THE STUDY OF CHARGE TRANSFER IN SHORT DNA …snsm/group/dissertations/dissertation_alexey.pdf1. Krasnoslobodtsev, Alexey, Smirnov, Sergei, “Effect of a single locked nucleotide modification

THE STUDY OF CHARGE TRANSFER IN SHORT DNA

OLIGONUCLEOTIDES-HAIRPINS AND SURFACE

BOUND ORGANIC MOLECULES

BY

ALEXEY VIKTOROVICH KRASNOSLOBODTSEV, M.S.

A dissertation submitted to the Graduate School

in partial fulfillment of the requirements

for the degree

Doctor of Philosophy

Major Subject: Chemistry

New Mexico State University

Las Cruces, New Mexico

August 2005

ii

“The Study of Charge Transfer in Short DNA Oligonucleotides-Hairpins and Surface

Bound Organic Molecules,” a dissertation prepared by Alexey Viktorovich

Krasnoslobodtsev in partial fulfillment of the requirements for the degree, Doctor of

Philosophy, has been approved and accepted by the following:

Linda Lacey Dean of the Graduate School Sergei Smirnov Chair of Examining Commitee Date

Committee in charge:

Dr. Sergei Smirnov, Chair

Dr. Igor Sevostianov

Dr. Jeremy Smith

Dr. Haobin Wang

iii

ACKNOWLEDGEMENTS

Although my education at New Mexico State University has come to an end, I

am indebted to many people who have aided my efforts here at the University. First

and foremost, I would like to thank Dr. Sergei Smirnov, whose inspiration and

support have impacted my life greatly. I will always remember being his advisee. He

has introduced me to many things which I had no idea about before. His supervision

made my Ph.D. program both intellectually stimulating and challenging.

Department of Chemistry and Biochemistry at New Mexico State University

should be acknowledged separately for the financial support over my entire Ph.D.

program. I have never heard about NMSU and Las Cruces before I moved here. But

it turned out to be a very nice place to live. Thanks to all the professors at the

Department, especially professors of physical chemistry Dr. Haobin Wang and Dr.

David Smith.

Members of my committee need special recognition. I thank Dr. J. Smith, Dr.

H. Wang and Dr. I. Sevostianov for reading my project and for valuable comments.

Present and former members of the Electron Transfer Group: Dr. B. Tadjikov

and graduate students: Ivan Vlassiouk, Pavel Takmakov and Kirill Velizhanin. Also I

want to thank undergraduate students who worked with us on the project: Ilia

Shalaev, Lisken Mason and Eric Rodrigez.

Although life in graduate school is hard, it is a lot easier with friends. I would

like to express special thanks to my Russian, American and from all over the world

iv

friends for nice conversations, drinks and fun, who made easier my studying when I

did not feel like continuing at NMSU and was feeling discouraged.

Special thanks go to Dr. Karen Shaefer, who has helped me a lot in coping

with the situation during last month of writing the dissertation. Not only I owe her

my appreciation, but also I thank her for a great revelation in my life.

The last but not least I would like to extend my greatest gratitude to my

parents and my wife. I would like to thank my wife separately for having enough

strength to go along with me through the process of finishing the project. She had a

patience to listen to my explanations when I had a hard time in formulating my

thoughts. She probably became an expert in how rotation time influences the dipole

signal amplitude in the case of noninstantaneous excitation.

v

VITA

April 7 1975 Born at Bobrovka, Novosibirsk region, Russia

June 1992 Graduated from Myshlanskaya Middle School

1992-1995 Student at Novosibirsk State Pedagogical University

1995-2000 Student at Novosibirsk State University

1998-2000 Research assistant at Novosibirsk Research Institute of Catalysis

2000 B.S. degree in Chemistry, Novosibirsk State University

2000-2005 Research and Teaching Assistant, Department of Chemistry and Biochemistry, New Mexico State University

August 2004 Dale Alexander Outstanding Teaching Award December 2004 M.S. degree in Chemistry, New Mexico State University

Publications

1. Krasnoslobodtsev, Alexey, Smirnov, Sergei, “Effect of a single locked nucleotide modification on stacking of bases and charge transfer efficiency in DNA-hairpins.” in press 2. Krasnoslobodtsev, Alexey V., Smirnov, Sergei N., “Temperature dependence of 2-aminopurine fluorescence quenching in short hairpins and its relation to electron transfer.” in press 3. Vlassiouk, Ivan, Krasnoslobodtsev, Alexey, Smirnov, Sergei and Germann, Markus, “’Direct’ Detection and Separation of DNA Using Nanoporous Alumina Filters.” Langmuir, 2004, 20, 9913-9915. 4. Krasnoslobodtsev, Alexey V., Smirnov, Sergei N. “Effect of water on silanization of silica by trimethoxysilanes.” Langmuir, 2002, 18(8), 3181-3184. 5. Krasnoslobodtsev, Alexey, Smirnov, Sergei, “Surface assisted intermolecular interactions in self-assembled coumarin submonolayers.” Langmuir, 2001, 17(24), 7593-7599.

vi

Field of Study

Major field: Chemistry

Physical chemistry

vii

ABSTRACT

THE STUDY OF CHARGE TRANSFER IN SHORT DNA

OLIGONUCLEOTIDES-HAIRPINS AND SURFACE

BOUND ORGANIC MOLECULES

BY

ALEXEY VIKTOROVICH KRASNOSLOBODTSEV, M.S.

Doctor of Philosophy

New Mexico State University

Las Cruces, New Mexico, 2005

Dr. Sergei N. Smirnov, Chair

Surface-assisted photoinduced transient displacement charge technique

(SPTDC) was developed to allow direct measurements of photoinduced charge

transfer in oriented by the surface molecules without need of external electric field.

For that purpose, a protocol of self assembled monolayers (SAM) formation using

alkoxy aminosilanes was optimized. Coumarin (D-1412) was used as a model system

to test SAM properties. Very high surface density (3×1014 cm-2) was achieved by

introducing a novel two-step silanization process with intermediate hydrolysis of

alkoxy groups. Such high surface concentrations were sufficient for observing

stimulated emission from optically excited SAM. The orientation of molecules on the

surface and the intermolecular interactions were shown to vary with solvent, linker

and surface concentration of coumarin.

viii

Modification of surface assisted photoinduced transient displacement charge

(SPTDC) technique was realized and tested on coumarin self-assembled monolayers

by comparing it with standard PTDC approach. A theoretical basis for evaluating the

charge separation extent from the amplitude of photovoltage was developed. Effects

of solvent and surrounding gas on the signal were investigated.

Photoinduced charge transfer in DNA was investigated via fluorescence

quenching of 2-aminopurine (2AP) by distant guanine (G) as compared with similar

by inosine (I), instead of G, containing oligonucleotide. The studied DNA

oligonucleotides were a series of 31-mer hairpins with 2AP separated from guanine

by different number of adenines An (n = 0-3). The fluorescence quenching yield of

2AP by G was found to fall off exponentially in accordance with distance: kCT ~ exp(-

βR). The efficiency of charge transfer was shown to be sensitive to base stacking in a

complex way: both static structural perturbations of base stacking and their dynamics

contribute to the overall effect. From one side, thermal fluctuations in DNA

backbone provide necessary structural heterogeneity to reach optimal conformations

for charge transfer. As a result, the apparent β decreased from 0.41 Å-1 at 5oC to 0.25

Å-1 upon temperature increase towards the hairpin melting temperature but rose up

dramatically above denaturing DNA. From another side, modification of intervening

adenosine between 2AP and G with the so-called locked DNA (LNA) sugar interrupts

base stacking in an asymmetric manner: the overall quenching yield of 2AP*

fluorescence drops primarily due to a weaker coupling with the opposite adenine but

allowing greater charge transfer yield to G.

ix

Neutralization of DNA by substituting counterions with cationic amphiphiles

results in the formation of DNA-amphiphile complex, which is soluble in organic

solvents. Amphiphiles, with hydrophilic head group and hydrophobic tail, induce

hydrophobic environment around the DNA helix and reduce the amount of water

within DNA duplex. This leads to a lower thermal stability of the DNA duplex in

organic solution. Among the studied amphiphiles only those with polyether moieties

next to the cationic head demonstrated formation of hybridized DNA duplex at room

temperature.

Combination of these results provides a solid basis for successful application

of SPTDC technique for studying charge transfer in DNA.

x

LIST OF TABLES

2.1 Wavelengths of absorption maxima of coumarin-A and coumarin-B in solutions ............................................................................................ 30

2.2 Solvent dependence of the optical absorption parameters of coumarin

immobilized on silica for two different linkers..................................... 31 2.3 Surface density of dyes on silica and their dependence on the order of

immobilization sequence....................................................................... 50 3.1 Characteristics of the ground and the first excited state of coumarin

molecules calculated with semiempirical method AM1 ....................... 79 3.2 Fluorescence properties of dyes in toluene solution under different

excitation densities ................................................................................ 85 3.3 Experimental values fitting parameters for SPTDC dipole signal of

coumarin in different environment........................................................ 93 4.1 Melting temperatures, Tm, and fluorescence quantum yields, Φ,

of 2-aminopurine in different hairpins at temperatures below and above Tm......................................................................................... 130

4.2 Comparison of several characteristics for four DNA and LNA

modified oligonucleotides..................................................................... 145 4.3 The computed energies of interaction between 2AP and neighboring

adenine bases......................................................................................... 155 5.1 Fluorescence quantum yields, Φ, of 2-aminopurine in 0sG and 0sI

hairpins at 25°C and melting temperatures, Tm..................................... 178 A.1 Conformational analysis of the tail of long aminosilane, AENPS ....... 203

A.2 Conformational analysis of the tail of short aminosilane, APS............ 207

D.1 Atomic charges of 2-aminopurine ........................................................ 219

D.2 Atomic charges of modified locked nucleotide .................................... 220

xi

LIST OF FIGURES

1.1 Energy diagram for the reactants (D/A) and products (D+/A-) as a function of nuclear configuration........................................................ 4

2.1 Schematic representation of a silane reacting with hydroxylated

surface ................................................................................................... 15 2.2 AFM images of silica substrate A) fresh and B) after multiple

recycling................................................................................................ 20 2.3 Structures of silanes, coumarin-A, coumarin-B, DNP and their

abbreviations ......................................................................................... 21 2.4 Illustration of the background subtraction procedure in absorption

spectra ................................................................................................... 25 2.5 The two-step procedure for coumarin self assembly ............................ 27

2.6 Absorption spectra in solution of coumarin-A and coumarin-B........... 30

2.7 Absorption spectra of coumarin stained slides in toluene and in ethanol at different light polarizations with respect to the surface ....... 32

2.8 Ball–and-stick representation of coumarin-B molecule and its

transition dipole moment orientation .................................................... 34 2.9 Absorption spectra of slides with different surface coumarin

concentrations corresponding to different ratio of octylsilane/aminosilane ([C8]/[AM] in the silanization step................. 38

2.10 Variation of the maximum absorption wavelength, λmax, with the

solvent polarity factor, (ε –1)/(2ε+1), for two light polarization........... 42 2.11 Variation of the absorbance for immobilized coumarin as a function

of time of treatment by aminosilane solution........................................ 43 2.12 Schematic representation of the double silanization with intermediate

water treatment...................................................................................... 44 2.13 Absorption spectra of slides in air at different light polarizations with

respect to the surface............................................................................. 46

xii

2.14 Schematic representation of the construction of alternating monolayer via double silanization with intermediate water treatment................................................................................................ 49

2.15 Absorption spectra of DNP and coumarin molecules immobilized

on a silica slide in the double silanization reaction............................... 50 3.1 Schematic representation of photoinduced transient displacement

current setup (PTDC) ............................................................................ 61 3.2 Illustration of a dipole moment in a spherical cavity immersed in

dielectric (ε) .......................................................................................... 64 3.3 Illustration of experimentally relevant models of dipoles in

self-assembled monolayer..................................................................... 66 3.4 Structures of coumarin molecules......................................................... 69

3.5 Schematic representation of photoinduced displacement current (charge) experimental setup .................................................................. 70

3.6 Schematic representation of the surface assisted photoinduced

transient displacement current setup ..................................................... 72 3.7 Schematic sketch of the sample cell designed for the study of charge

transfer in molecules assembled in monolayers.................................... 74 3.8 Transient displacement current (“dipole”) signals of coumarin-A and

coumarin-B in toluene solution............................................................. 77 3.9 SPTDC dipole signals (normalized for the same incident laser

energy) for coumarin immobilized on one side of different types of substrates in ethanol .............................................................................. 81

3.10 Dipole signals of coumarin immobilized on one side of glass

substrate in ethanol, glycerol and squalane........................................... 83 3.11 Normalized fluorescence spectra of two quartz slides with

self-assembled coumarin-B monolayers measured parallel to the slides............................................................................................ 84

3.12 Time-resolved fluorescence kinetics for the same sample as in

Figure 3.9 recorded at different geometries .......................................... 86

xiii

3.13 Normalized fluorescence spectra of coumarin-B in toluene solution... 87

3.14 Normalized fluorescence spectra of coumarin 153 in toluene solution 88 3.15 Dependence of the photovoltage signal on orientation of SAM inside

the cell ................................................................................................... 90 3.16 The photovoltage signal for 6 silica substrates with coumarin SAM

immersed in toluene .............................................................................. 91 3.17 Normalized dipole signals covalently bound coumarin-B on silica

substrates in toluene, ethanol and hexane as well as in the atmosphere of different gases (argon, nitrogen and helium) and under vacuum........................................................................................ 94

3.18 Schematic representation of orientation of coumarin molecules in

covalently linked layer of coumarin-B.................................................. 97 3.19 Dependence of average dipole moment change on collision rate for

different gases ....................................................................................... 98 3.20 Photovoltage signals from coumarin-B in air (0.21 atm of oxygen)

and in flow of pure oxygen (1 atm) after excitation by 416 nm from Raman shifted laser pulse...................................................................... 100

3.21 Photovoltage signals from coumarin-460 in air (0.21 atm of oxygen)

and in pure oxygen (1 atm) after excitation with nithrogen laser at 337 nm............................................................................................... 102

4.1 Schematic representation of the hairpins .............................................. 125

4.2 Base pairing in studied DNA of regular and modified bases ............... 131

4.3 Temperature variation of the absorption intensity at 330 nm for three hairpins.................................................................................................. 132

4.4 Fluorescence intensity variation with temperature for four hairpin

pairs with λFluor = 370 nm...................................................................... 135 4.5 Dependence of fluorescence quenching efficiency on distance ........... 136

4.6 Distance dependence of the charge transfer yield at room temperature............................................................................................ 137

xiv

4.7 Temperature dependence of the charge transfer rate constant.............. 139

4.8 Temperature dependence of the charge transfer decay constant, β ...... 142

4.9 The change of absorption at 330 nm with temperature for DNA_1sG and LNA_1sG ....................................................................................... 148

4.10 Fluorescence excitation spectra ............................................................ 149

4.11 Temperature dependence of cumulative molar circular-dichroic absorptivity of the first peak (275 nm) and the trough (247 nm).......... 150

4.12 Temperature dependence of the 2AP fluorescence yield, Φfl, for the

four oligonucleotides studied ................................................................ 152 4.13 Temperature dependence of the relative fluorescence yield of 2AP,

γ=ΦLNA/ΦDNA, in LNA and DNA oligonucleotides .............................. 154 5.1 Structures of crosslinking agents used in this study ............................. 165

5.2 Structures of cationic amphiphiles used in this study........................... 166

5.3 The three-step procedure for DNA immobilization.............................. 168

5.4 Schematic illustration of DNA-amphiphile1 complex formation......... 171

5.5 1H NMR spectra of amphiphile3 in CDCl3........................................... 173

5.6 AFM image of DNA-amphiphile1 complex species deposited onto modified with octylsilanes mica surface from c hloroform/ethanol/water (1:0.25:0.1) solution...................................... 175

5.7 Fluorescence “melting curves” of 0sG(I) ............................................. 177 5.8 2AP absorption spectra at room temperature........................................ 179 5.9 The change of absorption at 260 nm with temperature for

DNA-amphiphile1 complex .................................................................. 181 5.10 Effect of water content on CD spectra of DNA-amphiphile1 complex

in chloroform/ethanol (1/0.25) solution at 25°C................................... 183 5.11 The temperature dependence of ACott value for DNA- amphiphile1

complex in “dry” solution and after addition of 7%v.v. water ............. 184

xv

5.12 Absorption spectra of DNA-amphiphile1 complex immobilized on

silica surface at vertical polarization and horizontal polarization ........ 187 5.13 Photoresponse from DNA (2sG)-amphiphile1 complex in chloroform

and calculated traces for three charge recombination times ................. 191 5.14 Three solid lines represent expected photoinduced transient

displacement charge signal profiles for 2sG oligonucleotide for various recombination times ................................................................. 193

C.1 Absorption spectra of the sample cell, filled with toluene, with and

without 6 substrates of silica with immobilized coumarin ................... 216

xi

LIST OF FIGURES

1.1 Energy diagram for the reactants (D/A) and products (D+/A-) as a function of nuclear configuration........................................................ 4

2.1 Schematic representation of a silane reacting with hydroxylated

surface ................................................................................................... 15 2.2 AFM images of silica substrate A) fresh and B) after multiple

recycling................................................................................................ 20 2.3 Structures of silanes, coumarin-A, coumarin-B, DNP and their

abbreviations ......................................................................................... 21 2.4 Illustration of the background subtraction procedure in absorption

spectra ................................................................................................... 25 2.5 The two-step procedure for coumarin self assembly ............................ 27

2.6 Absorption spectra in solution of coumarin-A and coumarin-B........... 30

2.7 Absorption spectra of coumarin stained slides in toluene and in ethanol at different light polarizations with respect to the surface ....... 32

2.8 Ball–and-stick representation of coumarin-B molecule and its

transition dipole moment orientation .................................................... 34 2.9 Absorption spectra of slides with different surface coumarin

concentrations corresponding to different ratio of octylsilane/aminosilane ([C8]/[AM] in the silanization step................. 38

2.10 Variation of the maximum absorption wavelength, λmax, with the

solvent polarity factor, (ε –1)/(2ε+1), for two light polarization........... 42 2.11 Variation of the absorbance for immobilized coumarin as a function

of time of treatment by aminosilane solution........................................ 43 2.12 Schematic representation of the double silanization with intermediate

water treatment...................................................................................... 44 2.13 Absorption spectra of slides in air at different light polarizations with

respect to the surface............................................................................. 46

xii

2.14 Schematic representation of the construction of alternating monolayer via double silanization with intermediate water treatment................................................................................................ 49

2.15 Absorption spectra of DNP and coumarin molecules immobilized

on a silica slide in the double silanization reaction............................... 50 3.1 Schematic representation of photoinduced transient displacement

current setup (PTDC) ............................................................................ 61 3.2 Illustration of a dipole moment in a spherical cavity immersed in

dielectric (ε) .......................................................................................... 64 3.3 Illustration of experimentally relevant models of dipoles in

self-assembled monolayer..................................................................... 66 3.4 Structures of coumarin molecules......................................................... 69

3.5 Schematic representation of photoinduced displacement current (charge) experimental setup .................................................................. 70

3.6 Schematic representation of the surface assisted photoinduced

transient displacement current setup ..................................................... 72 3.7 Schematic sketch of the sample cell designed for the study of charge

transfer in molecules assembled in monolayers.................................... 74 3.8 Transient displacement current (“dipole”) signals of coumarin-A and

coumarin-B in toluene solution............................................................. 77 3.9 SPTDC dipole signals (normalized for the same incident laser

energy) for coumarin immobilized on one side of different types of substrates in ethanol .............................................................................. 81

3.10 Dipole signals of coumarin immobilized on one side of glass

substrate in ethanol, glycerol and squalane........................................... 83 3.11 Normalized fluorescence spectra of two quartz slides with

self-assembled coumarin-B monolayers measured parallel to the slides............................................................................................ 84

3.12 Time-resolved fluorescence kinetics for the same sample as in

Figure 3.9 recorded at different geometries .......................................... 86

xiii

3.13 Normalized fluorescence spectra of coumarin-B in toluene solution... 87

3.14 Normalized fluorescence spectra of coumarin 153 in toluene solution 88 3.15 Dependence of the photovoltage signal on orientation of SAM inside

the cell ................................................................................................... 90 3.16 The photovoltage signal for 6 silica substrates with coumarin SAM

immersed in toluene .............................................................................. 91 3.17 Normalized dipole signals covalently bound coumarin-B on silica

substrates in toluene, ethanol and hexane as well as in the atmosphere of different gases (argon, nitrogen and helium) and under vacuum........................................................................................ 94

3.18 Schematic representation of orientation of coumarin molecules in

covalently linked layer of coumarin-B.................................................. 97 3.19 Dependence of average dipole moment change on collision rate for

different gases ....................................................................................... 98 3.20 Photovoltage signals from coumarin-B in air (0.21 atm of oxygen)

and in flow of pure oxygen (1 atm) after excitation by 416 nm from Raman shifted laser pulse...................................................................... 100

3.21 Photovoltage signals from coumarin-460 in air (0.21 atm of oxygen)

and in pure oxygen (1 atm) after excitation with nithrogen laser at 337 nm............................................................................................... 102

4.1 Schematic representation of the hairpins .............................................. 125

4.2 Base pairing in studied DNA of regular and modified bases ............... 131

4.3 Temperature variation of the absorption intensity at 330 nm for three hairpins.................................................................................................. 132

4.4 Fluorescence intensity variation with temperature for four hairpin

pairs with λFluor = 370 nm...................................................................... 135 4.5 Dependence of fluorescence quenching efficiency on distance ........... 136

4.6 Distance dependence of the charge transfer yield at room temperature............................................................................................ 137

xiv

4.7 Temperature dependence of the charge transfer rate constant.............. 139

4.8 Temperature dependence of the charge transfer decay constant, β ...... 142

4.9 The change of absorption at 330 nm with temperature for DNA_1sG and LNA_1sG ....................................................................................... 148

4.10 Fluorescence excitation spectra ............................................................ 149

4.11 Temperature dependence of cumulative molar circular-dichroic absorptivity of the first peak (275 nm) and the trough (247 nm).......... 150

4.12 Temperature dependence of the 2AP fluorescence yield, Φfl, for the

four oligonucleotides studied ................................................................ 152 4.13 Temperature dependence of the relative fluorescence yield of 2AP,

γ=ΦLNA/ΦDNA, in LNA and DNA oligonucleotides .............................. 154 5.1 Structures of crosslinking agents used in this study ............................. 165

5.2 Structures of cationic amphiphiles used in this study........................... 166

5.3 The three-step procedure for DNA immobilization.............................. 168

5.4 Schematic illustration of DNA-amphiphile1 complex formation......... 171

5.5 1H NMR spectra of amphiphile3 in CDCl3........................................... 173

5.6 AFM image of DNA-amphiphile1 complex species deposited onto modified with octylsilanes mica surface from c hloroform/ethanol/water (1:0.25:0.1) solution...................................... 175

5.7 Fluorescence “melting curves” of 0sG(I) ............................................. 177 5.8 2AP absorption spectra at room temperature........................................ 179 5.9 The change of absorption at 260 nm with temperature for

DNA-amphiphile1 complex .................................................................. 181 5.10 Effect of water content on CD spectra of DNA-amphiphile1 complex

in chloroform/ethanol (1/0.25) solution at 25°C................................... 183 5.11 The temperature dependence of ACott value for DNA- amphiphile1

complex in “dry” solution and after addition of 7%v.v. water ............. 184

xv

5.12 Absorption spectra of DNA-amphiphile1 complex immobilized on

silica surface at vertical polarization and horizontal polarization ........ 187 5.13 Photoresponse from DNA (2sG)-amphiphile1 complex in chloroform

and calculated traces for three charge recombination times ................. 191 5.14 Three solid lines represent expected photoinduced transient

displacement charge signal profiles for 2sG oligonucleotide for various recombination times ................................................................. 193

C.1 Absorption spectra of the sample cell, filled with toluene, with and

without 6 substrates of silica with immobilized coumarin ................... 216

xvi

TABLE OF CONTENTS

Page

LIST OF TABLES............................................................................................ x

LIST OF FIGURES .......................................................................................... xi

1 INTRODUCTION ................................................................................ 1

1.1 Basic electron transfer theory ............................................................... 3

1.2 Approaches to study DNA mediated photoinduced charge transfer..... 6

1.3 The dissertation overview..................................................................... 9

1.4 References............................................................................................. 12

2 SELF-ASSEMBLED MONOLAYERS ON SILICA SURFACES AND THEIR PROPERTIES................................................................. 14

2.1 Introduction........................................................................................... 14

2.2 Experimental Section............................................................................ 17

2.2.1 Materials ............................................................................................... 17

2.2.2 Procedure .............................................................................................. 19

2.2.2.1 Preparation of substrates....................................................................... 19

2.2.2.2 Silanization ........................................................................................... 19

2.2.2.3 Immobilization of a dye ........................................................................ 22

2.2.2.4 Measurements of absorption spectra..................................................... 23

2.2.2.5 Orientation analysis .............................................................................. 24

2.3 Results and Discussion ......................................................................... 26

2.3.1 Effect of solvent.................................................................................... 29

2.3.2 Effect of a linker ................................................................................... 34

xvii

2.3.3 Effect of concentration.......................................................................... 36

2.3.4 Effect of water on silanization .............................................................. 40

2.3.5 Lateral alternation of molecules in a monolayer .................................. 47

2.4 Conclusions........................................................................................... 51

2.5 References............................................................................................. 52

3 CHARGE SEPARATION IN SELF-ASSEMBLED MOLECULAR FILMS................................................................................................... 55

3.1 Introduction........................................................................................... 55

3.2 Theory of SPTDC ................................................................................. 62


3.3.1 Materials ............................................................................................... 68

3.3.2 Experimental setup ............................................................................... 69

3.3.3 Dipole measurements in solution, PTDC.............................................. 71

3.3.4 Dipole measurements on surface, SPTDC............................................ 72

3.3.5 Fluorescence measurements.................................................................. 75


3.4.1 Charge transfer of coumarins in toluene solution................................. 76

3.4.2 Semiempirical calculations ................................................................... 78

3.4.3 Photoinduced charge transfer in self-assembled coumarin monolayer.............................................................................................. 80

3.4.4 Charge transfer in coumarin monolayer ............................................... 89

3.4.5 Photoinduced charge transfer in coumarin in the presence of oxygen ................................................................................................... 98

3.5 Conclusions........................................................................................... 104

xviii

3.6 References............................................................................................. 105

4 CHARGE TRANSFER IN DNA: STUDY OF 2AP QUENCHING IN SHORT OLIGONUCLEOTIDES......................................................... 107

4.1 Introduction........................................................................................... 107

4.1.1 Models of charge transfer in DNA ....................................................... 108

4.1.2 2-aminopurine in studies of photoinduced charge transfer in DNA..... 110

4.1.3 Base fluctuations and its influence on charge transfer in DNA............ 112

4.1.4 Structural perturbations and efficiency of charge transfer in DNA...... 115

4.1.5 Polymorphism of DNA and charge transfer ......................................... 118

4.1.6 Theoretical studies of the base stacking effect on charge transfer in DNA ...................................................................................................... 121

4.2 System Design ...................................................................................... 123


4.3.1 Materials ............................................................................................... 125

4.3.2 Absorption measurements..................................................................... 126


4.3.4 Circular dichroism measurements......................................................... 127

4.3.5 Molecular modeling.............................................................................. 127


4.4.1 Data analysis ......................................................................................... 128

4.4.2 Thermal stability of studied oligonucleotides....................................... 129

4.4.3 Absorption of 2-aminopurine................................................................ 131

4.4.4 Fluorescence quenching of 2-aminopurine in DNA oligonucleotides.. 133

xix

4.4.5 Effect of LNA modification.................................................................. 144

4.5 Conclusions........................................................................................... 155

4.6 References............................................................................................. 157

5 TOWARDS PTDC STUDY OF DNA: DNA-AMPHIPHILE COMPLEXES AND THEIR IMMOBILIZATION ON SURFACES.. 162

5.1 Introduction........................................................................................... 162


5.2.1 Materials ............................................................................................... 164

5.2.2 Preparation of DNA-amphiphile complexes......................................... 165

5.2.3 Immobilization of DNA-amphiphile complex on silica surface........... 166

5.2.4 Measurements of absorption spectra..................................................... 167


5.2.6 Circular dichroism measurements......................................................... 168

5.2.7 Proton NMR of DNA-amphiphile complexes ...................................... 169

5.2.8 AFM of DNA-amphiphile complexes on mica..................................... 169

5.2.9 FTIR measurements .............................................................................. 170

5.2.10 PTDC measurements ............................................................................ 170


5.3.1 DNA-amphiphile complexes ................................................................ 170

5.3.2 Immobilization of DNA-amphiphile complex on silica surface........... 186

5.3.3 Application of PTDC for studying charge transfer in DNA-amphiphile complex .................................................................... 188

5.4 Conclusions........................................................................................... 194

xx

5.5 References............................................................................................. 195

6 SUMMARY AND PERSPECTIVES ................................................... 197

APPENDICES

A THE ENERGIES AND ANGLES OF CONFORMATIONS OF THE COUMARIN LINKERS .............................................................. 202

B EVALUATION OF THE STRAY CAPACITANCE FOR THE

DESIGNED CELL................................................................................ 210 C DETERMINATION OF THE NUMBER OF EXCITED

MOLECULES....................................................................................... 213 D ATOMIC CHARGES OF MODIFIED LOCKED NUCLEOTIDE

AND 2-AMINOPURINE...................................................................... 218

1

1 INTRODUCTION

Electron transfer reactions are widespread in nature and play crucial role in

maintaining life on the Earth. Examples of such electron transfer processes include

photosynthesis in plants and bacteria or oxidative phosphorylation occurring in

membrane of mitochondria.

Since the discovery of the double helical structure of DNA by Watson and

Crick, scientists have been wondering whether the DNA duplex is capable of charge

transport (conductivity). The polymeric nature of DNA, consisting of a negatively

charged sugar-phosphate backbone outside the duplex and aromatic nucleobases

stacked on top of each other inside the duplex, leads to the appearance of a well-

organized DNA structure. DNA forms a continuous π-stacked combination of four

bases, adenine (A), guanine (G), that belong to purine family, and pyrimidines,

cytosine (C) and thymine (T). The secondary structure of DNA is a double helix,

which is stabilized by hydrogen bonding between the complementary bases, A-T and

G-C, and base stacking interactions. This stacking and overlapping of bases strongly

resembles aromatic crystals and similarly may provide an effective path for electron

transfer. This strong resemblance and the fact that aromatic crystals can be quite

conductive instigate the propositions that DNA is capable of conducting charges.

In the early 90s Professor J. Barton announced stunning results about using

DNA as a “conductive” bridge between an organometallic donor and acceptor

complexes positioned far from each other. She claimed that the photoinduced

2

electron transfer through DNA could occur over distances far beyond the tens of

angstroms (as much as 40 Å). This provocative claim launched a number of research

projects over the last decade, which made the charge transfer in DNA molecule a

subject of intensive investigation. A certain degree of excitement was introduced by

a potential use of DNA in nanotechnology, if it could act as a molecular wire. DNA

with its highly specific recognition between complementary nucleotides and the

ability to self-assemble may have a new technological potential in constructing

complex nano-wire networks.

Another valuable outcome from the study of charge transfer in DNA could

come through understanding the mechanism of oxidative DNA damage. The DNA

molecule, as a carrier of genetic information, is a very important part of the genetic

apparatus and, therefore, it is crucial to maintain its structural integrity. DNA

exposure to various damaging factors may result in its damage. Oxidative damage of

DNA involves a migration of charge to trap sites, primarily guanine and guanine

dimers, which possess the lowest oxidation potentials among the natural nucleobases.

Fortunately, enzymes efficiently repair the damage shortly after it occurs. However,

if the enzymatic repair fails for some reasons, the damage of DNA can result in

cancer. Studying charge transfer in DNA should provide a deeper insight into

understanding of how DNA gets damaged and the mechanisms of its repair.

3

1.1 Basic electron transfer theory

Semiclassical theory of the so-called non-adiabatic electron transfer,

developed by Marcus,1 is often applied for analyses of the charge transfer rate

constants. The model presumes that the overlap between relevant electronic orbitals

of the donor (D) and acceptor (A) is small. The initial and final states of the system

can be represented as two harmonic free energy curves, corresponding to two states–

before (D+A) and after (D++A-) the electron transfer, as shown in Figure 1.1. The

expression for the rate constant (Equation 1.1) can be separated into two terms: one

that describes electronic interaction between donor and acceptor of electron (kel) and

another one that contains the dependence on nuclear reorganization and free-energy

effects (kn). The rate constant of charge transfer for such system is given by the

following equation:2

nelCT kkvk = (1.1)

where ν is the frequency of nuclear motion along the reaction coordinate.

( )

+∆−=

TkGk

Bn λ

λ4

exp20

(1.2)

here ∆G0 is the free-energy of the reaction and λ is the reorganization energy, as

shown in Figure 1.1.

When the probability of electron transfer at the activated complex is high

(kel~1) the case is referred to as adiabatic regime, in other words, all reactants that

become products cross over the top of the barrier. In nonadiabatic regime for which

4

the probability of electron transfer is low (kel<<1), not all of the reactants that reach

the top of the barrier become products, many relax without reacting.

Figure 1.1 Energy diagram for the reactants (D/A) and products (D+/A-) as a function of nuclear configuration.

At the nonadiabatic limit, product ν kel is given by Equation 1.3:2

TkHkv

BDAel πλ

π4

12 2

h= (1.3)

5

Combination of (1.2) and (1.3) with (1.1) provides the overall expression for

electron transfer rate constant:

( )

+∆−=

TkG

TkHk

BBDACT λ

λπλ

π4

exp4

12 202

h (1.4)

The magnitude of the electronic coupling matrix element, HDA, depends on the

overlap of donor and acceptor wave functions and determines whether the reaction is

at adiabatic or nonadiabatic regime. HDA is strongly dependent on distance between

donor and acceptor and at longer separations falls off exponentially:

( )rHrH DADA β−= exp)( 202 (1.5)

The distance dependence of electron transfer rate originates from the distance

dependence of HDA. The electronic matrix element can not be measured directly but

is usually recovered from the charge transfer rate constant. For weak coupling, the

distance dependence of the charge transfer rate constant is often “fitted” with the

following equation:

)exp(0 rkkCT β−= (1.6)

where the beta decay parameter reflects the distance dependence of charge transfer.

This procedure provides a concise way of comparing charge transfer rates for

different D-A bridges and intervening media. To date, experimentally measured beta

values in DNA were reported to vary in a range between 0.1 and 1.4 Ǻ-1. Low beta

values reported by Barton3 were surprising because they appeared to be substantially

lower than those reported for proteins, which typically range within 0.9-1.2 Ǻ-1.4

6

However, recent studies suggest a more shallow distance dependence of electron

transfer in DNA. Although beta varies for different systems, its values tend to

converge within the range of 0.4-0.7Ǻ-1.5,6,7

1.2 Approaches to study DNA mediated photoinduced charge transfer

The first evidence of charge transfer facilitation by DNA was provided in

photochemical experiments on quenching of the excited state of Ru(phen)32+ by

Co(II) and Rh(III) complexes intercalated in DNA.8 Since then, many D/A systems

were developed for characterization of charge transfer in DNA. Originally, mostly

metallointercalators were used because of their ability to intercalate in the DNA

duplex, long excited state lifetimes and rich redox properties. To allow control over

the location of donors and acceptors along the duplex, DNA assemblies were

synthesized with metal complexes covalently tethered at the opposite ends of

oligonucleotides.9 In such systems, electron transfer from the intercalated Ru(II)-

donor to Rh(III)-acceptor was found to be extremely fast and efficient over long

distances, with kET ≥ 109 s-1 for 26 Å.9 The ability of donor and acceptor to

intercalate in DNA was shown to be critical for effective charge transfer in DNA.

Employment of nucleobases themselves and their analogs was a natural

important step in studying electron transfer in DNA. Almost all systems recently

employed for studying photoinduced charge transfer in DNA utilized guanine or its

analogs 8-oxoguanine, and 7-deazaguanine as hole traps and other molecules serving

7

as the oxidizing agents. Charge transfer events can be monitored by either

biochemical or photophysical measurements. In the biochemical method, the guanine

radical cation generated in the charge transfer event is detected by its reaction with

water, which leads to selective strand cleavage after treatment with piperidine.10

Polyacrylamide gel electrophoresis (PAGE) on DNA oligomers that contain a 32P

radiolabel detects those damaged sites.

Schuster and coworkers studied charge transfer in DNA assemblies equipped

with anthraquinone derivatives covalently attached to the duplex.11 Photoirradiated

anthraquinone injects a radical cation into DNA, which can migrate over a long

distance before it gets trapped at guanine sites. Giese and his group investigated

oxidative cleavage of guanine sites in a DNA duplex followed by hole injection from

glycosyl radical.12 Saito and coworkers have used as electron acceptor uridine

modified with p-cyanobenzophenone.13 Photoexcited p-cyanobenzophenone triggers

hole injection into a DNA duplex accepting electron and becoming dCNBPU anion

radical.

The described above methods utilize nucleobases, modified or natural, as

electron donors and acceptors and allow their positioning at predetermined sites

without perturbing the π-stacking of the B-form of DNA. The biochemical

measurements, which usually visualize the strand breaks selectively occurring at

guanine by polyacrylamide gel electrophoresis, allow measurements of the yields of

guanine damage at various sites. The strand cleavage studies are not direct and do not

8

detect the charge transfer intermediates. They also do not provide the absolute rate

constants for charge transfer processes.

Photophysical methods, introduced into studying CT in DNA, brought about

powerful tools advantageous for fast processes and charge transport phenomena in

particular. Both steady-state and time-resolved absorption and emission have been

employed.

Lewis and coworkers have extensively examined photoinduced charge

separation between guanine and photoexcited stilbene derivatives in a series of

synthetic DNA hairpins.14 In their structures, DNA hairpins were capped with

stilbene derivatives, which linked two complementary strands of the hairpin.

The stilbene (Sa) chromophore was shown to be well stacked with the adjacent base

pair in the double helix.15 Fluorescence of Sa* was quenched in DNA due to

electron-transfer from guanine to stilbene and formation of Sa-·-G+· radical ion pair.

Both, the excited singlet state, Sa*, and the anion radical of stilbene, Sa-·, have strong

absorptions, which makes it possible to follow kinetics for these species by their

transient absorption. This method enabled obtaining the rate constants of charge

separation and recombination by measuring lifetimes of Sa* and Sa-·, respectively.

The rate constants were shown to decrease with increasing distance between the

stilbene and guanine in DNA duplexes. The value of β was found to be dependent on

the donor-bridge-acceptor energetics and ranged from 0.4 to 1.1Ǻ-1.14

Direct base-to-base electron transfer was first employed by Barton.3

Fluorescence quenching of adenine analog, 2-aminopurine, by guanine and 7-

9

deazaguanine were examined by using steady state fluorescence and ultrafast

spectroscopy. 3,16

DNA assemblies, where both donor and acceptor are nucleobases or their

analogs, are more suitable for understanding the nature of photoinduced charge

transfer in DNA. The advantages include:

- minimal structural perturbations due to incorporation of probes in DNA,

- ability to design assemblies with well defined positions of donor and

acceptor using standard methods.

To date, many research groups use the 2-aminopurine fluorescent analog of

adenine nucleobase for studying charge transfer in DNA.

1.3 The dissertation overview

Among all the methods used so far in studying charge transfer in DNA, none

addresses charge separation directly. For example, studies of charge separation by

strand cleavage only allow measuring the yields of damage caused by charge transfer

to guanine sites. The most thorough study on charge separation in DNA was done by

Lewis and coworkers and employed the transient absorption method. Advantages of

time-resolved transient absorption include its ability to identify spectrally the charge

transfer intermediates and high time resolution. Unfortunately, it has some

limitations. The low extinction coefficients of nucleobases and their analogs prevent

study of charge transfer without introducing an external probe. Only molecules with

10

high extinction coefficients of its intermediate species, such as stilbene, can be used

by transient absorption. Even though the kinetics of Sa* and Sa- decay were

measured, information on the extent of the hole movement was not available directly.

We have been developing a new method based on photoinduced transient

displacement current, which could allow measuring the extent of charge separation in

DNA as well as the lifetimes of charge separated species. The PTDC technique is a

direct method for obtaining the charge separation distance in photoexcited molecules.

This technique has been successfully used in a variety of systems to study both

intramolecular and intermolecular photoinduced charge transfer.17,18,19 Less

ambiguous data interpretation in PTDC, as compared to other methods, makes this

technique very useful.20 Although the time resolution of the technique (ca. 0.5 ns)

does not allow measuring charge separation rates greater than 2×109 s-1, it is sufficient

for studying most charge transfer species and their recombination rates.

In this work, we attempt to utilize the PTDC technique for studying

photoinduced charge transfer in DNA modified with 2-aminopurine. Several issues

had to be resolved first because of the intrinsic restrictions in this technique, and thus,

three chapters are dedicated to answering these issues.

The major development of the PTDC technique necessary for studying charge

transfer in DNA involves elimination of the external electric field. In the standard

geometry, the applied electric field is used to orient dipolar molecules. DNA, due to

its polyanionic nature, normally is neutralized by small counterions, which make the

solution highly conductive. First, alternative means of orienting molecules involving

11

their covalent immobilization on flat surfaces should be developed. Second,

“neutralization” of DNA by substituting small counterions with cationic amphiphiles

and making DNA soluble in organic solvents, should be investigated and issues of

DNA duplex stability should be resolved. Although PTDC is not limited to nonpolar

solvents only, very polar solutions with a substantial dark current cannot be studied

with a high load resistance.

The dissertation consists of four individual chapters that address these

different issues. Therefore, they have own introductions describing the relevant

background information as well as questions to be addressed.

Chapter 2 describes the optimization of the silane-based self-assembly method

for covalent immobilization of molecules onto oxide surfaces. The resulting self-

assembled monolayers (SAM) produce unidirectional orientation of molecules

induced by the surface, without a need of external electric field. The Chapter

discusses the properties of coumarin SAM, which was used as a model system.

Questions related to surface density, orientation of molecules in self-assembled

monolayer, and intermolecular interactions, are addressed.

Chapter 3 details the evaluation of the modified PTDC technique for charge

transfer study in self-assembled monolayers on the example of coumarin molecules

and its comparison with the standard version. The modification of photoinduced

transient displacement charge technique (PTDC) is shown to allow direct

measurements of photoinduced charge transfer in oriented by the surface molecules at

solid-liquid as well as at solid-gas interfaces.

12

Chapter 4 discusses study of charge transfer in DNA short oligonucleotides by

means of 2-aminopurine fluorescence quenching. Owing to the structural flexibility

of DNA, the efficiency of charge transfer in DNA is susceptible to factors altering

base – base interactions. The results described in the Chapter emphasize the

sensitivity of charge transfer to dynamical and structural properties of DNA.

Chapter 5 is dedicated to the attempt of studying charge transfer in DNA by

means of the PTDC technique. The replacement of counterions in DNA by cationic

amphiphiles results in the formation of DNA-amphiphile complex, which is soluble in

organic solvents. Structure and thermal stability of DNA-amphiphile complexes in

organic solvents is described. Immobilization protocol for both DNA and DNA-

amphiphile complexes onto silica surface is analyzed. Attempts to combine all

developments into measurements of photoinduced charge transfer in DNA using

PTDC are described.

1.4 References 1 Marcus, R., J. Phys. Chem., 1956, 24, 966-979. 2 Sutin, N., Electron Transfer in Inorganic, Organic, and Biological Systems, in Advances in chemistry series, V. 228, Bolton, J.R., Mataga, N., McLendon, G., Ed., Washington DC, 1991, pp. 25-43. 3 Kelley, S.O., Barton, J.K., Science, 1999, 283, 375-381. 4 Langen, R., Colon, J.L., Casimiro, D.R., Karpishin, T.B., Winkler, J.R., Gray, H.B., J. Biol. Inorg. Chem., 1996, 1(3), 221-225. 5 Lewis, F.D., Wu, Y., Zhang, L., Zuo, X., Hayes, R.T., Wasielewski, M.R., J. Am. Chem. Soc., 2004, 126, 8206-8215.

13

6 O'Neill, M.A., Barton, J.K, Top. Curr. Chem., 2004, 236, 67-115. 7 Takada, T., Kawai, K., Cai, X., Sugimoto, A., Fujitsuka, M., and Majima, T., J. Am. Chem. Soc., 2004, 126, 1125-1129. 8 Barton, J.K., Kumar, C. V., Turro, N.J., J. Am. Chem. Soc., 1986, 108, 6391-6393; Purugganan, M.D., Kumar, C. V., Turro, N.J., Barton, J.K., Science, 1988, 241, 1645-1649. 9 Murphy, C. J., Arkin, M. A., Jenkins, Y., Ghatlia, N. D., Bossmann, S. H., Turro, N.J., Barton, J. K., Science, 1993, 262, 1025-1029. 10 Meggers, E., Michel-Beyerle, M.E., Giese, B., J. Am. Chem. Soc., 1998, 120, 12950- 12955. 11 Schuster G., Landman, U., Top. Curr. Chem., 2004, 236, 139–161. 12 Giese, B., Top. Curr. Chem., 2004, 236, 27–44. 13 Nakatani, K., Saito, I., Top. Curr. Chem., 2004, 236, 163–186. 14 Lewis F.D., Liu X., Wu Y., Miller S.E., Wasielewski M.R., Letsinger R.L., Sanishvili R., Joachimiak A., Tereshko V., Egli M., J. Am. Chem. Soc., 1999, 121, 9905-9906. 15 Lewis F.D., Liu X., Wu Y., Miller S.E., Wasielewski M.R., Letsinger R.L., Sanishvili R., Joachimiak A., Tereshko V., Egli M., J. Am. Chem. Soc., 1999, 121, 9905-9906. 16 Wan, C., Fiebig, T., Schiemann, O., Barton, J.K., Zewail, A.H., Proc. Natl. Acad. Sci. USA, 2000, 97, 14052-14055. 17 Smirnov, S.N., Braun C.L., J. Phys. Chem., 1994, 98, 1953-1961. 18 Mylon, S.E., Smirnov, S.N., Braun C.L., J. Phys. Chem., 1998, 102, 6558-6564. 19 Smirnov, S.N., Braun C.L., Anker-Mylon, S.E., Grzeskowiak, K.N., Greenfield, S.L., Wasielewski, M.R., Mol. Cryst. Liq. Cryst., 1996, 286, 243-248. 20 Smirnov, S.N., Braun C.L., Rev. Sci. Instrum., 1998, 69(8), 2875-2887.

14

2 SELF-ASSEMBLED MONOLAYERS ON SILICA SURFACES AND THEIR

PROPERTIES

2.1 Introduction

Thin molecular films on solid substrates, particularly self-assembled

monolayers (SAM), have generated substantial interest in recent years. SAM films

are spontaneously formed when an appropriate substrate is immersed in a solution

containing “active” molecules. Desired chemical functionalities can be introduced at

the terminus of SAM and bring unique physical and chemical properties to a variety

of new SAM applications in microelectronics,1 light emitting diodes (OLED),2

photovoltaics, biosensors,3,4 and molecular catalysis.5

Our interest in SAM construction was motivated by a desire to make

assemblies with a unidirectional molecular orientation. This would give an

opportunity to study charge separation in organized in such a way molecules by

means of photoinduced transient displacement charge technique (PTDC), which will

be described in detail in Chapter 3. Briefly, the PTDC technique allows direct study

of charge separation in oriented molecules. In its original form, PTDC implies the

use of external electric field to slightly orient molecules due to interaction of electric

field with molecular dipole moment. Our goal was primarily to apply the transient

displacement charge technique to investigate charge migration phenomenon along the

base-stack in DNA oligonucleotides-hairpins. However, the DNA molecule carries

negative charges and is neutralized by small inorganic counterions, which all cause a

15

dark conductivity signal under an applied external electric field. Alternative means of

orienting DNA molecules can eliminate the use of external electric field.

Constructing SAM of DNA on a solid substrate should produce a desired

unidirectional orientation of DNA molecules on a surface.

There are many methods developed for surface immobilization of molecules.

The procedures can vary with the substrate used for immobilization. The most

reliable route is covalent bonding. In order to study DNA, the substrate should be

transparent in UV region so that the absorption at 260nm of unmodified DNA can be

monitored. Silica substrates meet this requirement; they also possess plenty of

surface hydroxyl groups, which can be used for chemical modification. The

hydroxylated surfaces such as silica, alumina, glass, etc. are quite reactive towards

chlorosilanes and alkoxysilanes.6 If a hydroxylated surface is contacted with a

solution containing X-silane (X=chloro or alkoxy) the silane reacts with formation of

-Si-O-Si- bond.

Si OH Si

R

H3C-O R

R

+ + CH3OHSi O Si R

R

R

Figure 2.1 Schematic representation of a silane reacting with hydroxylated surface.

Immobilization of silanes onto silica substrates was believed to result in a self-

assembled monolayer with close-packed and aligned molecules.7 Surfaces silanized

16

with aminosilanes (NH2-R-Si-X3), where R is a hydrocarbon chain, can be further

modified by using the reactivity of the amine. Aminated surfaces can be modified

with reagents containing succinimidyl ester (refer to Figure 2.3), gluteraldehyde or

other active to amine group.

In spite of extensive studies conducted over the past two decades, the detailed

mechanism of silanization has been controversial.6,7,8,9,10,11 Different reaction

conditions, including the nature of the silane, its concentration, solvent, duration and

temperature of the reaction, water content, and the temperature of postcuring, were

found to affect the quality of the resultant film.10,11,12,13,14,15 The following questions

should be addressed for a detailed description of self-assembled molecular films:

1) How many surface Si-O-Si bonds are formed?

2) What is the surface density of molecules in the film?

3) What is the average conformation of the silane chains?

Answering these questions should aid in understanding the mechanism of self-

assembled monolayer formation on oxide surfaces, and guide construction of

molecular films optimized for study of charge transfer by optical and displacement

current techniques.

In order to compare the photoinduced transient displacement charge technique

on surfaces (SPTDC) with its standard realization in solution, molecules that can be

studied by both techniques are required. The choice of a coumarin derivative as a

model system for calibration was motivated by unique properties of coumarins: they

have high extinction coefficients and fluorescence yields, and are very sensitive to

17

changes in the molecular environment.16,17 Most importantly, coumarins possess

dipole moments in both the ground and excited states, which is necessary for

calibration of the photoinduced transient displacement charge technique.

Conveniently, coumarin derivatives modified with succinimidyl ester ready for

binding to aminated surfaces, such as D-1412 (7-diethylaminocoumarin-3-carboxylic

acid, succinimidyl ester) are commercially available (see Figure 2.3).

This chapter discusses:

a) optimization of the protocol for covalent immobilization of succinimidyl

ester molecules on flat silica surfaces

b) optical investigation of immobilized molecules orientation on the surface as

a function of molecule surface concentration, solvent and the length of aminosilanes

c) optical study of intermolecular interactions in dense films of coumarin self-

assembled monolayers

d) preparation of mixed monolayers constructed by two-step silanization with

intermediate water treatment.

2.2 Experimental Section

2.2.1 Materials

Polished 25 × 13 mm2 quartz slides (Quartz International) with 0.3 mm

thickness were used as substrates. These quartz slides show a high UV transparency

down to 200 nm and, thus, can be used for optical study of DNA with its absorption

18

in UV region. Topography image acquired with AFM (Molecular Imaging) revealed

that the quartz surface has a quite high quality: the surface roughness measured with a

fresh tip was within ±4Å. Figure 2.2 shows an AFM image of one quartz substrate

taken before and after a number of reuses: surface roughness gets enhanced due to

active chemicals used in the cleaning procedure (see below). Thus, to guarantee

quality of the results, we limited the use of slides to a few recyclings.

Three types of silanes were investigated for surface silanization. Two

aminosilanes: 3-amino-propyltrimethoxysilane (APS) and N-[3-(trimethoxysilyl)

propyl-ethylenediamine (AENPS) were purchased from Aldrich, and octylsilane

(with a saturated octyl hydrocarbon tail) was obtained from Fluka.

7-diethylaminocoumarin-3-carboxilic acid succinimidyl ester (“coumarin-A”),

was obtained from Molecular Probes (commercial name, D-1412). Its carboxamide

analogue (“coumarin-B”, see Figure 2.3) was formed via covalent immobilization

(refer to Figure 2.5) on the surface. To imitate similar molecule for measurements in

solution, coumarin-A was reacted with propylamine (see Figure 2.3 for details).

Another dye, 6-(2, 4-dinitrophenyl) aminohexanoic acid succinimidyl ester (“DNP”)

was also purchased from Molecular probes. The structures of these molecules are

shown in Figure 2.3.

HPLC grade solvents: acetone, acetonitrile, dimethylsulfoxide (DMSO),

ethanol, methanol, hexane, methylene chloride and toluene, from Aldrich, were used

without further purification.

19

Deionized (DI) water (with resistivity 18 MΩ cm) was obtained from

nanopure system (Barnstead).

2.2.2 Procedure

2.2.2.1 Preparation of the substrates

The quartz slides were first immersed in 0.1N NaOH solution for 30 min.

This removes covalently bound organic impurities due to hydrolysis of a Si-O bond in

basic media.7 In the next step, the slides were further cleaned in MeOH/HCl (1/1)

solution for 30 min, which was followed by rinsing in a copious amount of DI water.

The slides were then heated in concentrated H2SO4 for 2 hours and rinsed in DI water.

The final step of cleaning was done immediately prior to silanization – the slides were

boiled in DI water, rinsed in acetone and dried at 100°C.

This cleaning procedure has proven to be effective and reproducible, and did

not show any apparent presence of trace molecules from previous immobilizations

according to absorption measurements.

2.2.2.2 Silanization

The silanization procedure is based on a property of trimethoxysilane to react

spontaneously with surface hydroxyl groups resulting in a formation of self-

assembled monolayer. Cleaned quartz slides were immersed in a 2% v/v acetone

20

Figure 2.2 AFM images of silica substrate A) fresh and B) after multiple recycling. Vertical scale, 10nm.

21

SiO

OO

N H

NHH

CH3CH3

CH3

SiO

OO

N H

CH3CH3

CH3

H

SiO

OO

CH3CH3

CH3

N

O

O O

O O

O

N

Et

Et

NH2 O

O NH

O

N

Et

Et

OO

O2N

NO2

NO

O

AENPS APS C8 DNP

coumarin-A coumarin-B

Figure 2.3 Molecules used in this study and their abbreviations: N-[3-(Trimethoxysilyl)propyl]-ethylenediamine (AENPS), 3-amino-propyltrimethoxysilane (APS), and octyltrimethoxysilane (C8), ), 6-(2, 4-dinitrophenyl)aminohexanoic acid succinimidyl ester (DNP), 7-diethylaminocoumarin-3-carboxylic acid succinimidyl ester (coumarin-A), its carboxamide analogue (coumarin-B).

22

solution of trimethoxyaminosilane (APS or AENPS) or its mixture with

trimethoxyoctylsilane (C8) in an appropriate ratio. The latter was used when the

surface concentration of aminogroup needed to be varied. The duration of

silanization step was usually 3 min. Longer exposures were performed in the study of

the effect of silanization duration on the surface coverage. For a multistep

silanization, intermediate hydrolysis in DI water was performed between silanization

steps. Each silanization was finished by washing with acetone and baking/drying at

100°C in oven for 5 min, followed by cooling in the oven for 5 min.

2.2.2.3 Immobilization of a dye

The staining of the silanized slides by dye molecules (either coumarin-A or

DNP) was performed only on one side of a substrate. For that, a drop of 0.6 mM

DMSO solution of a dye was placed between the slide and the surface of a plastic

Petri dish. Reproducible results for the amount of immobilized dye were achieved

when 2 hours were provided for completing this step. The stained slides were washed

in acetone and dried in an oven at ca. 100°C. No noticeable deterioration of the

surface concentration of a dye molecule within a month after staining was observed

when stored under dark in dry conditions.

23

2.2.2.4 Measurements of absorption spectra

Absorption spectra were measured using Perkin Elmer Lamda 40 UV/VIS

spectrometer equipped with a homemade plastic polarizer. A stained slide was placed

vertically in a 1 cm quartz cuvette at 45° angle with respect to the incident light. The

two linear polarizations of excitation light were applied: vertical or horizontal.

Vertical polarization in the described geometry corresponds to the

measurement of optical absorption with light polarization parallel to the surface (A||).

Absorption polarized perpendicular (A⊥) to the surface substrate was calculated as the

difference between the double horizontal absorption and the vertical absorption

spectra on the same slide.

A|| = Av; A⊥ = 2Ah - Av (2.1)

Clean slides showed some apparent optical density, as a background, due to

wavelength dependence of the refractive index mismatch between the solvent and

quartz as well as due to absorption from minor impurities in quartz. The background

signal was subtracted from each spectrum of a slide. Since there is a slight variation

of the background with a solvent, light polarization, and from one slide to another, the

background absorption was each time simulated (by fitting over the regions to the red,

λ > 470 nm, and to the blue, from either coumarin (λ < 330 nm,) or DNP (λ < 310

nm) absorption as a smooth function of the wavelength, f(λ) = c1/(c2+λ)+c3. Figure

2.4 illustrates that the fitting of the background for the bare (2) and stained (1) slides

24

agree well. The result of background subtraction is given in a form of the processed

spectrum (4).

Surface concentration of a dye, ns, was calculated from the average

absorbance, A, using Lambert-Beer’s law and the extinction coefficients of coumarin

B (ε=47000 M-1cm-1) and DNP (ε=16760 M-1cm-1):

εAcm 202

s 106)(n ×=− (2.2)

where A quantifies the absorption of molecules irrespective of their orientation and is

calculated using parallel and perpendicular polarized absorptions:

3)+

= ⊥A (2A A ||

(2.3)

2.2.2.5 Orientation analysis

The orientation of immobilized molecules cannot be perfectly perpendicular to

the surface for the whole ensemble. The experimental data from optical absorption

were compared with those from molecular modeling. The orientation of coumarin

with respect to the quartz surface was evaluated using molecular mechanics (MM+)

and semiempirical (AM1) computational tools of HyperChem Pro 6. The transition

dipole moment of coumarin molecule was calculated using the AM1 method. The

coumarin molecule with either short (APS) or long (AENPS) silane tail was first

geometry optimized by the semiempirical AM1 method. The various conformations

of the molecules were obtained by rotating around the carbon-carbon or carbon-

25

nitrogen bonds in the tail of the original semiempirically optimized molecule. The

rotated part of the molecule was then geometry optimized using molecular mechanics.

The molecule’s energy was calculated using AM1 method at the optimized single

point. All conformations of the molecule with the energy higher by 5 kcal/mol than

the energy of the most stable conformation were discarded from further analysis as

being thermally inaccessible.

300 350 400 450 500

0.000

0.002

0.004

0.006

0.008

0.010

B

A

4

32

1

Abso

rban

ce

Wavelength, nm

Figure 2.4 Illustration of the background subtraction procedure in absorption spectra: 1--original spectrum of a slide with immobilized coumarin, 2--the same slide naked, dots, 3--the best fit to the baseline of spectrum 1 using function of a wavelength λ (see text for details); 4--the “processed” spectrum is a result of subtracting the curve 2 from 1.

26

2.3 Results and Discussion

In the first step of surface modification, described in the experimental section,

the hydroxylated surface is modified with aminosilanes to produce aminogroups

accessible for further alteration. In the next step, amines are utilized to immobilize

dye molecules by reacting with succinimidyl ester group of a dye (Figure 2.5). This

two-step procedure results in a formation of self-assembled monolayer of a dye. Out

of the two steps, the latter one is the least vulnerable to reaction conditions. The

immobilization step was performed for a long enough time to allow all accessible

amines to fully react. Good reproducibility and the highest yield were achieved when

the reaction was carried out for no less than 2 hours.

The first step can be affected by many factors: the quality of self-assembled

monolayers and the mechanism of SAM formation during silanization are influenced

by such factors as duration of the silanization step, the nature of a silane, temperature,

moisture and other, less controllable aspects.

Optical absorption of the immobilized dye molecules in the resultant

monolayer provided a measure of the density of aminosilanes. This method is

indirect in evaluating the density of silanes because it measures density of moieties

that were immobilized at the later step. However, due to the high reproducibility and

the perfect yield of the last reaction of dye molecule immobilization, the method

seems to be quite sufficient for analysis of the aminosilane density.

27

N

O

OO

OO

O

N

OH

O

CH3

OCH3

OCH3

Si N HH

N HH

SiO O

O

CH3CH3

N H

O

OO

N

SiO O

O

CH3CH3

Et

EtEt

+1 2

Et

+

in acetone, 3 min

in DMSO, > 2 h

Si SiSi

Figure 2.5 The two-step procedure for coumarin self assembly: 1 – silanization of hydroxylated silica surface by trimethoxyaminosilanes, 2 – functionalization of amines by coumarin-A to yield immobilized coumarin.

In the two-step immobilization procedure either of the two silanes (APS or

AENPS) provides maximum absorption of coumarin no greater than 0.016, which

corresponds to the surface concentration of 2.1×1014 molecules per cm2. The surface

concentration of molecules was calculated using Equation 2.2. The reaction of a dye

molecule modified with succinimidyl ester (coumarin-A) results in formation of an

amide bond (Figure 2.5). The alteration in structure of a molecule slightly changes

optical properties of the dye, which can be imitated in solution by reacting the dye-

ester with propylamine. For example, coumarin-A upon amination by propylamine

produces coumarin B (see Figure 2.3). Absorption spectra of both, coumarin-A and

coumarin-B, in ethanol and toluene are shown in Figure 2.6. Clearly, upon

28

amination, the absorption maximum wavelength, λmax, shifts to the blue and the

extinction coefficient at λmax drops. The value of extinction coefficient at λmax

changes from 56000 M-1cm-1 for coumarin-A, to 47000 M-1 cm-1 for coumarin-B.

The extinction coefficient, determined in such a way, was used for calculating the

surface concentration of molecules. The same method was used to determine

extinction coefficient for DNP molecule, although the change was not as significant,

18000 M-1 cm-1 before and 16760 M-1 cm-1 after amination.

The maximum surface concentration achieved by the two-step immobilization,

2.1×1014 cm-2, is lower than the reported density of hydroxyl groups on silica surface

– 5.0×1014 cm-2.12,13,15,18 However, the surface concentration is almost identical to

what has been reported for the surface density of molecules when monofunctional

silanes have been used (ca. 2×1014 cm-2).19 The silanes with a single alkoxy group

can form only one Si-O-Si bond with the surface. Similarly, one can speculate that

trimethoxysilanes form crowded but not fully packed monolayer with only one

methoxy group reacted per silane and the other two remained intact. This explanation

will be further discussed later, in 2.3.3, where it will be also applied for increasing the

surface density of immobilized molecules and alternating the structure of SAM.

It should be noted that such high surface concentration of coumarin molecules

results in significant interactions between them in the monolayer. Also, since

molecules are placed at the interface between silica surface and the surroundings,

they interact with solvent molecules as well. Both types of intermolecular

29

interactions are significant and have comparable energies; their corresponding

features can be identified in absorption spectra.

2.3.1 Effect of solvent

The absorption spectrum of a dye is solvent dependent because of interactions

between the solute and solvent molecules. This interaction changes upon excitation

because of different dipole moments of a dye in the ground and excited states. In

addition, the transition moment interaction with the solvent also contributes to the

solvent effect.

For a molecule with the ground state dipole moment µg, the energy of

solvation can be estimated by the following equation:20

121E 3

2

solv +−

−=ε

εµa

g (2.4)

using the dielectric constant of a solvent (ε) and describing the molecule as a sphere

with radius a. Coumarin derivatives usually possess substantial ground state dipole

moments.17,21 For a typical ground state dipole moment of coumarin, µg ~ 7 D22 and

the molecular radius a ~ 5.4 Ǻ, the solvation energies in two solvents, hexane and

acetonitrile, differ by over 2kBT. As a consequence of such difference, coumarin is

more soluble in acetonitrile than in hexane, which also results in more red-shifted

absorption spectrum in acetonitrile. Absorption maximum wavelengths for coumarin-

A and coumarin-B are given in table 2.1.

30

300 350 400 450 5000.00

0.03

0.06

0.09

0.12

0.15

0.18

DC

B

A

ethanoltoluene

Abso

rban

ce

wavelength (nm)

Figure 2.6 Absorption spectra in solution for coumarin-A (solid) and its aminated analog, coumarin-B (dashed), in two solvents, toluene (spectra A and B) and ethanol (spectra C and D).

Table 2.1 Wavelengths of absorption maxima (nm) of the two coumarins in solutions measured at ca. 3×10-5 M.

a coumarin B was prepared directly in a cuvette by adding 50 µL of propylamine to 4 mL solution of coumarin A. b Prepared in CH2Cl2 and dissolved in hexane.

Solvent

Solute hexane toluene acetonitrile methylene chloride ethanol water

Coumarin A N/A 426 431 435 431 444

Coumarin Ba 401b 411 414 417 417 427

31

The shift in absorption spectra is not solely described by the difference in

solvation energy but also includes dispersion interaction of the transition dipole

moment, µ, with a solvent:

121E 2

2

3

2

disp +−

−=n

naµ

(2.5)

where n is the refractive index of a solvent.

Table 2.2 Solvent dependence of the optical absorption parameters of coumarin immobilized on silica for two different linkers.

a 3-amino-propyltrimethoxysilane, b N-[3-(Trimethoxysilyl)propyl]-ethylenediamine, c 1 nm is the typical accuracy, d 10-4 is the typical accuracy, e calculated from the

following equation: II

2

2cos

AAA+

=⊥

⊥θ .

Solvent Hexane (ε = 1.9)

Toluene (ε = 2.4)

CH2Cl2 (ε = 8.9)

Ethanol (ε =24.6)

Acetonitrile (ε =37.5)

⊥ 405±1c 408 411 408 406 λmax

(nm) 414 415 419 417 417

⊥ 4.7±0.1d 6.1 6.8 6.2 6.3 Amax

×103 7.9 8.4 7.7 6.9 6.9

APS linker

<cos2θ>e 0.23 0.27 0.31 0.31 0.31

⊥ 408 410 408 407 407 λmax (nm) 416 420 420 419 420

⊥ 5.0 5.6 6.5 6.0 5.6 Amax

×103 6.2 5.8 6.1 6.1 5.5

AENPS linker

<cos2θ> 0.29 0.33 0.35 0.33 0.34

32

Figure 2.7 Absorption spectra of coumarin stained slides in toluene (A) and in ethanol (B) at different light polarizations with respect to the surface. Two concentrations are given for each solvent: without dilution (⊥--perpendicular and ||--parallel polarizations) and with dilution ratio of aminosilane: octylsilane equal to 1:10 (⊥1/10 and || 1/10 respectively).

0.000

0.002

0.004

0.006

0.008

0.010

A⊥

⊥ 1/10

||

|| 1/10

Abso

rban

ce

350 400 450 500

0.000

0.002

0.004

0.006

0.008

0.010

Abso

rban

ce

B⊥

⊥ 1/10|| 1/10

||

Wavelength (nm)

33

The transition moment µ can be estimated from absorption spectrum, ε(ν),

using:23

ννεν

µ ~)~(~109.2 -3

2 ∫×

= d (2.6)

and equals ca. 8.0 D and 7.7 D for coumarin A and B, respectively. Combination of

equations (2.4) and (2.5) produces reasonable description of the spectral shift of either

coumarin in solution.

The surface immobilized coumarin demonstrates a much weaker solvent

dependence of its maximum wavelength (see Table 2.2 and Figure 2.7). Moreover,

λmax is strongly dependent on coumarin concentration (see below) and varies with

light polarization.

The resonance light absorption by a molecule is maximal when the transition

dipole moment, µ, is parallel with the electric component of the light wave (µ || E).

The transition dipole moment in coumarin molecule is oriented along the axis

approximately connecting the carbonyl carbon and the nitrogen of amino group, as

shown in Figure 2.8.24,25 By comparing absorption at different light polarizations,

one can monitor orientation of the dipoles with respect to the surface. Two

polarizations of incident light were used to probe the orientation, perpendicular (⊥)

and parallel (||). Perpendicular polarized light is absorbed by molecules mostly

oriented perpendicular to the surface, whereas molecules oriented along the surface

absorb mostly parallel-polarized light.

Figure 2.7 demonstrates the effect of light polarization and solvent polarity on

the absorption intensity for coumarin on silica. Nonpolar environment, such as

34

toluene, favors parallel orientation of molecules due to the less preferable solvation of

polar coumarin in non-polar media. More polar solvents, such as ethanol, however,

favor solvation of coumarin more and, as a result, there is a greater absorption of

perpendicular polarized light in ethanol. Table 2.2 shows a gradual increase of

preference for the perpendicular orientation with increasing solvent polarity (ε).

Although the trend is evident, the preference in molecular orientation is also

dependent on the linker.

Figure 2.8 Ball–and-stick representation of coumarin-B molecule and its transition dipole moment orientation.

2.3.2 Effect of a linker

The length of a linker should also affect the ability of molecules to adopt

different orientations with respect to the surface due to their different flexibilities.

35

Out of the two linkers used, APS is the shortest and thus limits the flexibility for

coumarin orientations. Table 2.2 shows that there is a small preference of parallel

orientation with respect to the surface with this linker, and although solvent polarity

affects the orientation, the parallel bias remains even in the most polar solvent,

acetonitrile. The solvation effect for the longer linker, AENPS, is not as dramatic,

displaying a smaller difference in the absorption for two polarizations. Longer linker

provides more possibilities for various chain conformations, which in case of

AENPS, corresponds to almost random distribution of conformations. However, the

solvation effect is identifiable, with parallel orientation being preferable in non-polar

solvents, hexane, toluene, and perpendicular orientation--more favorable in polar

solvents. When two linkers are compared in the same solvents, a greater bias towards

the perpendicular orientation is evident for the long linker.

A simplified conformational search performed using HyperChem Pro 6,

provided further confirmation of different molecular orientations depending on the

length of a linker. The average molecular orientation was assessed via evaluation of

all possible orientations of the transition dipole moment with respect to the surface.

The line between ring carbonyl carbon and nitrogen of the amino group were

representing the coumarin’s transition dipole moment (see Figure 2.8). The silane

was assumed to be linked by a single Si-O-Si bond to flat silica surface. The angle

between the Si-O-Si bond of the silane group and the line connecting carbonyl carbon

and nitrogen atoms was calculated and averaged for all conformations within

5kcal/mol from the most stable. Only conformations with coumarin above the

36

“plane” of silica surface were included. As expected, the calculated angle was larger

for APS than AENPS. “All-trans” conformations of APS and AENPS form angles of

55° and 24° to normal axis, respectively. The calculated averaged angles were 69°

for APS and 47° for AENPS, which agreed well with experimental observation of a

greater preference towards parallel orientation with the short linker. The energies and

geometries of different conformations of silane tails are provided in Appendix A.

2.3.3 Effect of concentration

The concentration of coumarin molecules realized in a monolayer is high

enough to cause molecules to interact with each other. These interactions are

primarily of the dipole-dipole nature and can be divided into two categories: dipole-

dipole interaction of the ground state dipole moments and the interaction between

transition dipole moments, which takes place upon excitation. The latter one is also

called intermolecular excitonic coupling. The energy of the former interaction in

vacuum is described by the following equation:

( )( )5

213

21d-d

3E

RRR

R

rrrrrrµµµµ

−= (2.7)

where µ1 and µ2 are two interacting dipole moments, which are separated by the

distance R.

Obviously, the energy of dipole-dipole interaction is positive for collinear

dipoles, which makes perpendicular orientated to the surface molecules energetically

37

less favorable. More likely, a tilted orientation is realized when the concentration of

molecules is very high. However, the dipole-dipole interaction competes with

solvation.

Another type of interaction, intermolecular excitonic coupling, takes place

only upon excitation. Its energy is given in a similar manner to the Equation 2.7:

( )( )5

213

21exc

3E

RRR

R

rrrrrrΜΜ

−ΜΜ

= (2.8)

Now M1 and M2 are transition dipole moments of two interacting molecules. Again,

the molecules’ orientation perpendicular to the surface increases the energy of

transition due to the repulsion between M1 and M2. For that reason, the absorption

maximum for perpendicular polarization is always blue shifted with respect to that of

parallel polarization as well as absorption maximum in solution. Both types of

intermolecular interactions can be reduced by diluting aminosilane with octylsilane at

the silanization step, which results in decreasing concentration of immobilized

coumarin. Figure 2.9 shows coumarin absorption spectra for different dilution ratios

of aminosilane to octylsilane: AM/C8 = 1/0, 1/10, 1/100, 1/250. In spite of apparent

decline in surface concentration of coumarin, the reduction is not equal to the dilution

ratio. The discrepancy can be assigned to a greater reactivity of aminosilane towards

surface hydroxyls than that of octylsilane. If KAM and KC8 are equilibrium constants

for the reaction of aminosilane and octylsilane, respectively, then surface

concentration of coumarin molecules (i.e. corresponding absorbance) can be related

to their concentrations, [AM] and [C8], in solution from the following equation:

38

+=

][][111 88

0 AMC

KK

AA AM

C (2.9)

Figure 2.9 Absorption spectra of slides with different surface coumarin concentrations corresponding to different ratio of octylsilane/aminosilane ([C8]/[AM] in the silanization step: A--0/1, B--10/1, C--100/1, and D--250/1. The inset shows linear dependence of the absorbance reciprocal on [C8]/[AM].

Here A0 is the absorbance of coumarin when no dilution with octylsilane was

done, which corresponds to the maximum surface coverage of coumarin. The inset in

Figure 2.9 shows that 1/A depends linearly on the dilution ratio, [C8]/[AM]. From the

slope the ratio KAM/KC8 ≈ 40 can be estimated.

350 400 450 500 550

0.000

0.002

0.004

0.006

C

D

B

A

Abso

rban

ce

Wavelength, nm

0 50 100 150 200 2500

400

800

1200

1600

1/A

[C8] / [AENPS]

39

What makes aminosilanes so different from octylsilane? It is known that

presence of amines in the reaction mixture accelerates rate of the reaction between

methoxysilanes and surface hydroxyl groups of silica.8 But, there is no agreement for

aminosilanes whether it is an effect of autocatalysis, where products accelerate the

reaction or a self-catalysis, that is, the amino group of aminosilane accelerates only

reaction of the molecule itself. Aminosilanes have a possibility to be physisorbed

with formation of H-bonding by all three methoxy groups as well as the

aminogroup.26 Amines compete with methoxy moieties for the surface OH-sites,

being more advantageous in adsorption due to their higher heat of H-bond interaction

with isolated OH group then those of alkoxy groups.27 An alkoxy group, then, reacts

with Si-OH site which has H-bonded amine resulting in formation of siloxane bond

(Si-O-Si) between silane and the surface. Based on the study of aminosilane and

octylsilane mixtures, it is unambiguous to conclude that autocatalysis plays

insignificant role in silanization, whereas self-catalysis results in 40 fold reactivity of

aminosilane compare to octylsilane. Indeed, if the aminogroup of already

immobilized molecule was capable of accelerating the reaction of oncoming silane,

there would be no difference observed in reactivity between aminosilane and

octylsilane. Also, the flexibility of the alkyl chain bearing the amino group allows

aminosilanes to access OH sites by both amine and methoxy group simultaneously, as

shown in Figure 2.5. However, it is not clear whether the presence of aminogroup at

any position in the silane chain or specific γ-position is necessary for self-catalytic

effect, but the fact of self-catalysis seems to be definite. Therefore, amine of

40

aminosilane can self-catalyze the reaction, making silanes containing aminogroups to

immobilize on silica more efficiently.

Although the dilution of aminosilane with octylsilane causes a nonlinear

decrease in surface concentration of coumarin, it is still accompanied by a reduction

of intermolecular interactions between coumarin molecules in the monolayer. Figure

2.7 illustrates that for two solvents, non-polar toluene and polar ethanol, the

anisotropy of absorption, which reflects the orientation of molecules, remains almost

the same upon dilution. The parallel orientation is slightly preferred in toluene for

both maximal concentrated and diluted monolayers, whereas in ethanol the absorption

is greater at perpendicular polarization in both cases.

On the other hand, the effect of dilution is more pronounced in the position of

absorption maxima. The wavelengths of maxima red-shift upon dilution, and as one

would expect, the shift is greater in the case of perpendicular polarization. Also the

difference between maxima for two polarizations becomes smaller, which illustrates

weakening of interactions between molecules upon dilution. Figure 2.10 shows that

such behavior is observed in all solvents.

2.3.4 Effect of water on silanization

Various factors in preparation conditions affect the resulting properties of self-

assembled monolayers: the type of aminosilane reagent, the concentration of surface

hydroxyl groups given by the type of a substrate, temperature, and water content are

41

among the most crucial ones. The density of immobilized dye and the orientation of

molecules in the monolayer dramatically depend on these factors. In most cases well-

ordered monolayers are desired for a variety of applications.28

Among the silanizing agents, only di- and trifunctional silanes provide a

possibility of lateral polymerization and formation of multiple bonds with the surface.

Because two out of three methoxy groups in each silane remain intact after

silanization in anhydrous solution, they hinder access for other silanes to neighboring

hydroxyl groups. Most nearest neighbor hydroxyl groups become inaccessible, and

no continuous film can be form by fusing neighboring silanes. As a result, maximum

surface density of molecules is not achieved.

Water appears to play an important role in formation of a dense monolayer by

hydrolyzing unreacted methoxy groups of silane and activating them for further

reactions. Figure 2.11 demonstrates the dependence of the absorption of immobilized

coumarin on the duration of silanization. Absorption of immobilized coumarin

rapidly increases, within first few minutes of silanization. After 3 minutes, the

absorption reaches ca. 80 % of its maximum value and after 6-10 min of silanization

it displays no further change. The reaction was carried out in a glove box under

atmosphere of argon at otherwise ambient conditions. Complete elimination of water

vapor from the reaction conditions did not affect the final concentration, and the

density of the monolayer was the same for both ambient and argon atmosphere.

However, if a monolayer formed from the anhydrous solvent consequently treated

with water, then the density of the monolayer can be increased. Figure 2.12 shows

42

the double silanization procedure, which was introduced in order to enhance the

surface concentration. Intermediate water treatment hydrolyzes methoxy groups that

remained unreacted after the first silanization. This creates hydroxyl groups in

addition to the existing surface hydroxyls, which were hindered from reacting with

oncoming silanes. The second silanization performed after intermediate water

treatment increases the total surface concentration by 20%. The maximum absorption

of 0.024 corresponds to the surface density of 2.7×1014 cm-2.

0.20 0.25 0.30 0.35 0.40 0.45 0.50

400

405

410

415

420

⊥

⊥1/10

||

||1/10

λ max

, nm

(ε-1)/(2ε+1)

Figure 2.10 Variation of the maximum absorption wavelength, λmax, with the solvent polarity factor, (ε –1)/(2ε+1), for two light polarization. The concentrations are identified as following: without dilution (⊥--perpendicular and --parallel polarizations) and with dilution ratio of aminosilane/ octylsilane equal to 1:10 (⊥1/10 and 1/10, respectively).

43

As one can see from Figure 2.11, the absorption reaches its maximum value

within first 10 minutes in both cases. Lines 1 and 2 are drawn as guides for an eye

and each point in the figure is a result of averaging of at least two samples with error

bars representing the spread.

0 10 20 30 400.000

0.005

0.010

0.015

0.020

0.025

1

2

Abso

rban

ce

time (min)

Figure 2.11 Variation of the absorbance for immobilized coumarin as a function of time of treatment by aminosilane solution: () dry solution, (⋇) the same solution but with washing in water after each 3 minutes of silanization.

44

N HH

SiO O

OCH3

CH3

N HH

SiO OO CH3

CH3

OH

N HH

SiO OO

CH3

CH3

N HH

SiO

O

N HH

SiO O

O

N HH

Si O

O O CH3

Si

N HH

O Si

N HH

O

O

N HH

SiO O

OHH

N HH

SiO OO H

H

OH

N HH

SiO OO

HH

Si SiSi Si

Si SiSi

silane

Si

H2O

Si SiSi Si

H2O

silane

Figure 2.12 Schematic representation of the double silanization with intermediate water treatment.

It appears that one water treatment is enough to produce the maximal increase

of surface concentration with no identifiable change after a few consecutive similar

treatments. Also, the vertical polymerization, which could in theory start from

unreacted methoxy groups after the second silanization, does not take place.

Intermediate hydrolysis, therefore, eliminates unwanted polymerization both in the

solution and propagating away from the surface. Thus, the incomplete monolayer

45

builds up by embedding silanes between previously immobilized ones on subsequent

steps of surface modification leading to the formation of two-dimensional siloxane

network.

Orientation of molecules in the monolayer as well as intermolecular

interactions should be altered as a result of increasing density in the double

silanization. As was shown above, the absorption anisotropy offers the information

about orientation of molecules with respect to the surface. Figure 2.13 reveals that

the orientation of the molecules in monolayer has a preference for molecular

orientation parallel to the surface for both cases with and without additional

silanization. Note that these spectra were taken in air, which makes solvation

contribution very small. The anisotropy changes only slightly towards preference of

parallel orientation with increased density of the monolayer.

The spectral shift, as was discussed before, can be related to intermolecular

interactions, which are dependent on the density and molecular orientation within a

monolayer. The increased density of molecules in the film after treatment with water

results in an additional hypochromic shift due to increased interactions between

molecules on the surface, but the effect is rather small (see Figure 2.13).

An alternative explanation to the water treatment effect might imply that,

instead of steric hindrance, it could arise from different ways silanes physisorb to the

surface. Aminosilanes are known for forming hydrogen bonding with surface

hydroxyls using all four active groups: three methoxy-groups and amine. It was

claimed that, depending on aminosilane concentration, the orientation of physisorbed

46

silane can change from primarily methoxy group attached at low concentration, to

amine bonded orientation at high concentration.26 One may argue that the additional

treatment with water primarily eliminates improperly oriented aminosilanes, which

did not succeed in forming Si-O-Si bond with the surface. That could be a part of the

story but since dry organic solvents did not produce such an effect, the correctness of

the presented interpretation seems to be more reasonable.

300 350 400 450 500 5500.000

0.004

0.008

0.012

0.016

0.020

0.024

⊥1⊥2

|| 2

|| 1

Abso

rban

ce

nm

Figure 2.13 Absorption spectra of slides in air at different light polarizations with respect to the surface (⊥--perpendicular and ||--parallel polarizations): 1--for the point at 30 min on curve 1, Figure 2.11; and 2--for the point at 39 min on curve 2, Figure 2.11.

47

The effect of water should be considered as the low limit because of probable

presence of traces of water in the dry solution, although water concentration should

be extremely low due to typical weather conditions in Las Cruces--humidity usually

is very low.

2.3.5 Lateral alternation of molecules in a monolayer

The fact that the two steps in double silanization are separated in time can be

used to alternate the active groups and molecules in a monolayer using different

moieties before and after the water treatment. The double silanization was utilized to

construct a monolayer containing two different alternating functional groups. The

two molecules that were used: coumarin-A and DNP (see Figure 2.2). Both

molecules possess succinimidyl ester linker, which allows facile reaction with

aminated surfaces and formation of amide bond. Both molecules absorb in the UV-

VIS range and have relatively high extinction coefficients. Their absorption maxima

are different, which simplifies tracking the changes in surface concentration for both

dyes separately.

Figure 2.14 schematically represents the method, which was utilized for

immobilization of two dyes. First silanization results in the aminated silica surface

and formation of a single Si-O-Si bond per silane (2). Then, it was followed by a dye

immobilization (3). Subsequent hydrolysis (4) provides additional activation of

unreacted methoxy groups and preparing the surface for further modification. The

48

second silanization (5) utilizes hydroxyl groups formed at the previous step of

hydrolysis. At the last step, second dye reacts with freshly aminated surface (6). The

dyes were placed in different order, either immobilizing coumarin first and DNP

second or vice versa.

Figure 2.15 shows the absorption spectra of a silica slide with immobilized

dye molecules, curve 1 is the total absorption after placing both molecules, curve 2 is

the absorption of immobilized DNP before second staining, and curve 3 is the

difference between two, which corresponds to the spectrum of immobilized coumarin

alone.

The surface coverage of immobilized coumarin and DNP molecules on quartz

is shown in Table 2.4. Upon hydrolysis, the absorption drops insignificantly, and

does the same after the second silanization. Usually surface concentration changes

after these two steps do not exceed 20% (see Table 2.4) of the initial values. A

possible contribution to these changes can arise from incomplete washing off the

physisorbed dye molecules. The second staining always significantly increases the

density with almost no effect from repeating the steps indicating that two steps

provide almost full coverage.

Immobilization of DNP in the first step results in a greater surface coverage

when compared to that of coumarin being first (Table 2.4). A smaller size of DNP

minimizes hinderance and allows better reactivity with the surface hydroxyl groups

spatially unavailable for bigger molecules such as coumarin.29 The longer

hydrocarbon linker on DNP also provides more flexibility. When DNP is the first

49

O

C

H

Si Si Si Si

SiHO OHO

NH

O

O

N

O

CH3CH3

SiHO OH

O

NH

O

O

N

O

CH3CH3

SiHO OHO

NH

O

O

N

O

CH3 CH3

Si Si Si Si

NHO

O

N

O

NH

O

O

N

O

O CH3

Si O SiO

OC

ONH

SiOO

C SiO O

O

Si OO

NH NHO

O

N

OO

NH

O2N

NO2

CH3 CH3CH3

CH3CH3

CH3

NH

O2N

NO2

4

6

DNP

H2O

silane

coumarin A

OH

OH

OH

OH

Si Si Si Si

2

silane

OH

Si Si Si Si

SiO OO

CH3CH3

NH

O

O

N

O

CH3 CH3

SiO OO CH3

CH3

NH

O

O

N

O

CH3CH3

SiO OO

CH3CH3

NH

O

O

N

O

CH3CH3

3

Si Si Si Si

NHO

O

N

O

SiOO

C

NH

O

O

N

O

O CH3

Si O SiO

OC

NH

H

SiO O

O

Si O

O

NH HNH

O

O

N

O

CH3CH3CH3 CH3

CH3 CH3

5

N HH

SiO O

O

CH3CH3

Si

N HH

SiO O

O CH3

CH3

OH

N HH

SiO OO

CH3CH3

Si SiSi

1

Figure 2.14 Schematic representation of the construction of alternating monolayer via double silanization with intermediate water treatment.

immobilized dye, coumarin increases the total coverage by a factor of 1.35 only,

which is lower compared to other sequences of dye immobilization. The shorter

linker for coumarin and its larger size contribute to that. In all other cases, the

50

315 350 385 420 455 490

0.000

0.003

0.006

0.009

0.012

0.015

32

1Ab

sorb

ance

wavelength (nm)

Figure 2.15 Absorption spectra of DNP (2) and coumarin (3) molecules immobilized on a silica slide in the double silanization reaction.

Table 2.4 Surface density of dyes on silica and their dependence on the order of immobilization sequence. Order of dye immobilization

1st immobili zation, n1

After hydrolysis

After 2nd silanization,

n’1

After 2nd immobili zation, n2

Ratio n2/n’1

Coumarin 2.7

Coumarin-coumarin 1.79a 1.66 1.53 3.2 2.08

Coumarin-DNP 1.80 1.55 1.50 3.1 2.09

DNP-coumarin 3.57 3.18 3.00 4.1 1.35

DNP-DNP 2.87 2.80 2.73 5.8 2.12 a all values are in units of 1014 cm-2

51

ratio of molecular concentrations for two immobilization steps is close to 2 (Table

2.4). The surface coverage is maximal when DNP is used at both immobilization

steps. Also worthy of noticing is that the density of DNP after double silanization,

>5×1014 cm-2, appears to be even greater than that of surface hydroxyls, ~5×1014 cm-2.

It can never be achieved with one or more hydroxyls per silane but, according to

Figure 2.14, can be realized in the double silanization process.

2.4 Conclusions

1) A protocol for covalent immobilization of molecules based on

aminoalkoxylane and succinimidyl ester chemistry is optimized. The highest surface

density of coumarin molecules achieved via this two-step procedure using self-

assembly of trimethoxyaminosilanes from dry solvents is ~2×1014 cm-2, which is

lower than the surface density of hydroxyls.

2) The surface concentration of molecules is dependent on the presence of

water in the reaction mixture and can be further improved using double silanization

with intermediate hydrolysis. The first silanization step results in a loosely packed

monolayer, where only one, rarely two covalent bonds are formed between silane and

the surface. The introduction of intermediate hydrolysis of unreacted methoxy groups

improves the quality of a monolayer by the formation of 2-dimensional siloxane

network as well as increasing the surface concentration of immobilized molecules to

52

~3×1014 cm-2 for coumarin and >5×1014 cm-2 for DNP. Such a procedure also allows

construction of SAM with alternating dye molecules across the surface.

3) Such high surface concentration of molecules results in strong

intermolecular interactions, which are reflected in the optical absorption shift and its

linear anisotropy. The intermolecular interactions can be effectively reduced by

dilution of the functional silane with dull alkylsilane bearing no aminogroup in the

reaction mixture. The orientation of molecules in a monolayer (evaluated by linear

dichroism measurements and supported by conformational analysis) is weakly

dependent on solvent polarity and can be approximately described as almost random

distribution on a semisphere with increasing preference towards perpendicular

orientation in more polar solvents. Solvent free film shows even greater tendency

away from perpendicular molecule orientation.

2.5 References

1 Sung, M. M., Kluth, G. J., Maboudian, R., J. Vac. Sci. Technol. A, 1999, 17(2), 540-544. 2 Wu, A., Kakimoto, M.A., Adv. Mater., 1995, 7, 812. 3 O’Regan, B., Grätzel, M., Nature, 1991, 353, 737-740, Bach, U., Lupo, D., Comte, P., Moser, J. E., Weissortel, F., Salbeck, J., Spreitzer, H., Grätzel, M., Nature, 1998, 395, 583-585. 4 Paddeu, S., Ram, M.K., Nicolini, C., J. Phys. Chem. B, 1997, 101, 4759-4766. 5 McKittrick, M. W., Jones C. W., J. Am. Chem. Soc., 2004, 126, 3052-3053.

53

6 Murray, R. W., Chemically modified electrodes, in Electrochemical Chemistry, Vol. 13, A.J. Bard, Ed., M. Dekker, NY, 1984, pp. 191-368. 7 Ulman A., An Introduction to ultrathin organic films from Langmuir-Blodgett to Self-Assembly, Academic Press, Inc., 1991. 8 White, L.D., Tripp, C.P., J. Colloid Interface Sci., 2000, 232, 400-407. 9 Stronther, T., Cai, W., Zhao, X., Hammers, R.J., Smith, L.M., J. Am. Chem. Soc., 2000, 122, 1205-1209. 10 Brunner, H., Vallant, T., Mayer, U., Hoffman, H., Basnar, B., Vallant, M., Friedbacher, G., Langmuir, 1999, 15, 1899-1901. 11 Vallant, T., Brunner, H., Mayer, U., Hoffman, H., Leitner, T., Resch, R., Friedbacher, G., J. Phys. Chem. B, 1998, 102, 7190-7197. 12 Lygin, V.I., Russ. J. Phys. Chem., 1997, 71, 1557-1564. 13 Tuel, A., Hommel, H., Legrand, A.P., Kovats, E., Langmuir, 1990, 6, 770-775. 14 Sfez, R., De-Zhong, L., Mandler, D., Yitschaik, Sh., Langmuir, 2001, 17, 2556-2559. 15 Vigne-Maeder, F., Sautet, P., J. Phys. Chem. B, 1997, 101, 8197-8203. 16 Ghosh H. N., J. Phys. Chem. B, 1999, 103, 10382-10387. 17 Samanta, A., Fessenden R.W., J. Phys. Chem. A, 2000, 104, 8577-8582. 18 Wasserman, S.R., Tao, Y.T., Whitesides, G.M., Langmuir, 1989, 5, 1074-1087. 19 J. H. Moon, J.W. Shin, J.W. Park, Mol. Cryst. Liq. Cryst., 1997, 295, 185-188. 20 Böttcher, C.J.F., Theory of Electric Polarization, Elsevier, Amsterdam, 1973. 21 Rechtaler, K., Kohler, G., Chem. Phys., 1994, 71, 1557-1564. 22 The values of molecular dipole moments are usually expressed in Debye units abbreviated as D. For two point charges +e and –e at a distance a, the electric dipole moment is µ = e×a. When the charges are separated by 1 Ǻngstrom, the dipole moment equals 4.8 D.

54

23 Klessinger, M., Michl, J., Excited states and photochemistry of organic molecules, VCH Publishers, New York, 1994. 24 Smirnov, S., Braun, C., Rev. Sci. Instum., 1998, 69, 2875-2887. 25 The orientation of transition dipole moment of coumarin molecule is parallel to the line connecting C2 carbon atom and nitrogen of amine in AM1 optimized structure. The orientation of transition dipole moment agrees well for both AM1 and ZINDO/S. The transition dipole moment is also parallel to both, the ground and excited state dipole moments of coumarin. 26 Piers, A.S., Rochester, C.H., J. Colloid Interface Sci., 1995, 174, 97-103. 27 White, L.D., Tripp, C.P., J. Colloid Interface Sci., 2000, 227, 237-243. 28 Swalen, J.D., Allara, D.L., Andrare, J.D., Chandross, E.A., Garoff, S., Israelachvili, J., McCarthy, T.T.J., Murray, R., Pease, R.F., Rabolt, J.F., Wynne, K.J., Yu, H., Langmuir, 1987, 3, 932-950. 29 Molecular volume was calculated by HyperChem6 Pro, VCOUMARIN = 686.69 Å3, VDNP= 454.37 Å3, tail length and its volume was not taken into account.

55

3 CHARGE SEPARATION IN SELF-ASSEMBLED MOLECULAR FILMS

3.1 Introduction

The photoinduced transient displacement current technique, PTDC, is the

most direct method for studying the extent of charge separation. It measures the

change in dipole moments by monitoring transient dc currents caused by

photoinduced charge transfer in molecules in solution. The PTDC technique has been

successfully used in a variety of systems to study both intramolecular and

intermolecular photoinduced charge transfer.1,2,3 The measured signal in PTDC is

proportional to the change in the squared dipole moment of a molecule that happens

when molecule undergoes transition from the ground state to its excited state upon

photoexcitation. Certain advantages of the PTDC technique over other methods such

as transient absorption and transient microwave conductivity make this method very

useful for studying the extent of charge separation.4 The transient absorption

technique can identify the charge separated states and follow their evolution but is

often limited in providing information about the charge separation distance. The

transient microwave conductivity technique, that permits the determination of the

dipole moments of transient charge separated states, is restricted to nonpolar solvents

and has intrinsic limitations in time resolution. Moreover, it requires independent

estimate of the rotation time for calculating the dipole moment. The PTDC technique

requires no additional information and allows direct measure of the dipole moment

56

change upon photoexcitation, kinetics of charge recombination, and is not restricted

by solvent polarity.

The experimental setup of the PTDC technique is sketched in Figure 3.1:4

external voltage is applied across the two parallel electrodes cell (with solution

inside) in series with the load resistor, R. The molecules in solution are excited by a

laser pulse causing them to change the ground state dipole moment to (usually) a

larger dipole moment in their excited state. The interaction of a dipole with external

electric field, E, depends on the angle between electric field and the dipole moment.

Therefore, the newly formed dipoles reorient towards new equilibrium under applied

voltage with a rotation time, τr. The rotation changes the angular distribution of

dipoles causing a displacement current.

Orientation of dipoles in the cell can be achieved by either external electric

field by the surface. In standard PTDC, where solute dipoles in solution are oriented

by the field, the net equilibrium electric polarization from solutes, Psolute, represents

the average projection of molecules’ dipole moments, <µeff>, multiplied by their

concentration, n. At equilibrium in the limit of small concentration of dipoles and

small electric field, when Boltzmann distribution can be linearized, it equals:

( )

nTkEd

TkE

ndennPBB

TkUeffsolute

B

31

2/)( µϕ

µµϕµµ θ =Ω

⋅+=Ω== ∫∫ −

rrrr

(3.1)

where factor ϕ includes contributions from solvent molecules.

Upon excitation, some dipole moments change. If we assume that molecular

reorientation happens quickly (τr is close to zero), then the angular distribution of

57

each sort of dipole is always at equilibrium, and electric polarization can be

approximated from Equation 3.1. Assuming that there is only one sort of excited

dipole, µexc, the solute electric polarization change, ∆Psolute, depends on time only

through the concentration of excited states, nexc(t):

( )Tk

EtntPB

gexcexcsolute 3)()( 22 ϕµµ −=∆ (3.2)

Here µg and µexc denote the ground and excited state dipole moments, respectively.

Obviously, evolution of solute polarization in this case can be referred to a single

artificial effective dipole moment with the dipole moment µ calculated from:

222gexc µµµ −= (3.3)

The total electric polarization of the solution, P, comes from polarization of

solvent and the solutes:

P = Psolv+ Psolute (3.4)

The former can be estimated from the dielectric constant as Psolv= E/4π(ε -1) and the

latter is calculated from angular dipole distribution and their interaction with the

solvent. For calculating the dipole signal, i.e. the voltage drop across load resistor,

one calculates charge, Q, at the electrodes first, which is given by the product of

displacement, D = E + 4πP, and the electrode area, S, normalized by 4π:

Q = E + 4πPS/4π = εE/4π + Psolute S (3.5)

The electric field inside the cell, E, is given by the voltage drop across the cell, vcell,

divided by the cell gap, E = vcell /d. The voltage, v, measured across the load resistor,

R, in the measuring circuit, arises from the displacement current drop, v = R(dQ/dt).

58

The applied voltage, Vo (which could also be zero), is applied across the cell and the

load resistor in series, thus:

v = Vo - vcell, (3.6)

and the equation for time variation of v can be written as:4

dt

dPRS

dtdvv solute

RC =+τ (3.7)

Here the RC time of the circuit is introduced:

τ εεπRC R C RSd

= =0 4 (3.8)

Note that equation 3.5 is valid not only for the PTDC technique, where dipoles are

oriented by external voltage, but in other cases with different means of orienting

dipoles as well; they would differ by how Psolute is calculated.

The time evolution of Psolute is determined by two factors. The first one arises

from changing dipole concentration, nexc, (or n for simplicity) due to their

recombination with the time constant, τCR:

CRndtdn τ/−= , (3.9)

The other one is due to reorientation of dipoles that sometimes is slow enough to

make the average projection of the dipole moment, <µeff>, evolve more gradually.

Under the applied electric field, the resulting time dependence of <µeff> can be

described by the following equation:5

CReffrB

effeff

eff nTkE

dtd

τµτµ

µµ

// −

⋅+−= (3.10)

59

where kB is Boltzmann’s constant and T is temperature.

If molecular rotation, described by the rotational time, τr, is fast, quasi-

equilibrium treatment for the solute polarization Psolute (Equation 3.1) can be used,

simplifying the equation into:

dtdn

kTdVSR

dtd

RC 3

20 µϕντν ⋅⋅⋅

=+ (3.11)

where d is the gap between electrodes. The coefficient ϕ takes into account the

change in the electric field around a dipole due to the presence of solvent molecules.

Polarization of solvent molecules by solute can be accounted for using Onsager’s

semicontinuum model.6 According to the model, a molecule with dipole moment is

represented as a point dipole placed in a center of spherical cavity with a radius a, and

surrounded by continuous medium with dielectric constant ε. As a result of solvent

polarization by the dipole, the reaction field of the solvent induces an increase of the

dipole moment due to its polarizability, α. The dipole moment becomes larger than

its gas phase value, µo.

12)1(21 3 +

−−

=

εεα

µµ

a

o (3.12)

This value can be significantly greater than µo especially for small molecules with

high polarizability. Since the reaction field is much greater than any accessible

external fields, µ of Equation 3.12, not µo, represents the meaningful description of

dipole moment in solution.4 Combination of other contributions to the overall electric

60

polarization due to dipole interaction with solvent is combined in the parameter ϕ,

which for a spherical cavity, is:4

( )( )42

222

232

123

D

D

nn+

+

+=

εε

εεϕ (3.13)

where nD is solvent refractive index.

Two modes of PTDC can be distinguished depending on the load resistor, R:

displacement current and displacement charge. The displacement current mode is

realized with a 50 Ω load resistor, in which case the circuit RC time is short and the

signal is primarily proportional to time derivative of dipole concentration. In the

charge displacement mode, with a large load resistor (we use 1 MΩ or 20 kΩ) of a

high impedance probe, the resulting circuit RC time is greater than the lifetime of

excited state dipolar species, and the signal is primarily proportional to the dipole

concentration instead.

When studying charge transfer in large molecules or complexes, such as DNA

and proteins, one should be aware that rotation can be slower than recombination. In

standard geometry, such a condition prevents the signal from full development

because its decline is faster than rise. To overcome this shortcoming and eliminate

the use of the external electric field, we propose an alternative means of dipole

orientation – by immobilizing molecules on flat surfaces. In order to ensure that the

modification can be used to obtain quantitative data, self-assembled systems of

coumarin molecules were used as a model for technique evaluation.


61

a) development of the photoinduced displacement charge technique for

studying charge transfer in molecules organized in self-assembled films with

projected unidirectional molecular orientation – surface assisted photoinduced

transient displacement charge, SPTDC;

b) evaluation of SPTDC in quantitative analysis of charge transfer in surface

immobilized molecular films;

c) investigation of intrinsic advantages and disadvantages of SPTDC.

Rµexc

V 0

µg

Figure 3.1 Schematic representation of photoinduced transient displacement current setup (PTDC). Ground, µg, and excited state, µexc, dipole moments illustrate greater alignment with the field for µexc.

62

3.2 Theory of SPTDC

The dipole signal obtained by the displacement current/charge technique is a

capacitive measure of charge displacement within the cell caused by the

intramolecular charge transfer after laser excitation. In essence, the intramolecular

charge transfer results in formation of excited state dipole moments. The

photoinduced voltage signal from a monolayer in SPTDC is proportional to the

perpendicular component of the net change in molecular dipole moment in the

monolayer upon photoexcitation. The Helmholtz equation relates the surface

potential with the average dipole moment of molecules in a monolayer.7 The

monolayer of molecules with collinear dipole moments, µ, may be viewed as two

sheets of charges separated by distance l = µ/e. Likewise, the charge density, σ,

relates to surface density of molecules, n: σ = ne. The potential difference across the

monolayer is:

V = 4π·l·σ (3.14)

The dipole signal (photovoltage), v – the quantity we measure in SPTDC, can

be similarly expressed by the following equation:

v = ∆µ⊥·N/S (3.15)

where the surface density of excited molecules is given as the ratio between the

number of excited molecules, N, and the electrode area, S. The change of the dipole

moment perpendicular to the surface, ∆µ⊥ = q∆l, due to displacement of charge q

63

along distance ∆l upon photoexcitation can be calculated as a difference between the

average perpendicular projections of the dipole moments in excited and ground states:

∆µ⊥ = µexc<cosθ>exc − µg<cosθ>g (3.16)

where θ is the angle between the direction of the dipole moment and the normal axis

of the surface. As can be seen from Equation 3.15, photovoltage is independent of

the gap between the electrodes. However, it is dependent on the number of the

excited molecules N, orientation of molecules in the monolayer in ground and excited

state and the dipole moments of those states.

The average orientations of dipoles in the ground and excited states are not

necessarily the same and light polarization during excitation makes the distributions

different from that of the ground state dipole. The transition moments and the dipole

moments of the two states are also not perfectly aligned within the molecule.

Moreover, the molecules themselves have sufficient freedom to rotate, as seen in

absorption spectra. Polar solvents screen better repulsive interaction between

neighboring dipoles and aid to orienting them perpendicular to the surface.

Nevertheless, if assumed that the distribution of ground state dipoles is uniform

throughout a semisphere and the dipole moments are collinear with transition

moment, for the geometry used, i.e. light polarization perpendicular to the surface,

one can evaluate equation 3.16 as:

( ) ( ) µµµ

θθ

θθθ

µµµ ∆=−=−=∆

∫

∫⊥ 8

383

cossin

cossincos

1

0

2

1

0

2

gexcgexc

d

d

(3.17)

64

Equation 3.15 is given for vacuum, whereas in reality the cell is filled with

medium, usually a solvent, with characteristic dielectric constant ε. Following

Onsager’s semicontinuum model, a molecule with nonzero ground state dipole

moment may be represented as a point dipole, placed in a center of spherical cavity

and surrounded by continuous medium with dielectric constant ε.6 Equations 3.12

and 3.13 were derived under this approximation. Following the same model, one can

realize that a point dipole moment, µ, in a spherical cavity creates an electric field

outside the cavity identical to that created by a point dipole µeff:8

µε

µ12

3+

=eff (3.18)

ε µ

µeff = µ 32ε+1

Figure 3.2 Illustration of a dipole moment in a spherical cavity immersed in dielectric (ε).

This dipole moment is smaller than µ because of the canceling effect of

solvent, which would reduce µeff to almost zero in a high polarity solvent. The value

65

of µ itself increases with increasing polarity (see Equation 3.12) but to a lesser

extent.

Combining both these assumptions: on the dipole distribution (Equation 3.17),

and their interaction only with the solvent but not with each other (Equation 3.18),

and plugging them into equation 3.15, one finds:

SNv ⋅

+⋅∆=

123

834

εµπ (3.19)

The number of molecules, N, excited upon laser illumination can be measured

experimentally from absorption, allowing determination of ∆µ.

All three assumptions for equation 3.19 are not flawless: angular distribution

is not uniform, dipoles are not independent and there is simple modification of

individual spherically shaped dipoles into the Helmlotz double charge layer. The

latter seems to be the most confusing and require an alternative representation

because there is no transparent transition from a point dipole in a spherical cavity to

Helmholtz layer of charges. Previous attempts to accomplish that,7,8 don’t seem to be

convincing and as we will see later, arrive at slightly different conclusion.

As an alternative approach let us consider equation 3.7, where polarization,

Psolute, is calculated based on solute dipoles orientation. We presume again that the

dipoles in their spherical cavities are far spaced (l>>a) and do not experience any

effect from the substrate (h>>a). We will not require them to be in a sheet or any

kind of arrangement and will calculate the resulting polarization, Psolute. The nice

property of a point dipole in a spherical cavity is that there is a zero net polarization

induced in the solvent by the solute. Indeed, the integral of the field from a point

66

dipole, Eµ, over the region outside a spherical cavity is exactly zero due to the

spherical symmetry:9

( ) 0==∫ ∫> e eVV S

e SdrdVErr

ϕµ (3.20)

ε<µeff>⊥

µ µµ

l h

Substrate

Solvent

a

Figure 3.3 Illustration of experimentally relevant models of dipoles in self-assembled monolayer.

This enormously simplifies everything because now Psolute is just a product of

solute dipole concentration and ∆µ⊥:

Psolute = <∆µ⊥>·n (3.21)

When this is plugged into Equation 3.7, in the limit of charge displacement (large

τRC) we get:

67

dtdnRS

dtdv

RCτµ >∆=< ⊥ (3.22)

If one integrates Equation 3.8 and recalls τRC from Equation 3.8, the following

equation:

S

tNtv )(4)( εµ

π>∆<

= ⊥ (3.23)

very similar to that of equation 3.19 arises. The result almost coincides with previous

derivations7 that Helmholtz layer potential is given by µ/ε but with a different

interpretation and, apparently, from a completely different model. The dielectric

constant, ε, in equation 3.23 is that for a bulk solution where dipoles are randomly

distributed, while Demchak and Fort10 and then Taylor and Bayes7 arrived at a similar

formula with ε being the dielectric constant of the Helmholtz layer only.

Equation 3.23 will be the basis of our further SPTDC dipole signal

evaluations. In standard PTDC used for solutions under applied field the assumption

of each dipole interacting only with solvent and external field worked quite well. It

allowed straight forward calculation of average dipole moment projection, making

PTDC so powerful for providing unambiguous data. In SPTDC, at least in the

realized form, additional information in greater details is required. First of all, the

system is intrinsically anisotropic, substrate and the linker have different dielectric

properties from those of the solvent above SAM. Second, in dense SAM films, the

amount of solvent protruding in between molecules of SAM depends on surface

density of molecules, their size and the solvent, and thus is difficult to control. Third,

closely packed dipoles interact with each in the ground and excited states, as well

68

during their excitation (excitonic coupling) and strengths of these interactions are

different and dependent on solvent. Thus, the interpretation of < ∆µ.⊥> is dependent

on the model.


3.3.1 Materials

Three types of substrates, glass slides (Corning cover glass), mica, and

polished quartz slides (12 x 25 x 0.3 mm) from Quartz International, were modified

by trimethoxy aminosilanes, and then stained by a dye on one side to form a

monolayer using the procedure described in Chapter 2. The cleaning of the substrates

was performed as described in experimental section of Chapter 2 for glass and quartz

substrates. Mica was prepared by peeling off the upper layer of the substrate prior to

use, followed by boiling it in distilled water.

7-diethylaminocoumarin-3-carboxilic acid succinimidyl ester (D-1412)-

“coumarin-A” from Molecular Probes, was used without purification. Aminated

coumarin (coumarin-B) was prepared by reacting D-1412 with 1-propylamine (see

Figure 2.3). Coumarin 153 and coumarin 460 were obtained from Exciton, Inc.

(Figure 3.4).

69

OO

NEt

Et

CH3

OO

N

F3C

OO

NEt

Et

coumarin 153 coumarin 460 coumarin

12

345

6

7 8

Figure 3.4 Structures of coumarin molecules.

3.3.2 Experimental setup

Figure 3.5 gives a schematic representation of the experimental setup for the

PTDC (“dipole”) and fluorescence measurements. Picosecond pulses, with duration

ca. 20ps at 5kHz, were generated using Nd:YAG laser (“Orion SB-R” from MPB).

The third harmonic shifted on either H2 or CH4 (to make 416 nm or 396 nm,

respectively) was used for excitation. VSL-337 nitrogen laser (337 nm, 4ns long)

was also used as a source of excitation in the experiments with coumarin 460.

The signal was digitized by a 1 GHz digital oscilloscope (DO1), TDS 684A,

from Tektronix. Two pyroelectric detectors, Molectron J4-09, were used to measure

energy of the incident (PD1) and the transmitted (PD2) light allowing calculation of

70

the absorbed energy. The signals from the detectors were digitized by a digital

oscilloscope (DO2), TDS 220, also from Tektronix.

The rest of the setup consists of an electrical shutter (SH) – UniBlitz,

triggering photodiode (T), ET2000, from EOT, a neutral density filter (F), and the

sample cell (S). High voltage power supply (HV) was used in the standard PTDC

displacement current measurements and signal was measured across the 50Ω input

resistor of the TDS 684. In the modified surface assisted PTDC (SPTDC) version, a

dipole signal was measured across the input 20 kΩ resistance of a P6249 high

impedance probe (P),11 from Tektronix.

Laser System (Nd:YAG or Nitrogen)

Computer

S

PD1

PD2

TFPD

Fluorescence

dipole

P

F

DO1

DO2

SHHV

C

LG

Figure 3.5 Schematic representation of photoinduced displacement current (charge) experimental setup. The inset shows a part of the setup alternative for the fluorescence measurements.

71

Modifications for the fluorescence kinetics setup are shown in the inset of

Figure 3.5, where fluorescence from quartz cuvette (C) with a sample was collected

using quartz guide (LG) and simultaneously focused onto an InGaAs photodiode

(FPD) (model No 1437, 25 GHz, from New Focus). The rest of the setup was

identical to that used in dipole measurements.

3.3.3 Dipole measurements in solution, PTDC

Excited state dipole moments of both coumarin-A and coumarin-B in toluene

solution were measured using standard time-resolved photoinduced transient

displacement current (PTDC) setup for the purpose of calibrating. A solution of a

dye with a typical concentration 10-4 M was circulated through a dipole cell. The

sample cell had two parallel flat stainless steel electrodes and quartz windows for

laser excitation. The experiments were done at room temperature, and external

voltage 600V applied across 0.65 ± 0.05 mm gap between the electrodes with area 1.3

cm2.

The photoresponse, ν, is a result of displacement current, which was measured

across 50Ω input resistor, and digitized on the TDS 684A oscilloscope as a function

of time. Since the rotation time of coumarin molecules is fast (ca. 0.2ns), the dipole

signal is fairly well described by Equation 3.7. This simplification left three

parameters for describing the signal--the dipole moment change, µ, excited state life

time, τF, and the RC time, τRC, where µ described the signal magnitude, while τF and

72

τRC determined the shape of the signal. All three parameters were varied in order to

fit the signal. Optimized fittings,12 thus, provided the values for µ, τF and τRC.

Inclusion of the estimated rotation time, τr ~ 0.2 ns, insignificantly improved the

fittings and had no effect on the dipole moment value.

3.3.4 Dipole measurements on surface, SPTDC

Figure 3.6 shows a schematic representation of surface assisted photoinduced

transient displacement charge (SPTDC) setup. This method differs from standard

PTDC by a larger load resistor (as a high impedance probe) and the cell design.

l

hv solvent

ITO

R

d

D Ae

D Ae

D Ae

D Ae

Figure 3.6 Schematic representation of the surface assisted photoinduced transient displacement current setup.

73

The 50Ω cable was replaced with a high impedance probe (P6249 active probe

from Tektronix, 20kΩ input impedance and 4 GHz bandwidth). The sample cell was

designed to suit the modification of the SPTDC technique for studying photoinduced

charge transfer in surface immobilized molecules. The designed sample cell is

sketched in Figure 3.7. Two identical ITO (indium-tin-oxide) semitransparent

electrodes with resistivity 10 Ω/square (1) are placed inside the cell body (2). The

electrodes face each other so that the overlapping area is ca. 2.5×2.5 cm2. The gap

between the electrodes can be adjusted to the number of slides used by varying the

thickness of a dielectric spacer (4). Ideally, the dipole signal in SPTDC is

independent of the gap between the electrodes (see Equation 3.23) as long as the size

of the electrodes is much greater than the distance between them, so that the fringe

effects can be neglected. However, in reality the cell capacitance (and thus its ratio to

stray capacitance) varies with the distance between the electrodes. For the sake of

simplicity, the gap was maintained the same for all the experiments, ca. 3mm.

It allowed easy evaluation of stray capacitance once for all experiments. The sample

cell has a significant stray capacitance, 4.3 pF, see Appendix B, comparable with that

of the cell itself. Therefore, the correction with regard to this parameter should be

introduced in Equation 3.23. Stray capacitance affects the amplitude of the dipole

signal, especially in solvents of low polarity because the signal drops across both

stray capacitance, Cs, and cell capacitance. The correction automatically gets

included if the RC time of the circuit combines both capacitances, of the cell and

stray capacitance:

74

( )sRC CCR += 0ετ (3.24)

1

2

3

4

5

Figure 3.7 Schematic sketch of the sample cell designed for the study of charge transfer in molecules assembled in monolayers. 1--glass electrodes covered with conductive layer of ITO (indium doped tin oxide), surface resistance ~10Ω/square, 2--two part body made of Plexiglass, 3--holes for the cell body screws, 4--dielectric spacer (rubber sheet), 5--windows for laser excitation.

There is a critical limitation for dipole measurements at interface (SPTDC

technique). The amount of molecules that can be placed in SAM is very small, even

when using a few slides in the dipole cell. As a result, the signal is small as well.

75

Due to a poor electromagnetic shielding, the cell has noticeable noise pick up that

interferes with measurements. In order to reduce the noise and enhance the capability

of measuring small signals, an additional shielding case made of aluminum metal

sheets was applied.

3.3.5 Fluorescence measurements

Fluorescence spectra were detected using a fiber optic spectrometer, SD2000

from Ocean Optics, CCD of which was triggered by the laser. Two slides in contact

by their stained sides were placed vertically at 45° with respect to the incoming laser

beam in a 1 cm quartz cuvette. To minimize scattering, the gap between the slides

was filled with toluene. The liquid served not only as a refractive index matching

fluid but also minimized potential photooxidation of coumarin. Luminescence was

collected by a fiber via a short focal length lens. A glass filter with the short

wavelength cutoff at 435 nm and placed between fiber and the detector shielded

undesired laser scattering. The luminescence was measured at different incoming

laser light polarizations and at two different positions of fiber with respect to the

slides. Parallel polarization was applied with the fiber placed under the sample and

perpendicular one with the fiber oriented at 90° with respect to the laser beam (behind

the slides). The laser intensity was varied by neutral density filters.

76


3.4.1 Charge transfer of coumarins in toluene solution

Before coumarin can be used for evaluation of the surface assisted

photoinduced transient displacement charge method, it is necessary to characterize its

dipolar properties in solution. Coumarin possesses significant dipole moments in

both ground and excited states. To mimic the transformation that occurs with

coumarin-A, equipped with succinimidyl ester group in the 3-position, upon its

immobilization onto aminated surface, coumarin-B was studied as well (see Figure

2.3). Coumarin-B is synthesized by reacting with propylamine and is presumed to

have the same properties as coumarin immobilized on the surface. Since substitution

in the aromatic ring of coumarin affects quite significantly the value of dipole

moment, especially that of the excited state,13,14 some variations between coumarin-A

and coumarin-B were expected.

The excited state dipole moments and their lifetimes for coumarin-A and

coumarin-B were measured in toluene solution using standard PTDC setup. Figure

3.8 shows transient displacement current signals (dipole signals) for coumarin-A and

coumarin-B in toluene (solid lines). The best-fit signals (points) were calculated with

the ground state dipole moments presumed zero. Therefore, the excited state dipole

moment values, which are equivalent to µ (Equation 3.3), were calculated 10.8 D and

11.9 D, for coumarin-A and coumarin-B, respectively. The excited state lifetimes, τf,

were also obtained from the fitting as 3.5 ns (coumarin-A) and 3.0 ns (coumarin-B).

77

0 5 10 15 20-0.1

0.0

0.1

0.2

0.3

0.4

Coumarin-B

µ = 11.9 Dτ = 3.0 ns

Time (ns)

Phot

ores

pons

e (m

V)

0 5 10 15 20-0.1

0.0

0.1

0.2

0.3

0.4

Coumarin-A

µ = 10.8 Dτ = 3.5 ns

Phot

ores

pons

e (m

V)

Figure 3.8 Transient displacement current (“dipole”) signals of coumarin-A and coumarin-B in toluene solution.

78

3.4.2 Semiempirical calculations

Since the ground state dipole moment of coumarin is nonzero, the results of

PTDC and SPTDC cannot be directly compared. In order to calculate the dipole

moment change upon photoexcitation, it is necessary to know coumarin’s ground

state dipole moment, µg. The value of µg can be estimated using semiempirical

calculations, particularly AM1, with high degree of accuracy.13,14,15 Anticipating

similar accuracy for our coumarin molecules, we performed semiempirical

calculations (AM1) to estimate the dipole moments. The calculated values of the

ground state as well as excited state dipole moments are summarized in Table 3.1.

The ground state geometries were optimized with AM1 method and two energetically

close conformers, with syn- and anti- alkyl group orientations on amines were

analyzed. For comparison, ZINDO/S was also used to calculate the dipole moments

and spectra for which standard overlap weighting factors (σ−σ =1.267 and

π−π = 0.585) were used.16

The results of semiempirical calculations can be summarized as follows: 1) in

AM1 the syn- and anti- conformers have very close energies and their optical and

charge distribution properties in both ground and excited states are not dramatically

different; 2) ground state dipole moments are not very different from those of other 7-

aminocoumarins obtained both experimentally as well as using semiempirical

calculations; 3) excited state dipole moments are always greater than the ground state

moments and point in the same direction; 4) transition moments are almost parallel

79

for both the ground and excited state dipole moments (i.e. charge transfer transitions);

5) predicted absorption of coumarin B is slightly blue-shifted from coumarin A, in

agreement with experiment; and 6) the dipole moment change (either ∆µ or µ) is

relatively small compared to most experimental values.13,14,15

Table 3.1 Characteristics of the ground and the first excited state of coumarin molecules calculated with semiempirical method (HyperChemPro 6), AM1, with configurational interactions using 16 orbitals.

AM1 ZINDO/S

µga µexc

b ∆µc µd λmaxe ∆Ef µg

a µexcb ∆µc µd λmax

e ∆Ef

g 6.0 8.0 2.0 5.3 370 7.8 9.2 1.4 4.9 327 Coumarin h 6.2 8.4 2.2 5.7 371 -0.13 7.9 11.3 3.4 8.1 330 5.5

g 6.2 11.0 4.8 9.1 389 7.3 11.3 4.0 8.6 346 Coumarin 153 h 6.4 11.2 4.8 9.2 389 0.05 7.5 11.6 4.1 8.8 350 2.8

g 6.2 8.0 1.8 5.1 368 8.3 11.1 2.8 7.4 323 Coumarin 460 h 6.3 8.4 2.1 5.6 369 -0.15 8.4 11.5 3.1 7.9 324 5.5

g 6.8 9.5 2.7 6.6 377 9.2 12.1 2.9 7.9 352 Coumarin A h 7.0 9.9 2.9 7.0 380 0.01 9.3 12.3 3.0 8.0 355 4.3

g 6.2 9.0 2.8 6.5 374 7.3 9.6 2.3 6.2 350 Coumarin B h 6.4 9.5 3.1 7.0 379 -0.03 7.5 10.3 2.8 7.1 353 5.0a ground state dipole moment in Debye; b excited state dipole moment in Debye; c dipole moment change: ∆µ = µexc - µg, in Debye; d effective dipole moment change according to Equation 3.3, in Debye; e absorption wavelength of the lowest allowed transition in nm, f energy difference between syn- and anti- conformations in kcal/mol, g syn-conformation of diethylamine, h anti-conformation of diethylamine.

The discrepancy should not be surprising since experimentally measured are

not the gas phase dipole moments that were obtained theoretically, as illustrated in

Equation 3.12. One can mimic the experimental situation better by applying Equation

3.12 with calculated polarizability of coumarin. Using ZINDO/S at electric field

80

close to the anticipated reaction field (~0.001 a.u.) excited state dipole polarizability

along its main axis was calculated α = 73 Å3 and 75 Å3 for coumarin-B and

coumarin-A, respectively. Using Equation 3.12 with these values of α and taking

molecular radius ca. 5.4 Å, one obtains over 25% increase in the dipole moment in

toluene. Additional correction arises from nonsphericity of the molecule, it is slightly

of a prolate shape.

One can conclude that the change in dipole moment amplitude:

∆µ =µexc -µg, (3.25)

can be scaled similarly to that of µ, i.e. values in Table 3.1, ∆µ (gas) ~ 3 D, should be

multiplied by 11 D/7 D, making expected value in solution, ∆µ (solution) ~ 4-5 D.

3.4.3 Photoinduced charge transfer in self-assembled coumarin monolayer

Figure 3.9 shows photovoltage signals from coumarin SAM immobilized onto

three different substrates--glass slides, mica and silica slides. All three substrates

have surface hydroxyl groups that were utilized for coumarin SAM construction.

The signals were normalized to the number of substrates and incident energy used in

the experiment. Thus, the amplitude of each signal corresponds to the signal obtained

from one substrate.

Figure 3.9 shows that the biggest amplitude of the dipole signal was obtained

on glass, whereas silica substrate gave the smallest amplitude. However, the

quantitative evaluation of the PTDC technique requires information about

81

0 5 10 15 200.00

0.05

0.10

0.15

0.20

A B C

Pho

tore

spon

se (m

V)

Time (ns)

Figure 3.9 SPTDC dipole signals (normalized for the same incident laser energy) for coumarin immobilized on one side of different types of substrates in ethanol: A--glass, B--mica and C--quartz.

concentration of molecules and their orientation. The amplitude of the “dipole

signal” depends on the number of excited molecules as well as on the average angle

of their orientation with respect to normal axis of the surface. The necessary

information can be obtained from absorption spectra. Only quartz substrates possess

high transparency in the region of interest to use for aforementioned evaluation.

82

The first surprising outcome of experiments with coumarin SAM was that the

dipole signal had much shorter decay, indicating that the lifetime of the excited state

was shorter than for the same coumarin molecule in toluene solution. We

hypothesized that fast rotation of the amino group in the 7-position of coumarin,

which results in the formation of twisted intramolecular charge transfer (TICT)

state,14 might be a reason for time shortening. In order to verify the hypothesis, we

measured SPTDC dipole signal in solvents of various polarity and viscosity aiming at

two questions. First, the increase of solvent viscosity could suppress charge

transitions induced by conformational changes, such as amino-group rotation.

Second, solvent polarity stabilizes either intramolecular charge transfer (ICT) or

TICT states, due to their higher polarity character.14

Figure 3.10 shows SPTDC dipole signals of coumarin SAM in glycerol

(dielectric constant, ε = 42.5, viscosity – η = 945 cP), squalane (ε = ~2, η = 116 cP),

and in ethanol (ε =24.5, η = 1.08 cP). As one can see, the dipole signal is short lived

in all solvents regardless of their viscosity or polarity. This indicates that the

conformational changes have minimal effect on the form of the signal.

Fluorescence measurements from coumarin SAM led us to the conclusion that

the observed effect of the lifetime shortening should be attributed to stimulated

emission of molecules in monolayer. Figure 3.11 shows fluorescence spectra of

coumarin SAM measured parallel to the substrate surface at different laser energy

densities: 33, 200, 250 µJ/cm2 at 396 nm. Two slides were in contact by their stained

sides were placed in a 1 cm quartz cuvette vertically at 45o with respect to the

83

0 5 10 15 200.00

0.04

0.08

0.12

0.16

0.20

Pho

tore

spon

se (m

V)

Time (ns)

ethanol glycerol squalane

Figure 3.10 Dipole signals of coumarin immobilized on one side of glass substrate in ethanol (dash-dot), glycerol (solid) and squalane (dash).

incoming laser beam. To minimize refractive scattering, the gap between the slides

was filled with toluene. The liquid served not only as a refractive index matching

fluid but also minimized potential photooxidation of coumarin. Low pass 435nm

glass filter (absorption is shown by the dashed line, F, in Figure 3.9) was used to

screen out the laser scattering when measuring fluorescence spectra. Due to this fact,

the high-energy wings of the spectra are partially cut off. On the other hand, the

84

relative suppression of the low energy wings of the fluorescence spectra indicates the

redistribution of spectral energy.

400 450 500 550 600 6500

10

20

30

40

50

60

0 50 100 150 200 2500

2

4

6

8

0

1

2

3

Wavelength (nm)

F

33 µJ/cm2

200 µJ/cm2

250 µJ/cm2

Abs

orba

nce

Fluo

resc

ence

Inte

nsity

(arb

. uni

ts)

Rel

ativ

e in

tens

ity

Energy density, µJ/cm2

58

60

62

FW

HM

(nm

)

Figure 3.11 Normalized fluorescence spectra of two quartz slides with self-assembled coumarin-B monolayers measured parallel to the slides.

As one can see in Figure 3.11, the observed emission intensity is relatively

linear function of the incident laser energy but the spectra are narrowed with

increasing energy. The narrowing is manifested by the decrease of the full width at

half maximum (FWHM)--63, 61 and 59 nm, respectively. The effect of spectra

narrowing is not very dramatic and does not noticeably vary with light polarization.

85

Nevertheless, the assignment of the effect to stimulated emission is supported by

pronounced changes in the time resolved fluorescence.

Figure 3.12 illustrates that fluorescence decay from coumarin SAM has two

components, fast and slow. For the time resolved fluorescence measured parallel to

the film’s surface, ||, the decay is fast (B). For the same intensity of incident energy

(250 µJ/cm2), fluorescence kinetics “perpendicular” to the surface, ⊥, has two clearly

distinguishable components (A): the fast one is similar to that for the parallel

orientation, and the slow one, resembling fluorescence via only spontaneous

emission. Measurements at lower intensities could provide additional proof of the

stimulated emission interpretation (only slow fluorescence component would be

expected). Unfortunately, it was not possible due to the sensitivity limitations.

Table 3.2 Fluorescence properties of dyes in toluene solution under different excitation densities.

Excitation energy (µJ/cm2) Molecule 33 80 250

τF (ns) 3.6 3.1 2.7 λmax (nm) 453 454 455

Coumarin-B

∆λ (nm) 37 36 32 τF (ns) 5.3 5.0 4.7 λmax (nm) 492 494 494

Coumarin 153

∆λ (nm) 55 37 17

86

0 5 10 15 20

0

20

40

60

||

⊥B

A

Fluo

resc

ence

inte

nsity

Time (ns)

Figure 3.12 Time-resolved fluorescence kinetics for the same sample as in Figure 3.9 recorded at different geometries.

Nevertheless, we are confident in the interpretation because the same phenomenon of

stimulated emission was observed in liquid solutions of the same molecule at similar

conditions. Figure 3.13 illustrates that in toluene solution of coumarin-B spectrum

narrowing was observed at similar excitation intensities as in the case of SAM. The

same low pass 435nm glass filter was used for screening out the laser scattering.

87

420 440 460 480 500 520 5400

25

50

75

100

33 µJ/cm2

80 µJ/cm2

250 µJ/cm2

Fluo

resc

ence

Inte

nsity

(arb

. uni

ts)

Wavelength (nm)

Figure 3.13 Normalized fluorescence spectra of ~2×10-5 M coumarin-B in toluene solution.

However, since the maximum of fluorescence of coumarin-B is red-shifted in

solution as compared to the blue-shifted in surface immobilized molecules, now one

can see suppression of both low-energy and high energy wings of fluorescence

spectrum. The absorbance of coumarin-B in solution was A396 = 0.9 at 396 nm,

which corresponded to the concentration of coumarin ca. 2×10-5 M.

The fluorescence kinetics also indicated an enhanced rate of deactivation due

to stimulated emission. Shortening of the fluorescence lifetime parallels the spectra,

narrowing upon increase of the excitation density (Table 3.2).

88

440 480 520 560 6000

20

40

60

33 µJ/cm2

80 µJ/cm2

250 µJ/cm2

Fluo

resc

ence

Inte

nsity

(arb

. uni

ts)

Wavelength (nm)

Figure 3.14 Normalized fluorescence spectra of ~2×10-5 M coumarin 153 in toluene solution.

The demonstrated effect of stimulated emission from a single monolayer of

coumarin SAM can be noticeably improved by making a multilayer assembly and by

a better choice of dye. For example, a popular dye coumarin 153 showed much more

dramatic nonlinear behavior at the same conditions. Superior high intensity

narrowing of coumarin 153 (see Figure 3.14) correlates to its greater fluorescence

quantum yield due to a structurally rigid amino group.15

High molecular concentration of coumarin in SAM causes modification of its

optical properties, including enhanced excitonic interaction between molecules and

89

probably greater rates of energy transfer. Surprisingly, high density of SAM allowed

observation of stimulated emission from the films upon laser excitation even without

a resonator cavity.

To the best of our knowledge, lasing from self-assembled organic monolayers,

such as coumarin SAM, was not demonstrated before. The approach can offer an

interesting route for developing SAM dye lasers.

3.4.4 Charge transfer in coumarin monolayer

Preceding section discussed the lifetime shortening of excited coumarin

molecules assembled in monolayers. While the characteristic time of the “dipole

signal” decay reflects primarily the lifetime of charge transfer species, the amplitude

characterizes the extent of charge separation.

For coumarin-B molecule the direction of dipole moment does not change

significantly upon excitation. According to AM1 calculations for coumarin-B, dipole

moments of ground, excited state, and the transition moment are all within ~7°.17

First of all, we showed that the origin of the photovoltage signal arises from

intramolecular charge transfer. The simplest way to do that is to change the direction

of charge separation. Due to the unidirectional asymmetry of molecular orientation

induced by the surface, the direction of current displacement can be changed by

flipping the substrates inside the cell. This resulted in the opposite sign of the signal

and no change in amplitude.

90

lhv

solvent

ITO

R

d

l

hv

ITO

R

d

solvent

0 5 10 15 20-0.10

-0.05

0.00

0.05

0.10Ph

otov

olta

ge (m

V)

time (ns)

Figure 3.15 Dependence of the photovoltage signal on orientation of SAM inside the cell.

Figure 3.15 shows the two signals obtained for two different geometries, with

stained sides of substrates facing different electrodes. As one can see, the signals are

almost mirror images of each other with respect to zero line indicating that the origin

of photovoltage is indeed due to intramolecular charge transfer in surface bound

coumarin.

91

0 5 10 15 20

0.0

0.1

0.2

0.3

0.4

0.5

Phot

ovol

tage

(mV)

time (ns)

Figure 3.16 The photovoltage signal for 6 silica substrates with coumarin SAM immersed in toluene. The dashed line is the laser pulse, dash-dot line is the experimental curve and the solid line is the best fit. The slow decaying part at long time is due to the triplet states populated via intersystem crossing.

Figure 3.16 illustrates SPTDC signal from 6 substrates with coumarin SAM

immersed in toluene. The best fit provided <∆µ⊥> = 0.3 D and τF = 0.5ns. We also

introduced into the fitting scheme additional route with intersystem crossing to the

triplet state, which is realized in the long-lived component of the signal. Presuming

the same <∆µ⊥> for triplets, the yield was estimated to be ~4%, which reasonably

agrees with reported triplet yields for aminocoumarins.15 Under the assumptions of

92

equation 3.17 and using ∆µ from Table 3.2, the value of <∆µ⊥> in solution can be

estimated as 3/8∆µ = 1.5 – 1.9 D. The discrepancy with experimental value is not as

dramatic in ethanol, while in toluene and hexane the disparity is significant (see Table

3.3). This correlates with a greater degree of parallel to the surface orientation

observed optically in nonpolar solvents (Chapter 2). Unfortunately, even without any

rotation of coumarin, the second moment of angular distribution obtained in optical

measurements (A ∝ <cos2θ>) cannot be quantitatively related to the first moment of

distribution in SPTDC (<∆µ⊥> ∝ <cosθ>). For example, molecules oriented at

average 60° and 120° with respect to the normal would show the same absorption

anisotropy. On the other hand, their dipole signal would be distinguished: 120°-

average dipoles would give rise to the signal of the same amplitude but with negative

sign when compared to that of 60°.

In any case, the overall comparison for SPTDC of coumarin’s SAM in

solution fares well with anticipated values for ∆µ.

A greater “discrepancy” was observed when dipole signal was measured

without a solvent: the signals were negative and dependent on gas! Figure 3.17 and

Table 3.3 illustrate this behavior. As was discussed above, the polarity of signal is

defined by the direction of charge separation in monolayer (direction of the dipole

moment change). Thus in the gas phase, dipoles in the SAM have to be oriented in

the opposite direction despite being tethered to the surface by one end through a

linker. As discussed in Chapter 2, the greatest degree of parallel orientation of

93

coumarin was observed in air but it is not possible to separate optically between

orientations along and opposite to the normal axis of the surface.

Table 3.3 Experimental values fitting parameters for SPTDC dipole signal of coumarin in different environment.

Vacuum Ar N2 He Hexane Toluene Ethanol

<∆µ⊥> (D) -2.4 -1.9 -1.5 -0.9 ~0.07 0.3 1.1

τF, nsa 0.2 0.2 0.4 0.4 0.5 0.75

τisc, nsb 10 17

τcol, nsc ∞ 0.86 0.67 0.27

a lifetime of singlet state; b intersystem crossing time; c collision time with surface estimated using equation 3.19

As one can see from Figure 3.17, dipole signals measured in various gaseous

media, helium (ε = 1.00015), nitrogen (ε = 1.00059), argon (ε = 1.0057), as well as

under vacuum all have negative signs but different amplitudes. The effect of medium

on the sign and the amplitude of the dipole signal is reproducible, its amplitude as

well as the sign restored after changing solvents or drying the substrates and changing

gas during the experiment. Also noteworthy, that the magnitude of <∆µ⊥> increases

with solvent polarity but the effect of gas has no correlation with the dielectric

constant.

The phenomenon is not fully understood but it is certain that dipole

distribution, at least in gas phase, is nowhere close to a positive semisphere.

94

0 5 10 15 20

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5HexaneEthanolToluene

He

N2

Ar

Vacuum

Phot

ovol

tage

(mV)

time (ns)

Figure 3.17 Normalized dipole signals covalently bound coumarin-B on silica substrates in toluene, ethanol and hexane as well as in the atmosphere of different gases (argon, nitrogen and helium) and under vacuum.

Apparently, the linker’s length and the surface roughness (as little as a few Å)

provide sufficient room and flexibility for molecules to find the lowest energy

orientation, which without solvent happens to have coumarin aminogroup pointing to

the surface as shown in Figure 3.18. Such an orientation is supported by van der

Waals interactions with the surface. To corroborate this interpretation, noncovalently

95

deposited coumarin 460 was studied. It was deposited on quartz substrates from

ethanol solution with surface density close to that of covalently tethered coumarin-B.

It was achieved by adjusting concentration of coumarin 460 in the solution. The

dipole signal from the deposited layers of coumarin 460 showed similar negative

signal proving that there is a net asymmetry with opposite change in dipole moment.

The biased molecular orientation is provided by preferential physisorption of

coumarin 460 with aminogroup attracted to surface hydroxyl groups, as shown

schematically in Figure 3.18 together with that for coumarin-B. Both covalently

linked coumarin-B and physisorbed coumarin 460, have similar orientation in gas

phase which is opposite to that in solution. Arrows in Figure 3.18 represent

directions of dipole moments. It appears that the presence of the amino group in

coumarins plays significant role in the behavior of this molecule in monolayer.

Strong van der Waals interactions between aminogroup and surface hydroxyls can be

a reason they become oriented in the opposite direction with no solvent present.

One can see from Figure 3.17 that without solvent, the highest dipole signal

amplitude is in vacuum and decreases for lighter gases. Interestingly enough, the

average time between collisions at ambient conditions (680 torr in Las Cruces) is

comparable with the lifetime of coumarin’s excited state, and the rate of collisions

between gas molecules and the surface increases in the same order as the decline of

the dipole signal, as shown in Figure 3.19. The time between collisions was

calculated using Equation 3.26:

96

Tkm

nssn BTcol

πτ 21v4

== (3.26)

where s is the area occupied by one coumarin molecule, n--the density of gas

molecules, m--their mass, and vT--their average velocity. Assuming s = 50 Å2, which

is the area per molecule for the surface coverage of coumarin molecules 2×1014 cm-2,

and n = 2.4 ×1019 cm-3 (for 680 torr), one calculates τcol listed in Table 3.3 and in

Figure 3.19.

Based on this, one can conclude that there is some relaxation process involved

in signal evolution. It could be either intramolecular relaxation, transition between

states with different dipole moments, or “intermolecular” one, reorientation of

molecules with respect to the surface without significant change of their dipole

moments. Currently, we cannot unambiguously choose between them. Nevertheless,

semiempicial calculations had no indication of any excited state with either small or

opposite dipole moment in the excited state but relaxation to the ground state cannot

be discarded.

If one embraces the origin for negative signal as being to hydrogen bonding of

diethylamine end of coumarin with (hydroxylated and aminated) surface, it would

seem logical to extend the interpretation further and presume that increased charge

transfer from amine causes weakening of its hydrogen bonding and reorientation.

The latter requires effective energy exchange that is brought by colliding with

gaseous molecules.

97

O

O

N

CH3

O

O

N

CH3

Si

OH

SiO2Si

OH

Si

OH

Si

OH

Si

O

SiOHOH

NH O

O

N

O

B

C

O

O

N

CH3O

O

N

CH3

A

Si

O

SiOH OH

NH

OO

N

O

Si

O

SiOHOH

NH

OO

N

O

Si

OSiOH OH

NH

O

O

O

N

Si

OSiOH OH

NHO

O

O

N

Si

O

SiOH OH

NH O

O

O

N

Figure 3.18 Schematic representation of orientation of coumarin molecules in covalently linked layer of coumarin-B: in solution (A) and in gas (B), as well as physisorbed coumarin 460 (C).

98

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

-2.5

-2.0

-1.5

-1.0

-0.5

Ar

N2

He

Vacuum

<∆µ ⊥

> (D

)

Rate of collisions, ns-1

Figure 3.19 Dependence of average dipole moment change on collision rate for different gases.

3.4.5 Photoinduced charge transfer in coumarin- in the presence of oxygen.

7- aminocoumarins are well-known laser dyes.18 This is because of their high

fluorescence efficiency and low intersystem crossing yield into triplet state under

ordinary conditions. However, presence of molecular oxygen can induce intersystem

crossing, since molecular oxygen is a paramagnetic species (3O2) in its ground state.19

99

The yield of fluorescence quenching and triplet yield depend on the frequency of

collisions, and therefore to the concentration (pressure) of oxygen in the media.

The following scheme represents competitive routes of deactivation for

excited singlet state of coumarin molecule:

kq[O2]

1C 1C* + 3O2

hv

k F

3C* + 1O2* (3.28)

1C + hv (3.27)

3C* + 3O2 (3.29)

Coumarin can either fluoresce with rate constant kF (reaction 3.27) or convert to a

triplet state whenever collision with oxygen occurs with rate kq[O2] (reaction 3.29).

Reaction 3.28 is not energetically feasible, since S-T gap in coumarin is smaller than

the excited state energy of 1O2*. Figures 3.20 and 3.21 show the dipole signals of

coumarin B and coumarin 460 deposited on quartz substrates in air and in oxygen.20

Two distinct components can be seen--“fast” due to the singlet excited state and

“slow” due to the triplet state. Decay time of the slow component is supposed to be

equal the RC time of the circuit. The experimental value agrees well with the

calculated one using cell capacitance and stray capacitance of 4.3 pF.

It is clear from the Figures that the more oxygen is present, the greater

contribution of the slow component appears in the kinetics. The triplet yield, ϕT, due

to intersystem crossing induced by collisions with oxygen can be described with

following equation:

100

0 50 100 150 200

0.0

0.1

0.2

0.3

0.4

O2

Air

Phot

ovol

tage

(mV)

Time (ns)

Figure 3.20 Photovoltage signals from coumarin-B in air (0.21 atm of oxygen) and in flow of pure oxygen (1 atm) after excitation by 416 nm from Raman shifted laser pulse. The best fits correspond to τF = 0.5 ns, and τisc = 2.5 ns and 16 ns for pure oxygen and air, respectively. Both signals were fitted with τRC = 173 ns.

2

2

OqF

OqT pkk

pk+

=ϕ (3.30)

where τF is fluorescence time and 2

1

Oqisc pk

=τ is the fluorescence quenching time

of coumarin by oxygen that induces intersystem crossing.

101

The observed apparent difference in behavior of two coumarins may be

explained by competitive nature of the deactivation pathways. It resembles

previously observed effect of oxygen induced intersystem crossing in solution.19

Fluorescence and quenching due to the oxygen induced intersystem crossing have

comparable rates for coumarin dye molecules. Thus, the fluorescence time would

have a significant impact on how much triplet would form. When intersystem

crossing rate is greater than the rate of fluorescence, more triplet states form within

the lifetime of the excited singlet state (coumarin 460). Whereas, for short lived

species, such as coumarin-B, deactivation via fluorescence would prevail because

intersystem crossing due to collisions with oxygen is not fast enough to compete with

the fluorescence. Therefore, less number of molecules undergoes intersystem

crossing and fluorescence becomes main deactivation pathway.

Since coumarin B and coumarin 460 are similarly sized molecules, one

would expect the same rate constant for oxygen induced intersystem crossing (with

the rate constant kq) and be close to the rate constant of collisions, kcol. The latter can

be calculated similarly to equation 3.26, as follows:

sk Tcol 4

v= (3.31)

where s is the area occupied by one coumarin molecule and vT is the average velocity

of oxygen. Assuming s = 50 Å2, which is the area per molecule for the surface

coverage of coumarin molecules 2×1014 cm-2, and taking vT = 4.45 ×104 cm/s, one

calculates kcol = 1.9×106 s-1torr-1 (collision frequency vcol = 1.3×109 s-1 at 680 torr of

pure oxygen).

102

0 50 100 150 200

0.0

0.1

0.2

0.3

0.4

O2

AirPhot

ovol

tage

(mV)

Time (ns)

Figure 3.21 Photovoltage signals from coumarin-460 in air (0.21 atm of oxygen) and in pure oxygen (1 atm) after excitation with nithrogen laser at 337 nm. The best fit curves were calculated with τF = 4.3 ns, and τisc = 2.5 ns and 16 ns for pure oxygen and air, respectively. Both signals were fitted with τRC = 173 ns, corresponding to overall capacitance of 8.7 pF almost equally split between calculated cell capacitance and measured stray capacitance (4.3 pF).

The resulting value is the high limit estimate since the area of a single

coumarin molecule is close to 50 Å2 only when it stretches out flat on the surface.

The experimental quenching rate in pure oxygen is ~4×108 s-1 (kq =6×105 s-1torr-1),

for both coumarin 460 and coumarin-B.

103

As was discussed before,19 the intersystem crossing induced by a radical

(oxygen) results in a formation of transition state that is an equilibrium between two

complexes of oxygen with coumarin either in its singlet, 1C*, or triplet, 3C**, state.

Two asterisks for 3C** refer to excess vibrational energy in the triplet. Both

complexes identified in reaction 3.33 have overall triplet state and possess the same

spin projections, thus allowing interconversion between them. If vibrationally excited

triplet, 3C**, relaxes (with the constant krelax), according to reaction 3.32, faster than

oxygen escapes from the complex (reaction 3.33), then all complexes transform into

3.32 and the overall intersystem crossing rate becomes equal to the rate of encounters.

Otherwise, when kdis is faster than krelax, only half of 1C* become 3C*.

3 (3C* + 3O2) (3.32)

3 (1C* + 3O2) 3 (3C** + 3O2) (3.33)kdis

1C* + 3O2 3 (3C* + 3O2) (3.34)

krelax

kdis

Experimental rate constants are close to half of the collision rate constant, kq/

kcol ~ 0.4, which would be expected for a very brief encounter between coumarin and

gaseous oxygen (kdis >> krelax).

This example clearly demonstrates intramolecular contribution to relaxation in

excited states of surface bound coumarins. Oxygen plays here a role of spin

scrambler that allows intersystem crossing by virtue of its nonzero spin. As a result,

104

intersystem crossing proceeds with conservation of total spin (and projection)

yielding the triplet excited state only in half of the cases, due to spin statistics.

3.5 Conclusions

1) High surface concentration of coumarin molecules covalently immobilized

on the surface in the form of self-assembled monolayer was sufficient for observing

stimulated emission from optically excited SAM. The stimulated emission of excited

coumarin resulted in the lifetime shortening and fluorescence spectrum narrowing for

molecules in monolayers without resonant cavity. The effect of stimulated emission

from coumarin monolayer correlated with similar phenomenon in solution.

Coumarin SAM can be applied for constructing miniature devices with lasing action.

2) Surface assisted photoinduced transient displacement charge technique

(SPTDC) was developed and employed for studying charge transfer processes in

coumarin molecules assembled into oriented layers on solid surfaces such as silica,

glass and mica. Effect of the solvent above SAM was investigated and compared

with theoretical predictions and measurements in solution PTDC.

3) Theoretical basis for evaluating the dipole signal in SPTDC technique was

suggested that would allow quantitative analysis of the charge transfer dipole moment

change and its kinetics in SAM.

4) Surprisingly, gaseous media exhibited a profound effect on covalently

immobilized coumarin molecules in a monolayer--the orientation was opposite to that

105

in solution and depended on gas. The amplitude declined with increasing the rate of

collisions, suggesting that there is a relaxation mechanism most likely due to

reorientation of excited molecules. The presence of oxygen leads to formation of

long-lived triplet (also dipolar) state of coumarin with the rate constant close to half

of the collision rate in agreement with spin scrambling mechanism with short lived

transition state.

3.6 References

1 Smirnov, S.N., Braun C.L., J. Phys. Chem., 1994, 98, 1953-1961. 2 Mylon, S.E., Smirnov, S.N., Braun C.L., J. Phys. Chem., 1998, 102, 6558-6564. 3 Smirnov, S.N., Braun C.L., Anker-Mylon, S.E., Grzeskowiak, K.N., Greenfield, S.L., Wasielewski, M.R., Mol. Cryst. Liq. Cryst., 1996, 286, 243-248. 4 Smirnov, S.N., Braun C.L., Rev. Sci. Instrum., 1998, 69(8), 2875-2887. 5 Smirnov, S.N., Liddell, P.A., Vlassiouk, I.V., Teslja, A., Kuciauskas, D., Braun, C.L., Moore, A.L., Moore, T.A., Gust, D., J. Phys. Chem. A, 2003, 107, 7567-7573. 6 Onsager, L., J. Am. Chem. Soc., 1936, 58, 1486-1493. 7 Taylor, D.M., Bayes, G.F., Phys. Rev. E, 1994, 49(2), 1439-1449. 8 Böttcher, C.J.F., Theory of Electric Polarization, Elsevier: Amsterdam, 1973, Vol I, Chapter 1. 9 Vlassiouk, I., Smirnov, S., J. Phys. Chem. A, 2003, 107, 7561-7566. 10 Demchak, R.J., Fort, Jr, T.J., J .Colloid Interface Sci., 1974, 46, 191. 11 Out of the two active probes from Tektronix, P6249 active probe with 20kΩ input impedance and 4 GHz bandwidth was chosen over P6245 active probe, with 1MΩ

106

input impedance and 1.5 GHz bandwidth. P6249 probe provided better signal to noise ratio due to a lesser attenuation factor, 5X, instead of 10X for P6245. 12 The fitting protocol uses the Marquardt optimization technique (ref. 4). 13 Moylan, C.R., J. Phys. Chem., 1994, 98, 13513-13516. 14 Rehthaler, K., Köhler, G., Chem. Phys., 1994, 189, 99-116. 15 Samanta, A., Fessenden, R.W., J. Phys. Chem. A, 2000, 104, 8577-8582. 16 Del Bene, J., Jaffe, H.H., J. Phys. Chem., 1968, 48, 1807. 17 According to AM1 calculations, the dipole moments for coumarin-B are: ground state dipole moment, µg = 6.4 (-1.36, -5.99, -1.79), and excited state dipole moment, µexc = 9.1 (-0.88, -8.47, -3.1). The angle between two vectors was calculated using

following equation: zyxzyx

zzyyxx

VVVVVV

VVVVVVVVVV

22

22

22

12

12

12

212121

21

21cos++⋅++

⋅+⋅+⋅=

⋅⋅

=λ

18 Sahyun, M.R.V., Sharma D.K., Chem. Phys. Lett., 1992, 189, 571-576. 19 Smirnov, S., Vlassiouk, I., Kutzki, O., Wedel, M., Montforts, F.P., J. Am. Chem. Soc., 2002, 124, 4212-4213; Vlassiouk, I., Smirnov, S., Kutzki, O., Wedel, M., Montforts, F.P., J. Phys. Chem. A, 2002, 106, 8657-8666. 20 Here and further all dipole signals in gaseous phase are inverted to positive for the convenience.

107

4 CHARGE TRANSFER IN DNA: STUDY OF 2AP QUENCHING IN SHORT

OLIGONUCLEOTIDES

4.1 Introduction

Since early investigations of Barton et al.1 nearly two decades ago the

conductive properties of DNA have been under intensive study. The possibility to

use DNA as a conductive wire has stirred a great deal of controversy and launched

numerous research projects. Various techniques were introduced to probe the charge

transfer properties of DNA. Among them, fluorescence quenching is the most

common method in studying photoinduced charge transfer (CT) between electron

donor and acceptor. Various types of intercalators as well as end-capping fluorescent

molecules have been used in these studies. Nevertheless, donor and acceptor

covalently bound to the duplex are believed to provide more reliable results because

of minimal disturbance of DNA structure. However, even in these systems the results

were not always consistent. The controversial value of the exponential fall-off

parameter, β, in the coupling matrix element of Equation 1.5 has been reported

varying from as low as 0.14 Å-1 to as high as 1.4 Å-1.1,2 The value of β has been

shown to depend on the base sequence,3 duplex conformation (A, B or Z)4,5 and

subtle structural perturbations.6

108

4.1.1 Models of charge transfer in DNA

It has come to a consensus that DNA double helix can facilitate relatively

efficient charge transfer over substantial distances. The question of how electrons

migrate through DNA has prompted a considerable interest and generated a great deal

of controversy. Various models for the mechanism of charge transfer in DNA have

been proposed recently in order to explain the experimental data.

The superexchange mechanism implies a single step charge tunneling between

the localized donor and acceptor sites.2,7 In this mechanism the charge does not

physically reside on the DNA bridge during the charge transfer process. Owing to

this exponential distance dependence (see Equation 1.5), only a short-range electron

transfer is expected to be effective.

A mechanism of hopping was introduced to explain a weak distance

dependence of charge transfer in DNA observed experimentally by Giese.8 This

mechanism implies a multistep process in which the charge “hops” between localized

sites (base pairs) in a random walk fashion until it reaches the acceptor.9,10 In the

multistep hopping mechanism the charge migration rate depends weakly on distance

and it is usually described by the following equation:9

Nln -ln η∝CTk (4.1)

where N is the number of hopping steps and parameter η is between 1 and 2 for an

acceptor-direction-biased random walk process. Each single step (hop) is viewed as

an individual superexchange process.

109

Jortner and coworkers proposed a generic mechanism of charge transfer as a

mixture of the two: it changes from super-exchange tunneling at short distances, to

multistep hopping at long D-A separation.7,11

The phonon-assisted polaron hopping mechanism was proposed by Schuster.12

It implies the existence of the so-called polarons, where the hole wavefunction and

the accompanying nuclear distortion is spread over a number of bases. The charge

moves by hopping from one set of bases to another set of different bases provided

that they have similar (or lower) oxidation potentials. This process was termed

“phonon-assisted polaron-like hopping”.

Another model of charge propagation along DNA has been recently proposed

by O'Neill et al.4 It suggests that charge moves by hopping between “domains”. The

formation of these domains is strongly dependent on conformational dynamics and

the structure of nucleobase sequences. DNA is a dynamic molecule, which possesses

a great degree of conformational freedom even at low temperatures. Dynamic

processes can affect donor/acceptor coupling as well as the interactions between D-

bridge and A-bridge units by alteration of their relative position and orientation. The

bridge dynamics, thus, should be taken into account in the mechanistic description of

charge transfer in DNA.

110

4.1.2 2-aminopurine in studies of photoinduced charge transfer in DNA

2-aminopurine (2AP) is one of a few known fluorescent analogs of natural

nucleobases. It has gained its popularity in studying DNA structure, dynamics as

well as electron transfer through DNA due to its unique properties.13,14,15 Owing to

its structural similarity with adenine, 2AP forms two hydrogen bonds with

complementary thymine, and is well stacked inside DNA duplex without distortion of

its structural integrity.16 Due to a large area and its aromatic character, 2Ap is

inclined towards stacking with nucleobases, especially purines, adenine and guanine,

which have the highest stacking ability.17,18 More importantly, 2AP has a high

fluorescence yield (Φ = 0.68 in solution) and its absorption inside DNA duplex is red-

shifted (λmax ~ 320 nm) from that of natural bases (ca. 260 nm), which conveniently

allows selective excitation and fluorescence measurements of 2AP.19

Fluorescence of 2AP* is quenched when the molecule is incorporated into

DNA. Since the energy transfer from excited 2AP* to other bases is not feasible, the

fluorescence quenching is attributed to charge transfer between 2AP and neighboring

bases.20 Based on the oxidation potentials of nucleobases (G=1.29V < A=1.42V <

C=1.6V < T=1.7V vs NHE)21 and the reduction potential of 2AP* (1.5V vs NHE),1

one would expect a positive driving force for 2AP* quenching via hole transfer to

purines. Pyrimidines are more easily reduced (G=-2.76V<A=-2.45V<C=-2.14V<T=-

2.14V vs. NHE)22 and thus quench 2AP* via electron transfer.

111

Recently, Zewail et al.23 used femtosecond transient absorption and

fluorescence up-conversion to observe a reduction in fluorescence time for 2AP*

upon its complexation with all natural nucleobases and inosine. Based on the

measured rates of 2AP* decay, the authors suggested that both electron transfer (ET)

and hole transfer (HT) mechanisms of 2AP* fluorescence quenching were possible.

They estimated the free energies for hole transfer from 2AP* to G and A: ∆GHT 2AP*-G

= -0.53eV, ∆GHT 2AP*-A = -0.3eV, and for electron transfer to T and C: ∆GET 2AP*-T = -

0.54eV, ∆GET 2AP*-C = -0.45eV, and proposed that adenine and inosine may exhibit

ambivalent behavior owing to their redox properties. The same experimental method

was used to study kinetics of 2AP* fluorescence quenching in a series of DNA

duplexes.24 The decay of excited 2AP* was found to depend on composition of the

oligonucleotide, in particular on the position of guanine with respect to 2AP. The

decay of 2AP* becomes fast (ca. 10 ps lifetime) with guanine located next to 2AP,

and it slows down gradually with increasing distance between G and 2AP. The

authors confirmed that the charge transfer can occur through both oxidative and

reductive reaction of 2AP* with nucleobases depending on their redox properties.23

Free 2AP* in solution has a single exponential fluorescence decay with

characteristic lifetime ~ 10 ns.25 Upon incorporation of 2-aminopurine into DNA its

decay kinetics becomes heterogeneous, which was attributed to variations of 2AP

stacking with neighboring bases.25 As many as four time constants were needed for

describing decay kinetics of 2AP. These time constants lie in a range from few tens

of picoseconds, < 50 ps, up to ~ 10 ns. The short decay time of 2AP* in DNA duplex

112

was attributed to a fully stacked 2-aminopurine, while the longest time was assigned

to an unstacked state.26 Zewail and coworkers attributed the shortest component in

the 2AP* decay to the optimal charge transfer conformation.23 Larsen et al. also

observed large amplitude of short decay components (a few tens of picoseconds),

which they assigned to hole transfer from excited 2AP to guanine.14

The fluorescence decay of 2AP has a complex nature even in dinucleotides27

and trinucleotides,28 where 2AP is flanked by purine and pyrimidine bases. Both

studies found that at least four time constants were required to describe the

multiexponetial behavior of 2AP* fluorescence decay, similar with previously

observed heterogeneity in larger oligonucleotides. However, Jean et al. 28 did not

observe the long lifetime component (~10 ns) in any of the studied trimers. They

attributed the observed complexity to rapid stacking-unstacking motions of 2AP with

respect to the neighboring bases, occuring on picosecond to nanosecond timescales.

These fast conformational fluctuations reflect the competition between molecular

motions and charge transfer in stacked conformation.28

4.1.3 Base fluctuations and its influence on charge transfer in DNA

Previously, Wan et al. suggested that molecular motions within DNA duplex

may play an important role in regulating the rate of charge transfer.29 Their study of

charge transfer between intercalated ethidium bromide and deazaguanine revealed

that the ultrafast charge transfer has two time constants, 5 ps and 75 ps. The former

113

one has been assigned to direct charge transfer. It was concluded from the anisotropy

measurements that 75 ps component corresponded to a CT process, which required

reorientation of the reactants before charge transfer.

O’Neill et al.30 compared charge transfer in DNA at room temperature and

77K through the fluorescence yields of 2AP* placed at different distances from G

inside DNA. At low temperature the base motions were effectively slowed down and

the fluorescence yield of 2AP* was dramatically altered. Only the duplex with

neighboring guanine and 2AP showed some fluorescence quenching, while in others,

even for G separated from 2AP by a single adenine, no significant quenching was

observed. The study indicates that CT processes in DNA are indeed dynamic and the

quenching of 2AP* is suppressed at low temperature due to restricted conformational

motions.

Jean et al.28 examined the effect of solvent viscosity on fluorescence of 2AP*

in DNA trinucleotides by varying the content of glycerol in solution. The effect of

viscosity provided evidence that the fluorescence quenching mechanism is coupled to

conformational fluctuations, at least in trinucleotides. In viscous solutions at high

glycerol content (60%), the authors observed increase of the fluorescence time

constants. The long component increased from 3.5 ns to 6.9 ns, while the shortest

decay component also increased from 200 ps to 460 ps when the glycerol content was

changed from 0% to 60%.

All these studies show pronounced dependence of charge transfer rates on

temperature, which have to be attributed to thermally activated conformational

114

fluctuations in DNA. A study of O’Neill et al.30 revealed that electron transfer rate

constant in 2AP-G and 2AP-A-G containing duplexes increased with temperature in

accordance with Marcus equation (Equation 1.4). On the other hand, the relative

amplitudes of short (ET) and long components of time-resolved decay of excited

2AP* showed more gradual dependence on temperature. The short decay component

was assigned to the ET process based on its absence in redox inactive

oligonucleotides, in which guanine is replaced with inosine, and only long decay

component was observed. The behavior of the steady state fluorescence yield was

parallel to the temperature dependence of the charge transfer rate constant. The yield

of charge transfer was extracted from comparison of the fluorescence yield in

oligonucleotides containing guanine to those where G was replaced by ionosine. The

CT yield decreased gradually with temperature when 2AP and guanine were in direct

contact, while it increased for a greater separation between guanine and 2AP.

Efficient charge transfer was observed for DNA duplexes where 2AP and guanine

were separated by up to 34Ǻ. The authors also showed that temperature had a greater

impact on CT yields for the duplexes with greater 2AP-G separation. They arrived at

the conclusion that thermally induced base motions active for charge transfer

conformations and allow charge transfer to occur over longer distances than in

rigidified DNA.30

Jean et al.28 showed the influence of conformational fluctuations on dynamics

of charge transfer by studying temperature dependence of 2AP fluorescence lifetime

in DNA trimers. The trimers are expected to exhibit greater structural flexibility

115

compare to the duplex, and, thus the enhanced conformational disorder should lead to

long component in the fluorescence decay (similar to free 2AP). However, the

characteristic long time constant was absent in the time-resolved data suggesting

significant fluctuations of 2AP in trimers on pico to nanosecond timescale. These

rapid fluctuations preclude the existence of unstacked 2AP* long enough to exhibit its

characteristic lifetime (~10ns). Unstacked 2AP conformations due to fast diffusion

become stacked with neighboring bases in which 2AP* is quenched via charge

transfer.

Although the mechanism of CT in DNA remains vague, the described above

experimental evidences indicate that the role of base motions plays an important role

in charge transport through DNA, and, thus, should be incorporated into mechanistic

description.

4.1.4 Structural perturbations and efficiency of charge transfer in DNA

Charge transfer depends strongly on how well the bases are stacked within

DNA. One way to disrupt the charge transfer between donor and acceptor in the helix

is to perturb the π stack. There could be many ways to introduce perturbations, which

affect charge transport. The single base mismatch would be the first to come in one’s

mind. The mismatch does not provide good stacking with neighboring bases within

DNA duplex and results in effective shut off of charge transfer reaction.

116

The sensitivity of guanine oxidative damage to introduction of a mismatch

between donor and acceptor has demonstrated the critical requirement of the integrity

of π-stack for an effective charge transfer over long distance.

For example, efficient charge transfer is observed between 2AP and guanine

through four adenine residues.30 However, when a single A-A mismatch is

introduced at the third position between 2AP and G, the efficiency of charge transfer

becomes effectively zero. Although the mismatch did not change the sequence

through which charge transfer occurs, the stacking was disrupted by the mismatch

which resulted in such significant difference between modified and original DNA.

Another fluorescence analog of adenine 1-N6-ethenoadenine (Aε) is capable

of oxidizing guanine residues after excitation.1 The reactivities of 2AP and Aε were

studied with regards to efficiency of charge transfer to guanine. These two molecules

do not show any difference when reaction is carried out in solution. However,

striking differences are observed once they are incorporated into DNA duplex. Aε

exhibits much slower charge transfer (~100 times) and steeper distance dependence

compare to 2AP. The difference in charge transfer efficiency arises from their

structures. 2AP forms two hydrogen bonds with complementary T and very well

stacked within the DNA helix, while Aε is sterically bulky, does not undergo

hydrogen bonding with thymine and poorly stacked in DNA (provided by NMR

studies).26 Thus, the only difference between these two molecules--efficiency of

stacking within the DNA helix, resulted in such significant difference in their

reactivity.

117

The comparison of interstrand versus intrastrand charge transfer between

guanine and 2AP may also provide an understanding of the effect base-base stacking

on the efficiency of charge transfer. Barton et al.1 showed that the efficiency of

charge transfer between 2AP and guanine proceeding through interstrand reaction is

~100 times slower than intrastrand reaction.

Lewis et al.31 also showed less effective charge transfer for interstrand

reaction. They introduced a penalty factor for the reaction to proceed via interstrand

route between G sites. For their assemblies (GACC(GG)), where GG hole trap is

located at complementary strand the penalty factor was found to be 6 which agrees

well with theoretical estimation. They also pointed out that the kinetics through

GTGG is 50-100 times slower than in GAGG sequence clearly indicating that

sequence matters.

Many experimental as well as theoretical studies pointed out the dependence

of charge transfer on particular sequence.32,33,34,35,36 Adenine as a bridge was shown

to be more effective in facilitating the charge transfer from 2AP to distant guanines.

O’Neill et al.30 reported that if in A4 bridge separating 2AP and G one adenine is

replaced by either thymine or inosine, the charge transfer becomes essentially zero.

Interactions of DNA with proteins, drugs induce structural deviation from

canonical B-form as well as perturb π-stack of base pairs. It was shown that several

proteins disturb base stacking within DNA helix upon binding site-specifically.

Charge transfer was inhibited upon binding of such proteins.37 However, for some

proteins there was an enhancement of CT upon binding.38 These proteins do not

118

perturb the base stack significantly and also DNA-protein binding results in insertion

of tryptophan residue at 3’ end of guanine which perhaps lowers the oxidation

potential of the trap site.

Our investigations with altered stacking between donor and acceptor show that

the charge transfer is closely related to the efficiency of base-base stacking within

DNA duplex. In order to study the effect of base-base stacking, we deliberately

introduced a modification into DNA. A specifically synthesized oligonucleotide was

designed in such a way that locked nucleotide, LNA, was introduced next to 2-

aminopurine position, between 2Ap and G. LNA nucleotide is an analog of a regular

nucleotide with sugar moiety restrained in C3’-endo conformation (N-type) by a 2’-

O, 4’-C-methylene bridge. Insertion of a single locked nucleotide modification can

increase the melting temperature by up to 8°C per modification.39 Structural

constrain of deoxyribose conformation ultimately leads to the alteration of base

stacking. The alteration of base overlap and flexibility affects fluorescence

quenching efficiency. The comparison of modified oligonucleotide with regular B-

form shows that even single modification results in significant alterations of structure

and properties. See results and discussion of this chapter for more details.

4.1.5 Polymorphism of DNA and charge transfer

When talking about DNA, one usually imagines double stranded molecule

with right-handed helicity as it was proposed by Watson and Crick.40 However, DNA

119

exhibits rather remarkable polymorphism. Depending on conditions the double helix

can adopt A, B, C or Z-form of DNA.41 The structure of DNA can be changed in a

variety of ways: a) selecting particular base sequence, b) chemical alteration of

natural bases, c) using ribose instead of deoxyribose, d) replacing counterions, e)

changing ionic strength of the solution.

Base to base distance, angle between bases and their overlap differ from one

DNA conformation to another. Structural differences result in differences in

properties, particularly base-base overlap. With regards to charge transfer, different

conformations of DNA were shown to exhibit different degree of charge transfer

effeciency.42,43

For example, DNA:RNA hybrids where complementary deoxyribo-T were

replaced by ribo-U adopt A-form of nucleic acid conformation characteristic for RNA

duplexes.43 Comparison of charge transfer efficiencies in regular DNA and

DNA:RNA hybrids reveal that distance dependence of charge transfer in A-form of

DNA:RNA hybrids exhibits more shallow behavior than in B-DNA. Moreover, the

interstrand charge transfer was found to be as efficient as inrastrand one in hybrids,

while, in B-DNA, CT has much steeper distance dependence for interstrand reaction.

The effect was attributed to better overlap in A-conformation for both inter and

intrastrand base-base interactions. The authors also pointed out that the effect may be

partly due to greater flexibility of DNA:RNA hybrids which have lower melting

temperatures compare to corresponding DNA duplex.

120

Schuster has studied charge migration in hairpins consisting of (CG)n

sequence which adopts the Z-form of DNA at high salt concentration.44 The

irradiation of an anthraquinone derivative covalently attached to a 5’end is followed

by injection of radical cation into duplex DNA. The charge transfer is monitored by

strand cleavage at GG site in the hairpin loop. It was shown that charge transport

through Z-form DNA is more effective than through B-DNA for these particular

systems and it occurs over distances greater than 30 Å.

Another example is the study of charge transfer between BrU and interstrandly

placed guanine in Z-DNA. Photoirradition of BrU which is followed by the charge

transfer was shown to be efficient in Z-DNA but not in B-DNA.45 Conformational

change from B to Z form alters base stacking within duplex. With Z-form formation

G and BrU separated by 5 base pairs and located on different strands acquire better

stacking which facilitates the charge transfer.

Charge transfer was also found to occur in single stranded DNA. The fact that

ssDNA can mediate charge transfer seems rather strange from intuitive point of view.

After so many studies showing how sensitive the charge transfer is to subtle

alterations in structural integrity of DNA helix, one would expect no effective charge

transfer in single stranded DNA. However, charge transfer was observed for ssDNA

by Schuster et al.,46 by O’Neill et al.,1 and we also observed effective charge transfer

between 2AP* and guanine at high temperature above melting for our studied

oligonucleotides-hairpins where they exist in single stranded form (see Results and

121

Discussion section of this chapter). The charge transfer in ssDNA is less effective

than in dsDNA, however, the fact of its appearance is worth attention.

4.1.6 Theoretical studies of the base stacking effect on charge transfer in DNA

Theoretical studies have been extremely helpful in explaining seemingly

inexplicable observations from different laboratories.9,10,36,47,48,49,50 However, the

more we study CT in DNA the more questions rise. DNA can adopt a variety of local

conformations undergoing dynamic motions, which depend on various factors

including temperature, chemical modification and sequence content. These

conformations and transitions between them govern charge transfer by affecting

coupling and even energies of charge transfer states. In fact, if we could predict

quantitatively the energetics and base-base couplings in DNA as a function of those

parameters affecting local conformations, we might be able to utilize charge transport

in DNA in more effective way.

Recently, a theoretical prediction for structural dynamic disorder has been

made.51 The authors considered the charge transfer rate between two guanine bases

separated by adenine/thymine bridge. It was found that while for short bridges the

transition rate decays exponentially with the number, n, of the AT pairs in the bridge,

for bridges with n larger than ~ 4, the transfer rate in the systems with dynamic

disorder no longer depends on n. This result suggests that dynamic disorder is an

essential component of the charge transfer process in the DNA molecules.

122

Structural fluctuations determine the effective electronic coupling between

bases (Heff).52 The CT rate can be increased by as much as 2 orders of magnitude by

these geometry fluctuations for guanine sites separated by 4 intervening adenines.

The authors also identified base motions, which have the greatest contribution to Heff

change. These motions can be described as sliding of bases parallel to their plane

occurring on picosecond timescale. Other base motions were found to be relatively

ineffective.

Besides electronic coupling between D and A, the CT energetics between

nucleobases can be affected by structural perturbations. The CT driving force was

found to fluctuate with the standard deviations of ∆G ~ 0.3-0.4 eV, however these

fluctuations are mainly due to molecular vibrations of the donor and acceptor sites

and motions of counterions as well as water molecules.53 The relative motions of

adjacent nucleobases play insignificant role in the CT energetics, whereas they do

substantially affect the electronic coupling between bases.54

It is apparent that charge transfer in DNA has a complex nature and its full

description should include such effects as base-base coupling in DNA, energetics and

their dependence on structural fluctuations.

This chapter discusses experimental results on photoinduced charge transfer in

DNA hairpins, measured with 2AP, as electron acceptor, which fluorescence is

quenched by guanine. The following results will be discussed:

a) distance dependence of charge transfer by guanine placed at different

position within DNA

123

b) temperature dependence of charge transfer for the same systems

c) effect of base stacking on charge transfer introduced by a single locked

nucleotide modification

4.2 System Design

Fluorescence of 2AP* is greatly quenched upon its incorporation into DNA. The

quenching of 2AP is attributed to charge (not energy) transfer to neighboring bases, since

2AP has lower excited state energy than natural DNA bases. Guanine is considered to be

the most favorable candidate for oxidative quenching of 2AP fluorescence owing to its

lowest oxidation potential among all natural bases. However, other bases were shown to

quench fluorescence of 2AP as well.23 Based on the oxidation potentials for guanine (G),

adenine (A), cytosine (C) and thymine (T) (1.29 V, 1.42 V, 1.6 V and 1.7 V vs. NHE,

respectively),21 and the reduction potential of 2AP* (1.5V vs NHE)1, quenching of 2AP*

fluorescence by G is expected to be an electron transfer from guanine. In order to

discriminate that from other modes of quenching, comparison with a reference system,

where guanine is replaced with inosine, can be used.1 Inosine participates in similar

hydrogen bonding as G (see Figure 4.2), and was shown to be relatively inert towards

oxidation by 2AP*.1 Using this approach, photoinduced charge transfer between 2AP*

and G in DNA can be investigated as a function of distance, temperature and alteration of

DNA conformation.

124

Following this scheme, a series of hairpins, 31-mer oligomers were designed,

which are schematically shown in Figure 4.1. They differ by position of G within the

duplex: it is separated from 2AP by various number of adenines (from 0 to three)

either on the same strand DNA_(0sG-3sG) or on the opposite strand DNA_1oG.

Similar oligomers, with I instead of G (DNA_0sI-3sI and DNA_1oI), were used as

the reference sequences, where the route of quenching by G was eliminated. Each

oligonucleotide has two 14-base complementary strands linked by a triple thymine

loop. DNA hairpins are more suitable for the purpose of temperature dependent

studies: they have higher melting temperature and their hybridization is independent

of DNA concentration. Thymines were chosen for the three-base loop because of

their small size.

In order to investigate the effect of base stacking in greater details, LNA modified

oligonucleotides were investigated for comparison. We took 1sG and 1sI and modified

adenine (A) in between 2AP and G/I with ribose locked in c3’-endo conformation (N-

type) by a 2’-O, 4’-C-methylene bridge, AL (see Figure 4.1). We shall refer to these

oligonucleotides as LNA in the text with the corresponding abbreviation, LNA_1sG, and

LNA_1sI, for guanine and inosine contaning oligonucleotides, respectively.

125

----------------

--------------------

--------------------

5'3'

(I)

T

T

T

T

ATAT

ATA

T

TCTTTAATT

AAGAAp

ATTAA

1sG(I)

DNALNA

L

----------------

----

----

----

--------------------

5'3'

(I)

ApATTAA

TTAA

TT

T

T

T

T

A

TA

T

C

ATA

G

(T)n (A)n

----------------

--------------------

--------------------

5'3'

(I)

T

T

T

T

A

TA

T

ATA

T

TAA

G

TTTAA

TT

CAAp

ATTAA

nsG(I) 1oG(I)

OG(I)O

POO-

O

OA

O

O

O2Ap

O

POO-

PO4-

OG(I)O

OA

O

PO O-

O

O2Ap

O

POO-

PO4-

Figure 4.1 Schematic representation of the hairpins. See section “System design” for details. 4.3 Experimental Section

4.3.1 Materials

All solutions of DNA oligonucleotides used in this study, were prepared in 6

mM phosphate buffer saline (PBS) at pH = 7.2 and 0.15 M NaCl immediately prior to

use. The typical concentration of DNA in fluorescence and absorption measurements

was approximately 20 µM except for melting temperature determination, where

concentration was usually 5-8 µM. Concentrations were calculated from the

absorbance at 260 nm using 3.05×105M-1cm-1 extinction coefficients. Melting

126

temperatures, Tm, were determined from the derivative’s maxima of the melting

curves monitored by absorption at 260 nm.

4.3.2 Absorption measurements

Absorption spectra and melting curves (temperature dependences of

absorption) were measured in 40 µL cuvettes with 1 cm path length using Cary 100

Bio UV-Vis spectrophotometer. Melting curves were measured at 260 nm by

lowering the temperature from 85 to 5 °C at the rate of 1°C per minute. Absorption

spectra were measured every 10 degrees in the same range from 85°C to 5°C.

Absorption spectra were normalized to the absorption at 285 nm, which was more

convenient than at 260 nm because of high concentration, but seemed to work just as

well.


Fluorescence spectra and fluorescence melting profiles were also measured in

40 µL cuvettes with 1 cm pathlength using Varian Cary Eclipse fluorescence

spectrophotometer. The fluorescence “melting curves” were monitored at 370 nm

(wavelength of 2-aminopurine fluorescence maximum) at the rate 1°C per minute

with excitation at 316 nm. The fluorescence intensities were normalized to the

absorbance at 316 nm, the wavelength close to the isosbestic point for all hairpins.

127

Fluorescence quenching yield, η, was calculated using following equation:

η = 1 - ΦG/ΦI (4.2)

where fluorescence yields of a guanine containing oligonucleotide, ΦG, and that for

inosine containing oligonucleotide, ΦI, were calculated relative to the fluorescence

intensity of free 2-aminopurine (2AP) in aqueous solution.

4.3.4 Circular dichroism measurements

Circular dichroism (CD) spectra were measured in l = 0.1 cm pathlength

cuvettes using Jasco J-810 Spectropolarimeter and a typical oligonucleotide

concentration ca. 25 µM. CD spectra were recorded in the temperature range 5-85°C

with 10°C increment. ∆ε was calculated using the following equation:

cl ⋅⋅=∆

32980θε (4.3)

where θ is measured ellipticity, l is the path length and c is the concentration of

nucleobases.

4.3.5 Molecular modeling

All calculations were performed with HyperChem Pro6. Oligonucleotides were

initially constructed in standard double-stranded B-form using HyperChem Nucleic

Acids Databases. The appropriate nucleotides were modified to LNA nucleotides, and

128

adenine at 6th position was modified to 2-aminopurine. Atomic charges for the modified

structures of nucleotides were replaced with charges given in Appendix D. DNA duplex

was immersed in periodic box (24Å×24Å×56Å) filled with 1068 molecules of water.

Counterions were also added to neutralize negative charges of phosphate groups.

Initially, molecules of water and counaterions were optimized by energy minimization

using molecular mechanics AMBER force field method with amber94 parameter set

using Polak-Ribiere Conjugate Gradient algorithm and 0.003 kcal/Å mol convergence

limit. Oligonucleotide inside the periodic box was subjected to 100 ps of molecular

dynamics “heating” from 10 to 290K in time step 1 fs, followed by 10 ps simulation at

290K. The geometry of oligonucleotides was then optimized with AMBER method.

Energies were calculated using ZINDO/S.


4.4.1 Data analysis

Since fluorescence yield relates to the rate of charge transfer indirectly, it is

important to establish a reliable procedure of how this relationship should be

evaluated.

Fluorescence of 2AP* proceeds with the rate constant kF, and competes with

various nonradiative decay routes, which result in lowering the fluorescence yield.

Presuming that the fluorescence yield of an inosine containing duplex, QF

FI kk

k+

=Φ ,

129

differs from that with guanine at the same position, CTQF

FG kkk

k++

=Φ , only due to

the additional charge transfer route from guanine to 2AP*, one can estimate the rate

constant, kCT, of the latter using ΦI/ΦG ratio:

( )

−

ΦΦ

=

−

ΦΦ

+= 111G

I

IG

IQFCT kkk

τ (4.4)

where kQ is the rate constant of all deactivation processes except for that of charge

transfer between 2AP and G, and τI is the lifetime of 2AP* in inosine containing

duplex:

QF

I kk +=

1τ (4.5)

The fluorescence kinetics of 2AP* are not single exponential in DNA duplexes13 and

thus cannot be characterized by a single kQ. Nevertheless, the relative variation of kCT

with separation distance between 2AP and G, a dependence of kCT on temperature and

upon conformational alteration can be analyzed.

4.4.2 Thermal stability of studied oligonucleotides

Absorption at 260 nm of the studied hairpins exhibits hyperchromic increase

(ca. 20%) for temperatures above melting. Upon inspecting the melting temperatures

(Tm) of the studied oligonucleotides that are listed in Table 4.1, one can see that

inosine containing hairpins have melting temperatures consistently lower by ca. 4

degrees when compared to the analogous guanine containing oligonucleotides. The

130

difference can be attributed to fewer hydrogen bonds in the I-C base pair: it forms

only two hydrogen bonds with cytosine (C), instead of the three in regular G-C base

pair (see Figure 4.2).

Table 4.1 Melting temperatures, Tm, and fluorescence quantum yields, Φ, of 2-aminopurine in different hairpins at temperatures below and above Tm.

T = 25 oC T = 80 oC

Sequence Tm(oC) Φ ΦG/ΦI Φ ΦG/ΦI

0sG 59.9 0.0085 0.064

0sI 54.3 0.052 0.16

0.160 0.40

1sG 59.5 0.0204 0.116

1sI 55.9 0.060 0.34

0.195 0.59

2sG 58.8 0.036 0.168

2sI 54.8 0.054 0.67

0.187 0.90

3sG 59.7 0.072 0.166

3sI 55.0 0.088 0.82

0.158 1.05

1oG 60.6 0.029 0.179

1oI 54.3 0.055 0.53

0.183 0.98

131

Figure 4.2 Base pairing in studied DNA of regular and modified bases.

4.4.3 Absorption of 2-aminopurine

One of the reasons 2-aminopurine is widely used in study of DNA is that its

optical properties are sensitive to the environment. Absorption maximum of 2AP in

DNA duplex, λmax ~ 320 nm, exhibits more than 10 nm shift when compared to that in

ss-DNA, λmax ~ 310 nm, and free 2AP in solution, λmax ~ 305 nm. This shift of λmax,

as well as the reduction in fluorescence yield, is attributed to stacking interactions

with nearest neighbor nucleobases. Both absorption and fluorescence, have been

used to probe the equilibrium stacking properties of DNA duplexes.55,56

NN

O

NH

N

N

N

N

O

N HH

H

H

NN

O

NH

N

N

N

N

O

H

H

Guanine - Cytosine

Inosine - Cytosine

N

NN

N

N H

N

O

NO

H

H

N

NN

N

N HH

N

O

NO

H

Adenine - Thymine

2-Aminopurine - Thymine

132

Figure 4.3 shows that changes in the absorption spectra of 2AP in DNA

hairpins are gradual--the peak at 320 nm does not saturate right below the melting

temperature but continues growing even 50 degrees below melting. The changes can

be interpreted in a two state model, there is a distinct isosbestic point at 316 nm

observed for all DNA hairpins that it was used for excitation in fluorescence

measurements. It should be noted, that excitation at three different wavelengths -

316, 320, 324 nm results in the same fluorescence yields all throughout the

temperature range when normalized to the absorption at corresponding wavelength.

0 20 40 60 80 1000.00

0.01

0.02

0.03

0.04

0.05

300 320 340 3600.00

0.02

0.04

0.06

0.08

0SGAbs

orba

nce

Wavelength (nm)

5oC 15oC 25oC 35oC 45oC 55oC 65oC 75oC 85oC

300 320 340 3600.00

0.05

0.10

0.15

0.20

Wavelength (nm)

1oI

Abs

orba

nce

5oC 15oC 25oC 35oC 45oC 55oC 65oC 75oC 85oC

1oI0sI0sG

∆A 33

0nm

T oC

Figure 4.3 Temperature variation of the absorption intensity at 330 nm for three hairpins. Insets: Red wing of absorption spectra of hairpins with 2-aminopurine recorded at various temperatures.

133

The 316 nm wavelength appears to be the most convenient for excitation,

since it does not require normalization for 2AP absorption at different temperatures

and the absorbance of all natural bases is already negligible at this wavelength.

4.4.4 Fluorescence quenching of 2-aminopurine in DNA oligonucleotides

The fluorescence spectrum of 2AP is less sensitive to the environment than

absorption. Its maximum stays nearly constant around 370 nm and practically

independent of temperature and hybridization. The fluorescence yield (Φ), on the

other hand, changes dramatically due to quenching. The fluorescence yield is much

lower for 2AP incorporated into single stranded DNA (ss-DNA) as compared to free

2AP and suffers even greater reduction upon DNA hybridization. At room

temperature, when hairpins are hybridized, the fluorescence yield is particularly

sensitive to the distance between 2AP and guanine. Guanine has the lowest oxidation

potential among natural nucleobases and thus, should be the most effective quencher

of 2AP* fluorescence by charge transfer. Its efficiency of quenching should vary

with the distance between 2AP and G. Fluorescence quenching efficiency, η, is

plotted as a function of separation distance between 2AP and G in Figure 4.5.

Three important observations should be noted about this figure. First, the

fluorescence quenching yield drops upon distancing guanine away from 2AP.

Second, the quenching yield for the interstrand charge transfer diminishes to zero

above the melting temperature, while it is comparable to the intrastrand quenching

134

with G at the same distance from 2AP. Third, quenching of 2AP fluorescence by

intrastrand guanines is effective even above melting temperatures (black bars). Let us

discuss these observations in their reverse order.

Persistence of intrastrand quenching above the duplex melting should not be

totally surprising after all, donor and acceptor remain in proximity of each other. The

case with neighboring 2AP and G can be reasonably explained by this but farther

separated donor and acceptor would require some persistent effective bridge. The

effect can be attributed to the presence of poly-adenine (multiple neighboring

adenines), which is known to stay in a single stranded helical form due to its high

stacking ability.57,58

At temperatures above melting, when DNA hairpin transforms into the single

stranded form, and guanine located on the opposite (complementary) strand from that

with 2AP becomes completely separated from it. As a result, no quenching due to

charge transfer between guanine and 2AP can occur. However, at room temperature,

DNA oligonucleotide forms a duplex that brings guanine to close proximity, and

fluorescence of 2AP* is effectively quenched. Also fluorescence melting curves

reveal that inosine containing oligonucleotides have the same fluorescence yield

throughout the temperature range regardless of inosine position with respect to 2AP,

see Figure 4.4. On the other hand, fluorescence yields for guanine containing

oligonucleotides differ from each other. It is apparent from the figure that the

difference depends on the distance between G and 2AP.

135

20 40 60 8020 40 60 800

200

400

600

800

0

200

400

600

800

2sG 2sI

T, oC

1oG 1oI

T, oC

Nor

m. F

luor

esce

nce

1sG 1sI

Nor

m. F

luor

esce

nce 0sI

0sG

Figure 4.4 Fluorescence intensity variation with temperature for four hairpin pairs with λFluor = 370 nm. XsI--red, XsG--black.

The charge transfer rate calculated from the yield declines exponentially with

the separation distance and is commonly described by the following expression:

)exp(0 rkkCT ⋅−= β (4.6)

Its characteristic parameter, β, is often used to characterize the charge transfer

efficiency of a “bridge” linking donor and acceptor. The smaller β is the higher is

“conductivity” of the bridge. According to Equation 4.4, the variation of the charge

transfer rate constant, kCT, can be evaluated through [ΦI/ΦG – 1]. By plotting

136

ln[ΦI/ΦG – 1] vs distance (Figure 4.6), one can estimate the parameter β as a slope of

the plot, since ( )rG

I ⋅−∝

−

ΦΦ βexp1 .

3.4 5.1 6.8 8.5 10.2 11.9 13.60.0

0.2

0.4

0.6

0.8

1.0

Interstrand

η=1−

ΦG/Φ

I

Distance (A)

Figure 4.5 Dependence of fluorescence quenching efficiency (η) on distance; white bars--at room temperature (25°C), black bars--at temperature above melting (80°C).

At room temperature this fit for intrastrand 2AP/G pairs results in β = 0.32 Å-

1, which is quite small compared to the range of reported values. The value of β for

137

charge transfer in DNA has been reported from as low as 0.14 Å-1 to as high as 1.4 Å-

1. Nevertheless, our value is close to those measured using fluorescence quenching of

2AP in similar systems.30 For simplicity, we estimated the separation distance here

by multiplying each interbase distance by 3.4 Å. From a similar plot at 80°C, which

is above the DNA melting temperature, β increases to ~0.5 Å-1 if one assumes the

same distances between bases.

0 1 2 3 4 5

-2

-1

0

1

2

0.0 3.4 6.8 10.2 13.6 17.0R, A

1oX

3sX

2sX

1sX

0sX

ln[(Φ

I /ΦG) -

1]

Ap-X base separation

Figure 4.6 Distance dependence of the charge transfer yield at room temperature for intrastrand (squares) and interstrand (circle) 2AP/G pair.

138

Charge transfer rate constant, kCT, which, according to Equation 4.4 is

proportional to [ΦI/ΦG-1], exhibits pronounced temperature dependence for all

oligonucleotides in the temperature range from 5 to 85°C (see Figure 4.7). One can

distinguish three temperature regimes for the yields in that graph. At temperatures

above melting transition (>65°C), no significant variation is observed for all

oligonucleotides. Artificial peaks in the second region near melting (45-65°C) are

primarily due to different melting temperatures for inosine and guanine containing

oligonucleotides. The explanation contradicts with the speculation in a recent study

of O’Neil et al.,30 where similar peaks were assigned to a better facilitation of charge

transfer from enhanced motions in DNA.

In the third region, at low temperatures, the slopes of temperature dependence

for kCT differ between the oligonucleotides. The charge transfer rate declines with

increasing temperature for 0sX and 1sX but rather increases with temperature for 2sX

and 3sX. It has to be noted that 1sX oligonucleotide exhibits lesser decline compared

to 0sX.

These unparallel changes in kCT with temperature for different

oligonucleotides correspondingly result in different values of β for different

temperatures. Instead of calculating β from a slope, one can analyze a pair wise

derived, βn,n+1, that is calculated for the cases with guanine quencher shifted by one

base position:

βn,n+1 = - ln([ΦI/ΦG-1]n+1/[ΦI/ΦG-1]n) (4.7)

The values of βn,n+1 can be related to β through dividing them by 3.4 Å:

139

β = βn,n+1/3.4 Å (4.8)

0 20 40 60 800

1

2

3

4

5

6

7

3sX

1sX2sX

0sX[ΦI/Φ

G -

1]

Temperature (0C)

Figure 4.7 Temperature dependence of the charge transfer rate constant (kCT ~ [ΦI/ΦG-1]) for four pairs of oligonucleotides: 0sX (), 1sX (), 2sX (), 3sX ().

The dependence of β on temperature exhibits some interesting features.

Figure 4.8 shows it for β01 and β12 determined this way. The apparent values of β

appear to have minima near the melting temperature: both β01 and β12 reach minima

at as low as 0.25 Å-1. The values gradually increase upon lowering temperature and

140

grow to as high as ~0.4 Å-1 at 5°C. Above melting, β values also increase with

temperature but noticeably different for β01 and β12. Proximal quenchers show weak

dependence on temperature (β01). Increase in the separation distance for a quencher

even by a single base makes β12 vary to a greater extent. Notably, the temperature

dependence of β displays the opposite behavior to the temperature dependence of

base stacking. Base stacking efficiency improves with lowering temperature as

indicated by absorption of 2AP at 330 nm (Figure 4.2) and circular dichroism (Figure

4.11). This trend correlates with contemporary observations suggestive that efficient

stacking in the B-form helix is counterproductive in quenching by charge transfer.

For example, stilbene capped hairpins of Lewis et al.59 are well stacked and

show a relatively large value of β~0.65. More loose, DNA:RNA hybrids, that usually

do not adopt B-form, exhibited smaller β in studies with 2AP.30 It was noted in

theoretical analyses that the arrangement of bases in the B-form duplex could have as

much as an order of magnitude less efficient coupling from the maximum possible.60

These observations lead to introduction of a fluctuation gated or polaron-like hopping

mechanism of charge transport emphasizing the importance of structural fluctuations

related to electronic coupling in DNA. The concept of a conformational gating

mechanism presumes existence of various conformations at the same time which may

facilitate charge transfer with different from each other efficiencies.

The mobility seems to rule the rate of charge transfer even far below the

melting temperature of oligonucleotides. The charge transfer is activated thermally

141

through conformational fluctuations of bases. This results in decreasing distance

dependence of charge transfer (βeff) with increasing temperature.

Charge transfer yield is proportional to the charge transfer rate constant, kCT,

and thus its temperature dependence should follow the expression for kCT:

( )

+∆−=

λλ

πλπ

TkG

TkHk

BB

DACT 4

exp4

2 22

h (4.7)

where HDA is the electronic coupling matrix element, kB is the Boltzmann constant, T

is temperature and λ is reorganization energy. ∆G--is the free energy change for

charge transfer reaction.

If all parameters are assumed to be temperature independent, the rate, kCT,

according to Equation 4.7, becomes dependent on temperature primarily due to the

activation barrier, Ea:

TkE

CTB

a

eTAk

−

~ (4.8)

where ( )

λλ

4~

2+∆ CTa

GE . The free energy of charge transfer, ∆GCT, can be

estimated using Weller’s equation:

CEEEG oxredSCT +++−=∆ )( (4.9)

where ES is the 2AP excited singlet energy (3.74 eV),23 Ered is its reduction potential,

Eox is guanine oxidation potential and C is the coulombic interaction term. The latter

should be on the order of 0.05eV for a pair of charges separated by 3.4 Å in a solvent

dielectric constant of water and is even less for greater separation distances, thus it is

142

usually neglected.61 Therefore, for ∆GCT ~ -0.6 eV and λ ~ 1.2 eV,62 Ea is small,

0.075eV, which would result in slightly increasing with temperature kCT. However,

our experimental results demonstrate different behaviour from what is suggested by

Marcus theory. Charge transfer rate constant does increase with temperature when

donor and acceptor are separated by more than two bases. While, for 0sX and 1sX

oligonucleotides the reverse dependence is observed (see Figure 4.5).

0 20 40 60 800.00

0.34

0.68

1.02

1.36

0.0

0.1

0.2

0.3

0.4

∆Tm

β12

β01

β, A

-1

β N

T oC

Fugure 4.8 Temperature dependence of the charge transfer decay constant, β, calculated using Equations 4.7 and 4.8, for oligonucleotides 0sG-1sG (β01)--(), and 1sG-2sG (β12)--().

143

The more studies on DNA structure become available, the more apparent it

becomes that DNA is a dynamic molecule and its conformational flexibility should

not be neglected. The conformational changes in DNA duplex include shifting and

sliding of bases along their planes that have a great effect on the electronic matrix

element, HDA.60 These motions occurring on picosecond timescale affect greatly base

overlap and the overall base stacking in DNA.

According to McConnell,63 the electronic coupling matrix element between

donor and acceptor, HDA, can be approximated using following equation:

1−

∆∆=

NBBBADB

DA EV

EVVH (4.13)

where VDB and VBA are the electronic coupling strengths between donor(D) and

bridge(B), bridge and acceptor(A), respectively, VBB is the electronic coupling

between bridge sites, ∆E is the energy gap between donor and bridge, and N is the

number of identical bridge sites.

When both electron tunneling and vibrational motion occur on the same time

scale, HDA in Equation 4.13 can become dependent on nuclear coordinates resulting

in a breakdown of Condon approximation. The dependence of HDA on thermally

induced conformational fluctuations is a premise of the so called “conformationally

gated” charge transfer mechanism,15,62,64 which presumes that the electronic coupling

strength in the lowest energy conformation of a molecule is modulated by thermal

excitation to conformations with different HDA. As a result, the temperature

dependence of kCT under the gated mechanism can noticeably alter.

144

It is not clear whether the DNA conformation can be optimized throughout the

helix in a particular form to maximize the rate of charge transfer but currently it

appears that flexibility in the helix stacking (induced thermally or otherwise) is

crucial in improving the rate of charge transfer.

4.4.5 Effect of LNA modification

To further clarify the role of base-stacking efficiency on charge transfer in

DNA, local alteration of base stacking was employed. It was achieved by introducing

single LNA modification to interfere stacking between G and 2AP (Figure 4.9). LNA

nucleotide is an analog of a regular nucleotide with sugar moiety restrained in C3’-

endo conformation (N-type) by a O2’-C4’ methylene bridge.

Insertion of a single locked nucleotide modification can increase the melting

temperature by up to 8°C per modification.39 We chose 1sG and 1sI and introduced

LNA modification on adenine, AL, between 2AP and G/I. Thermodynamic

parameters ∆H°, ∆S° and Tm (listed in table 4.2) were measured from absorbance

melting curves. Melting temperatures, Tm, of inosine containing hairpins for LNA is

lower than that of the analogous guanine containing one, similar to what was seen in

DNA, and can be attributed to fewer hydrogen bonds for inosine. One may even

argue that locked nucleotide induces greater discrimination: melting temperature

difference of G and I containing oligonucleotides, ∆Tm = TmG - TmI, is greater for the

case of LNA (5.2°) than for DNA (4.6°). But most noticeably, the locked nucleotide

145

modification (even on a single nucleotide) increases melting temperature. In the

recent analysis of the thermodynamic data for a series of LNA containing duplexes it

was concluded that LNA can stabilize the duplex “by either preorganization or

improved stacking, but not by both simultaneously”.65 For base sequence fragments

similar to ours--AALG with the central LNA modified adenine, AL, the change in

thermodynamic parameters, as compared to analogous DNA, were found to be mostly

due to the decrease in the entropy of melting.

Table 4.2 Comparison of several characteristics for four DNA and LNA modified oligonucleotides. 5° C 85° C

Sequence Tm ∆H°, kJ/mol

∆S°, J/mol K

λmax, nm Φ λmax,

nm Φ

DNA_1sG 59.6 -214.8 -640.4 - 0.006 - 0.066 DNA_1sI 55.0 - - 315 0.012 310 0.135 LNA_1sG 61.5 -206.4 -609.9 - 0.012 - 0.131 LNA_1sI 56.3 - - 320 0.033 310 0.220

Similarly for our hairpins, the enthalpy (∆∆H°) and entropy (∆∆S°) changes

calculated using equations:

DNALNA HHH ooo ∆−∆=∆∆ ; oooDNALNA SSS ∆−∆=∆∆ , (4.11)

are both positive: ∆∆H° = 8.4 kJ/mol and ∆∆S° = 30.5 J/mol K.

By measuring ∆∆H° and ∆∆S° it is possible to establish the nature of

stabilizing effect for LNA modified duplex. Positive ∆∆H° represents a less

146

favorable stacking interaction in LNA that destabilizes the duplex, when compared to

analogous DNA. On the other hand, positive change in entropy, ∆∆S°, indicates that

the structural constrains of the deoxyribose conformation make up enough stabilizing

contribution to ∆∆G° to result in more stable LNA duplex. Positive ∆∆S° most likely

occurs due to preorganization of unhybridized LNA. Therefore, LNA modification is

stabilized entropic contribution and leads to a less effective base stacking, as

indicated by positive enthalpy change (∆∆H°), since the main contribution to the

enthalpy comes from stacking energy.18 Weaker stacking interaction should be

accompanied by altered base-base overlap.

As was discussed above, optical properties of 2AP are sensitive to local

environment was and can be employed for monitoring local structural properties at

the charge transfer site. The absorption maximum of 2AP, λmax, is red shifted in a

single-stranded nucleic acid (λmax ~ 310 nm) and even further, to > 320 nm, upon

duplex hybridization. However, as seen from Figure 4.9, absorption of 2AP in LNA

is not as apparently red shifted, it is too close to absorption of regular bases.

Nevertheless, it can be revealed in the excitation spectrum (see Figure 4.10). The

wavelengths of excitation maxima for both, hybridized and unhybridized, forms are

given in Table 4.2. The absorption shift, which is also accompanied by reduction in

the fluorescence yield, is attributed to stacking interactions with neighboring

nucleobases.66 Apparently there is some degree of stacking even in ss-DNA, since

the absorption maximum of 2AP is red shifted there as well.

147

In the simplest two-state model, where only “fully-stacked” and “less-stacked”

conformations can exist, there should be an isosbestic point in the variation of

absorption spectrum with temperature. Absorbance on the red side of the isosbestic

point (e.g. at 330 nm) would indicate a portion of fully-stacked 2AP in the DNA

duplex. Two insets in Figure 4.9 show absorption spectra of DNA and LNA at

temperatures 5 and 85 degrees. It is apparent that both LNA and DNA oligomers

have the isosbestic point, which justifies the two-state model. Absorbance at 330nm,

∆A330, gradually increases upon lowering temperature in accordance with improved

stacking of 2AP within duplex of both LNA and DNA. The transition extends to a

lower temperature than that observed at 260 nm, but the most noticeable difference is

that the absorbance at 330 nm does not saturate even far below the melting

temperature suggesting that there is still significant local adjustment of 2AP

conformation towards improved stacking. Another noticeable feature in the spectra is

that LNA and DNA oligomers follow similar temperature dependence but ∆A330 in

LNA is always smaller than in DNA, indicating a poorer stacking of 2AP within LNA

duplex. Red absorption band of 2-aminopurine in LNA does not become fully

pronounced even at the lowest temperature measured, 5°C.

Additional information about secondary structure of DNA is provided by

circular dichroism (CD) spectra. CD of DNA and LNA oligonucleotides are very

similar above melting temperature (Figure 4.11) and correspond to a conformation

that is different from the canonical B-form observed at low temperature.

148

0 20 40 60 800.000

0.005

0.010

0.015

0.020

300 320 340 3600.00

0.02

0.04

0.06

0.08

0.10

5OC

85OC

DNA

A

wavelength (nm)

300 320 340 3600.00

0.02

0.04

0.06

0.08

0.10

5OC

85OC

LNA

A

wavelength (nm)

LNA

DNA∆A

330

Temperature (oC)

Figure 4.9 The change of absorption at 330 nm with temperature for DNA_1sG and LNA_1sG. The insets show corresponding spectra.

Above Tm the negative trough at 210 nm is more pronounced and the trough at

247 nm is weaker than in the B-form seeing below melting (Figure 4.11). These

spectral features are representative of the A-form of DNA.67 It confirms previous

findings that above Tm single stranded oligonucleotides remain in a helical form with

right-handed chirality and a high degree of base stacking.57,58 At low temperatures

the differences in CD spectra become more apparent - mainly in the amplitudes of

peaks and troughs. The amplitude difference between the first peak, ∆ε275, and the

second trough, ∆ε247, the so-called amplitude of Cotton effect, ACott:68

149

ACott = (∆ε275nm - ∆ε247nm) (4.12)

characterizes excitonic splitting that in our case can be related to the degree of base

stacking in DNA as a whole.68 Figure 4.11 shows that ACott decreases with

temperature in both oligonucleotides but it is always lower for LNA, which confirms

a less degree of stacking in modified oligonucleotide.

290 300 310 320 330 340 3500.0

0.5

1.0

290 300 310 320 330 340 350

290 300 310 320 330 340 3500.0

0.5

1.0

850C

50C DNA

Fluo

resc

ence

inte

nsity

LNA

850C

50C

wavelength (nm)

2AP

Fluo

resc

ence

inte

nsity

wavelength (nm)

Figure 4.10 Fluorescence excitation spectra. DNA and LNA at temperatures from 85°C to 5°C (10° increment). Free 2AP at three temperatures, 5°C, 25°C and 85°C.

150

This correlates very well with absorption measurements. It should be noted

that absorption at 330 nm reflects local environment of 2AP, while ACott in CD

spectra reveals features of the global structure of oligonucleotides.

0 20 40 60 80

20

22

24

26

28

30

32Acott

Acott

220 240 260 280 300

-10

-5

0

5

10

∆ε

wavelength (nm)

220 240 260 280 300

-20

-10

0

10

∆ε

wavelength (nm)

5 OC

85 OCLNA

DNA

A Cot

t (M

-1cm

-1)

Temperature (oC)

Figure 4.11 Temperature dependence of cumulative molar circular-dichroic absorptivity of the first peak (275 nm) and the trough (247 nm). The insets show CD spectra of DNA and LNA at temperatures below 5°C and above 85°C melting of a duplex.

The observation of different stacking by 2AP absorption is not a surprise since

the LNA modification was intentionally placed next to 2AP. As one can see from CD

spectra, there are noticeable differences in global hairpin structure despite the small

151

fraction of modified bases (1/30). Thus, the altered stacking of 2AP affects the global

stacking as well. However, the differences in CD spectra are small which

demonstrate that the global stacking is altered insignificantly and most changes occur

near the locked nucleotide modification.

It has been reported that a single LNA modification can force the DNA duplex

to adapt an A-form.69 Petersen et al., however, showed using NMR that structural

changes are rather local and do not extend substantially beyond the vicinity of LNA

modification.70 Circular dichroism and optical absorption measurements confirm that

a single LNA modification affects hairpins’ structure mostly in proximity of the

modified nucleotide. Although CD reveals some structural distortion in the LNA

case, the overall structure of a hairpin duplex remains close to the standard B-form

below melting temperature. Furthermore, CD of both DNA and LNA

oligonucleotides look almost identical above melting and have a signature that was

previously assigned to single stranded oligonucleotides with bases arranged in an A-

form like fashion. Optical absorption of 2AP confirms that its stacking with

neighboring bases is weaker in our single LNA-modified oligonucleotides that in

corresponding DNA despite greater thermal stability of the LNA-modified form.

This correlates with thermodynamical calculations which suggest that entropy is the

main factor in LNA stabilization not enthalpy.

Figure 4.12 shows temperature dependences of the fluorescence yield, Φfl

(normalized to fluorescence yield of free 2Ap at each temperature), for four

oligonucleotides, DNA-1sG, DNA-1sG I, LNA-1sG and LNA-1sI. Four important

152

0 20 40 60 800

50

100

150

0.00

0.05

0.10

0.15

0.20

0.25

T (oC)

LNA_1sG

DNA_1sI

DNA_1sG

LNA_1sI

B

1/Φ

flA

DNA_1sI

LNA_1sI

LNA_1sG

DNA_1sG

Φfl

Figure 4.12 A) Temperature dependence of the 2AP fluorescence yield (normalized to fluorescence of free 2AP in solution), Φfl, for the four oligonucleotides studied. See Figure 4.1 and text for details. B) Temperature dependence of the reciprocal of fluorescence yields, 1/Φfl.

153

remarks can be made from inspecting Figure 4.12. First, the fluorescence of 2AP* is

quenched in all four oligonucleotides (see also Table 4.2) at all temperatures, above

and below melting. Second, a dramatic decline in all fluorescence yields is observed

upon hairpin hybridization (i.e. below melting temperature). In Figure 4.12B the

differences below melting temperature are emphasized by plotting reciprocal of Φfl.

Third, the quenching in LNA is less efficient than in analogous DNA. Forth, the

fluorescence yield of 2AP* is noticeably smaller in guanine containing

oligonucleotides compare to inosine containing ones, which is assigned to the charge

transfer contribution since G has smaller oxidation potential than I.

It is apparent from the figure that reciprocal of Φfl for DNA_1sG is the largest

(mostly quenched fluorescence yield) among all four oligonucleotides. Also, it is the

only one constantly rising upon temperature lowering. Φfl–1 of other oligonucleotides

have different amplitudes, whereas their temperature dependence exhibit somewhat

similar behavior. After initial rise just below Tm, Φfl–1 saturates and even starts to

gradually decline upon further temperature lowering.

For delineating subtle differences between LNA and DNA, it seems

appropriate to evaluate the ratio:

γ = ΦLNA/ΦDNA (3)

between the fluorescence yields of corresponding LNA and DNA oligonucleotides

(see Figure 4.13). Above melting, the temperature dependence of γ for guanine

containing LNA and DNA, γG, is relatively flat, while just below melting it drops and

gradually increases upon further temperature decrease. The drop is mostly artificial

154

and is due to the difference in melting temperatures of LNA and DNA hairpins. The

ratio for the inosine containing oligonucleotides, γI, is smaller than γG above the

melting temperature but dramatically increases below Tm, reaching almost a factor of

3 at 5oC.

0 20 40 60 801.0

1.5

2.0

2.5

γG

γI

γ

Temperature (oC)

Figure 4.13 Temperature dependence of the relative fluorescence yield of 2AP, γ=ΦLNA/ΦDNA, in LNA and DNA oligonucleotides.

Since the differences in DNA and LNA oligonucleotides with identical base

sequences are due to peculiarities of their structural assembly, understanding subtle

structural variations should be addressed. The molecular dynamics simulation

155

revealed that the overall geometry of the oligonucleotide is not substantially altered,

which coincides with the conclusion drawn from absorption and circular dichroism

measurements. Only local changes, near modified LNA nucleotide, are observed

between DNA and LNA. The subtle structural variations manifest themselves in

different distances between bases. As a result the interactions between 2AP and

neighboring bases differ. The computed interaction energies between 2AP and

neighboring adenines are listed in Table 4.3. It is apparent from the Table that the

interactions between 2AP and neighboring adenine bases are better in DNA in both

directions. More pronounced shift in 2AP absorbance and greater quenching of 2AP*

fluorescence result from such a better interaction exhibited by bases in DNA as

compared to LNA.

Table 4.3 The computed energies of interaction between 2AP and neighboring adenine bases.

4.5 Conclusions

1) Study of photoinduced charge transfer between photoexcited 2-aminopurine

and guanine provided quite small value of β at room temperature (0.32Å-1). The

DNA LNA

Direction A3’(G) A5’ AL3’(G) A5’

Energy (kcal/mol) 8.2 9.7 10.8 4.9

156

method of assessment of the charge transfer rate by comparing 2AP* fluorescence

quenching in guanine containing oligonucleotides with a reference system, where

guanine was replaced with inosine, was validated in oligonucleotides with interstrand

charge transfer pathway at temperatures above duplex melting.

2) Temperature dependence of photoinduced charge transfer between 2-

aminopurine and guanine at various positions within DNA hairpins has been studied

and correlated with thermal fluctuations of bases in the DNA duplex. The results

support the gated mechanism, where thermally induced conformational fluctuations

control the rate of charge transfer.

3) Modification of a single nucleotide with locked sugar moiety (LNA) causes

mostly local changes in conformation of a 31-mer DNA hairpin below melting

temperature, as seen in optical absorption of 2AP neighboring to LNA modification.

The global changes, according to CD, are identifiable but not significant – the

characteristic B-form persists. Above the melting temperature, the difference in

global structure of LNA and DNA diminishes: they both possess helicity and chirality

characteristic of A-form.

4) Despite a greater thermal stability of LNA containing oligonucleotide, it

has a lower efficiency of 2AP* fluorescence quenching by G separated from 2AP by

a single LNA modified nucleotide (adenine). A lower efficiency of charge transfer

between 2AP* and guanine in LNA modified hairpin agrees with a poorer stacking

identified by 2AP absorption and CD.

157

4.6 References

1 Kelley, S.O., Barton, J.K., Science, 1999, 283, 375-381. 2 Priyadarshy S., Beratan D.N., Risser, S.M., J. Phys. Chem., 1996, 100, 17678–17682. 3 see for example Williams, T.T., Odom, D.T., Barton, J.K., J. Am. Chem. Soc., 2000, 122, 9048-9049. 4 O'Neill, M.A., Barton, J.K., J. Am. Chem. Soc., 2004, 126(37), 11471-11483. 5 Abdou, I.M., Sartor, V., Cao, H., Schuster, G.B., J. Am. Chem. Soc., 2001, 123, 6696-6697. 6 Bloom, L.B., Otto, M.R., Beechem, J.M., Goodman, M.F., Biochemistry, 1993, 32, 11247-11258. 7 Meggers, E., Michel-Beyerle, M. E., Giese, B., J. Am. Chem. Soc., 1998, 120, 12950-12955. 8 Meggers, E., Kusch D., Spichty, M., Wille, U., Giese, B., Angew. Chem. Int. Ed. Engl., 1998, 37, 460-462. 9 Jortner, J., Bixon, M., Langenbacher, T., Michel-Beyerle, M. E., Proc. Natl. Acad. Sci. USA, 1998, 95, 12759-12765. 10 Bixon, M., Giese, B., Wessely, S., Langenbacher, T., Michel- Beyerle, M. E., Jortner, J., Proc. Natl. Acad. Sci. USA, 1999, 96, 11713- 11716. 11 Bixon, M., Jortner, J., Chem. Phys., 2002, 281, 393-408. 12 Henderson, P.T., Jones, D., Hampikian, G., Kan, Y., Schuster, G.B., Proc. Natl. Acad. Sci. USA, 1999, 96, 8353-8358. 13 Nordlund, T.M., Andersson, S., Nilsson, L., Rigler, R., Gräslund, A., McLaughlin, L.W., Biochemistry, 1989, 28, 9095-9103. 14 Larsen, O.F.A., van Stokkum, I.H.M., Gobets, B., van Grondelle, R., van Amerongen, H., Biophysical Journal, 2001, 81, 1115-1126.

158

15 see for example O’Neil, M.A., Barton, J.K., Proc. Natl. Acad. Sci. USA, 2002, 99(26), 16543-16550. 16 Sowers, L.C., Fazakerley, G.V., Eritja R., Kaplan, B.E, Goodman, M.F., Proc. Natl. Acad. Sci. USA, 1986, 83(15), 5434-5438. 17 Guckian, K.M., Schweitzer, B.A., Ren, R.X.-F., Sheils, Ch.J., Tahmassebi, D.C., Kool, E.T., J. Am. Chem. Soc., 2000, 122, 2213-2222. 18 van Holde, K.E., Johnson, W.C., Ho, P.Sh., Principles of Physical Biochemistry, Prentice-Hall Inc., New Jersey, 1998, pp. 122-125. 19 Ward, D.C., Reich, E.S., Stryer, L., J. Biol. Chem., 1969, 244, 1228-1237. 20 Jean, J.M., Hall, K.B., Proc. Natl. Acad. Sci. USA, 2001, 98, 37-41. 21 Steenken, S., Jovanovic, S.V., J. Am. Chem. Soc., 1997, 119, 617–618. 22 Carell, T., Behrens, C., Gierlich, J., Org. Biomol. Chem., 2003, 1, 2221-2228. 23 Fiebig, T., Wan, Ch., Zewail, A.H., Chem. Phys. Chem., 2002, 3, 781-788. 24 Wan, Ch., Fiebig, T., Schiemann, O., Barton, J.K., Zewail, A.H., Proc. Natl. Acad. Sci. USA, 2000, 97(26), 14052-14055. 25 Rachofsky, E.L., Osman, R., Ross, J.B.A., Biochemistry, 2001, 40, 946-956. 26 Nordlund, T.M., Andersson, S., Nilsson, L., Rigler, R., Gräslund, A., McLaughlin, L.W., Biochemistry, 1989, 28, 9095-9103. 27 Somsen, O.J.G., van Hoek, A., van Amerongen, H., Chem. Phys. Lett., 2005, 402, 61–65. 28 Jean, J.M., Hall, K.B., Biochemistry, 2004, 43, 10277-10284. 29 Wan, Ch., Fiebig, T., Kelly, Sh.O., Treadway, C.R., Barton, J.K., Zewail, A.H., Proc. Natl. Acad. Sci. USA, 1999, 96, 6014-6019. 30 O’Neill, M.A., Barton J.K., J. Am. Chem. Soc., 2004, 126, 13234-13235. 31 Lewis, F.D., Wasielewski, M.R., Top. Curr. Chem., 2004, 236, 45–65. 32 Sanii, L., Schuster, G.B., J. Am. Chem. Soc., 2000, 122, 11545-11546.

159

33 Williams, T.T., Odom, D.T., Barton, J.K., J. Am. Chem. Soc., 2000, 122, 9048-9049. 34 Giese, B., Acc. Chem. Res., 2000, 33(9), 631-636. 35 Lewis, F.D., Liu, X., Miller, S.E., Hayes, R.T., Wasielewski, M.R., J. Am. Chem. Soc., 2002, 124, 14020-14026. 36 Berlin, Yu.A., Burin, A.L., Ratner, M.A., J. Phys. Chem. A, 2000, 104(3), 443-445. 37 Rajski, S.R., Barton, J.K., Biochemistry, 2001, 40, 5556-5564. 38 Rajski, S.R., Kumar, S., Roberts, R.J., Barton J.K., J. Am. Chem. Soc. 1999, 121, 5615-5616. 39 Proligo LLC, web publications. http://www.proligo.com/index.html. 40 Watson, J.D., Crick, F.H.C., Nature, 1953, 171, 737. 41 Mathews, C.K., van Holde, K.E., Ahern, K.G., Biochemistry, 3rd ed., Addison Wesley Longman, Inc., San Francisco, 1999. 42 Boon, E.M., Barton J.K., Bioconjugate Chem., 2003, 14, 1140-1147. 43 O’Neill, M.A., Barton J.K., J. Am. Chem. Soc., 2002, 124, 13053-13066. 44 Abdou, I.M., Sartor, V., Cao, H., Schuster, G.B., J. Am. Chem. Soc., 2001, 123, 6696-6697. 45 Tashiro, R., Sugiyama, H., J. Am. Chem. Soc., 2003, 125, 15282-15283. 46 Kan, Y., Schuster, G.B., J. Am. Chem. Soc., 1999, 121, 10857- 10864. 47 Grozema, F.C., Berlin, Y.A., Siebbeles, L.D.A., J. Am. Chem. Soc., 2000, 122, 10903-10909. 48 Bixon, M., Jortner, J., J. Phys. Chem. B., 2000, 104, 3906-3913. 49 Voityuk, A.A., Rosch, N., Bixon, M., Jortner, J., J. Phys. Chem. B., 2000, 104, 9740-9745. 50 Grozema, F.C., Siebbeles, L.D.A., Berlin, Y.A., Ratner, M.A., Chem. Phys. Chem., 2002, 3(6), 536-539.

160

51 Matulewski, J., Baranovski, S. D., Thomas, P., Phys. Chem. Chem. Phys., 2005, 7, 1514–1517. 52 Troisi, A., Orlandi, G., J. Phys. Chem. B, 2002, 106, 2093-2101. 53 Voityuk, A.A., Siriwong, Kh., Rösch, N., Angew.Chem.Int.Ed., 2004, 43, 624-627. 54 Voityuk, A.A., Siriwong, Kh., Rösch, N., Phys.Chem.Chem.Phys., 2001, 3, 5421-5425. 55 Nordlund, T.M., Xu, D., Evans, K.O., Biochemistry, 1993, 32(45), 12090-12095. 56 Xu, D., Evans, K.O., Nordlund, T.M., Biochemistry, 1994, 33(32), 9592-9599. 57 Saenger, W., Riecke, J., Suck, D., J. Mol. Biol., 1975, 93, 529-534. 58 Aida, M., Nagata, Ch., Chem. Phys. Lett., 1982, 86(1), 44-46. 59 Lewis, F.D., Wu, T., Zhang, Y., Letsinger, R.L., Greenfield, S.R., Wasielewski, M.R., Science, 1999, 277, 673-676. 60 Troisi, A., Orlandi, G., J. Phys. Chem. B, 2002, 106, 2093-2101. 61 Netzel, T.L., J. Chem. Ed., 1997, 74(6), 646-651. 62 O’Neill, M.A., Becker, H-C., Wan, C., Barton, J.K., Zewail, A.H., Angew. Chem. Int. Ed., 2003, 42, 5896-5900. 63 McConnell, H. M. J. Chem. Phys. 1961, 35, 508-515. 64 Davis, W.B., Ratner, M.A., Wasielewski, M.R., J. Am. Chem. Soc. 2001, 123, 7877-7886. 65 McTigue, P.M., Peterson, R.J., Kahn, J.D., Biochemistry, 2004, 43, 5388-5405. 66 Stivers, J.T., Nucleic Acids Res., 1998, 26(16), 3837-3844. 67 Berova, N., Nakanishi, K., Woody, R.W., Circular Dichroism. Pronciples and Applications, 2nd ed., Wiley-VCH, Inc., New York, 2000, pp. 703-718. 68 Harada, N., Nakanishi, K., Circular dichroic spectroscopy: exciton coupling in organic stereochemistry, University Science Books: Mill Valley, CA, and Oxford University Press: Oxford, 1983.

161

69 Egli, M., Minasov, G., Teplova, M., Kumar, R., Wengel, J., J.Chem.Soc., Chem. Commun., 2001, 7, 651-652. 70 Petersen, M., Nielsen, C.B., Nielsen, K.E., Jensen, G.A., Bondensgaard, K., Singh, S.K., Rajwanshi, V.K., Koshkin, A.A., Dahl, B.M., Wengel, J., Jacobsen, J.P., J. Mol. Recognit., 2000, 13, 44-53.

162

5 TOWARDS PTDC STUDY OF DNA: DNA-AMPHIPHILE COMPLEXES AND

THEIR IMMOBILIZATION ON SURFACES

5.1 Introduction

The study of DNA complexed with cationic amphiphiles was primarily

motivated by the necessity of neutralizing DNA and making it soluble in non-polar

solvents. One of the limitations of photoinduced displacement charge technique is

that only organic solvents can be used in the experiment. DNA is a polyanionic

molecule with negatively charged sugar-phosphate backbone. Normally, DNA is

soluble only in aqueous solutions, and it is neutralized with sodium ions. This would

make its solution fairly conductive. However, if the counter ions of phosphate

negative charges are replaced with cationic amphiphiles, the resulting DNA-

amphiphile complex becomes soluble in organic media. Such a complex could be

studied by means of either photoinduced displacement current, if the complex is

dissolved in organic solvent, or by the displacement charge method, if it is

immobilized in oriented films on a silica surface.

Charge transfer is sensitive to how well the bases overlap with each other, as

was discussed in Chapter 4. Variations of base overlap due to temperature changes,

and the alteration of DNA conformation by LNA modification, revealed strong

dependence of the charge transfer efficiency on base stacking along the double helix

of DNA. Complexation of DNA with amphiphiles perturbs its standard B-form, and

thus the base stacking when the complex is dissolved in organic solvents. Schuster et

163

al.1 reported that the yield of strand cleavage at guanine sites, caused by oxidation of

guanine via photoinduced charge transfer, was significantly reduced in DNA-

amphiphile complexes.

Hydration of DNA within the complex increases its thermal stability and

improves base stacking.2 The water content is known to affect DNA structure, for

example, at high alcohol concentration in aqueous solution B-form transforms into C-

form.3 Tanaka et al.4 reported that conformation of DNA changes from the C-form in

dry organic solvents to the B-from upon addition of water. They also showed that

base stacking within DNA-amphiphile complex varies with water content. In their

experiment, DNA-amphiphile complex was aligned in one direction by stretching a

cast film. In the “wet” sample, intercalated ethidium bromide exhibited a strong

linear dichroism with a greater absorption at polarization of light perpendicular to the

stretching direction. However, little anisotropy was observed in a dry film, indicating

that the base pairs, as well as the intercalated dyes become “slant” to the axis of the

strands. Yang et al.5 showed that base stacking in a similar aligned film of DNA is

not planar in a dehydrated sample; they speculated that bases are rather positioned

edge to edge with respect to each other. Hydration causes bases to rotate from the

edge-to-edge to planar stacking, increasing π-overlap.

In this study we show that a DNA-amphiphile complex soluble in organic

solvents exists, but not necessarily in the hybridized double stranded form. The DNA

duplex requires the presence of water. The duplex’s thermal stability, although

improved by water, is nevertheless weaker than that exhibited by DNA in buffer

164

solution. The low thermal stability of hybridized DNA-amphiphile complexes

imposes limitations on their application for PTDC study at room temperature.


a) DNA-amphiphile complexes, their preparation, structure and thermal

stability

b) immobilization of DNA and DNA-amphiphile complexes on silica surfaces

c) possibility of using PTDC technique to study charge transfer in DNA-

amphiphile complexes


5.2.1 Materials

DNA oligonucleotides, 31-mer hairpins designed for photoinduced charge transfer

study in Chapter 4, were obtained from IDT and stored at -10°C before use.

Oligonucleotides used in DNA immobilization on the surface had a C6-aminolinker at 5’-

position. Aqueous solutions of oligonucleotides (~20 µM) were prepared immediately

before the experiments.

Disuccinimidyl ester crosslinking agents, disuccinimidyl suberate (DSS), from

Pierce, and disuccinimidyl carbonate (DSC), from Aldrich, were used without

purification. Structures of the compounds are shown in Figure 5.1.

Three different cationic amphiphiles were tested for DNA-amphiphile

complexation: N,N,N-trimethyl-N-(3,6,9,12-tetraoxadocosyl)ammonium bromide

165

(amphiphile1) generously provided by Professor A. Gopalan, decyltrimethylammonium

bromide, DTAB (amphiphile2), and didodecyldimethylammonium bromide, DDAB

(amphiphile3), from Aldrich. Structures of these amphiphiles are shown in Figure 5.2.

HPLC grade solvents: chloroform, ethanol, dimethylsulfoxide (DMSO) and

toluene, from Aldrich, were used without further purification.

N OO N

O

O

O

O

O

O

N O

OO

O

O N

O O

O

DSS

DSC

Figure 5.1 Structures of crosslinking agents used in this study.

5.2.2 Preparation of DNA-amphiphile complexes

Aqueous solution of DNA oligonucleotide was mixed with aqueous solution of a

cationic amphiphile in a desired proportion of DNA to amphiphile. The precipitate of a

resulting complex was gathered by centrifugation at 6000 RPM, thoroughly washed with

distilled water to eliminate salt and any unreacted amphiphiles or oligonucleotides, and

vacuum-dried. The DNA-amphiphile complex was then dissolved in a mixture of

166

solvents, either toluene/ethanol (1:0.25) or chloroform/ethanol (1:0.25). It should be

noted that only thoroughly desalted complexes dissolve completely in organic solvents.

CH3(CH2)9 O

CH2 CH2N

+

CH3

CH3

CH3

4

CH3 (CH2)11 N+

CH3

CH3CH3

CH3 (CH2)11

CH3 (CH2)11

N+

CH3

CH3

amphiphile 1

amphiphile 2

amphiphile 3

Br-

Br-

Br-

Figure 5.2 Structures of cationic amphiphiles used in this study.

5.2.3 Immobilization of DNA-amphiphile complex on silica surface

Silica substrates were cleaned and modified with aminosilane (APS) as described

in experimental section of Chapter 2. Amines of the modified surface were activated

with a cross linker, DSC or DSS, by immersing the aminated substrates in 1mM DMSO

solution of disuccinimidyl ester for 2 hours at room temperature. The activated substrates

which were thorough rinsed in ethanol and dried at 100°C, were then modified by

167

aminated at 5’-end DNA oligonucleotide or its complex with amphiphile from either

aqueous or chloroform/ethanol solution (~10-6 M) overnight at room temperature. Then,

the substrates were rinsed with the solvent and dried in the air. Figure 5.3 sketches the

described immobilization procedure.

5.2.4 Measurements of absorption spectra

Absorption spectra of DNA-amphiphile complexes in organic solvents and

mixtures were measured in 40 µL cuvettes with 1 cm path length using Cary 100 Bio

UV-Vis spectrophotometer.

Absorption spectra of DNA immobilized on silica substrate were measured

using Perkin Elmer Lamda 40 UV/VIS spectrometer, equipped with a Glen-Tompson

polarizer allowing two polarizations of light (vertical and horizontal).


Fluorescence melting profiles were also measured in 40 µL cuvettes with 1 cm

pathlength using Varian Cary Eclipse fluorescence spectrophotometer. The

fluorescence ‘melting curves’ were monitored at 370 nm (wavelength of 2-

aminopurine fluorescence maximum) at the rate 1°C per minute with the excitation at

316 nm. Fluorescence intensities were further normalized to the temperature

variation of 2AP absorbance at 316 nm.

168

N O

O

O

NO O

O

O

O

OH

O

CH3

OCH3

OCH3

Si

N

HH

N HH

SiO O

O

CH3CH3

N H

O

SiO O

O

CH3CH3

NO O

O

O

N H

O

SiO O

O

CH3CH3

N H

DNA

O

+1

2

+

in acetone, 3 min

Si

Si

Si

3

in CHCl3/C2H5OH overnight

Si

in DMSO, > 2 h

Figure 5.3 The three-step procedure for DNA immobilization: 1 – silanization of hydroxylated silica surface by trimethoxyaminosilanes, 2 – functionilization of amines by dissucinimidyl cross-linker, 3 – immobilization of 5’-NH2-C6-DNA.

5.2.6 Circular dichroism measurements

CD spectra were measured in 0.1 cm cuvettes using Jasco J-810

Spectropolarimeter and a typical concentration of complexes was ~25 µM. CD

169

spectra were recorded at various temperatures in the range 5-55°C. ∆ε was calculated

using equation 4.2.

5.2.7 Proton NMR of DNA-amphiphile complexes

1H NMR spectra of amphiphile and DNA-amphiphile complexes dissolved in

CDCl3 were recorded using Varian XL-200 NMR Spectrometer. The chemical shifts

were measured relative to TMS.

5.2.8 AFM of DNA-amphiphile complexes on mica

AFM images were obtained using a PicoSPM microscope from Molecular

Imaging. Freshly cleaved mica surface was modified with octyltrimethoxysilane

from its anhydrous acetone solution, as described in experimental section of Chapter

2. A solution of DNA-amphiphile complex in chloroform/ethanol/water mixture

(1:0.25:0.1) at a concentration of ~30 µM was deposited on octyltrimethoxysilane

modified mica for 5 min. Then mica substrate was washed with solvent and dried in

the air for 5 min.

170

5.2.9 FTIR measurements

Appropriate amounts of the amphiphile1 and DNA-amphiphile1 complex

were evenly deposited onto PTFE-IR card from chloroform solution. Spectra were

recorded using Perkin-Elmer Spectrum One FT-IR spectrometer against air.

5.2.9 PTDC measurements

The experiments were carried out following the procedure described in the

experimental section of Chapter 3. Nitrogen laser (337 nm) delivering 100 µJ energy

per pulse at 10 Hz repetition rate was used for excitation. All experiments were

performed at room temperature.

5.3 Results and discussion

5.3.1 DNA-amphiphile complexes

All three cationic amphiphiles shown in Figure 5.2 form complexes with DNA

oligonucleotides when mixed together in aqueous solution. Formation of DNA-

amphiphile complexes (schematically represented in Figure 5.4) is manifested by its

precipitation, and thus, indicating very poor solubility of the resulting complex in

aqueous solution. In order for precipitation to occur, the product of concentrations of

DNA phosphate groups and amphiphile must be at a certain value. By analogy with

171

solubility products for slightly soluble salts, one can introduce a solubility product,

KSP, for DNA-amphiphile1 complex.

DNA-PO4- (aq) + amphiphile1 (aq) <==> DNA-PO4

--amphiphile1 (s), KSP

OO

O

B

..

...

P-O O

.

.

ASE

n

Amphiphile BromideO

O

O

B

..

...

P-O O

.

.

ASE

n

Na+ N

+

CH3 CH3

CH3

OCH2

CH3

49

Figure 5.4 Schematic illustration of DNA-amphiphile1 complex formation.

By addition of small amounts of amphiphile1 to the solutions of different

DNA concentrations, we found that the product of concentrations should be

1.5(±0.2)×10-7 M2 for the formation of precipitate. This value was calculated for our

31mer-oligonucleotide and it could be different for oligonucleotides with different

lengths.

Depending on the initial concentration of DNA, the precipitation occurred at

different +/- charge ratios. At first, it seemed confusing since the formation of the

complex was reported to occur at the isoelectric point (1:1 ratio).4 This made us wonder

whether the complex is completely neutralized or its structure is more complicated than

simply equal amounts of phosphate groups and amphiphile molecules. The solubility of

172

the complex in non-polar solvents would be affected, if the complex carries charges

which are not neutralized. To understand the structure of the complex we used another

avenue. We compared the amount of the amphiphile1 obtained through IR spectrum with

the amount of phosphate groups obtained through absorption. By calibrating the intensity

of IR bands to the number of moles of amphiphile1, we were able to use the intensity of

IR band characteristic of CH2 group asymmetric stretch (2925 cm-1) as a measure of the

amphiphile1 amount in the complex. The amount of phosphate groups (31 per one

oligonucleotide) was calculated using absorption of DNA at 260nm. The comparison of

the amount of the amphiphile1 obtained through IR spectrum with the amount of

phosphate groups obtained through absorption proved that the DNA-amphipihle1

complex had 1:1 ratio, indeed.

1H NMR signals from protons of amphiphiles were broadened upon

complexation with DNA as compared with those of free amphiphile in solution.

Figure 5.5 shows 1H NMR spectra of amphiphile3 in CDCl3 before (A) and after (B)

complexation with DNA. The signals from N-methyl protons of the cationic head

group (3.4 ppm), as well as protons of neighboring methylene groups CH2 (3.52 ppm,

1.62 ppm) become broader upon complexation, whereas little change was observed in

the line widths and chemical shifts for the rest of the amphiphile (0.88 ppm, 1.26

ppm, 1.35 ppm). The broadening of the signals was previously attributed to the

fluctuation of an anisotropic local magnetic field induced by the reduction of

amphiphile molecules motion.6 Therefore, after the complexation with DNA,

173

3.5 3.0 2.5 2.0 1.5 1.0

0.00

0.05

0.10

0.15

0.20

0.25

3.52

3.41

1.62

1.35

1.26

0.88

Am phiphile head

Am phiphile ta il

A

3.5 3.0 2.5 2.0 1.5 1.0

0.00

0.05

0.10

0.15

3.45

3.36

1.66

1.34

1.25

0.88

ppm

B

Figure 5.5 1H NMR spectra of amphiphile3 in CDCl3: free (A) and after complexation with DNA (B).

cationic head groups of the amphiphile are tightly fixed, while the binding to DNA

has no apparent effect on mobility of the hydrocarbon chain tails.

174

Possible aggregation between complexes would affect their immobilization on

silica via covalent attachment. In order to covalently link DNA to the surface, 5’-end

aminogroups of oligonucleotides should be accessible for chemical reaction with the

activated surface. Aggregation, however, may conceal the amine linkers of DNA,

preventing them from reaction with the surface. Also, aggregation would increase the

size of the species and, consequently, their rotation times. The photovoltage signal in

the standard PTDC evolves due to the rotation of species with charge separation

directed by the external electric field. Thus, slow rotation of large aggregates would

make the signal too small when using standard PTDC.

AFM analysis supported that there is no aggregation between DNA-

amphiphile complexes neither in “dry” nor in “wet” forms. Figure 5.6 shows AFM

image of DNA-amphiphile1 complex species deposited onto mica. Freshly cleaved

mica was modified with trimethoxyoctylsilane to form hydrophobic surface for better

adsorption of hydrophobic DNA-amphiphile complex. Figure 5.6 shows that the

complexes are distributed randomly on the surface in the form of particles of ~10nm

in size. This size is close to the estimated size of individual DNA-amphiphile1

complexes, therefore suggesting that there is no aggregation.

After thorough desalting and drying, the complexes readily dissolve in such

organic solvents as toluene, chloroform as well as in their mixtures with ethanol and

water. Solubility, however, strongly depends on the nature of the solvent. For

example, toluene dissolves DNA-amphiphile complexes better than chloroform.7

Still, in toluene solution, the characterization of the complex is limited to

175

fluorescence only because of strong toluene absorption at 260 nm. This leaves out

the use of such powerful techniques as circular dichroism and absorption

spectroscopy.

Figure 5.6 AFM image of DNA – amphiphile1 complex species deposited onto modified with octylsilanes mica surface from chloroform/ethanol/water (1:0.25:0.1) solution.

The fluorescence study revealed that DNA in the complex with amphiphile1

in dry toluene/ethanol solution is not hybridized. Figure 5.7-A shows fluorescence

176

“melting profiles” of oligonucleotide-amphiphile1 complex in toluene-ethanol

solution. No apparent melting transition appeared within the 25-70°C temperature

range, indicating that there is no hybridization in dry toluene/ethanol mixture.

The quenching is surprisingly very weak for the complex. Even for the

oligonucleotide with guanine placed next to 2-aminopurine (0sG), which exhibited

strong quenching in both double stranded and single stranded forms in buffer

solution, the fluorescence yield of 0sG-amphiphile complex is almost the same as the

yield of 0sI-amphiphile complex. The resulting quenching yield, η = 0.07, is even

lower than for 3sX in the buffer! This result is probably the most striking result and

can be caused by change in energetics of the reaction (∆G and λ) and/or by change in

geometry of base-base orientation/interaction.

Evident melting appears only upon addition of a small amount of water (see

Figure 5.7-B). Based on fluorescence ‘melting curves’, the melting temperatures Tm

increase in solutions with 7 % v.v. of water, and reach 35°C and 34°C for 0sG and

0sI, respectively. Ethanol serves as a buffer between organic solvent and water, since

it is miscible with both. The maximum amount of water which can be added to

chloroform or toluene mixed with ethanol in 1/0.25 proportion is about 7% v.v.

Above this percentage of water, the mixture becomes immiscible with a visible

stratification into two layers. Less amount of water drops Tm dramatically. The

decreasing of the water content to 6 % v.v. reduces the Tm by 20 degrees to only

15°C! It should be noted that due to the dramatic sensitivity of melting temperature

towards water and small volumes used in the study (1 µL of water at a time was

177

Figure 5.7 Fluorescence “melting curves” of 0sG(I), A) in toluene/ethanol (1/0.25), B) in toluene/ethanol/water (1/0.25/0.1).

0.15

0.20

0.25

0.30

0.35

0.40

0.45

A

ΦG

ΦIΦ

30 40 50 60 700.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

B

ΦG

ΦI

Temperature

Φ

178

added), the reproducibility of melting point was rather poor: the observed Tm varied

between 20°C and 35°C for the same amount of water in 1/0.25 mixture of

chloroform/ethanol. Thus, the observed melting temperatures of DNA amphiphile

complexes in organic solvents never reached the Tm for these oligonucleotides in

buffer solution (~56-60 °C).

The presence of water appears not only to increase the melting temperature of

DNA in complexes with amphiphiles dissolved in organic solvents, but it also

“improves” fluorescence quenching. Although 2-aminopurine fluorescence quantum

yields in hairpins are still greater than in buffer solution, the η value becomes closer

to what was observed in buffer (see Table 5.1).

As was described in Chapter 4, the absorption of 2AP at 330 nm can be used

to characterize stacking of 2-aminopurine within DNA. Absorption of 2AP in DNA-

amphiphile1 complex, reveals poor stacking of 2-aminopurine with other bases at

room temperature (see Figure 5.8).

Table 5.1 Fluorescence quantum yields, Φ, of 2-aminopurine in 0sG and 0sI hairpins at 25°C and melting temperatures, Tm.

Toluene/ethanol (1 : 0.25)

Toluene/ethanol/water(1 : 0.25 : 0.1) PBS buffer

Sequence Tm , °C Φ η a Tm, °C Φ η a Tm, °C Φ η a 0sG N/A 0.39 35 0.10 59.9 0.0085 0sI N/A 0.42 0.07 34 0.19 0.47 54.3 0.052 0.84

a quenching yield, η = 1-ΦG/ΦI

179

300 310 320 330 340 350 3600.00

0.01

0.02

0.03

0.04

0.05

0.06Ab

sorp

tion

wavelength (nm)

Figure 5.8 2AP absorption spectra at room temperature: in DNA (PBS solution)--dash-dot, in DNA-amphiphile complex (toluene/ethanol=1/0.25)--solid, and (toluene/ethanol/water=1/0.25/0.1)--dash lines.

Chloroform is a more convenient solvent than toluene for studying DNA

because of a better transparency in the UV region. In chloroform, absorption and

circular dichroism spectroscopy can be used for DNA-amphiphile complex structural

characterization. Figure 5.9 shows the absorption melting profile for DNA-

amphiphile1 complex in “dry” chloroform/ethanol (1:0.25) solution and after addition

of water (1:0.25:0.1). The same as in toluene/ethanol solution, no apparent melting

transition is observed within the temperature range 2-55 °C, without water in the

180

chloroform/ethanol solution. However, addition of water results in a pronounced

melting transition with Tm ≈ 20°C.

Only the complex of DNA with amphiphile1 was found to be responsive to

addition of water in the solution. Other amphiphiles (2 and 3) studied did not show

any sign of hybridization with or without water. The efficiency with which water

molecules could be expected to approach DNA in the complex would be influenced

by the hydrophobicity of an amphiphile. Obviously, the presence of ethylene glycol

groups makes the amphiphile1 quite hydrophilic in the proximity of cationic head

group, and therefore, next to DNA. Other amphiphiles, such as DTAB (amphiphile2)

and DDAB (amphiphile3), have hydrophobic tails and do not sustain as much water

near DNA in the complex as amphiphile1 does. This is consistent with the

observation of Tanaka et al.4 who studied DNA complexes with different cationic

amphiphiles and found that DNA duplex was preserved only when DNA was

complexed with single chain ethyleneglycol containing amphiphile. The presence of

the oxygen atom increases the hydrophilic properties of cationic amphiphile mainly

by solvation of oxygen and, possibly,8 by disrupting the hydrophobicity of adjacent

methylene groups.

Additionally, above the point of stratification when mixture coexists in two

different phases, organic and aqueous, DNA-amphiphile complexes have different

behavior depending on the nature of amphiphile. When DNA is complexed with

amphiphile1, the concentration of DNA in the organic phase gradually decreases.

181

0 10 20 30 40 50 600.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35∆A

260n

m

Temperature

Figure 5.9 The change of absorption at 260 nm with temperature for DNA-amphiphile1 complex in chloroform/ethanol(1/0.25)--, chloroform/ethanol/water(1/0.25/0.1)--.

This leads to the weaker signal in either absorption or CD spectra. While,

amphiphiles 2 and 3 do not produce such an effect, DNA complexes with these

amphiphiles stay in organic phase. These observations also explain different degree

of hydrophobicity of studied amphiphiles.

The effect of water on DNA conformation can be clearly seen in organic

solutions. Figure 5.10 shows circular dichroism spectra of DNA-amphiphile1

complex in chloroform/ethanol mixture upon increasing of water content. In “dry”

182

solution, the spectrum demonstrates spectral features of the C-form of DNA.4 It is

well known that native B-form of DNA undergoes conversion to the C-form in

aqueous solutions at high salt concentration or high content of ethanol.9 Water has

been shown to play a crucial role in maintaining the native B-form by interacting with

atoms in the minor groove via formation of the so-called “water spine”.10

The CD spectra in Figure 5.10 reveal the change in DNA conformation upon

addition of water. With increasing water content, the positive peak near 270 nm blue

shifts and overall value of ACott increases, eventually bringing the CD spectrum to

become very similar to the spectrum of DNA in PBS buffer solution. It should be

noted that the measurements were performed at ambient temperature which was

slightly higher than Tm. Therefore, changes in CD spectrum should be attributed to

the conformational changes of single-stranded DNA.

Circular dichroism provides information not only about DNA conformation,

but also about the efficiency of base stacking through ACott value, as discussed in

Chapter 4. Figure 5.11 shows that base stacking efficiency improves slightly with

lowering temperature even in ‘dry’ solution. However the effect is more pronounced

when water is added. The temperature dependence of ACott value parallels that of the

absorption melting profile, indicating an improvement of base stacking upon

hybridization.

183

240 260 280 300-6

-4

-2

0

2

4

6water percentage

2%4%5%6 %7 % 0 %∆ε

wavelength (nm)

Figure 5.10 Effect of water content on CD spectra of DNA-amphiphile1 complex in chloroform/ethanol (1/0.25) solution at 25°C.

CD spectra of DNA-amphiphile1 complex in chloroform(1)/ethanol(0.25)/

water(0.1) mixture and DNA in buffer solution were compared above melting, 55°C

and 85°C, respectively (upper inset in Figure 5.11) as well as below melting, 2°C and

25°C (lower inset in Figure 5.11). CD spectra coincide for both cases, therefore,

providing an evidence for the conformational similarity between DNA molecules

184

both in buffer solution and in the complex with amphiphile1 in organic solution,

when mixed with appropriate amount of water.

0 10 20 30 40 500123456789

101112131415

240 260 280 300 320

-8

-6

-4

-2

0

2

4

6

∆ε

wavelength (nm)

240 260 280 300 320-6

-4

-2

0

2

4

∆ε

wavelength (nm)

+H2O

A Cot

t(∆ε 28

0nm-∆ε 24

7nm)

Temperature (OC)

Figure 5.11 The temperature dependence of ACott value for DNA- amphiphile1 complex in “dry” solution () and after addition of 7%v.v. water (). Upper inset: CD spectrum of DNA oligonucleotide in buffer solution (solid) at 85°C and DNA-amphiphile1 complex in chloroform/ethanol/water (1/0.25/0.1) solution at 55°C. Lower inset: CD spectra of DNA in buffer at 25°C and DNA-amphiphile complex in chloroform/ethanol/water (1/0.25/0.1) solution at 2°C.

185

The difference between CD spectra of DNA in buffer solution and DNA in the

complex is even less noticeable than between DNA and LNA modified

oligonucleotides (Chapter 4). This suggests very small structural perturbations of

DNA structure in the complex as compared to DNA in buffer solution.

Although the structure of DNA in the complex is similar to the structure of

DNA in buffer solution, its thermal stability is lower by ~25°C. The thermal stability

depends not only on the duplex itself, namely its sequence and length, but also on the

concentration and nature of counterions. The negatively charged phosphate groups of

complementary strands in DNA duplex repel each other unless they are neutralized.

In aqueous buffer solution the DNA molecule is surrounded by a “cloud” of cations,

which reduce the repulsion between strands. The greater the concentration of counter

ions in the buffer the more stable the DNA duplex is. In the DNA-amphiphile

complex positive charge density is not that high, only one cation per one negatively

charged phosphate group. Therefore, one should expect the reduction of melting

temperature in the complex.

Besides the nature of the counter ion, the presence of organic solvent is a very

well known factor which contributes to the lowering of duplex thermal stability. For

example, formamide or urea has been used for years to reduce melting temperature of

DNA.11 Hence, the reduced thermal stability of DNA-amphiphile1 complex in

organic media should not be surprising.

186

5.3.2 Immobilization of DNA and DNA-amphiphile complex on silica surface

As was discussed in Chapter 2, the construction of self-assembled monolayers

using trimethoxysilanes on silica can provide quite high surface density of covalently

immobilized molecules. Similar aminosilane modified films were used for the

immobilization of DNA on silica. Aminated silica slides were treated with a

crosslinker, disuccinimidyl ester, which possesses two succinimidyl ester groups, one

at each end, and reactive towards primary amines. A crosslinker reacts with the

surface amine as shown in Figure 5.2, forming one covalent amide bond and leaving

another succinimidyl group for further reaction with amine of DNA. The last step

involves reaction of 5’ aminated (5’NH2-C6-DNA) oligonucleotide with the

succinimidyl group of a cross linker, providing covalent attachment of DNA. Both

the aminated DNA by itself or in the complex with amphiphile can be covalently

immobilized in this manner.

The density of DNA molecules in a monolayer was tested by measuring

optical absorption of immobilized DNA-amphiphile complex. Figure 5.12 shows

absorption spectra of a silica slide with immobilized DNA-amphiphile1 complex.

The three-step immobilization procedure provides absorption of DNA no less than

0.02, which corresponds to the surface concentration of 3×1013 molecules per cm2.

The surface concentration of molecules was calculated using Equation 2.2.

The extinction coefficient (ε) of oligonucleotide was converted to ε for single-

stranded form, expecting 20% increase in absorption for ss-DNA vs. ds-DNA

187

(3.06×105 M-1cm-1/0.8 per oligonucleotide),12 since DNA most probably has single

stranded form with no water present. It should be noted that cross-linking agents,

either disuccinimidyl suberate or disuccinimidyl carbonate, produce the same surface

density of DNA-amphiphile complexes.

240 260 280 300 3200.00

0.02

0.04

0.06

Abso

rptio

n

wavelength (nm)

Figure 5.12 Absorption spectra of DNA-amphiphile1 complex immobilized on silica surface at vertical polarization (solid line) and horizontal polarization (dashed line).

It is worth mentioning that the immobilization of bare, not complexed DNA

onto activated with succinimidyl surface from PBS buffer solution results in lower

density of bound molecules, 9×1012 cm-2. Lower surface concentration is a result of

188

competitive nature of the reaction of primary amine with succinimidyl groups and

their hydrolysis, when water is present. In organic solvents we exclude the hydrolysis

by eliminating water from reaction solution. This results in the increase of DNA

surface concentration approximately 3 times.

5.3.3 Application of PTDC for studying charge transfer in DNA-amphiphile complex

Initially our plan for studying charge transfer in DNA included the application

of two techniques. The first one was photoinduced displacement current in its regular

mode to study charge transfer in solution. The second technique was photoinduced

displacement charge, which was modified purposely for the study of charge transfer

events at interfaces.

Although very powerful, photoinduced displacement current technique has

two major limitations with respect to the study of charge separation extent in DNA.

The first one, as it was mentioned earlier in the introduction to this chapter, stems

from the fact that this technique is restricted to nonconductive solvents only. In order

to overcome this difficulty we replaced inorganic counter ions with cationic

amphiphiles. By doing this, we managed to make DNA complexes soluble in non-

polar solvents. The other limitation is related to the size of the DNA-amphiphile

complex. Since in this technique, the signal evolves with the rotation of species

directed by external electric field, the rotation time becomes a determining factor. At

189

the same time the rotation is directly impacted by the size of the molecule, the smaller

the molecule the faster the rotation.

The rotational diffusion time can be calculated using the Einstein law for a

sphere of volume V in a medium of viscosity η:13

TkV

D B ⋅⋅

==ητ

61

(5.1)

For the estimated volume of DNA-amphiphile1 complex V = 3×105 Å3,14 and

η = 0.58 cP15 for chloroform solvent viscosity at room temperature (298K), the

calculated rotation time is about 4.4 ns. However, DNA-amphiphile1 complex is not

a spherical molecule. The complex can be viewed as an ellipsoid of revolution,

where semiaxis a is greater than other two semiaxes, b=c. According to Perrin,13

there are two diffusion coefficients for an ellipsoid of revolution, DL for the rotation

about the longitudinal semiaxis and DE for the rotation about equatorial semiaxis. In

our case the charge separation occurs along the long axis of the molecule. Therefore,

the rotation time of charge separated state is determined by the rotation about

equatorial semiaxis. This type of rotation can be approximated by the following

equation in the case of small γ:13

)ln2.0(361

2 γγτ

τ−

≈= spher

EE D

(5.2)

The aspect ratio γ=b/a is equal to 0.35 for the DNA-amphiphile1 complex.16 The

approximation provided by Equation 5.2 gives ~10ns for the rotation time of DNA-

amphiphile1 complex.

190

Among all studied oligonucleotides, 2sG is the most appropriate for the

application of PTDC technique. It is the suitable charge recombination time of this

oligonucleotide which makes it preferable to other DNA molecules. Lewis et al.17

reported that when donor (G) and acceptor (stilbene-Sa) are separated by 2 adenine

bases, the observed charge recombination time was 1.9 ns. Taking into account that

∆G for the charge recombination in the case of 2AP- and G+ is almost the same as for

Sa--G+ pair, respectively -3.14eV and -3.1eV, one should expect similar rate constants

for charge recombination. Therefore, the charge recombination time in 2sG

oligonucleotide is expected to be ~2 ns. Other oligonucleotides with closer position

of donor relative to acceptor exhibit much faster recombination times, which are

beyond the time resolution of our PTDC method (~0.5ns).

The number of the excited molecules depends on the concentration of

molecules, the yield of charge transfer which for 2sG oligonucleotide is 33%, and the

energy of the excitation source. VSL 337 nitrogen laser is the only source of

excitation with the proper wavelength that we have in our disposal. Owing to the low

energy pulses of this laser, ~100µJ (4ns long), the number of excited molecules

should be relatively low.

For the charge separation extent in 2sG oligonucleotide of 10.2Å (assuming

3.4 Å base-base separation), for optical density, A337=0.14 (~10-4 M), of 2AP in

DNA-amphiphile1 molecules in the solution which would result in 3µJ of energy

absorption, for the rotation time of ~10ns, and for the recombination times of infinity,

10ns, and 1ns, the expected signals are depicted in the Figure 5.13. Depiction of

191

three traces for three different recombination times would show where to expect our

signal.

0 10 20 30 40 50-20

-10

0

10

20

30

40τCR=infinity

τCR=10nsτ

CR=1ns

Time (ns)

Phot

ores

pons

e (µ

V)

laser

Figure 5.13 Photoresponse from DNA (2sG) – amphiphile1 complex in chloroform and calculated traces for three charge recombination times (1, 10 ns and ∞).

As one can see from Figure 5.13, the amplitudes of the expected signals for

the conditions described above are small and near the detection limit. This situation

is caused by the low laser energy and long rotation time of the complex. While the

192

latter is a factor that cannot be changed for this specific oligonucleotide, the former

can be replaced with a different laser.

There are two aspects that should be considered while choosing the excitation

source--high energy, delivered in short pulses, and more appropriate excitation

wavelength. 337nm, the wavelength of nitrogen laser, is at the edge of the 2AP

absorption. Because of that this laser is able to excite only a small portion of 2AP

molecules. The situation worsens in organic solvents due to the blue shifted

absorption of 2AP in DNA-amphiphile complex dissolved in organic media at room

temperature. Thus, a more preferable laser would be the one, which wavelength is

near to the maximum of 2AP absorption spectrum, around 320 nm.

The modified PTDC method, discussed in details in Chapter 3, allows

studying charge transfer in molecular films at interfaces. It provides reliable results

for measuring charge separation extent, and charge recombination time. And above

all, the big rotation time ceases to be a limiting factor for this technique. We use the

molecular immobilization on the surface as a means of orienting molecules, instead of

using external electric field.

At this point, the technique seems to be very suitable to study charge transfer

in DNA covalently immobilized on transparent silica surface. For signal

amplification, several substrates, each of them with a maximum concentration of

3×1013 DNA amphiphile complexes per cm2, can be stacked on top of each other. For

six such substrates, taking into account 33% yield of charge transfer in 2sG

oligonucleotide, the expected photovoltage is ~0.37 mV (see Appendix C.2).

193

0 5 10 15 20

0.0

0.1

0.2

0.3

0.4Ph

otor

espo

nse

(mV

)

Time (ns)

Figure 5.14 Three solid lines represent expected photoinduced transient displacement charge signal profiles for 2sG oligonucleotide for various recombination times: ∞ (black), 10 ns (green) and 1 ns (blue), while the red dashed line shows the laser pulse profile.

This photovoltage corresponds to the maximum possible amplitude, in the

case when the excitation is instantaneous and all molecules are excited at once.

However, due to long pulse (4ns long) of our laser the excitation is not instantaneous.

Therefore the rise of the signal is slow. When the charge recombination time is long

(assumed infinity), the charge separated species live long enough for the photovoltage

to reach the maximum possible value as depicted in the Figure 5.14. On the other

hand, for shorter charge recombination times, the amplitude would be smaller

because some of the species would relax before all the molecules are excited and the

194

photovoltage reaches its maximum. As it is demonstrated in Figure 5.14, for short

charge recombination times, the signal amplitude decreases and approaches the

detection limit of this method.

Although the technique is applicable in its current configuration, still

improvement with regard to the excitation source is essential: more powerful laser,

with shorter pulses at the most appropriate wavelength (320nm) would be preferable.

5.4 Conclusions

1) Complexation of DNA with cationic amphiphiles allows dissolving DNA in

organic solvents. Among three amphiphiles compared with regard to thermal

stability, only complexes with amphiphile1 containing ethyleneglycol moieties

exhibit melting transitions when the water is present. DNA has different

conformation while in dry solution and adopts close to native B-form after addition of

water. The thermal stability is a direct dependent of the amount of water present in

the solution. The melting transitions of DNA in the complexes with amphiphile1 are

observed at temperatures lower than those for DNA in buffer solution. The DNA-

amphiphile1 complexes are the most suitable to study charge transfer because they

are the only ones that can form duplex at room temperature.

2) Immobilization procedure based on self-assembly of aminosilanes and

disuccinimidyl crosslinking was shown to provide quite high surface density of DNA.

195

As many as 3×1013 DNA-amphiphile complexes per cm2 can be covalently

immobilized with either of the two disuccinimidyl linkers.

3) The study of charge transfer in DNA-amphiphile1 complexes using either

photoinduced displacement current or modified displacement charge techniques is

possible. However, the application of both techniques will be improved with an

excitation source which has short pulses of high energy, and wavelength around

320nm.

5.5 References

1 Huachuan, C., Schuster, G.B., Charge transfer in DNA-lipid complex, Abstracts of Papers, 227th ACS National Meeting, Anaheim, CA, United States, March 28-April 1, 2004. 2 Yang, C., Moses, D., Heeger, A.J., Adv. Mat., 2003, 15, 1364-1367. 3 Girod, J.C., Johnson, W.C. Jr., Huntington, S.K., Maestre, M.F., Biochemistry, 1973, 12, 5092-5096. 4 Tanaka, K., Okahata, Y., J. Am. Chem. Soc., 1996, 118, 10679-10683. 5 Yang, C., Moses, D., Heeger, A.J., Adv. Mat., 2003, 15, 1364-1367. 6 Akao, T., Ito, A., J. Chem. Soc., Perkin Trans., 1997, 2, 213-218. 7 Mel’nikov, S. M., Lindman, B., Langmuir, 1999, 15, 1923-1928. 8 Menger, F.M., Chlebowski, M.E., Langmuir, 2005, 21, 2689-2695. 9 Hanlon, S., Brundo, S., Wu, T.T., Wolf, B, Biochemistry, 1975, 14, 1648-1660. 10 van Holde, K.E., Johnson, W.C., Ho, P.Sh., Principles of Physical Biochemistry, Prentice-Hall Inc., New Jersey, 1998.

196

11 Hutton, J.R., Nucl. Acids Res., 1977, 4(10), 3537–3555. 12 The extinction coefficient of ds-DNA oligonucleotide was calculated using SciTools: Oligoanalyser 3.0, http://scitools.idtdna.com/analyzer. 13 Smirnov, S.N., Liddell, P.A., Vlassiouk, I.V., Teslja, A., Kuciauskas, D., Braun, C.L., Moore, A.L., Moore, T.A., Gust, D., J. Phys. Chem. A, 2003, 107, 7567-7573. 14 The volume of DNA-amphiphile1 complex was estimated using HyperChem Pro6. 15 Handbook of Organic Solvents, Lide, D.R., Ed., CRC Press: Boca Raton, FL, 1995. 16 The aspect ratio γ=b/a can be calculated using moments of inertia obtained with HyperChem Pro6. For DNA amphiphile1, the moments of inertia for the rotation about equatorial semiaxes are 4.5 times greater than the moment of inertia about longitudinal semiaxis. Taking the ratio between equatorial, IE, and longitudinal, IL,

moments of inertia for prolate ellipsoid ( )

2

22

52

5

bM

baM

II

L

E+

= , one calculates γ=0.35.

17 Lewis, F.D., Liu, J., Zuo, X., Hayes, R.T., Wasielewski, M.R., J. Am. Chem. Soc., 2004, 126, 8206-8215.

197

6 SUMMARY AND PERSPECTIVES

Since the discovery of DNA structure by Watson and Crick in 1953, the

ability of DNA duplex to facilitate charge transport through π-stack array has been

teasing scientists’ brains for decades. It has come to a consensus that DNA double

helix can facilitate relatively efficient charge transfer over substantial distances. The

attention has shifted from the question of whether or not DNA can facilitate charge

transfer to the question of how charges migrate through DNA. This has prompted a

considerable interest and generated a great deal of controversy. Various models for

the mechanism of charge transfer in DNA have been proposed in order to explain the

experimental data. The work described in this dissertation commenced in this highly

controversial situation. The existing controversy originates from indirect methods

which are used to study charge transfer in DNA. The need for unambiguous

measurements of charge separation in DNA has stimulated this project.

The work described in this thesis is dedicated to the development of an

appropriate method to study charge transfer in DNA molecules. The development

was made on the basis of transient photoinduced displacement charge technique

(PTDC), which addresses directly the charge separation extent in molecules via

measuring transient photovoltage.

Several issues had to be resolved because of the intrinsic restrictions in this

technique. Standard photoinduced displacement charge technique uses the applied

electric field to orient dipolar molecules, which is not applicable directly to DNA

198

studies. DNA, due to its polyanionic nature, normally is neutralized by small

counterions, which make the solution highly conductive. Two major modifications

necessary to study photoinduced charge transfer in DNA were accomplished. These

modifications included: 1) modification of PTDC technique eliminating the use of

external electric field by orienting molecules via their covalent immobilization on flat

surfaces, i.e. development of surface assisted PTDC (SPTDC) technique, and 2)

‘neutralization’ of DNA with the purpose of making it soluble in organic solvents, by

substituting small counterions with cationic amphiphiles.

Silane-based self-assembly method for covalent immobilization of molecules

onto oxide surfaces was optimized. Molecules in the resulting self-assembled

monolayers (SAM) possessed preferential orientation induced by the surface. A

model system of coumarin SAM was employed evaluating issues of surface density,

orientation of molecules in self-assembled monolayer, and intermolecular

interactions. Introduction of a novel two-step silanization protocol with intermediate

hydrolysis of alkoxy groups provided the possibility to increase surface concentration

of molecules in a monolayer and allowed construction of self-assembled monolayers

with alternating dye molecules across the surface. High surface concentration of

coumarin molecules in self-assembled monolayer (~3×1014 cm-2) was sufficient for

observing stimulated emission from optically excited SAM. The stimulated emission

resulted in lifetime shortening and narrowing the fluorescence spectrum of coumarin

in monolayers. To the best of our knowledge, the observation of lasing from self-

199

assembled coumarin films was the first demonstration of such a possibility in organic

monolayers. The approach could be used for developing SAM dye lasers.

The modified surface assisted photoinduced transient displacement charge

technique (SPTDC) applied for coumarin SAM was shown to be suitable for studying

charge transfer in molecules, assembled into oriented layers on solid surfaces.

SPTDC technique provides quantitative results, which include charge separation

extent and dipole orientation as well as charge recombination rates. A theoretical

basis for evaluating SPTDC dipole signals was developed.

Study of photoinduced charge transfer between 2-aminopurine and guanine

provided evidence of a quite shallow distance dependence of charge transfer in DNA

at room temperature (β = 0.32Å-1). The study of temperature dependence of

photoinduced charge transfer between 2-aminopurine and guanine at various

positions within DNA hairpins revealed that thermal fluctuations of bases play an

important role in governing charge transfer in DNA. The results support the gated

mechanism, where thermally induced conformational fluctuations control the rate of

charge transfer. Modification of a single nucleotide with locked sugar moiety (LNA)

next to 2AP causes local changes in DNA conformation. This leads to a poorer base-

base stacking which is identified by 2AP absorption and circular dichroism.

Correspondingly, a lower efficiency of 2AP* fluorescence quenching by guanine,

which is separated from 2AP by a single LNA modified nucleotide (adenine) is

observed. The experimental evidences obtained in this work emphasize the

sensitivity of charge transfer to static and dynamic base stacking within DNA.

200

The substitution of counterions in DNA with cationic amphiphiles results in

the formation of DNA-amphiphile complex, which is soluble in organic solvents.

DNA in the complex with amphiphiles does not form duplex while in dry organic

solution. However, the thermal stability of DNA duplex was shown to be directly

dependent on the amount of water present in the solution. DNA in the complex has

different conformation while in dry organic solution and adopts close to native B-

form after addition of water. Only complexes of DNA with amphiphile containing

hydrophilic PEG moieties are suitable to study charge transfer in DNA because only

these complexes can form duplex at room temperature upon addition of water.

Immobilization of DNA-amphiphile complexes onto silica surface via the procedure

based on self-assembly of trimethoxyaminosilanes was shown to provide quite high

surface concentration of DNA molecules (~3×1013 cm-2).

The work presented in this dissertation has built a solid foundation for

extending charge transfer study in molecules organized in oriented molecular films

and DNA in particular. While, the answer to the question of the applicability of

modified PTDC method for studying charge separation in molecular films is “yes”,

one final touch to the experimental setup of PTDC technique is left. A more suitable

source of excitation is required for studying charge transfer in DNA equipped with 2-

aminopurine.

Documents

THE STUDY OF CHARGE TRANSFER IN SHORT DNA …snsm/group/dissertations/dissertation_alexey.pdf1. Krasnoslobodtsev, Alexey, Smirnov, Sergei, “Effect of a single locked nucleotide modification