EB + 1–3 (0–200lM)] was submitted to UV–Vis scanning (see SupplementaryMaterial). The spectral feature over the range of 5–200 lM of 1–3 appeared to be similar in trend to the spectra of the compounditself in phosphate buffer at 0 h, with both Rh(III) complexes hav-ing higher absorption and Ir(III) complex having lowest absorption.As the concentration of each compound increased, there wasincrease in absorbance as well. Since excess amount of the com-pounds of interest was used, major absorption was most likelydue to the presence of complex rather than due to CT-DNA or EB.UV–Vis spectra of [CT-DNA + EB ± 1–3], having same spectral fea-tures as complexes alone, suggest that there was no change inthe structure of each compound under the scanned condition.

4. Conclusion

We investigated the efficiency of the thioether crown-contain-ing Rh(III) and Ir(III) compounds in terms of cleaving pBR322DNA upon irradiation with two wavelengths at 254 and 350 nm,respectively. Furthermore, we evaluated the binding tendency ofthe compounds of interest by monitoring ethidium bromide dis-placement efficiency using fluorescence spectroscopy. Our findingsuggests [Rh(9S3)Cl3] was the most efficient photocleaver at both254 and 350 nm, while [Ir(9S3)Cl3] was the least activated oneeven at a higher concentration at both wavelengths. [Rh(9S2O)Cl3] exhibited a mild-level of photocleavage activity especiallyupon exposure at 254 nm. The photoinduced cleavage activity of[Rh(9S3)Cl3] is promising since it shows the mild level cleavageof plasmid DNA at 350 nm which could be a better choice for pho-totherapeutic/photodiagnostic applications than 254 nm. Theranking order in photocleavage efficiency of the Rh(III) and Ir(III)complexes was in good agreement with the binding interactionwith CT-DNA, with [Rh(9S3)Cl3] being a stronger binder and [Ir(9S3)Cl3] being a poorest binder.

Acknowledgements

The study was supported in part by Grote Fund and Provost Stu-dent Research Award at the Department of Chemistry and Physicsat the University of Tennessee at Chattanooga.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at https://doi.org/10.1016/j.ica.2017.10.005.

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1

Supplementary Material for

Interaction with Calf-thymus DNA and Photoinduced

Cleavage of pBR322 by Rhodium(III) and Iridium(III)

Complexes Containing Crown Thioether Ligands

Jisook Kim,* Ashley D. Cardenal, Hendrik J. Greve, Weinan Chen, Hitesh Vashi, Gregory Grant, and Titus V. Albu

Department of Chemistry and Physics, University of Tennessee at Chattanooga, Chattanooga, TN 37403, USA

In this supplementary material, we present:

– UV-Vis spectra of CT-DNA + EB in the presence of complexes (Figure 1A-1C)

– Figure with HOMO and LUMO for complexes 1-3 determined at the mPW1PW91/6-31G(d,p)

level of theory

– Cartesian coordinates for the optimized geometries of complexes 1-3 at various levels of

theory

– Cartesian coordinates for the optimized geometries of complexes obtained by replacing a

chloride ligand by H2O or OH– at HF/6-31G(d,p) and mPW1PW91/6-31G(d,p) levels of

theory

– Table with calculated energies of reaction for chloride replacement reactions at HF/6-31G(d,p)

and mPW1PW91/6-31G(d,p) levels of theory

– Table with calculated dipole moments at HF/6-31G(d,p) and mPW1PW91/6-31G(d,p) levels

of theory for complexes 1-3 and the complexes obtained by replacing chloride ion by

hydroxide ion

2

Figure 1A. UV-Vis spectra of CT-DNA + EB in the presence of [Rh(9S3)Cl3] 1 (0, 5, 10, 25,

50, 75, 100, 125, 150, 175, 200 M) in SPB. The arrow notes the direction of

increasing concentration of the compounds.

3

Figure 1B. UV-Vis spectra of CT-DNA + EB in the presence of [Rh(9S2O)Cl3] 2 ( (0, 5, 10, 25,

50, 75, 100, 125, 150, 175, 200 M) in SPB. The arrow notes the direction of

increasing concentration of the compounds.

4

Figure 1C. UV-Vis spectra of CT-DNA + EB in the presence of [Ir(9S3)Cl3] 3 ( (0, 5, 10, 25,

50, 75, 100, 125, 150, 175, 200 M) in SPB. The arrow notes the direction of

increasing concentration of the compounds.

5

Figure 2. HOMO and LUMO for complexes 1-3 calculated at the mPW1PW91/6-31G(d,p)

level of theory

[Rh(9S3)Cl3] - HOMO [Rh(9S3)Cl3] - LUMO

[Rh(9S2O)Cl3] - HOMO [Rh(9S2O)Cl3] - LUMO

[Ir(9S3)Cl3] - HOMO [Ir(9S3)Cl3] - LUMO

6

[Rh(9S3)Cl3] 1 geometry – HF/6-31G(d,p) Rh1 -0.602824 0.139861 0.315803

S2 1.645315 0.480735 1.224267

S3 0.160945 -2.041817 -0.492012

S4 0.209333 1.093445 -1.788170

C5 2.531511 -1.042796 0.728692

C6 2.253485 1.720863 0.038667

C7 0.897629 -1.649339 -2.121535

C8 1.615019 -2.257088 0.581656

C9 1.972707 1.438855 -1.437042

C10 0.273655 -0.424796 -2.790290

H11 3.258465 -1.257520 1.501680

H12 3.082408 -0.848997 -0.180805

H13 3.318276 1.855527 0.187405

H14 1.751547 2.625515 0.354379

H15 0.734760 -2.506408 -2.762614

H16 1.966436 -1.537072 -2.006306

H17 2.188882 -3.105724 0.228758

H18 1.176434 -2.519224 1.535039

H19 2.248414 2.315808 -2.008895

H20 2.571814 0.618003 -1.805151

H21 0.793357 -0.211583 -3.716836

H22 -0.767086 -0.607639 -3.021454

Cl23 -1.105490 2.329567 1.069469

Cl24 -1.208139 -0.955186 2.328256

Cl25 -2.669322 -0.218289 -0.787288

[Rh(9S3)Cl3] 1 geometry – HF/6-311G(d,p) Rh1 -0.489329 0.232336 0.419779

S2 1.891101 0.031635 0.921923

S3 -0.369531 -1.983155 -0.598120

S4 0.138272 1.163416 -1.747727

C5 2.331714 -1.599809 0.211513

C6 2.551423 1.209637 -0.300825

C7 0.148745 -1.624441 -2.319500

C8 1.163673 -2.583900 0.181122

C9 1.970286 1.111007 -1.710161

C10 -0.296355 -0.248978 -2.813123

H11 3.115150 -2.016323 0.831472

H12 2.747950 -1.447838 -0.773846

H13 3.628798 1.101040 -0.340409

H14 2.324480 2.170312 0.140691

H15 -0.302296 -2.378317 -2.951827

H16 1.220158 -1.743801 -2.390957

H17 1.468345 -3.498918 -0.312855

H18 0.845071 -2.831297 1.184428

H19 2.325200 1.955058 -2.287412

H20 2.301847 0.214622 -2.213987

H21 0.100290 -0.069897 -3.805443

H22 -1.374934 -0.191112 -2.864662

Cl23 -0.395769 2.415958 1.346539

Cl24 -0.979220 -0.850991 2.474590

Cl25 -2.751754 0.394438 -0.280931

7

[Rh(9S3)Cl3] 1 geometry – HF/6-311+G(d,p) Rh1 -0.526921 0.208132 0.392718

S2 1.836364 0.177440 1.010828

S3 -0.207219 -2.014527 -0.569414

S4 0.146311 1.135788 -1.764672

C5 2.419602 -1.434056 0.359843

C6 2.475930 1.373642 -0.205375

C7 0.370571 -1.655825 -2.271871

C8 1.322036 -2.494711 0.296554

C9 1.973806 1.208127 -1.638596

C10 -0.140806 -0.323497 -2.816883

H11 3.198095 -1.784350 1.025616

H12 2.872801 -1.273487 -0.607774

H13 3.558973 1.338039 -0.190492

H14 2.163166 2.325298 0.202295

H15 0.003083 -2.451115 -2.907856

H16 1.449860 -1.703760 -2.288941

H17 1.710863 -3.397067 -0.160652

H18 0.971491 -2.742508 1.289217

H19 2.300140 2.062465 -2.217927

H20 2.388633 0.326310 -2.105255

H21 0.290936 -0.137457 -3.793334

H22 -1.217203 -0.340178 -2.920602

Cl23 -0.614370 2.408015 1.272613

Cl24 -1.040861 -0.873021 2.439257

Cl25 -2.751446 0.205627 -0.427547

[Rh(9S3)Cl3] 1 geometry – mPW1PW91/6-31G(d,p) Rh1 -0.620677 -0.000119 0.000050

S2 0.700402 -0.824338 -1.740290

S3 0.699329 1.920503 0.156189

S4 0.700687 -1.094697 1.584048

C5 2.032107 0.441949 -1.856376

C6 1.541497 -2.179484 -0.847526

C7 2.032333 1.388201 1.310033

C8 1.539208 1.824544 -1.464454

C9 2.033393 -1.828462 0.546348

C10 1.540349 0.356833 2.311216

H11 2.370424 0.463656 -2.895276

H12 2.878585 0.122873 -1.244847

H13 2.361077 -2.560371 -1.463018

H14 0.757590 -2.940189 -0.799515

H15 2.370607 2.277083 1.848243

H16 2.878273 1.018551 0.726859

H17 2.358120 2.548728 -1.487863

H18 0.754411 2.162002 -2.146916

H19 2.370399 -2.739498 1.047023

H20 2.880677 -1.140171 0.518144

H21 2.359661 0.015197 2.949671

H22 0.755602 0.778561 2.945132

Cl23 -1.863201 -2.000624 -0.221543

Cl24 -1.865226 1.190872 -1.620894

Cl25 -1.864805 0.807044 1.842759

8

[Rh(9S3)Cl3] 1 geometry – mPW1PW91/6-311G(d,p) Rh1 0.614843 0.000000 0.000185

S2 -0.707047 1.089984 1.584957

S3 -0.706119 0.827762 -1.736792

S4 -0.705899 -1.919011 0.151554

C5 -2.039513 1.825443 0.547896

C6 -1.550942 -0.360330 2.308999

C7 -2.037917 -0.438586 -1.855012

C8 -1.550925 2.179885 -0.843607

C9 -2.039105 -1.387976 1.305848

C10 -1.548695 -1.820962 -1.467101

H11 -2.376166 2.731980 1.053870

H12 -2.881685 1.133491 0.519924

H13 -2.372271 -0.012350 2.939332

H14 -0.774102 -0.780282 2.951037

H15 -2.375551 -0.453398 -2.892771

H16 -2.879351 -0.117180 -1.240706

H17 -2.372152 2.551142 -1.460665

H18 -0.774424 2.946188 -0.800889

H19 -2.377035 -2.279198 1.837515

H20 -2.879890 -1.016987 0.719152

H21 -2.369297 -2.541640 -1.481945

H22 -0.770901 -2.165749 -2.151372

Cl23 1.883208 -0.817793 1.824812

Cl24 1.882163 1.989891 -0.202879

Cl25 1.884188 -1.169782 -1.620665

[Rh(9S3)Cl3] 1 geometry – mPW1PW91/6-311+G(d,p) Rh1 0.617881 -0.000205 0.000087

S2 -0.701763 -0.905805 -1.695618

S3 -0.702587 -1.015477 1.632437

S4 -0.700709 1.922243 0.063442

C5 -2.035389 -1.751413 -0.748220

C6 -1.545973 0.615280 -2.254790

C7 -2.035067 0.228876 1.890433

C8 -1.547412 -2.259741 0.594774

C9 -2.033544 1.525083 -1.143515

C10 -1.545668 1.645727 1.659745

H11 -2.373965 -2.594959 -1.352744

H12 -2.876222 -1.065297 -0.642681

H13 -2.368384 0.338430 -2.918489

H14 -0.769957 1.104144 -2.847353

H15 -2.374331 0.127336 2.923031

H16 -2.875867 -0.021722 1.243003

H17 -2.369606 -2.696206 1.166603

H18 -0.771538 -3.017387 0.466985

H19 -2.370996 2.470495 -1.572459

H20 -2.875178 1.091788 -0.602466

H21 -2.367120 2.359963 1.752337

H22 -0.769369 1.912729 2.379764

Cl23 1.873046 1.014531 -1.730832

Cl24 1.871513 -2.007565 -0.013659

Cl25 1.872919 0.990778 1.744831

9

[Rh(9S2O)Cl3] 2 geometry – HF/6-31G(d,p) Rh1 -0.652324 -0.002493 0.018205

O2 0.794188 0.211099 -1.663879

S3 0.945218 1.591181 0.999201

S4 0.926579 -1.803354 0.586041

C5 1.736105 1.271087 -1.687740

C6 1.266595 -1.060487 -2.085274

C7 2.359136 0.513754 1.439363

C8 1.438270 2.299254 -0.603425

C9 1.949749 -1.802627 -0.934422

C10 1.948860 -0.912885 1.801519

H11 1.691401 1.754985 -2.655832

H12 2.732643 0.862852 -1.563550

H13 1.955920 -0.938376 -2.912935

H14 0.396857 -1.597727 -2.423667

H15 2.844672 0.956428 2.299876

H16 3.075163 0.524639 0.630009

H17 2.295219 2.948033 -0.468253

H18 0.583728 2.901826 -0.874993

H19 2.103165 -2.834689 -1.221317

H20 2.919114 -1.382852 -0.699017

H21 2.833784 -1.501478 2.012621

H22 1.328329 -0.915071 2.688620

Cl23 -1.965748 -1.577635 -1.145424

Cl24 -1.956993 1.844700 -0.646191

Cl25 -1.718433 -0.314652 2.070218

[Rh(9S2O)Cl3] 2 geometry – HF/6-311G(d,p) Rh1 -0.644128 -0.003360 0.023249

O2 0.793840 0.221543 -1.649947

S3 0.949260 1.581704 1.012054

S4 0.927108 -1.807187 0.576274

C5 1.723203 1.290901 -1.683167

C6 1.266970 -1.040554 -2.093524

C7 2.369280 0.500728 1.433218

C8 1.436313 2.309272 -0.586045

C9 1.943565 -1.804510 -0.952548

C10 1.961520 -0.926786 1.791011

H11 1.658043 1.781114 -2.647063

H12 2.725361 0.890819 -1.576397

H13 1.961179 -0.903079 -2.914790

H14 0.401557 -1.572992 -2.449050

H15 2.863343 0.941424 2.289685

H16 3.072385 0.516128 0.613237

H17 2.298266 2.949731 -0.444450

H18 0.584517 2.921210 -0.842322

H19 2.080219 -2.836195 -1.247665

H20 2.917642 -1.398625 -0.713113

H21 2.846600 -1.520291 1.987205

H22 1.348443 -0.936913 2.683061

Cl23 -1.969299 -1.569686 -1.147657

Cl24 -1.961723 1.837952 -0.646338

Cl25 -1.736595 -0.316506 2.061531

10

[Rh(9S2O)Cl3] 2 geometry – HF/6-311+G(d,p) Rh1 -0.648001 -0.003140 0.022083

O2 0.792865 0.215675 -1.661045

S3 0.946322 1.584358 1.005565

S4 0.922817 -1.803322 0.582579

C5 1.730102 1.278180 -1.686094

C6 1.268687 -1.050454 -2.089715

C7 2.363179 0.504844 1.440599

C8 1.441684 2.301814 -0.594691

C9 1.945352 -1.803396 -0.941992

C10 1.952084 -0.921113 1.800324

H11 1.677851 1.767168 -2.651963

H12 2.728772 0.871788 -1.570425

H13 1.963374 -0.920696 -2.912273

H14 0.404465 -1.588694 -2.440517

H15 2.852093 0.948843 2.298539

H16 3.071500 0.517419 0.624967

H17 2.305807 2.939581 -0.453225

H18 0.592859 2.915678 -0.857023

H19 2.088890 -2.835942 -1.231376

H20 2.916452 -1.391368 -0.700893

H21 2.835678 -1.515207 2.002388

H22 1.334502 -0.928030 2.689380

Cl23 -1.965575 -1.573077 -1.146900

Cl24 -1.955219 1.841924 -0.649027

Cl25 -1.730259 -0.311227 2.061582

[Rh(9S2O)Cl3] 2 geometry – mPW1PW91/6-31G(d,p) Rh1 -0.591111 0.000659 0.010557

O2 0.772277 0.168787 -1.726396

S3 0.854660 1.595060 0.948860

S4 0.841713 -1.747738 0.648244

C5 1.784439 1.178098 -1.666085

C6 1.253316 -1.143260 -2.035729

C7 2.282157 0.557941 1.478316

C8 1.413973 2.249552 -0.661679

C9 1.929785 -1.798557 -0.835825

C10 1.860538 -0.848644 1.871299

H11 1.880990 1.632071 -2.657929

H12 2.747943 0.720583 -1.414613

H13 1.943659 -1.091003 -2.884442

H14 0.359583 -1.699029 -2.322457

H15 2.736709 1.049110 2.342182

H16 3.028138 0.548244 0.681252

H17 2.244671 2.944950 -0.517759

H18 0.530803 2.799730 -0.997925

H19 2.125946 -2.848627 -1.062621

H20 2.885213 -1.331795 -0.585390

H21 2.735050 -1.454336 2.124739

H22 1.200158 -0.822501 2.742575

Cl23 -1.910532 -1.592453 -1.097134

Cl24 -1.914231 1.800044 -0.708026

Cl25 -1.721373 -0.240256 2.007103

11

[Rh(9S2O)Cl3] 2 geometry – mPW1PW91/6-311G(d,p) Rh1 -0.585989 0.000090 0.015451

O2 0.771119 0.178054 -1.714355

S3 0.863472 1.587949 0.958777

S4 0.849864 -1.750876 0.639379

C5 1.776745 1.193620 -1.662769

C6 1.253326 -1.127553 -2.043185

C7 2.296163 0.547316 1.467004

C8 1.413260 2.256652 -0.649613

C9 1.929038 -1.796337 -0.852455

C10 1.880142 -0.858826 1.857575

H11 1.856985 1.651303 -2.653011

H12 2.743696 0.739633 -1.423682

H13 1.943268 -1.060490 -2.889595

H14 0.364117 -1.680923 -2.341422

H15 2.759888 1.037264 2.324939

H16 3.027443 0.542655 0.658615

H17 2.246417 2.945761 -0.499571

H18 0.532978 2.814860 -0.973985

H19 2.115621 -2.845317 -1.084898

H20 2.885498 -1.336394 -0.599728

H21 2.755851 -1.467863 2.092985

H22 1.231838 -0.842105 2.736356

Cl23 -1.921265 -1.584293 -1.094089

Cl24 -1.927057 1.789349 -0.705768

Cl25 -1.738059 -0.240897 2.000015

[Rh(9S2O)Cl3] 2 geometry – mPW1PW91/6-311+G(d,p) Rh1 -0.587639 0.000109 0.013645

O2 0.765863 0.168297 -1.721573

S3 0.855777 1.592029 0.948530

S4 0.843346 -1.744466 0.647489

C5 1.775216 1.180680 -1.667753

C6 1.251890 -1.139742 -2.037010

C7 2.287105 0.555800 1.468973

C8 1.413768 2.250120 -0.660964

C9 1.927175 -1.796747 -0.840117

C10 1.870481 -0.848388 1.865065

H11 1.861656 1.633844 -2.660002

H12 2.739278 0.723660 -1.423858

H13 1.943206 -1.078383 -2.883136

H14 0.364926 -1.699360 -2.331396

H15 2.747306 1.051097 2.326025

H16 3.021587 0.547353 0.663461

H17 2.251968 2.933628 -0.511730

H18 0.538529 2.813463 -0.990384

H19 2.117679 -2.846790 -1.065469

H20 2.881623 -1.332316 -0.588041

H21 2.746006 -1.456628 2.104274

H22 1.220338 -0.828177 2.742514

Cl23 -1.913514 -1.593926 -1.090628

Cl24 -1.919184 1.794062 -0.710773

Cl25 -1.723599 -0.234488 2.005569

12

[Ir(9S3)Cl3] 3 geometry – HF/6-31G(d,p) Ir1 0.000075 0.002179 0.561870

S2 0.842672 -1.756929 -0.840431

S3 -1.940216 0.143669 -0.847430

S4 1.097205 1.603403 -0.853497

C5 -0.436184 -1.888658 -2.146217

C6 2.187472 -0.854670 -1.671833

C7 -1.410745 1.309296 -2.158489

C8 -1.828618 -1.476993 -1.668882

C9 1.846065 0.554306 -2.155996

C10 -0.359526 2.312178 -1.683774

H11 -0.473004 -2.925558 -2.454669

H12 -0.123411 -1.310464 -3.004359

H13 2.549811 -1.450068 -2.501358

H14 2.959697 -0.819605 -0.915108

H15 -2.289337 1.857779 -2.472977

H16 -1.063695 0.744254 -3.012202

H17 -2.522794 -1.498021 -2.500494

H18 -2.186752 -2.158797 -0.909226

H19 2.761351 1.039087 -2.470478

H20 1.185898 0.531197 -3.011676

H21 -0.028023 2.918926 -2.517957

H22 -0.773313 2.967722 -0.929297

Cl23 2.035834 -0.199649 1.824230

Cl24 -1.196578 -1.652426 1.830296

Cl25 -0.838524 1.873293 1.816575

[Ir(9S3)Cl3] 3 geometry – HF/6-311G(d,p) Ir1 -0.002765 -0.038459 -0.554171

S2 -0.950459 -1.628732 0.968619

S3 1.946762 0.077198 0.835774

S4 -0.983629 1.727822 0.735845

C5 0.323954 -1.751298 2.282555

C6 -2.231253 -0.584731 1.735576

C7 1.503994 1.369725 2.060073

C8 1.737397 -1.468336 1.777094

C9 -1.795732 0.829653 2.114017

C10 0.518940 2.401950 1.514582

H11 0.289668 -2.763214 2.664640

H12 0.051913 -1.090083 3.092731

H13 -2.621279 -1.096956 2.606983

H14 -3.005069 -0.558160 0.980642

H15 2.420599 1.876705 2.332004

H16 1.123708 0.889144 2.950028

H17 2.433397 -1.466547 2.607411

H18 2.050448 -2.225312 1.071115

H19 -2.673407 1.399169 2.390562

H20 -1.130672 0.826109 2.965651

H21 0.225389 3.085166 2.302547

H22 0.968428 2.975521 0.715584

Cl23 -2.046446 -0.187516 -1.820087

Cl24 1.062370 -1.857356 -1.719714

Cl25 0.956616 1.662952 -1.963490

13

[Ir(9S3)Cl3] 3 geometry – HF/6-311+G(d,p) Ir1 0.004883 -0.052335 -0.556296

S2 -0.674042 -1.737831 1.009937

S3 1.906147 0.414628 0.830090

S4 -1.254354 1.561638 0.694439

C5 0.600152 -1.618271 2.324426

C6 -2.109676 -0.898361 1.753408

C7 1.255431 1.647251 2.022968

C8 1.949591 -1.120467 1.809937

C9 -1.912145 0.577180 2.095830

C10 0.116039 2.490854 1.454098

H11 0.730789 -2.612378 2.732133

H12 0.221289 -0.990491 3.118227

H13 -2.412646 -1.445309 2.638429

H14 -2.876572 -1.017219 1.000063

H15 2.076202 2.303807 2.281443

H16 0.956812 1.132471 2.924962

H17 2.633518 -0.984551 2.639380

H18 2.384302 -1.833936 1.123080

H19 -2.871724 1.002298 2.360450

H20 -1.257011 0.703684 2.945867

H21 -0.286746 3.135807 2.226113

H22 0.468005 3.111201 0.641033

Cl23 -1.982788 -0.568153 -1.805977

Cl24 1.358802 -1.696761 -1.670467

Cl25 0.672068 1.749638 -2.000722

[Ir(9S3)Cl3] 3 geometry – mPW1PW91/6-31G(d,p) Ir1 -0.514279 0.000065 0.000068

S2 0.787596 -0.640399 1.801628

S3 0.787146 -1.240485 -1.455976

S4 0.787806 1.881247 -0.346935

C5 2.126917 -1.628765 1.003896

C6 1.641403 0.942680 2.140105

C7 2.127681 -0.056118 -1.910141

C8 1.639897 -2.326138 -0.254219

C9 2.127823 1.683863 0.907150

C10 1.640537 1.381936 -1.887224

H11 2.449410 -2.378294 1.730245

H12 2.979021 -0.975394 0.804719

H13 2.464026 0.763819 2.837529

H14 0.863447 1.514652 2.653460

H15 2.453538 -0.311108 -2.921172

H16 2.977321 -0.209559 -1.241602

H17 2.462191 -2.841265 -0.757773

H18 0.861427 -3.056066 -0.015309

H19 2.450369 2.687577 1.193355

H20 2.979790 1.185019 0.440307

H21 2.462727 2.075468 -2.082434

H22 0.861994 1.538967 -2.638983

Cl23 -1.809186 1.264520 1.557573

Cl24 -1.809763 -1.980508 0.316612

Cl25 -1.809998 0.716615 -1.873270

14

[Ir(9S3)Cl3] 3 geometry – mPW1PW91/6-311G(d,p) Ir1 0.509969 0.000024 0.000152

S2 -0.792760 -0.018113 -1.909791

S3 -0.792618 -1.645672 0.971123

S4 -0.792962 1.663064 0.939528

C5 -2.132514 -1.211936 -1.476522

C6 -1.650459 1.586585 -1.716146

C7 -2.133928 -0.673536 1.785663

C8 -1.648502 -2.280632 -0.516337

C9 -2.133404 1.884902 -0.310433

C10 -1.650691 0.692265 2.231920

H11 -2.454985 -1.678897 -2.408264

H12 -2.979124 -0.654746 -1.074382

H13 -2.474329 1.633302 -2.431269

H14 -0.879354 2.295986 -2.023476

H15 -2.458351 -1.246986 2.655196

H16 -2.978886 -0.603550 1.100006

H17 -2.471592 -2.924816 -0.200301

H18 -0.876198 -2.899847 -0.977280

H19 -2.454991 2.925583 -0.249290

H20 -2.980515 1.258913 -0.028341

H21 -2.474475 1.288021 2.630397

H22 -0.879730 0.602852 3.000020

Cl23 1.827535 1.701315 -1.044782

Cl24 1.828849 -1.754485 -0.950849

Cl25 1.828637 0.054817 1.995197

[Ir(9S3)Cl3] 3 geometry – mPW1PW91/6-311+G(d,p) Ir1 0.513030 -0.000126 0.000049

S2 -0.790348 -0.116646 1.905465

S3 -0.789183 1.709461 -0.852119

S4 -0.789985 -1.591974 -1.054123

C5 -2.130061 1.103954 1.557356

C6 -1.649330 -1.702692 1.599441

C7 -2.129911 0.798141 -1.733910

C8 -1.646558 2.237995 0.675105

C9 -2.131127 -1.900458 0.175717

C10 -1.647728 -0.533341 -2.275031

H11 -2.454348 1.503775 2.519471

H12 -2.975797 0.575822 1.115847

H13 -2.474795 -1.797929 2.308209

H14 -0.880446 -2.433999 1.857569

H15 -2.454628 1.431448 -2.561057

H16 -2.974948 0.680555 -1.054909

H17 -2.470848 2.901297 0.404227

H18 -0.876114 2.825100 1.179506

H19 -2.455178 -2.933649 0.040923

H20 -2.976627 -1.254125 -0.062059

H21 -2.472869 -1.099141 -2.713002

H22 -0.878081 -0.390786 -3.036598

Cl23 1.817817 -1.772195 0.929068

Cl24 1.819365 1.689274 1.070111

Cl25 1.819400 0.080937 -1.998092

15

[Rh(9S3)Cl2H2O]+ geometry – HF/6-31G(d,p) Rh1 0.693956 -0.001703 -0.237102

S2 -0.243942 -0.046943 1.939089

S3 -0.951483 1.713435 -0.841789

S4 -0.968759 -1.667937 -0.924029

C5 -1.601008 1.183705 1.865273

C6 -1.100096 -1.655090 1.887666

C7 -2.425132 0.743562 -1.333563

C8 -1.352596 2.288932 0.841398

C9 -1.910879 -1.939635 0.625500

C10 -2.073042 -0.618564 -1.925609

H11 -1.668150 1.632672 2.847830

H12 -2.532347 0.667581 1.683064

H13 -1.738634 -1.729153 2.759527

H14 -0.296081 -2.369604 2.002045

H15 -2.951148 1.320558 -2.083226

H16 -3.084040 0.650412 -0.482990

H17 -2.217072 2.938339 0.784690

H18 -0.496969 2.890765 1.115807

H19 -2.208313 -2.979877 0.634670

H20 -2.813101 -1.346886 0.584529

H21 -2.979406 -1.173619 -2.134356

H22 -1.541851 -0.506407 -2.862854

Cl23 2.160991 -1.733188 0.378385

Cl24 2.152894 1.738418 0.374323

O25 1.623502 -0.003977 -2.139139

H26 2.217726 -0.747586 -2.177350

H27 2.160739 0.777988 -2.221046

[Rh(9S3)Cl2H2O]+ geometry – mPW1PW91/6-31G(d,p) Rh1 0.631501 -0.002406 -0.232055

S2 -0.195603 -0.044337 1.895248

S3 -0.898052 1.687688 -0.833620

S4 -0.916894 -1.643026 -0.910749

C5 -1.578939 1.172489 1.849122

C6 -1.068785 -1.651427 1.878179

C7 -2.397042 0.736500 -1.314943

C8 -1.322388 2.278805 0.842901

C9 -1.887671 -1.918086 0.629237

C10 -2.047676 -0.613747 -1.913684

H11 -1.649870 1.601663 2.851689

H12 -2.511698 0.641664 1.651547

H13 -1.691457 -1.714608 2.774843

H14 -0.251998 -2.370600 1.979480

H15 -2.930802 1.334084 -2.058216

H16 -3.049988 0.641127 -0.446006

H17 -2.186046 2.944748 0.770204

H18 -0.450747 2.876925 1.120487

H19 -2.204352 -2.963609 0.621488

H20 -2.787274 -1.301679 0.586346

H21 -2.953714 -1.190950 -2.118175

H22 -1.513792 -0.501122 -2.861097

Cl23 2.127069 -1.712438 0.358886

Cl24 2.119893 1.713374 0.363087

O25 1.637664 0.003346 -2.113254

H26 2.241454 -0.753360 -2.006353

H27 2.206854 0.788880 -2.030213

16

[Rh(9S2O)Cl2H2O]+ geometry – HF/6-31G(d,p) Rh1 0.640730 -0.112661 0.177251

O2 -0.760971 1.211017 1.153787

S3 -0.434726 0.714877 -1.761703

S4 -1.211208 -1.696407 0.488041

C5 -1.452713 2.211484 0.396692

C6 -1.532120 0.517399 2.133659

C7 -2.015042 -0.216555 -1.798622

C8 -0.891122 2.315943 -1.017170

C9 -2.310173 -0.636518 1.502427

C10 -1.904465 -1.614374 -1.196825

H11 -1.336030 3.160914 0.901111

H12 -2.505365 1.961556 0.376497

H13 -2.205335 1.209012 2.623605

H14 -0.824606 0.153279 2.863080

H15 -2.297997 -0.309483 -2.839242

H16 -2.777388 0.371544 -1.308691

H17 -1.600292 2.817401 -1.664016

H18 0.035389 2.871052 -1.029198

H19 -2.729919 -1.257353 2.282936

H20 -3.129261 -0.288584 0.888395

H21 -2.877361 -2.089345 -1.191466

H22 -1.233134 -2.236453 -1.774869

Cl23 2.293905 1.539473 -0.012532

Cl24 1.847720 -1.734450 -0.967416

O25 1.617785 -0.723181 1.953246

H26 2.299302 -0.084930 2.144107

H27 2.055760 -1.556744 1.811930

[Rh(9S2O)Cl2H2O]+ geometry – mPW1PW91/6-31G(d,p) Rh1 0.594147 -0.094010 0.180518

O2 -0.755149 1.303792 1.128389

S3 -0.379829 0.603640 -1.761193

S4 -1.137359 -1.659844 0.550824

C5 -1.519999 2.170031 0.262030

C6 -1.535395 0.596967 2.107501

C7 -1.967162 -0.336026 -1.802422

C8 -0.890584 2.236960 -1.113950

C9 -2.286802 -0.577808 1.493990

C10 -1.845030 -1.692341 -1.133989

H11 -1.532977 3.168417 0.707426

H12 -2.550346 1.806873 0.209138

H13 -2.229685 1.291619 2.589426

H14 -0.813970 0.257176 2.851350

H15 -2.221886 -0.470823 -2.856535

H16 -2.749545 0.275521 -1.350236

H17 -1.567314 2.711234 -1.829377

H18 0.049098 2.793522 -1.094345

H19 -2.728095 -1.184413 2.287963

H20 -3.097707 -0.260696 0.835591

H21 -2.816195 -2.192384 -1.095632

H22 -1.152599 -2.342623 -1.675418

Cl23 2.261932 1.522756 -0.070312

Cl24 1.843062 -1.725678 -0.855546

O25 1.631374 -0.598635 1.970768

H26 2.305506 0.105362 1.991136

H27 2.119257 -1.407341 1.739481

17

[Ir(9S3)Cl2H2O]+ geometry – HF/6-31G(d,p) Ir1 0.576246 -0.001847 -0.177931

S2 -0.427402 -0.039996 1.954516

S3 -1.002086 1.704000 -0.842226

S4 -1.018154 -1.660559 -0.916244

C5 -1.781200 1.190349 1.830164

C6 -1.282728 -1.646812 1.885583

C7 -2.459309 0.742093 -1.398986

C8 -1.486496 2.292202 0.814912

C9 -2.031875 -1.940164 0.588099

C10 -2.090648 -0.626333 -1.966433

H11 -1.885453 1.640807 2.808588

H12 -2.706044 0.676614 1.610939

H13 -1.961885 -1.711885 2.726750

H14 -0.485144 -2.360699 2.043215

H15 -2.944044 1.320322 -2.174822

H16 -3.157357 0.658120 -0.579159

H17 -2.348001 2.939459 0.709999

H18 -0.644756 2.896223 1.126056

H19 -2.319373 -2.983034 0.581589

H20 -2.935642 -1.354153 0.503705

H21 -2.990134 -1.184562 -2.195295

H22 -1.530269 -0.524785 -2.887693

Cl23 2.065294 -1.745293 0.475685

Cl24 2.063141 1.744949 0.473582

O25 1.617121 -0.001964 -2.031301

H26 2.209070 -0.749569 -2.039948

H27 2.163566 0.777792 -2.077139

[Ir(9S3)Cl2H2O]+ geometry – mPW1PW91/6-31G(d,p) Ir1 0.534850 -0.002897 -0.169629

S2 -0.395003 -0.029913 1.910967

S3 -0.946560 1.673233 -0.843900

S4 -0.962882 -1.638662 -0.899546

C5 -1.776539 1.189990 1.801489

C6 -1.273986 -1.635165 1.878057

C7 -2.429251 0.727886 -1.397673

C8 -1.472403 2.286406 0.798674

C9 -2.019427 -1.918020 0.588538

C10 -2.061737 -0.631637 -1.962245

H11 -1.885311 1.625673 2.797311

H12 -2.701599 0.659778 1.568467

H13 -1.947538 -1.677426 2.737994

H14 -0.468111 -2.355879 2.039571

H15 -2.914573 1.324679 -2.173487

H16 -3.126627 0.647313 -0.562241

H17 -2.336012 2.942330 0.663901

H18 -0.622873 2.896205 1.116961

H19 -2.321747 -2.967135 0.564930

H20 -2.920510 -1.309877 0.491389

H21 -2.960211 -1.215129 -2.180761

H22 -1.498179 -0.536615 -2.894080

Cl23 2.056878 -1.708063 0.460852

Cl24 2.053845 1.706248 0.460940

O25 1.654139 0.000797 -1.993402

H26 2.249798 -0.761782 -1.866837

H27 2.225807 0.784093 -1.886317

18

[Rh(9S3)Cl2OH] geometry – HF/6-31G(d,p) Rh1 0.742335 0.003931 -0.279047

S2 -0.285637 -0.052985 2.043839

S3 -0.923634 1.717446 -0.817546

S4 -0.912229 -1.680291 -0.905701

C5 -1.652905 1.155023 1.887981

C6 -1.127576 -1.662157 1.896853

C7 -2.357641 0.726989 -1.372521

C8 -1.385400 2.254474 0.858358

C9 -1.912214 -1.913452 0.606836

C10 -1.946055 -0.621049 -1.963614

H11 -1.783397 1.625789 2.854134

H12 -2.569186 0.624324 1.670283

H13 -1.785365 -1.797462 2.746994

H14 -0.314343 -2.370788 1.986914

H15 -2.865988 1.294985 -2.141409

H16 -3.051871 0.609911 -0.552267

H17 -2.248471 2.906480 0.792813

H18 -0.535948 2.854059 1.158978

H19 -2.252326 -2.941429 0.614268

H20 -2.793216 -1.289891 0.552074

H21 -2.827285 -1.172170 -2.269261

H22 -1.313595 -0.475286 -2.830158

Cl23 2.237028 -1.742066 0.336867

Cl24 2.207494 1.787418 0.305011

O25 1.186042 -0.017700 -2.162698

H26 2.125433 -0.060455 -2.249418

[Rh(9S3)Cl2OH] geometry – mPW1PW91/6-31G(d,p) Rh1 0.646204 0.002702 -0.279935

S2 -0.113068 0.003829 1.981039

S3 -0.900370 1.653843 -0.827186

S4 -0.859652 -1.682955 -0.821559

C5 -1.523765 1.183522 1.899672

C6 -0.965237 -1.613672 1.963353

C7 -2.381466 0.657999 -1.274472

C8 -1.313471 2.258573 0.844922

C9 -1.816572 -1.894408 0.733096

C10 -1.978006 -0.683509 -1.863068

H11 -1.614444 1.660433 2.878611

H12 -2.445036 0.622885 1.727444

H13 -1.562885 -1.721570 2.872578

H14 -0.127448 -2.314616 2.016634

H15 -2.945166 1.223419 -2.020660

H16 -3.022375 0.546108 -0.397339

H17 -2.185236 2.915978 0.784602

H18 -0.437291 2.867077 1.085728

H19 -2.163558 -2.930201 0.768247

H20 -2.702630 -1.256898 0.699927

H21 -2.858006 -1.274535 -2.130088

H22 -1.361859 -0.541402 -2.756291

Cl23 2.209231 -1.680865 0.283504

Cl24 2.125958 1.793400 0.183288

O25 1.060442 -0.076175 -2.220768

H26 2.005539 -0.268472 -2.239356

19

[Rh(9S2O)Cl2OH] geometry – HF/6-31G(d,p) Rh1 -0.707634 -0.138075 -0.217891

O2 0.781784 1.182653 -1.159207

S3 0.485745 0.796942 1.839101

S4 1.169437 -1.722398 -0.422984

C5 1.503634 2.154540 -0.425244

C6 1.500872 0.444882 -2.136725

C7 2.045777 -0.162774 1.817024

C8 0.919414 2.339174 0.970032

C9 2.263673 -0.715334 -1.493208

C10 1.898428 -1.571031 1.238087

H11 1.453828 3.098629 -0.954928

H12 2.545368 1.859762 -0.368310

H13 2.185391 1.101958 -2.661580

H14 0.750694 0.076716 -2.817138

H15 2.383050 -0.256661 2.841541

H16 2.800540 0.402287 1.288665

H17 1.604309 2.923697 1.572301

H18 -0.026388 2.860005 0.915126

H19 2.644200 -1.364029 -2.271345

H20 3.109814 -0.375895 -0.909830

H21 2.863693 -2.063611 1.242080

H22 1.225502 -2.160058 1.849074

Cl23 -2.349384 1.566216 -0.087145

Cl24 -1.951638 -1.747955 0.952612

O25 -1.240671 -0.714988 -1.974658

H26 -2.181703 -0.670619 -2.040228

[Rh(9S2O)Cl2OH] geometry – mPW1PW91/6-31G(d,p) Rh1 0.639955 -0.010178 -0.236540

O2 -0.397433 0.065584 1.781313

S3 -0.925311 1.652377 -0.765364

S4 -0.920231 -1.698445 -0.681394

C5 -1.387826 1.078021 1.949182

C6 -0.834501 -1.263120 2.065844

C7 -2.442821 0.663619 -1.096517

C8 -1.169166 2.204416 0.958126

C9 -1.727886 -1.831227 0.964596

C10 -2.110719 -0.710776 -1.654660

H11 -1.315420 1.478680 2.966446

H12 -2.389158 0.646342 1.836737

H13 -1.360962 -1.285886 3.026911

H14 0.086949 -1.842163 2.143176

H15 -3.038745 1.213783 -1.828881

H16 -3.034550 0.596778 -0.181425

H17 -1.988060 2.926218 1.010762

H18 -0.223347 2.713878 1.163414

H19 -1.905898 -2.890843 1.160447

H20 -2.702275 -1.339149 0.920850

H21 -3.022728 -1.293126 -1.812273

H22 -1.593506 -0.620237 -2.614164

Cl23 2.154473 -1.682118 0.430467

Cl24 2.139180 1.737278 0.250408

O25 1.166277 -0.136878 -2.109772

H26 2.113054 -0.322106 -2.066154

20

[Ir(9S3)Cl2OH] geometry – HF/6-31G(d,p) Ir1 0.592890 0.002709 -0.223332

S2 -0.349740 -0.042512 2.047357

S3 -1.001984 1.696667 -0.795141

S4 -0.989474 -1.671733 -0.863226

C5 -1.722953 1.164968 1.911788

C6 -1.206415 -1.647207 1.936918

C7 -2.445800 0.716854 -1.348945

C8 -1.474733 2.257383 0.870121

C9 -1.991883 -1.909664 0.649326

C10 -2.039419 -0.637326 -1.929602

H11 -1.834352 1.638941 2.878582

H12 -2.641676 0.630933 1.712728

H13 -1.864610 -1.758074 2.790338

H14 -0.398926 -2.360744 2.038291

H15 -2.945336 1.286134 -2.122386

H16 -3.142665 0.609654 -0.529275

H17 -2.348196 2.894175 0.793699

H18 -0.633408 2.872576 1.161670

H19 -2.323895 -2.940098 0.658250

H20 -2.876533 -1.291201 0.592398

H21 -2.921216 -1.196578 -2.218276

H22 -1.411759 -0.500744 -2.801253

Cl23 2.128613 -1.750755 0.394923

Cl24 2.099216 1.794244 0.362185

O25 1.069450 -0.020889 -2.144191

H26 2.007749 -0.054594 -2.237011

[Ir(9S3)Cl2OH] geometry – mPW1PW91/6-31G(d,p) Ir1 0.542574 0.000158 -0.210142

S2 -0.276774 -0.003074 1.987235

S3 -0.951262 1.647017 -0.814150

S4 -0.931113 -1.661301 -0.817526

C5 -1.682510 1.190516 1.884462

C6 -1.157679 -1.608743 1.962481

C7 -2.432960 0.675697 -1.327692

C8 -1.440365 2.264004 0.836612

C9 -1.955763 -1.887648 0.697733

C10 -2.024787 -0.671554 -1.898355

H11 -1.784230 1.662639 2.864212

H12 -2.603871 0.636652 1.691385

H13 -1.797637 -1.687224 2.845430

H14 -0.336662 -2.323835 2.064650

H15 -2.950193 1.253661 -2.096993

H16 -3.112272 0.574359 -0.478416

H17 -2.315338 2.911281 0.732504

H18 -0.580547 2.882328 1.109332

H19 -2.296552 -2.925601 0.707687

H20 -2.841357 -1.252146 0.629114

H21 -2.900661 -1.263660 -2.175725

H22 -1.388822 -0.540020 -2.779257

Cl23 2.118066 -1.691778 0.385808

Cl24 2.070649 1.763640 0.306944

O25 1.075267 -0.058839 -2.148959

H26 2.030386 -0.187489 -2.138503

21

Table A. Zero-point-exclusive energy of reaction (E) and zero-point-inclusive energy of

reaction (H0) in kcal/mol for various chloride replacements reactions calculated at

HF/6-31G(d,p) and mPW1PW91/6-31G(d,p) levels of theorya

Reaction and Theoretical Method E H0

HF/6-31G(d,p)

[Rh(9S3)Cl3] + H2O [Rh(9S3)Cl2H2O]+ + Cl– 93.8 96.6

[Rh(9S2O)Cl3] + H2O [Rh(9S2O)Cl2H2O]+ + Cl– 93.9 96.8

[Ir(9S3)Cl3] + H2O [Ir(9S3)Cl2H2O]+ + Cl– 93.2 96.0

[Rh(9S3)Cl3] + H2O [Rh(9S3)Cl2OH] + HCl 18.3 16.6

[Rh(9S2O)Cl3] + H2O [Rh(9S2O)Cl2OH] + HCl 16.1 14.5

[Ir(9S3)Cl3] + H2O [Ir(9S3)Cl2OH] + HCl 19.2 17.6

mPW1PW91/6-31G(d,p)

[Rh(9S3)Cl3] + H2O [Rh(9S3)Cl2H2O]+ + Cl– 99.9 102.5

[Rh(9S2O)Cl3] + H2O [Rh(9S2O)Cl2H2O]+ + Cl– 99.7 102.4

[Ir(9S3)Cl3] + H2O [Ir(9S3)Cl2H2O]+ + Cl– 99.9 102.6

[Rh(9S3)Cl3] + H2O [Rh(9S3)Cl2OH] + HCl 18.5 16.8

[Rh(9S2O)Cl3] + H2O [Rh(9S2O)Cl2OH] + HCl 15.9 14.2

[Ir(9S3)Cl3] + H2O [Ir(9S3)Cl2OH] + HCl 18.3 16.6

a The basis set is for all atoms except the central metal, Rh or Ir, for which LANL2DZ basis set

was used.

Table B. Dipole moments calculated at HF/6-31G(d,p) and mPW1PW91/6-31G(d,p) levels of

theorya

Complex HF/6-31G(d,p) mPW1PW91/6-31G(d,p)

[Rh(9S3)Cl3] 18.6 16.6

[Rh(9S2O)Cl3] 18.0 16.0

[Ir(9S3)Cl3] 19.2 16.9

[Rh(9S3)Cl2OH] 16.2 14.4

[Rh(9S2O)Cl2OH] 15.5 14.1

[Ir(9S3)Cl2OH] 16.7 14.7

a The basis set is for all atoms except the central metal, Rh or Ir, for which LANL2DZ basis set

was used.