Towards binuclear metal complexes as probes for DNA : a study of bis phenanthroline ruthenium (II) dipyrido[3,2 d:2'3' f]quinoxaline and its derivatives

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Phenanthroline Ruthenium (II)

Dipyrido [3,2-d:2 ',3 '-fjquinoxaline and

its derivatives.

BY

Karen A. O ’Donoghue

A THESIS SUBM ITTED TO THE UNIVERSITY OF DUBLIN FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY.

DEPARTM ENT OF CHEM ISTRY,

UNIVERSITY OF DUBLIN,

This thesis has not been submitted as an exercise for a degree at any other

University. Except w here otherwise indicated, the w ork described herein has been

carried out by the author alone.

I give perm ission for the Library to lend or copy this thesis upon request.

Towards Binuclear M etal Complexes as Probes f o r DNA: A Study o f Bis-Phenanthroline

Ruthenium (II) D ipyrido[3,2-d:2',3’-f]quinoxaline and its derivatives.

Karen A. O ’Donoghue

The synthesis o f the novel ligand methyldipyrido[3,2-f:2',3'-h]quinoxaline-2-carboxylate (mdpqc), with an electrophilic site for the attachment o f a flexible tail, is presented. The synthesis, purification and characterisation o f five dpq based mononuclear ruthenium complexes is described - [Ru(phen)2(dpq)]^",^ [Ru(phen)2(Medpq)]^^ [Ru(phen)2(Me2dpq)]^^ [Ru(phen)2(mdpqc)]^'' and [Ru(phen)2(dpqa)]^''. [Ru(phen)2(mdpqc)]^^ was found to decompose to [Ru(phen)2(dpq)]^’" in an aqueous environment. Although the synthesis o f the targeted bridging ligand was not accomplished, the central aim of synthesising a ligand with the potential for the selective synthesis o f both homonuclear and heteronuclear metal complexes was still achieved through the synthesis of 2- dipyrido[3,2-f:2',3'-h]quinoxaline acid amino-pentylamide. The presence o f the free amino group provides the potential for the future selective synthesis o f both homonuclear and heteronuclear complexes.

The ground state pKjS for the mono-protonation o f the [Ru(phen)2(Rdpq)]^‘" family were determined. The electron donating methyl groups increase the electron density available for protonation and so the order o f basicity was determined to be [Ru(phen)2(M e2dpq)]^^ (pK^ = -1.9) > [Ru(phen)2(Medpq)]-^ (pKa = -2.3) > [Ru(phen)2(dpq)]^^ (pKa = -2.7).

The photophysical properties o f the [Ru(phen)2(Rdpq)]^"^ family in DMSO, M eCN, MeOH, EtOH, H2O and pH 7 tris buffer were established. The trend in Xdeg (and (jideg) o f the [Ru(phen)2(Rdpq)]‘'" family in organic solvents correlates well with the solvent polarity param eter %*, DMSO > MeCN > MeOH > EtOH. There is a decrease in Tjeg on going from [Ru(phen)2(dpq)]^^ to [Ru(phen)2(Medpq)]^'^ to [Ru(phen)2(Me2dpq)]‘^ due to the increase in !<„. The trend in tjeg in water is [Ru(phen)2(M e2dpq)]^'^ > [Ru(phen)2(Medpq)]^'^ > [Ru(phen)2(dpq)]^”^. The increased steric hindrance o f the successive methyl groups is proposed to inhibit the deactivation by hydrogen bonding interactions with solvent water molecules.

The emitting ^MLCT excited state o f the [Ru(phen)2(Rdpq)]^* family in solution is deactivated by internal conversion to the thermally accessible metal centred (^MC) state. In degassed MeCN, Eact exhibits the following trend [Ru(phen)2(dpq)]^'^ (28 kJmoK') > [Ru(phen)2(Medpq)]^"^ (25 kJmol’') > [Ru(phen)2(Me2dpq)]^'^ (21 kJmol'').

[Ru(phen)2(dpqa)]^'^ exhibits luminescence in organic solutions but does not emit in an aqueous environment. This is first example o f this effect being activated by a single substitution - [Ru(phen)2(Medpq)]^^ luminescent in an aqueous environment while [Ru(phen)2(dpqa)]^'^ is not.

Binding o f the rac [Ru(phen)2(Rdpq)]^'^ family to salmon sperm DNA was investigated. At P/D values o f 100, a general broadening of the M LCT band and some hypochromicity is observed in the absorption spectra of [Ru(phen)2(Rdpq)]^"^. The excited state lifetimes and luminescence intensities are substantially increased when [Ru(phen)2(Rdpq)]^”^ interacts with DNA. Analysis of the binding data suggests that the strength o f binding decreases on increasing the num ber o f methyl substituents - the values o f Kapp are 4.1 x 10^ M‘' (± 0.2 x 10^ M '') for [Ru(phen)2(dpq)]^'^, 2.6 x 10^ M‘' ( ± 2 X 10^ M-') for [Ru(phen)2(Medpq)]^^ and 1.3 x 10® M ' (± 2 x 10^ M ') for [Ru(phen)2(Me2dpq)]^"^.

Binding o f the A and A enantiomers o f [Ru(phen)2(dpq)]'‘^ (provided by Dr. Janice Aldrich- Wright) to salmon sperm DNA, [poly(dA-dT).poly(dA-dT)] and [poly(dG-dC).poly(dG-dC)] was studied. Both emission intensity and excited state lifetime are significantly increased when either enantiomer is bound to DNA or the polynucleotides. Analysis o f the spectroscopic data demonstrated little enantioselectivity between the A and A enantiomers o f [Ru(phen)2(dpq)]^'^ on binding DNA (Kapp ~ 10® M ‘').

A bbreviations

bpy

2 ,2 ’-bipyridine

dafo

4,5-diazafluoren-9-one

dppz

[dipyrido [2,3 -a:2 ’ ,3 ’ -cjphenazine]

dpq

dipyrido [3,2-f; 2 ’ ,3 ’ -h] quinoxaline

dpqa

2-dipyrido[3,2-f:2’,3 ’-h]quinoxaline acid pentylam ide

hat

1,4,5,8,9,12-hexaazatriphenylene

mdpqc

m ethyldipyrido [3,2-f:2 ’ ,3 ’ -h]quinoxaline-2-carboxylate

M edpq

2-m ethyldipyrido [3,2-f:2 ’ ,3 ’ -h]quinoxaline

Me2dpq

2,3-dim ethyldipyrido[3,2-f;2’,3’-h]quinoxaline

phen

1,10-phenanthroline

phendione

1,10-phenanthroline-5,6-quinone

HPLC

High perform ance liquid chrom atography

IR

Infrared

N M R

N uclear m agnetic resonance

SPC

Single photon counting

UV/Vis

Ultra-violetA^isible

DM SO

Dimethyl sulfoxide

EtOH

Ethanol

M eCN

Acetonitrile

MeOH

M ethanol

A

Delta

A

Lamda

CT-DNA

C alf thym us deoxyribonucleic acid

DNA

D eoxyribonucleic acid

A

Adenine

G

Guanine

T

Thymine

C

Cytosine

P

[DNA]

D

[dye]

W avelength (nm)

s

Extinction coefficient (d m ^ m o r'cm '')

T

Lifetim e (ns)

<1)

Q uantum Y ield

c

concentration (moldm'^)

Abs

Absorbance

Em

Em ission

IC

Internal conversion

IL

Intraligand

ISC

Intersystem crossing

MC

M etal centred

M LCT

M etal-to-ligand charge transfer

kdd

Decay rate constant o f MC state

knr

N on-radiative rate constant

kr

Radiative rate constant

Summary

The aim o f this research p roject was to d ev e lo p a novel, versatile bridging ligand that w ould enable binuclear ruthenium com plexes to be m ade but also have th e potential for th e selective synthesis o f a heteronuclear species.

T ow ards this end the synthesis o f the novel ligand m ethyldipyrido[3,2-f:2',3'-h]quinoxaline- 2-carboxylate (m dpqc), with an electrophilic site fo r th e attachm ent o f a flexible tail, is presented. The prim ary focus o f this research centred on the d evelopm ent o f a novel, versatile bridging ligand and subsequent binuclear com plex. H ow ever, it was considered beneficial to initially exam ine the photophysical behaviour o f the sim pler m ononuclear ruthenium dpq based com plexes. T he synthesis, purification and characterisation o f five dpq based m ononuclear ruthenium com plexes is described - [Ru(phen)2(dpq)]^'', [Ru(phen)2(M edpq)]^'', [Ru(phen)2(M e2dpq)]^’', [R u(phen)2(m dpqc)]^'' and [Ru(phen)2(dpqa)]^''. [Ru(phen)2(dpq)]^'', [Ru(phen)2(M edpq)]-^ and [Ru(phen)2(M e2dpq)]^'' constitute the [Ru(phen)2(Rdpq)]^'" family. [Ru(phen)2(m dpqc)]^^ was found to decom pose to [Ru(phen)2(dpq)]^'^ in an aqueous environm ent.

The synthesis o f the targeted bridging ligand was not accom plished. H ow ever, the central aim o f synthesising a ligand with the potential for the selective synthesis o f both h om onuclear and heteronuclear m etal com plexes was still achieved through the synthesis o f 2-d ip y rid o [3,2-f:2',3'- hjquinoxaline acid am ino-pentylam ide. The presence o f the fi'ee am ino group provides the potential for the future selective synthesis o f both hom onuclear and heteronuclear com plexes.

The ground state pKjS for the m ono-protonation o f the [R u(phen)2(R dpq)]^’^ fam ily w ere determ ined. The electron donating m ethyl groups increase the electron density available for protonation and so the order o f basicity is [Ru(phen)2(Me2dpq)]^^ (pKa = -1.9) > [R u(phen)2(M edpq)]'^ (pKa = -2.3) > [Ru(phen)2(dpq)]'-^ (pK^ = -2.7).

The em itting ^MLCT excited state o f the [R u(phen)2(Rdpq)]"'" fam ily in solution is deactivated by internal conversion to the therm ally accessible m etal centred (^MC) state. In degassed M eC N , Eac, exhibits the follow ing trend [R u(phen)2(dpq)]^'' (28 k J m o l'') > [R u(phen)2(Medpq)]^'" (25 k Jm o l'') > [R u(phen)2(M e2dpq)]‘^ (21 k Jm o l'').

C onverting [R u(phen)2(M edpq)]^'' to [R u(phen)2(dpqa)]^'" involves changing a single functional group from a sim ple alkyl group to an am ide. T his m odification has a m inim al effect on the photophysical properties o f [R u(phen)2(dpqa)]^'' in organic solvents relative to the [R u(phen)2(Rdpq)]^'' family. W hile [R u(phen)2(dpqa)]^^ exhibits photolum inescence in organic solutions it does not em it in an aqueous environm ent. This is first exam ple o f this effect being activated by a single substitution - [R u(phen)2(Medpq)]^'" lum inescent in an aqueous environm ent w hile [R u(phen)2(d p q a )]'‘^ is not.

B inding o f the rac [R u(phen)2(Rdpq)]^'" fam ily to salm on sperm DNA was investigated. At P/D values o f 100, a general broadening o f the M L C T band and som e hypochrom icity is observed in the absorption spectra o f [R u(phen)2(Rdpq)]^^. The excited state lifetim es and lum inescence intensities are substantially increased w hen [R u(phen)2(Rdpq)]^'^ interacts with DNA. This produces an increase o f approxim ately six tim es in the lum inescence intensity o f [R u(phen)2(dpq)]"'^ w hen bound to DNA relative to the initial intensity in aerated 50 m M aqueous tris buffer. An increase o f approxim ately three tim es the initial intensity is produced for ra c [R u(phen)2(M edpq)]^^ and rac [R u(phen)2(M e2dpq)]^'^. A nalysis o f the binding data suggests that the strength o f binding decreases on increasing the num ber o f m ethyl substituents - the values o f K^pp are 4.1 x 10® M"' (± 0.2 x 10^ M '') for [R u(phen)2(d p q )]‘^, 2.6 x 10^ M '' (± 2 x 10^ M"') for [R u(phen)2(Medpq)]^'^ and 1.3 x 10^ M ‘‘ (± 2 X 10^ M ‘‘) for [R u(phen)2(M e2dpq)]^^.

B inding o f the A and A enantiom ers o f [R u(phen)2(dpq)]^'^ (pro v id ed by Dr. Janice A ldrich- W right) to salm on sperm DNA, [poly(dA -dT ).poly(dA -dT )] and [poly(dG -dC ).poly(dG -dC )] w ere studied. Both em ission intensity and excited state lifetim e are significantly increased w hen either [R u(phen)2(dpq)]^'^ enantiom er is bound to DNA or the polynucleotides. A nalysis o f the sp ectroscopic data dem onstrated that little enantioselectivity is evident betw een th e A and A enantiom ers o f [R u(phen)2(dpq)]"'" on binding DNA (K app- 10^ M '').

I w ould to thank my two supervisors Prof. John M. Kelly and Dr. Paul E.

Kruger. For financial assistance, I w ould like to thank Trinity College (U ssher

Fellowship), Enterprise Ireland and Kerry County Council.

I w ould also like to acknow ledge to the rest o f the academ ic staff in the

Chem istry D epartm ent - especially the organic chem ists w hose brains I picked on

several occasions regarding the “art” o f synthesis. For kindly allow ing me to use his

HPLC, I would like to thank Dr. Thorfinnur G unnlaugsson. I w ould also like to give

a special m ention to P ro f John Corish and Dr. D avid H. Grayson.

Regarding all the technical staff that keep the departm ent going - a special

thanks goes to Dr. M artin Feeney for his tim e and help with so many things. A lso I’d

like to express my thanks to Patsy, Brendan, Fred, John Kelly “the glassblow er”,

John O ’Brien for running countless N M R spectra and Paul Byrne.

At times this was an unbelievably soul destroying experience and I would

like to express my gratitude to all the people that helped m e through it - none more

so than the members o f both the Kelly and K ruger groups both past and present.

Particularly Aine, Aoife, Carlos, M ichelle, Phil, Ruth, Sarah, Suresh and o f course

M ichael who has had to put up w ith me the longest!!! N o doubt I will probably

forget someone but I would m ost definitely like to show my appreciation to all my

fellow chem istry post graduates who give the departm ent that special som ething.

l . I DNA 1

1.1.1 T he Prim ary Structure o f DNA 1-2

1.1.2 The Secondary Structure o f DNA 2-3

1.2 Why transition m e ta lp o ly p y rid y l com plexes? 3

1.2.1 R uthenium polypyridyl com plexes 3-4

1.2.2 [Ru(phen)3]^'‘ 4-5

1.2.3 [R u(bpy)2(dppz)]^* 5-7

1.2.4 [R u(tap)2(bpy)]^'^ and [R u(hat)2(bpy)]^'^ 7

1.2.5 B inuclear R uthenium com plexes 8-9

1.2.6 H eteronuclear com plexes 9

1 3 P hotophysical properties o f ruthenium p o ly p yrid yl com plexes 10-13

1.4 A im s o f this study 13-16

R eferences 17-18

C hapter 2 - Synthesis

2.1 P revious Synthesis o f B inuclear Ruthenium C om plexes 19-20 2.2 The O riginal Idea: D iazafluorenone

2.2.1 D iazafluorenone : Im ine Form ation, The W ang and R illem a A pproach 20-23 2.2.2 D iazafiuorenone : Im ine Form ation, The Tai M ethod 23 2.2.3 D iazafluorenone: R eductive A m ination 24-25 2.2.4 The O riginal Idea: D iazafluorenone, P resent Status 25

2.3 The new strategy

2 .3 .2 c M edpq: O xidation using Selenium D ioxide 32-33

2.3.3 Synthesis o f 2-dipyrido[3,2-f:2',3'-h]quinoxaline acid pentylam ide 34-35

2.4 The M o nonuclear R uthenium dpq b a se d fa m ily

2 .4 .1 Synthesis o f [R u(phen)2(Rdpq)]^’^ 36-38

2.5 H PLC P urification - [R u(phen)2( d p q a )f* 39-40 2.6 H P L C P urification - [R u(phen)2( m d p q c )f* 41-43 2 .7 A tte m p ted synthesis o f bridging lig a n d 44-45

2.8 C onclusions 45

R eferences 46

Chapter 3 - Solvent and Substituent Effects

3.1 Introduction

3.2 A bsorption spectra o f [R u(phen)2(Rdpq)]^*

3.3 D eterm ination o f G round S ta te pKa o f [R u(phen)2( R d p q ) f a m i l y 3.3.1a A nalysis o f T itration C urves

3.3.1b A nalysis o f Titration C urves - C oncentration estim ation 3.3.1c D eterm ination o f n and ground state pK^

3.4 P h otophysical A nalysis o f [R u(phen)2(Rdpq) f *

3.4.1 Photophysical Analysis o f [R u(phen)2(dpq)]^'^ 55-59 3.4.2 P hotophysical Analysis o f [R u(phen)2(Medpq)]^'^ 60-62 3.4.3 Photophysical A nalysis o f [R u(phen)2(M e2dpq)]^^ 63-66 3.4.4 Photophysical Analysis o f [R u(phen)2(Rdpq)]^'^ - C orrelation with 67-70

3.6 P hotophysical analysis o f [R u(phen)2( d p q a ) f* 79-83

3.7 C onclusions 84

R eferences 85

Chapter 4 - DNA

4.1 DNA binding studies - Introduction 86

4.2 A an d A E nantiom ers o f [R u(phen)2(dpq)]^*

4.2.1 A and A Enantiom ers o f [R u(phen)2(dpq)]^'^: A bsorption 87-88 4.2.2 A and A E nantiom ers o f [R u(phen)2(dpq)]^^ : L um inescence 89-90 4.2.3 A and A E nantiom ers o f [R u(phen)2(dpq)]^'^; Lum inescence - DNA 91-95 4.2.4 A and A Enantiom ers o f [R u(phen)2(dpq)]^'^: Lum inescence - DNA 95

A nalysis o f B inding C urves 4.2.4 a A nalysis o f B inding C urves - The Scatchard M odel 96 4.2.4b A nalysis o f B inding Curves - The M cG hee/von H ippel m odel 97 4.2.3 c A nalysis o f B inding C urves - C oncentration estim ation 98-100

4.2.5 A and A Enantiom ers o f [R u(phen)2(dpq)]^'^: Lum inescence -[poly(dA -dT ).poly(dA -dT )]

101-103

4.2.5 a A and A E nantiom ers o f [R u(phen)2(dpq)]^'^: L um inescence [P oly(dA -dT ).P oly(dA -dT ]. A nalysis o f B inding Curves

104-105

4.2.6 A and A Enantiom ers o f [R u(phen)2(dpq)]^”^ : L um inescence - [poly(dG -dC ).poly(dG -dC )]

106-107

4.2.6 a A and A E nantiom ers o f [R u(phen)2(dpq)]^‘^ : L um inescence [P oly(dG -dC ).Poly(dG -dC )] A nalysis o f B inding Curves

108-109

4.2.7 C om parison o f the M cGhee / von H ippel m odel to the B ard m odel 110-112

4.3.2a Racem ic [R u(phen)2(R dpq)]C l2 Family: D eterm ination o f 122-124 binding data

4.3.3 R acem ic [R u(phen)2(R dpq)]C l2 Fam ily: D iscussion 124-126

4.3.4 R acem ic [R u(phen)2(dpqa)]C l2: L um inescence 126-129

4.4 DNA bind in g studies - C onclusion 130

R eferences 131-132

Chapter 5 - Experim ental

5.1 M aterials an d M ethods

5.1.1 R eagents 133

5.1.2 C haracterisation o f ligands and com plexes 133

5.1.3 H PLC Purification 134

5.1.4 Solutions 135

5.1.5 DNA titrations. 136

5.1.6 A bsorption Spectrocopy. 137

5.1.7 Steady State Em ission Spectroscopy. 13 8 5.1.8 T im e-C orrelated Single P hoton C ounting (SPC ) 139-140

5.2 Procedures:

5.2.1 l,10-P henanthroline-5,6-quinone (phendione) 141

5.2.2 4,5-D iazafluoren-9-one (dafo) 141

5.2.3 D ip y rid o [3 ,2 -f2 ',3 '-h ]q u in o x alin e (dpq) 142

5.2.7a l,10-P henanthroline-5,6-dioxim e 145 5.2.7b l,1 0-P henanthroline-5,6-diam ine 145-146 5.2.7c 2,3-D im ethyldipyrido[3,2-f:2',3'-h]quinoxaline 146

5.2.8. [Ru(phen)2(Me2dpq)](PF6)2 147

5.2.9a M ethyldipyrido[3,2-f:2',3'-h]quinoxaline-2-carboxylate (m dpqc) 147-148 5.2.9b M ethyldipyrido[3,2-f:2',3'-h]quinoxaline-2-carboxylate (m dpqc) 148-149

5.2.10 [Ru(phen)2(mdpqc)](PF6)2 149-150

5.2. J I 2-D ipyrido[3,2-f:2',3'-h]quinoxaline acid pentylam ide (dpqa) 150-151

5.2.12. [Ru(phen)2(dpqa)](Pp6)2 151-152

5.2.13 2-D ipyrido[3,2-f:2',3'-h]quinoxaline acid n-am inopentylam ide 152

R eferences 153

Chapter 6 - Future W ork

6 .1 F uture Work

6.1.1 W hy does [R u(phen)2(dpqa)]^"'behave as a light sw itch? 154

6.1.2 [R u(phen)2(mdpqc)]^^ 155

6.1.3 Synthesis o f bridging ligand 156

A ppendix

'H N M R and ES M S o f [R u(phen)2(dpq)]^'" 'H N M R and ES M S o f [R u(phen)2(Medpq)]^'" 'H N M R and ES M S o f [R u(phen)2(M e2dpq)]^'^

Fig. 1.3. R ep resen tatio n o f A -, B - and Z - fo rm s o f D N A .

Fig. 1.4. M odel o f b in d in g m o d es to D N A . ([R u (p h e n )3]^^: p artial in te rc a la tio n and g roove b in d in g , [R u (b p y )3]^'^: e le c tro sta tic b in d in g .)

Fig. 1.5. S ch em atic m o d el o f th e b in d in g o f [R u (p h e n )3]^^. T h e A e n a n tio m e r is p ro p o sed to bind p re ferab ly to D N A re la tiv e to th e A en a n tio m e r.

Fig. 1.6. S chem atic m odel o f [R u (b p y )2(dppz)]^'^ in te rc a la tin g D N A . Fig. 1.7. T he c o m p lex es [R u (ta p )2(bpy)]^'^ and [R u (h at)2(bpy)]"'^.

Fig. 1.8. B in u clear co m p le x [(p h en )2R u (M e b ip y )-(C H2)n -(b ip y M e )R u (p h e n )2]'*'^. Fig. 1.9. B is-in te rc a la to r [|j,-C4(cpdppz)2-(phen)4Ru2]^^.

Fig. 1.10. H e te ro aro m atic b rid g in g ligands.

Fig. 1.11. P o ssib le e le c tro n ic tra n sitio n s in an o c ta h e d ra l co o rd in a tio n co m p le x . Fig. 1.12. S chem atic o f en erg y level d iag ram o f ru th e n iu m p o ly p y rid y l co m p le x e s. Fig. 1,13. dpqa based b rid g in g ligand.

Fig. 1.14. M e th y ld ip y rid o [3 ,2 -f 2 ',3 '-h ]q u in o x a lin e -2 -c a rb o x y la te (m d p q c). Fig. 1.15. 2 -D ip y rid o [3 ,2 -f;2 ',3 '-h ]q u in o x a lin e acid n -a m in o p en ty lam id e. Fig. 1.16. F am ily o f dpq b ased m o n o n u c le a r ru th en iu m co m p lex es.

Fig. 2.1. B in u clear co m p le x [(p h en )2R u (M e b ip y )-(C H2)n -(b ip y M e )R u (p h e n )2]"^. Fig. 2.2. B ip y rd in e based b rid g in g ligand.

Fig. 2.3. Initial sy n th etic stratgey.

Fig. 2.4. D esired S c h iff base co n d e n sa tio n reactio n . Fig. 2.5. M ech an ism o f acid c a taly sed im in e fo rm atio n . Fig. 2.6. [R u (p h en )2(dafo)]^’^.

Fig. 2.7. M ech an ism o f re d u ctiv e am in atio n . Fig. 2.8. R ed u ctiv e am in atio n o f dafo.

Fig. 2.9. 2 -d ip y rid o [3 ,2 -f:2 ',3 '-h ]q u in o x a lin e acid d o d ecy lam id e.

Fig. 2.10. R eactio n sch em e fo r the p re p a ra tio n o f 2 ,3 -d ia m in o p ro p io n ic acid dodecylam ide.

Fig. 2.11. Synthesis o f 2 -d ip y rid o [3 ,2 -f;2 ',3 '-h ]q u in o x a lin e acid d o d e c y la m id e .

Fig. 2.16. O xidation o f 4-m ethylquinoline.

Fig. 2.17. O xidation o f M edpq followed by esterification.

Fig. 2.18. 'H N M R spectrum (d^-MeCN) o f the desired ester follow ing oxidation (using selenium dioxide) then esterification o f M edpq.

Fig. 2.19. Synthesis o f 2-dipyrido[3,2-f:2',3'-h]quinoxaline acid pentylam ide from m dpqc and am ylam ine.

Fig. 2.20. 2,3-D im ethyldipyrido[3,2-f:2',3'-h]quinoxaline (M e2dpq). Fig. 2.21. Form ation o f l,10-phenanthroline-5,6-dioxim e (phendioxim e). Fig. 2.22. Form ation o f l,10-phenanthroline-5,6-diam im e (phendiam im e).

Fig. 2.23. Form ation o f 2,3-D im ethyldipyrido[3,2-f;2’,3 ’-h]quinoxaline (M c2dpq). Fig. 2.24. Fam ily o f dpq based m ononuclear ruthenium com plexes.

Fig. 2.25. HPLC chrom atogram o f the [R u(phen)2(dpqa)]^^ m onitored at 444 nm. Fig. 2.26. UV spectrum at 13.48 m inutes o f [R u(phen)2(dpqa)]^'^ chrom atogram

m onitored at 444 nm.

Fig. 2.27. C hrom atogram o f the [R u(phen)2(mdpqc)]^^ m onitored at 444 nm. Fig. 2.28. M ass spectrum o f post-HPLC [R u(phen)2(mdpqc)]^^.

Fig. 2.29. Possible reaction scheme for the decom position o f [R u(phen)2(mdpqc)]^'^ post-HPLC.

Fig. 2.30. dpqa based bridging ligand.

Fig. 2.31. 2-D ipyrido[3,2-f;2',3'-h]quinoxaline acid n-am inopentylam ide Fig. 3.1. A bsorption spectrum o f [R u(phen)2(dpq)]^'".

Fig. 3.2. T itration o f [R u(phen)2(M e2dpq)]^^ w ith HCl. Fig. 3.3. pH titration curve o f [R u(phen)2(Rdpq)]^’^.

Fig. 3.4. D oes mono- or di- protonation o f [R u(phen)2(Rdpq)]^^ occur ? Fig. 3.5. Plot to determ ine n and pKa for [R u(phen)2(dpq)]^'^.

Fig. 3.14. Fig. 3,15.

Fig. 3.16.

Fig. 3.17.

Fig. 3.18. Fig. 3.19. Fig. 3.20. Fig. 3.21. Fig. 3.22. Fig. 3.23.

Fig. 3.24. Fig. 3.25. Fig. 3.26.

Fig. 3.27.

Fig. 3.28.

Fig. 3.29. Fig. 4.1.

Fig. 4.2.

Fig. 4.3.

Fig. 4.4.

E m issio n o f [R u (p h en )2(M e2dpq)]^'" in aerated so lu tio n s.

C o m p ariso n o f lifetim es o f [R u (p h e n )2(R dpq)]^‘' in d iffe re n t d e g assed solvents.

P lo t o f lifetim es o f [R u (p h e n )2(Rdpq)]^'^ in d iffe re n t d e g assed so lv en ts a g ain st

P lo t o f q u an tu m y ield s o f [R u (p h e n )2(Rdpq)]^'" in d iffe re n t d e g assed so lv en ts a g ain st ti*.

P lo t o f k „ o f [R u (p h e n )2(R dpq)]^’" in d iffe re n t d e g a sse d so lv en ts a g a in st 7t*. S ch em atic o f e n erg y level d iag ram fo r ru th en iu m p o ly p y rid y l c o m p lex es. E ffe c t o f tem p e ra tu re on o f [R u (p h e n )2(R d p q )](P F6 ) 2 fa m ily in M eC N . F itted ex p erim en tal d ata o f [R u (p h e n )2(R d p q )](P p6 ) 2 in d eg assed M eC N . E ffe c t o f tem p e ra tu re on o f [R u(phen)2(R dpq)]C l2 fa m ily in d e g a sse d H2O. E x p erim en tal d ata o f [R u(phen)2(R dpq)]C l2 in d e g assed H2O fitted to eq u atio n : k = ko + kie'^^'^’^'^^.

E m issio n o f [R u (p h e n )2(dpqa)]^^ in d eg assed so lu tio n s. E m issio n o f [R u (p h e n )2(dpqa)]^^ in aerated so lu tio n s.

S ingle fu n ctio n al g ro u p su b stitu tio n a lterin g p h o to p h y sic a l b e h a v io u r in w ater.

H y d ro g en b o n d in g from am id e g ro u p to u n c o o rd in a te d n itro g en a to m s in [R u (p h e n )2(dpqa)]^''.

C o m p ariso n o f lifetim es o f [R u (p h e n )2(M edpq)]^^ and [R u (p h e n )2(dpqa)]^'^ in d iffe re n t d eg assed solvents.

[R u (b p y )2(m N H C H3)]^^ and [R u (b p y )2(m N E t2)]^".

A b so rp tio n titratio n o f 5 |J,M A [R u (p h e n )2(dpq)]^^ w ith salm on sp erm D N A in aerated 50 m M a q u e o u s pH 7.1 tris buffer.

L u m in escen ce titra tio n o f 5 ^.M A [R u (p h en )2(dpq)]^'^ w ith salm o n sperm D N A in aerated 50 m M a q u eo u s pH 7.1 tris buffer.

C o m p arin g lu m in esen ce titra tio n re su lts o f A and A [R u (p h e n )2(dpq)]^'^ (5 |o,M) w ith salm on sp erm D N A in aerated 50 m M a q u e o u s pH 7.1 tris b u ffe r

Fig. 4.7.

Fig. 4.8.

Fig. 4.9.

Fig. 4.10.

Fig. 4.11.

Fig. 4.12.

Fig. 4.13.

Fig. 4.14.

Fig. 4.15.

Fig. 4.16.

Fig. 4.17.

Fig. 4.18.

Fig. 4.19.

Fig. 4.20.

Fig. 4.21.

enantiom er with salm on sperm DNA.

Selected M cG hee / von Hippel binding curve for A [R u(phen)2(dpq)]^'^ enantiom er with salm on sperm DNA.

C om paring lum inesence titration results o f A and A [R u(phen)2(dpq)]^’" (5 fJ-M) w ith [poly(dA -dT).po!y(dA -dT)] in aerated 50 mM aqueous pH 7.1 tris buffer.

Plot to determ ine kq for A and A [R u(phen)2(dpq)]^'‘ (5 |iM ) w ith [poly(dA - dT). poly(dA -dT)] (P/D = 100) in 50 mM aqueous pH 7.1 tris buffer according to the Stern-V olm er equation:

x^li

= 1 + kqXo[02].

M cG hee / von H ippel binding curve for A [R u(phen)2(dpq)]"'^ enantiom er with [poly(dA -dT).poly(dA -dT)].

M cG hee / von H ippel binding curve for A [R u(phen)2(dpq)]^'^ enantiom er with [poly(dA -dT).poly(dA -dT)].

C om paring lum inesence titration results o f A and A [R u(phen)2(dpq)]^'" (5 p,M) with [poly(dG -dC ).poly(dG -dC )] in aerated 50 mM aqueous pH 7.1 tris buffer.

M cG hee / von Hippel binding curve for A [R u(phen)2(dpq)]^'" enantiom er with [poly(dG -dC ).poly(dG -dC )].

M cG hee / von H ippel binding curve for A [Ru(phen)dpq]^^ enantiom er with [poly(dG -dC ).poly(dG -dC )].

B inding curve for A [R u(phen)2(dpq)]^'^ enantiom er with salm on sperm DNA fitted using the Bard equation.

B inding curve for A [R u(phen)2(dpq)]^^ enantiom er with salm on sperm DNA fitted using the Bard equation.

A bsorption titration o f

rac

[R u(phen)2(dpq)]^^ (6 fiM) w ith salm on sperm DNA in aerated 50 m M aqueous pH 7.1 tris buffer.

Lum inescence titration o f

rac

[R u(phen)2(Medpq)]^* (2 |j.M) with aerated salm on sperm DNA in 50 mM aqueous pH 7.1 tris buffer.

C om paring lum inescence titration results o f

rac

[R u(phen)2(Rdpq)]^^ (2 |iM ) with salm on sperm DNA in aerated 50 m M aqueous pH 7.1 tris buffer.

B i-exponential excited state decay o f (2 |j,M) [R u(phen)2(M e2dpq)]^^ with salm on sperm DNA (P/D = 100) in oxygenated 50 m M aqueous pH 7.1 tris buffer.

Fig. 4.24.

Fig. 4.25.

Fig. 4.26.

F ig S .l. Fig. 6.1.

Fig. 6.2. Fig. 6.3. Fig. 6.4. Fig. 6.5. Fig. 6.6.

L um inescence titration o f

rac [R u(phen)

2(dpqa)]^'^ (5 |J.M) w ith aerated salm on sperm D N A in 50 mM aqueous pH 7.1 tris buffer.

T he titration results o f rac [R u(phen)2(dpqa)]^^ (5 |iM ) with salm on sperm DNA in aerated 50 mM aqueous pH 7.1 tris buffer.

D ecrease in lum inescence intensity o f

rac [R u(phen)

2(dpqa)]^^ after m axim um intensity is achieved is accom panied by slight blue shift.

Schem atic o f tim e correlated single photon counting instrum ent.

Single functional group substitution altering photophysical behaviour in w ater.

[R u(phen)2(dpqa)]^’*^ m inus the alkyl tail.

D ecom position o f [R u(phen)2(mdpqc)]^^ to [R u(phen)2(dpq)]^’^. R uthenium com plex o f hindered ester,

dpqa based bridging ligand.

Table 3.2. Photophysical properties o f [R u(phen)2(dpq)]^^. Table 3.3. kr and knr values for [R u(phen)2(dpq)]^’".

Table 3.4. Literature values o f excited state lifetim es in degassed solvents o f other ruthenium polypyridyl com plexes ( t ± 1 0 %).

Table 3.5. Photophysical properties o f [R u(phen)2(Medpq)]^^. Table 3.6. kr and k„r values for [R u(phen)2(M edpq)]^’^.

Table 3.7. Photophysical properties o f [R u(phen)2(M e2dpq)]^'^. Table 3.8. kr and k„r values for [R u(phen)2(M e2dpq)]^"^.

Table 3.9. Solvent param eters.

T able 3.10. V ariation o f the lifetim es o f [R u(phen)2(R dpq)](P p6 ) 2 in degassed M eCN w ith tem perature.

T able 3.11. Param eters determ ined from fitting plots o f I/x versus 1/RT o f [R u(phen)2(R dpq)](P p6 ) 2 family in degassed M eC N to: k = ko + k ie‘^“ '^‘^^. Table 3.12. V ariation o f the lifetim es o f [R u(phen)2(R dpq)]C l2 in degassed H2O with

tem perature.

Table 3.13. Param eters determ ined from fitting plots o f 1/t versus 1/RT o f [R u(phen)2(R dpq)]C l2 fam ily in degassed H2O to: k = ko +

Table 3.14. Photophysical properties o f [R u(phen)2(dpqa)]^^. Table 3.15. kr and knr values for [Ru(phen):(dpqa)]^'^.

Table 4.1. A bsorption data for A and A [R u(phen)2(dpq)]^^ (5 |iM ) in aerated 50 mM aqueous pH 7.1 tris buffer.

T able 4.2. Lum inescence data for A and A [R u(phen)2(dpq)]^^ (5 p,M) in 50 mM aqueous pH 7.1 tris buffer (P/D = 100).

T able 4.3. A and A [R u(phen)2(dpq)]^^ (5 )iM) w ith salm on sperm DNA in 50 mM aqueous pH 7.1 tris buffer (P/D = 100)

T able 4.4. Lifetim e data o f A and A [R u(phen)2(dpq)]^'" (5 ju,M) with salm on sperm DNA in aerated 50 mM aqueous pH 7.1 tris buffer, (error ± 1 0 % )

T able 4.5. B inding data determ ined for [R u(phen)2(dpq)]^'^ enantiom ers (5 (iM) bound to salm on sperm DNA in aerated 50 mM aqueous pH 7.1 tris buffer.

Table 4.6. A and A [R u(phen)2(dpq)]^* (5 |J.M) w ith [poly(dA -dT). poly(dA -dT )] in 50 mM aqueous pH 7.1 tris buffer (P/D = 100).

T a b le 4 .1 0

T a b le 4.11 T a b le 5.1.

S um m ary o f b in d in g d ata d e te rm in e d fo r A and A [R u (p h e n )2(dpq)]^'" u sin g the B ard eq u atio n .

1.1

DNA

Deoxyribonucleic acid, DNA, encodes and preserves the hereditary

information o f life. This nucleic acid enables the genetic information to be stored and

transmitted from one generation o f cells to the next. The proteins that a cell will

produce and the functions that these proteins will perform are all determined by this

biological polymer.'

1.1.1

The Primary Structure o f DNA

The primary structure o f DNA is composed o f a sequence o f nucleotides.

Each nucleotide consists o f a five-carbon sugar (deoxyribose) bonded to a phosphate

group and a heterocyclic base. Phosphodiester linkages bond two neighbouring

nucleotides together resulting in a “backbone” consisting o f alternating sugar and

phosphate groups. There are four heterocyclic bases in DNA, two purines (adenine

and guanine) and two pyrimidines (thymine and cytosine) that are arranged as side

O . H N

\ / \ /

1 c c c c

I

/

\

/■

\

/ c N — H • • • IM- C — H

C l \ /

N C C - N1

\

/

\

N H • • • o c r

Fig. 1.1. G=C and A=T base pairs (• • • hydrogen bond).

cr

o

H H, /

C

\ /

c — c

/ \

N c —

\ /

c N1

/ \

o

[image:24.510.44.489.26.626.2]

HC,

Fig. 1.2. The primary structure o f DNA.

1.1.2

The Secondary Structure o f DNA

The ladder-like double chain molecule then adopts a three-dimensional

structure by twisting to form a double helix. The right-handed helical B-DNA is the

most com mon form, how ever right-handed A -DNA and left-handed Z-DNA

constitute tw o other principal secondary DNA structures.'

Minor Groove

Major Groove

Minor Groove'

A-form RNA

B-form DNA

Z-form DNA

[image:25.510.35.499.33.707.2]

Ten consecutive nucleotide pairs give rise to one complete turn o f the B-DNA helix

in 3.4 nm. The B-DNA helix has a diameter o f 2 nm and consists o f a wide major

groove and narrow minor groove that are both well solvated by water molecules. The

helix is further stabilised by stacking interactions between the base pairs.' Other

local irregular forms also exist, for example cruciforms and hairpin loops, which are

reasonably assumed to have some function in the recognition, regulation or

expression o f particular genes.

1.2

Why transition metal polypyridyl complexes?

In the last two decades, the potential o f polypyridyl transition metal

complexes as probes for nucleic acids has been recognised.

The positively charged

transition metal complexes can interact strongly with the DNA polyanion.^"^

The three-dimensional structure o f these coordinatively saturated, rigid metal

complexes makes them attractive spatial molecular tools for DNA. Through variation

o f the central metal it is possible to change not only the photophysical,

photochemical and electrochemical properties but also the geometry (tetrahedral,

octahedral, square planar). Subtle changes in the attached ligands can also facilitate

further modifications in the properties o f the complex.

The water-soluble metal complexes are often spectroscopically active. The

transition metal complexes typically undergo electronic transitions in the visible and

some subsequently display luminescence. In the excited state, the complexes are

better oxidising and reducing agents than the ground state.

1.2.1 Ruthenium polypyridyl complexes

The size, shape, photophysical, photochem ical and redox properties o f ruthenium

com plexes can be fine-tuned by modification o f the ligand.^"^ Here we present some

examples to illustrate the rich and diverse chemistry o f ruthenium polypyridyl

com plexes and their potential as probes for DNA.

1.2.2

[Ru(phen)sf^

Early studies on [Ru(phen)

3

]^^ established that binding o f [Ru(phen)

3

]^^ to

the DNA double helix resulted in absorption hypochromicity and luminescence

enhancement, with a binding constant o f - 10^ M

A lthough this is one o f the

simplest ruthenium polypyridyl complexes, the m ode o f binding still remains

controversial. Partial intercalation, major o r minor groove-binding and electrostatic

interactions have all been proposed m ethods o f binding for the positively charged

[Ru(phen)3]^^.^**

Fig. 1.4. Model o f binding modes to DNA.

[Ru(phen)

3

]^^: partial intercalation and groove binding, [Ru(bpy)j]^^: electrostatic binding.

[image:27.510.31.493.53.661.2]

enantioselectivity in their interactions with DNA.^ Barton

et al found a greater

^ j

luminescence enhancement o f A [Ru(phen)

3

]

relative to the A enantiomer in the

« 9+

presence o f DNA and in equilibrium dialysis experiments on

rac [Ru(phen)

3

] , the

dialysates were found to be optically enriched in the A enantiomer.^'^ In general,

although some degree o f preferential binding o f the A enantiomer is predicted for B-

DNA, where binding constants have been determined the degree o f enantioselectivity

o

is usually modest.

A

A

Fig. 1.5. Schematic model of the binding o f |Ru(phen)

3

]^^. The A enantiomer is proposed to

bind preferably to DNA relative to the A enantiomer.

1.2.3 [Ru(bpy)

2(dppz)

[Ru(bpy)

2

(dppz)]^^ is described as a “molecular light switch” - it displays no

photo luminescence in aqueous solution but emits on intercalating DNA.^ This light

switch effect is attributed to protection o f the extended aromatic dppz ligand from

[image:28.510.30.495.30.590.2]

F ig. 1.6. S c h e m a tic m o d e l o f [R u (b p y )2(dppz)]^^ in terca la tin g D N A .

[image:29.510.39.494.39.608.2]

M LCT" interconversion. Therefore the light switch effect is attributed to fast M LCT'

—>■

M LCT" interconversion prom oted by interactions w ith w ater and followed by

rapid M LCT" radiationless decay. In DNA, [Ru(bpy)2(dppz)]^"^ is protected from

w ater and thus M LCT' ^ M LCT" conversion is restrained and em ission is observed

from the M LCT' sta te .''

1.2.4

[Ru(tap)2

( b p y ) a n d [Ru(hat)2

(bpy)

The excited states o f some ruthenium polypyridyl com pounds are

photochem ically active and exploitation o f these properties offers the possibility to

develop novel chem otherapeutic agents that are subject to control by light.

F ig . 1.7. T h e c o m p le x e s [R u (tap )2(bpy)]^'^ and [R u (h a t)2(bpy)]^'^.

In the excited state, com plexes o f the type [Ru(tap)2(bpy)]

and [Ru(hat)2(bpy)]

12

[image:30.510.40.500.30.591.2]

1.2.5 B in u c le a r Ruthenium complexes

The binuclear complexes

[(pp)

2

R u(M ebipy)-(C H

2

)n-(bipyM e)Ru(pp)

2

]'*^,

where pp = bpy or phen, were synthesised previously in this laboratory.'^ As

m olecular probes for D N A , these binuclear compounds were found to have several

advantageous properties as compared to the simple mononuclear complexes. The

binuclear ruthenium complexes exhibited a higher binding a ffin ity fo r D N A , were

less sensitive to ionic strength and produced more effective photocleavage o f

plasmid D N A.

13

..vCHj H,C„ ' ■ " ' V

-Fig. 1.8. Binuclear com plex [(phen)2R u (M e b ip y)-(C H2)n-(bipyM e)R u(phen)2] 4+

Recently Norden and co-workers reported the synthesis o f a dppz based

binuclear complex.'"' When bound to D N A , the A-A and A -A enantiomers o f [jj.-

C4(cpdppz)2-(phen)4Ru2]''^ exhibit luminescence very sim ilar to that o f the

corresponding parent [Ru(phen)2(dppz)]^'^

enantiomer. Both

A-A

and

A -A

,CN

HN

NH

N

Ru(phen) 2

NC _N

[image:31.510.45.498.28.790.2]

enantiom ers display extrem ely high affinity for D NA and N orden et al propose a bis-

intercalative binding mode where the com plex acts as a “staple” holding the bases

firm ly stacked together. Therefore binding o f this bis-intercalator m ust involve a

kind o f threading m echanism - the bulky R u(phen

) 2

moiety possibly passing through

the D NA strands.'"'

1.2.6

H eteronuclear complexes

In

the

hom onuclear

com plexes

[(pp)

2

R u(M ebipy)-(C H

2

)n-

(bipyM e)Ru(pp)

2

]'''^ (where pp = bpy or phen) synthesised in this laboratory before,

the ruthenium metal atoms are linked a flexible alkyl chain. Therefore to a large

degree the metal centres are independent o f each other. This is in contrast to the

many exam ples in the literature o f heteronuclear com plexes bridging by extended

arom atic ligands w here good electronic com m unication is expected.

quaterpyridine hat dpp tpphz

F ig. 1.10. H e teroarom atic b rid g in g ligan d s.

[image:32.510.41.492.35.581.2]

7, i

P hotophysical properties o f ruthenium polypyridyl com plexes

D uring the formation o f a ruthenium polypyridyl com plex, the non-bonding

electrons o f the polypyridyl ligands are attracted to and directed tow ards the

positively charged metal centre. Crystal field theory provides a sim ple model to

describe these bonding interactions. The energy o f the d orbitals that point tow ards

the electron donating ligands (eg*) is increased relative to the d orbitals that are

directed between the ligands (t

2

g). This results in a splitting o f the d orbital energies

and w hen com bined with the ligand orbital diagram produces a com posite orbital

energy diagram for the complex.

d orbitals

I

TTl’'

e g ’'

t2g

a L •

7 1 *

K

metal

complex

ligand

Fig. 1.11. Possible electronic transitions in an octahedral coordination complex.

[image:33.510.35.497.30.539.2]

Excitation o f a typical ruthenium (II) polypyridyl com plex in any o f its

absorption bands initially produces an excited state that is largely singlet in

character, 'M LCT. The heavy atom effect results in m ixing o f the singlet and triplet

states by spin-orbit coupling and this leads to rapid intersystem crossing (ISC)

producing the em itting triplet M LCT state (hence a large Stokes shift). Intersystem

crossing from 'M L C T to ^MLCT is efficient with a quantum yield close to unity.

The em itting M LCT excited state is actually a com posite o f closely spaced

energy levels. The three lowest lying states are predom inantly triplet in character and

the energy difference am.ong them is small (~ 100 cm"'), hence at room tem perature

they are in therm al equilibrium. The energy gap to the fourth state is higher (~ 600

1 i n

cm ' ) and is short lived because o f its greater singlet character.

>MLCT

ISC

3MC

act

abs

nr em

Ground State

Fig. LI 2. Schem atic o f energy level diagram o f ruthenium polypyridyl com p lexes.

Three main pathways exist for the deactivation o f the ^MLCT excited state -

radiative

( k r )

and non-radiative

( k n r )

decay, internal conversion to the therm ally

accessible ^MC state and quenching by m olecular oxygen:

k = kf + k j y . + k ( ] j + k q [ 0 2 ]

accessible metal centred (^MC) state follows the expression;

kdd =

where : Eact is defined as the energy gap between the ^M LCT state and the ^MC state

(kJ m o l''), ki is the pre-exponential factor, R is the gas constant (J K'^ m o l'') and T is

I 8 9 0 1

the temperature in Kelvin. '

The MC (or dd) excited state geometry is strongly

distorted relative to the ground state as it effectively involves the promotion o f an

electron to an anti-bonding orbital. Therefore, it undergoes very fast non-radiative

decay that can include ligand substitution and racemization reactions.'*

The values o f kr and k„r can be established from the

experimentally

determined emission quantum yield and the lifetime data using:

1 / x = k r + k n r a n d k r = ( ) ) e m / ' C .

For

ruthenium polypyridyl complexes, k„r and kdd are usually two orders o f

magnitude greater than kr, therefore the lifetimes o f these ^MLCT excited states are

predominantly controlled by non-radiative processes.

Molecular oxygen efficiently quenches excited state ruthenium polypyridyl

1 o

complexes.

Varying the dissolved molecular oxygen concentration allows the

pseudo first order rate constant, kq(02), to

be

determined

according

to the

Stern Volmer equation:

T q /t =

1

+ k q X o [0 2 ]

The emission properties o f ruthenium polypyridyl complexes are sensitive to

the local environment due to the charge transfer nature o f the lowest energy ^MLCT

excited state.'* For charge transfer excited states there is a considerable change in

dipole moment upon excitation. The distribution o f solvent molecules that gave the

most stable configuration in the ground state does not necessarily minimise the

energy in the excited state. The absorption process in virtually instantaneous

compared w ith molecular motion therefore the solvent molecules subsequently re­

orientate to stabilise the excited state, resulting in a reduction in energy and thus a

1.4

A im s o f this study

The binuclear ruthenium m etal com plexes

[(pp)

2

Ru(M ebipy)-(CH

2

)n-

(bipyM e)R u(pp)

2

]''^, where pp = bpy or phen, were synthesised previously in this

laboratory.'^ As m olecular probes for DNA, these binuclear com pounds w ere found

to have several advantageous properties as com pared to the sim ple m ononuclear

com plexes. The synthetic strategy o f [(pp)

2

R u(M ebipy)-(CH

2

)n-(bipyM e)Ru(pp)

2

]'*^

had som e advantages - it allowed for variation in the alkyl chain length and o f the

term inal chelate ligands.'^

The aim o f this research project was to continue this work and devise a

synthetic strategy that would allow the synthesis o f heteronuclear com plexes. This

w ould require the developm ent o f a novel, versatile bridging ligand that should

enable binuclear ruthenium com plexes to be m ade but also have the potential for the

selective synthesis o f a heteronuclear species.

C hapter 2 details the synthetic strategies investigated. D espite num erous

attem pts, the initial synthetic route based on diazafluorenone did not prove

successful. The selected alternative to a dafo based bridging ligand w as a novel dpq

based bridging ligand shown below.

NH(CH2)5HN

Fig. 1.13. dpqa based bridging ligand.

Fig. 1.14. M e th y ld ip y r id o [3 ,2 - f: 2 ',3 '-h ] q u in o x a l in e -2 - c a r b o x y la te ( m d p q c ) .

Subsequent reaction o f two equivalents o f m dpqc w ith one equivalent 1,5-

diam inopentane, how ever, did not allow the desired bridging ligand to be synthesised

in adequate yields. A lthough the synthesis o f the targeted ligand was not

accom plished, the central goal o f synthesising a ligand with the potential for the

selective synthesis o f both hom onuclear and heteronuclear metal com plexes was still

achieved through the synthesis o f 2-dipyrido[3,2-f;2',3'-h]quinoxaline acid amino-

pentylam ide from m dpqc and an excess o f 1,5-diaminopentane.

H

C ^(CH2)5NH2

O

Fig. 1. 15. 2 - D ip y r id o [ 3 ,2 -f :2 ' ,3 '- h ] q u in o x a lin e acid n -a m in o p e n t y la m id e .

The presence o f the free am ino group on the aliphatic tail provides the potential for

the future selective synthesis o f both hom onuclear and heteronuclear metal

The prim ary focus o f this research centred on the developm ent o f a novel,

versatile bridging ligand that could enable the selective synthesis o f both

hom onuclear and heteronuclear com plexes. However, it was considered constructive

to initially exam ine the photophysical behaviour o f the m ononuclear ruthenium

com plexes o f the sim pler dpq ligands. C hapter

2

therefore describes the synthesis,

purification and characterisation o f a family o f dpq based m ononuclear ruthenium

com plexes.

2+

[Ru(phen)2(Me2dpq)]^'^

[Ru(phen)2(M edpq)]'

^OMe

N C

II

O

[Ru(phen)2(mdpqc)]^'^

H

N C (CH2)4CH3

O [R u(phen)2(dpqa)]

Fig. 1.16. F a m ily o f dpq b ased m o n o n u c le a r ruthenium c o m p l e x e s .

o f the photophysical properties o f [Ru(phen)2(Rdpq)]^’^ was investigated in both

acetonitrile and water.

In Chapter 4, binding o f the racemic [Ru(phen)2(Rdpq)]^'^ family to salmon

sperm DNA was examined by UV-Vis absorption and steady state luminescence.

Excited state lifetimes, binding constants and sites sizes are determined. The A and A

2_|_

enantiomers of [Ru(phen)2(dpq)]

were provided by Dr. Janice Aldrich-Wright.

Binding of the A and A enantiomers o f [Ru(phen)2(dpq)]^"^ to salmon sperm DNA,

[poly(dA-dT).poly(dA-dT)] and [poly(dG-dC).poly(dG-dC)] is studied. Binding was

monitored by both absorption and luminescence techniques and single photon

counting is used to determine the excited state lifetimes o f the fully bound

complexes. Analysis o f the spectroscopic data allows the binding constants and site

sizes to be estimated. Chapter 4 concludes with the study o f the binding o f

rac-

[Ru(phen)2(dpqa)]^’^ to salmon sperm DNA.

This illustrates an extraordinary

transformation that converting a methyl group to an amide has on the ability of

[Ru(phen)2(dpqa)]^'^ to act as a photophysical probe for DNA.

Chapter 5 reports the experimental conditions employed. Experimental

synthetic procedures, subsequent purification techniques and characterisation details

are presented. Sample preparation and the apparatus used for the photophysical

analysis o f the ruthenium complexes are described. The thesis concludes with

References

1.

Voet, D.; Voet, J.G.

‘‘B iochem istry"

2"^* Edition, 1995, John W iley and

Sons Inc.

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M oucheron, C.; K irsch-De M esm aeker, A.; Kelly, J.M .

"Structure and

Bonding”

Vol. 92.

"Photophysics a n d Photochem istry o f M etal P olypyridyl

and R elated Com plexes with N ucleic Acids",

1998.

3.

Norden, B.; Lincoln, P.; A kerm an, B.; Tuite, E.

"Metal Ions in Biological

System s"

Vol. 33.

"Probing o f N ucleic A cids by M etal Ion C om plexes o f

Sm all M olecules"

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Satyanarayana, S.; Dabrowiak, J.C.; Chaires, J.B.

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2.1

Previous Synthesis o f Binuclear Ruthenium Complexes

In an effort to extend the vast am ount o f research on m ononuclear ruthenium

com plexes, binuclear ruthenium metal com plexes were successfully synthesised

previously in this laboratory. The com plexes w ere o f the type [(pp)2Ru(M ebipy)-

(C H

2

)n-(bipyM e)Ru(pp)2]''^, where pp = bpy or phen.' It was hoped the binuclear

species would produce superior interactions w ith DNA. Indeed, as D NA probes, the

com pounds were found to possess several favourable characteristics relative to their

m ononuclear cousins. The binuclear ruthenium com plexes exhibited a higher binding

affinity for DNA, resulted in more effective photocleavage o f plasm id DNA and

were less sensitive to ionic strength.’

Fig. 2.1. B inu cle ar c o m p l e x [(p hen)2R u ( M e b i p y ) - ( C H2)n - (b ip y M e )R u (p h e n )2]'*^.

Synthesis o f the above binuclear com plex firstly involved the treatm ent o f

4,4'-dim ethyl-2,2'-bipyridine with lithium diisopropylam ide (LDA), a strong base

generated by the addition o f butyllithium to diisopropylam ide dissolved in dry THF.^

LDA converts 4,4'-dim ethyl-2,2'-bipyridine to the corresponding carbanion w hich is

then reacted with the appropriate dibrom oalkane to produce a bridging ligand w hich

is bipyridine based with the linkage provided by m eans o f an alkyl chain.

CH: _{CH CH:}

LDA

_{BrH2C(CH2)nCH2Br}

►

CH

The binuclear com plex was then formed by reacting the bridging ligand with two

equivalents o f Ru(pp)

2

Cl

2

, w here pp = bpy or phen. Synthetically the above strategy

boasts some advantages - it allow s for variation in the alkyl chain length and o f the

term inal chelate ligands.

2.2

The Original Idea: Diazafluorenone

2.2.1

Diazafluorenone : Imine Formation, The Wang and Rillema Approach

The focus o f this research program m e was to extend this w ork through the

developm ent o f a novel, versatile bridging ligand. This novel bridging ligand should

enable binuclear ruthenium com plexes to be made but also have the potential for the

selective synthesis o f heteronuclear species.

The initial synthetic route investigated did not prove successful despite

repeated efforts on various different synthetic methods. It was centred on the

previously know n ligand diazafiuorenone (dafo).

D iazafluorenone is obtained

N-N'

phen

KBr

phendione

Fig. 2.3. Initial synthetic stratgey.

O H

-dafo

^0

from the reaction o f aqueous sodium hydroxide w ith l,10-phenanthroline-5,6-

quinone (phendione).^ Phendione is readily prepared in alm ost quantitative yields by

the oxidation o f phenanthroline with B ri and H NO

3

(the m olecular brom ine is

generated in situ from

H2SO4

and KBr).^ The attractiveness o f this system was the

presence o f an exocyclic ketone. Hence, by means o f a S chiff base condensation,

potentially only a single reaction step separated the precursor, dafo, from the desired

resulting ligand would be symmetric thus minimising the amount o f potential

diastereoisomers in the final ruthenium complex. Amylamine (

1

-pentamine) was the

aliphatic amine o f choice as the previous work on the binuclear ruthenium

complexes, [(pp)2Ru(Mebipy)-(CH2)n-(bipyMe)Ru(pp)2]''^ (where pp = bpy or phen),

indicated that five carbon atoms provided the optimum chain length for DNA

mteraction.

a m y l a m i n e

Fig. 2.4. Desired Schiff base condensation reaction.

Wang and Rillema have described the synthesis o f similar imines.^ Their

emphasis was on rigid bridging ligands and so they employed aromatic amines.

Application o f their experimental procedure,^ refluxing dafo and amylamine in

ethanol using glacial acetic acid as the catalyst, did not result in the condensation of

dafo and amylamine. The starting material, dafo, was the only substance recovered

from the reaction mixture. Consideration o f the reaction mechanism and inherent

g

properties o f imines can account for this.

R

\

/

R

C = 0 + NH2R

H2O, H

R

\

C=

f/

R

NHR

Schiff base condensation involves the nucleophilic attack o f an amine on a

o

carbonyl carbon, this is followed by proton transfer and the eventual loss o f water.

As im ine form ation is acid catalysed, glacial acetic acid was added to act as a

catalyst by prom oting the loss o f the leaving group, water. This is achieved in W ang

and R illem a’s system, where an aromatic am ine is used. A m ylam ine is an aliphatic

amine and thus is a much more basic amine

eg.

pKa o f propylam ine 10.67 and pKa o f

aniline 4.58 . It is therefore highly probable that the am ylam ine becom es protonated

thereby reducing the nucleophilicity o f the amine.

Further com pounding the situation is the stability o f the resulting imine.

Substituent groups have a large effect on the position o f the equilibrium show n in

8 X

Fig. 2.4. Aryl am ines as a rule form quite stable S chiff bases. In contrast, simple

alkyl amines result in imines w hich are unstable and known to rapidly decom pose or

o

polymerise. Secondary, tertiary and aromatic aldehydes react readily to form imines.

Ketones, in particular aromatic ketones, react m uch m ore slowly.* As dafo is a

sterically hindered conjugated ketone, it is not expected to undergo a S chiff base

reaction easily. It was now apparent that am ylamine and dafo were not ideal

precursors for a S chiff base condensation and that other avenues w ould have to be

explored.

Co-ordination o f dafo to [Ru(phen)

2

Cl

2

] to form the ruthenium com plex

was then considered. It was hoped that the positive metal centre w ould create an

inductive effect and enhance the electrophilicity o f the carbonyl carbon o f dafo.

2

+

S+

c=o

Fig. 2.6. [R u(phen)2(dafo)]^^.

However, Wang and Rillem a have found that the presence o f the ruthenium metal