Ru(II) COMPLEXES AS PHOTOACTIVATED CISPLATIN ANALOGS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Tanya N. Singh, M.S.

* * * * *

The Ohio State University 2006

Dissertation Committee: Approved by Professor Claudia Turro, Adviser

Professor Malcolm Chisholm ______Professor Yiying Wu Adviser Graduate Program in Chemistry

ABSTRACT

Cisplatin is an anticancer drug that is used in the treatment of various cancers;

however, its toxicity towards healthy cells and its acquired resistance are problems that

need to be overcome. Photodynamic therapy (PDT) is a treatment that combines the

action of a light source and a photosensitizing agent to kill cancerous cells. Typical PDT

methods require the photoreaction of singlet oxygen through energy transfer from an

excited state, which results in oxidative damage to the cells. The need for molecular

oxygen in the vicinity of the drug is a major drawback, since malignant and drug resistant

cells are often hypoxic. Hence the design and DNA interactions of Ru(II) complexes that

combines the mode of action of cisplatin with PDT will be investigated.

2+ 2+ Cis-[Ru(bpy)2(NH3)2] and cis-[Ru(bpy)2(CH3CN)2] were synthesized and their

DNA binding to single stranded and double stranded DNA and to the DNA bases; 9-

ethylyguanine and 9-methylguanine were investigated. Irradiating cis-

2+ [Ru(bpy)2(CH3CN)2] in water resulted in the formation of the bisaqua complex, cis-

2+ [Ru(bpy)2(OH)2] with quantum yields of φ350 = 0.377(3), φ400 = 0.207(1), φ450 =

2+ 0.222(18) whereas irradiation of cis-[Ru(bpy)2(NH3)2] in water resulted in quantum

yields of φ350 = 0.024(2) and φ400 = 0.018. The higher quantum yield of photoaqaution

2+ observed upon irradiation of cis-[Ru(bpy)2(CH3CN)2] resulted in greater covalent DNA

binding to both single and double stranded DNA. Binding of both complexes to 9-

ii ethylguanine and 9-methyl guanine was also observed upon irradiation but not in the

dark.

+ + The photolysis of cis-[Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpy)CH3CN)4] in

(II),(II) aqueous solution with λirr> 420 nm results in the formation of μ-oxo dimers of Ru of

II II + molecular formula [(H2O)(bpy)(phpy)Ru ORu (phpy)(bpy)(OH2)]H .H2O and

(II) (II) + [(CH3CN)(H2O)(phpy)Ru ORu (phpy)(H2O)(CH3CN)]H .H2O respectively. Cis-

+ + [Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpy)CH3CN)4] bind to 9-ethylguanine and 9-

+ methylguanine upon irradiation. Binding of [Ru(phpy)CH3CN)4] to single stranded 15

mer DNA sequences was observed upon irradiation with λirr> 420 nm, however, no

binding to single stranded DNA was observed upon irradiation of cis-

+ [Ru(phpy)(bpy)(CH3CN)2] . No binding to double stranded DNA was observed upon

+ + irradiation of cis-[Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpy)(CH3CN)4] with λirr> 420 nm.

Compound containing oxygen in the coordination sphere of ruthenium was studied as PDT agents. Cis-[Ru(quo)2(py)2] was observed to bind to 15-mer DNA sequences upon irradiation in aqueous solution with λirr > 345 nm. Binding to duplex

DNA was observed upon irradiation of cis-[Ru(quo)2(py)2] with λirr> 345 nm. Irradiation

of cis-[Ru(quo)2(py)2] with 395 nm < λirr < 420 nm also resulted in the metal complex binding to DNA, however, to a lesser extent. Theses results provide the way for the development of new metal complexes that can be better utilized to achieve the wavelength required for PDT.

iii

Dedicated to my Family, especially to my Loving Mother who passed away on June 30, 2003.

iv

ACKNOWLEDGMENTS

The encouragement, advice and support from various people have this dissertation

possible, without them there is no way that I would have been able to have made such an

accomplishment.

First and foremost, I want to express my greatest appreciation to my adviser, Dr.

Claudia Turro, for her patience, guidance and support in making this dissertation possible

and for spending her timeless effort in correcting and making suggestions to improve my

scientific writing. She always instills in me and my group members to think

independently and critically and to try and solve problems concerning our research,

which are major components of attaining this level of confidence which is worthwhile for

achieving this degree and for that I will always be thankful.

I also want to thank Dr. Latif Chouai from Dr. Kim Dunbar’s group at Texas A &

M University who synthesized some of the compounds that I worked with for my

dissertation.

I also want to thank Dr. Yoa Lui for intelligent discussions and for teaching and helping me to understand and do electrochemical measurements, low temperature emission and lifetime measurements. He was always willing to help me whenever I asked for help.

v I have to say a big thank you to my family and friends who have supported me in

my decision to continue graduate school and for there encouragement and kind words

when things got rough especially having to deal with the death of my Mom during my

third year here at OSU and my Dad having a heart attack shortly after that. The one thing

that my Mom had always longed for and hoped for was to be there when I graduate with

my PhD. Today she is not here in flesh but I know that her spirit is here and will always

be with me and she will forever hold a special place in my heart. I am so grateful to my

sister, Nardia, and brother, Garry who always seems to be the stronger one emotionally

even though they are the youngest and for helping me to deal with hard times and for my

Dad who always puts on a brave face to put a smile on my face in difficult times.

Lastly, I would like to thank Aaron Rachford for his patience and understanding

in listening to my research frustrations and for his suggestions. He was always kind and

very warm hearted and always takes the time to listen to me without any complaint even though I am sure he must have been dealing with his own research frustrations because being a graduate student himself, it is almost impossible to not have any research frustrations. Throughout all this, he always cares and supports me in my decisions.

vi

VITA

March 21, 1977…………………………. Born - St. Elizabeth, Jamaica

1995-1999………………………………... B.S. Chemistry and Biochemistry,

University of the West Indies (UWI)

1999-2000……………………………….... Organic Chemistry Graduate Student (UWI)

2000-2002 M.S. Chemistry, The Ohio State University

2000-Present………………………….…… Graduate Teaching and Research Associate, The Ohio State University

PUBLICATIONS

Research Publication

1. Photoinduced DNA Binding by a Ru(II) Complex: A Photo-Cisplatin Analog" Tanya N. Singh and Claudia Turro, Inorg. Chem., 2004, 43, 7260-7262.

FIELDS OF STUDY

Major Field: Chemistry

vii

TABLE OF CONTENTS

Abstract...... ii Acknowledgments...... v Vita ...... vii List of Tables ...... xi List of Scheme ...... xiii List of Figures...... xiv Chapters 1. Introduction...... 1 1.1. Cisplatin ...... 1 1.2. Interaction of cisplatin with DNA...... 2 1.3. Photodynamic Therapy ...... 4 References...... 10

2. Background...... 14 2.1. Photophysical Properties and Photochemistry of Ru(II) polypyridyl complexes ...... 14 2.2. Photochemistry of Ruthenium Ammine Complexes ...... 21 2.3. Covalent DNA Binding...... 29 References...... 34

3. Experimental Section...... 39 3.1. Materials...... 39 3.2. Instrumentation ...... 44 3.3. Methods...... 46 viii References...... 50

2+ 2+ 4 Photoreactivity of cis-[Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] ……...51 4.1. Electronic Absorption and Emission………………………………………..51 4.2. Photochemistry in Solution...... 52

4.2.1. Photochemistry in H2O ...... 52 4.2.2. Photochemistry with other ligands...... 75 4.2.3. Photoinduced Binding to DNA Bases...... 82 4.2.4. Binding to Double Stranded DNA...... 94

4.3 Conclusion ...... 105

References………………………………………………………………………107

+ 4. Photochemistry and DNA Studies of cis-[Ru(phpy)(bpy)(CH3CN)2] and cis- + [Ru(phpy)(CH3CN)4] ...... 110

5.1. Introduction...... 110 5.2. Background...... 111 5.3. Results and Discussions...... 114 5.3.1. Photophysical Properties...... 114 5.3.2. Photochemistry in H2O ...... 117 + 5.3.3. Photochemistry of cis-[Ru(phpy)(bpy)(CH3CN)2] in CH3CN ...... 123 + 5.3.4. Photochemistry of cis-[Ru(phpy)(bpy)(CH3CN)2] in MeOH ...... 127 5.3.5. Reaction with bpy and py ligands...... 129 5.3.6. Reaction with 9-MG and 9-EG ...... 133 5.3.7. Reaction with oligonucleotide ...... 136 5.3.8. Binding to double stranded DNA ...... 141 5.4. Conclusion...... 147 References...... 150

+ complexes and the 6. Photophysical and electronic properties of [Ru(bpy)2(L)] photoinitiated DNA binding of [Ru(quo)2(py)2]...... 15 6.1. Introduction...... 153 6.2. Background...... 154 6.3 Results and Discussion ...... 157 + 6.3.1. Electronic and Photophysical properties of [Ru(bpy)2(L)] ...... 157

6.4. Results and Discussions of [Ru(quo)2(py)2] ...... 161 ix 1 6.4.1. Interpretation of H NMR spectra of [Ru(quo)2(py)2] ...... 161

6.4.2. Electronic Properties of [Ru(quo)2(py)2] ...... 163

6.4.3. Photochemistry of [Ru(quo)2(py)2] in H2O...... 164

6.4.4. Photoinitiated Binding of [Ru(quo)2(py)2] to single stranded oligonucleotide...... 165

6.4.5 Photoinitiated Binding of [Ru(quo)2(py)2] to double stranded oligonucleotide...... 167

6.5. Conclusion ...... 172 References...... 174

Complete Bibliography...... 175

x

LIST OF TABLES

Table Page

2.1 Charge Transfer Maxima, Emission Maxima, E1/2(II/III), and Quantum Yield for the n+ Photoanation of cis-[Ru(bpy)2XY] in CH2Cl2 with (C4H9)4NCl…………………18

2.2 Luminescence Quantum Yields, Excited State Lifetimes, and Quantum Yields of Photoanation of Cl- for Ru(II) Complexesa………………………………………...20

2.3 Photosubstitution Quantum Yields, Lowest Energy Absorption Maximum (λmax) of 2+ [Ru(NH3)5(L)] Complexes in Aqueous Media with the Wavelength of Irradiation

(λirr)…………………………………………………………………………………23

2.4 Photosubstitution Quantum Yields and Electronic Absorption Data of cis- 2+ [Ru(NH3)4LL’] Complexes in Aqueous Media.b………………………………...27

2+ 4.1 Low temperature emission (77 K) data collected for cis-[Ru(bpy)2(CH3CN)2] and 2+ cis-[Ru(bpy)2(NH3)2] compared to other known Ru(II) compounds……………...57

2+ 4.2 Room temperature emission data collected for cis-[Ru(bpy)2(CH3CN)2] and cis- 2+ [Ru(bpy)2(NH3)2] compared to other known Ru(II) compounds…………………58

4.3 Absorption maxima of MLCT transition, quantum yield of photoanationc with Cl- (tetrabutyl ammonium chloride) anion and E1/2 (II/III) values obtained for cis- 2+ 24 [Ru(bpy)2(L)2] complexes ………………………………………………………65

2+ 4.4 Quantum yield of ammonia photosubstitution of cis-[Ru(bpy)2(NH3)2] and related compounds at various wavelengths………………………………………………...71

4.5 1H NMR chemical shifts obtained for the reactions of 5 mM of cis- 2+ [Ru(bpy)2(CH3CN)2] at λirr > 420 nm for 6 hr in the presence of 2.5 eq. of 9-EG

and 9-MG in D2O to those previously reported…………………………………….84 xi 4.6 Thermal denaturation results for Covalent Adducts of Ru(II) Aqua complexes with calf thymus DNA…………………………………………………………………...96

6.1 Absorption (λabs) and Emission (λem) maxima, Estimated Excited-State Energy (E00) + and Ground State Oxidation Potentials (vs NHE) of the [Ru(L)2quo] complexes in H2O………………………………………………………………………………..155

+ 6. Absorption maxima of the [Ru(bpy)2(L)] complexes in CH3CN……………...... 158

1 6.3 H NMR chemicals shifts of [Ru(quo)2(py)2] and [Sn(quo)2] obtained in d6- DMSO…………………………………………………………………………….162

xii

LIST OF SCHEMES

Scheme Page

2+ 4.1 . Photolysis of cis-[Ru(bpy)2(L)2] (L = CH3CN, NH3) in water……………….63

2+ 4.2 Proposed mechanism for the photolysis of cis-[Ru(bpy)2(L)2] (L = CH3CN,

NH3) in the presence of 1 eq of bpy ligand in dichloromethane………………...79

2+ 4.3 The reaction scheme of of cis-[Ru(bpy)2(L)2] (L = CH3CN, NH3) with guanine

in D2O irradiating with λirr > 420 nm and λirr > 345 nm…………………………83

+ 5.1 Reaction Schemes for the reaction of cis-[Ru(phpy)(bpy)(CH3CN)2] ………120

xiii

LIST OF FIGURES

Figure Page

1.1 Sequential aquation of the chloride ligands of cisplatin resulting in the formation 2+ of the active species cis-[Pt(NH3)2(OH2)2] , along with subsequent deprotonation steps………………………………………………………………………………..3

1.2 Formation of the products from cisplatin with DNA: (a) intrastrand and (b) interstrand crosslink of cisplatin with ds-DNA...... 3

1.3 The molecular structure of hematoporphyrin……………………………………..5

1 1.4 Schematic representation of the production of O2 by typical PDT agents, where 1S represents the molecule in its ground state, 1*S represents the singlet excited state, and 3*S is the triplet excited state of the molecule following intersystem crossing (ISC). ……………………………………………………………………6

2.1 Schematic representation of the processes that occur upon excitation of Ru(II) complex with a photon of light (hν)……………………………………………..15

2.2 Schematic representation of the “tuning model” proposed by Ford:33 (a) “reactive complexes”; (b) “unreactive complexes”………………………………………...28

2.3 Structure of the DNA bases (a) guanine and (b) adenine………………………..29

xiv 2+ 2+ 4.1 Uv-vis spectra of the cis-[Ru(bpy)2(CH3CN)2] (⎯ ) and cis-[Ru(bpy)2(NH3)2]

(− − − −) in H2O…………………………………………………………………...52

4.2 Excitation (λem = 542 nm, ----) and emission (λexc = 420 nm, ⎯) spectra of cis- 2+ [Ru(bpy)2(CH3CN)2] at 77 K in (4:1 v:v) EtOH:MeOH……………………….53

2+ 4.3 Emission spectrum of cis-[Ru(bpy)2(NH3)2] (λexc = 450 nm) in water………....54

4.4 Changes to the electronic absorption spectrum of cis-[Ru(bpy)2(CH3CN)2](PF6)2

in water during photolysis (λirr > 420 nm) in water at t = 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 min……………………………………………………………………59

2+ 4.5 Photolysis of cis-[Ru(bpy)2(NH3)2] in H2O (λirr > 375 nm) at (a) showing early times 0, 1,2, 3, 4, 5 min and (b) showing later times 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 min…………………………………………………………….………….62

2+ 4.6 Cyclic voltammogram of cis-[Ru(bpy)2(CH3CN)2] in 0.1 M TBAPF6 in

deaerated CH3CN at a Pt electrode at 50 mV / sec. * denotes impurity that may have been in the sample………………………………………………………….69

2+ 4.7 Cyclic voltammogram of cis-[Ru(bpy)2(CH3CN)2] in 0.1 M TBAPF6 upon the

addition of excess pyridine in deaerated CH3CN at a Pt electrode at 50 mV / sec. * 2+ indicates the formation of cis-[Ru(bpy)2(py)2] ………………………………...69

4.8 Changes observed in the electronic absorption spectrum of cis-

[Ru(bpy)2(CH3CN)2](PF6)2 with (a) excess bpy ligand in CH2Cl2, λ > 420 nm.for t

= 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 min. (b) with 2 eq of TBACl in CH2Cl2, λ > 420 nm.for t = 0, 2, 4, 6, 8, 10, 12, 14,16, 18, 20 min…………………………...76

xv 2+ 4.9 Photolysis of cis-[Ru(bpy)2(NH3)2] and 1 eq of bpy ligand in CH2Cl2, λirr > 455 nm for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 min………………….77

2+ 4.10 Changes in the absorption of cis-[Ru(bpy)2(NH3)2] at (a) 490 nm and (b) at 453 nm in the presence of 1 eq. bpy ligand in dichloromethane as a function of irradiation time at λ > 455 nm and in the dark…………………………………..78

2+ 4.11 Emission spectra of cis-[Ru(bpy)2(NH3)2] + 1 equivalent of bpy ligand in

CH2Cl2 (λexc = 450 nm) for 2 minutes interval up to 12 minutes………………...80

2+ 4.12 Absorption spectra of 25 μM of cis-[Ru(bpy)2(NH3)2] with 50 μM of TBACl in

CH2Cl2 (λexc > 375 nm) for 5 minutes interval up to 35 minutes………………...82

1 2+ 4.13 H NMR spectra of the aromatic region of the reaction of cis-[Ru(bpy)2(NH3)2]

with (a) 9-EG and (b) 9-MG in D2O when irradiated at λirr > 345 nm…………..85

2+ 4.14 MALDI mass spectroscopy of the reaction of cis-[Ru(bpy)2(CH3CN)2] with 2.5

eq of (a) 9-EG, λirr > 420 nm for over 24 hours (b) 9-MG, λirr > 420 nm for 36

hours in D2O……………………………………………………………………..87

2+ 4.15 Electrospray mass spectroscopy of the reaction of cis-[Ru(bpy)2(NH3)2] with (a)

9-MG and (b) 9-EG in D2O when irradiated at λirr > 345 nm for 18 hours……...88

2+ 4.16 MALDI mass spectrometry of the reaction of cis-[Ru(bpy)2(CH3CN)2] with the

15-mer oligonucleotide, 5’-AGTGCCAAGCTTGCA-3’, λirr > 420 nm for15 min in 5 mM Tris, 50 mM NaCl, pH = 7.5…………………………………………90

4.17 Electrospray ionization mass spectra of solutions containing 10μM of cis- 2+ [Ru(bpy)2(NH3)2] with 10μM of single stranded DNA (a) strand 1 (b) strand 2 xvi when irradiated with λirr > 345 nm in 5 mM Tris Buffer (pH = 7.5 mM, 50 mM NaCl)……………………………………………………………………………..92

4.18 Ethidium bromide stained agarose gels of 50 μM linearised pUC18 ( 10 mM phosphate, pH = 7.5) in the presence of various ratios of (a) cisplatin incubated o 2+ for 4 hr at 37 C, (b) cis-[Ru(bpy)2(CH3CN)2] irradiated (λirr > 420 nm) for 15 o 2+ min at 25 C. and (c) cis-[Ru(bpy)2(NH3)2] irradiated (λirr > 345 nm) for 15 min at 25 oC. Lanes 1 and 8: DNA molecular weight standard (1 kb). Lanes 2 and 7: Linearized plasmid alone, Lanes 3-6: [DNA bp]/[Complex] = 100, 20, 10, 5…..98

4.19 Ethidium bromide stained agarose gels of 50 μM linearized pUC18 (10 mM phosphate, pH = 7.5) in the presence of various ratios of (a)cis- 2+ [Ru(bpy)2(CH3CN)2] kept in the dark for 20 minutes and (b) cis- 2+ [Ru(bpy)2(NH3)2] kept in the dark for 30 minutes. Lanes 1 and 8: DNA molecular weight standard (1 kb). Lanes 2 and 7: Linearized plasmid alone, Lanes 3-6: [DNA bp]/[Complex] = 100, 20, 10, 5…………………………………...100

1/3 4.20 Relative viscosity measurements of the plot of (ηDNA / ηo) vs R = [probe]:[DNA] 2+ for ethidium bromide (●), hoecht (x), cisplatin (▲), cis-[Ru(bpy)2(CH3CN)2] , 2+ λirr> 420 nm for 15 min (▼), cis-[Ru(bpy)2(CH3CN)2] dark (■),cis- 2+ 2+ [Ru(bpy)2(NH3)2] , λirr> 345 nm for 15 min (♦), cis-Ru(bpy)2(NH3)2 dark (∗)……………………...103

+ 5.1 UV-vis spectrum of cis-[Ru(phpy)(bpy)(CH3CN)2] (⎯ ⎯) and + [Ru(phpy)(CH3CN)4] (…..) in H2O……………………………………………115

5.2 Emission (λexc = 480 nm, ⎯) and excitation (λexc = 790 nm, ----) spectra of cis- + [Ru(phpy)(bpy)(CH3CN)2] in H2O at room temperature………………………116

xvii + 5.3 Low temperature emission spectra of cis-[Ru(phpy)(bpy)(CH3CN)2] (⎯ ⎯) and + [Ru(phpy)(CH3CN)4] (…..) EtOH:MeOH (4:1 v/v) at 77 K…………………..117

+ 5.4 Absorption spectrum of the photolysis reaction of cis-[Ru(phpy)(bpy)(CH3CN)2]

in H2O with irradiated with λ > 345 nm at t = 0, 10, 20, 30, 40, 40, 60, 70 min up to 4 hours with spectrum taken every 10 minutes……………………………...118

5.5 Overlaid aromatic region of the 1H NMR spectra of the reaction of cis- + [Ru(phpy)(bpy)(CH3CN)2] in D2O when irradiated with λ > 345 nm for (a) 0 min (b) 4.5 hours and (c) 6.5 hours………………………………………………….120

+ 5.6 Absorption spectrum of the photolysis reaction of cis-[Ru(phpy)(CH3CN)4] in

H2O with irradiated with λ > 345 nm at t = 0, 10, 20, 30, 40, 40, 60, 70 min up to 2 hours with spectrum taken every 10 minutes…………………………………123

5.7 Overlaid aromatic region of the 1H NMR spectra of the reaction of cis- + [Ru(phpy)(CH3CN)4] in D2O when irradiated with λ > 345 nm for (a) 0 min (b) 1hr and (c) 6 hrs………………………………………………………………...124

+ 5.8 Absorption spectrum of the photolysis reaction of cis-[Ru(phpy)(bpy)(CH3CN)2]

in CH3CN with irradiated with λ > 420 nm at t = 5, 10, 15, 20, 25, 30, 35, 40 min up to 2 hours with spectrum taken every 5 minutes…………………………….125

5.9 Overlaid aromatic region of the 1H NMR spectra of the reaction of cis- + [Ru(phpy)(bpy)(CH3CN)2] in CH3CN when irradiated with λ > 420 nm for (a) 0 min (b) 1 hours and (c) ~24 hours. * denotes protons from unreacted cis- + [Ru(phpy)(bpy)(CH3CN)2] …………………………………………………….126

xviii 5.10 Time of flight electrospray ionization mass spectrometry of the reaction of cis- + [Ru(phpy)(bpy)(CH3CN)2] in CH3CN when irradiated with λ > 420 nm for ~24 hours…………………………………………………………………………….127

5.11 Overlaid aromatic region of the 1H NMR spectra of the reaction of cis- + Ru(phpy)(bpy)(CH3CN)2 in CD3OD (a) t = 0 min (b)irradiated with λ > 420 nm for 20 min……………………………………………………………………….128

5.12 Absorption spectrum of the photolysis reaction of (a) cis- + + [Ru(phpy)(bpy)(CH3CN)2] and (b) [Ru(phpy)(CH3CN)4] with 1 eq of bpy ligand

in CH2Cl2, irradiated with λ > 420 nm for t = 0, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60 min……………………………………………………………………………...131

5.13 Absorption spectrum of the photolysis reaction of (a) cis- + [Ru(phpy)(bpy)(CH3CN)2] with excess py in CH2Cl2, irradiated with λ > 420 nm for t = 0, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60 min………………………………….133

5.14 Overlaid aromatic region of the 1H NMR spectra of the reaction of (a) cis- + + [Ru(phpy)(bpy)(CH3CN)2] in D2O (b) cis-[Ru(phpy)(bpy)(CH3CN)2] + 9-MG, t

= 0 min (c) λirr > 420 nm for 3 hrs (d) λirr > 420 nm for 5 hrs …………………135

5.15 MALDI mass spectrometry results of the product of the photolysis reaction of 5 + mM of cis-[Ru(phpy)(bpy)(CH3CN)2] with 2.5 eq of 9-MG, λirr > 420 nm for 24 hours…………………………………………………………………………….136

5.16 Overlaid aromatic region of the 1H NMR spectra of the reaction of (a) 5 mM cis- + [Ru(phpy)(CH3CN)4] + 2.5 eq of 9-EG in D2O, t = 0 min (b) t = 1 hr, λirr > 420

nm (c) t = 3.5 hr λirr > 420 nm (d) t = 6 hr λirr > 420 nm. * denotes H8 proton of free guanine……………………………………………………………………..137

xix 5.17 MALDI mass spectrometry results of the reaction of cis- + [Ru(phpy)(bpy)(CH3CN)2] with the 15-mer oligonucleotide sequences (a) 5’- TGCAAGCTTGGCACT-3’ (strand 1, S1) and (b) 5’- AGTGCCAAGCTTGCA-

3’ (strand 2, S2) after irradiation with λirr > 420 nm for 30 mins………………138

+ 5.18 MALDI mass spectrometry results of the reaction of [Ru(phpyCH3CN)4] with the 15-mer oligonucleotide sequences (a) 5’- TGCAAGCTTGGCACT-3’ (strand 1, S1) and (b) 5’- AGTGCCAAGCTTGCA-3’ (strand 2, S2) after irradiation with

λirr > 420 nm for 30 mins……………………………………………………….140

5.19 Ethidium stained agarose gel electrophoresis of 50 μM linearized plasmid (10 mM phosphate buffer, pH 7.5) in the presence of various ratios of (a) cisplatin, + incubated for 3 hours (b) cis-[Ru(phpy)(bpy)(CH3CN)2] , λirr> 420 nm for 30 min + (c) [Ru(phpy)(CH3CN)4] , λirr> 420 nm for 30 min. Lanes 1 and 8: DNA molecular weight standard, Lanes 2 and 7: linearized plasmid only; Lanes 3-6 [DNA bp] : [Complex] = 100, 20, 10, 5………………………………………..143

5.20 Ethidium stained agarose gel electrophoresis of 50 μM linearized plasmid (10 mM phosphate buffer, pH 7.5) in the presence of various ratios of (a) cis- + + [Ru(phpy)(bpy)(CH3CN)2] and (b) [Ru(phpy)(CH3CN)4] in the dark. Lanes 1 and 8: DNA molecular weight standard, Lanes 2 and 7: linearized plasmid only; Lanes 3-6 [DNA bp] : [Complex] = 100, 20, 10, 5……………………………..145

1/3 5.21 Relative viscosity measurements of the plot of (ηDNA / ηo) vs R = [probe]:[DNA] for ethidium bromide (●), hoecht (x), cisplatin (▲), cis- + + [Ru(phpy)(bpy)(CH3CN)2] , λirr> 420 nm for 30 min (♦) [Ru(phpy)(CH3CN)4] ,

λirr> 420 nm for 30 min(*)……………………………………………………...146

xx 2+ 6.1 Simplified molecular orbital (a) and energy state (b) diagram of [Ru(bpy)3] and + [Ru(bpy)2(quo)] showing the electronic configuration and state energies relative to each other…………………………………………………………………….154

+ 6.2 Structures of the ligands of [Ru(bpy)2(L)] ...... 157

+ 6.3 Uv-vis absorption spectra of [Ru(bpy)2(2-aminophenol)] (⎯), [Ru(bpy)2(3- + + amino-2-naphthol)] (⎯ ⎯ ), [Ru(bpy)2(8-hydroxyquinoline)] (− − −) and + [Ru(bpy)2(8-quinolinethiol)] (…..) in CH3CN…………………………………158

6.4 Room temperature () and low temperature (77K) (….) emission (λexc = 500 nm) + and excitation spectra(λmax emis) of (a) [Ru(bpy)2(2-aminophenol)] , (b) + + [Ru(bpy)2(3-amino-2-naphthol)] and (c) [Ru(bpy)2(8-hydroxyquinoline)] …..160

1 6.5 H NMR spectrum of [Ru(quo)2(py)2] in d6-DMSO……………………………162

6.6 Uv-vis absorption spectrum of [Ru(quo)2(py)2] in H2O………………………..163

6.7 Uv-vis absorption spectra of [Ru(quo)2(py)2] in H2O, λirr > 345 nm for t = 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 min…………………………………..164

6.8 MALDI mass spectrometry results of the reaction of [Ru(quo)2(py)2] with the 15- mer oligonucleotide sequences (a) 5’- TGCAAGCTTGGCACT-3’ (strand 1, S1)

and (b) 5’- AGTGCCAAGCTTGCA-3’ (strand 2, S2) after irradiation with λirr > 345 nm for 30 mins……………………………………………………………..166

6.9 Ethidium stained agarose gel electrophoresis of 50 μM linearized plasmid (10 mM phosphate buffer, pH 7.5) in the presence of various ratios of (a)cisplatin, xxi o incubated for 3 hours (b) [Ru(quo)2(py)2], λirr> 345 nm for 30 min at 25 C: Lanes 1 and 8: DNA molecular weight standard, Lanes 2 and 7: linearized plasmid only; Lanes 3-6 [DNA bp] : [Complex] = 100, 20, 10, 5……………...168

6.10 Ethidium stained wavelength dependence agarose gel electrophoresis of 50 μM linearized plasmid (10 mM phosphate buffer, pH 7.5) in the presence of various

ratios of [Ru(quo)2(py)2] (a) λirr > 395 nm and (b) λirr > 420 nm (c) dark. Lanes 1 and 8: DNA molecular weight standard, Lanes 2 and 7: linearized plasmid only; Lanes 3-6 [DNA bp] : [Complex] = 100, 20, 10, 5……………………………..169

6.11 Relative viscosity measurements of ethidium bromide (●), hoecht (x), cisplatin

(▼), [Ru(quo)2(py)2] (λirr> 345 nm, 15 min) (♦) and Ru(quo)2(py)2 dark (■)….171

xxii

CHAPTER 1

INTRODUCTION

1.1 Cisplatin

Cisplatin, cis-[Pt(NH3)2Cl2], first synthesized by M. Peyrone in 1847, is

one of the most effective and potent anticancer drugs.1 In the 1960’s Barnett

Rosenberg serendipitously discovered its chemotherapeutic cancer activity.1,2

Cisplatin was further shown by Hill et al in 1971 to be active against malignant lymphoma, Hodgson’s disease, and certain other cancers.3 Cisplatin has also been

used in the treatment of epithelial malignancies such as lung, head and neck,

ovarian, bladder, and testicular cancers.4,5 It is interesting to note that its

stereoisomer, trans-[Pt(NH3)2Cl2] (transplatin), does not exhibit the anticancer

activity of cisplatin. The difference between the two isomers is the ability of

cisplatin to form intra-strand DNA crosslinks upon binding to double-stranded

DNA, whereas transplatin is incapable of forming cross links that block

replication.6 The continued clinical use of cisplatin is hampered by its severe

adverse reactions.1,2 Other drawbacks of cisplatin include its acquired cellular

resistance and its toxicity towards healthy cells.2 Therefore, there is the need to

1 discover new compounds with antitumor activity that circumvent these problems.7- 9

1.2 Interaction of cisplatin with DNA

The mode of action of cisplatin has been shown to depend on hydrolysis,

where each chloride ion is replaced by a water molecule, resulting in a positively

2+ charged molecule, cis-[Pt(NH3)2(OH2)2] . The sequential thermal ligand loss of

the labile chloride groups and substitution by water molecules to generate the

active species is shown in Figure 1.1.10 The hydrolyzed product, believed to be

the active species, reacts mainly with gluthathione in the cytoplasm and with

DNA in the nucleus, thus inhibiting replication, transcription, and other nuclear

functions and arresting proliferation and tumor growth.4,5 Cis-

2+ [Pt(NH3)2(OH2)2] , has been shown to bind specifically to the N7 position of

guanine and adenine bases of ds-DNA, thereby forming 1,2 intrastrand crosslinks

(Figure 1.2).11 Cisplatin is also known to form minor products such as interstrand

crosslinks to guanine and adenine (Figure 1.2) as well as monoadducts with

nucleophilic sites on the DNA, including thiols, amino, and hydroxyl groups.

2 2+ + - OH H N Cl - H3N Cl +H2O, -Cl H3N 2 3 +H2O, -Cl Pt Pt Pt - - +Cl , -H2O +Cl , -H2O H3N OH H3N OH2 H3N Cl 2

Cisplatin + -H+ pK = 5.37 -H pKa = 6.41 a

+ H N OH H3N Cl 3 Pt Pt H N OH H3N OH 3 2

+ -H pKa = 7.21

H3N OH Pt

H3N OH

Figure 1.1. Sequential aquation of the chloride ligands of cisplatin resulting in the 2+ formation of the active species cis-[Pt(NH3)2(OH2)2] , along with subsequent deprotonation steps (from ref 10).

(a) (b) Figure 1.2. Formation of the products from cisplatin with DNA: (a) intrastrand and (b) interstrand crosslink of cisplatin with ds-DNA.

3 1.3 Photodynamic Therapy

Photodynamic therapy (PDT) is a treatment that uses a drug, called a

photosensitizer, and light of a particular wavelength to effect cell death. In PDT,

molecules are activated with low energy light, thereby localizing the action of a

drug to the irradiated area.12,13 The wavelength of activation determines how far light can travel through tissue. For example, excitation with 690 nm light results in tissue penetration of about 1 cm whereas excitation at 630 nm results in a penetration depth of 0.5 cm as previously determined using benzoporphyrin derivatives and porfimer sodium, respectively.14,15 16

The first step of PDT for cancer treatment involves the injection of a

photosensitizing agent into the bloodstream, which is absorbed by cells

throughout the body. Some agents are retained by cancer cells longer than in

normal cells, at which time the drug is photoactivated by visible light.16 However,

longer retention or localization in cancerous cells is not necessary, since

selectivity can be achieved by irradiating only the affected areas. In general, PDT

results in the selective death of cancerous cells without affecting normal tissue.

To date, the U.S. Food and Drug Administration (FDA) has approved the

photosensitizer porfimer sodium, or Photofrin® (a mixture of hematoporphyrin

and its derivatives), for use in the treatment of esophageal and lung cancers.17 The

® 1 structure of Hematoporphyrin is shown in Figure 1.3. Photofrin produces O2 upon excitation with λ > 650 nm, which is the species that induces cell death

(Figure 1.4). This scheme is general for PDT agents, where following the

4 absorption of light, the sensitizer is excited from its ground state (1S) into a long-

lived excited triplet state (3*S) with typical lifetimes in the microsecond to

millisecond timescale,18,,19 20 via a short-lived singlet excited state (1*S, τ ~ 1-12

ns).21 The triplet excited state can transfer its energy directly to molecular oxygen

3 1 1 ( O2) to form singlet oxygen ( O2). The lifetime of the reactive O2 species is 3 μs

in water and ~ 200 ns within cells.16 Therefore, the efficacy of PDT depends on

1 the yield of O2 produced in the tumor, which in turn depends on the concentration of molecular oxygen in the tissue.22- 24 Thus, hypoxic cells are resistant to PDT,

and since some of the most malignant and drug resistant cancer cells are oxygen

deficient, this dependence on oxygen represents a drawback.25- 27 In addition to

directly killing cancer cells, PDT appears to shrink or destroy tumors in two other ways, the photosensitizer can damage blood vessels in the tumor, thereby preventing the cancer from receiving necessary nutrients. In addition, the presence of the PDT agent in the cancer cells may activate the immune system to attack the

tumor cells.12-15

OH

COOH

NH N

N HN HO COOH

Figure 1.3. The molecular structure of hematoporphyrin.

5 1*S

3*S hv 3 λ > 650 nm O2

1 O2 1 S

1 Figure 1.4. Schematic representation of the production of O2 by typical PDT agents, where 1S represents the molecule in its ground state, 1*S represents the singlet excited state, and 3*S is the triplet excited state of the molecule following intersystem crossing (ISC).

The action of almost all PDT drugs currently being used or investigated

are oxygen dependent. Therefore, there is a need to design new drugs that can be

activated with low energy light whose toxicity does not depend on oxygen. The

development of metal complexes for use as PDT agents seems promising since

they may react with double-stranded DNA directly from their excited states28,29 or

via the production of various reactive species, such as OH•.30- 32

In a manner similar to that of cisplatin, several transition metals have been shown to covalently bind to DNA through the displacement of labile ligands from the coordination sphere of the metal. These include metal complexes of Ru(II),33-

36 Rh(III),37- 39 and Ni(II).40 The effect of light on the interactions of chromium,41 cobalt,42 rhodium,33c,43,44 and ruthenium45 complexes that cleave DNA have also

been investigated. Covalent interactions of ruthenium(II) complexes with DNA

6 2+ were studied using [Ru(NH3)2] fragments coordinated to guanosine derivatives.46 Thorp and coworkers have shown the covalent binding of

2+ [Ru(L)(L’)(H2O)] (L,L’= 2,2’-bipyridine, terpyridine, trimethylethylenediammine, 1,10-phenanthroline, pyridine, dipyrido[3,2-a:2’,3’-

c]phenazine) to DNA via formation of a monoadduct by thermal denaturation

studies.33 The adducts of the monofunctional complexes show small positive shifts in DNA melting temperature (ΔTm = 0.8-3.5). The binding of 9-

ethylgaunine to [Ru(bpy)2(Cl2)] to yield the displacement product to [Ru(bpy)2(9-

EG)Cl]+ has also been reported47.

2+/3+ Rhodium ammine complexes, [Rh(NH3)5X] (X = halides) are

kinetically inert under thermal conditions, but upon irradiation into the ligand

48,49 + field band photosubstitution occurs. Cis-[Rh(Cl)2(polypyridyl)2] is known to covalently bind to DNA when irradiated.50 For example, Morrison and coworkers

+ have shown that cis-[Rh(Cl)2(phen)2] can act as a photoactivated cisplatin analog

and that the compound photometalates nucleobase nitrogen.49,51 The reaction of

the complex with guanine involves reductive quenching of a ligand field excited

state by the nucleobase, resulting in the formation of Rh(II). The latter is

reoxidized to Rh(III) upon covalent binding of the metal to the N7 position.52

Other rhodium compounds whose photochemistry was investigated with

DNA include Rh(III) tris (polypyridyl) complexes and related

metallointercalators, as well as dinuclear rhodium(II) carboxylates, which exhibit oxidative DNA strand scission originating from an electronic excited state.53,54

7 Rhodium (III) complexes possessing phi ligands (phi = 9,10-phenanthrenequinone

diimine) have been shown to bind to DNA (K > 106 M-1) and irradiation with uv

52 light (λirr = 313 nm) results in DNA cleavage at the complex’s binding site.

3+ [Rh(phi)2(bpy’)3] (bpy’ = 4-butyric acid-4’-dimethyl-2,2’-bipyridine) is known

to covalently bind to DNA, and long-range photooxidative damage to guanine

52 bases at 5’-GG-3’ and 5’-GGG-3’ sites has been observed with λirr ≥ 365 nm. It

3+ 3+ has also been shown that irradiation of [Rh(phen)(phi)] and [Rh(phi)2(bpy)]

(phen = 1,10-phenanthroline) with uv light (λirr = 313 nm) in the absence of DNA results in an irreversible loss of phi ligand in basic media possibly via a ligand to metal charge transfer excited state.55 Irradiation of [Rh(phen)(phi)]3+ and

3+ [Rh(phi)2(bpy)] intercalated in DNA leads to direct strand scission with products consistent with 3’-hydrogen abstraction from the deoxyribose backbone

of the DNA.56 The mechanism of direct DNA photocleaveage was believed to be

associated with the photoinduced ligand-loss reactivity, since the amount of DNA

cleavage correlates with the quantum yield of phi ligand loss observed in the

absence of DNA. The high energy of excitation required for the DNA cleavage by

Rh(III) complexes is a major drawback for the use of these systems as PDT

agents. A range of simple octahedral inorganic complexes was later studied and was observed to show single-strand scission by photoexcitation of different

3+ 3+ absorption bands of complexes that include [Cr(phen)3] and [Co(en)3] . These

complexes show photoactivated DNA cleavage from excitation of intraligand and

ligand field transitions57.

8 The goal of this dissertation is to design potential anticancer drugs that can be photoactivated with low energy light and whose toxicity does not require the presence of oxygen. To this end, the focus is to use metal complexes designed to combine the mode of action of cisplatin with the absorption of light, such that these metal complexes bind covalently to DNA after photolysis.

9 References

1. Rosenberg, B. Adv. Exp. Med. Biol. 1977, 91,129-150

2. Rosenberg, B.; Van Camp, L.; Trosko, J. L.; Mansour, V. H. Nature 1969, 222, 385-392.

3. Hill, J. M.; Speer, R. J. Anticancer Res. 1982, 2, 173-186.

4. Oliver, T.; Mead, T. Curr. Opin. Oncol. 1993, 5, 559-567.

5. Stathopoulous, G. P.; Rigatos, S.; Melamos, N. A. Oncol. Rep. 1999, 6, 797- 800.

6. Bruhn, S. L.; Toney, J. H.; Lippard, S. J. Prog. Inorg. Chem. 1990, 38, 477- 516.

7. Jakupec, M. A.; Galanski, M.; Keppler, B. K. Rev. Physiol. Biochem. Pharmacol. 2003, 146, 1-53.

8. Fuertes, M. A.; Alonso, C.; Pérez, J. M. Chem. Rev. 2003, 103, 645-62.

9. Farrell, N. Coord. Chem. Rev. 2002, 232, 1-4

10. Platinum-based drugs in cancer therapy 2000, Humana Press, N. J.

11. Sherman, S. E.; Lippard, S. J. Chem. Rev. 1987, 87, 1143.

12. DeRosa, M. C.; Crutchley, R. J. Coord. Chem. Rev 2002, 233-234, 351-371.

13. (a) Dalla Via, L.; Magno, S. M. Curr. Med. Chem. 2001, 8, 1405-1418. (b) McCaughan, J. S., Jr Drugs Aging 1999, 15, 49-68. (c) Allen, C. M.; Sharman, W. M.; van Lier, J. E. Tumor Targeting Can Ther. 2002, 329-361.

14.Vrouenraets, M. B.; Visser, G. W. M.; Snow, G. B.; van Dongen, G. A. M. S.; Anticancer Research 2003, 23, 505–522.

15. Dougherty, T. J.; Gomer, C. J.; Henderson, B. W. J. Natl Cancer Inst. 1998, 90, 12, 889–905.

16. His, A.; Rosenthal, D. I.; Glatstein, E. Drugs 1999, 57, 5, 725-734.

17. Dolmans, D. E. G. J.; Fukumura, D.; Jain, R. K. Nature Rev. 2003, 3, 380.

10

18. Kalyanasundaram, K. “Photochemistry of Polypyridine and Porphyrin Complexes,”Academic Press: San Diego, 1992.

19. Foley, S.; Jones, G.; Liuzzi, R.; McGarvey, D. J.; Perry, M. H.; Truscot, T. G. J. Chem. Soc. Perkin Trans.2 1997, 1725-1730.

20. Kobayashi, N.; Ishizaki, T.; Ishii, K.; Konami, H. J. Am. Chem. Soc. 1999, 121,9096-9110.

21. Henderson, B. W.; Dougherty, T. J. Photochem. Photobiol. 1992, 55, 145- 157.

22. Henderson, B. W.; Dougherty, T. J. Photochem. Photobiol, 1992, 55,145-157.

23. Moan, J.; Sommer, S. Cancer Res, 1985, 1608-1610.

24. Tromberg, B. J.; Orensten, A.; Kimel, S.; Barker, S. J.; Hyatt, J.; Nelson, J. S.; Berns, M. W. Photochem. Photobiol., 1990, 52, 375-385.

25. Guppy, M. Biochem. Biophys. Res. Comm. 2002, 299, 679-680.

26. Harris, A. L. Nature Rev. Cancer 2002, 2, 38-47.

27. Knowles, H. J.; Harris, A. L. Breast Cancer Res. 2001, 3, 318-322.

28. Fu, P. K.-L. Bradley, P. M.; van Loyen, D.; Durr, H.; Bossmann, S. H.; Turro, C. Inorg. Chem. 2002, 41, 3808.

29. Lecomte, J.-P; Kirsch-De Mesmaeker, A.; Feeney, M. M.; Kelly, J. M. Inorg. Chem. 1995, 34, 6481.

30. Pogozelski, W. K.; Tullius, T. D. Chem. Rev. 1998, 98, 1089.

31. Armitage, B. Chem. Rev. 1998, 98, 1171.

32. Erkkila, K. E.; Odom, D. T.; Barton, J. K. Chem. Rev. 1999, 99, 2777.

33. Grover, N.: Welch, T. W.; Fairley, T. A.; Cory, M.; Thorp, H. H. Inorg. Chem. 1994, 33, 3544-3548.

34. Grover, N.; Gupta, N.; Thorp, H. H. J. Am. Chem. Soc. 1992, 114, 3390-3393.

35. Chen, H.; Parkinson, J.A.; Parsons, S.; Coxall, R. A.; Gould, R.O.; Sadler, P. J. J. Am. Chem. Soc. 2002, 124, 3064-3082. 11

36. Zhao, M.; Clarke, M. J. J. Biol. Inorg. Chem. 1999, 4, 325-340.

37. Barry, C. G.; Turney, E. C.; Day, C. S.; Saluta, G.; Kucera, G. L.; Bierbach, U. Inorg. Chem. 2002, 41, 7159-7169.

38. Morrison, H. F.; Harmon, H. Photochem. Photobiol. 2000, 72, 731-738.

39. Mohammad, T.; Morrison, H. F. Photochem. Photobiol. 2000, 71, 369-381.

40. Muller, J. G.; Kayser, L. A.; Paikoff, S. J.; Durante, V.; Tang, N.; Perez, R. J.; Rokita, S. E.; Burrows, C. J. Coord. Chem. Rev. 1990, 185-186, 761-774.

41. Billadeau, M. A.; Morrison, H. J. Inorg. Biochem. 1995, 57, 249-270.

42. (a) Chang, C. H.; Meares, C. F. Biochemistry 1984, 23, 2268-2274. (b) Barton, J. K.; Rapheal, A. L. J. Am. Chem. Soc. 1984, 106, 2246-2468. (c) Fleisher, M. B.; Waterman, K. C.; Turro, N. J.; Barton, J. K. Inorg. Chem. 1986, 25, 3549-3551.

43. (a) Mahnken, R. E.; Bina, M.; Debel, R. M.; Leubek, K.; Morrison, H. Photochem. Photobiol. 1989, 49, 519-522. (c) Mahnken, R. E.; Billadeau, M. A.; Nikonowicz, E. P.; Morrison, H. J. Am. Chem. Soc. 1992, 114, 9253-9265.(d) Harmon, H. L.; Morrison, H. Inorg. Chem. 1995, 34, 4937-4938.

44. Sitalni, A.; Long, E. C.; Pyle, A. M.; Barton, J. K. J. Am. Chem. Soc. 1992, 114, 2303-2312.

45. (a) Kelly, J. M.; Feeney, M. M.; Tossi, A. B.; Lecomte, J.-P.; Kirsch,-De Mesmaeker, A. Anti-Cancer Drug Des. 1990, 5, 69-75. (b) Lecomte, J.-P.; Kirsch-De Mesneaker, A.; Feeney, M. M.; Kelly, J. M. Inorg. Chem. 1995, 34, 6481-6491.

46. Clarke, M. J.; Taube, H. J. Am. Chem. Soc. 1974, 96, 5413.

47. Van Vliet, P. M.; Haasnoot, J. G.; Reedijk, J. Inorg. Chem. 1994, 33, 1934- 1939.

48. Kelly, T. L. J. Phys. Chem. 1972, 76, 1937.

49. Magde, D.; Rojas, G. E.; Skibsted, L. H. Inorg. Chem. 1988, 27, 2900.

50. Billadeau, M. A.; Morrison, H. F. Metal Ions Biol. Syst. 1996, 33, 269.

12

51. Mahnken, R. E.; Billadeau, M. A.; Niknowicz, E. P.; Morrison, H. F. J. Am. Chem. Soc. 1992, 114, 9253.

51. Harmon, H. L.; Morrison, H. F. Inorg. Chem. 1995, 34, 4937.

53. (a) Erkkila, K. E.; Odom, D. T.; Barton, J. K. Chem Rev. 1999, 99, 2777. (b) Turro, C.; Hall, D. B.; Chen, W.; Zuilhof, H.; Barton, J. K.; Turro, N. J. J. Phys. Chem. A. 1998, 102, 5708.

54. Bradley, P. M.; Fu, P. K.; Turro, C. Comments Inorg. Chem. 2001, 22, 393.

55. (a) Pyle, A. M.; Chiang, M. Y.; Barton, J. K. Inorg. Chem. 1990, 29, 4487- 4495. (b) Krotz, A. H.; Kuo, L. Y.; Barton, J. K. Inorg. Chem. 1993, 32, 5963- 5974.

56. Sitlani, A.; Long, E. C.; Pyle, A. M.; Barton, J. K. J. Am. Chem. Soc. 1992, 114, 2303-2312.

57. Fleisher, M. B.; Waterman, K. C.; Turro, N. J.; Barton, J. K. Inorg. Chem. 1986, 25, 20, 3549-3551.

13

CHAPTER 2

BACKGROUND

2.1 Photophysical Properties and Photochemistry of Ru(II) polypyridyl complexes

2+ 4 [Ru(bpy)3] has intense absorption in the visible region (λabs = 453 nm, ε = ~10

M-1cm-1) which has been assigned as a metal to ligand charge transfer (1MLCT) transition which is singlet in character from the ground state (1GS), as shown in Figure 2.1.1 The compound has an emissive excited state (3MLCT) with a lifetime of 0.6 μs in water at

298 K. 2,3 In general, Ru(II) complexes with polypyridyl and related ligand exhibit long-

lived triplet 3MLCT excited states that can be accessed upon absorption of a photon.4 The

3MLCT excited state can typically be deactivated by the following processes (Figure 2.1): radiative emission of light (kr), non-radaitive decay (knr), and by the population of a

higher energy metal-centered excited state (MC). The MC excited state has contributions

from d-orbitals with antibonding character, therefore, population of this state weakens

3,7 2+ one or more of the Ru-N bonds, resulting in ligand loss. For [Ru(bpy)3] , the MC

excited states are populated thermally from the 3MLCT, and undergo rapid non-radiative

decay to the ground state. It has previously been shown that photosubstitution in

2+ [Ru(bpy)3] occurs via a dissociative mechanism involving the thermally accessible

ligand field excited states where k2 and k-2 shown in Figure 2.1 represents the reaction 14 rate for the forward and reverse reaction, respectively.5- 8 According to this mechanism,

the thermally activated 3MC excited state leads to cleavage of a Ru-N bond, with the

formation of a five-coordinate square pyramidal species. In the presence of coordinating

anions this species forms a hexacoordinated monodentate bpy intermediate. Once formed,

the monodentate bpy species can undergo loss of bpy and formation of Ru(bpy)2X2 (X = coordinating anions) or a self-annealing process (chelate ring closure) with reformation

2+ 9 of[Ru(bpy)3] . The self-annealing protective step is favored in aqueous solution,

2+ presumably because of stabilization of the cationic [Ru(bpy)3] , whereas formation of

the neutral Ru complexes is favored in low polarity solvents.9

3 MC Ligand loss k 1 MLCT 2 k-2 ISC

3 MLCT

hν k k r n r kr kn r

1 GS

Figure 2.1. Schematic representation of the processes that occur upon excitation of Ru(II) complex with a photon of light (hν).

2+ Gleria and coworkers, reported that irradiation of [Ru(bpy)3] with λ = 436 nm light results in the formation of [Ru(bpy)2Cl2] as the major product in chlorinated solvents, such as chloroform, 1,2-dichloromethane, and trichloroethane with Φ = 0.02 in 15 10,11 CH2Cl2. It was not clear in the report whether the origin of the chloride ligand was from the chloride counterion or from the solvent. It appears, however, that the quantum yield obtained was very sensitive to the amount of ethanol in solution and it was reported to be drastically reduced by increasing the ethanol precipitation.11 Photochemical studies

2+ 11 were also conducted with [Ru(bpy)3] in potentially coordinating solvents such as H2O,

12 and CH3CN, and the results showed that the complex is essentially photochemically inert at room temperature with λirr = 436 nm. However, Hoggard and Porter have reported the existence of a relatively inefficient photosubstitution reaction in DMF, where upon irradiation with visible or UV light, the quantum yield obtained for the disappearance of

2+ -4 13 [Ru(bpy)3] is less than 10 . The chemical stability and photoexcited states of

2+ [Ru(bpy)3] has led to the investigation of the photochemistry of a series on compounds

2+ of the type [Ru(bpy)2(L)2] where L represents monodentate ligands including CH3CN

- - - and py, and of the type Ru(bpy)2(L)2 where L = Cl , Br and NCS .

Meyer and coworkers have reported the photochemical synthesis of complexes of

+ - - the type cis-[Ru(bpy)2LX] and cis-[Ru(bpy)2X2] (L = py, CH3CN and X= ClO4 , NO3 ,

- - 2+ NCS , Br ) from the photosubstitution reactions of the parent complexes [Ru(bpy)2L2] , most notably where L = pyridine in solvents of low polarity, such as dichloromethane.14

Mechanistically he proposed that the reactions appear to involve a dissociative step at the metal center resulting in a quantum yield for monosubstitution of 0.18 for

2+ [Ru(bpy)2(py)2] , which was found to be independent of the chemical identity and concentration of the entering ligand, X-.14

16 Durham and coworkers have also synthesized a series of complexes of the type

n+ [Ru(bpy)2XY] (n = 0, 1, 2) where the ligands X and Y span the range of the spectrochemical series from Cl- to CO, and have studied the photosubstitution reactions of chloride from tertabutylammonium chloride using several irradiation wavelengths.15

The correlation between the energy of the lowest energy charge transfer transition and the quantum yield of photoanation with chloride ions from (C4H9)4NCl in CH2Cl2 (Table 1), was interpreted in terms of the relative energies of the MLCT and MC excited states in each complex.22

Durham observed a linear relationship between quantum yield for the photosubstitution of chloride ion and the energy of the lowest energy charge transfer transition. The quantum yields were also observed to correlate well with the emission energies and E1/2(II/III) couples of the complex, where plots of Φsusb versus the lowest energy MLCT transition the emission energy, E1/2(II/III) were linear with correlation coefficients of 0.93, 0.92, and 0.99, respectively. From Table 1 is it also apparent that the bipyridine ligands play a dominant role in the energy of the emission energy and

n+ absorption, and on the electrochemical potentials of [Ru(bpy)2XY] complexes, since the lowest energy excited state in all the complexes is believed to derive from the electronic configuration d5π*(bpy)1.22 The monodentate ligands in Table 1 also have a profound influence over the energy of the lowest MLCT transition, but do not appear to change the nature of the lowest energy excited state. The correlation of the quantum yields in Table 1 with emission energies, lowest MLCT transition energy, and E1/2(II/III) also follows the

2+ 16 model proposed by Watts and coworkers for [Ru(bpy)3] , which suggests that

17

a b a λmax, abs Emission Φ -1 -1 E1/2 X Y cm x cm x c photoanation (II/III) 103 103 pyridine Cl 0.04 19.8 15. 2 0.79

4-acetylpyridine Cl 0.07 20.2 15.3 0.82

N- N- <0.001 20.7 15.6 0.94 methylimidazole methylimidazole imidazole imidazole <0.001 20.5 15.9 1.02

CH3CN Cl 0.12 20.8 16.0 0.86 pyridine pyridine 0.20 22.0 17.1 1.30

4,4’-bpy 4,4’-bpy 0.20 22.4 17.3 1.32

4-acetylpyridine 4-acetylpyridine 0.29 22.6 17.4 1.45

3-iodopyridine 3-iodopyridine 0.24 22.6 17.4 1.36 pyridazine pyridazine 0.06 22.7 17.5 1.42

(P(C6H5)2CH3) (P(C6H5)2CH3) ~0 23.3 18.3 1.52

CH3CN CH3CN 0.31 23.5 18.5 1.44

CO CO 0.05 32.78 22.6 >1.9 a b Determined in CH2Cl2. Highest energy maxima at 77 K in 1:1 methanol-ethanol glass. c Determined in CH3CN with TBAH (from ref 22) vs SCE.

Table 2.1. Charge Transfer Maxima, Emission Maxima, E1/2(II/III), and Quantum Yield n+ for the Photoanation of cis-[Ru(bpy)2XY] in CH2Cl2 with (C4H9)4NCl.

18 2+ photosubstitution in [Ru(bpy)3] is a result of thermal population of a MC excited state from the 3MLCT. Although it was difficult to dtermine the effect of the monodentate ligands on the energy of the MC state since there was no good measure of the σ- bonding abilities of the ligands used in the study one factor that influenced Φ of photosubstitution was the electron density around the metal. Therefore, as a monodentate ligand is replaced by increasingly better π-acceptor ligands, the electron density on the metal is reduced, resulting in greater quantum yield of photosubstitution. Therefore, it appears that in these systems the charge on the metal is the dominant factor that determines the energy of the

MC excited state.

Meyer and coworkers have also reported that in mixed chelates [Ru(L-L)3-

2+ n(bpy)n] (n = 1,2; L-L = 2,2’-bipyrazine, 2,2’-bipyrimidine, 2,3-bis(2- pyridyl)quinoxaline, 2,2’-biisoquinoline), ligand loss takes place in CH3CN containing 2

2+ 17 mM (C4H9)4NCl but is quenched for [Ru(L-L)(bpy)2] . The authors suggested that the quenching in these systems is related to the relative energy of the d-d state compared to the 3MLCT state. The combination of the low-lying π* level localized on the L-L in

2+ [Ru(L-L)(bpy)2] and the average ligand field strength may result in greater spacing

18,19 between two energy levels, thereby reducing ligand loss. White and coworkers investigated the role of the lowest-lying π* energy level localized on the L-L ligand in directing photosubstitution in mixed-ligand ruthenium(II) complexes. 20 They studied the photosubstitution reaction of a series of ruthenium(II) complexes containing the ligands

- bpy, 2,2-bipyrimidine (bpm), 2,2’-bipyrazine (bpz), py, CH3CN, and Cl in N2-degassed acetonitrile solutions containing 1 mM (TEA)Cl (Table 2).27

19

Abs Em E Ligand 1/2 Compound / / τ / ns Φ c Φ d (II/II λmax λmax 0 p sub loss nma nmb I)g,h 2+ e e,f [Ru(bpy)3] 451 620 800 0.042 .0021 bpy 1.27

2+ [Ru(bpy)2(py)2] 457 610 0.00086 .0059 py 1.27

2+ e e,f [Ru(bpy)2(bpz)2] 710 376 0.0056 0.0002 bpz 1.49

2+ e e,f [Ru(bpy)2(bpm)2] 710 76 0.0011 0.0003 bpm 1.40

2+ e e,f [Ru(bpm)3] 454 639 131 0.0028 0.043 bpm 1.69

2+ e e,f [Ru(bpm)2(bpz)] 454 655 338 0.032 0.091 bpz 1.78

2+ e e,f [Ru(bpm)2(bpy)] 670 182 0.0038 0.0016 bpm 1.55

+ [Ru(bpm)2(CH3CN)Cl] 487 788 < 7 0.00002 0.0007 CH3CN 1.15

2+ f e,f [Ru(bpz)3] 440 610 795 0.034 0.35 bpz 1.98

2+ e,f e,f [Ru(bpz)2(bpy)] 654 1099 0.040 0.0049 bpz 1.72

2+ [Ru(bpz)2(py)] 472 665 570 0.0156 0.070 py 1.76

+ [Ru(bpz)2(CH3CN)Cl] 492 766 56 0.00018 0.0052 CH3CN 1.37 a 0 b c In CH3CN unless otherwise stated; T = 25 ± 1 C. λex = 436 ± 1 nm. Phosphorescence d quantum yield, ± 5 %; λex = 436. Photochemical quantum yield, ± 5 %; λex = 436 nm; 1 mM (TEA)Cl. e In propylene carbonate. f Data from 24. g Potentials are in V vs SSCE. h 0 Solutions were 0.10 M in TBAH, with CH3CN as solvent, T = 25 ± 1 C. (from ref 27)

Table 2.2. Luminescence Quantum Yields, Excited State Lifetimes, and Quantum Yields of Photoanation of Cl- for Ru(II) Complexesa

20 A plot of EMLCT (eV) vs Eem (eV) for the compounds in Table 2 was linear with a correlation coefficient of 0.97. A linear relationship was also observed between EMLCT

(eV) and E1/2(II/III) with a correlation coefficient of 0.99. A plot of the logarithm of the observed photochemical quantum yield log (Φ)obs for the complexes in Table 2 also resulted in a linear function of the energy when plotted against ΔE1/2 , where ΔE1/2 is the positive potential difference between the first oxidation and first reduction, the 1MLCT absorption energy maxima, the emission maxima, and the photochemical quantum yield.

The correlation of ln Φp (obs) with ΔE1/2 occurred in two series: one with complexes containing bpm and bpz ligands and the other with complexes containing only bpy-type ligands. Within each class, the higher the energy diffenence between the 3MLCT and GS, the more susceptible the complex is to photosubstitution. Therefore, ligand loss was determined by bond strength in the excited state rather than by the loss of the ligand with the lowest energy π* level

2.2 Photochemistry of Ruthenium Ammine Complexes

At the end of the 1960’s that Peter C. Ford published a series of fundamental papers on the photochemistry of ruthenium(II) compounds.21 Since then, there have been a number of achievements in the photochemistry of ruthenium(II) pentammine and tetraammine complexes, in which photoreactions are dominated by substitution processes.22 It was shown that in these systems the wavelength dependence of the quantum yields (Φ) is sensitive to the relative energies of the ligand field (LF) dd metal – centered excited states and the metal to ligand charge transfer excited state 21 (MLCT).23,24 ,25 The photosubstitution reactions of these complexes have MLCT and LF states as the lowest energy excited states (LEES) which have been rationalized in terms of the excited state “tuning” model.26 The model which was originally proposed to explain the photosubstitution chemistry of pentaammineruthenium(II) complexes

2+ [Ru(NH3)5L] (L = an aromatic nitrogen ligand) has been extended to other systems

2+ 2+ including [Ru(bpy-X)y] , [Ru(bpy)x(X)] (X = 4-acetylpyridine, 2-cyanopyridine, 4- cyanopyridine) where the MLCT states are usually the LEES .27 According to the model, the relatively photosubstitution “reactive” complexes have a LF state as the LEES, and those that are relatively “unreactive” have a MLCT state as the LEES.

Ruthenium(II)ammine complexes of unsaturated aromatic nitrogen heterocycles have LF and MLCT as the LEES of comparable energies whose energy is dependent on the substituents on the coordinated heterocycle on the solvent.28

The photochemistry of photosubstitution of various pentammine and

2+ tetraammineruthenium (II) complexes, [Ru(NH3)5L] (L= py, isonicotinamide (isn), pyrazine (pz), 4-acetylpyridine (4-acpy), 2-cyanopyridine (2-NCpy) and , CH3CN)and

2+ 29,30 [Ru(NH3)4L’L] (L’L = py, 4-acpy, isn) have been extensively investigated. The wavelength dependence of the Φ was shown to be sensitive to the relative energies of the

LF state and MLCT state (Table 3).31- 33

22

-3 -3 L λmax / nm λirr / nm ΦNH3 (10 ) ΦL (10 ) py(a) 407 405 45 ± 2 63 ± 5 449 49 ± 1 63 ± 1

isn(a) 479 405 4.5 ± 0.1 22 ± 2 479 1.07 ± 0.04 5.3 ± 0.2 500 0.37 ± 0.02 2.4 ± 0.1 546 0.30 ± 0.02 0.7 ± 0.1

pz(a) 472 479 1.4 ± 0.1 1.8 ± 0.2

4-acpy(a) 523 405 4.5 ± 0.7 27 ± 1 449 1.4 ± 0.1 8.6 ± 0.5 520 0.25 ± 0.06 0.9 ± 0.1

2-NCpy(b) 424 365 71 ± 1 56± 2 404 72 ± 2 40 ± 1 436 65 ± 2 59 ± 1

(c) CH3CN 354 313 130 ± 10 100 ± 20 366 160 ± 10 100 ± 20 a From ref 21. b From ref 19. c From ref 20.

Table 2.3. Photosubstitution Quantum Yields, Lowest Energy Absorption Maximum 2+ (λmax) of [Ru(NH3)5(L)] Complexes in Aqueous Media with the Wavelength of

Irradiation (λirr).

23 The energy of the 3MLCT excited state on the ruthenium(II) ammine complexes

2+ [Ru(NH3)5L] , related to the absorption maximum (λmax) listed in Table 3, depends on

34 2+ the nature of the ligand L. In [Ru(NH3)5L] complexes with L = substituted pyridine or piperazine (pz), the energy of the MLCT band varies with the nature and the position of the substituent on the aromatic heterocycle of ligand L.11 Electron donating groups destabilize the charge transfer state resulting in lower energy charge transfer transition, as

2+ seen in [Ru(NH3)5(4-acpy)] (λmax = 523 nm). Electron withdrawing groups stabilize the

MLCT state and result in higher energy MLCT transition as is the case in [Ru(NH3)5(2-

2+ NCpy)] (λabs = 424 nm). The maximum of the MLCT absorption band is observed at

2+ 407 nm for [Ru(NH3)5(py)] , which is blue shifted to 354 nm when py is replaced by

2+ 8b 2+ CH3CN in [Ru(NH3)5(CH3CN)] . [Ru(NH3)5L] (L = py, isn, pz, 4-acpy, 2-NCpy,

CH3CN) complexes generally display only one MLCT band. For the above complexes, it is generally accepted that photosubstitution reactions require the thermal population of the MC state, which lies above the lowest energy excited state, the 3MLCT state.35 It is

2+ shown in Table 3 that irradiation of solutions of [Ru(NH3)5(py)] with visible light with wavelengths corresponding to the MLCT band (λirr = 405 nm) leads to the photoaquation

2+ of both the pyridine and ammonia ligands. Upon irradiation of [Ru(NH3)5(py)] with lower energy light (λirr = 449 nm) no significant pyridine or ammonia photoaqaution was observed (Table 1). In contrast, an independence of the quantum yield of pyridine and ammonia photosubstitution as a function of irradiation wavelength was observed for

2+ 2+ [Ru(NH3)5(2-NCpy)] and [Ru(NH3)5(CH3CN)] , indicating that the MC transition is

2+ close in energy to the MLCT transition. However, both [Ru(NH3)5(isn)] and

24 2+ [Ru(NH3)5(2-acpy)] showed a wavelength dependence in the quantum yield of both

2+ pyridine and ammonia photosubstitution. Irradiation of [Ru(NH3)5(isn)] with 405 nm, resulted in a greater quantum yield of isonicotinamide and ammonia photosubstitution,

-3 -3 Φisn = 22 ± 2 x 10 and 4.5 ± 0.1 x 10 , respectively, compared to irradiation with lower

-3 -3 energy (λirr = 546 nm) which resulted in Φisn = 0.7 ± 0.1 x 10 and 0.3 ± 0.02 x 10 respectively. These results indicate that the energy of the MC excited state is significantly higher than that of the 3MLCT state. Such large energy difference would preclude the thermal population of the reactive MC state when the irradiating energy is low, populating only the MLCT. In these systems, high energy irradiation is necessary for photosubstitution to occur. From Table 3 it can also be concluded that complexes displaying a charge transfer maximum at wavelengths lower than ~ 460 nm are relatively reactive towards photoaquation (ΦL = 0.02 - 0.05 mol / einstein) when irradiated at wavelengths approximating to λmax of charge transfer.

2+ Cis-[Ru(NH3)4LL’] (LL’ = py, 4-acpy, isn) complexes exhibit two broad, intense (ε = 104 M-1cm-1) MLCT absorption bands in the UV-vis region of the spectrum which generally overlap. The range of absorption is usually larger when L ≠ L’ as shown in Table 4.36,37 In these complexes, the obscured LF absorption bands are assumed to be near 390 nm, and thus, the LF state may lie below both MLCT states, between them, or above them in energy.

2+ The wide range of absorption in the cis-[Ru(NH3)4L2] (λmax = 313 - 518 nm) complexes allowed the study of the quantum yield on irradiation wavelength with a large

2+ number of irradiation wavelengths. The irradiation of cis-[Ru(NH3)4(py)2] at various

25 irradiation wavelengths leads to relatively high quantum yields of photosubstitution when compared to the other metal complexes (Table 4). The photosubstitution quantum yield in this complex is independent of irradiation wavelength. In contrast, cis-[Ru(NH3)4(4-

2+ 2+ acpy)2] and cis-[Ru(NH3)4(isn)2] displayed irradiation wavelength dependent and relatively lower yields of substitution. It should also be noted that cis-

2+ 2+ [Ru(NH3)4(isn)(py)] and cis-[Ru(NH3)4(isn)(4-acpy)] shows ammonia photoaquation, as well as the preferential photosubstitution of one of the unsaturated ligands as the major products. As indicated by the decreasing energy of the lowest energy MLCT band, the π- acceptor ability of the ligands follows the order py < isn < 4-acpy.38 Hence, for the

2+ complexes cis-[Ru(NH3)4(isn)L] (L = 4-acpy, py), the ligand that has the lower π- acceptor ability are preferentially labilized; these ligands have the weakest π-back bonding ability and are aquated with the higher quantum yield.30 For these complexes the photosubstitution is independent of irradiation energy, which indicates a common reactive excited state as a precursor to photosubstitution. Therefore, it is believed that irradiation of higher energy states is followed by deactivation to one common reactive state, which presumably is the triplet ligand field excited state. An exact description of the electronic configurations and states of these complexes is not simple, since the LF absorptions are not observed in the spectra. However, the aqaution of different ligands for the different cis complexes is indicative of reactive LF excited states with different electronic configurations in each complex, which agrees well with the model proposed by

Ford (see Figure 2.2).31 For the complexes listed in Table 4 there is a crossover in the

26 -3 -3 -3 L L’ λmax /nm λirr /nm ΦL (10 ) ΦL’ (10 ) ΦNH3 (10 ) py py 366 (MLCT2) 313 32 ± 3 53 ± 4 407 (MLCT1) 365 28 ± 3 53 ± 5 405 26 ± 4 57 ± 4 436 32 ± 1 66 ± 3

4-acpy 4-acpy 442 (MLCT2) 405 3.8 ± 0.2 39 ± 1 518 (MLCT1) 436 3.7 ± 0.2 9.7 ± 0.3 480 1.3 ± 0.1 3.6 ± 0.2 519 0.55 ± 0.05 1.2 ± 0.1

isn isn 413 (MLCT2) 365 8.5 ± 0.1 38 ± 1 478 (MLCT1) 405 7.8 ± 3 34 ± 1 436 2.4 ± 0.1 10 ± 1 480 1.5 ± 0.1 4.5 ± 0.3

isn py 378 (MLCT2) 365 3.0 ± 0.2 12.5 ± 0.4 11.5 ± 0.5 466 (MLCT1) 405 2.5 ± 0.1 9.7 ± 0.7 9.5 ± 0.4 436 2.5 ± 0.1 8.4 ± 0.2 8.2 ± 0.1 480 1.1 ± 0.1 3.8 ± 0.1 8.7 ± 0.3

isn 4-acpy 426 (MLCT2) 405 2.6 ± 0.1 0.9 ± 0.1 6.2 ± 0.1 503 (MLCT1) 480 0.7 ± 0.1 0.04 ± 0.01 1.3 ± 0.1 519 < 0.4 ± 0.1 < 0.01 < 0.7 ± 0.1 b Data from ref 24.

Table 2.4 Photosubstitution Quantum Yields and Electronic Absorption Data of cis- 2+ b [Ru(NH3)4LL’] Complexes in Aqueous Media.

27 reactivity of the complex from been “reactive” to “unreactive” based on the energy of the lowest energy MLCT excited state (MLCT-1). It is determined from the complexes listed in Table 4 that crossover is in the 407 -466 nm region, since the MLCT-1 band of the lowest energy in the “reactive” complex lies at 407 nm and the highest energy of the

“unreactive” complex lies at 466 nm.

A schematic representation for a “reactive” complex is shown in Figure 2.2a, where initial excitation into the MLCT states is followed by efficient internal conversion to the lower reactive LF excited state. Figure 2.2b also depicts the energy diagram for

“unreactive” complexes. For these complexes, initial excitation to higher energy states involves relaxation to the lowest energy MLCT states, and wavelength dependence of ligand loss can be explained by the internal conversion from MLCT state to LF along modes competitive in rate with 3MLCT relaxation.

3 MLCT-2 3 LF products 3MLCT-1 3MLCT-2 3LF products 3MLCT-1

GS GS (a) (b)

Figure 2.2. Schematic representation of the “tuning model” proposed by Ford:33 (a) “reactive complexes”; (b) “unreactive complexes”. 28 2.4 Covalent DNA binding

The synthetic chemistry of ruthenium ammine, ammine and imine ligands are well developed and provide a means for the development of metallopharmaceuticals.

Ruthenium (II) ammine complexes are known to covalently bind to imine sites in biomolecules, which unlike amine sites, are not protonatated at neutral pH, leaving their nitrogen lone pairs available for coordination to the metal ion.39,,40 41 On this basis ruthenium complexes selectively bind to the histidyl imidazole nitrogen on proteins2,3 and to the N7 site on the imidazole rings of the purine bases, guanine and adenine (Figure

2.3).2,3 For ammine complexes of Pt(II) and Ru(II) containing either labile chloro or aqua ligands, the pyrimidine N7 sites of DNA are the targets because they are easily accessible in the major groove of the DNA.42

(a) (b) O NH2

N 6 N 6 HN 5 N 1 7 1 5 7 2 8 8 2 4 4 9 3 9 3 N N N H2N N H H

Figure 2.3. Structure of the DNA bases (a) guanine and (b) adenine

Covalent binding of metal complexes to DNA involves the replacement of a labile ligand of the metal complex by a nucleophile on the DNA.43 Barton and Lolis have

29 reported a chiral selectivity for the covalent binding of cis-[Ru(phen)2Cl2] to DNA that favors covalent binding of the Λ isomer to B-DNA.48 The Λ-enantiomer is known to selectively associate with B-DNA through electrostatic and hydrophobic interactions before coordinating to the N7 position of guanine.44, Thorp and coworkers have also

2+ reported covalent binding of complexes of the type [Ru(tpy)(L)(H2O)] (L = bpy, phen,

2+ 2+ 45 tmen), [Ru(bpy)2(L)(H2O)] (L = py, ph) and [Ru(phen)2(L)(H2O)2] (L = py, ph).

The results obtained were similar to those reported by Baron and Lois,48 where the reaction proceeds with stereoselectivity that favors covalent binding of the Λ isomer to B-

DNA. The degree of selectivity was greater for bis-1,10-phenanthroline complexes compared to those of bis-2,2’-bipyridine and a factor of two greater for

2+ 2+ [Ru(L)2(py)(OH2)] (L = phen, tmen, bpy) and [Ru(tpy)(py)(OH2)] compared to

2+ [Ru(L)2(OH2)2] complexes. The selectivity can be explained in terms of the size of the complexes, since the larger phen complexes are more sterically demanding and have

2+ more easily recognized chirality. Similarly, [Ru(L)2(py)(OH2)] (L = phen, tmen, bpy) complexes are somewhat larger than the diaqua complexes and thus exhibit greater

51 2+ selectivity. Covalent binding of cis-[Ru(bpy)2(OH2)2] to DNA has been previously reported as an undesired side effect of probing DNA structure with a derivative of

2+ 46 [Ru(bpy)3] . Studies have also shown that bpy induces only minimal intercalative

47 2+ binding with DNA. The binding of cis-[Ru(bpy)2(H2O)2] and cis-Ru(bpy)2Cl2 with the alkylated 6-ketopurines, 9-methylhypoxanthine, and 9-ethylgaunine was investigated by the thermal reaction of the DNA bases with the metal complex at 37 oC.48 A 1:1 binding stoichiometry was observed between the DNA bases and the cis-Ru(bpy)2

30 moiety, even under strong heating conditions. A crystal structure showed that in these adducts the purine base is positioned with the keto group between the pyridyl rings of the cis configurated bpy ligand.52 However, it has been shown by Alberto and coworkers that

o refluxing cis-[Ru(bpy)2(O3SCF3)2] with 2.4 eq of 9-methylgaunine (9-MG) at 37 C for 3

49 days affords cis-[Ru(bpy)2(9-MG)2. A crystal structure reveals that the two bases are in a head-to-tail orientation with the base-base dihedral angle of 60.4 o.53 The above results led to the study of the DNA binding of a polypyridyl complex containing three potential

50 binding sites, mer-[Ru(terpy)Cl3]. It should be noted that the oxidation state of the metal in the latter complex is +3, Ru(III). Ru(III) complexes are usually considered to be kinetically inert, and activation mechanisms involving in vivo reduction to Ru(II) are

51,52 often implicated in their biological activity. Covalent binding of mer-[Ru(tpy)(Cl)3] was observed to the N7 position of guanine resulting in the formation of an interstrand

54 crosslink with 2 % yield. Mer-[Ru(terpy)Cl3] was also reacted with 9- methylhypoxanthine (9mhpy) and 9-ethylguanine (9-EG), where the corresponding

7 complex trans-[Ru(terpy)(B-N )(H2O)](PF6)2, where B = 9mhpy or 9-EG, was isolated upon refluxing in ethanol:methanol (7:3 % v/v) for 16 hours.54 1H NMR indicates that each base was symmetrically arranged around the metal center and involved hydrogen bonding with the water ligand via their O6 atoms.54

Other Ru(III) complexes that are known to covalently bind to DNA includes

2+ 53 - trans-[Ru(NH3)4(SO2)(Cl)] , mer-Ru(Me2SO)3Cl3], trans-[Ru(Me2SO)2Cl4] , and mer-

54 [Ru(Me2SO)(H2O)2Cl3], all of which produce DNA interstrand crosslinks. Intrastrand croslinking to guanine N7 may be possible in these systems but it is sterically more

31 55,56 ,57 - crowded by the octahedral geometry. For example, trans-[Ru(Cl)4(Me2SO)2] reacts with d(GpG) to yield a macrocyclic chelate with the formulation cis-

N7 N7 [Ru(II)d(G pG )Cl(H2O(Me2SO)2] ,in which the sugars are in anti-configurations and the guanines are destacked in a head-to-head arrangement.58 The anticancer complex

- trans-[Ru(Cl)4(Im)2] (Im= imidazole) has also been shown to covalently bind to DNA by forming croslinks to the N7 position of guanine bases upon aquation.59 In this complex aquation occurs by the sequential loss of the two chlorides that are cis to the Im ligand with initial rates of 9.6 x 10-6 s-1 at 25 0C and 5.26 x 10-5 s-1 at 37 0C.59 The ease of

- reduction of trans-[Ru(Cl)4(Im)2] (-0.24 V vs SCE) may allow in vivo reduction, which causes the chlorides to dissociate more rapidly. In this complex, a mechanism of

60 activation by reduction has been proposed. Trans-[Ru(9-MeAde)(dmtp)2Cl3] (dmtp =

5,7-dimethyl[1,2,4]-triazolo[1,5-a]pyrimidine, 9-MeAde = 9-methyladenine) has also been studied and it was shown that the Ru(III) to 9-Me-Ade coordinates through the exocyclic N6 nitrogen. Two intramolecular hydrogen bonds stabilize the coordination of the neutral adenine ligands with the proton at N1 in this complex.61

2+ Ruthenium(II) and (III) ammine complexes such as [Ru(NH3)5(OH2)] and

2+ [Ru(NH3)5Cl] , respectively, are also known to bind readily to surface accessible imidazole nitrogens on a number of proteins.54 The loss of ammines and heterocyclic

2+ nitrogen bases from [Ru(NH3)5L] (L = OH2 or nitrogen heterocycle) is usually faster than for the corresponding Ru(III) complexes, but still proceeds slowly with a half-life on the order of a day under physiological conditions.62The cationic metal complex

2+ [Ru(NH3)5(H2O)] is also believed to have electrostatic attraction to polyanionic nucleic

32 acids and, therefore, its rate of nucleic acid binding to the guanine N7 sites proceeds with a rate constant of 6.0 ± 0.9 M-1s-1 and is strongly dependent on ionic strength.63 The

2+ -1 affinity binding constants for [Ru(NH3)5(H2O)] are 5100 and 7800 M for double-

64 2+ stranded and single-stranded DNA, respectively. Unlike [Ru(NH3)5(H2O)] , covalent

2+ binding of trans-[Ru(NH3)4(py)(H2O)] to the N7 position of guanine is slower with a pseudo first order rate constant of 5.6 x 10-4 s-1 at 25 oC.57 Compounds with pyridine

2+ ligands, such as trans-[Ru(NH3)4(py)(H2O)] , bind slowly to DNA relative to

2+ 65 [Ru(NH3)5(H2O)] and favor Ru(III/II) reduction. It has also been demonstrated in vivo

2+ that the reduction of [Ru(NH3)5Cl] is catalyzed by subcellular components of the rat liver cells.66 This reduction facilitates DNA binding since the aqua ligand in

2+ [Ru(NH3)5(H2O)] exchanges with a half life of 0.1 s, which is considerably more rapid than that for the corresponding Ru(III) complex.57

The Ru(II) complexes cis- and trans-[Ru(dmso)4Cl2] (dmso = dimethylsulfoxide) were also shown to bind in vitro to plasmid DNA, causing inhibition of restriction enzymes which normally cut the DNA at guanine-rich sites.67 The reaction of trans-

[Ru(dmso)4Cl2] with 2’-deoxyguanosine or d(GpG) results in the formation of a cis configurated N(7) bis adduct68 and a Ru(2’-deoxyguanylyl-N7-(3’-5’)-2-deoxyguanosine-

N7) chelate, respectively, which like cisplatin, is capable of binding two guanine ligands at the N(7) sites in a cis-configuration.52

33 References

1. (a) Lytle, F. E.; Hercules, D. M. J. Am. Chem. Soc. 1969, 91, 253. (b) Klassen, D. M.; Crosby, G. A. J. Chem. Soc. 1968, 48, 1853. (c) Harrigan, R. W.; Hager, G. D.; Crosby, G. A. Chem Phys. Lett. 1973, 21, 487. (d) Baker, D. C.; Crosby, G. A. Chem. Phys. 1974, 4, 428. (e) Harrigan, R. W.; Crosby, G. A. J. Chem. Phys. 1973, 59, 3468.

2. McCusker, J. K. Acc. Chem. Res. 2003, 36, 876-887.

3. Van Houten, J.; Watts, R. J. J. Am. Chem. Soc. 1976, 98, 4853.

4. Moucheron, C.; C.-M. Kirsch-De Mesmaeker, A.; Kelly, J. M. J. Photochem. Photobiol. B 1997, 40, 91.

5. Van Houten, J.; Watts, R. J. Inorg. Chem. 1978, 17, 3381-3385.

6. Xu, J.; Porter, G. B. Can. J. Chem. 1982, 60, 2856-2858.

7. Durham, B.; Caspar, J. V.; Nagle, J. K.; Meyer, T. J. J. Am. Chem. Soc. 1982, 104, 4803-4810.

8. McMillin, D. R.; Kirchhoff, J. R.; Goodwin, K. V. Coord. Chem. Rev. 1985, 64, 83-92.

9. Juris, A.; Balzani, V. Coord. Chem. Rev. 1988, 84, 85-277.

10. Gleria, M.; Minto, F.; Beggiato, G.; Bortolus, P. J. Chem. Soc., Chem. Comm. 1978, 285.

11. Van Houten, J.; Watts, R. J. J. Am. Chem. Soc. 1976, 98, 4853.

12. Durham, B.; Walsh, J. L.; Carter, C. L.; Meyer, T. J. Inorg. Chem. 1980, 19, 860-865.

13. Hoggard, P. E.; Porter, G. B. J. Am. Chem. Soc. 1978, 100, 1457.

14. Durham, B.; Walsh, J. L.; Carter, C. L.; Meyer, T. J. Inorg. Chem. 1980, 19, 860-865.

15. Pinnick, D. V.; Durham, B. Inorg. Chem. 1984, 23, 1440-1445.

16. Van Houten, J.; Watts, R. J. Inorg. Chem. 1978, 17, 3381.

17. (a) Allen, G. H.; White, R. P.; Rillema, D. P.; Meyer, T. J. J. Am. Chem. Soc. 1984, 106, 2613. (b) Rillema, D. P.; Taghdiri, D. G.; Jones, D. S.; Keller, C. D.; Worl, L. A.; Meyer, T. J.; Levy, H. A. Inorg. Chem. 1987, 26, 578.

34

18. Meyer, T. J. Pure Appl. Chem. 1986, 58, 1193.

19. Barigelletti, F.; Juris, A.; Balzani, V.; Belser, P. Inorg. Chem. 1983, 22, 3335.

20. Ross, H. B.; Boldaji, M.; Rillema, D. P.; Blanton, C. B.; White, R. P. Inorg. Chem. 1989, 28, 1013-1021.

21. (a) Ford, P. C.; Struermer D. H.; McDonald D. P. J. Am Chem. Soc. 1969, 91, 6209. (b) Ford, P. C.; Chaisson, D. A.; Stuermer, D. H. J. Am. Chem. Commun. 1971, 530. (c) Chaisson, D. A.; Hintze, R. E.; Stuermer, D. H.; Petersen, J. D.; McDonald, D. P.; Ford, P. C. J. Am. Chem. Soc. 1972, 94, 6665.

22. Tfouni, E. Coord. Chem. Rev. 2000, 196, 281-305.

23. a) Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1974, 96, 601-603. (b) Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1977, 99, 7313- 7221. (c) Durante, V. A.; Ford, P. C. Inorg. Chem. 1979, 18, 588-593.

24. (a) Ford, P. C. Coord. Chem. Rev. 1970, 5, 75-99. (b) Ford, P. C. Coord. Chem. Rev. 1982, 44, 61-82. (c) Ford, P. C.; Wink, D.; Dibenedetto, J. Progress Inorg. Chem. 1983, 3 0, 213-271. 25. (a) Tfouni, E. Coord. Chem. Rev. 2000, 196, 281-305.(b) Martinez, M. S.; J. Photochem. Photobiol. A: Chemistry 1999, 122, 103-108. (c) Pavanin, L. A.; da Rocha, Z. N.; Giesbrecht, E.; Tfouni, E. Inorg. Chem. 1991, 30, 2185, 2190.

26. (a) Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1977, 99, 7213. (b) Malouf, G.; Ford, P. C. Ibid 1974, 96, 601.

27. (a) Wrighton, M. S.; Abrahamson, H. B.; Morse, D. L. J. Am. Chem. Soc. 1976, 98, 4105. (b) Abrahamson, H. B.; Wrighton, M. S. Inorg. Chem. 1979, 17, 3385. (c) Figard, J. E.; Petersen, J. D. Ibid 1978, 17, 1059.

28.Pavanin, L. A.; Novais da Rocha, Z.; Giesbrechat, E.; Tfouni, E. Inorg. Chem. 1991, 30, 2185-2190.

29. Silva, M.; Tfouni, E. J. Photochem. Photobiol. A. 1999, 122, 103.

30. Hintze, R. E.; Ford, P. C. J. Am. Chem. Soc. 1975, 97, 2664.

31. (a) Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1974, 96, 601-603. (b) Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1977, 99, 7213-7221. (c) Durante, V. A.; Ford, P. C. Inorg. Chem. 1979, 18, 588-593.

35

32. (a) Ford, P. C. Coord. Chem. Rev. 1970, 5, 75-99. (b) Ford, P. C. Coord. Chem. Rev. 1982, 44, 61-82. (c) Ford, P. C.; Wink, D.; Dibenedetto, J. Progress Inorg. Chem. 1983, 30, 213-271.

33. Tfouni, E. Coord. Chem. Rev. 2000, 196, 281-305. (b) Martinez, M. S. J. Photochem. Photobiol. A: Chemistry 1999, 122, 103-108. (c) Pavanin, L. A.; da Rocha, Z. N.; Giesbrecht, E.; Tfouni, E. Inorg. Chem. 1991, 30, 2185-2190.

34. Ford, P. C.; Rudd, DeF. P.; Gaunder, R.; Taube, H. J. Am. Chem. Soc. 1968, 90, 1187.

35. (a) Durham, B.; Casper, J. V.; Nagle, J. K.; Meyer, T. J. J. Am. Chem. Soc. 1982, 104, 4803-4810. (b) Allen, G. H.; White, R. P.; Rillema, D. P.; Meyer, T. J. J. Am. Chem. Soc. 1984, 106, 2613-2620. (c) Rillema, D. P.; Taghdri, D. G.; Jones, D. S.; Keller, C. D.; Worl, L. A.; Meyer, T. J.; Levy, H. A. Inorg. Chem. 1987, 26, 578-585.

36. Bento, M. L.; Tfouni, E. Inorg. Chem. 1988, 27, 3410.

37. Tfouni, E.; Ford, P. C. Inorg. Chem. 1980, 19, 72.

38. Pavanin, L. A.; Rocha, Z. N.; Giesbrecht, E.; Tfouni, E. Inorg. Chem. 1991, 30, 2185.

39. Gray, H. B.; Winkler, J. R.; Ann. Biochem. 1996, 65, 537.

40. Messori, L.; Vilchez, G. G.; Vilaplana, R.; Piccioli, F.; Aleesio, E.; Keppler, B. Met. Based Drugs 2000, 7, 335.

41. Messori, L.; Orioli, P.; Vullo, D.; Alessio, E.; Iengo, E. Eur. J. Biochem. 2000, 267, 1206.

42. Clarke, M. J. Clin. BiIochem. Med. 1989, 10, 25.

43. Clarke, M. J. Met. Ions Biol. Syst. 1980, 11, 231.

44. Barton, J. K.; Lolis, E. J. Am. Chem. Soc. 1985, 107, 708.

45. Grover, N.; Gupta, N.; Thorp, H. J. Am. Chem. Soc. 1992, 114, 3390-3393.

46. Telser, J.; Cruickshank, K. A.; Schanze, K. S.; Netzel, T. L. J. Am. Chem. Soc. 1989, 111, 7221.

47. Morgan, R. J.; Chatterjee, S.; Baker, A. D.; Strekas, T. C. Inorg. Chem. 1991, 30, 2687.

36

48. Van Vliet, M. P.; Haasnoot, J. G.; Reedijk, J. Inorg. Chem. 1994, 33, 1934-1939.

49. Zobi, F.; Hohl, M.; Zimmerman, I.; Alberto, R. Inorg. Chem. 2002, 43, 2771-2772.

50. Van Vliet, P. M.; Toeimin, S. M.S.; Haasnoot, J. G.; Reedijk, J.; Novakova, O.; Brabec, V. Inorg. Chim. Acta. 1995, 57-64.

51. (a) Clarke, M. J. Metal Ions Biol. Syst. 1980, 11, 231. (b) Clarke, M. J. Metal Complexes in Cancer Chemotherapy 1993, 129-156.

53 Isied, S.; Taube, H. Inorg. Chem. 1976, 15, 3070.

54. Pacor, S.; Sava, G.; Ceschia, V.; Bregant, F.; Mestroni, G.; Alessio, E. Chem. Biol. Interact. 1991, 78, 223.

55. Marzilli, L. G.; Iwamoto, M.; Alessio, E.; Hansen, L.; Calligaris, M. J. Am. Chem. Soc. 1994, 116, 815.

56. Alessio, E.; Zangrando, E.; Roppa, R.; Marzilli, L. G. Inorg. Chem. 1998, 37, 2458.

57. Iwamoto, M.; Alessio, E.; Marzilli, L. G. Inorg. Chem. 1996, 35, 2384.

58. Esposito, G.; Cauci, S.; Fogolari, F.; Alessio, E.; Scocchi, M.; Quadrifoglio, F.; Viglino, P. Biochemistry 1992, 31, 7094.

59.Dhubhghaill, O. M.; Hagen, W.; Keppler, B. K.; Lipponer, K.-G.; Saddler, J. J. Am. Chem. Soc. 1994, 3305.

60. Hartmann, M.; Lipponer, K.-G.; Keppler, B. K. Inorg. Chim. Acta 1998, 267, 137.

61 .Velders, A. H.; Geest, B.; Kooijman, H.; Spek, a. L Haasnoot, J. G.; Reedijk, J. Eur. Inorg. Chem. 2001, 369.

62. Clarke, M. J.; Jansen, B. Inorg. Chim. Acta 1986, 124, 13-28.

63. McNamara, M. J.; Clarke, M. J. Inorg. Chim. Acta 1992, 175.

64.Clark, M. J.; Janse, B.; Marx, K. A.; Kruger, R. Inorg. Chim. Acta, 1986, 124, 13.

65. Zhao, M. Clarke, M. J. J. Biol. Inorg. Chem. 1999, 325.

66. Clarke, M. J.; Bitler, S.; Rennert, D.; Buchbinder, M.; Kelman, A. D. J. Inorg. Biochem. 1980, 12, 79.

37

67. Loseto, F.; Alesio, E.;Mestroni, G.; Lacidogna, G.; Nassi, A.; Giordano, D.; Coluccia, M. Anticancer Res. 1991, 1549.

68. Cauci, S.; Viglio, P.; Esposito, G.; Quadrifoglio, F. J. Inorg. Biochem. 1991, 43, 739.

38

CHAPTER 3

EXPERIMENTAL SECTION

3.1. MATERIALS

RuCl3.3H2O, 2,2’-bipyridine, 8-hydroxyquinoline, and pyridine were purchased

from Aldrich and used without further purification. Sephadex C-25, Sephadex G-15 sodium chloride, sodium phosphate, sodium hydrogen phoshate, Tris base/HCl, ethidium bromide, gel loading buffer (0.25 % (w/v) bromophenol blue, 40 % sucrose, 0.1 M

EDTA (pH = 8.0), 9-ethyl guanine (9-EG) and 9-methyl guanine (9-MG), DNA

Molecular weight standard (10000 kbase), Hoecht 33258 were purchased from Sigma and used as received. The Sma I restriction enzyme was purchased from invitrogen and used as received. The pUC18 plasmid was purchased from Bayou Biolabs and purified using the Qiaprep spin Miniprep system (250) from Life technology. Herring sperm DNA was

purchased from Invitrogen and used as received. Ru(II) complexes were prepared as described below. Water was deionised using a Barnstead Fi-stream filter system to 18

MΩ.

Synthesis of cis-Ru(bpy)2Cl2

Cis-[Ru(bpy)2Cl2] was synthesized by modification to an already published

procedures.1,2 Ruthenium(III) chloride (1.001 g, 0.0013 moles), 2,2’-bypyridine (1.20 g,

39 0.0077 moles) and lithium chloride (1.61 g, .038 moles) were refluxed in N,N-

dimethylformamide (DMF, 10.00 ml) for 8 hours under argon. The reaction was stirred

magnetically during this time. The starting brown-black solutions turned red-purple. The

mixture was allowed to cool to room temperature and 20 ml of reagent grade acetone:water was added (1:1,v:v). The solution was filtered and the green black solid was stirred in 20 ml of water overnight. The compound was further purified by washing with 3 x 5 mL portions of CH2Cl2 and ether respectively. The compound was dried by

1 suction on a vacuum. H NMR in DMSO-d6, δ (int., mult): 10.02 (2H, d), 8.7 6 (2H, d),

8.56 (2H, d), 8.18 (2H, t), 7.82, 7.78 (4H, t), 7.6 (2H, d), 7.2 (2H, t).

2+ Synthesis of cis-[Ru(bpy)2(NH3)2]

The compound was prepared by a modification of a reported method.2 Cis-

Ru(bpy)2Cl2 (1.0 mmol) was stirred in a solution of anhydrous methanol with 2 eq of

AgBF4 for 1 hr, followed by filtration to remove the solid AgCl. Reduction of the solvent

resulted in the formation of a red/purple solid that was filtered and washed with ether and

dichloromethane. A 2.0 M NH3 solution (10 mL) was added to the solid and the solution

was stirred at room temperature for 1 hour, after which time the product was passed

through a Sephadex SP C-25 column and eluted with 0.2 M HCl. The brown eluant was

evaporated to dryness and the solid was precipitated from acetone/ether with 40 % yield.

1 H NMR in DMSO-d6, δ (int., mult): 9.0 (2H, d), 8.6 (2H, d), 8.4 (2H, d), 8.0 (2H, t), 7.6

+ (4H, t), 7.3 (2H, d), 7.0 (2H, t), 2.9 (NH3). ESMS (m/z, ion): 445, [Ru(bpy)2(NH3)2] .

40 2+ Synthesis of [Ru(bpy)2(en)]

The compound was prepared by a procedure similar to that previously described

2+ 1,3 for cis-[Ru(bpy)2(NH3)2] using cis-[Ru(bpy)2(Cl2)] and ethylenediammine (en). The

complex cis-Ru(bpy)2Cl2 (0.500 g, 1.0 mmol) was suspended in 30 ml of a 1:1 (v/v)

water: methanol solution and excess ethylenediammine added (5 ml). The mixture was

heated at reflux for 1 hour under an argon atmosphere. The deep red solution was cooled

and filtered and the filtrate evaporated to dryness. The red crystals were recrystallized

from acetone/ether before they were dissolved in water and was passed through a C-25 sephadex column. The bright red complex was eluted with 0.2 M HCl. The solution was

1 collected and dried. H NMR in DMSO-d6, δ (int., mult): 9.3 (2H, d), 8.8 (2H, d), 8.3

(2H, t), 7.9 (4H, t), 7.6 (2H, d), 7.3 (2H, t), 4.8 (2H, t). ES-MS: m/z = 235.0 for cis-

2+ [Ru(bpy)2(en)] .

Synthesis of cis-[Ru(bpy)2(CH3CN)2](PF6)2

A solution of cis-[Ru(bpy)2Cl2] (0.5 g, 1.0 mmol) was refluxed for 1.5 hr in 30 mL of a 1:1 (v/v) mixture of acetonitrile and water. The solution was then filtered while still hot and reheated to the point of boiling on a hot plate. A 5 mL amount of a saturated aqueous solution of NH4PF6 was added, the reaction mixture was allowed to cool slowly to room temperature, and was then placed in an ice bath. The precipitate was collected by filtration, washed with 30 mL of cold water, and was air-dried to afford a red solid with

1 70 % yield. H NMR in DMSO-d6, δ (int., mult): 9.3(2H, d), 8.6 (2H, d), 8.4 (2H, d), 8.2

41 (2H, t), 7.9 (2H, t), 7.8 (2H,t), 7.6(2H, d), 7.3 (2H, t), 2.3 (3H,s). ESMS (m/z, ion): 641,

[Ru(bpy)2(CH3CN)2](PF6).

Synthesis of cis-[Ru(quo)2(py)2]

RuCl3.3H2O (0.2665 g, 1.0 mmol) was refluxed in dearated ethanol until the

solution turned blue. A solution of 8-hydroxyquinoline (0.3135 g, 2.2 mmol) in dearated

ethanol was added and the solution refluxed for 6 hours under an argon atmosphere.

Excess dearated pyridine was then added (10.0 ml) and the solution was refluxed for

another two hours. The solution was then cooled under an argon atmosphere and was

evaporated to dryness by rotovapping. The compound was precipitated from CH2Cl2

1 /Hexane to give a green solid. H NMR (400 MHz) in d6-DMSO, δ/ppm (multi, int, assignment) 7.2 (1H, d, quo), 7.58 (2H, m, quo), 7.75 (3H, 2H, t, py, 1H quo), 8.18 (1H, t, py), 8.6 (1H, d, quo), 8.8 (2H, d, py), 8.95 (2H, d, quo). Mass spectrum m/z = 548

+ -1 -1 ([Ru(quo)2(py)2 + H] ).Uv-vis in CH3CN, λ/ nm (ε/ M cm ): 243 (89 618), 253 (77

268), 321 (15 438), 426 (16 071).

Synthesis of [Ru(phpy)(CH3CN)4](PF6)

6 [Ru(phpy)(CH3CN)4](PF6) was prepared by cycloruthenation of the ammine [η -

4 C6H6)RuCl(μ-Cl)]2 as previously described. The ruthenium complex (1.5 g, 3.01 mmol),

KPF6 (2.22 g, 12.04 mmol), NaOH (0.48 g, 6.02 mmol) and arylpyridine (6.02 mmol)

0 were stirred in 50 mL of CH3CN at 45- 50 C for 20 hours. The resulting yellow slurry

42 was evaporated to dryness under reduced pressure, and the residue was purified by

column chromatography on Al2O3 using CH3CN as the eluent. Bright yellow crystals

were obtained by diffusion of ethyl ether into a concentrated solution of the yellow solid

1 in a mixture of CH2Cl2: CH3CN (1:1 v/v). H NMR (d3-CD3CN) , δ (int., mult): 8.89 (1H,

d), 7.95 (1H, dd), 7.86 (1H, d), 7.72 (1H, td), 7.70 ((1H, d), 7.15 (1H, td), 7.07 (1H, td),

6.95 (1H, td), 2.49 (3H, s), 2.13 (3H, s), 1.94 (6H, s). MS: 419 (5 %) [M + H]+, 379 (63

%) [(M + H)- MeCN]+, 338 (37 %) [(M + H) – 2MeCN]+, 297 (45 %) [(M + H) - 3

MeCN]+, 256 [(M + H) - 4 MeCN]+.

Synthesis of cis-[Ru(phpy)(bpy)(CH3CN)2](PF6)

Cis-[Ru(phpy)(bpy)(CH3CN)2](PF6) was synthesized following a known

5 procedure. A solution of [Ru(phpy)(CH3CN)4](PF6) (0.865 mmol) and 2,2-bipyridine

(143 mg, 0.796 mmol) was stirred at room temperature in 30 mL of CH2Cl2. The resulting deep purple solution was evaporated to dryness under reduced pressure and the residue was purified by column chromatography on Al2O3 using CH2Cl2 as an eluent. The

purple crystals were ontained by slow diffusion of diethyl ether into a concentrated

1 solution of toe purple solid in a mixture of CH2Cl2: CH3CN (1:1 v/v). H NMR (d3-

CD3CN) , δ (int., mult):9.35 (1H, d), 8.44 (1H, d), 8.20 (2H, m), 7.90-7.75 (4H, m), 7.65

(1H, td), 7.50 (1H, td), 7.43 (1H, dd), 7.28-7.14 (2H, m), 7.08-6.98 (2H, m), 6.72 (1H,

+ + td), 2.21 (3H, s), 2.14 (3H, s) MS (FAB ): 653 (29 %) [(M + H) + PF6] , 508 (28 %) [(M

+ H)]+, 467 (30 %) [(M + H)- MeCN]+, 426 (98 %) [(M + H)- 2 MeCN]+, 270 (20 %)

[(M + H)- 2 MeCN- bpy]+, 43 Synthesis of [Ru(Phpy)(bpy)2](PF6)

6 [Ru(Phpy)(bpy)2](PF6) was synthesized using a reported procedure. Cis-

[Ru(bpy)2Cl2] (0..968 g, 0.2 mmol) and 2-phenylpyridine (0.154 g, 0.1 mmol) was

refluxed in CH2Cl2 in the presence of silver(I). The crude material was purified by

column chromatography on Al2O3 eluting with toluene-CH3CN (4:1). The complex was

1 obtained as red solid (74%). %). H NMR in CD3CN -d6, δ (int., mult): 8.43 (1H, d), 8.36

(1H, d), 8.30 (2H, t), 8.05-7.94 (3H, m), 7.85-7.77 (5H, m), 7.74-7.66 (3H, m), 7.56 (1H,

dd), 7.40 (1H, dd), 7.32-7.17 (3H,m), 6.94-6.79 (3H,m), 6.42 (1H, dd,); ESI MS m/z

568.12 [M – PF6].

3.2 INSTRUMENTATION

The photolysis of Ru(II) complexes was carried out on a 150 W Xe arc lamp (PTI

220) housed in a Milliarc compact arc lamp housing powered by a Model LPS-220 lamp power supply or on a 450 W Xe lamp Housing A6000 powered by a Model LPS-500

lamp power supply which was cooled by a continuous flow of water through a quartz glass chamber with a minimum flow rate of 500 ml / min which was positioned at 412

mm from the front of the lamp. The wavelength was controlled by using either a long-

pass colored glass filter or by using bandpass filters of wavelengths 350 nm (10LF10,

Newport), 400 nm (57510, Oriel), and 450 nm (57 530, Oriel). Photolysis experiments

were conducted in 1 x 1 x 4 cm quartz cuvette. The cuvette was positioned in the focal

point of a either the 150W or 450 W lamps.

44 Emission lifetime measurements were conducted either on an Edinburgh nF900 single photon counting instrument with fitting software or on a home-built time-resolved system using the frequency doubled (532 nm) or tripled (355 nm) output of a Spectra-

Physics GCR-150 Nd:YAG laser (fwhm ~8 ns, ~5 mJ / pulse) as the excitation source.

The emission wavelength was controlled using a Jobin Yvon monochromator and was detected with a red-sensitive PMT (Hamamatsu R928). The signal was digitized on a

Tektronics 400 MHz oscilloscope (TDS 380), and the data were collected on a PowerMac

7600/132 (Apple) equipped with a National Instruments GPIB interface (NI-488.2) and a

National Instruments data acquisition board (PCI-1200) programmed with Labview 4.1 software.

Electronic absorption spectra were recorded using a Hewlett Packard Diode Array

Spectrometer (HP 8453) with HP 8453 Win System software and emission spectra were collected on ISA Spex-Flouromax-2 spectrometer with a 90o optical geometry equipped with a 150 W Xenon source. DataMax-Std software on a Pentium microprocessor was utilized for data acquisition and analysis. 1H NMR spectra were recorded on a 400 MHz

Bruker istrument. Matrix-assisted laser desorption/ionization (MALDI) measurements were performed on a Bruker Reflex III mass spectrometer operated in the reflection negative ion mode with a N2 laser. The ethidium stained agarose gels were image using a

GelDoc 2000 illuminator (Biorad) equipped with Quality One software.

45 3.3. METHODS

Solutions were dearated with argon by passing the gas through a syringe needle

into the reaction vessel that was sealed from the atmosphere using a rubber septum. The pressure developed during this time was allowed to escape through a needle outlet placed at the top of the reaction vessel in another rubber septum.

Quantum yield measurements were taken relative to potassium ferrioxalate, by a

previously reported method.7 Quantum yields measurements were calculated from

absorbance data collected at several wavelengths, λ = 350, 400, and 450 nm. The photon

2+ flux was first determined by calculating the moles of Fe , nFe2+, produced when a solution

3+ of K3Fe(C2O4)3 in aqueous sulphuric acid was irradiated by light. The Fe ions are

reduced to Fe2+ via the following reaction:

3+ 3- hν 2+ 2- - [Fe (C2O4)3] [Fe (C2O4)3] + C2O4

2+ The product Fe(C2O4)2 does not absorb incident light and the Fe ions can be

spectrophotometrically determined as a red colored complex with 1, 10-phenanthroline

2+ which absorbs at λmax = 510nm. The moles of Fe produced upon irradiation is

therefore calculated according to the following equation:

20 2+ nFe = 6.023 x 10 V1V3A/ V2lε (1)

where V1 is the volume of actinometer solution irradiated (2 ml), V2 is the volume of

aliquot taken for analysis (1 ml), V3 is the final volume to which aliquot V2 is diluted (10

4 ml), ε is the molar extinction coefficient of potassium ferrioxalate(1.15 x 10 lmol-1cm-1), and A is the absorbance of potassium ferrioxalate at 510 nm. K3Fe(C2O4)3 is used as the

46 standard due to its wide spectral range of quantum yield and wavelength that is

8 known. The quanta absorbed by the actinometer (na) can be calculated using equation 2:

+2 na = nFe / φλ (2)

8 In eq 2, φλ is the quantum yield at 350 nm,1.21. Since the photon flux is the number of

photons absorbed per second, a plot of na versus irradiation time results in a straight line

graph, slope of which is equal to the photon flux. The changes in the absorbance due to

the formation of each Ru(II) complex was used to determine the concentration of the

product at a give time with irradiation of a certain wavelength. Since the quanta absorbed

at a given irradiation time is known (na)t. Equation 3 can then be applied to calculate the

+2 quantum yield where the slope of a plot of nRu vs. photon absorbed is the quantum yield

of the reaction.

+2 Φλ = (nRu )t / (na)t (3)

Cyclic voltammetry measurements were performed in dry CH3CN at a platinum-

disk working electrode with 0.1 M [Bu4N][BF4] as the supporting electrolyte on a BAS

CV-50W voltammetric analyzer. The measurements were made vs the saturated sodium

+/0 calomel electrode (SSCE). The scan rate used was 100 mV/s. Ferrocene (E1/2(Fc ) =

0.425 V vs SCE in CH3CN) was added as an internal standard following each experiment.

Emission spectra of samples in methanol:ethanol (1:4 v/v) were obtained by

degassing the solution with nitrogen for 20 minutes prior to each experiment. Spectra at

77 K were recorded using a liquid nitrogen Dewar with quartz windows and the sample

was either a solid or in a 4:1 mixture of ethanol:methanol in an NMR tube inside the

dewar. The quantum yield of emission at room temperature were calculated relative to

47 2+ that of [Ru(bpy)3] as the standard using equation 4; where As is the absorbance of the

standard, Aem is the area of emission of the standard, and ηs is the refractive index of the

solvent that is used for the standard.

-As -A s 2 φem = φstd x (1-10 )/ (1-10 ) x (Aem / Aem ) x (η / ηs) (4)

The quantum yield of emission of the Ru(II) complexes at low temperature (77 K) was

calculated according to equation 5.

LT RT φLT = Aem / Aem x φRT (5)

Annealing of two complementary single stranded 15 mers oligonucleotides was

performed by heating a mixture of 1 mM of each strand (5 mM Tris, pH = 7.5, 50 mM

NaCl) to 90 oC for 5 min and allowing the mixture to cool to room temperature over a

period of 3 hrs inside the heating block. Melting Temperature experiments were

performed by heating 50 μM of double stranded DNA with 5 μM metal complex in 2 mM NaCl and 1 mM Tris Buffer pH = 7.2 and the reaction followed on a Hewlett-

Packard diode array spectrometer (HP 8453) equipped with an HP 89090A temperature controller and HP 8453 Win System software monitored at 260 nm.

Gel mobility experiments were carried out by first linearizing 100 μM pUC18

plasmid DNA with 10 units of Sma I in 100 mM Tris, pH = 7.6, 100 mM MgCl2, 150 mM NaCl at 37 oC for 1 hr, followed by deactivation at 65 oC for 5 min. The

concentration of plasmid DNA was spectrochemically determined using an extinction coefficient of 6 600 M-1cm-1 at 260 nm. The ratio of DNA base pairs (bp) to metal

complex (mc) was varied from ranging from (bp):(mc) 100 to 5. For cisplatin, the

solutions containing metal were incubated at 40 oC for 4 hrs in 5 mM phosphate buffer,

48 pH = 7.2 before adding 4 μL of loading dye to each sample. Control samples containing

molecular weight standard and the linearized plasmid were not incubated. The solutions

were loaded onto a 0.8 % agarose mixture containing 0.09 M Tris, 0.002 M disodium salt of EDTA, 0.09 M boric acid, pH 7.5.The gel electrophoresis was conducted at 105 V for

1.5 hrs. The gels were stained with 0.5 μg / ml EtBr for 30 min and were kept in a water

bath for 15 min before imaging on a Gel Doc 2000 (Biorad) transilluminator equipped

with Quality One software. The Ru(II) complexes were irradiated at different

wavelengths ranging from 15 to 30 min at 25 0C depending on the Ru(II) complex used

before loading each sample on the gel.

Viscosity measurements were performed using 1 mM sonicated sperm herring

DNA in 5 mM Tris buffer pH = 7.5 and 50 mM NaCl. The relative concentration of the

metal complexes to the DNA was varied so that the final ratio of metal complex to DNA

(R) was 0.05, 0.1, 0.15, 0.20, 0.25, 0.30. Relative viscosity measurements were plotted as

1/3 (η / ηo) vs R, where η = t – to and ηo = tDNA – to, t is the observed flow time for each ratio of metal complex to DNA, tDNA is the flow time of the DNA and buffer only and to

is the flow time of the buffer alone. The flow time was recorded three times for each

DNA:metal ratio and an average flow time was calculated.

49 References

1. Bryant, G. M.; Ferguson, J. E.; Powell, H. K. Aust. J. Chem. 1971, 24, 257-273.

2. Asesefa, Z.; Stanbury, D. J. Am. Chem. Soc. 1997, 119, 521-530.

3. Brown, G. M.; Weaver, T. R.; Keene, F. R.; Meyer, T. J. Inorg. Chem. 1970, 15, 189- 196.

4. Fernandez, S.; Pfeffer, M.; Ritleng, V.; Sirlin, C. Organometallics 1999, 18, 2390- 2394.

5. (a) Ryabov, A. D.; Le Lagadec, R.; Estevez, H.; Toscano, R. A.; Hernandez, S.; Alexandrova, L.; Kurova, V. S.; Fischer, A.; Sirlin, C.; Pfeffer, M. Inorg. Chem. 2005, 44, 1626-1634. (b) Ryabov, A. D.; Sukharev, V. S.; Alexandrova, L.; Le Lagadec, R.; Pfeffer, M. Inorg. Chem. 2001, 40, 6529-6532.

6. Constable, E. C.; Holmes, J. M. J. Organomet. Chem. 1986, 301, 203-208.

7. Murov, S. L.; Carmichael, I.; Hug, G. L. “Handbook of Photochemistry” Second edition, Marcel Dekker: New York; 1993.

8. Demas, J. N.; Bowman, W. D. J. Phys. Chem. 1981, 85, 2771-2777.

50

CHAPTER 4

2+ PHOTOREACTIVITY OF cis-[Ru(bpy)2(CH3CN)2] AND cis- 2+ [Ru(bpy)2(NH3)2]

4.1 Photophysical Properties

4.1.1 Electronic Absorption and Emission

2+ The electronic absorption spectrum of cis–[Ru(bpy)2(CH3CN)2] was previously

1 reported and is shown in Figure 4.1 in H2O. The two intense bands observed in the

ultraviolet (uv) region at 243 nm (19,900 M-1cm-1) and at 283 nm (57,200 M-1cm-1) have been previously assigned as arising from π π* intraligand transitions of the bpy

2+ ligand. Similarly, cis-[Ru(bpy)2(NH3)2] exhibits two intense bands at 243 nm (ε = 20

-1 -1 -1 -1 600 M cm ) and 290 nm (ε = 55 500 M cm ) in H2O which have also been assigned to

intraligand transitions of the bpy ligand (Figure 4.1).2 The latter complex was previously

synthesized and reported by T. J. Meyer, and the results obtained here agree well with

those previously reported.1,2 Peaks of lower intensity were observed for cis–

2+ -1 -1 -1 -1 [Ru(bpy)2(CH3CN)2] at 425 nm (8,900 M cm ) and at 490 nm (ε = 8,210 M cm ) for

2+ cis-[Ru(bpy)2(NH3)2] , which can be assigned to Ru bpy(π*) metal-to-ligand charge

51 0.20

0.15

0.10

Absorbance (a.u.) Absorbance 0.05

0.00 300 400 500 600 700 800 Wavelength (nm)

2+ Figure 4.1. Electronic absorption spectra of cis-[Ru(bpy)2(CH3CN)2] (⎯⎯) and cis- 2+ [Ru(bpy)2(NH3)2] (− − − −) in H2O.

transfer (MLCT).1,2 An additional peak was also observed at 345 nm (ε = 7, 340 M-1cm-1)

2+ 1 for cis-[Ru(bpy)2(NH3)2] in water, which was also assigned as a MLCT transition. For

2+ comparison, [Ru(bpy)2(en)] was also synthesized here and exhibits ππ* transitions at

243 nm (ε = 19,600 M-1cm-1) and 291 nm (ε = 57,500 M-1cm-1), as well as an MLCT

transition at 485 nm (ε = 9,750 M-1cm-1). These assignments are consistent with those for

2+ the homoleptic [Ru(bpy)3] compound, where peaks observed in the uv region at 243 nm

(ε = 27,245 M-1cm-1) and at 290 nm (ε = 74,056M-1cm-1) are assigned as ligand centered transitions, and the MLCT transition in the visible region appears at 453 nm (ε = 14,600

-1 -1 3,4 2+ M cm ). The large blue shift in the MLCT band of cis–[Ru(bpy)2(CH3CN)2]

2+ compared to cis-[Ru(bpy)2(NH3)2] can be explained in terms of the greater ligand field 52 stabilization energy of the CH3CN ligands in the former complex compared to the NH3 in

the latter. Acetonitrile ligands are strong π-acceptors, and hence are expected to be

stronger field ligands, whereas the ammines are σ-donor only ligands and would be

expected to be weaker field ligands than CH3CN.

2+ Cis–[Ru(bpy)2(CH3CN)2] exhibits very weak emission at room temperature in

methanol with maximum at 610 nm (φ = 0.0001, τ = <10 ns), whereas at 77 K in

ethanol:methanol (4:1 v:v) the compound exhibits strong emission with maximum at 540

nm (φ = 0.227, τ = 5.4 μs) and a shoulder at 582 nm (Figure 4.2). These results agree well

with those in the literature for this complex, where a strong emission was reported at 540

nm with a shoulder at 580 nm in methanol:ethanol (5:1 v/v) at 77 K.5 The peak positions

1.2

1.0

0.8

0.6

Intensity (AU) 0.4

0.2

0.0 300 400 500 600 700 800 Wavelength (nm)

Figure 4.2. Excitation (λem = 542 nm, ----) and emission (λexc = 420 nm, ⎯) spectra of 2+ cis-[Ru(bpy)2(CH3CN)2] at 77 K in (4:1 v:v) EtOH:MeOH.

53 2+ in the excitation spectrum of cis–[Ru(bpy)2(CH3CN)2] (Figure 4.2) coincides with those

in the absorption spectrum (Figure 4.1) indicating that emission arises from the

2+ 2+ photoexcited cis–[Ru(bpy)2(CH3CN)2] complex. Cis-[Ru(bpy)2(NH3)2] exhibits very

weak emission in H2O with a maximum at λ = 710 nm at room temperature (Figure 4.3).

2+ Cis-[Ru(bpy)2(NH3)2] was previously reported to have a weak MLCT emission at 741

nm (φ = 0.002, τ = 52 ns) and at 715 nm (φ = 0.003, τ = 173 ns) in methanol:ethanol glass

5 2+ at 157 K. A weak emission was also reported for cis-[Ru(bpy)2(NH3)2] in

5 CH3OH:EtOH at 77 K at 690 nm (φ = 0.015, τ = 369 ns), and is characterized by a well-

defined vibronic progression with Δν~1132 cm-1. Similarly, vibronic progression was

2+ -1 also observed for cis-[Ru(bpy)2(CH3CN)2] with Δν ~ 1,336 cm . Emission spectra for

2+ these complexes at 77 K are consistent with that of the prototypical [Ru(bpy)3] complex, with MLCT emissive excited states and bipyridine as the chromophoric ligand.6

500000

450000

400000

350000

300000

250000

Intensity (cps) Intensity 200000

150000

100000

50000

0 400 450 500 550 600 650 700 750 800 850 Wavelength (nm)

2+ Figure 4.3. Emission spectrum of cis-[Ru(bpy)2(NH3)2] (λexc = 450 nm) in water at room temperature. 54 2+ 3,4 [Ru(bpy)3] exhibits strong emission at λ = 605 nm in methanol. The vibronic

2+ progression observed in the 77 K emission spectra of cis-[Ru(bpy)2(NH3)2] and cis-

2+ [Ru(bpy)2(CH3CN)2] is assignable to a normal mode of the bpy ligand and agrees well

2+ -1 3- 8 2+ with related compounds and with [Ru(bpy)3] (Δν ~1350 cm ). [Ru(bpy)3] possesses

a long lived emissive 3MLCT excited state at low temperature (τ = 5.21 μs), which can be deactivated by processes that include radiative decay (kr), radiationless decay (knr) and

the thermal population of non-emissive metal-centered lignad-field (LF) excited states

located at energies greater than that of the 3MLCT.

2+ The emission lifetime observed of cis-[Ru(bpy)2(CH3CN)2] at room temperature

2+ is <10 ns while that observed for cis-[Ru(bpy)2(NH3)2] is 64 ns. These lifetimes are

3 2+ 9 significantly shorter than that of the MLCT state of [Ru(bpy)3] at 298 K, 0.6 μs. The

2+ shorter emission lifetime observed for cis-[Ru(bpy)2(CH3CN)2] compared to cis-

2+ 2+ 3 [Ru(bpy)2(NH3)2] and [Ru(bpy)3] implies that the MLCT of the former is subject to

2+ 2+ more efficient non-radiative decay relative to cis-[Ru(bpy)2(NH3)2] and [Ru(bpy)3] .

Van Houten and Watts calculated the energy difference between the MLCT state and the deactivating dd LF excited state to be 3600 cm-1 in water.28 The values of the radiative

and non-radiative rate constants, kr and knr, respectively were calculated for cis-

2+ 2+ [Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] using eq 1 and 2 from the emission

lifetime (τ) and the quantum yield of emission (φem) at 77 K. The values of kr and knr

obtained from eq 1 and 2 were 4.2 x 104 s-1 and 1.4 x 105 s-1 for cis-

2+ 4 -1 6 -1 [Ru(bpy)2(CH3CN)2] respectively, and 3.9 x 10 s and 2.7 x 10 s for cis-

2+ [Ru(bpy)2(NH3)2] respectively (Table 1), while the values of kr and knr obtained for

55 2+ 4 -1 [Ru(bpy)3] from τ of 5.21 μs and Φem of 0.35 reported by Ford are 6.7 X 10 s and 1.8

x 105 s-1, respectively.28

kr = φem / τ (1)

φem = kr / (knr + kr) (2)

2+ 2+ The values obtained for cis-[Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] here are consistent with emission data collected for other Ru(II) complexes at low temperature (Table 1).10- 12 The non-radiative decay rate constant obtained for the

complexes here can be explained in terms of the energy gap law,13,14 which states that

increasing the energy of the MLCT excited state is accompanied by a decrease in the

non-radiative decay constant, which results in an increase in the emission quantum yield and lifetime. The energy gap law has been successfully applied to nonradiative decay in various polypyridyl complexes including those of Ru(II), Re(I) and Os(II).15 Analysis of lifetimes based on the energy gap law is valid only for nonradiative decay from the lowest MLCT state. Hence, the enhanced non-radiative decay observed for cis-

2+ [Ru(bpy)2(NH3)2] relative to the other complexes in Table 1, can be explained by a

lowering of the MLCT excited state of the complex compared to cis-

2+ 2+ 2+ [Ru(bpy)2(CH3CN)2] , [Ru(bpy)3] , and [Ru(bpy)2(pic)2] which qualitatively

translates to a decrease in the emission quantum yields and emission lifetime. Table 1

3 2+ shows that the energy of the emission from the MLCT state of cis-[Ru(bpy)2(NH3)2] is

–5 lower than those of the other complexes, resulting in a greater values of knr (26.5 X 10 s-1) and a lower emission quantum yield (φ = 0.0145) and lifetime (τ = 0.369 μs)

2+ 2+ compared to cis-[Ru(bpy)2(CH3CN)2] and [Ru(bpy)3] , in agreement with the energy

56 2+ gap law. The emission properties observed for cis–[Ru(bpy)2(CH3CN)2] and

2+ –5 -1 –5 -1 [Ru(bpy)3] at 77 K are very similar, with knr values of 1.4 X 10 s and 1.8 X 10 s , respectively (Table 1). However, at room temperature significant differences are observed between the two complexes. The primary reason for observing strong low temperature emission (77K) for these complexes and very weak room temperature emission is attributed to the fact that at low temperature there is less deactivation of the

3MLCT by thermal population of the dd state from the 3MLCT and hence deactivation is

by emission of the triplet excited state to the ground state. Another reason for observing low temperature emission is that at low temperature the solvent is a solid and hence there is negligible vibrational movement of the solvent resulting in very small energy transfer

from the metal complex to the solvent hence the molecule becomes more emissive.

Emission Metal complexes 4 -1 5 -1 λmax (nm) τ (μs) φ kr (10 s ) knr (10 s ) cis-[Ru(bpy) (CH CN) ]2+ 2 3 2 540 5.41 0.227 4.20 1.43

cis-[Ru(bpy) (NH ) ]2+ 2 3 2 690 0.369 0.0145 3.93 26.7

[Ru(bpy) ]2+ 3 578 (a) 5.21(a) 0.350 (a) 6.72 1.85

[Ru(bpy) (pic) ]2+(b) 2 2 592 (c) 5.6 (c) 0.410 (c) 7.32 1.05 a From ref 28, b From ref 29

Table 4.1. Low temperature emission (77 K) data collected for cis- 2+ 2+ [Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] compared to other Ru(II) compounds.

57 2+ The emission maximum at room temperature for cis–[Ru(bpy)2(CH3CN)2]

2+ occurs at 610 nm in methanol, whereas [Ru(bpy)3] emits at 630 nm at room temperature

28 the same solvent (Table 2). According to the energy gap law, the knr value obtained for

2+ 2+ cis–[Ru(bpy)2(CH3CN)2] should be lower than that of [Ru(bpy)3] , since the energy of

2+ 2+ the triplet excited state is higher in cis–[Ru(bpy)2(CH3CN)2] than [Ru(bpy)3] .

Therefore, it would be expected that the emission lifetime and quantum yield of cis–

2+ 2+ [Ru(bpy)2(CH3CN)2] would be greater than that observed for [Ru(bpy)3] . However, a

lower quantum yield of emission was observed at room temperature for cis-

2+ 2+ [Ru(bpy)2(CH3CN)2] (φ = 0.227) than for [Ru(bpy)3] (φ = 0.350). This result can be

2+ explained by the ligand loss from cis–[Ru(bpy)2(CH3CN)2] , that will be explained in a

later section.

Emission Metal complexes 4 -1 7 -1 λmax (nm) τ (ns) φ kr (10 s ) knr (10 s ) cis-[Ru(bpy) (CH CN) ]2+ 2 3 2 610 < 10 .0001 < 1.11 < 11.1

cis-[Ru(bpy) (NH ) ]2+ 2 3 2 700 64 0.001 1.50 1.49

[Ru(bpy) ]2+ 3 630 (a) 600(a) 0.042 (a) 7.00 0.160

a from ref 28

2+ Table 4.2. Room temperature emission data collected for cis-[Ru(bpy)2(CH3CN)2] and 2+ cis-[Ru(bpy)2(NH3)2] compared to other known Ru(II) compounds.

58 4.2 Photochemistry in solution

4.2.1 Photochemistry in H2O

2+ 2+ The photolysis of cis–[Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] in water

(350 nm < λirr < 450 nm) results in the sequential loss of the two CH3CN and NH3 ligands, respectively. The changes to the electronic absorption spectrum of cis–

2+ [Ru(bpy)2(CH3CN)2] in water show the formation of an intermediate species upon

photolysis, which absorbs at 450 nm after 2 min of irradiation (Figure 4.4). Continued photolysis leads to an increase in the absorption at 490 nm (Figure 4.4). The former species is believed to correspond to the loss of a single acetonitrile ligand, resulting in the

2+ formation of the monosubstituted compound, cis-[Ru(bpy)2(CH3CN)(OH2)] . This

2

1

Absorbance (a.u.)

*

0 200 300 400 500 600 700 Wavelength (nm) Figure 4.4. Changes to the electronic absorption spectrum of cis-

[Ru(bpy)2(CH3CN)2](PF6)2 in water during photolysis (λirr > 420 nm) in water at t = 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 min. The * denotes the spectrum collected at 2 min.

59 2+ complex can lose another CH3CN ligand and generate cis-[Ru(bpy)2(OH2)2] which is

known to absorb at 490 nm. The assignment of the identity of the cis-

2+ [Ru(bpy)2(CH3CN)(OH2)] species was based on the comparison of its absorption

2+ spectrum to those of cis-[Ru(bpy)2(CH3CN)(OH2)] and trans-

2+ 16 [Ru(bpy)2(CH3CN)(OH2)] previously reported by Durham in 1 N H2SO4 . Absorption

2+ maxima of 445 nm and 465 nm were reported for cis-[Ru(bpy)2(CH 3CN)(OH2)] , and

2+ 21 trans-[Ru(bpy)2(CH 3CN)(OH2)] , respectively. The absorption observed at 450 nm

2+ upon irradiation of cis–[Ru(bpy)2(CH 3CN) 2] in water with (350 nm < λirr < 450 nm) at

2+ early times corresponds well to that reported for cis-[Ru(bpy)2(CH 3CN)(OH2)] and was

therefore assigned as this complex. The assignment is reasonable since continued

2+ irradiation leads to the formation of the cis-bis-aqua complex, cis-[Ru(bpy)2(OH 2) 2] .

This interpretation is consistent with the known molar absorption coefficients of cis-

2+ -1 -1 2+ [Ru(bpy)2(NH 3) 2](ε490 nm= 8, 900 M cm ) and cis-[Ru(bpy)2(OH 2) 2](ε490 nm= 9, 300

± 200 M-1cm-1).

2+ The photolysis of cis-[Ru(bpy)2(NH3)2] in water with 350 nm < λirr < 400 nm

also resulted in the sequential loss of the NH3 ligands and the formation of the bis-aqua

2+ complex. Irradiating cis-[Ru(bpy)2(NH3)2] with high energy in the near-uv region (λirr >

375 nm) resulted in significant changes in the absorption spectrum. Figure 4.5a shows the changes in the absorption spectrum when the compound is irradiated at early times with

λirr > 375 nm. Isosbestic points are observed at 410 nm and 580 nm, which is indicative

of the formation of single species from the starting material. Further irradiation with λirr >

60 375 nm, resulted in significant changes in the absorption spectrum, which indicates that

an additional new species is formed (Figure 4.5b).

Irradiating the complex with higher energy light (λirr > 345 nm), resulted in more pronounced changes in the absorbance spectrum to those obtained with λirr > 375 nm. A

significant increase in the absorbance at 490 nm was observed when the complex was

photolyzed at λirr > 345 nm, which is indicative of the formation of cis-[Ru

2+ (bpy)2(OH2)2] . The formation of the mono-substituted intermediate species was not observed at this irradiation wavelength (λirr > 345 nm), suggesting that excitation with

higher energy light is necessary for the direct loss of both ammine groups. It is known

2+ 17 that complexes of the type [Ru(L)2(OH2)2] can undergo cis-trans isomerisation, which can be readily detected through changes in the absorption spectra of the metal complexes.18 However, cis-trans isomerization was not detected in the experiments

presented here.

2+ Cis-[Ru(bpy)2(OH2)2] was independently synthesized and is known to have an

absorption maximum at 490 nm (ε = 9,300 M-1cm-1).19 No changes were observed in the

2+ electronic absorption spectrum when cis–[Ru(bpy)2(CH3CN)2] or cis-

2+ [Ru(bpy)2(NH3)2] were kept in the dark in water over a 24 hour period. Cis-trans

isomerization can also be ruled out since no changes are observed in the dark following

2+ 2+ photolysis for 24 hours. Cis-[Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] are resistant to thermal substitution since no changes were observed in the absorption spectrum upon refluxing the complexes in water for 3 hours. Thermal resistance and

2+ 20 photochemical reactivity of cis-[Ru(bpy)2(CH3CN)2] was also reported by Meyer.

61 (a)

0.2

Absorbance 0.1

0 300 400 500 600 700 800 Wavelength (nm)

(b) 0.3

0.2

Absorbance 0.1

0 300 400 500 600 700 800 Wavelength (nm)

2+ Figure 4.5. Changes in the absor[tion spectrum of cis-[Ru(bpy)2(NH3)2] in H2O upon irradaition (λirr > 375 nm) at (a) early times 0, 1,2, 3, 4, 5 min and (b) later times 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 min. 62 2+ The mechanism for the photolysis reactions of cis-[Ru(bpy)2(CH3CN)2] and cis-

2+ [Ru(bpy)2(NH3)2] in water appears to be similar, and is proposed to occur when

irradiated with wavelengths 345 nm < λirr < 395 nm. A proposed mechanism is shown in

Scheme 1.

2+ hν 2+ cis-[Ru(bpy)2(L)2] cis-[Ru(bpy)2(L)] -L

H2O

2+ cis-[Ru(bpy)2(L)(OH2)]

hν hν -H2O -L H2O

2+ 2+ cis-[Ru(bpy)2(OH2)] cis-[Ru(bpy)2(L)]

H2O

2+ cis-[Ru(bpy)2(OH2)2]

2+ Scheme 4.1. Proposed mechanism for the formation of cis-[Ru(bpy)2(H2O)2] upon

2+ continuous irradiation of cis-[Ru(bpy)2(L)2] (L = CH3CN, NH3) in water.

2+ As shown in Scheme 1, irradiation of cis-[Ru(bpy)2(CH3CN)2] in water with λirr

> 420 nm results in the substitution of one of the CH3CN groups to form the mono-aqua

adduct, which upon continued irradiation leads to the loss of the second CH3CN ligand to

2+ form the bis-aqua adduct complex. Solutions containing cis-[Ru(bpy)2(CH3CN)2] in water were irradiated with λirr > 420 nm until the appearance of the peak at 450 nm was

observed, after which time the solution was kept in the dark. No changes in the 63 absorbance spectra were observed for these solutions over a one hour period in the dark,

2+ indicating that the formation of cis-[Ru(bpy)2(H2O)2] from cis-

2+ 2+ [Ru(bpy)2(CH3CN)(H2O)] requires a photon. For cis-[Ru(bpy)2(NH3)2] , the fast

increase in absorption at 450 nm within the first 2 min of irradiation (λirr > 375 nm) is

2+ consistent with the initial photoconversion of cis-[Ru(bpy)2(NH3)2] to cis-

2+ [Ru(bpy)2(NH3)(OH2)] . Continued irradiation eventually results in the formation of cis-

2+ [Ru(bpy)2(OH)2] . The fact that this is a slower process can be explained by the possible

2+ loss of either NH3 or H2O from cis-[Ru(bpy)2(NH3)(OH2)] upon absorption of a photon.

As shown in Scheme 1, the former results in the desired product, whereas the latter

2+ simply regenerates cis-[Ru(bpy)2(NH3)(OH2)] . The observation that irradiation with

higher energy light results in greater photochemical reaction indicates that population of

higher energy states is necessary for efficient photosubstitution to occur.

The results presented here are consistent with one photon necessary to remove

each CH3CN or NH3 ligand. Similar results were observed for the photoanation reaction

2+ 21 2+ 22 of cis-[Ru(bpy)2(py)2] , as well as the photoaquation of cis-[Ru(NH3)4(py)2] .

The quantum yield of the photosubstitution reaction of the following reaction was calculated at various wavelengths in water using potassium ferrioxalate as a standard for

2+ 23 cis-[Ru(bpy)2(L)2] (L = CH3CN, NH3).

cis-[Ru(bpy) (L) ]2+ hν 2+ 2 2 cis-[Ru(bpy)2(OH2)2]

2+ The quantum yield for the formation of cis-[Ru(bpy)2(OH2)2] upon irradiation of cis-

2+ [Ru(bpy)2(CH3CN)2] at various irradiation wavelengths in water were calculated,

resulting in φ350 = 0.377(3), φ400 = 0.207(1) and φ450 = 0.222(18), while those calculated 64 2+ upon irradiation of cis-[Ru(bpy)2(NH3)2] in water were φ350 = 0.024(2) and φ400 = 0.018

2+ (2). The values of the quantum yields measured here for cis-[Ru(bpy)2(CH3CN)2] are

1 similar to those previously reported for the complex, φ283 = 0.115 (9), φ414 = 0.199 (9)

1,6 and φ426 = 0.31 (1). The quantum yield of photoanation with chloride ions at various

2+ concentrations with complexes of the type cis-[Ru(bpy)2Y2] (Y = pyridine, 4-

acetylpyridine, 3-iodopyridine, 4,4’-bpy) have been reported in CH2Cl2 and the quantum

2+ yields obtained are similar to that calculated for cis-[Ru(bpy)2(CH3CN)2] in H2O (Table

3).6

Emission c λ , -1 E (III/II), Metal complex max 0-0, cm φa 1/2 cm-1 x103a V x 103b 2+ cis-[Ru(bpy)2(CH3CN2)] 23.47 18.42 0.31 1.44 2+ cis-[Ru(bpy)2(py)2] 22.03 17.10 0.20 1.30 2+ cis-[Ru(bpy)2(4,4’-bpy)2] 22.37 17.32 0.20 1.32 2+ cis-[Ru(bpy)2(4,4’-acpy)2] 22.62 17.36 0.29 1.45 2+ cis-[Ru(bpy)2(4,4’-iodopy)2] 22.62 17.40 0.24 1.36 a b determined in CH2Cl2 Highest energy maxima at 77 K in 1:1 methanol-ethanol glass c determined in CH3CN with TBAH vs. SSCE

Table 4.3. Absorption maxima of MLCT transition, quantum yield of photoanation with - Cl (tetrabutyl ammonium chloride) anion at 436 nm and E1/2 (II/III) values obtained for 2+ 24 cis-[Ru(bpy)2(L)2] complexes

Most members of the series in Table 3 exhibit absorption maxima that are similar to that

2+ 3 -1 of [Ru(bpy)3] (22.07 x 10 cm ), most specifically, the visible spectra are dominated

by single absorption bands. Other spectral similarities that are observed include nearly

constant differences between the emission energy and the absorption energy (4.96 ± 0.17 x 103 cm-1), as well as similar energy spacing in the vibrational progression of the

65 emission spectra. The quantum yield of emission was also observed to correlate linearly

with the oxidation potentials of the complexes. The interrelations of the absorption

energies, and the electrochemical potentials indicates that the bipyridine orbitals play a dominant role in the spectroscopy of the complexes in Table 3, and hence may also play a role in their photochemistry since the lowest energy excited state in all the molecules

5 1 derives from an excited state with electronic configuration (t2g) (π*-bpy) .

It was suggested that photosubstitution in the complexes listed in Table 3 took

place from a LF state that was thermally populated from a lower lying MLCT state.26

This interpretation was consistent with the photochemical model proposed by Watts and

2+ 24 coworkers for [Ru(bpy)3] . The nature of the monodentate ligand in the complexes in

Table 3 took place was also observed to influence the efficiency of populating the dd

state from the lower MLCT state. The most likely mechanism by which this population

can occur is by reducing the energy difference between the MLCT and the dd states.

III/II However, the large variation in the metal-centered oxidation potential E1/2(Ru ), and very small variation in bpy-centered reduction potential indicates that variations in the visible absorption and emission energies of the complexes can be attributed to variations

III/II in the metal-centered t2g orbital. Table 3 also shows that E1/2(Ru ) is influenced by the

nature of the monodentate ligands, which can be explained in terms of the electron

density on the metal. For example, as the electron density on the metal is reduced in cis-

2+ III/II 2+ [Ru(bpy)2(CH3CN2)] (E1/2(Ru ) = 1.44 V) compared to cis-[Ru(bpy)2(py)]

III/II (E1/2(Ru ) = 1.30 V), the quantum yield of photoanation is reduced. In general, as the

π-acceptor ability of the monodentate ligands is increased, the electron density on the

66 metal is reduced making the metal center harder to oxidize. Based on the dependency of

III/II the quantum efficiency of ligand photosubstitution on E1/2(Ru ) it was proposed by

Durham that the charge on the metal may be a dominant factor in the energy between the

MLCT state and the dd states.26 This work showed that the greater the oxidation

potential, the smaller the energy difference between the MLCT state and the dd states, a

finding that supports the quantum yield of photoanation obtained with cis-

2+ [Ru(bpy)2(CH3CN2)] complex.

2+ The quantum yields of photoanation of cis-[Ru(bpy)2(CH3CN2)] and cis-

2+ 25 [Ru(bpy)2(py)2] in CH2Cl2 were also reported by Durham. The temperature

dependence of the quantum yield for the photoanation of these complexes was

investigated in excess tetra-butylammonium chloride and was compared to that of

2+ [Ru(bpy)3] . The variation in the quantum yield for the photoanation of cis-

2+ 2+ [Ru(bpy)2(CH3CN2)] and cis-[Ru(bpy)2(py)2] with temperature was small, resulting in

an Arrhenius activation energy of 700 cm-1 for both complexes. For comparison, the

2+ -1 activation energy for the photosubstitution in [Ru(bpy)3] was reported to be ---- cm .

2+ The low activation energies observed for cis-[Ru(bpy)2(CH3CN2)2] and cis-

2+ 2+ [Ru(bpy)2(py)2] suggest that the model proposed by Watts for [Ru(bpy)3] where

photosubstitution is a result of the thermal population of the dd state from the lower

MLCT state, does not apply for these complexes.

2+ The luminescence lifetime of cis-[Ru(bpy)2(L)2] (L = py, CH3CN) was also

investigated as a function of temperature, where an increase in the temperature resulted in

a decrease in the emission lifetime. These results parallel those reported by Caspar and

67 26 2+ 26 Meyer for a series of complexes of the type cis-[Ru(bpy)2L2] . The temperature

dependence of the emission lifetimes contrast the dependence of the photoanation

quantum yields. A nonlinear relation was observed for the luminescence lifetime as a

function of temperature, while a linear relationship was observed with the quantum

32 2+ yields. Durham suggested that the photosubstitution in cis-[Ru(bpy)2(CH3CN2)] and

2+ cis-[Ru(bpy)2(py)2] is not a result of thermal population of the d-d state as is the case in

2+ [Ru(bpy)3] .

Electrochemical measurements were conducted here in order to further investigate

2+ the mechanism of photosubstitution in cis-[Ru(bpy)2(CH3CN2)] . In the MLCT excited

states of the complex, oxidation of the Ru(II) center to Ru(III) takes place. It is possible

that the Ru(II) center is not as good as the Ru(II) for strong coordination of π-acceptors

CH3CN ligands. If this is indeed the case, then electrochemical oxidation may provide

2+ more evidence for the mechanism of photosubstitution of cis-[Ru(bpy)2(CH3CN2)] .

2+ Cyclic voltammograms of cis-[Ru(bpy)2(CH3CN2)] in degassed CH3CN showed a

III/II reversible oxidation potential of E1/2(Ru ) = 1.45 V vs SCE (Figure 4.6). This value is

in agreement with that previously reported.24,27 However, upon the addition of excess

2+ pyridine to the solution of cis-[Ru(bpy)2(CH3CN2)] in deaerated CH3CN, the oxidation

becomes irreversible, with Ep = 1.55 V (Figure 4.7). Following oxidation of the complex,

2+ a small peak corresponding to cis-[Ru(bpy)2(py)2] was observed at 1.30 V. This

2+ assignment was based on the E1/2 value previously reported for cis-[Ru(bpy)2(py)2] ,

26 1.30 V in CH3CN. It should be noted from Figure 4.7 that the peak at 1.30 V increases

in intensity with additional cycles.

68 4e-5

2e-5

) * * 0

μΑ

-2e-5 ( Current

-4e-5

-6e-5 -1500 -1000 -500 0 500 1000 1500 2000 Potential (mV)

2+ Figure 4.6. Cyclic voltammogram of cis-[Ru(bpy)2(CH3CN)2] in deaerated CH3CN

(0.1 M TBAPF6) at a Pt electrode at 50 mV / sec, (* denotes impurity that may have been in the sample). 2.0e-5

0.0

-2.0e-5 * -4.0e-5

A)

μ -6.0e-5

-8.0e-5

Current ( Current -1.0e-4

-1.2e-4

-1.4e-4 -1.6e-4 0 500 1000 1500 2000 2500 Potential (mV) 2+ Figure 4.7. Cyclic voltammogram of cis-[Ru(bpy)2(CH3CN)2] in deareated CH3CN

(0.1 M TBAPF6) upon the addition of excess pyridine at a Pt electrode (50 mV / sec), 2+ where * indicates the formation of cis-[Ru(bpy)2(py)2] . The first cycle is labeled 1 and the second 2 in the figure.

69 A possible explanation for the above results is that upon undergoing the Ru-center

oxidation, the newly-generated Ru(III) center does not support a strong bond to CH3CN.

3+ The loss of a CH3CN ligand from oxidized cis-[Ru(bpy)2(CH3CN)2] in CH3CN as the

solvent results in the binding of another CH3CN ligand, regenerating the original complex. Therefore, in the electrochemical experiment, the molecule can be reduced to the starting Ru(II) complex, resulting in an apparent reversible oxidation. However, upon

2+ the addition of excess pyridine to the solution of cis-[Ru(bpy)2(CH3CN)2] in CH3CN, the loss of one of the CH3CN ligands upon oxidation results in the coordination of a

3+ pyridine molecule, generating cis-[Ru(bpy)2(py)2] . Once this complex is formed, it is

present in solution such that it can be oxidized in the second cycle. It should be noted,

3+ however, that the reduction of cis-[Ru(bpy)2(py)2] is not observed.

The cyclic voltammetry results support the idea that ligand loss may be occurring

from the MLCT state, and it also supports Durham statement that photosubstutution in

2+ cis-[Ru(bpy)2(CH3CN)2] is not a result of thermal population of the dd state from the lower lying MLCT state. In particular, the observation of irreversible oxidation in the presence of a coordinating ligand points at the fact that ligand substitution takes place upon the generation of Ru(III).The high quantum yields of photosubstitution that is observed for the various wavelengths of irradiation in water may also result from direct ligand loss from the MLCT state.

The higher quantum yield obtained at 350 nm is as a result of exciting the

molecule with higher energy light thus resulting in direct loss of the ligands from the dd

2+ excited state along with population of the MLCT state. When cis-[Ru(bpy)2(CH3CN)2]

70 was excited with lower energy light (λirr = 400 nm, 450 nm) quantum yield of

photosubstitution was the same (within experimental error). The lower quantum yields at

λirr = 400 nm, 450 nm relative to 350 nm may be explained by the photosubstitution with lower energy light taking place only from the MLCT, without involvement of the dd

states.

2+ The mechanism of photosubstitution of cis-[Ru(bpy)2(NH3)2] is more easily

2+ understood than that for cis-[Ru(bpy)2(CH3CN)2] . The quantum yield values of

2+ photosubstitution of compounds of the type cis-[Ru(NH3)4)(L)2] show close parallels to

2+ that of cis-[Ru(bpy)2(NH3)2] . For example, the quantum yields of NH3 photoaquation

2+ for cis-[Ru(NH3)4(isn)2] (isn = isonicotinamide) were reported to be φ365 = 0.029(1),

28,29 φ436 = 0.010(1), and φ480 = 0.00045(3) as listed on Table 4.4. For comparison, compounds whose quantum yield of photosubstitution is not dependent on the

2+ wavelength include cis-[Ru(NH3)4(py)2] (py = pyridine) with φ365 = 0.057(4) and φ436 =

Compound λmax / nm λirr / nm φNH3 350 0.024(2) cis-[Ru(bpy) (NH ) ]2+ 490, 345 2 3 2 400 0.018(2)

365 0.057(4) cis-[Ru(NH ) (py) ]2+(a) 407, 366 3 4 2 436 0.066(3)

365 0.039(1) cis-[Ru(NH ) (4acpy) ]2+(a) 518,442 3 4 2 405 0.030(1)

365 0.029(1) 2+(a) cis-[Ru(NH3)4(isn)2] 478, 413 436 0.0010(1) 480 0.0045(3) (a) from ref 14a

2+ Table 4.4. Quantum yield of ammonia photosubstitution of cis-[Ru(bpy)2(NH3)2] and related compounds at various wavelengths

71 2+ 0.066 (3), and cis-[Ru(NH3)4(4-acpy)2] (4-acpy = 4-acetylpyridine), with φ365 =

0.039(1) and φ436 = 0.030 (1) (Table 4.4).

The wavelength dependence of the photosubstitution quantum yield of cis-

2+ 30 [Ru(bpy)2(NH3)2] can be explained by the ‘tuning model’ proposed by Ford. The model proposes that ‘reactive’ complexes are those that have high energy MLCT absorption bands while ‘unreactive’ complexes are those which have low energy MLCT bands. The model also proposes that LF excited states are responsible for ligand labilization, and that the photoaquation quantum yields are dependent on the character of the lowest energy excited state (LEES), whether it is MLCT or LF. For the reactive

2+ complex cis-[Ru(NH3)4(py)2] the quantum yield of photosubstitution is observed to be

independent of wavelength (Table 4.4) as excitation into the MLCT band is followed by

efficient conversion to the reactive LF state. In low-spin octahedral Ru(II) complexes,

excitation into the LF state involves the population of M-L σ* orbital (eg) along the

2+ ligand axis which accounts for photosubstitution. For cis-[Ru(bpy)2(NH3)2] the

quantum yield of photosubstitution was observed to be dependent on the wavelength of

2+ excitation. As is shown in Table 4.4, exciting cis-[Ru(bpy)2(NH3)2] with low energy

light, λirr = 400 nm, resulted in a lower quantum yield of photosubstitution, whereas with

λirr = 350 nm, the quantum yield was observed to increase by 33 % indicating that the LF

state is at higher energy than the MLCT state in this complex. This interpretation is

consistent with those for other complexes where the quantum yield was dependent on

2+ 2+ wavelength including cis-[Ru(NH3)4(isn)2] and cis-[Ru(NH3)4(4-acpy)2] (Table 4.4).

In these complexes, higher energy excitation will result in the direct population of the

72 higher energy LF state from which photosubstitution occurs. However, since in these

complexes the LEES is MLCT, reactivity upon irrdiation with low energy light requires

thermal population of the LF state for ligand-loss to take place.

2+ 31,32 [Ru(NH3)5py] MLCT excitation results almost exclusively in substitution reactions.

(II) [Ru (NH3)5(L)] complexes show crossover from ‘reactive’ to ‘unreactive’ behaviour when the absorption maxima of the MLCT band falls above ~460 nm as a function of the ligands.24

2+ 1 The photostabilities of [Ru(bpy)2(pbz)] (pbz = 2,2 -bipyrazine) and

2+ [Ru(bpz)(bpy)(py)2] have been attributed to the position of LF state been higher in

2+ 2+ energy than MLCT. Previous work on [Ru(bpy)2(pbz)] and [Ru(bpy)2(bpm)] (bpm =

2,21-bipyrimidine) suggests that the MLCT state lies sufficiently below the LF state in

these compounds to permit sufficient photochemical ligand loss.33

As will be shown and discussed in the following chapter, Ru(II) complexes may

form μ-oxo dimers when irradiated in water. Oxo-bridged complexes, M-O-M, are

observed in complexes that have a short bridging distance with the possibility of strong π

overlap. Griffith34 has pointed out that oxo-bridged complexes of the first row transition

metals exhibit only weak metal-metal interactions whereas, metal-metal interactions was

much stronger for the second and third row metals. A series of oxo-bridged complexes of

n+ ruthenium(III) of the type, [(AA)2XRuORuX(AA)2] where AA is bpy or phen and X =

- 35 H2O, Cl- or NO2 have been synthesized. The chemical and physical properties of the

M-O-M complexes are unusual when compared to related monomeric complexes due to

the strong chemical interaction between the ruthenium ions across the oxide bridge. Oxo-

73 bridged dimer of ruthenium bipyridine compounds are known to have very strong

absorptions in the range 600-700 nm range,36 and no formation of the oxo-bridged dimer

was observed in any of the reactions.

The quantum yield of photoaquation is important since it represents the formation

of the diaqua activated complex, and therefore the amount of complex available for covalent binding to DNA. It is also important that the ligand exchange only takes place upon photoexcitation, a condition that is necessary for PDT for this particular system.

4.2.2 Photochemistry with other ligands

2+ To verify that both CH3CN ligands are displaced when cis-[Ru(bpy)2(CH3CN)2] is

irradiated with λirr > 420 nm, reactions were performed using 1 eq of bipyridine ligand

and 2 eq of (t-Bu)4NCl (TBACl) in a noncoordinating solvent such as dichloromethane

2+ (CH2Cl2). Irradiating 25 μM cis–[Ru(bpy)2(CH3CN)2] with 1 eq of bpy in CH2Cl2

2+ resulted in the formation of [Ru(bpy)3] , which is evident by the increase in absorbance

at 453 nm (ε = 14 600 M-1cm-1) and the growth in the emission peak at 620 nm (Figure

2+ 4.8a). Similar results was also observed upon irradiation of cis-[Ru(bpy)2(NH3)2] with bpy ligand. Control experiments were also performed when similar concentrations of cis–

2+ [Ru(bpy)2(CH3CN)2] and bpy ligand were kept in the dark for 1 hour, where no

apparent changes were observed in the absorption spectrum. These results show the

ability of the two photolabile CH3CN ligands to be substituted by a strong bidentate

ligand such as bpy. Similar results were obtained when 25 μM of cis–

2+ [Ru(bpy)2(CH3CN)2] was irradiated with 50 μM of TBACl in CH2Cl2 with λirr > 420.

74 An increase in the absorbance was observed at 553 nm, which is indicative of the

-1 -1 37 formation of cis-Ru(bpy)2Cl2 (ε = 8910 M cm ) (Figure 4.8b). Similar results were

observed with λirr > 345 nm. The reaction, however, goes to completion faster when the

sample was irradiated with λirr > 345 nm compared to λirr > 420 nm. This wavelength dependence is consistent with greater photoreactivity when the complex is irradiated with higher energy photons, since with higher energy greater ligand loss is expected based on the photoaquation quantum yields at 350, 400, and 450 nm (Table 4.3).

75 (a)

0.4

0.2

Absorbance (AU)

0 (b) 300 400 500 600 700 800 Wavelength (nm) 0.6

0.4

0.2 Absorbance (AU)

0 300 400 500 600 700 800 Wavelength (nm)

Figure 4.8. Changes observed in the electronic absorption spectrum of cis-

[Ru(bpy)2(CH3CN)2](PF6)2 with (a) excess bpy ligand in CH2Cl2, λ > 420 nm.for t = 0, 2,

4, 6, 8, 10, 12, 14, 16, 18, 20 min. (b) with 2 eq of TBACl in CH2Cl2, λ > 420 nm.for t = 0, 2, 4, 6, 8, 10, 12, 14,16, 18, 20 min.

76 Similarly, to ensure that both ammines were displaced photochemically from cis-

2+ [Ru(bpy)2(NH3)2] , 25 μM of the complex was irradiated with 1 eq of bpy λirr > 375 nm

and λirr > 455 nm in CH2Cl2 (Figure 4.9). A decrease in the absorbance was observed at

490 nm (Figures 4.10a), while an increase was observed at 453 nm (Figures 4.10b) and

530 nm during irradiation at both wavelengths. Control experiments in the dark under

similar experimental conditions do not result in spectral changes.

0.4

0.2

Absorbance (AU)

0 300 400 500 600 700 Wavelength (nm)

2+ Figure 4.9. Photolysis of cis-[Ru(bpy)2(NH3)2] and 1 eq of bpy ligand in CH2Cl2, λirr > 455 nm for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 min.

77

(a)

0.2

Absorbance (AU) Absorbance light dark 0.1 0481216

Time (min)

(b) 0.3

0.2

0.1

(AU) Absorbance light dark 0 0481216 Time(min)

2+ Figure 4.10. Changes in the absorption of cis-[Ru(bpy)2(NH3)2] at (a) 490 nm and (b) at 453 nm in the presence of 1 eq. bpy ligand in dichloromethane as a function of irradiation time with λirr > 455 nm and in the dark.

78 The decrease in absorbance at 490 nm resulted in a plateau after 5 min of

irradiation when irradiated with λirr > 375 nm, while the plateau is observed after 10 min

when irradiated at λirr > 455 nm. This result indicates that the completion of the reaction

is faster with higher energy irradiation. Similar trends are observed for the increase of the

2+ absorption at 453 nm, which corresponds to the formation of [Ru(bpy)3] , as a function

of irradiation wavelength. The increase in the absorbance at 530 nm is indicative of the

formation of an intermediate species, which likely corresponds to the η1 – bpy complex,

however, there is no definitive evidence for this assignment. A proposed reaction scheme

2+ 2+ for the reaction of cis-[Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] with bpy ligand

is shown in Scheme 2.

2+ bpy [Ru(bpy) (L)] 1 2+ 2 cis-[Ru(bpy)2(L)(η -bpy)

L hν hν L

2+ 2+ cis-[Ru(bpy)2(L)2] [Ru(bpy)3] ( λAbs = 453 nm)

2+ Scheme 4.2. Proposed mechanism for the photolysis of cis-[Ru(bpy)2(L)2] (L =

CH3CN, NH3) in the presence of 1 eq of bpy ligand in dichloromethane.

2+ From the absorption data and the known emission spectrum of [Ru(bpy)3] , it

2+ would be expected that upon photolysis of cis-[Ru(bpy)2(NH3)2] in CH2Cl2, the

2+ replacement by bpy would result in the characteristic emission spectrum of [Ru(bpy)3]

with maximum at 605 nm in CH2Cl2. The emission spectra were collected at 2 minute 79 intervals for a total of 12 minutes during photolysis. A strong emission band was observed with maximum at 605 nm after irradiation for 2 min (Figure 4.11). These results

2+ are in agreement with the formation of [Ru(bpy)3] upon irradiation with λirr > 455 nm and with the changes in absorption discussed above (Figure 4.9). The absorption and emission data shows that indeed both ammine ligands are photosubstituted by the bpy ligand, and that the process may proceed as shown in Scheme 2.

50000000

45000000

40000000 35000000

30000000

25000000

20000000 Intensity (cps) 15000000

10000000

5000000

0 450 500 550 600 650 700 750 800 850 -5000000 Wavelength (nm)

2+ Figure 4.11. Emission spectra of cis-[Ru(bpy)2(NH3)2] + 1 equivalent of bpy ligand in

CH2Cl2 (λexc = 450 nm) for 2 minutes interval up to 12 minutes.

2+ The reaction of cis-[Ru(bpy)2(NH3)2] with TBACl in CH2Cl2 at various wavelengths was also investigated. The results obtained, however, were interesting when irradiation was carried out with λirr > 375 nm compared to the results obtained upon

2+ irradiation with λirr > 345 nm and with cis–[Ru(bpy)2(CH3CN)2] with TBACl.

80 Irradiation of 25 μM of the metal complex with 50 μM of TBACl with λirr > 345 nm in

CH2Cl2 results in an increase in the absorption at 553 nm, which is consistent with the

- formation of cis-[Ru(bpy)2Cl2] with its characteristic maxima at 553 nm (ε = 8 910 M

1 -1 24 2+ cm ). Irradiation of cis-[Ru(bpy)2(NH3)2] , however with λirr > 375 nm with TBACl in CH2Cl2 results in the formation of an intermediate species with maximum at 540 nm, which may be attributed to the formation of the monosubstituted product, cis-

+ [Ru(bpy)2(NH3)Cl] after ~ 5 min of irradiation (Figure 4.12). Interestingly this intermediate species was not observed when the reaction was irradiated with lower energy where after ~ 2 min of irradiation with λirr > 375 nm, only the formation of the bisubstituted product, cis-[Ru(bpy)2Cl2] was observed. A possible explanation for these

2+ observations is that irradiation of cis-[Ru(bpy)2(NH3)2] with 345 nm > λirr > 455 nm

+ results in the formation of the monosubstituted compound, cis-[Ru(bpy)2(NH3)Cl] , which upon irradiation with high energy excitation results in preferential and direct loss of the chloride group, whereas lower energy excitation results in loss of the NH3 group or

- both Cl or NH3. The lack of photosubstitution with λirr > 455 nm is consistent with a LF state that is thermally inaccessible from the lower lying MLCT state. No changes in the absorption spectrum were observed when the reaction was kept in the dark. The

2+ photolysis of the control complex [Ru(bpy)2(en)] in CH2Cl2 in the presence of TBACl does not result in any changes in the absorption spectrum when irradiated with similar wavelength, likely due to the bidentate nature of the en ligand.

81

0.4

0.3

0.2

Absorbance (AU) 0.1

0 300 400 500 600 700 800 Wavelength (nm) 2+ Figure 4.12. Absorption spectra of 25 μM of cis-[Ru(bpy)2(NH3)2] with 50 μM of

TBACl in CH2Cl2 (λexc > 375 nm) for 5 minutes interval up to 35 minutes.

4.2.3 Photoinduced Binding to DNA Bases

Since the goal of this research is for the ruthenium complex to act as photoactivated cisplatin analog, it is important to show that the metal complex binds to

DNA bases upon irradiation. As described in deatail in Chapter 2, Ru(II) complexes are known to covalently bind to DNA at guanine rich sites.38 Clarke and coworkers have

2+ shown that DNA binding of [Ru(NH3)5(H2O)] occurs preferentially to guanine

39,40 bases. Compounds including mer-[RuCl3(terpy)] (terpy = 2,2’:6’2”-terpyridine), mer-

- [RuCl2(Me2SO)4] and trans-[RuCl4(Me2SO)2] have been shown to form interstrand cross–links in DNA, and have been shown to bind guanine derivatives in a trans

41,,,42 43 44 configuration, while trans-[RuCl2(Me2SO)4] forms a stable GpG adduct. The enantiomeric selectivity for the binding of the Δ and Λ isomers of cis-[Ru(phen)2Cl2] and

2+ 45 cis-[Ru(bpy)2(OH2)2] to B-DNA was investigated by Barton and Lolis and by Thorp 82 46 and coworkers. Reedijk and coworkers have shown that cis-[Ru(bpy)2Cl2] forms only a mono adduct with 9-ethylguanine even when the solution is refluxed under strong conditions for extended periods of time.47 However, Alberto and others recently reported the binding of 9-methyl guanine to cis-[Ru(2,2’-bpy)2(O3SCF3)2] to form the bisadduct,

2+ cis-[Ru(2,2’-bpy)2(9-MG)2] . Crystal structure data showed the binding of the bases to the metal center via the N7 atoms of the bases in a head to tail orientation.48

2+ Therefore, the photoinduced binding of cis-[Ru(bpy)2(CH3CN)2] and cis-

2+ [Ru(bpy)2(NH3)2] to relatively simple guanine derivatives, 9-MG and 9-EG, in aqueous solutions was investigated and compared to previous work (Scheme 4.3). These reactions were monitored by 1H NMR and the products subjected to mass spectrometry.

2+ 2+ 4a H NH2 O 3a 5a N 6 1 7 3a' O 1 5 N 2a' N HN 4a' 2a 6a N 8 N 2 N 9 5a' N N N L H2N N 4 N Ru 3 6a' Ru 6b' L H O N D2O, λirr > 420 nm 5b' N 2 N 2b' N 6b 4b' 2b 3b' 5b 3b 4b

2+ Scheme 4.3. The reaction scheme of of cis-[Ru(bpy)2(L)2] (L = CH3CN, NH3) with guanine in D2O (λirr > 420 nm or λirr > 345 nm).

2+ The photolysis of 5 mM of cis-[Ru(bpy)2(CH3CN)2] with λirr > 420 nm for 6 hr

1 in the presence of 2.5 eq of 9-EG in D2O was followed by H NMR, and it parallels the

2+ 49,50,49 thermal reaction of [Ru(bpy)2(OH2)2] with 9-MG and 9-EG. Table 5 compares 83 the chemical shifts of the bpy protons and bound H8 proton of guanine obtained by

Reedijk,48 and Alberto49 to those observed here upon irradiation of cis–

2+ [Ru(bpy)2(CH3CN)2] in D2O (λirr > 420 nm, 6 hr) with 9-EG and 9-MG.

δ / ppm bpy protons cis-[Ru(bpy)2- cis-[Ru(bpy)2- cis-[Ru(bpy)2- cis-[Ru(bpy)2- +(a) 2+(b) 2+(c) 2+(c) (scheme 3) (9-EG)Cl] (9MG)2] (9-EG)(OH2)] (9-MG)(OH2)] 6a 9.30 9.30 9.32 9.30 6b 8.78 8.80 8.98 8.95 3a 8.58 8.60 8.60 8.60 3b,3a’ 8.40 8.50 8.46 8.45 3b’ 8.32 8.40 8.38 8.35 4b 8.15 8.20 8.30 8.25 4a 8.05 8.15 8.15 8.10 4a’ 7.90 7.90 7.95 7.90 4b’,6b’,6a’ 7.80 7.85 7.84 7.80 5a 7.70 7.75 7.70 7.75 5b 7.62 7.65 7.62 7.65 5a’, 5b’ 7.18 7.20 7.30 7.20 H8(guanine) 6.8 6.8, 8.0 6.80 6.80 a ref 48, b ref 49, c This work. Numbering scheme shown in Scheme 4.

Table 4.5 1H NMR chemical shifts obtained for the reactions of 5 mM of cis- 2+ [Ru(bpy)2(CH3CN)2] at λirr > 420 nm for 6 hr in the presence of 2.5 eq. of 9-EG and 9-

MG in D2O to those previously reported.

2+ Similar results were observed when 5 mM of cis-[Ru(bpy)2(NH3)2] was irradiated with

λirr > 345 nm for 18 hours in the presence of 2.5 equivalen ts of 9- EG or 9-MG . The 84 reaction was followed by 1H NMR and the product subjected to positive mode electrospray mass spectrometry. The binding of one 9-EG and 9-MG was observed by 1H

NMR in D2O to the metal center as is evident from the changes in the chemical shifts observed upon binding of either DNA base (Figure 4.13).

*

(a) *

H8

3a 3b3a' 4b 4a 6a 6b 3b' 4a' 4b' 5a'5b' 5a5b

9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 (b)

H8

6b' 6a' 3b3a' 3a 4b 4a' 4b' 6a 6b 3b' 4a 5a 5b 5a' 5b'

9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2

Figure 4.13. 1H NMR spectra of the aromatic region of the reaction of cis- 2+ [Ru(bpy)2(NH3)2] with (a) 9-EG and (b) 9-MG in D2O when irradiated at λirr > 345 nm for 18 hours. The asterisk indicates H8 proton of free guanine. Numbering scheme shown in Scheme 4.

85 A 1:1 binding of 9-MG or 9-EG to the cis-[Ru(bpy)2] moiety was observed upon

2+ 2+ irradiation of cis–[Ru(bpy)2(CH3CN)2] (λirr > 420 nm, 6 hr) and cis-[Ru(bpy)2(NH3)2]

(λirr > 345 nm, 18 hours). The 1:1 binding of the guanine base to the Ru(bpy)2 fragment in each case, leads to an unsymmetrical complex, and therefore, two distinct bpy patterns are observed in the 1H NMR spectra of the products. Therefore, the aromatic region of the spectrum shows different chemical shifts for each bpy ligand. The resonance of bound water was not observed at room temperature, indicating fast exchange of the coordinated water on the NMR time scale with bulk water. It was previously shown, that the guanine cannot freely rotate about the Ru- N7 bond due to steric hindrance in these complexes.38

Therefore, the H8 protons of free guanine differ significantly in chemical shifts to bound guanine (Figure 4.13). The large upfield shift observed by the H8 proton is a result of the stronger influence of the bpy π clouds resulting from N7 coordination to the Ru(bpy)2 fragment.

+ In cis-[Ru(bpy)2(G)(H2O)] (G = 9-MG, 9-EG) the H6 protons of the different bpy ligand are near different ligands and hence the chemical shifts differ significantly from each other. The results are similar to that observed for the thermal reaction of cis-

[Ru(bpy)2(Cl)2] in water with 9-EG, where only one base was observed to bind to upon

o + 48 incubation for 24 hours at 37 C, resulting in cis-[Ru(bpy)2(9-EG)Cl] . Continued

2+ 2+ irradiation of cis–[Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] in D2O over 24 hours resulted in no further changes in the 1H NMR spectrum, indicating that there is no additional binding of another molecule of guanine to the metal complex. A possible explanation for the binding of only one guanine base to the metal complex is steric

86 hindrance of the bulky guanine base, precluding a second base from coordinating to the

48 same Ru(bpy)2 fragment. It was shown by Reedijk in a crystal structure of

+ [Ru(bpy)2(EG)Cl] that guanine binds to the Ru(II) metal with the keto group wedged between the bpy ligands, thereby creating a large barrier to rotation about the Ru-

N(guanine) bond. It was later shown by Alberto and others that the thermal reaction of

2+ cis-[Ru(bpy)2(O3SCF3)2] in ethanol with 9-MG affords cis-[Ru(bpy)2(9-MG)2] where binding of the second guanine was in a head to tail orientation to each other.51

Upon subjecting the product of the irradiation reaction of cis–

2+ [Ru(bpy)2(CH3CN)2] with 9-EG and 9-MG to positive mode MALDI mass

2+ spectrometry, peaks at m/z = 594 and m/z = 580 corresponding to cis-[Ru(bpy)2(9-EG)]

2+ and cis-[Ru(bpy)2(9-MG)] , respectively were observed (Figure 4.14a,b). Again only

(a) (b)

2+ Figure 4.14. MALDI mass spectroscopy of the reaction of cis-[Ru(bpy)2(CH3CN)2] with 2.5 eq of (a) 9-EG, λirr > 420 nm for over 24 hours (b) 9-MG, λirr > 420 nm for 36 hours in D2O.

87 one molecule of the DNA base was observed to bind to the Ru complex. It should be

2+ noted that when solutions containing cis–[Ru(bpy)2(CH3CN)2] and 9-EG or 9-MG were kept in the dark no binding of the DNA bases to the Ru complex was observed.

2+ Similar results were observed when cis-[Ru(bpy)2(NH3)2] was irradiated with 9-

EG and 9-MG. Positive electrospray mass spectroscopy on the products from these reactions resulted in parent ion peaks at m/z = 578.2 and m/z = 592.2 corresponding to of

2+ 2+ cis-[Ru(bpy)2(9-MG)] and of cis-[Ru(bpy)2(9-EG)] , respectively (Figure 4.15).

However, longer irradiation time was required at higher energy (λirr > 345 nm for 18

2+ hours) for efficient photosubstitution of cis-[Ru(bpy)2(NH3)2] by the DNA bases to take

2+ place relative to cis-[Ru(bpy)2(CH3CN)2] (λirr > 345 nm, 6 hours). These results are consistent with the photoaquation and other ligand-substitution reactions described for the two complexes above.

(a) (b)

2+ Figure 4.15. Electrospray mass spectroscopy of the reaction of cis-[Ru(bpy)2(NH3)2] with (a) 9-MG and (b) 9-EG in D2O when irradiated at λirr > 345 nm for 18 hours.

88 4.3 Photochemistry with DNA

4.3.1 Binding to single-stranded oligonucleotides

Cisplatin has been shown to covalently bind to adjacent purines in double stranded DNA, forming 1,2 intrastrand cross-links at GpG (65 %) and ApG (25 %)

52,53 2+ sites. In order to compare the binding of cis-[Ru(bpy)2(CH3CN)2] to that of cisplatin, the binding of the metal complex to the single-stranded 15-mer oligonucleotide sequence 5’-AGTGCCAAGCTTGCA-3’ was investigated in the dark and upon irradiation. Photolysis of 100 μM complex in the presence of 100 μM bases of the oligonucleotide (6.67 μM strands) with λirr > 420 nm for 15 min resulted in the covalent binding of the metal complex to the oligonucleotide. Negative mode MALDI mass spectrometry shows a peak corresponding to the calculated mass of the 15-mer at m/z =

4578, where the calculated mass obtained by the addition of the masses of the respective bases and phosphodiester backbone in the sequence results in m/z = 4577. This peak is labeled S2 in Figure 4.16. Binding of one, two, and three fragments of Ru(bpy)2 to the strand S2 was observed at m/z ratios of 4991.3, 5403.3 and 5814.4, labeled A, B, and C respectively, in Figure 4.18. Peaks labeled A2 and B2 and C2 correspond to the additional binding of one equivalent of triethanolammonium chloride to the fragments of mass A and B, with m/z ratios of 5130.0, 5542.2, and 5954.7 respectively. B3 corresponds to the binding of an additional equivalent of triethanolammonim chloride to the ion B2, with m/z = 5677.2. The experiments were conducted in NaCl, and the 15-mer was repeatedly washed with 1M triethanolammonium acetate (TEAA), 50 % CH3CN and

H2O before it was injected in the mass spectrometer. It should be noted that the metal 89 complex was in 15-fold excess in the photolysis experiments relative to the number of strands, and covalent binding of up to three molecules of Ru(bpy)2 fragment per DNA strand was observed.

A

B

B2

S2 A2

B3 C C2

4000 4500 5000 5500 6000 m /z

2+ Figure 4.16. MALDI mass spectrometry of the reaction of cis-[Ru(bpy)2(CH3CN)2] with the 15-mer oligonucleotide, 5’-AGTGCCAAGCTTGCA-3’(S2), λirr > 420 nm for 15 min in 5 mM Tris, 50 mM NaCl, pH = 7.5.

Similar reactions with single stranded DNA were also investigated with cis-

2+ 2+ [Ru(bpy)2(NH3)2] . The products of the photolysis of 10 μM cis-[Ru(bpy)2(NH3)2] in the presence of 10 μM of each single stranded 15–mer oligonucleotide sequences, 5’-

TGCAAGCTTGGCACT-3’ (S1) and 5’- AGTGCCAAGCTTGCA-3’ (S2), with λirr >

345 nm were monitored by positive ion ESMS, following repeated washings with 1 M

TEAA, 50 % CH3CN, and H2O. Peaks corresponding to the calculated masses of S1 (m/z

= 4568) and S2 (m/z = 4577) were observed at m/z = 4570.3 and m/z = 4578.8,

90 respectively and are labeled A in Figure 4.17a and 4.17b. The relative intensity pattern and position of the peaks labeled A, B and C in Figure 4.17 is observed in the ESMS collected for each single strand in the absence of the metal complex. The peaks labeled B and C correspond to the addition of one and two molecules of CH3CN, respectively. The peaks labeled A’, B’ and C’ correspond to the covalent binding of one cis-Ru(bpy)2 fragment to each single strand peak labeled A, B, and C, respectively, upon photolysis in

2+ water. In contrast, when solutions containing cis-[Ru(bpy)2(NH3)2] are kept in the dark with each strand for several hours and subjected to purification to remove complexes that are not covalently bound, no evidence of Ru(II) complex binding to either S1 or S2 was

2+ observed. In addition, when 10 μM of the control complex [Ru(bpy)2(en)] was photolyzed with 10 μM S1 and S2 (λirr > 345 nm, 10 min), no covalent binding of the

Ru(bpy)2 fragment was observed. These results are consistent with the lack of photoreactivity in water that was observed for this compound, which is attributed to the bidentate nature of the ethylenediammine ligand.

2+ It should also be noted that the concentration of cis-[Ru(bpy)2(NH3)2] in the photolysis experiments with each single stranded 15-mer is in 15 fold excess, however, only one Ru(bpy)2 fragment was observed to bind per DNA strand. Since cisplatin is known to covalently bind to the GG sequence,4,5,6 the S1 was deliberately chosen so as to have a GG site, whereas S2 does not. Greater binding of Ru(bpy)2 was observed for S1

(82 %) compared to its complementary strand, S2 (58 %). The position of the binding of

Ru(bpy)2 fragment on either DNA strand is unknown at this time.

91 (a)

A’

B’ C’

A B C

4000 4500 5000 5500 6000 m/z

(b) A’

A

B B’

C C’

4000 4500 5000 5500 6000 m/z

Figure 4.17. Electrospray ionization mass spectra of solutions containing 10 μM of cis- 2+ [Ru(bpy)2(NH3)2] with 10μM of single stranded DNA (a) S1 (b) S2 when irradiated with λirr > 345 nm in 5 mM Tris Buffer (pH = 7.5 mM, 50 mM NaCl).

92 2+ As shown, the photolysis of cis-[Ru(bpy)2(CH3CN)2] with single-stranded oligonucleotide results in the binding of one, two and three fragments of Ru(bpy)2 to S2.

However, only one Ru(bpy)2 fragment was observed to bind to either S1 or S2 upon

2+ irradiation of cis-[Ru(bpy)2(NH3)2] . This result agrees well with the greater quantum

2+ yiled of photoaquation measured for cis-[Ru(bpy)2(CH3CN)2] compared to cis-

2+ [Ru(bpy)2(NH3)2] .

4.3.2 Binding to double-stranded DNA

Since cellular DNA is found in duplex form, it is important for the complex to bind to ds-DNA upon photolysis. Since the early 1980’s the melting of short duplex oligonucleotides less than ~20 bases was used to evaluate the thermodynamic stability of the DNA.54,55 In a double helix, the bases stack close enough to exclude water and to let the aromatic ring π-clouds interact. The complementary hydrogen bonding patterns of

A:T and G:C allows DNA to form the double helix, and π-stacking of the base strongly contributes to the stability of the double helix. As the DNA is heated, it reaches a temperature where the double strands separate into single strands. The base pairs are separated as the hydrogen bonds between them are broken. The temperature at which the duplex separates into single strands is known as the melting temperature, Tm. Metal complexes can bind to ds-DNA, stabilizing or destabilizing the duplex, resulting in an increase or decrease of the melting temperature, respectively. Cisplatin was shown to

o 40 reduce the thermal stability of a 20-mer duplex by ΔTm = - 8 C. The decrease in the melting temperature upon covalent binding of cisplatin to DNA is as a result of cross-

93 linking by the complex, which perturbs the hydrogen-bonding and π-stacking in the duplex.

In order to investigate the binding of the metal complex to ds-DNA, DNA melting temperature (Tm) experiments were conducted with the annealed 15-mer ss-strands, S1 and S2, discussed in Section 4.2.3 thus forming duplex DNA. Gel mobility assays and relative viscosity measurements were also conducted. The melting temperature of the

2+ o o duplex in the absence of cis-[Ru(bpy)2(CH3CN)2] was measured at 57 C (± 2 C).

2+ Upon irradiation of 50 μM the duplex with 50 μM cis-[Ru(bpy)2(CH3CN)2] with λirr >

420 nm for 15 min, the observed melting temperature shifts to 51 oC (± 2 oC),

o o corresponding to ΔTm = -6 C (± 4 C). When solution containing 50 μM of the 15-mer

2+ duplex and 50 μM of cis-[Ru(bpy)2(CH3CN)2] are kept in the dark for 15 minutes, an

o o insignificant change of ΔTm = -1 C (± 4 C) was measured. When 50 μM of the double

2+ stranded 15-mer was photolyzed with 50 μM of cis-[Ru(bpy)2(NH3)2] (λirr > 345 nm, 10

o o min) a melting temperature of 52 (± 2 C) was measured, corresponding to ΔTm = -5 C

o (± 4 C). The shift of Tm of the duplex to lower temperatures observed upon irradiation

2+ 2+ with cis-[Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] is consistent with covalent binding of each complex to duplex DNA. In contrast no significant change in the melting temperature was observed when solutions containing 50 μM of the duplex and 50 μM of either metal complexes were left in the dark for several hours or when solutions

2+ containing similar concentrations of [Ru(bpy)2(en)] were irradiated with λirr > 345 nm for 10 min. It should also be noted that it was previously reported that the formation of interstrand cross-links by mono- and di- substituted Ru(II) complexes result in positive 94 shifts of the melting temperature when covalently bound to DNA . DNA binding of these compounds, however, was not initiated by light.56

In contrast, Ru(II) polypyridyl complexes possessing one or two H2O molecules in their ligation sphere which covalently bind to DNA, were shown to result in a positive shift in the melting temperature of DNA.57 Complexes with a single labile aqua ligand have little effect on the thermal denaturation of calf thymus DNA, with ΔTm values that

o range from + 0.8 to 3.5 C (Table 5). Comparing the ΔTm obtained for

2+ [Ru(tpy)(bpy)(OH2)] (tpy = terpyridine) (ΔTm = 0.8 ± 0.3) with that obtained for

2+ [Ru(tpy)(dppz)(OH2)] (dppz = dipyrido [3,2-a:2’,3’-c] phenazine) (ΔTm = 1.6 ± 0.2), it can be concluded that the bpy or the dppz ligand has no effect on the change in melting temperature, and therefore, that they do not interact appreciably with the DNA upon

2+ formation of the covalent adduct. Similarly, [Ru(L)(py)(OH2)] (L = bpy, phen) exhibits

ΔTm = 2.4, indicating little interaction with the double (Table 6). It is also shown in Table

2+ 5 that the binding of cis-[Ru(bpy)2(OH2)2] to DNA, resulted in a greater shift, with ΔTm

= 6.1 ± 0.5. It is believed that this difference is due to the formation of two covalent bonds to DNA, not just one as is the case with the complexes with a single labile water molecule. Platinum(II) complexes are also capable of forming monoadducts. One such

+ example is [Pt(dien)Cl] which shows a comparable shift in the melting temperature, ΔTm

o = +0.5 C, to those of the mono-adducts of Ru(II) complexes. The positive ΔTm indicates that the complex-DNA adduct is more difficult to melt than the DNA by itself. Trans-

o [Pt(NH3)2Cl2] was shown to have a positive shift in the Tm value, ΔT = +4.5 C. This can be attributed to the formation of an interstrand diadduct by the complex, thereby 95 covalently cross-linking the two DNA strands making them more difficult to separate.54

Farell et. al.,58 has shown that the diplatinum complex trans-

[{PtCl(NH3)2}2H2N(CH2)4NH2]Cl2 induces an interstrand crosslink and exhibits a ΔTm =

+9.3 oC. Ru(II) complexes are also known to form interstrand diadducts resulting in large

2+ o positive ΔTm. For example, [Ru(phen)2(OH2)2] was shown to have a ΔT = +12.9 C indicating the formation of interstrand diadducts.54

0 a Metal Complexes ΔTm / C 2+ [Ru(tpy)(bpy)(OH2)] 0.8 ± 0.3 2+ [Ru(tpy)(tmen)(OH2)] 0.8 ± 0.7 2+ [Ru(tpy)(phen)(OH2)] 3.5 ± 0.7 2+ [Ru(tpy)(dppz)(OH2)] 1.6 ± 0.2 2+ [Ru(bpy)2(py)(OH2)] 2.4 ± 0.3 2+ [Ru(phen)2(py)(OH2)] 2.4 ± 0.5 2+ [Ru(bpy)2(OH2)2] 6.1 ± 0.5 a from ref.44

Table 4.6. Thermal denaturation results for Covalent Adducts of Ru(II) Aqua complexes with calf thymus DNA.

2+ Additional experiments designed to determine whether cis–[Ru(bpy)2(CH3CN)2] covalently binds to ds-DNA upon photolysis, the shift in the mobility of linearized plasmid DNA was measured. The relative concentration of base pairs (bp) to metal complex (mc) was varied from 1:100 to 1:5 (bp:mc), and the results were analyzed by agarose gel electrophoresis. For comparison, incubations of the DNA were also

96 performed with the known covalent binder, cisplatin, with equivalent bp:mc ratios.

Cisplatin is known to form covalent bonds to DNA via the platinum center binding preferentially to two neighboring guanine bases on the same strand.6 As shown in Figure

4.18(a), increasing concentration of cisplatin results in a reduction in the mobility of the linearized plasmid on the agarose gel. Lanes 1 and 8 are molecular weight standard ranging from 1000 bp to 10 000 bp increasing in increments of 1000 bp, and Lanes 2 and

7 contain only the linearized plasmid as a control, with no metal complex added. Lanes 3-

6 contains the plasmid DNA with varying bp:mc ratios of cisplatin. These lanes show the reduced mobility of linearized plasmid after incubation with cisplatin as the concentration of metal complex is increased. The reduced mobility was most pronounced at the lowest ratio of metal to base pairs, or at the highest relative metal concentration. The retardation in the mobility of the DNA on the agarose gel may be due to several factors including molecular weight, molecular shape of the DNA, as well as the overall charge. The retardation of the gel observed as a result of increasing concentration of cisplatin may be due to the changes in the conformation of the DNA as cisplatin becomes covalently bound to the DNA creating kinks in the DNA, which leads to reduced migration through the gel. The molecular weight of the DNA is also altered as cisplatin binds to DNA. In

2+ addition, binding of the cationic cis-[Pt(NH3)2] fragment to DNA reduces the overall charge on the phosphate backbone of the DNA, resulting in lower attraction to the positive electrode and reduced mobility.

As shown in Figure 4.18b and 4.18c, similar reduced mobility of linearized

2+ plasmid was observed when various concentrations of cis-[Ru(bpy)2(CH3CN)2] and

97 1 2 3 4 5 6 7 8 (a)

1 2 3 4 5 6 7 8 (b)

1 2 3 4 5 6 7 8 (c)

Figure 4.18. Ethidium bromide stained agarose gels of 50 μM linearised pUC18 ( 10 mM phosphate, pH = 7.5) in the presence of various ratios of (a) cisplatin incubated for 4 hr at o 2+ o 37 C, (b) cis-[Ru(bpy)2(CH3CN)2] irradiated (λirr > 420 nm) for 15 min at 25 C. and 2+ o (c) cis-[Ru(bpy)2(NH3)2] irradiated (λirr > 345 nm) for 15 min at 25 C. Lanes 1 and 8: DNA molecular weight standard (1 kb). Lanes 2 and 7: Linearized plasmid alone, Lanes 3-6: [DNA bp]/[Complex] = 100, 20, 10, 5.

98 2+ cis-[Ru(bpy)2(NH3)2] were irradiated with 50 μM linearized plasmid (λirr > 420 nm, 15 minutes). A decrease in mobility was observed as a result of increasing concentration of

2+ both metal complexes indicating that cis–[Ru(bpy)2(CH3CN)2] and cis-

2+ [Ru(bpy)2(NH3)2] covalently bind ds-DNA. An interference with the staining of the intercalative dye, ethidium bromide (EtBr), was observed upon increasing concentration

2+ 2+ of cis-[Ru(bpy)2(CH3CN)2] to DNA, more so than for cis-[Ru(bpy)2(NH3)2] . The intensity of the fluorescence of EtBr decreases at higher complex concentrations for both

2+ cisplatin and cis–[Ru(bpy)2(CH3CN)2] . This decrease in EtBr emission may be due to the change in conformation of the DNA as the metal complex binds to it, thus making it difficult for the dye to intercalate. Intercalation of EtBr is essential for its emission to be observed in these experiments. It should also be noted that no change in the DNA mobility was observed when solutions of the complex with linearized DNA containing

2+ 2+ similar concentrations of cis-[Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] were kept in the dark for 30 min prior to loading onto the agarose gel (Figure 4.19). No shift in

DNA mobility was detected when solutions containing [Ru(bpy)(en)]2+ were irradiated

o with linearized plasmid with λirr > 345 nm for 15 min at 25 C under similar experimental conditions.

99 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

(a) (b)

Figure 4.19. Ethidium bromide stained agarose gels of 50 μM linearized pUC18 (10 mM 2+ phosphate, pH = 7.5) in the presence of various ratios of (a)cis-[Ru(bpy)2(CH3CN)2] 2+ kept in the dark for 20 minutes and (b) cis-[Ru(bpy)2(NH3)2] kept in the dark for 30 minutes. Lanes 1 and 8: DNA molecular weight standard (1 kb). Lanes 2 and 7: Linearized plasmid alone, Lanes 3-6: [DNA bp]/[Complex] = 100, 20, 10, 5.

Relative viscosity measurements were also performed to investigate the structural changes of the duplex DNA that take place upon covalent binding of the metal complexes, since changes in the length of the rod-like DNA have been shown to result in measurable viscosity changes.59,60 Compounds can bind to DNA covalently or noncovalently. Intercalation and groove binding are the most likely binding modes for the latter. Groove binding results in only subtle changes to the DNA and, therefore, has little effect on the viscosity of DNA. Intercalation results in substantial changes in the DNA structure as a planar ligand moiety is inserted between adjacent base pairs. Intercalation

100 results in lengthening and unwinding of the DNA helix thus causing an increase in its relative viscosity. Covalent binding to the DNA results in distortions to the double helix which affects the relative viscosity of the DNA. The changes in the viscosity of DNA solutions were investigated with ethidium bromide, a known DNA intercalator; Hoechst

33258, a minor groove binder; cisplatin, a compound that is known to covalently bind to

2+ DNA, and the compounds of interest cis-[Ru(bpy)2(CH3CN)2] and cis-

2+ [Ru(bpy)2(NH3)2] .

Figure 4.20 shows the relative changes in the viscosity of linearized Herring

Sperm DNA with increasing amounts of ethidium bromide, Hoechst 33258, cisplatin, and

2+ 2+ cis-[Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] (irradiated and in the dark). As expected for an intercalator, ethidium bromide increases the relative viscosity of the

DNA solution. In contrast, Hoechst 33258, a minor groove binder, has no effect on the relative viscosity of DNA. Cisplatin is known to covalently bind to DNA,61,,62 63 and it is shown in Figure 4.20 to result in a decrease in the relative viscosity of the solution. As the concentration of cisplatin increases there is a significant decrease in the viscosity of the solution to a ratio of [DNA]:[cisplatin] = 1:0.15, after which point there is a slight increase in the viscosity until a plateau is reached at [DNA]:[cisplatin] of 1:0.30. The decrease in viscosity for cisplatin is consistent with that previously reported, where it was explained in terms of a “shortening effect” on the DNA induced by the covalent binding of the metal complex.64,65 The length of the DNA was reported to shorten by as much as

50 % upon covalent binding of cisplatin to DNA.66 The precise nature of the metal-DNA interaction that produces this remarkable collapse of the double helix is not known.

101 2+ Similar results to that of cisplatin were obtained with cis-[Ru(bpy)2(CH3CN)2] following irradiation (λirr > 420 nm,15 minutes) in the presence of ds-DNA. A decrease in the relative viscosity of the solution was observed up to [DNA]:[complex] = 1:0.15 followed by an increase the relative viscosity until a plateau is reached at [DNA]:[ cis-

2+ [Ru(bpy)2(CH3CN)2] ] of 1: 0.30 (Figure 4.20). These results show a similar relative

2+ change in the viscosity of DNA induced by cis-[Ru(bpy)2(CH3CN)2] upon photolysis to

2+ cisplatin. When solutions of cis-[Ru(bpy)2(CH3CN)2] were kept in the dark no change in viscosity was observed (Figure 4.20). Similar results to that observed for cisplatin and

2+ 2+ photolyzed cis-[Ru(bpy)2(CH3CN)2] were also observed for cis-[Ru(bpy)2(NH3)2] when irradiated with λirr > 345 nm for 15 minutes. Increasing concentrations of irradiated

2+ DNA solutions of cis-[Ru(bpy)2(NH3)2] (λirr > 345 nm, 15 min) results in a significant decrease in the relative viscosity of the solution to a ratio of [DNA]:[complex] = 1: 0.15, after which point there is a slight increase in the viscosity until a plateau is reached at

[DNA]:[complex] of 1: 0.30 (Figure 4.22). In contrast, no change in the viscosity was

2+ observed when DNA solutions containing cis-[Ru(bpy)2(NH3)2] were kept in the dark

2+ for 30 min. The results are consistent with covalent binding of cis-[Ru(bpy)2(NH3)2] upon irradiation. However, the extent of covalent binding to Herring Sperm DNA is greater for cisplatin than for the Ru(II) complexes, as is evident by the greater decrease observed in the viscosity of the solution in the presence of the former. In addition,

2+ photoactivated cis-[Ru(bpy)2(CH3CN)2] appears to cause a greater structural change in

2+ the DNA than cis-[Ru(bpy)2(NH3)2] . The greater structural change observed for cis-

2+ [Ru(bpy)2(CH3CN)2] can also be attributed to the greater quantum yield of the bis-aqua

102 complex that was calculated for the complex compared to the smaller quantum yield

2+ obtained by cis-[Ru(bpy)2(NH3)2] . This difference may result in a greater number of

Ru(bpy)2 fragments binding to DNA, thus resulting in greater distortion to the double helix.

1.3

1.2

1.1

1.0 1/3 ) 0 η /

η 0.9 (

0.8

0.7

0.6 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 [DNA]:[Metal]

1/3 Figure 4.20. Relative viscosity measurements of the plot of (ηDNA / ηo) vs R = [probe]:[DNA] for ethidium bromide (●), hoecht (x), cisplatin (▲), cis- 2+ 2+ [Ru(bpy)2(CH3CN)2] , λirr> 420 nm for 15 min (▼), cis-[Ru(bpy)2(CH3CN)2] dark 2+ 2+ (■),cis-[Ru(bpy)2(NH3)2] , λirr> 345 nm for 15 min (♦), cis-Ru(bpy)2(NH3)2 dark (∗)

103 4.3 Conclusion

2+ Irradiating cis-[Ru(bpy)2(CH3CN)2] with λirr> 420 nm for 15 min in water

2+ resulted in the formation of the bisaqua complex, cis-[Ru(bpy)2(OH)2] with quantum yields of φ350 = 0.377(3), φ400 = 0.207(1), φ450 = 0.222(18), whereas irradiation of cis-

2+ [Ru(bpy)2(NH3)2] in water resulted in quantum yields of φ350 = 0.024(2) and φ400 =

0.018. High quantum yield of photoconversion to the bisaqua complex is important since this species is known to covalently bind to double strand DNA in a similar manner to that

2+ of cisplatin. Ligand loss in cis-[Ru(bpy)2(CH3CN)2] may arise from the MLCT state

2+ resulting in greater quantum yields, whereas ligand loss in cis-[Ru(bpy)2(NH3)2] arises from the thermal population of the LF dd states from the lower lying MLCT state.

Therefore, in the latter high energy is needed for efficient photolysis to occur.

2+ 2+ Binding of cis-[Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] to DNA bases,

9-methylguanine and 9-ethyl guanine was observed upon photolysis and was confirmed by 1H NMR and MALDI mass spectrometry. DNA binding was also observed with single-stranded 15-mer oligonucleotide sequence, 5’- AGTGCCAAGCTTGCA-3’, upon

2+ irradiation of cis-[Ru(bpy)2(CH3CN)2] , where up to three Ru(bpy)2 fragments bound to

2+ the DNA strand were observed. Binding of cis-[Ru(bpy)2(NH3)2] upon irradiation was also observed to the single stranded 15-mer oligonucleotide sequences, 5’-

TGCAAGCTTGGCACT-3’ and 5’- AGTGCCAAGCTTGCA-3’, where binding of only one Ru(bpy)2 fragment to each strand was observed. Greater binding was observed to the strand that possesses a GG sequence (82 %), as compared to the one that does not (58 %).

104 2+ Binding of photolyzed cis-[Ru(bpy)2(CH3CN)2] to duplex 15-mer oligonucleotide resulted in a shift of the melting temperature to lower temperature (ΔTm =

-6 0C), consistent with covalent binding of the activated metal complex to ds-DNA. A

o similar shift in the melting temperature (ΔTm = -5 C) was also observed upon covalent

2+ binding of photolyzed cis-[Ru(bpy)2(NH3)2] to the DNA. Binding of cis-

2+ 2+ [Ru(bpy)2(CH3CN)2] and cis-[Ru(bpy)2(NH3)2] to ds-DNA upon irradiation was also confirmed by gel mobility assays, which showed a decrease in the mobility of the DNA upon increasing concentration of both metal complexes when photolyzed, whereas no change in the mobility of the DNA was observed when either complex was kept in the

2+ dark. Irradiating cis-[Ru(bpy)2(CH3CN)2] with Herring Sperm DNA at λirr> 420 nm for

15 min decreases the viscosity of the DNA solution in a similar manner to cisplatin, a known covalent DNA binder. A similar decrease in the viscosity was also observed upon

2+ irradiation of DNA solutions containing cis-[Ru(bpy)2(NH3)2] at λirr > 345 nm for 15

2+ minutes. However, the decrease was greater for cis-[Ru(bpy)2(CH3CN)2] , than cis-

2+ [Ru(bpy)2(NH3)2] , which is consistent with the greater quantum yield of formation of

2+ the bis-aqua complex in the former. DNA binding by cis-[Ru(bpy)2(CH3CN)2] or cis-

2+ [Ru(bpy)2(NH3)2] was not observed in the dark.

2+ The above results indicate that cis-[Ru(bpy)2(CH3CN)2] and cis-

2+ [Ru(bpy)2(NH3)2] may be candidates as a photoactivated cisplatin analogs, since they are shown here to covalently bind to DNA only when irradiated.

105 References

1. Brown, G. M.; Callahan, R. W.; Meyer, T. J. Inorg. Chem. 1975, 14, 8, 1915-1921.

2. Assefa, Z.; Stanbury, D. M. J. Am. Chem. Soc. 1997, 119, 521.

3. Wiliams, R. J. P. J. Chem Soc 1953, 75, 2163.

4. Palmer, R. A.; Pipe, T. S. Inorg. chem. 1966, 5, 864.

5. Chen, Z. J. Chem. Chinese 1987, 8, 9, 839-841.

6. Casper, V. C.; Meyer, T. J. Inorg. Chem. 1983, 22, 2444-2453.

7. Bardley, P. G.; Kress, N.; Hornberger, B. A.; Dallinger, R. F.; Woodruff, W. H. J. Am. Chem. Soc. 1981, 103, 7441.

8. Walsh, J. L.; Durham, B. Inorg. Chem. 1982, 21, 329-337.

9. Wacholtz, W. M.; Auerbach, R. A.; Schmehl, R. H.; Ollino, M.; Cherry, W. R. Inorg. C hem. 1985, 24, 1758-1760. 10. Crosby, G. A.; Elfring, W. H. J. Phys. Chem. 1976, 80, 2206.

11. Elfring, W. H.; Crosby, G. A. J. Am. Chem. Soc. 1981, 103, 2683.

12. Henderson, L. J.; Ollino, M.; Gupta, V. K.; Newkome, G. R.; Cherry, W. R. J. Photochem. 1985, 31, 1999.

13. Meyer, T. J. Pure Appl. Chem. 1986, 58, 1193.

14. Kalyanasundaram, K.; Nazeeruddin, M. K. Chem Phys. Lett. 1992, 4, 292-297.

15. Treadway, J. A.; Lopez, R.; Anderson, P.A.;Keene, F. R.; Meyer, T. J. Inorg. Chem. 1996, 10, 35, 8, 2242-2246.

16. Walsh, J. L.; Durham, B. Inorg. Chem. 1982, 21, 329-337.

17. Durham, B.; Wilson, S. R.; Hodgson, D. J.; Meyer, T. J. Am. Chem. Soc. 1980, 102, 600-607.

18. Hecker, C. R., Franwick, P. E., McMillin, D. R. Inorg. Chem. 1991, 30, 659-666.

19. Durham, B.; Wilson, S. R.; Hodgson, D. J.; Meyer, T. J. J. Am. Chem. Soc. 1980, 102:2, 600-607. 106

20. Durham, B.; Walsh, J. L.; Carter, C. L.; Meyer, T. J. Inorg. Chem. 1980, 19, 860-865.

21. Pinnick, D. V.; Durham, B. Inorg. Chem. 1984, 23, 1440-1445.

22. Pavanin, L. A.; Rocha, Z. N.; Giesbrecht, E.; Tfouni, E. Inorg. Chem., 1991, 30, 2185-2190.

23. Murov, S. L.; Carmichael, I.; Hug, G. L. Handbook of Photochemistry, 2nd ed.; Marcel Dekker: New York, 1993. 24. Van Houten, J.; Watts, R. J. Inorg. Chem. 1978, 100, 1457.

25. Pinnick, D. V.; Durham, B. Inorg. Chem. 1984, 23, 3841-3842.

26. Casper, J. V.; Meyer, T. J. Inorg. Chem. 1983, 22, 2444.

27. Brown, G. M.; Callahan, R. W.; Meyer, T. J. Inorg. Chem. 1975, 14, 8, 1915-1921.

28. (a) Ford, P. C. Coord. Chem. Rev. 1970, 5, 75-99. (b) Ford, P. C. Coord. Chem. Rev. 1982, 44, 61-82. (c) Ford, P. C.; Wink, D.; Dibenedetto, J. Progress Inorg. Chem. 1983, 30, 213-271 (d) Pavanin.

29.(a) Tfouni, E. Coord. Chem. Rev. 2000, 196, 281-305.(b) Martinez, M. S.; J. Photochem. Photobiol. A: Chemistry 1999, 122, 103-108. (c) Pavanin, L. A.; da Rocha, Z. N.; Giesbrecht, E.; Tfouni, E. Inorg. Chem. 1991, 30, 2185, 2190.

30. Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1977, 99, 7213.

31. (a) Ford, P. C.; Stuer, D. H.; McDonald, D. P. J. Am. Chem. Soc. 1969, 91, 6209. (b) Ford, P. C.; Chaisson D. A.; Stuermer, D. H. Chem. Comm. 1971, 530.

32. Chaisson, D. A.; Hintze R. E.; Stuermer D. H.; Petersen, J. D.; McDonald D. P.; Ford P. C. J. Am. Chem. Soc., 1972, 94, 6665.

33. Allen, G. H.; White, R. P.; Rillema, D. P.; Meyer, T. J. J. Am. Chem. Soc. 1984, 106, 2613.

34. Griffith, W. P. Corrd. Chem Rev. 1970, 5, 459.

35. Weaver, T. R.; Meyer, T. J.; Adeyemi, S. A.; Brown, G. M.; Eckberg, R. P.; Hatfield, W. E.; Johnson, E. C.; Murray, R. W.; Untereker, D. J. Am. Chem. Soc. 1975, 97, 11, 3039-3048.

107

36. Gilbert, J. A.; Eggleston, D. S.; Murphy, W. A. Jr.; Geslowitz, D. A.; Gersten, S. W.; Hogson, D. J.; Meyer, T. J. J. Am. Chem. Soc. 1985, 107, 3855.

37. Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; Von, Zelewsy, A. Coord. Chem. Rev. 1988, 84, 85-277.

38. Van Vliet, P. M.; Haasnoot, J. G.; Reedijk, J. Inorg. Chem. 1994, 33, 1934-1939.

39. Clarke, M.; Jansen, B.; Marx, K. A.; Kruger, R. Inorg. Chim. Acta 1986, 124, 13-28.

40. McNamara, M.; Clarke, M. J. Inorg. Chim. Acta 1992, 195, 175-185.

41. Van Vliet, P.M.; Toekimin, S. M. S.; Haasnoot, J. G.; Reedijk, J.; Bradec, V. Biochemistry 1995, 34, 12369-12378.

42. Pacor, S.; Sava, G.; Ceschia, V.; Bregant, F.; Mestroni, G.; Alessio, E. Chem.-Biol. Interact. 1991, 78, 223-234.

43. Cauci, S.; Viglino, P.; Espoito, G.; Quadrigoglio, F. Inorg. Biochem. 1991, 43, 739- 751.

44. Esposito, G.; Cauci, S.; Fogolari, F.; Alessio, E.; Scocchi, M.; Quadrifoglio, F.; Viglino, P. Biochemistry 1992, 31, 7094-7103.

45. Barton, J. K.; Lolis, E. J. Am. Chem. Soc. 1985, 107, 708-708.

46. Grover, N.; Gupta, N.; Thorp, H. H. J. Am. Chem. Soc. 1992, 114, 3390-3393.

47. Van Vliet, P.M.; Haasnoot, J. G.; Reedijk, J. Inorg. Chem. 1994, 33, 1934-1939.

48. Zobi, F.; Hohl, M.; Zimmermann, I.; Alberto, R. Inorg. Chem. 2004, 43, 2771-2772.

49. Zhao, M.; Clarke, M. J. J. Biol. Inorg. Chem. 1994, 325-340.

50. Van Vliet, P. M.; Haasnoot, J. G.; Reedijk, J. Inorg. Chem. 1994, 33, 1934-1939.

51. Zobi, F.; Hohl, M.; Zimmermann, I.; Alberto, R. Inorg. Chem. 2004, 43, 2771-2772.

52. Burstyn, J. N.; Heiger-Bernays, W. J.; Cohen, S. M.; Lippard, S. J. Nucleic Acids Res. 2000, 28, 4237-4243.

53. Kankia, B. I.; Soto, A. M.; Burns, N.;Shikiya, R.; Tung, C-S.; Markey, L. A. Biopolymers 2002, 65, 218-227.

108

54. Breslauer, K. J.; Frank, R.; Blocker, H.; Marky, L. A. Proc. Natl. Acad. Sci. USA 1986, 83, 3746-3750.

55. Marky, L. A.; Breslauer, K. J. Biopolymers 1982, 2185-2194.

56. Grover, N.; Welch, T. W.; Fairley, T. A.; Cory, M.; Thprp, H. H. Inorg. Chem. 1994, 33, 3544-3548.

57. Grover, N.; Welch, T. W.; Fairley, T. A.; Cory, M.; Thorp, H. H. Inorg. Chem. 1994, 33, 3544-3548.

58. Farrell, N.; Qu, Y.; Feng, L.; Van Houten, B. Biochemistry 1990, 29, 9522-9531.

59. Suh, D.; Oh, Y.; Chaires, J. B. Process Biochem. 2001, 37, 521-525.

60. Satynarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Biochem. 1992, 31, 9319-9324.

61. Brown, S. J.; Kellet, P. J.; Lippard, S. J. Science 1993, 261, 603.

62. Takahara, P. M.; Rosenzweis, A. C.; Frederick, P. M.; Lippard, S. J. Nature 1995, 377, 649.

63. Sherman, S. E.; Lippard, S. J. Chem. Rev. 1990, 100, 293.

64. Butour, J.-L.; Macquet, J.-P. Biochim. Biophys. Acta 1981, 653, 305.

65. Mong, S.; Huang, C. H.; Prestayko, A. W.; Crooke, S. T. Cancer Res. 1980, 40, 3313.

66. Cohen, G. L.; Bauer, W. R.; Barton, J. K.; Lippard, S. J. Science, 1979, 203, 1014- 1016.

109

CHAPTER 5

+ + Cis-[Ru(phpy)(bpy)(CH3CN)2] and cis-[Ru(phpy)(CH3CN)4]

5.1. Introduction

The photochemistry and photophysics of d6 coordination complexes of ruthenium

containing nitrogen heterocyclic ligands such as 2,2’-bipyridine (bpy) and its derivatives

have been extensively investigated.1 The investigation has centered around prototypical

n+ compounds of the type, [M(bpy)3-xLx] where L is typically a bidentate ligand containing

nitrogen atoms for coordination. Recently, however, cyclometalated metal complexes

containing phenylpyridine ligands have been investigated.2,3 Cyclometalated complexes

of platinum were first reported in 1965,4 and were shown to be promising in various

fields of chemistry including organic synthesis,5 catalysis,6 and bioinorganic chemistry.7

It was shown that the replacement of a nitrogen donor by a carbanion donor effectively alters the electron density around the metal, resulting in variations in the ground state and excited state properties.7 The ligand field state of compounds containing carbanion will

also be of higher energy since the carbanion donor is a stronger ligand in the

spectrochemical series than aromatic nitrogen atoms in bpy. As a result the photophysical

and photochemical properties of Ru(II) complexes with carbanion coordination are

2+ substantially perturbed relative to [Ru(bpy)3] .

110 5.2 Background

Many cyclometalated complexes containing phenyl groups that are adjacent to a

8 good ligating functionality are known. They are of the type, [Ru(o-C6H4-

Z)(LL)(X2)](PF6) where ( Z = 2-pyridinyl, 2-imidazolyl, CH2NMe2; LL/LL’ = 2,2’- bipyridine (bpy), 1,10-phenanthroline (phen), X = pyridine or CH3CN). The

cyclometalated compounds previously reported have been used as mediators of electron

transfer to or from oxidized or reduced active sites of the redox enzymes.8 The complexes

were also shown to display unprecedented high reactivity towards horseradish peroxidase

(HRP) and glucose oxidase (GO) from Aspergillus niger.9 These complexes possess a

metal-carbon σ-bond, which was shown to lower the redox potentials of the Ru(II)

2+ complexes relative to [Ru(bpy)3] . The oxidation potential of ruthenacycles [Ru(o-C6H4-

Z)(LL)(LL’)](PF6) (Z = 2-pyridinyl, 2-imidazolyl, CH2NMe2; LL/LL’ = bpy, phen) were

found in the region 150- 400 mV (vs SCE), which permits electron exchange of the

ruthenacycles with glucose oxidase (GO),9 glucose dehydrogenase,10 or peroxidases from

horse radish, sweet potato and royal palm tree.9,11 The rate constants for the oxidation of

reduced GO and glucose dehyrogenase by the Ru(III) species was found to be 10-6-10-7 M-

1s-1.12

The ruthenacyles of cis-[Ru(o-C6H4-2-py)(LL)(CH3CN)2]PF6 (LL = bpy, phen)

were previously synthesized by ligand substitution with bpy and phen from [Ru(o-C6H4-

12 2-py)(CH3CN)4]PF6 in CH2Cl2 and CH3CN with 74 % and 83 % yields, respectively.

The reaction of [Ru(o-C6H4-2-py)(CH3CN)4]PF6 with bpy in CH3CN was unsuccessful,

but it worked quiet well in CH2Cl2. This was explained in terms of the fact that the

111 stability of phen is approximately an order of magnitude greater than that of the

corresponding bpy complexes. The X-ray structural characterization of cis-[Ru(o-C6H4-2-

py)(phen)(CH3CN)2]PF6 has been performed, where a cis configuration of the CH3CN groups has been determined.12 It was also observed that one of the phen nitrogens is

located trans to the carbon of the cyclometalated ligand, resulting in a longer Ru-N bond

length compared to the Ru-N bond length of the same phen ligand that is in a trans

position with respect to CH3CN. Careful inspection of bond lengths of cis-[Ru(o-C6H4-2-

py)(phen)(CH3CN)2]PF6 indicates that the Ru-Nphen bond distances are longer than the

Ru-NCH3CN distances due to a more pronounced back-bonding capability of the

12 coordinated acetonitrile as compared to phen. The cis geometry of the CH3CN ligands

has been confirmed by X-ray structural studies where the σ-bound sp2 carbon of the

metallated ring is trans to the LL nitrogen.

The cyclic voltammetry of cis-[Ru(o-C6H4-2-py)(phen)(CH3CN)2]PF6 and cis-

(II)/(III) [Ru(o-C6H4-2-py)(bpy)(CH3CN)2]PF6 reveals Ru quasy-reversible redox features at

573 and 578 mV (vs Ag/AgCl), respectively in MeOH in the dark. It was also reported that after a minute of irradiation of cis-[Ru(o-C6H4-2-py)(phen)(CH3CN)2]PF6 and cis-

[Ru(o-C6H4-2-py)(bpy)(CH3CN)2]PF6 in MeOH, the complexes were converted into new species with redox potentials of -230 and 270 mV, respectively. The large potential drop

experienced by cis-[Ru(o-C6H4-2-py)(phen)(CH3CN)2]PF6 was accounted for in terms of

1 photosubstitution of both CH3CN ligands by MeOH. ESR, H NMR, and electronic

absorption data indicate that the primary product upon photolysis is a Ru(III) species,

presumably cis-[Ru(o-C6H4-2-py)(phen)(MeOH)2]PF6. The primary product of cis-[Ru(o-

112 C6H4-2-py)(bpy)(CH3CN)2]PF6 was reported to be cis-[Ru(o-C6H4-2-

py)(bpy)(CH3CN)(MeOH)]PF6, which accounts for the lower redox potential relative to

the corresponding phen product. Irradiation of cis-[Ru(o-C6H4-2-py)(phen)(CH3CN)2]PF6 in the presence of added chloride ligands affords Ru-[(phen) (o-C6H4-2-

III IV py)ClRu ORu ClRu(o-C6H4-2-py)(phen)]PF6, the first μ-oxo-bridged dimer of a

cyclometalated compound with mixed valency.

The work presented in this chapter will focus on the photochemistry of the

ruthenacycles, cis-[Ru(phpy)(bpy)(CH3CN)2](PF6) (phpy = phenylpyridine) and

[Ru(phpy)(CH3CN)4](PF6) in H2O and other solvents where a photoinduced solvolytic

substitution of the CH3CN ligands is expected. The photoinduced substitution with strong

ligands such as bpy ligand will also be probed. The DNA binding to single stranded 15-

mer oligonucleotide and to double stranded DNA will be investigated by mobility-shift

gel electrophoresis experiment and viscosity.

113 5.3 Results and Discussion

5.3.1 Photophysical Properties

+ The absorption spectra of cis-[Ru(phpy)(bpy)(CH3CN)2] and

+ + [Ru(phpy)(CH3CN)4] in H2O are shown in Figure 5.1. Cis-[Ru(phpy)(bpy)(CH3CN)2]

exhibits absorption maxima at 290 nm (ε = 63 500 M-1cm-1), 370 nm (ε = 9 870 M-1cm-1)

and 488 nm (ε = 5 700 M-1cm-1). The position and intensity of the latter two absorption

bands are characteristic of metal-to-ligand charge transfer (MLCT), Ru → bpyπ*as reported for related complexes.5 The band centered at 290 nm is assigned as a ligand

centered transition (π → π*), since the absorption spectra of complexes with bpy ligand, typically exhibit this band, which is also observed in the free ligand.

+ -1 -1 [Ru(phpy)(CH3CN)4] exhibits absorption maxima at 242 nm (ε = 32 363 M cm ) and

378 nm (ε = 5 200 M-1cm-1), where the former band may be assigned as a ligand centered

transition and the latter band as an MLCT transition. The absence of the peak at 488 nm

+ in [Ru(phpy)(CH3CN)4] is consistent with the assignment of this peak in cis-

+ [Ru(phpy)(bpy)(CH3CN)2] as a MLCT transition from the ruthenium to the bipyridine

ligand. By comparison, the peaks at 370 nm and 378 nm for cis-

+ + [Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpy)(CH3CN)4] respectively, may be assigned as a

MLCT transition from the metal to the phenylpyridine ligand. These assignments seem

2+ reasonable since [Ru(bpy)3] is known to have MLCT transitions at 385 nm and 453

13,14 2+ nm. The shift in the MLCT transition from 453 nm in [Ru(bpy)3] to 488 nm in

114 2.5

2.0

1.5

1.0 Absorbance (a.u.) Absorbance

0.5

0.0 300 400 500 600 700 800 Wavelength (nm)

+ Figure 5.1. Electronic absorption spectra of cis-[Ru(phpy)(bpy)(CH3CN)2] (⎯ ⎯) and + [Ru(phpy)(CH3CN)4] (…..) in H2O.

+ cis-[Ru(phpy)(bpy)(CH3CN)2] can be explained by the lower oxidation potential of the metal center in the latter, resulting in a lower energy transition.

+ + The emission of cis-[Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpy)(CH3CN)4] was investigated at room temperature in CH3CN and H2O and at 77 K in EtOH:MeOH (4:1

+ v/v). Cis-[Ru(phpy)(bpy)(CH3CN)2] was weakly emissive at room temperature in

CH3CN and in H2O with an emission maximum at 780 nm (φ = 0.0023, τ = < 10 ns) in

H2O (Figure 5.2). The excitation spectra are consistent with the emission arising from the

+ complex (Figure 5.2). [Ru(phpy)(CH3CN)4] was non emissive at room temperature. The

+ intensity of the emission of cis-[Ru(phpy)(bpy)(CH3CN)2] at 790 nm in H2O was lower than that observed in CH3CN. However, at 77 K in EtOH:MeOH (4:1 v/v) both

115

1.2

1.0

0.8

0.6

0.4

Intensity (cps) 0.2

0.0

300 400 500 600 700 800 Wavelength (nm)

Figure 5.2. Emission (λexc = 480 nm, ⎯) and excitation (λexc = 790 nm, ----) spectra of + cis-[Ru(phpy)(bpy)(CH3CN)2] in H2O at room temperature.

compounds exhibit strong emission with maxima at 662 nm (φ = 0.35, τ = 0.92 μs) and

+ 542 nm (φ = 0.18, τ = 0.91 μs) for cis-[Ru(phpy)(bpy)(CH3CN)2] and

+ [Ru(phpy)(CH3CN)4] , respectively (Figure 5.3). The observation of the emission

+ maxima at 542 nm and 662 nm at 77 K for [Ru(phpy)(CH3CN)4] and cis-

+ 2+ [Ru(phpy)(bpy)(CH3CN)2] , which are shifted relative to [Ru(bpy)3] (λmax = 583 nm), is

consistent with the respective hypochromic and bathochromic shifts observed in the

absorption spectra of the complexes. The emission spectra also display the vibrational

fine structure that is characteristic of a Ru-bpy MLCT emission.15 In light of these

observations, the emission may reasonably be assigned as a MLCT transition localized

primarily on the phenylpyridine ligand for the former complex and on the bpy ligand for

116

1.2

1.0

0.8

0.6

0.4

Intensity (c.p.s) 0.2

0.0

500 600 700 800

Wavelength (nm) + Figure 5.3. Low temperature emission spectra of cis-[Ru(phpy)(bpy)(CH3CN)2] ((λexc = + 480 nm, ⎯ ⎯) and [Ru(phpy)(CH3CN)4] (λexc = 380 nm, …..) EtOH:MeOH (4:1 v/v) at 77 K.

2+ the latter complex. Similar results have also been reported for [Ru(bpy)2(biq)] and

2+ [Ru(biq)3] (biq = 2,2’-biquinoline) where in the mixed ligand complex distinct Ru →

bpy and Ru → biq charge transfer bands are present in the absorption spectrum, but

emission is only observed from the lowest excited state, which is Ru → biq MLCT.14,16

5.3.2 Photochemistry in H2O

+ The photolysis of cis-Ru(phpy)(bpy)(CH3CN)2 in H2O with λirr > 420 nm results

in the disappearance of the peak at 488 nm after the first 12 minutes of irradiation,

117 2

1.5

1 Absorbance (AU)

0.5

0 300 400 500 Wavelength (nm)600 700 800

Figure 5.4. Absorption spectrum of the photolysis reaction of cis- + [Ru(phpy)(bpy)(CH3CN)2] in H2O with irradiated with λ > 345 nm at t = 0, 10, 20, 30, 40, 40, 60, 70 min up to 4 hours with spectrum taken every 10 minutes.

followed by a growth in a new peak at 560 nm. The later step is slow and requires

irradiation for 4 hours. There is a blue shift in the peak at 370 nm to 350 nm after photolysis in H2O (Figure 5.4). Initial photolysis is believed to generate cis-

+ [Ru(bpy)(phpy)(CH3CN)2] . The second phase can be attributed to an interaction of O2

with the diaqua complex, resulting in the formation of a dinuclear μ-oxo species that will be discussed in more detail below. It was also reported recently that the photosolvolysis of Ru(o-C6H4-2-py)(LL)(CH3CN)2]PF6 (LL = phen, 4,4’-bpy) affected the electronic

spectra,19 for which irradiation induced a 20-40 nm blue shift of the band at ~ 370 nm,

but caused the disappearance of the lowest energy transition at ~ 470 nm.17,18 A much

118 slower step was also reported, which manifested itself in the growth of a new peak at

530-560 nm. This latter step was thought to be due to the formation of either a Ru(III) complex or a dinuclear μ-oxo species.19,20

+ 1 The photolysis of cis-[Ru(phpy)(bpy)(CH3CN)2] in D2O was followed by H NMR and

the final product obtained after irradiation for 6 hours was analyzed by mass

spectrometry. The 1H NMR signals in the aromatic region of cis-

+ [Ru(phpy)(bpy)(CH3CN)2] before and after photolysis are shown in Figure 5.5. The

+ peaks for cis-[Ru(phpy)(bpy)(CH3CN)2] were assigned based on the shown reaction in

Scheme 1, and were based on those previously published.12

The compound before irradiation shows sixteen distinct protons in the aromatic region of the 1H NMR, which correspond to the eight protons of the unsymmetrically- coordinated bipyridine ligand and eight protons from the phenylpyridine ligand. Two distinct peaks are observed at 2.17 ppm and 2.3 ppm, corresponding to the coordinated protons from CH3CN ligand before irradiation. The CH3CN ligands are observed at

different chemical shifts due to their different coordination environments. Upon

+ irradiation of cis-[Ru(phpy)(bpy)(CH3CN)2] for 4.5 hours in D2O (λirr > 345 nm), there

is a downfield shift in the resonances observed for bpy and phpy ligands, but the aromatic proton count remains the same, while the peaks corresponding to CH3CN are no longer observed. The resonance of bound water was not observed at room temperature, indicating fast ligand exchange on the NMR time scale with bulk water. However, a

broad peak was observed at 2.0 ppm, which might be attributed to free CH3CN. The

+ reaction of cis-[Ru(phpy)(bpy)(CH3CN)2] in D2O is complete after 4.5 hrs hours of

119 5' 6' 4' 5' 3 6' 4' 3 3' 4 C 3' 4 C C N 5 N 6 NCCH3 5 N λirr > 420 nm 6 O 10' NCCH N 3 D2O 9' N O O N N 7" N H2 H2 N 8' 7' 8" 10" 9"

+ Scheme 5.1. Reaction Schemes for the reaction of cis-[Ru(phpy)(bpy)(CH3CN)2] .

(c) 7” 3’

0.9 1.0 3.1 1.13.2 2.2 1.0 3.1 1.1

10.4 10.2 10.0 9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2

(b)

1.0 1.2 1.7 1.0 1.1 2.9 2.1 0.9 2.9 1.1

10.4 10.2 10.0 9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2

(a)

7” 3’ 10’ 9” 8’ 10’ 4 9” 5

1.0 1.2 2.3 1.1 4.1 1.1 1.2 1.1 1.1 1.1 1.0 1.0

10.4 10.2 10.0 9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2

Figure 5.5. Overlaid aromatic region of the 1H NMR spectra of the reaction of cis- + [Ru(phpy)(bpy)(CH3CN)2] in D2O when irradiated with λ > 345 nm for (a) 0 min, (b) 4.5 hours, and (c) 6.5 hours. 120 irradiation at λ > 345 as shown in Figure 5.5b, since no additional changes in the 1H

NMR were observed after this time (Figure 5.5c).The product of the reaction was also

subject to electrospray ionization mass spectrometry where peaks corresponding to

+ + [Ru(phpy)(bpy)(OH2)] , [Ru(phpy)(bpy)(OH2)2] ,

II II + [(H2O)(bpy)(phpy)Ru ORu (phpy)(bpy)(OH2)]H .H2O, and

II II + [(H2O)(bpy)(phpy)Ru ORu (phpy)(bpy)(OH2)]H .2H2O are observed at m/z = 428, 447,

890 and 908 respectively indicating the formation of the μ-oxo dimer. The 1H NMR

indicates the formation of a pure species, however the mass spectrometry data showed the

presence of a monomer of the photosubstitution reaction of cis-

+ [Ru(phpy)(bpy)(CH3CN)2] in D2O as well as the dimer. A possible explanation for the

observation of the monomer species in the mass spectrum is fragmentation of the dimer

that may have occurred upon electrospray or that a small impurity of the monomer is

present, but it is more volatile than the dimer.

Oxo-briged dimers of Ru(III) complexes are known to decompose upon titration

in 0.1 M HCl solution.20 Therefore, to further support the formation of μ-oxo dimer upon

+ irradiation of cis-[Ru(phpy)(bpy)(CH3CN)2] in aqueous solution, the compound was

irradiated in 0.1 M HCl solution and the reaction was followed by uv-vis. An immediate

increase in the absorbance was observed at 600 nm upon irradiations of cis-

+ [Ru(phpy)(bpy)(CH3CN)2] (λirr > 420 nm) in 0.1 M HCl. Upon mass spectrometry

anaylysis of the product of this reaction, a peak was observed at m/z = 447 corresponding

+ to the formation of cis-[Ru(phpy)(bpy)(OH2)2] . No peaks were observed in the region

that corresponds to the formation of a dimer. This evidence further support the formation

121 + of a μ-oxo dimer upon irradiation of cis-[Ru(phpy)(bpy)(CH3CN)2] in aqueous

media.The photochemistry was also investigated in phosphate buffer solutions of pH 5.2,

7.5 and 10.1, where similar changes were observed in the uv-vis spectrum to that observed in H2O, indicating that the reaction is independent of the pH.

+ For comparison, the photochemistry of [Ru(phpy)(CH3CN)4] was also studied.

+ The uv-vis spectra of the photochemistry of [Ru(phpy)(CH3CN)4] observed in H2O upon

irradiation with λirr > 345 nm is more complex than that observed for cis-

+ [Ru(phpy)(bpy)(CH3CN)2] . This complexity likely results from the fact that in

+ [Ru(phpy)(CH3CN)4] the photosubstitution of one, two, three or four CH3CN ligands

with H2O is possible, in addition to the possibility of dimer formation. Upon irradiation

+ of [Ru(phpy)(CH3CN)4] in H2O with λirr > 345 nm, broad peaks with λmax of 595 nm and

720 nm were observed (Figure 5.6). The product of he photolysis reaction was analyzed by electrospray ionization mass spectrometry and a m/z ratio of 661 was observed corresponding to the formation of

(II) (II) + [(CH3CN)(H2O)(phpy)Ru ORu (phpy)(H2O)(CH3CN)]H .H2O. The increase in the

(II),(II) peak observed at 595 nm is consistent with the formation of Ru2 dimers as observed

+ and explained in the reaction of cis-[Ru(phpy)(bpy)(CH3CN)2] in H2O.

+ The reaction of [Ru(phpy)(CH3CN)4] in D2O (λirr > 345 nm) was also followed

by 1H NMR where 8 distinct protons corresponding to the phpy ligands were observed in

the aromatic region, and the aliphatic regions shows two distinct peaks corresponding

to bound CH3CN ligands at 2.55 ppm (3H) and 2.0 ppm (9H) (Figure 5.7). However,

+ 1 upon irradiation of [Ru(phpy)(CH3CN)4] the aromatic region of the H NMR shows

122

2.5

2

1.5

1 (AU) Absorbance

0.5

0 250 350 450 550 650 750 850 Wavelength (nm)

+ Figure 5.6. Absorption spectrum of the photolysis reaction of cis-[Ru(phpy)(CH3CN)4]

in H2O with irradiated with λ > 345 nm at t = 0, 10, 20, 30, 40, 40, 60, 70 min up to 2 hours with spectrum taken every 10 minutes.

that different species were formed, since the intensity of the peaks observed in the

aromatic region are not consistent with the formation of a single product. Owing to the complexity of 1H NMR spectra, characterization of the product(s) formed upon irradiation was based on mass spectrometry.

+ 5.3.3 Photochemistry of cis-[Ru(phpy)(bpy)(CH3CN)2] in CH3CN

To further investigate the formation of the RuII/II dimer from the reaction of cis-

+ [Ru(phpy)(bpy)(CH3CN)2] in H2O, the compound was irradiated with λirr > 420 nm in

distilled CH3CN and the reaction was followed by uv-vis. A red shift was observed in the

+ MLCT peak at 488 nm to 530 nm upon irradiation of cis-[Ru(phpy)(bpy)(CH3CN)2] in 123

0.8 0.9 1.4 0.6 0.5 0.7 3.0 3.9 7.0 3.6 3.2 2.5 5.6 4.2

9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6

9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6

0.9 0.7 1.0 0.8 1.3 0.9 2.0 9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6

Figure 5.7. Overlaid aromatic region of the 1H NMR spectra of the reaction of cis- + [Ru(phpy)(CH3CN)4] in D2O when irradiated with λ > 345 nm for (a) 0 min (b) 1hr and (c) 6 hrs.

CH3CN (Figure 5.8). No changes in the absorption spectrum of the complex are expected in a ligand substitution reaction in CH3CN solvent since the product would be identical to the starting material. However, as shown in Figure 5.8, a red shift in the absorption spectrum was observed with an isosbestic point at 495 nm, indicating the formation of a

124 single product. In order to elucidate the observed reactivity, the reaction was followed by

1 + H NMR in CD3CN by irradiating cis-[Ru(phpy)(bpy)(CH3CN)2] with λ > 420 nm. After

1 hour of irradiation, the growth of several new peaks, along with peaks corresponding to the starting material, were observed in the 1H NMR spectrum (Figure 5.9b). However, upon continued irradiation for ~ 24 hours, the intensity of the peaks corresponding to the

0.8

0.6

0.4

Absorbance (AU)

0.2

0 300 350 400 450 500 550 600 650 700 750 800 Wavelength(nm)

Figure 5.8. Absorption spectrum of the photolysis reaction of cis- + [Ru(phpy)(bpy)(CH3CN)2] in CH3CN with irradiated with λ > 420 nm at t = 5, 10, 15, 20, 25, 30, 35, 40 min up to 2 hours with spectrum taken every 5 minutes. starting material decreases while the intensity of the new peaks increases. The product of the photolysis after 24 hours was subject to time of flight mass spectrometry (TOF MS), where peaks were observed at m/z = 964.6, 1001.2 and 1094.2, corresponding to

II II + [(phpy)(bpy)(CH3CN)Ru ORu (CH3N)(bpy)(phpy)]H + 1 CH3CN,

II II + [(phpy)(bpy)(CH3CN)Ru ORu (CH3N)(bpy)(phpy)]H + 2 CH3CN and 125 II II + [(phpy)(bpy)(CH3CN)Ru ORu (CH3N)(bpy)(phpy)]H + 4 CH3CN respectively (Figure

5.10). It is possible that the formation of μ-oxo dimer is a result of traces of H2O that may

be present in the solvent. These results support the formation of a RuII/II μ-oxo dimer.

(c)

* * * * *

1.0 0.9 1.2 1.3 1.8 2.0 1.9 1.6 0.9 1.1 1.0 0.9 0.9

10.0 9.8 9.6 9.4 9. 2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7. 6 7.4 7.2 7.0 6.8 6.6 6.4 6.2

(b)

1.0 0.7 0.9 1.3 0.8 3.8 1.8 4.5 2.5 2.5 0.8 0.8 1.1 2. 5 1.6 0.9 0.8 10.0 9.8 9.6 9.4 9. 2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7. 6 7.4 7.2 7.0 6.8 6.6 6.4 6.2

(a)

1.0 1.1 2.9 3.2 1.0 1.1 1.1 1. 1 2.1 1.0

10.0 9.8 9.6 9.4 9. 2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7. 6 7.4 7.2 7.0 6.8 6.6 6.4 6.2

Figure 5.9. Overlaid aromatic region of the 1H NMR spectra of the reaction of cis- + [Ru(phpy)(bpy)(CH3CN)2] in CH3CN when irradiated with λ > 420 nm for (a) 0 min (b) 1 hours and (c) ~24 hours. * denotes protons from unreacted cis- + [Ru(phpy)(bpy)(CH3CN)2] .

126

Figure 5.10. Time of flight electrospray ionization mass spectrometry of the reaction of + cis-[Ru(phpy)(bpy)(CH3CN)2] in CH3CN when irradiated with λ > 420 nm for ~24 hours.

+ 5.3.4 Photochemistry of cis-[Ru(phpy)(bpy)(CH3CN)2] in MeOH

+ Irradiation of cis-[Ru(phpy)(bpy)(CH3CN)2] in distilled MeOH with λirr > 420 nm for 1 hour results in a decrease in the peak at 488 nm and its eventual disappearance, together with a red shift in the peak at 376 nm to 386 nm. The reaction was also followed

1 by H NMR, where 20 min of irradiation in CD3OD resulted in the disappearance of the well-defined aromatic peaks of 2-phenylpyridine and bipyridine ligand, together with the appearance of a sharp singlet at 2.03 ppm, which is attributed to liberated CH3CN (Figure

127 5.11). The broad and ill defined aromatic peaks that are observed is an indication of

oxidation of a Ru(II) to Ru(III) species. The reaction observed in MeOH after 20 min

irradiation parallels those observed by Ryabov et al,12 where it was reported that

+ irradiation of cis-[Ru(phpy)(bpy)(CH3CN)2] in MeOH resulted in the oxidation of the ruthenium center.

(b) H2O CD3OD

MeCNfree

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5

(a) MeCNbound

1.0 1.2 1.1 1.9 1.1 3.3 1.1 1.1 1.0 1.2 2.0 1.1 5.7

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5

Figure 5.11. Overlaid aromatic region of the 1H NMR spectra of the reaction of cis- + Ru(phpy)(bpy)(CH3CN)2 in CD3OD (a) t = 0 min (b)irradiated with λ > 420 nm for 20 min.

128 The oxidizing agent could be dioxygen as was found in studies done by Ford et al.21

Upon continued irradiation in MeOH for one hour, no further change was observed in the proton NMR.

5.3.5 Reaction with bpy and py ligands

+ Reacting cis-[Ru(phpy)(bpy)(CH3CN)2] with 1 eq of bipyridine ligand (λirr> 420 nm) in the non-coordinating solvent CH2Cl2, resulted in the formation of

+ [Ru(phpy)(bpy)2] , characterized by the increase of the peaks at 404 nm and 546 nm

(Figure 5.12a). A small shoulder was also observed at 490 nm upon irradiation. The

asymmetric nature of the band centered at 546 nm suggests that more than one electronic

transition may be involved. In fact, electrochemical data of results from the related

+ complex, [Ru(bpy)2NPP] (NPP = 2-(3-nitrophenylpyridine) indicates that this band is

actually due to at least two MLCT transitions, one on the bpy and the other on the

phenylpyridine ligand. The bathochromic shift in the Ru → bpy MLCT band relative to

2+ that in [Ru(bpy)3] (λmax = 453 nm) is likely due to changes in energy of the metal

centered t2g orbital. The replacement of the nitrogen donor ligand on the bpy ligand by the

carbanion in phpy greatly increases the electron density around the metal, thereby

increasing the energy of the pseudo-t2g orbital. This destabilization of the metal centered

HOMO is evident in the electrochemistry, where the oxidation potential of

+ [Ru(bpy)2NPP] in CH2Cl2 was reported as 0.776 V (vs SCE), whereas that reported for

2+ 13 to [Ru(bpy)3] is 1.29 V. The close proximity of the asymmetric bands of

129 + * [Ru(phpy)(bpy)2] may also indicate that the π orbitals of bpy and phenylpyridine lie at

nearly identical energies since the two MLCT transitions are very close in energy.

+ Similar results to those described above for cis-[Ru(phpy)(bpy)(CH3CN)2] were

+ observed for the photolysis of [Ru(phpy)(CH3CN)4] with excess bipyridine ligand (λirr>

420 nm) in CH2Cl2. An increase in the absorption was observed at 404 nm and 546 nm,

+ which is characteristic of the formation of [Ru(phpy)(bpy)2] (Figure 5.12b). This result

+ indicates that all four CH3CN ligands on [Ru(phpy)(CH3CN)4] are displaced upon

irradiation.

+ [Ru(phpy)(bpy)2] was independently synthesized and characterized and was

shown to exhibit absorption maxima at 249 nm (ε = 47 420 M-1cm-1), 296 nm (ε = 81 320

M-1cm-1), 368 nm (ε = 18 080 M-1cm-1), 404 nm (ε = 13 950 M-1cm-1) and 546 nm (ε = 12

000 M-1cm-1), which corresponded well to the peaks observed in the photolysis reactions

+ + of cis-[Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpy)(CH3CN)4] in the presence of bpy

ligand (Figure 5.12). Control reactions upon the addition of bpy ligand to either cis-

+ + [Ru(phpy)(bpy)(CH3CN)2] or [Ru(phpy)(CH3CN)4] in CH2Cl2 in the dark resulted in no

changes in the absorption spectra of the complexes, indicating that light is necessary for

the displacement of the CH3CN ligands. It was shown in the above reactions of

+ [Ru(phpy)(CH3CN)4] with excess bpy ligand that the four CH3CN ligands are photodisplaced and photosubstitution ocurr with the strong bpy ligand. Hence to

+ determine the reactivity of [Ru(phpy)(CH3CN)4] with other π acceptor ligand, the

photolysis of the metal complex was carried out in excess pyridine.

130

(b)

0.8

0.6

0.4 Absorbance (AU)

0.2

0 300 400 500 600 700 800 Wavelength (nm) 0.2 (a)

)

0.1

Absorbance (AU

0

300 400 500 600 700 800

Wavelength(nm)

Figure 5.12. Absorption spectrum of the photolysis reaction of (a) cis- + + [Ru(phpy)(bpy)(CH3CN)2] and (b) [Ru(phpy)(CH3CN)4] with 1 eq of bpy ligand in

CH2Cl2, irradiated with λ > 420 nm for t = 0, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60 min.

131 + The photolysis reaction of [Ru(phpy)(CH3CN)4] (λirr > 420 nm) with excess pyridine was followed by observing the changes in the absorption spectra upon photolysis by uv- vis spectroscopy. The absorption spectra show an increase in the peak at 387 nm and at

2+ 640 nm (Figure5.13). The absorption spectra of [Ru(bpy)(py)4] has been reported to

have absorption maxima at 285 nm, 360 nm and 500 nm.22 The former peak is assigned

as a ligand centered transition on the bpy ligand (π→π*) whereas the peak at 360 nm is

assigned as a MLCT transition involving the pyridine ligand and the peak at 500 nm as a

MLCT peak involving the bpy ligand.21 Based on the assignments of the absorption peaks

2+ + for [Ru(bpy)(py)4] , the peaks observed upon irradiation of [Ru(phpy)(CH3CN)4] with

λirr > 420 nm with excess pyridine are assigned in a similar manner. Hence the peak

observed at 387 nm is assigned as a MLCT transition involving the py ligand whereas,

the peak observed at 640 nm would be assigned as a MLCT transition involving the

phenylpyridine ligand. The bathochromic shift in the MLCT transitions observed upon

+ 2+ the irradiation of [Ru(phpy)(CH3CN)4] with excess pyridine relative to [Ru(bpy)(py)4]

can be explained in terms of changes in the metal centered t2g orbital. The replacement of

the nitrogen donor by a carbon donor greatly increases the electron density on the metal,

which raises the energy of the d orbitals thereby lowering the energy separation between the metal and the ligand.

132

0.9

0.8 0.7

0.6

0.5

0.4

Absorbance (A.U.) 0.3

0.2

0.1

0 300 400 500 600 700 800 900 WAvelength (nm)

Figure 5.13. Absorption spectrum of the photolysis reaction of (a) cis- + [Ru(phpy)(bpy)(CH3CN)2] with excess py in CH2Cl2, irradiated with λ > 420 nm for t = 0, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60 min.

5.3.6 Reaction with 9-MG and 9-EG

+ The photolysis of 5 mM cis-[Ru(phpy)(bpy)(CH3CN)2] in the presence of 2.5 eq.

1 of 9-EG or 9-MG, (λirr> 420 nm, 6 hours) was followed by H NMR and the products

were analyzed by MALDI mass spectrometry. The 1H NMR did not show the growth of

the bound H8 proton of either 9-MG or 9-EG as was described previously for other

complexes. As shown in previous chapters, the chemical shift of the H8 protons of

guanine differ significantly when the guanine is bound to the ruthenium (Figure 5.14).

The changes observed in the 1H NMR spectrum shown in Figure 5.14 are consistent with

+ the formation of cis-[Ru(bpy)(phpy)(H2O)2] which was described in detail above. 133 + However, when the product of photolysis of 5 mM cis-[Ru(phpy)(bpy)(CH3CN)2] in the presence of 2.5 eq. of 9-EG and 9-MG (λirr > 420 nm, 6 hours) was subject to MALDI mass spectrometry analysis, the results indicate the binding of 9-MG and 9-EG to the

+ metal complex, resulting in the formation of cis-[Ru(phpy)(bpy)(9-EG)(H2O)] and cis-

+ [Ru(phpy)(bpy)(9-MG)(H2O)] with m/z at 606 and 592, respectively (Figure 5.15).

Peaks corresponding to the μ-oxo dimer were also observed with the irradiation of cis-

+ [Ru(phpy)(bpy)(CH3CN)2] with the guanine derivatives, at m/z of 893 and 910

corresponding most specifically to

II II + [(phpy)(bpy)(H2O)Ru ORu (phpy)(bpy)(H2O)]H .H2O and

II II + [(phpy)(bpy)(H2O)Ru ORu (phpy)(bpy)(H2O)]H .2H2O respectively.

+ The reaction of 5 mM [Ru(phpy)(CH3CN)4] in the presence of 2.5 eq. of 9-EG

1 and 9-MG (λirr> 420 nm) were also followed by H NMR and the product of the

photolyzed reaction was analyzed by MALDI mass spectrometry. The proton NMR was

very similar to that observed when the complex was irradiated at the same wavelength in

D2O but the chemical shift of the H8 proton of bound guanine was observed at 7.9 ppm

(Figure 5.16), which is similar to that observed by Reedijk and Alberto.23, 24 Reedijk and

coworkers have reported a chemical shift of 7.8 ppm when cis-[Ru(bpy)2Cl2] is refluxed

for extended periods of time with 9-EG forming only the monoadduct. Alberto and

others, however, recently reported the binding of two equivalents of 9-methyl guanine in

the thermal reaction with cis-[Ru(bpy)2(O3SCF3)2], forming the bisadduct, and reported the chemical shift of the bound guanine at 6.8 and 8.0 ppm. The guanines exhibit different chemical shifts because the two guanines are in a head-to-tail orientation

134

(d)

1.0 2.2 3.8 7.5 1.9 1.8 3.5 2.5 2.5 2.1 1.9

9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 8. 2 8.0 7.8 7.6 7. 4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 (c)

1.0 2.2 3.5 6.4 4.5 2.4 2.0 2.6 1.0 1.5

9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0

(b)

1.0 1.2 1.9 1.4 2.7 1.2 2.9 1.1 1.1 1.3 1.6 1.2 1.4 9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 (a)

1.0 1.0 2.1 1.0 3.5 1.2 1.0 0.8 0.9 0.8 1.0 0.9

9.8 9.6 9. 4 9.2 9.0 8.8 8.6 8.4 8. 2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0

Figure 5.14 Overlaid aromatic region of the 1H NMR spectra of the reaction of (a) cis- + + [Ru(phpy)(bpy)(CH3CN)2] in D2O (b) cis-[Ru(phpy)(bpy)(CH3CN)2] + 9-MG, t = 0 min

(c) λirr > 420 nm for 3 hrs (d) λirr > 420 nm for 5 hrs

135

Figure 5.15. MALDI mass spectrometry results of the product of the photolysis reaction + of 5 mM of cis-[Ru(phpy)(bpy)(CH3CN)2] with 2.5 eq of 9-MG, λirr > 420 nm for 24 hours.

determined by x-ray crystallography. MALDI mass spectrometry data for the reaction of

+ [Ru(phpy)(CH3CN)4] with 9-EG shows peaks at m/z = 586 and 627, corresponding to

+ + the formation of [Ru(phpy)(9-EG)(ACN)2THF] and [Ru(phpy)(9-EG)(ACN)3THF] , respectively. Similar peaks were also observed for the reaction of 9-MG with

+ [Ru(phpy)(CH3CN)4] at m/z ratios = 572 and 613, corresponding to [Ru(phpy)(9-

+ + MG)(ACN)2THF] and [Ru(phpy)(9-MG)(ACN)3THF] , respectively.

5.3.7 Reaction with Oligonucleotides

In order to investigate the effect of the photolysis of cis-

+ + [Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpy)(CH3CN)4] in the presence of DNA, the

136 reactivity of the complex towards two different 15-mer oligonucleotide sequences (5’-

TGCAAGCTTGGCACT-3’ (S1) and 5’- AGTGCCAAGCTTGCA-3’ (S2) were undertaken using MALDI mass spectrometry. The products of the photolysis of 100 μM

+ cis-[Ru(phpy)(bpy)(CH3CN)2] in the presence of 100 μM S1 or S2 (λirr > 420 nm, 30 min) were subjected to mass spectrometry analysis. Peaks corresponding to the calculated masses of S1 (m/z = 4568) and S2 (m/z = 4577) were observed at m/z = 4567.4, and m/z

= 4578.4, respectively (Figure 5.17).

*

(d) H8

9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6

(c) H8

1.0 0.6 1.4 3.3 5.3 2.3 3.3

9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6

(b) H8

9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6

(a) 1.0 1.1 1.2 1.3 1.8 1.1 2.4

9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6

Figure 5.16 Overlaid aromatic region of the 1H NMR spectra of the reaction of (a) 5 mM + cis-[Ru(phpy)(CH3CN)4] + 2.5 eq of 9-EG in D2O, t = 0 min (b) t = 1 hr, λirr > 420 nm

(c) t = 3.5 hr λirr > 420 nm (d) t = 6 hr λirr > 420 nm. * denotes H8 proton of free guanine. 137

(a)

(b)

Figure 5.17. MALDI mass spectrometry results of the reaction of cis- + [Ru(phpy)(bpy)(CH3CN)2] with the 15-mer oligonucleotide sequences (a) 5’- TGCAAGCTTGGCACT-3’ (S1) and (b) 5’- AGTGCCAAGCTTGCA-3’ (S2) after irradiation with λirr > 420 nm for 30 mins.

138 However, photoinduced binding of the metal complex was not observed to either

oligonucleotide sequence. It is possible that binding was not observed due to the

formation of μ-oxo dimer, a reaction that is known to take place upon irradiation of cis-

+ [Ru(phpy)(bpy)(CH3CN)2] in water.

+ [Ru(phpy)(CH3CN)4] was also irradiated with both 15-mer sequences, S1 and S2

(λirr > 420 nm, 30 min) and the resulting solutions were submitted to MALDI mass

+ spectrometry analysis. Upon irradiation of [Ru(phpy)(CH3CN)4] with S1 (λirr > 420 nm,

30 min) peaks labeled A, B, C, D were observed (Figure 5.18a) corresponding to the

mass of two, three, and four, Ru(phpy) fragments bound to S1 observed at m/z of 5073.3,

+ 5333.9, 5593.9 and 5845.8, respectively. Irradiation of [Ru(phpy)(CH3CN)4] with S2 results in peaks corresponding to the binding of one, two, three, four, Ru(phpy) fragments, in addition to a molecule and of triethanolammonium choride at m/z of

4973.9, 5228.5, 5657.3 and 5929.0 respectively and are labeled as labeled A’, B’, C’ and

D’ in Figure 5.18b. A peak corresponding to the actual mass of S2 was also observed

(Figure 5.18b). In contrast, a peak for the actual mass of S1 was not observed, indicating complete binding of the metal complex to S1. It should be noted that although the metal complex is in 15-fold excess in the photolysis experiments, covalent binding of up only to four molecules of Ru(phpy) fragment per DNA strand was observed. with 5’-

TGCAAGCTTGGCACT-3’ (strand 1, S1) and 5’- AGTGCCAAGCTTGCA-3’ (strand 2,

S2), that greater covalent binding occurred for the DNA strand containing the GG

sequence since the mass corresponding to the 15-mer sequence of the oligonucloetide

only was not observed for strand 1 compared to strand 2.

139

(a) B

C D

A

(b) S2

A

B’ C’ D’

+ Figure 5.17. MALDI mass spectrometry results of the reaction of [Ru(phpyCH3CN)4] with the 15-mer oligonucleotide sequences (a) 5’- TGCAAGCTTGGCACT-3’ (strand 1,

S1) and (b) 5’- AGTGCCAAGCTTGCA-3’ (strand 2, S2) after irradiation with λirr > 420 nm for 30 mins.

140 5.3.8 Binding to double stranded DNA

The photoinduced binding of the metal complexes was also investigated with

double stranded DNA. The complementary 15-mer oligonucleotide sequences, S1 and

S2, were annealed resulting in the formation of a double stranded 15-mer duplex. A

melting temperature of 57 oC was measured for the 15-mer duplex in the absence of the

metal complex (2 mM NaCl, 5 mM phosphate buffer, pH = 7.2). Irradiation of 50 μM

+ cis-[Ru(phpy)(bpy)(CH3CN)2] with 50 μM ds-15-mer did not result in a shift of the

+ melting temperature, indicating that cis-[Ru(phpy)(bpy)(CH3CN)2] did not covalently

+ bind to the duplex. However, upon irradiation of 50 μM [Ru(phpyCH3CN)4] with the ds-

15-mer, a melting temperature of 55 0C was obtained, resulting in a ΔT = -2 0C which

+ indicates that there may be a small degree of covalent binding of [Ru(phpyCH3CN)4] to

the duplex. However, this shift is within experimental error, and is therefore inconclusive.

Control experiments were also conducted when similar concentrations of cis-

+ + [Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpyCH3CN)4] were kept in the dark with the duplex resulting in no shift in the melting temperature. Cisplatin was previously reported to covalently bind to a 20-mer duplex resulting in a ΔT = -8 oC.25 Therefore, from the melting temperature data it can be concluded that the metal complexes are not covalently binding to DNA in a manner similar to that observed for cisplatin. Again, this observation may be due to the formation of dinuclear μ-oxo dimer upon irradiation in aqueous media.

Covalent binding of cisplatin to linearized DNA has been shown to result in the

retardation of the mobility of the DNA on the agarose gel. Increasing concentration of the covalent cross linker, cisplatin, results in a reduction in the mobility of the linearized 141 plasmid as the relative concentration of base pairs (bp) to metal complex (mc) was varied

from 1 bp:100 mc to 1 bp:5 mc (Figure.5.19a). Lanes 1 and 8 are molecular weight

standard ranging from 1000 bp to 10 000 bp with increasing increments of 1000 bp.

Lanes 2 and 7 contain the linearized plasmid as a control, with no metal complex added.

Lanes 3-6 contain the plasmid DNA with varying bp to mc ratios of cisplatin after incubation for 3 hours at 37 oC, ranging from 1 bp:100 mc to 1bp:5 mc, respectively. As

shown in Figure 5.19a, reduction in the mobility of the DNA on the agarose gel was most

pronounced at the lowest ratio of cisplatin to base pairs, or at the highest relative metal

+ concentration (Lane 6). When various concentrations of cis-[Ru(phpy)(bpy)(CH3CN)2] were irradiated with 50 μM linearized plasmid (λirr > 420 nm, 30 min), a very small

decrease in the mobility of the DNA was observed (Figure 5.19b) compared to those

recorded with equivalent concentrations of cisplatin (Figure 5.19a). A decrease in EtBr

+ emission was observed for both cisplatin and cis-[Ru(phpy)(bpy)(CH3CN)2] with

increasing bp:mc. Intercalation of EtBr is essential for its emission to be observed in

these experiments. The decrease in the emission may be due to the change in

conformation of the DNA as the metal complex binds to it, thus making it difficult for

interaction of the dye. The small decrease in the retardation and the decrease in the

+ emission of EtBr observed for cis-[Ru(phpy)(bpy)(CH3CN)2] may be due to the changes

in the conformation of the DNA as the μ-oxo dimer formed from the reaction of cis-

+ [Ru(phpy)(bpy)(CH3CN)2] in water covalently interacts with the DNA. Alternatively, no

covalent binding is present, and either the mononuclear or dimeric Ru complexes quench the emission from EtBr.

142 1 2 3 4 5 6 7 8

(a)

1 2 3 4 5 6 7 8

(b)

1 2 3 4 5 6 7 8

(c)

Figure 5.19. Ethidium stained agarose gel electrophoresis of 50 μM linearized plasmid (10 mM phosphate buffer, pH 7.5) in the presence of various ratios of (a) cisplatin, + incubated for 3 hours (b) cis-[Ru(phpy)(bpy)(CH3CN)2] , λirr> 420 nm for 30 min (c) + [Ru(phpy)(CH3CN)4] , λirr> 420 nm for 30 min. Lanes 1 and 8: DNA molecular weight standard, Lanes 2 and 7: linearized plasmid only; Lanes 3-6 [DNA bp] : [Complex] = 100, 20, 10, 5.

143 Irradiation of 50 μM of linearized plasmid with varying ratios of bp:mc with

+ [Ru(phpy)(CH3CN)4] (λirr> 420 nm, 30 min) also resulted in an interference in the

staining of the intercalative dye, ethidium bromide. Although no retardation in the mobility of the DNA was observed with increasing ratio of bp:mc of

+ [Ru(phpy)(CH3CN)4] upon irradiation, a decrease in the intensity of the fluorescence was observed with the increase in bp:mc (Figure 5.19c). Lane 6 in Figure 5.19c contains the highest relative metal concentration and shows a complete disappearance of the emission of EtBr. This decrease in EtBr emission may be due to the change in

+ conformation of the DNA as [Ru(phpy)(CH3CN)4] binds to it upon irradiation, thus

making it difficult for interaction of the dye, however, there is no other evidence for such

binding. The decrease in EtBr emission is likely due to quenching by the photolytically

generated μ-oxo Ru dimer on or near the DNA. No change in the DNA mobility was

observed when solutions containing relative concentration of base pairs (bp) to metal

+ complex (mc) varying from 1 bp: 100 mc to 1bp: 5 mc of cis-[Ru(phpy)(bpy)(CH3CN)2]

+ and [Ru(phpy)(CH3CN)4] with 50 μM linearized DNA were kept in the dark (Figure

5.20).

Structural changes of the duplex DNA that take place upon covalent binding of

the metal complex to the DNA were also investigated by relative viscosity measurements.

Changes in the length of the rod-like DNA are known to produce viscosity changes that are measurable. 26,27 Cisplatin is known to covalently bind to DNA,28,,29 30 and is shown in Figure 5.21 to result in a decrease in the relative viscosity of the solution upon covalent

144

(a) (b)

Figure 5.20. Ethidium stained agarose gel electrophoresis of 50 μM linearized plasmid (10 mM phosphate buffer, pH 7.5) in the presence of various ratios of (a) cis- + + [Ru(phpy)(bpy)(CH3CN)2] and (b) [Ru(phpy)(CH3CN)4] in the dark. Lanes 1 and 8: DNA molecular weight standard, Lanes 2 and 7: linearized plasmid only; Lanes 3-6 [DNA bp] : [Complex] = 100, 20, 10, 5.

binding to the DNA. As the ratio of [DNA]:[cisplatin] is increased there is a significant

decrease in the viscosity of the solution to a ratio of [DNA]: [cisplatin] of 1: 0.15, after

which point there is a slight increase in the viscosity until a plateau is reached at [DNA]:

[cisplatin] of 1: 0.30. The decrease in viscosity observed for cisplatin is likely due to a

“shortening effect” on the DNA, which is induced by the covalent binding of the metal

complex. Macquet and Crook have also observed and reported similar results upon

covalent binding of cisplatin to DNA.31,32 For comparison, relative viscosity

measurements were also conducted for Hoechst 33258, a minor groove binder and EtBr, 145 a known DNA intercalator. Hoechst 33258, being a minor groove binder, has no effect on the relative viscosity of DNA, whereas EtBr, upon intercalation into the DNA, increases the length of the DNA and results in an increase in the viscosity of the solution (Figure

+ 5.21). Irradiation of increasing concentrations of cis-[Ru(phpy)(bpy)(CH3CN)2] (λirr >

420 nm, 30 min) results in no change in the relative viscosity of the solution. The results

are consistent with the lack of covalent binding of the complexes with DNA. A slight

+ decrease in the relative viscosity was observed upon irradiation of [Ru(phpyCH3CN)4] , however, the change is much smaller than that measured for cisplatin and may not be significant.

1.3

1.2

1.1

1.0 1/3 ) o

η/η 0.9 (

0.8

0.7

0.6 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

[Metal]:[DNA]

1/3 Figure 5.21. Plot of the relative viscosity measurements of the plot of (ηDNA / ηo) , as a function of R = [probe]:[DNA] for ethidium bromide (●), HOECHT (x), cisplatin (▲), cis-

146 + + [Ru(phpy)(bpy)(CH3CN)2] (λirr> 420 nm, 30 min) (♦) [Ru(phpy)(CH3CN)4] (λirr> 420 nm,r 30 min) (*). 5.4 Conclusions

+ Cis-[Ru(phpy)(bpy)(CH3CN)2] exhibits absorption maxima at 290 nm (ε = 63

500 M-1cm-1), 370 nm (ε = 9 870 M-1cm-1), and 488 nm (ε = 5 700 M-1cm-1), while

+ -1 -1 [Ru(phpy)(CH3CN)4] exhibits absorption maxima at 242 nm (ε = 32 363 M cm ) and

378 nm (5 200 M-1cm-1). The absorption at 488 nm arises from a Ru → bpy (π*) MLCT

transition, while those at 370 nm and 378 nm can be attributed to Ru → phpy MLCT.

The band centered at 290 nm is assigned as a bpy-centered transition (π → π*), and those

+ at 242 nm for [Ru(phpy)(CH3CN)4] is phpy ππ*in nature. Cis-

+ [Ru(phpy)(bpy)(CH3CN)2] is weakly emissive at room temperature in CH3CN and H2O,

with emission maximum at 780 nm (φ = 0.0023, τ = < 10 ns) in H2O.

+ [Ru(phpy)CH3CN)4] is non emissive at room temperature. However, at 77 K in

EtOH:MeOH (4:1 v/v) both compounds exhibit strong emission with maxima at 662 nm

(φ = 0.35, τ = 0.92 μs) and 542 nm (φ = 0.18, τ = 0.91 μs) for cis-

+ + [Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpy)(CH3CN)4] , respectively.

+ + The photolysis of cis-[Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpy)CH3CN)4] in

(II),(II) water (λirr> 420 nm) results in the formation of μ-oxo dimers of Ru with molecular

II II + formula [(H2O)(bpy)(phpy)Ru ORu (phpy)(bpy)(OH2)]H .H2O and

(II) (II) + [(CH3CN)(H2O)(phpy)Ru ORu (phpy)(H2O)(CH3CN)]H .H2O, respectively.

147 + + Both complexes, cis-[Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpy)CH3CN)4] react

with bipyridine ligand upon irradiation (λirr> 420 nm) in the non-coordinating solvent,

+ CH2Cl2, resulting in the formation of [Ru(phpy)(bpy)2] . The results of the reaction of the

metal complexes with bpy indicates that there is photosubstitution of both CH3CN

+ ligands of cis-[Ru(phpy)(bpy)(CH3CN)2] and photosubstitution of all four CH3CN

+ ligands of [Ru(phpy)CH3CN)4] .

+ Cis-[Ru(phpy)(bpy)(CH3CN)2] reacted with 9-EG and 9-MG upon irradiation resulting in the covalent binding of only one molecule of each guanine base, resulting in

+ the formation of cis-[Ru(phpy)(bpy)(9-EG)(H2O)] , and cis-[Ru(phpy)(bpy)(9-

+ + MG)(H2O)] respectively. [Ru(phpy)(CH3CN)4] also reacted with 9-EG generating with

+ + [Ru(phpy)(9-EG)(ACN)2THF] and [Ru(phpy)(9-EG)(ACN)3THF] , respectively upon

photolysis. Similar results were also observed for the photolysis reaction of 9-MG with

+ + [Ru(phpy)(CH3CN)4] resulting n the formation of [Ru(phpy)(9-MG)(ACN)2THF] and

+ [Ru(phpy)(9-MG)(ACN)3THF] . No reactivity was observed for the complexes with 9-

MG and 9-ET in the dark.

+ + The reactions of cis-[Ru(phpy)(bpy)(CH3CN)2] and [Ru(phpy)(CH3CN)4] with

two different 15-mer oligonucleotide sequences (5’- TGCAAGCTTGGCACT-3’ (S1)

and 5’- AGTGCCAAGCTTGCA-3’ (S2) were investigated upon irradiation (λirr > 420

nm). No binding of either strand of the 15-mer oligonucleotide was observed with cis-

+ [Ru(phpy)(bpy)(CH3CN)2] which may be as a result of the formation of the μ-oxo dimer

descried above in H2O. However, binding of up to four Ru(phpy) fragments were

observed for S1 and S2 upon irradiation.

148 + + Binding of either cis-[Ru(phpy)(bpy)(CH3CN)2] or [Ru(phpy)(CH3CN)4] to double stranded DNA was not observed upon photolysis. No change in the melting

+ temperature was observed upon irradiation of cis-[Ru(phpy)(bpy)(CH3CN)2] with the ds-15-mer duplex. Whereas, a ΔT = -2 0C was observed upon irradiation of the duplex

+ with [Ru(phpy)(CH3CN)4] which is within experimental error and is insignificant

compared to the ΔT = -8 0C that was observed upon covalent binding of cisplatin to a 20- mer duplex. Binding of both metal complexes to ds-DNA was also investigated with mobility gel experiments and viscosity measurements. Both complexes resulted in no significant change in the mobility of DNA throughout the agarose gel, signifying that covalent binding of either metal complex to the DNA did not occur upon irradiation (λirr

> 420 nm). Likewise, viscosity measurements resulted in no significant decrease in the

+ viscosity of the solution upon irradiation of either cis-[Ru(phpy)(bpy)(CH3CN)2] or

+ [Ru(phpy)(CH3CN)4] (λirr > 420 nm) with Herring Sperm DNA.

These results indicate that complexes that form μ-oxo dimers upon irradiation with DNA, do not covalently bind to duplex DNA and therefore, are unlikely candidates as photo-activated cisplatin analogs.

149

References

1. Seddon, K. R. Coord. Chem. Rev. 1982, 41, 79.

2. Reveco, P.; Schmehl, R. H.; Cherry, W. R.; Fronczek, F. R. Selbin, J. Inorg. Chem. 1985, 24, 4078-4082.

3. (a) Watts, R. J.; Harringhton, J. S.; Van Houten, J. J. Am. Chem. Soc. 1977, 99, 2179. (b) Nord, G.; Hazell, A. C.; Hazell, R. G.; Farver, O. Inorg. Chem. 1983, 22, 4060.

4. Cope, A. C.; Siekman, R. W. J. Am. Chem. Soc. 1965, 87, 7272-3273.

5. (a) Ryabov, A. D.; Synthesis 1985, 233-252. (b0 Pfeffer, M. Recl. Trav. Chim. Pays- Bas 1990, 109, 567-576. (c) Spencer, J.; Pfeffer, M. Adv. Metal-Org. Chem. 1998, 6, 103- 1 44. 6. (a) Hermann, W. A.; Bohm, V.P.W.; Reisinger, C. J. Organomet. Chem. Rev. 1999, 576, 23-41. (b) Beletskaya, I. P.; Cheprakov, A. V. Chem. Rev. 2000, 100, 3009-3066.

7. (a) Krooglyak, E. V.; Kazankov, G. M.; Kurzeev, S.A.; Polyakov, V. A.; Semenov, A. N.; Ryabov, A. D. Inorg. Chem. 1996, 35, 4804-4806. (b) Ryabov, A. N.; Kazankov, G. M.; Kurzeev, S. A.; Samuleev, P. V.; polyakov, V. A. Inorg. Chim. Acta 1998, 280, 57- 61. (c) Alexandra, L.; D’yachenko, O. G.; Kazankov, G. M.; Polyakov, V. A.; Samuleev, P. V.; Sansores, E.; Ryabov, A. D. J. Am. Chem. Soc. 2000, 122, 5189-5200.

8.(a) Nanoyama, M. Bull. Chem. Soc. Jpn. 1974, 47, 767. (b) Nanoyama, M.; Yamasaki, K. Inorg. Nucl. Chem. Lett. 1971, 7, 943. (c) Yin, C.C.; Deeming, A. J.J. Chem. Soc.; Dalton Trans. 1975, 2091.(d) Hiraki, K.; Obayashi, Y.; Oki, Y. Bull. Chem. Soc. Jpn. 1979, 52, 1372.

9. Ryabov, A. D.; Sukharev, V. S.; Alexandrova, L.; Le Lagadec, R.; Pfeffer, M. Inor. Chem. 2001, 40, 6529-6532.

10. Le Lagadec, R.; Rubio, L.; Alexandrova, L.; Toscano, R. A.; Ivanova, E. V.; Meskys, R.; Laurinavicius, V.; Pfeffer, M.; Ryabov, A. D. J. Organomet. Chem. 2004, 689, 4820- 4832.

11. Alpeeva, I. S.; Soukharev, V. S.; Alexandrova, L.; Shilova, N. V.; Bovin, N. V.; Csoregi, E.; Ryabov, A. D.; Sakharov, I. Y.J. Biol. Inorg. Chem. 2003, 8, 683-688. 150

12. Ryabov, A.D.; Le Lagadec, R.; Estevez, H.; Toscano, R. A.; Herandez, S. Alexandrova, l.’ Kurova, V. S.; Fischer, A.; Sirlin. C.; Pfeffer, M. Inorg. Chem. 2005, 44, 1626-1634.

13. Reveco, P.; Schmehl, R. H.; Cherry, W. R.; Fronczek, F. R.; Selbin, J. Inorg. Chem. 1985, 24, 4078-4082.

14. Bryant, G. M.; Ferguson, J. E.; Powell, H. K.; J. Aust. J. Chem. 1971, 24, 257. 15. Barigelletti, F.; Juris, A.; Balzani, V.; Belser, P. Inorg. Chem. 1983, 22, 3335-3339.

16.Anderson, S.; Seddon, K. R.; wright, R. D.; Cocks, A. T. Chem. Phys. Lett. 1980, 71, 383.

17 Ryabov, A. D.; Firsova, Y. N.; Goral, V. N.; Sukharev, V. S.; Ershov, A. Y.; Lejbolle, C.; Bjerrum, M. J.; Eliseev, A. V. Inorg. React. Mech. 2000, 2, 343-360.

18. Schoonover, J. R.; Ni, J. F.; Roecker, L.; White, P. S.; Meyer, T. J. Inorg. Chem. 1996, 35, 5885-5892.

19. Schoonover, J. R.; Ni, J.-F.; Roecker, L.; White, P.S.; Meyer, T. J. Inorg. Chem. 1996, 35, 5885-5892.

20. Weaver, T. R.; Meyer, T. J.; Adeyemi, S. A.; Brown, G. M.; Eckberg, R. P.; Hatfield, W. E.; Johnson, E. C.; Murray, R. W.; Untereker, D. J. Am. Chem. Soc. 1975, 97,11, 3039-3048.

21.(a) Hintze, R. E.; Ford, P. C. J. Am. Chem. Soc. 1975, 97, 2664-2671. (b) Matsubara, T.; Ford, P. C. Inorg.Chem. 1978, 17, 1747-1752.

22. Wallan, S.; Davidson, J.; Modin, J.; Hammarstrom, L. J. Phys. Chem. A. 2005, 109, 4697-4704.

23. Van Vliet, P. M.; Haasnoot, J. G.; Reedijk, J. Inorg. Chem. 1994, 33, 1934-1939.

24. Zobi, F.; Hohl, M.; Zimmermann, I.; Alberto, R. Inorg. Chem. 2004, 43, 2771-2772.

25. Poklar, N.; Pilch, D. S.; Lippard, S. J.; Redding, E. A.; Dunham, S. U.; Breslauer, K. Proc. Natl. Acad. Sci. 1996, 93, 7606-7611.

26. Suh, D.; Oh, Y.; Chaires, J. B. Process Biochem. 2001, 37, 521-525.

27. Satynarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Biochem. 1992, 31, 9319-24.

151

28. Brown, S. J.; Kellet, P. J.; Lippard, S. J. Science 1993, 261, 603.

29. Takahara, P. M.; Rosenzweis, A. C.; Frederick, P. M.; Lippard, S. J. Nature 1995, 377, 649.

30. Sherman, S. E.; Lippard, S. J. Chem. Rev. 1990, 100, 293.

31. Butour, J.-L.; Macquet, J.-P. Biochim. Biophys. Acta 1981, 653, 305.

32. Mong, S.; Huang, C. H.; Prestayko, A. W.; Crooke, S. T. Cancer Res. 1980, 40, 3313.

152

CHAPTER 6

Ru(II) Complexes with Oxygen-coordinating ligands

6.1 Introduction

The photochemical ligand substitution of Ru(II) bpy and related complexes are

known to proceed via a thermal population of the low-lying LF dd-states from the lowest

energy MLCT excited state. 1,2 ,3 MLCT states of Ru(II) complexes have been utilized in

pioneering studies of the excited state properties of metal complexes.4 The introduction

of an oxygen atom in the coordination sphere of a Ru(II) complex is expected to shift the

MLCT transition to lower energy. Comparing the electronic configuration of the

2+ + homoleptic [Ru(bpy)3] to [Ru(bpy)2(quo)] (bpy = 2,2’-bipyridine, quo = 8-

6 0 hydroxyquinoline), both complexes are expected to have a ground state of (t2g) (π*) , however, the transition from the ground state to the MLCT state with configuration

5 1 + (t2g) (π*) is expected to take place at lower energy (red shifted) in [Ru(bpy)2(quo)]

2+ compared to [Ru(bpy)3] . This shift can be attributed to the smaller ligand field splitting

that is experienced by the compound containing a coordinated oxygen. This difference in

orbital energies and corresponding states is illustrated in Figure 6.1 assuming an

octahedral symmetry for both complexes. Other oxygen containing ligands such as H2O

153 (a) (b)

eg (d σ∗) LF LF bpy π∗ MLCT MLCT

GS t2g (d π) GS 2+ + 2+ [Ru(bpy) ] [Ru(bpy) (quo)] [Ru(bpy) ] + 3 2 3 [Ru(bpy)2(quo)]

2+ Figure 6.1. Simplified (a) molecular orbital and (b) energy state diagrams of [Ru(bpy)3] + and [Ru(bpy)2(quo)] showing the relative orbital and state energies.

and OH- are also known to result in a lower ligand field splitting when compared to those

containing nitrogen.5

6.2 Background

Several Ru(II) complexes containing a single oxygen atom in the coordination

sphere have been synthesized and their photophysical properties characterized, however,

their photochemistry has not been investigated.6 Table 1 compares the photophysical and

+ electrochemical properties of these complexes which, include the series [Ru(quo)(L)2]

(L = bpy, phen, (1, 10-phenanthroline), dmphen (4,7-dimethyl-phen, tmphen (3,4,7,8,- tetramethyl-phen)). As shown in Table 1, both the MLCT absorption and emission of each complex containing the quo ligand shifts to lower energy relative to the

154 6 2+ 2+ corresponding homoleptic parent complex. For example, [Ru(phen)3] and [Ru(bpy)3] absorb at 450 nm and 453 nm and emit at 590 nm and 610 nm, respectively, whereas

+ + [Ru(phen)2(quo)] and [Ru(bpy)2(quo)] absorb at 490 nm and 460 nm and emit at 783

nm and 767 nm respectively in water.7 Similar results were also observed for

+ - - + [Ru(bpy)2(L)] (L = 3-COO-iqu , 1-COO-iqu ). For example, [Ru(bpy)2(1-COO-iqu]

+ exhibits λabs = 480 nm and λem = 764 nm while [Ru(bpy)2(3-COO-iqu] exhibits

III/II L λabs / nm λem E00 E1/2 (Ru ) (ε / X 103 M-1cm-1) / nm a / eV / V + b [Ru(bpy)2quo] 245 (66.2), 290 (86.5), 460 (15.8) 767 1.85 +0.69

+ b [Ru(phen)2quo] 265 (78.4), 445 (14.4), 490 (12.1) 783 1.82 +0.62

+ c [Ru(dmphen)2quo] 271 (69.2), 453 (14.1) 757 1.88 +0.69

+ c,d [Ru(tmphen)2quo] 264 (89.4), 459 (16.8) 776 1.83 +0.70

+ b [Ru(bpy)2(1-COO-iqu)] 239 (27.6), 290 (33.4), 480 (7.86) 764 2.10 +0.74

+ b [Ru(bpy)2(3-COO-iqu)] 238 (30.0), 290 (35.9), 470 (5.77) 697 2.17 +0.75

a b c Emission corrected for instrument and detector response. In H2O, 1 M KCl. In DMF with 0.1 M (t-Bu)4NPF6. dApproximate value.

Table 6.1. Absorption (λabs) and Emission (λem) maxima, Estimated Excited-State Energy + (E00) and Ground State Oxidation Potentials (vs NHE) of the [Ru(L)2quo] complexes in H2O

λabs = 470 nm and λem = 697 nm. The metal-centered oxidation potential in these

complexes are at lower potentials to those of the corresponding parent complexes by

III/II 2+ 2+ ~0.65 V. For example, the measured E1/2 (Ru ) for both [Ru(phen)3] and [Ru(bpy)3]

155 9 + is 1.26 V vs NHE in water, whereas those observed for [Ru(phen)2(quo)] and

+ [Ru(bpy)2(quo)] are 0.62 and 0.69 V, respectively. The red shift observed in the MLCT

transition and lower oxidation potential is consistent with a smaller ligand-field in the

oxygen containing complexes relative to the corresponding parent complexes as shown in

+ Figure 6.1. A similar shift has also been reported for the complex [Ru(bpy)2L] (L =

RN(O)=NHH-p-CH3C6H4 (R = Et, Ph) that chelate the metal center through coordination

III/II by the oxygen and deprotonated NH atoms. The E1/2(Ru ) values reported for these complexes are +0.40 and +0.49 V vs NHE in acetonitrile respectively.8

The work described in this chapter will center around the synthesis of complexes

+ of the type [Ru(bpy)2(L)] (L = 2-aminophenol, 3-amino-2-naphthol, 8-hyroxyquinoline,

8-quinolinethiol). The electronic properties of the metal complexes will be investigated

2+ and compared to those of the homoleptic [Ru(bpy)3] . The synthesis of these complexes

will provide insight into the possible preparation of [Ru(quo)2(py)2]. This complex

contains two monodentate ligands and may be useful in ligand-loss photochemistry with

light of lower energy than required for complexes with nitrogen coordination.

156 6.3 Results and Discussion

6.3.1 Photophysical Properties

In an attempt to shift the metal to ligand charge transfer transition to lower

+ energy, a series of Ru(II) complexes of the type [Ru(bpy)2(L)] (L = 2- aminophenol,3-

amino-2-napthol, 8-hydroxyquinoline, 8-quinolinethiol) (Figure 6.2) were synthesized

and their photophysical and redox properties were investigated.

NH2 2-aminophenol 8-hydroquinoline (quoH) OH N OH NH2 3-amino-2-napthol OH N 8-quinolinethiol SH

+ Figure 6.2. Structures of the ligands (L) of [Ru(bpy)2(L)] .

+ The electronic absorption spectra of [Ru(bpy)2(L)] (L = 2- aminophenol,3-

amino-2-napthol, 8-hydroxyquinoline, 8-quinolinethiol) in acetonitrile are shown in

Figure 6.3 and the maxima are listed in Table 2. The complexes exhibit LC π → π*

transition in the r242-295 nm region which is consistent with the presence of bpy

9 ligands. In addition, t2g→ π* (MLCT) transitions were observed in the uv and visible region of the spectrum.

157 1.4

1.2

1.0

0.8

0.6 Absorbance (A.U.) 0.4

0.2

0.0 300 400 500 600 700 800

Wavelength (nm)

+ Figure 6.3. Absorption spectra of [Ru(bpy)2(2-aminophenol)] (⎯), [Ru(bpy)2(3-amino- + + 2-naphthol)] (⎯ ⎯ ), [Ru(bpy)2(8-hydroxyquinoline)] (− − −) and [Ru(bpy)2(8- + quinolinethiol)] (…..) in CH3CN.

3 -1 -1 L λabs /nm (ε / 10 M cm )

2-amino-phenol 243 (22.6), 295 (48.8), 356 (7.63), 505 (7.86) 3-amino-2-naphthol 242 (4.40), 292 (4.40), 346 (0.750), 500 (0.480) 8-hydroxyquinoline 250 (72.5), 293 (71.0), 374 (15.9), 508 (18.0) 8-quinolinethiol 249 (29.5), 293 (37.1), 345 (7.60), 501 (7.90)

+ Table 6.2. Absorption maxima of the [Ru(bpy)2(L)] complexes in CH3CN.

158 A shift to lower energy were observed in the MLCT transitions of the complexes of

+ 2+ [Ru(bpy)2(L)] relative to [Ru(bpy)3] (λabs = 453 nm). This can be explained by a

smaller ligand field splitting induced by the ligands: 2-amino-phenol, 3-amino-2- naphthol, 8-hydroxyquinoline and 8-quinolinethiol compared to bpy ligand since as shown in 6.1 nitrogen chelators that are apart of an aromatic rings are known to result in larger filed splitting than neutral and anionic oxygen containing ligands.10 Hence, the

introduction of oxygen and sulfur atom in the ligation sphere will cause the ligand field

+ 2+ stabilization energy to be smaller in [Ru(bpy)2(L)] than [Ru(bpy)3] .

+ All the compounds except for [Ru(bpy)2(8-quinolinethiol)] exhibit room temperature emission in various solvents and their emission spectrum is shown in CH3CN along with low temperature emission in EtOH:MeOH glass (4:1 v/v) and the results are shown in Figure 6.3. Inspection of room temperature emission reveals a large shift in the

2+ emission maxima of ~90-150 nm relative to [Ru(bpy)3] . Low temperature emission

spectra shows vibrational progression for all three emissive complexes which are very

2+ similar to that observed for [Ru(bpy)3] . These results indicate that emission may be occurring from a MLCT state, form Ru(II) → bpy ligand. The bathochromic shift that are

+ shown in the emission spectra of the [Ru(bpy)2(L)] complexes are consistent with a

2+ lower energy LFSE relative to [Ru(bpy)3] .

159 (a) 1.2

1.0

0.8

0.6

0.4 Intensity / a.u. 0.2

0.0 300 400 500 600 700 800

1.2 Wavelength / nm

(b) 1.0

0.8

0.6 0.4 Intensity / a.u. 0.2

0.0 300 400 500 600 700 800 Wavelength / nm

1.2

(c) 1.0

0.8

0.6

0.4 Intensity / a.u. 0.2

0.0 300 400 500 600 700 800 Wavelength / nm

Figure 6.4. Room temperature () and low temperature (77K) (….) emission (λexc = 500 + nm) and excitation spectra(λmax emis) of (a) [Ru(bpy)2(2-aminophenol)] , (b) [Ru(bpy)2(3- + + amino-2-naphthol)] and (c) [Ru(bpy)2(8-hydroxyquinoline)] . 160 6.4 Results and Discussions of [Ru(quo)2(py)2]

+ From the synthesis of the oxygen containing ligand of [Ru(bpy)2(L)] complexes,

a red shift was observed in the MLCT transition by ~ 50 nm when compared to

2+ [Ru(bpy)3] . Hence this indicates that complexes containing oxygen in the coordination

sphere will result in lower energy ligand field stabilization energy and cause a higher

energy filled metal center (t2g) orbital and lower energy eg-type orbital. Since the photochemical ligand substitution of Ru(II) bpy and related complexes are known to occur from the lowest energy excited MLCT state, the introduction of lower field atoms to the octahedral coordination sphere is expected to result in higher photoaquation quantum yields. Hence, [Ru(quo)2(py)2] was synthesized and the photochemistry in water

and with single and double stranded DNA was investigated.

1 6.4.1 Interpretation of H NMR spectra of [Ru(quo)2(py)2]

1 The H NMR spectra of [Ru(quo)2(py)2] is shown in Figure 6.4 where

assignments of the protons corresponding to the pyridine and 8-hydroxyquinoline (quo)

ligands are shown. The signals due to Hα, Hβ and Hγ protons of the coordinated pyridine

2+ ligands are easily identifiable when the spectrum was compared to [Ru(bpy)2(py)2] in

11 (CD3)2SO which was previously studied, and occurred at δ 8.76, 7.70 and 8.26 ppm

respectively. This leaves six proton signals that are identifiable with the quo ligand.

Assignments of the quo ligands are based on the assignment of bis(8-quinolinato)tin(II) complex that has already been synthesized and reported by Jean-Michel Kauffman

(Figure 6.5).12 The basis to the former assignment arises from the well known feature that 161

4 3 2 5 Hα H2, Hβ 6 1 N α β O N γ α β Hγ O py N

H1 H4, H5 H6

H3

0.8 2.5 0.7 1.6 3.5 1.7 0.8

9.4 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8

1 Figure 6.5. H NMR spectrum of [Ru(quo)2(py)2] in d6-DMSO.

the coupling constant of an aromatic proton in the ortho position to a pyridine atom is

smaller than that observed in a substituted phenyl ring.13 The remaining atoms were

assigned from a 2D 1H{13C} HMQC-COSY spectrum. The assignments are summarized

in Table 3 and compared to that of the tin(II) complex.

δ/ ppm Proton a [Ru(quo)2(py)2] [Sn(quo)2] H1 8.65 (d) 8.95 (d) H2 7.70 (d) 7.68 (d) H3 8.58 (d) 8.49 (d) H4 7.54 (d( 7.17 (d) H5 7.50 (d) 7.46 (d) H6 7.20 (d) 6.87 a from ref 13, Proton scheme from Figure 6.4

1 Table 3. H NMR chemicals shifts of [Ru(quo)2(py)2] and [Sn(quo)2] obtained in d6- DMSO. 162 6.4.2 Electronic Properties of [Ru(quo)2(py)2]

The electronic absorption spectrum of [Ru(quo)2(py)2] is shown in Figure 6.6 in

-1 -1 H2O. [Ru(quo)2(py)2] has absorption maxima at 243 (ε = 89 618 M cm ), 253 (s) (ε = 77

270 M-1cm-1), 321 (ε = 15 438 M-1cm-1), 426 (ε = 16, 100 M-1cm-1), 736 (ε = 2 700 M-

1cm-1) nm. The transitions in the visible region are probably due to MLCT transitions while that observed in the 240-300 nm region may be assigned as ππ* transition.

Assignments are based on similar spectral behavior that has been previously reported for

14,15 [Ru(quo)2(bpy)] complexes. The electronic spectra of [Ru(quo)2(bpy)] in acetonitrile

shows three intense absorptions in the visible region at 570 (ε = 8 000 M-1cm-1), 450 (ε =

12 000 M-1cm-1) and 370 nm (ε = 9 300 M-1cm-1) which were assigned as probably

MLCT transitions by Bhattacharya.3,4

The emission of [Ru(quo)2(py)2] was investigated in various solvents and the

compound was non emissive at room temperature.

2.5

2

1.5

1 Absorbance (A.U.)

0.5

0 200 300 400 500 600 700 800 900 Wavelength (nm)

Figure 6.6. Uv-vis absorption spectrum of [Ru(quo)2(py)2] in H2O. 163 6.4.3 Photochemistry of [Ru(quo)2(py)2] in H2O

Irradiating [Ru(quo)2(py)2] in H2O with λirr > 345 nm results in a general decrease

in the absorbance of the uv-vis spectrum with time (Figure 6.7) indicating the formation

of a new species. Irradiation of [Ru(quo)2(py)2] in water was also done at longer

wavelengths of irradiation, λirr > 395 nm and 420 nm, but the changes that are observed in the uv-vis spectra were very small compared to that observed upon irradiation with λirr >

345 nm.

1.4

1.2

1

0.8

0.6 Absorbance (AU)

0.4

0.2

0 200 300 400 500 600 700 800 900 1000 Wavelength (nm)

Figure 6.7. Uv-vis absorption spectra of [Ru(quo)2(py)2] in H2O, λirr > 345 nm for t = 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 min.

164 6.4.4 Photoinitiated Binding of [Ru(quo)2(py)2] to single stranded oligonucleotide

The product of the photolysis of 100 μM of [Ru(quo)2(py)2] in the presence of

100 μM (6.7 μM strands) of single stranded 15-mer oligonucleotides, 5’-

TGCAAGCTTGGCACT-3’ (strand 1) and 5’- AGTGCCAAGCTTGCA-3’ (strand 2)

(λirr > 420 nm,30 min) were monitored by MALDI mass spectrometer. Upon irradiation

of [Ru(quo)2(py)2] with λirr > 345 nm for 3 hours, peaks corresponding well to the calculated mass of strand 1 (4568) and strand 2 (4577) were observed at m/z of 4570.5

and at 4583, respectively. Binding of Ru(quo)2 fragment to strand 1 and strand 2, labeled

A and B was also observed at m/z of 4958.7 and 4973.3 respectively, (Figure 6.8). In

contrast, when solutions containing similar concentration of [Ru(quo)2(py)2] with either

DNA strand were kept in the dark for several hours no binding of the Ru(II) fragment

was observed. It should be noted that [Ru(quo)2(py)2] is in 15 fold excess in the

experiments but only one Ru(quo)2 fragment is observed to bind to either DNA strand.

165 (a) (S1)

A

(S2) (b)

B

Figure 6.8. MALDI mass spectrometry results of the reaction of [Ru(quo)2(py)2] with the 15-mer oligonucleotide sequences (a) 5’- TGCAAGCTTGGCACT-3’ (strand 1, S1) and

(b) 5’- AGTGCCAAGCTTGCA-3’ (strand 2, S2) after irradiation with λirr > 345 nm for 30 mins.

166 6.4.5 Photoinitiated Binding of [Ru(quo)2(py)2] to double stranded oligonucleotide

Annealing of the two complimentary single stranded 15 mer DNA strands

described above (strands 1 and 2) resulted in the formation of a double stranded duplex to

which DNA binding experiments were performed with [Ru(quo)2(py)2] complex. The

0 duplex by itself melts at 57 C. However, upon irradiation (λirr > 345 nm) of 50 μM of the

ds-duplex with 50 μM [Ru(quo)2(py)2] a shift in the melting temperature of the ds-15-mer

to lower temperature (ΔT = -4 0C) occurred resulting in a 53 0C. No shift in the melting

temperature was observed when solutions containing [Ru(quo)2(py)2] and ds-15-mer are

kept in the dark. The shift observed upon irradiation of the duplex with [Ru(quo)2(py)2] is consistent with intrastrand covalent binding of the complex to double stranded DNA, since a ΔT = -8 0C was previously reported for a 20-mer duplex upon covalent binding to

cisplatin.16

Additional evidence for the photoinduced binding of [Ru(quo)2(py)2] to double

stranded DNA is shown in Figure 6.9 with linearized pUC18. The covalent binding of

cisplatin to ds-DNA is known to result in reduced mobility of linearized plasmid on the

agarose gel. The reduction in mobility increases with increasing cisplatin concentration

(Figure 6.8a). The photolysis of [Ru(quo)2(py)2] (λirr > 345 nm, 30 min) in the presence

of linearized plasmid also results in a decreased in mobility of the DNA (Figure 6.9b).

The photolysis of [Ru(quo)2(py)2] with linearized plasmid was also done by irradiation

with λirr > 395 and 420 nm, 30 min and the results are compared to that obtained when irradiated at λirr > 345 nm (Figure 6.9). A decrease in the mobility of DNA on the agarose

gel was observed at all wavelengths of irradiation but the effect was more pronounced 167

1 2 3 4 5 6 7 8 (a)

1 2 3 4 5 6 7 8 (b)

Figure 6.9. Ethidium stained agarose gel electrophoresis of 50 μM linearized plasmid (10 mM phosphate buffer, pH 7.5) in the presence of various ratios of (a)cisplatin, incubated o for 3 hours (b) [Ru(quo)2(py)2], λirr> 345 nm for 30 min at 25 C: .Lanes 1 and 8: DNA molecular weight standard, Lanes 2 and 7: linearized plasmid only; Lanes 3-6 [DNA bp] : [Complex] = 100, 20, 10, 5.

when the complex was irradiated with λirr > 345 nm. Irradiation of the complex with λirr >

395 nm also seemed to cause a greater reduction in the mobility of linearized plasmid on

the agarose gel as compared to irradiation with λirr > 420 nm (Figure 6.9 a,b) respectively.

A possible explanation for the large reduction in mobility obtained upon irradiation with

λirr > 345 nm compared to the reduction that was observed upon irradiation with λirr > 395 and 420 nm (Figure 6.8b,c), is that pyridine is not a very good leaving ligand and hence, 168 high energy is needed for efficient photolysis of [Ru(quo)2(py)2] in water to occur

forming the active species, [Ru(quo)2(OH2)2] which then covalently binds to the DNA. In contrast, no change in the mobility is observed for samples that are exposed to similar

concentrations of [Ru(quo)2(py)2] in the dark.

1 2 3 4 5 6 7 8

(a)

1 2 3 4 5 6 7 8

(b)

1 2 3 4 5 6 7 8

(c)

Figure 6.10. Ethidium stained wavelength dependence agarose gel electrophoresis of 50 μM linearized plasmid (10 mM phosphate buffer, pH 7.5) in the presence of various ratios of [Ru(quo)2(py)2] (a) λirr > 395 nm and (b) λirr > 420 nm (c) dark. Lanes 1 and 8: DNA molecular weight standard, Lanes 2 and 7: linearized plasmid only; Lanes 3-6 [DNA bp] : [Complex] = 100, 20, 10, 5. 169 Viscosity measurements were also conducted with Herring Sperm DNA to further

provide more evidence for photoinduced binding of [Ru(quo)2(py)2] to double stranded

DNA. It is well know that covalent binding of cisplatin to ds-DNA results in a decrease in the viscosity of the solution with Herring Sperm DNA. The decrease in viscosity as a function of increasing relative concentration of [cisplatin]:[DNA] occurs at a ratio of

[cisplatin]:[DNA] of 1:0.15 after which there is a slight increase in the viscosity until there is a plateau with [cisplatin]:[DNA] of 1:0.30 as shown in Figure 6.11. Ethidium bromide is shown in Figure 6.10 to result in an increase in the viscosity of the solution as the length of the DNA increases as the relative concentration of [EtBr]:[DNA] increases.

In contrast, bound HOECHST 33258, results in no change in the viscosity of the solution

(Figure 6.11). Irradiation of [Ru(quo)2(py)2] (λirr > 345 nm, 30 min) is also shown in

Figure 6.10 to result in a decrease in the relative viscosity of the solution similar to that observed upon covalent binding by cisplatin. The decrease in viscosity observed by

[Ru(quo)2(py)2] is not as large as that observed by cisplatin. Irradiated [Ru(quo)2(py)2]

results in a decrease in the viscosity of the solution at [metal]:[DNA] of 1:0.05 followed

by a slight increase in the viscosity resulting in a plateau at [metal]:[DNA] of 1:0.30. In contrast, when similar relative concentrations of [Ru(quo)2(py)2]:[DNA] are kept in the

dark, no change in the viscosity was observed (Figure 6.11).

170 1.3

1.2

1.1

1.0 1/3 ) 0 η /

η 0.9 (

0.8

0.7

0.6 0 0.050.10.150.20.250.3 [DNA]:[Metal]

Figure 6.11. Relative viscosity measurements of ethidium bromide (●), hoecht (x), cisplatin (▼), [Ru(quo)2(py)2] (λirr> 345 nm, 15 min) (♦) and Ru(quo)2(py)2 dark (■).

171 Conclusions

Binding of cis-[Ru(quo)2(py)2] upon irradiation was observed to the single

stranded 15-mer oligonucleotide sequences, 5’- TGCAAGCTTGGCACT-3’ and 5’-

AGTGCCAAGCTTGCA-3’ where binding of only one Ru(quo)2 fragment was observed.

Binding was also observed upon irradiation of cis-[Ru(quo)2(py)2] to duplex 15-mer

oligonucleotide where a shift of the melting temperature to lower temperature (ΔTm = -4

0 C) was observed upon irradiation with λirr > 345 nm. The result is consistent with

covalent binding of the activated metal complex to ds-DNA since a similar shift in the

o melting temperature (ΔTm = -8 C) was also observed upon covalent binding of cisplatin.

Binding of cis-[Ru(quo)2(py)2] to ds-DNA upon irradiation was also confirmed by gel mobility assays, which showed a decrease in the mobility of the DNA upon increasing concentration of the metal complex when photolyzed with various irradiation

wavelengths. The greatest decreased in mobility was observed upon irradiation with high

energy light, λirr > 345 nm, whereas no change in the mobility of the DNA was observed

when the complex was kept in the dark. Irradiating cis-[Ru(quo)2(py)2] with Herring

Sperm DNA at λirr> 345 nm for 15 min decreases the viscosity of the DNA solution in a

similar manner to cisplatin, a known covalent DNA binder.

These results indicate that the introduction of oxygen containing ligands in the

coordination sphere of ruthenium will shift the MLCT transition to lower energy for

eventual utilization as PDT agents. As shown in the results and discussion, binding of cis-

[Ru(quo)2(py)2] occurs to both single and double stranded DNA, thus providing the way

172 for the development of new metal complexes that can be better utilized for the wavelength required for PDT.

173 References

1. Kelly, J. M.; Moucheron, C.-M. Kirsch-De Mesmaeker, A. Struct. Bond. 1999, 92, 163.

2. Krause, R. A. Structure. Bonding (Berlin) 1987, 67, 1.

3.Tfouni, E. Coord. Chem. Rev. 2000, 196, 281.

4. (a) Casper, J. V.; Meyer, T. J. J. Am. Chem. Soc. 1983, 105, 5583. (b) Casper, J. V.; Meyer, T. J. Inorg. Chem. 1983, 22, 2444.

5. (a) Jorgensen, C. K. Absorption Spectra and Chemical Bonding in Complexes; Pergamon Press: London, 1962. (b) Huheey, J. E.; Keiter, E. A.; Keiter, R. L. Inorganic Chemistry; Principles of Structure and Reactivity, 4th ed.; HarperCollins: New York, 1993.

6. Warren, T. J.; Chen, W.; Johnston, D. H.; Turro, C. Inorg. Chem. 1999, 38, 6187-6192.

7. Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; Von Zelewsky, A. Coord. Chem. Rev. 1988, 84, 85-277.

8. Mukherjee, R.; Chakravorty, A. J. Am. Chem. Soc., Dalton Trans. 1983, 2197.

9.(a) Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; von Zelewsky, A. Coord. Chem. Rev. 1988, 84, 85.(b) Allen, G. H.; White, R. P.; Rillema, D. P.; Meyer, T. J. J. Am. Chem. Soc. 1984, 106, 2613.

11. Hitchcock, P. B.; Seddon, K. R.; Turp, J. P.; Yousif, Y. Z.; Zora, J. A. J. Chem. Soc. Dalton Trans 1988, 1837-1842.

12. Alafandy, M.; Willem, R.; Mahieu, B.; Alturky, M.; Gielen, M.; Biesemans, M.; Legros, F.; Camu, F.; Kaufman, J.-M. Inorg. Chim. Acta. 1997, 255, 175-179.

13. Silverstein, R. M.; Bassler, G.; Morrill, T. C.; Spectrometric Identification of Organic Compounds 1991, Wiley, New York, 5th ed, 221.

14. Pramanik, N. C.; Bhattacharya, S. J. Chem. Research (S) 1997, 98-99.

15. (a) Ghatak, N.; Chakravarty, J.; Bhattacharya, S. Polyhedron 1995, 14, 3591.

16. Poklar, N.; Pilch, D. S.; Lippard, S. J.; Redding, E. A.; Dunham, S. U.; Breslauer, K. Proc. Natl. Acad. Sci. 1996, 93, 7606-7611.

174 COMPLETE LIST OF REFERENCES

1.Vrouenraets, M. B.; Visser, G. W. M.; Snow, G. B.; van Dongen, G. A. M. S.; Anticancer Research 2003, 23, 505–522.

2.Tfouni, E. Coord. Chem. Rev. 2000, 196, 281.

3. Pavanin, L. A.; Novais da Rocha, Z.; Giesbrechat, E.; Tfouni, E. Inorg. Chem. 1991, 30, 2185-2190.

4. Dhubhghaill, O. M.; Hagen, W.; Keppler, B. K.; Lipponer, K.-G.; Saddler, J. J. Am. Chem. Soc. 1994, 3305.

5. Clark, M. J.; Janse, B.; Marx, K. A.; Kruger, R. Inorg. Chim. Acta, 1986, 124, 13.

6. Anderson, S.; Seddon, K. R.; wright, R. D.; Cocks, A. T. Chem. Phys. Lett. 1980, 71, 383.

7. (a) Tfouni, E. Coord. Chem. Rev. 2000, 196, 281-305.(b) Martinez, M. S.; J. Photochem. Photobiol. A: Chemistry 1999, 122, 103-108. (c) Pavanin, L. A.; da Rocha, Z. N.; Giesbrecht, E.; Tfouni, E. Inorg. Chem. 1991, 30, 2185, 2190.

8. (a) Nanoyama, M. Bull. Chem. Soc. Jpn. 1974, 47, 767. (b) Nanoyama, M.; Yamasaki, K. Inorg. Nucl. Chem. Lett. 1971, 7, 943. (c) Yin, C.C.; Deeming, A. J.J. Chem. Soc.; Dalton Trans. 1975, 2091.(d) Hiraki, K.; Obayashi, Y.; Oki, Y. Bull. Chem. Soc. Jpn. 1979, 52, 1372.

9. (a) Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; von Zelewsky, A. Coord. Chem. Rev. 1988, 84, 85.(b) Allen, G. H.; White, R. P.; Rillema, D. P.; Meyer, T. J. J. Am. Chem. Soc. 1984, 106, 2613.

10. (a) Hintze, R. E.; Ford, P. C. J. Am. Chem. Soc. 1975, 97, 2664-2671. (b) Matsubara, T.; Ford, P. C. Inorg.Chem. 1978, 17, 1747-1752.

11. (a) Ghatak, N.; Chakravarty, J.; Bhattacharya, S. Polyhedron 1995, 14, 3591.

12. Zobi, F.; Hohl, M.; Zimmermann, I.; Alberto, R. Inorg. Chem. 2004, 43, 2771-2772.

13. Zhao, M.; Clarke, M. J. J. Biol. Inorg. Chem. 1999, 4, 325-340.

14. Zarnegar, P. P.; Bock, C. R.; Whitten, D. G. J. Am. Chem. Soc. 1973, 95, 4367.

15. Xu, J.; Porter, G. B. Can. J. Chem. 1982, 60, 2856-2858.

16. Wiliams, R. J. P. J. Chem Soc 1953, 75, 2163. 17. Weaver, T. R.; Meyer, T. J.; Adeyemi, S. A.; Brown, G. M.; Eckberg, R. P.; Hatfield, W. E.; Johnson, E. C.; Murray, R. W.; Untereker, D. J. Am. Chem. Soc. 1975, 97, 11, 3039-3048.

18. Weaver, T. R.; Meyer, T. J.; Adeyemi, S. A.; Brown, G. M.; Eckberg, R. P.; Hatfield, W. E.; Johnson, E. C.; Murray, R. W.; Untereker, D. J. Am. Chem. Soc. 1975, 97:11, 3039-3048.

19. Warren, T. J.; Chen, W.; Johnston, D. H.; Turro, C. Inorg. Chem. 1999, 38, 6187- 6192.

20. Walsh, J. L.; Durham, B. Inorg. Chem. 1982, 21, 329-337.

21. Wallan, S.; Davidson, J.; Modin, J.; Hammarstrom, L. J. Phys. Chem. A. 2005, 109, 4697-4704.

22. Wacholtz, W. M.; Auerbach, R. A.; Schmehl, R. H.; Ollino, M.; Cherry, W. R. Inorg. Chem. 1985, 24, 1758-1760.

23. Van Vliet, P.M.; Toekimin, S. M. S.; Haasnoot, J. G.; Reedijk, J.; Bradec, V. Biochemistry 1995, 34, 12369-12378.

24. Van Vliet, P.M.; Haasnoot, J. G.; Reedijk, J. Inorg. Chem. 1994, 33, 1934-1939.

25. Van Vliet, P. M.; Toeimin, S. M.S.; Haasnoot, J. G.; Reedijk, J.; Novakova, O.; Brabec, V. Inorg. Chim. Acta. 1995, 57-64.

26. Van Vliet, P. M.; Haasnoot, J. G.; Reedijk, J. Inorg. Chem. 1994, 33, 1934-1939.

27. Van Houten, J.; Watts, R.J. Inorg. Chem. 1978, 17, 3381-3385.

28. Van Houten, J.; Watts, R. J. J. Am. Chem. Soc. 1976, 98, 4853.

29. Van Houten, J.; Watts, R. J. Inorg. Chem. 1978, 17, 3381-3385.

30. Van Houten, J.; Watts, R. J. Inorg. Chem. 1978, 100, 1457.

31. Tromberg, B. J.; Orensten, A.; Kimel, S.; Barker, S. J.; Hyatt, J.; Nelson, J. S.; Berns, M. W. Photochem. Photobiol., 1990, 52, 375-385.

32. Treadway, J. A.; Lopez, R.; Anderson, P.A.;Keene, F. R.; Meyer, T. J. Inorg. Chem. 1996, 10, 35, 8, 2242-2246.

33. Tfouni, E.; Ford, P. C. Inorg. Chem. 1980, 19, 72. 34. Tfouni, E. Coord. Chem. Rev. 2000, 196, 281-305. (b) Martinez, M. S. J. Photochem. Photobiol. A: Chemistry 1999, 122, 103-108. (c) Pavanin, L. A.; da Rocha, Z. N.; Giesbrecht, E.; Tfouni, E. Inorg. Chem. 1991, 30, 2185-2190.

35. Tfouni, E. Coord. Chem. Rev. 2000, 196, 281-305.

36. Telser, J.; Cruickshank, K. A.; Schanze, K. S.; Netzel, T. L. J. Am. Chem. Soc. 1989, 111, 7221.

37. Takahara, P. M.; Rosenzweis, A. C.; Frederick, P. M.; Lippard, S. J. Nature 1995, 377, 649.

38. Suh, D.; Oh, Y.; Chaires, J. B. Process Biochem. 2001, 37, 521-525.

39. Stathopoulous, G. P.; Rigatos, S.; Melamos, N. A. Oncol. Rep. 1999, 6, 797-800.

40. Sitlani, A.; Long, E. C.; Pyle, A. M.; Barton, J. K. J. Am. Chem. Soc. 1992, 114, 2303-2312.

41. Silverstein, R. M.; Bassler, G.; Morrill, T. C.; Spectrometric Identification of Organic Compounds 1991, Wiley, New York, 5th ed, 221.

42. Silva, M.; Tfouni, E. J. Photochem. Photobiol. A. 1999, 122, 103.

43. Sherman, S. E.; Lippard, S. J. Chem. Rev. 1990, 100, 293.

44. Seddon, K. R. Coord. Chem. Rev. 1982, 41, 79.

45. Schoonover, J. R.; Ni, J.-F.; Roecker, L.; White, P.S.; Meyer, T. J. Inorg. Chem. 1996, 35, 5885-5892.

46. Schoonover, J. R.; Ni, J. F.; Roecker, L.; White, P. S.; Meyer, T. J. Inorg. Chem. 1996, 35, 5885-5892.

47. Satynarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Biochem. 1992, 31, 9319-9324.

48. Ryabov, A.D.; Le Lagadec, R.; Estevez, H.; Toscano, R. A.; Herandez, S. Alexandrova, l.’ Kurova, V. S.; Fischer, A.; Sirlin. C.; Pfeffer, M. Inorg. Chem. 2005, 44, 1626-1634.

49. Ryabov, A. D.; Sukharev, V. S.; Alexandrova, L.; Le Lagadec, R.; Pfeffer, M. Inor. Chem. 2001, 40, 6529-6532.

50. Ross, H. B.; Boldaji, M.; Rillema, D. P.; Blanton, C. B.; White, R. P. Inorg. Chem. 1989, 28, 1013-1021. 51. Ross, H. B.; Boldaji, M.; Rillema, D. P.; Blanton C.B.; White, R. P., Inorg. Chem. 1989, 28, 1013-1021.

52. Rosenberg, B.; Van Camp, L.; Trosko, J. L.; Mansour, V. H. Nature 1969, 222, 385- 392.

53. Rosenberg, B. Adv. Exp. Med. Biol. 1977, 91,129-150

54. Reveco, P.; Schmehl, R. H.; Cherry, W. R.; Fronczek, F. R.; Selbin, J. Inorg. Chem. 1985, 24, 4078-4082.

55. Pramanik, N. C.; Bhattacharya, S. J. Chem. Research (S) 1997, 98-99.

56. Poklar, N.; Pilch, D. S.; Lippard, S. J.; Redding, E. A.; Dunham, S. U.; Breslauer, K. Proc. Natl. Acad. Sci. 1996, 93, 7606-7611.

57. Poklar, N.; Pilch, D. S.; Lippard, S. J.; Redding, E. A.; Dunham, S. U.; Breslauer, K. Proc. Natl. Acad. Sci. 1996, 93, 7606-7611.

58. Pogozelski, W. K.; Tullius, T. D. Chem. Rev. 1998, 98, 1089.

59. Platinum-based drugs in cancer therapy 2000, Humana Press, N. J.

60. Pinnick, D. V.; Durham, B. Inorg. Chem. 1984, 23, 1440-1445.

61. Pavanin, L. A.; Rocha, Z. N.; Giesbrecht, E.; Tfouni, E. Inorg. Chem. 1991, 30, 2185.

62. Palmer, R. A.; Pipe, T. S. Inorg. chem. 1966, 5, 864.

63. Pacor, S.; Sava, G.; Ceschia, V.; Bregant, F.; Mestroni, G.; Alessio, E. Chem. Biol. Interact. 1991, 78, 223.

64. Oliver, T.; Mead, T. Curr. Opin. Oncol. 1993, 5, 559-567.

65. Murov, S. L.; Carmichael, I.; Hug, G. L. Handbook of Photochemistry, 2nd ed.; Marcel Dekker: New York, 1993.

66. Murov, S. L.; Carmichael, I.; Hug, G. L. “Handbook of Photochemistry” Second edition, Marcel Dekker: New York; 1993.

67. Muller, J. G.; Kayser, L. A.; Paikoff, S. J.; Durante, V.; Tang, N.; Perez, R. J.; Rokita, S. E.; Burrows, C. J. Coord. Chem. Rev. 1990, 185-186, 761-774.

68. Mukherjee, R.; Chakravorty, A. J. Am. Chem. Soc., Dalton Trans. 1983, 2197.

69. Moucheron, C.; C.-M. Kirsch-De Mesmaeker, A.; Kelly, J. M. J. Photochem. Photobiol. B 1997, 40, 91. 70. Morrison, H. F.; Harmon, H. Photochem. Photobiol. 2000, 72, 731-738.

71. Morgan, R. J.; Chatterjee, S.; Baker, A. D.; Strekas, T. C. Inorg. Chem. 1991, 30, 2687.

72. Mong, S.; Huang, C. H.; Prestayko, A. W.; Crooke, S. T. Cancer Res. 1980, 40, 3313.

74. Mohammad, T.; Morrison, H. F. Photochem. Photobiol. 2000, 71, 369-381.

75. Moan, J.; Sommer, S. Cancer Res, 1985, 1608-1610.

76. Meyer, T. J. Pure Appl. Chem. 1986, 58, 1193.

77. Messori, L.; Vilchez, G. G.; Vilaplana, R.; Piccioli, F.; Aleesio, E.; Keppler, B. Met. Based Drugs 2000, 7, 335.

78. Messori, L.; Orioli, P.; Vullo, D.; Alessio, E.; Iengo, E. Eur. J. Biochem. 2000, 267, 1206.

79. McNamara, M.; Clarke, M. J. Inorg. Chim. Acta 1992, 195, 175-185.

80. McMillin, D. R.; Kirchhoff, J. R.; Goodwin, K. V. Coord. Chem. Rev. 1985, 64, 83- 92.

81. McCusker, J. K. Acc. Chem. Res. 2003, 36, 876-887.

82. Marzilli, L. G.; Iwamoto, M.; Alessio, E.; Hansen, L.; Calligaris, M. J. Am. Chem. Soc. 1994, 116, 815.

83. Marky, L. A.; Breslauer, K. J. Biopolymers 1982, 2185-2194.

84. Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1977, 99, 7213.

85. Mahnken, R. E.; Billadeau, M. A.; Niknowicz, E. P.; Morrison, H. F. J. Am. Chem. Soc. 1992, 114, 9253.

86. Magde, D.; Rojas, G. E.; Skibsted, L. H. Inorg. Chem. 1988, 27, 2900.

87. Loseto, F.; Alesio, E.;Mestroni, G.; Lacidogna, G.; Nassi, A.; Giordano, D.; Coluccia, M. Anticancer Res. 1991, 1549.

88. Lecomte, J.-P; Kirsch-De Mesmaeker, A.; Feeney, M. M.; Kelly, J. M. Inorg. Chem. 1995, 34, 6481.

89. Le Lagadec, R.; Rubio, L.; Alexandrova, L.; Toscano, R. A.; Ivanova, E. V.; Meskys, R.; Laurinavicius, V.; Pfeffer, M.; Ryabov, A. D. J. Organomet. Chem. 2004, 689, 4820- 4832. 90. Krause, R. A. Structure. Bonding (Berlin) 1987, 67, 1.

91. Kobayashi, N.; Ishizaki, T.; Ishii, K.; Konami, H. J. Am. Chem. Soc. 1999, 121,9096- 9110.

92. Knowles, H. J.; Harris, A. L. Breast Cancer Res. 2001, 3, 318-322.

93. Kelly, T. L. J. Phys. Chem. 1972, 76, 1937.

94. Kelly, J. M.; Moucheron, C.-M. Kirsch-De Mesmaeker, A. Struct. Bond. 1999, 92, 163.

95. Kankia, B. I.; Soto, A. M.; Burns, N.;Shikiya, R.; Tung, C-S.; Markey, L. A. Biopolymers 2002, 65, 218-227.

96. Kankia, B. I.; Soto, A. M.; Burns, N.;Shikiya, R.; Tung, C-S.; Markey, L. A. Biopolymers 2002, 65, 218-227.

97. Kalyanasundaram, K.; Nazeeruddin, M. K. Chem Phys. Lett. 1992, 4, 292-297.

98. Kalyanasundaram, K. “Photochemistry of Polypyridine and Porphyrin Complexes,”Academic Press: San Diego, 1992.

99. Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; Von, Zelewsy, A. Coord. Chem. Rev. 1988, 84, 85-277.

100. Jakupec, M. A.; Galanski, M.; Keppler, B. K. Rev. Physiol. Biochem. Pharmacol. 2003, 146, 1-53.

101. Iwamoto, M.; Alessio, E.; Marzilli, L. G. Inorg. Chem. 1996, 35, 2384.

102. Hoggard, P. E.; Porter, G. B. J. Am. Chem. Soc. 1978, 100, 1457.

103. Hitchcock, P. B.; Seddon, K. R.; Turp, J. P.; Yousif, Y. Z.; Zora, J. A. J. Chem. Soc. Dalton Trans 1988, 1837-1842.

104. His, A.; Rosenthal, D. I.; Glatstein, E. Drugs 1999, 57, 5, 725-734.

105. Hintze, R. E.; Ford, P. C. J. Am. Chem. Soc. 1975, 97, 2664.

106. Hill, J. M.; Speer, R. J. Anticancer Res. 1982, 2, 173-186.

107 Henderson, L. J.; Ollino, M.; Gupta, V. K.; Newkome, G. R.; Cherry, W. R. J. Photochem. 1985, 31, 1999.

108. Henderson, B. W.; Dougherty, T. J. Photochem. Photobiol. 1992, 55, 145-157. 109 Hecker, C. R., Franwick, P. E., McMillin, D. R. Inorg. Chem. 1991, 30, 659-666.

110. Hartmann, M.; Lipponer, K.-G.; Keppler, B. K. Inorg. Chim. Acta 1998, 267, 137.

111. Harris, A. L. Nature Rev. Cancer 2002, 2, 38-47.

112. Guppy, M. Biochem. Biophys. Res. Comm. 2002, 299, 679-680.

113. Grover, N.; Welch, T. W.; Fairley, T. A.; Cory, M.; Thprp, H. H. Inorg. Chem. 1994, 33, 3544-3548.

114. Grover, N.; Gupta, N.; Thorp, H. J. Am. Chem. Soc. 1992, 114, 3390-3393.

115. Grover, N.: Welch, T. W.; Fairley, T. A.; Cory, M.; Thorp, H. H. Inorg. Chem. 1994, 33, 3544-3548.

116. Griffith, W. P. Corrd. Chem Rev. 1970, 5, 459.

117. Gray, H. B.; Winkler, J. R.; Ann. Biochem. 1996, 65, 537.

118. Gleria, M.; Minto, F.; Beggiato, G.; Bortolus, P. J. Chem. Soc., Chem. Comm. 1978, 285.

119. Gilbert, J. A.; Eggleston, D. S.; Murphy, W. A. Jr.; Geslowitz, D. A.; Gersten, S. W.; Hogson, D. J.; Meyer, T. J. J. Am. Chem. Soc. 1985, 107, 3855.

120. Fuertes, M. A.; Alonso, C.; Pérez, J. M. Chem. Rev. 2003, 103, 645-62.

121. Fu, P. K.-L. Bradley, P. M.; van Loyen, D.; Durr, H.; Bossmann, S. H.; Turro, C. Inorg. Chem. 2002, 41, 3808.

122. Ford, P. C.; Rudd, DeF. P.; Gaunder, R.; Taube, H. J. Am. Chem. Soc. 1968, 90, 1187.

123. Ford, P. C. Coord. Chem. Rev. 1970, 5, 75

124. Foley, S.; Jones, G.; Liuzzi, R.; McGarvey, D. J.; Perry, M. H.; Truscot, T. G. J. Chem. Soc. Perkin Trans.2 1997, 1725-1730.

125. Fleisher, M. B.; Waterman, K. C.; Turro, N. J.; Barton, J. K. Inorg. Chem. 1986, 25, 20, 3549-3551.

126. Fernandez, S.; Pfeffer, M.; Ritleng, V.; Sirlin, C. Organometallics 1999, 18, 2390- 2394.

127. Farrell, N.; Qu, Y.; Feng, L.; Van Houten, B. Biochemistry 1990, 29, 9522-9531. 128. Farrell, N. Coord. Chem. Rev. 2002, 232, 1-4

129. Esposito, G.; Cauci, S.; Fogolari, F.; Alessio, E.; Scocchi, M.; Quadrifoglio, F.; Viglino, P. Biochemistry 1992, 31, 7094.

130. Esposito, G.; Cauci, S.; Fogolari, F.; Alessio, E.; Scocchi, M.; Quadrifoglio, F.; Viglino, P. Biochemistry 1992, 31, 7094-7103.

131. Erkkila, K. E.; Odom, D. T.; Barton, J. K. Chem. Rev. 1999, 99, 2777.

132. Elfring, W. H.; Crosby, G. A. J. Am. Chem. Soc. 1981, 103, 2683.

133. Durham, B.; Wilson, S. R.; Hodgson, D. J.; Meyer, T. J. J. Am. Chem. Soc. 1980, 102:2, 600-607.

134. Durham, B.; Wilson, S. R.; Hodgson, D. J.; Meyer, T. J. Am. Chem. Soc. 1980, 102, 600-607.

135. Durham, B.; Walsh, J. L.; Carter, C. L.; Meyer, T. J. Inorg. Chem. 1980, 19, 860- 865.

136. Durham, B.; Casper, J. V.; Nagle, J. K.; Meyer, T. J. J. Am. Chem. Soc. 1989, 111, 541.

137. Durham, B.; Caspar, J. V.; Nagle, J. K.; Meyer, T. J. J. Am. Chem. Soc. 1982, 104, 4803-4810.

138. Dougherty, T. J.; Gomer, C. J.; Henderson, B. W. J. Natl Cancer Inst. 1998, 90, 12, 889–905.

139. Dolmans, D. E. G. J.; Fukumura, D.; Jain, R. K. Nature Rev. 2003, 3, 380.

140. DeRosa, M. C.; Crutchley, R. J. Coord. Chem. Rev 2002, 233-234, 351-371.

141. Demas, J. N.; Crosby, G. A. J. Am. Chem. Soc. 1971, 93, 2841.

142. Demas, J. N.; Bowman, W. D. J. Phys. Chem. 1981, 85, 2771-2777.

143. Crosby, G. A.; Elfring, W. H. J. Phys. Chem. 1976, 80, 2206.

144. Cope, A. C.; Siekman, R. W. J. Am. Chem. Soc. 1965, 87, 7272-3273.

145. Constable, E. C.; Holmes, J. M. J. Organomet. Chem. 1986, 301, 203-208.

146. Cohen, G. L.; Bauer, W. R.; Barton, J. K.; Lippard, S. J. Science, 1979, 203, 1014- 1016. 147. Clarke, M.; Jansen, B.; Marx, K. A.; Kruger, R. Inorg. Chim. Acta 1986, 124, 13-28.

148. Clarke, M. J.; Taube, H. J. Am. Chem. Soc. 1974, 96, 5413.

149. Clarke, M. J.; Jansen, B. Inorg. Chim. Acta 1986, 124, 13-28.

150. Clarke, M. J.; Bitler, S.; Rennert, D.; Buchbinder, M.; Kelman, A. D. J. Inorg. Biochem. 1980, 12, 79.

151. Clarke, M. J. Met. Ions Biol. Syst. 1980, 11, 231.

152. Clarke, M. J. Clin. BiIochem. Med. 1989, 10, 25.

153. Chen, Z. J. Chem. Chinese 1987, 8, 9, 839-841.

154. Chen, H.; Parkinson, J.A.; Parsons, S.; Coxall, R. A.; Gould, R.O.; Sadler, P. J. J. Am. Chem. Soc. 2002, 124, 3064-3082.

155. Chaisson, D. A.; Hintze R. E.; Stuermer D. H.; Petersen, J. D.; McDonald D. P.; Ford P. C. J. Am. Chem. Soc., 1972, 94, 6665.

156. Cauci, S.; Viglio, P.; Esposito, G.; Quadrifoglio, F. J. Inorg. Biochem. 1991, 43, 739.

157. Cauci, S.; Viglino, P.; Espoito, G.; Quadrigoglio, F. Inorg. Biochem. 1991, 43, 739- 751.

158. Casper, V. C.; Meyer, T. J. Inorg. Chem. 1983, 22, 2444-2453.

159. Butour, J.-L.; Macquet, J.-P. Biochim. Biophys. Acta 1981, 653, 305.

160. Burstyn, J. N.; Heiger-Bernays, W. J.; Cohen, S. M.; Lippard, S. J. Nucleic Acids Res. 2000, 28, 4237-4243.

161. Bryant, G. M.; Ferguson, J. E.; Powell, H. K.; J. Aust. J. Chem. 1971, 24, 257.

162. Bruhn, S. L.; Toney, J. H.; Lippard, S. J. Prog. Inorg. Chem. 1990, 38, 477-516.

163. Brown, S. J.; Kellet, P. J.; Lippard, S. J. Science 1993, 261, 603.

164. Brown, G. M.; Weaver, T. R.; Keene, F. R.; Meyer, T. J. Inorg. Chem. 1970, 15, 189-196.

165. Brown, G. M.; Callahan, R. W.; Meyer, T. J. Inorg. Chem. 1975, 14, 8, 1915-1921. 166. Breslauer, K. J.; Frank, R.; Blocker, H.; Marky, L. A. Proc. Natl. Acad. Sci. USA 1986, 83, 3746-3750.

167. Bradley, P. M.; Fu, P. K.; Turro, C. Comments Inorg. Chem. 2001, 22, 393.

168. Billadeau, M. A.; Morrison, H. J. Inorg. Biochem. 1995, 57, 249-270.

169. Bento, M. L.; Tfouni, E. Inorg. Chem. 1988, 27, 3410.

170. Beley, M.; Collin, J.-P.; Sauvage, J-P. Inorg. Chem. 1993, 32, 4539-4543.

180. Beley, M.; Collin, J.-P.; Louis, R.; Metz, B.; Sauvage, J.-P. J. Am. Chem. Soc. 1991, 113, 8521-8522.

181. Barton, J. K.; Lolis, E. J. Am. Chem. Soc. 1985, 107, 708-708.

182. Barry, C. G.; Turney, E. C.; Day, C. S.; Saluta, G.; Kucera, G. L.; Bierbach, U. Inorg. Chem. 2002, 41, 7159-7169.

183. Barigelletti, F.; Juris, A.; Balzani, V.; Belser, P. Inorg. Chem. 1983, 22, 3335-3339.

184. Bardley, P. G.; Kress, N.; Hornberger, B. A.; Dallinger, R. F.; Woodruff, W. H. J. Am. Chem. Soc. 1981, 103, 7441.

185. Asesefa, Z.; Stanbury, D. J. Am. Chem. Soc. 1997, 119, 521-530.

186. Armitage, B. Chem. Rev. 1998, 98, 1171.

187. Alpeeva, I. S.; Soukharev, V. S.; Alexandrova, L.; Shilova, N. V.; Bovin, N. V.; Csoregi, E.; Ryabov, A. D.; Sakharov, I. Y.J. Biol. Inorg. Chem. 2003, 8, 683-688.

188. Allen, G. H.; White, R. P.; Rillema, D. P.; Meyer, T. J. J. Am. Chem. Soc. 1984, 106, 2613.

189. Alessio, E.; Zangrando, E.; Roppa, R.; Marzilli, L. G. Inorg. Chem. 1998, 37, 2458.

190. Alafandy, M.; Willem, R.; Mahieu, B.; Alturky, M.; Gielen, M.; Biesemans, M.; Legros, F.; Camu, F.; Kaufman, J.-M. Inorg. Chim. Acta. 1997, 255, 175-179.

191. (a) Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1974, 96, 601-603. (b) Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1977, 99, 7313- 7221. (c) Durante, V. A.; Ford, P. C. Inorg. Chem. 1979, 18, 588-593.

192. (a) Wrighton, M. S.; Abrahamson, H. B.; Morse, D. L. J. Am. Chem. Soc. 1976, 98, 4105. (b) Abrahamson, H. B.; Wrighton, M. S. Inorg. Chem. 1979, 17, 3385. (c) Figard, J. E.; Petersen, J. D. Ibid 1978, 17, 1059. 193. (a) Watts, R. J.; Harringhton, J. S.; Van Houten, J. J. Am. Chem. Soc. 1977, 99, 2179. (b) Nord, G.; Hazell, A. C.; Hazell, R. G.; Farver, O. Inorg. Chem. 1983, 22, 4060.

194. (a) Tfouni, E. Coord. Chem. Rev. 2000, 196, 281-305.(b) Martinez, M. S.; J. Photochem. Photobiol. A: Chemistry 1999, 122, 103-108. (c) Pavanin, L. A.; da Rocha, Z. N.; Giesbrecht, E.; Tfouni, E. Inorg. Chem. 1991, 30, 2185, 2190.

195. (a) Ryabov, A. D.; Synthesis 1985, 233-252. (b0 Pfeffer, M. Recl. Trav. Chim. Pays- Bas 1990, 109, 567-576. (c) Spencer, J.; Pfeffer, M. Adv. Metal-Org. Chem. 1998, 6, 103- 144.

196. (a) Ryabov, A. D.; Le Lagadec, R.; Estevez, H.; Toscano, R. A.; Hernandez, S.; Alexandrova, L.; Kurova, V. S.; Fischer, A.; Sirlin, C.; Pfeffer, M. Inorg. Chem. 2005, 44, 1626-1634. (b) Ryabov, A. D.; Sukharev, V. S.; Alexandrova, L.; Le Lagadec, R.; Pfeffer, M. Inorg. Chem. 2001, 40, 6529-6532.

197 (a) Pyle, A. M.; Chiang, M. Y.; Barton, J. K. Inorg. Chem. 1990, 29, 4487-4495. (b) Krotz, A. H.; Kuo, L. Y.; Barton, J. K. Inorg. Chem. 1993, 32, 5963-5974.

198. (a) Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1977, 99, 7213. (b) Malouf, G.; Ford, P. C. Ibid 1974, 96, 601.

199. (a) Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1974, 96, 601-603. (b) Malouf, G.; Ford, P. C. J. Am. Chem. Soc. 1977, 99, 7213-7221. (c) Durante, V. A.; Ford, P. C. Inorg. Chem. 1979, 18, 588-593.

200. (a) Mahnken, R. E.; Bina, M.; Debel, R. M.; Leubek, K.; Morrison, H. Photochem. Photobiol. 1989, 49, 519-522. (c) Mahnken, R. E.; Billadeau, M. A.; Nikonowicz, E. P.; Morrison, H. J. Am. Chem. Soc. 1992, 114, 9253-9265.(d) Harmon, H. L.; Morrison, H. Inorg. Chem. 1995, 34, 4937-4938.

201. (a) Lytle, F. E.; Hercules, D. M. J. Am. Chem. Soc. 1969, 91, 253. (b) Klassen, D. M.; Crosby, G. A. J. Chem. Soc. 1968, 48, 1853. (c) Harrigan, R. W.; Hager, G. D.; Crosby, G. A. Chem Phys. Lett. 1973, 21, 487. (d) Baker, D. C.; Crosby, G. A. Chem. Phys. 1974, 4, 428. (e) Harrigan, R. W.; Crosby, G. A. J. Chem. Phys. 1973, 59, 3468.

202. (a) Krooglyak, E. V.; Kazankov, G. M.; Kurzeev, S.A.; Polyakov, V. A.; Semenov, A. N.; Ryabov, A. D. Inorg. Chem. 1996, 35, 4804-4806. (b) Ryabov, A. N.; Kazankov, G. M.; Kurzeev, S. A.; Samuleev, P. V.; polyakov, V. A. Inorg. Chim. Acta 1998, 280, 57-61. (c) Alexandra, L.; D’yachenko, O. G.; Kazankov, G. M.; Polyakov, V. A.; Samuleev, P. V.; Sansores, E.; Ryabov, A. D. J. Am. Chem. Soc. 2000, 122, 5189-5200.

203. (a) Kober, E. M.; Meyer, T. J. Inorg. Chem. 1984, 23, 3377. (b) Kober, E. M.; Marshall, J. L.; Dressick, W. J.; Sullivan, B. P.; Casper, J. V.; Meyer, T. J. Inorg. Chem. 1985, 24, 2755. 204. (a) Kelly, J. M.; Feeney, M. M.; Tossi, A. B.; Lecomte, J.-P.; Kirsch,-De Mesmaeker, A. Anti-Cancer Drug Des. 1990, 5, 69-75. (b) Lecomte, J.-P.; Kirsch-De Mesneaker, A.; Feeney, M. M.; Kelly, J. M. Inorg. Chem. 1995, 34, 6481-6491. 1. (a) Jorgensen, C. K. Absorption Spectra and Chemical Bonding in Complexes; Pergamon Press: London, 1962. (b) Huheey, J. E.; Keiter, E. A.; Keiter, R. L. Inorganic Chemistry; Principles of Structure and Reactivity, 4th ed.; HarperCollins: New York, 1993.

205. (a) Hermann, W. A.; Bohm, V.P.W.; Reisinger, C. J. Organomet. Chem. Rev. 1999, 576, 23-41. (b) Beletskaya, I. P.; Cheprakov, A. V. Chem. Rev. 2000, 100, 3009-3066.

206. (a) Ford, P. C.; Stuer, D. H.; McDonald, D. P. J. Am. Chem. Soc. 1969, 91, 6209. (b) Ford, P. C.; Chaisson D. A.; Stuermer, D. H. Chem. Comm. 1971, 530.

207. (a) Ford, P. C.; Struermer D. H.; McDonald D. P. J. Am Chem. Soc. 1969, 91, 6209. (b) Ford, P. C.; Chaisson, D. A.; Stuermer, D. H. J. Am. Chem. Commun. 1971, 530. (c) Chaisson, D. A.; Hintze, R. E.; Stuermer, D. H.; Petersen, J. D.; McDonald, D. P.; Ford, P. C. J. Am. Chem. Soc. 1972, 94, 6665.

208. (a) Ford, P. C. Coord. Chem. Rev. 1970, 5, 75-99. (b) Ford, P. C. Coord. Chem. Rev. 1982, 44, 61-82. (c) Ford, P. C.; Wink, D.; Dibenedetto, J. Progress Inorg. Chem. 1983, 30, 213-271.

209. (a) Ford, P. C. Coord. Chem. Rev. 1970, 5, 75-99. (b) Ford, P. C. Coord. Chem. Rev. 1982, 44, 61-82. (c) Ford, P. C.; Wink, D.; Dibenedetto, J. Progress Inorg. Chem. 1983, 30, 213-271.

210 (a) Ford, P. C. Coord. Chem. Rev. 1970, 5, 75-99. (b) Ford, P. C. Coord. Chem. Rev. 1982, 44, 61-82. (c) Ford, P. C.; Wink, D.; Dibenedetto, J. Progress Inorg. Chem. 1983, 30, 213-271 (d) Pavanin.

211. (a) Erkkila, K. E.; Odom, D. T.; Barton, J. K. Chem Rev. 1999, 99, 2777. (b) Turro, C.; Hall, D. B.; Chen, W.; Zuilhof, H.; Barton, J. K.; Turro, N. J. J. Phys. Chem. A. 1998, 102, 5708.

212. (a) Durham, B.; Casper, J. V.; Nagle, J. K.; Meyer, T. J. J. Am. Chem. Soc. 1982, 104, 4803-4810. (b) Allen, G. H.; White, R. P.; Rillema, D. P.; Meyer, T. J. J. Am. Chem. Soc. 1984, 106, 2613-2620. (c) Rillema, D. P.; Taghdri, D. G.; Jones, D. S.; Keller, C. D.; Worl, L. A.; Meyer, T. J.; Levy, H. A. Inorg. Chem. 1987, 26, 578-585.

213. (a) Dalla Via, L.; Magno, S. M. Curr. Med. Chem. 2001, 8, 1405-1418. (b) McCaughan, J. S., Jr Drugs Aging 1999, 15, 49-68. (c) Allen, C. M.; Sharman, W. M.; van Lier, J. E. Tumor Targeting Can Ther. 2002, 329-361.

214. (a) Clarke, M. J. Metal Ions Biol. Syst. 1980, 11, 231. (b) Clarke, M. J. Metal Complexes in Cancer Chemotherapy 1993, 129-156. 215. (a) Chang, C. H.; Meares, C. F. Biochemistry 1984, 23, 2268-2274. (b) Barton, J. K.; Rapheal, A. L. J. Am. Chem. Soc. 1984, 106, 2246-2468. (c) Fleisher, M. B.; Waterman, K. C.; Turro, N. J.; Barton, J. K. Inorg. Chem. 1986, 25, 3549-3551.

216. (a) Casper, J. V.; Meyer, T. J. J. Am. Chem. Soc. 1983, 105, 5583. (b) Casper, J. V.; Meyer, T. J. Inorg. Chem. 1983, 22, 2444.

217. (a) Allen, G. H.; White, R. P.; Rillema, D. P.; Meyer, T. J. J. Am. Chem. Soc. 1984, 106, 2613. (b) Rillema, D. P.; Taghdiri, D. G.; Jones, D. S.; Keller, C. D.; Worl, L. A.; Meyer, T. J.; Levy, H. A. Inorg. Chem. 1987, 26, 578.

218. Meyer, T. J. Pure Appl. Chem. 1986, 58, 1193.

219. Ryabov, A. D.; Firsova, Y. N.; Goral, V. N.; Sukharev, V. S.; Ershov, A. Y.; Lejbolle, C.; Bjerrum, M. J.; Eliseev, A. V. Inorg. React. Mech. 2000, 2, 343-360.

220. Isied, S.; Taube, H. Inorg. Chem. 1976, 15, 3070.

221. Velders, A. H.; Geest, B.; Kooijman, H.; Spek, a. L Haasnoot, J. G.; Reedijk, J. Eur. Inorg. Chem. 2001, 369.